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Sample records for plasmin

  1. Heat Inactivation of Bovine Plasmin.

    NARCIS (Netherlands)

    Metwalli, A.A.M.; Jongh, de H.H.J.; Boekel, van M.A.J.S.

    1998-01-01

    The thermal behaviour of the protease plasmin was studied in the absence and presence of milk components. A global unfolding of the isolated plasmin occurred at 50-55oC, but secondary structure of plasmin increased with increasing temperature, possibly due to aggregation. Plasmin was reversibly inac

  2. Does plasmin have anticoagulant activity?

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    Jane Hoover-Plow

    2010-03-01

    Full Text Available Jane Hoover-PlowJoseph J Jacobs Center for Thrombosis and Vascular Biology, Departments of Cardiovascular Medicine and Molecular Cardiology, Lerner Research Institute Cleveland Clinic, Ohio, USAAbstract: The coagulation and fibrinolytic pathways regulate hemostasis and thrombosis, and an imbalance in these pathways may result in pathologic hemophilia or thrombosis. The plasminogen system is the primary proteolytic pathway for fibrinolysis, but also has important proteolytic functions in cell migration, extracellular matrix degradation, metalloproteinase activation, and hormone processing. Several studies have demonstrated plasmin cleavage and inactivation of several coagulation factors, suggesting plasmin may be not only be the primary fibrinolytic enzyme, but may have anticoagulant properties as well. The objective of this review is to examine both in vitro and in vivo evidence for plasmin inactivation of coagulation, and to consider whether plasmin may act as a physiological regulator of coagulation. While several studies have demonstrated strong evidence for plasmin cleavage and inactivation of coagulation factors FV, FVIII, FIX, and FX in vitro, in vivo evidence is lacking for a physiologic role for plasmin as an anticoagulant. However, inactivation of coagulation factors by plasmin may be useful as a localized anticoagulant therapy or as a combined thrombolytic and anticoagulant therapy.Keywords: thrombosis, anticoagulant, cardiovascular disease, plasminogen’s protease, blood

  3. Plasmin Regulation through Allosteric, Sulfated, Small Molecules

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    Rami A. Al-Horani

    2015-01-01

    Full Text Available Plasmin, a key serine protease, plays a major role in clot lysis and extracellular matrix remodeling. Heparin, a natural polydisperse sulfated glycosaminoglycan, is known to allosterically modulate plasmin activity. No small allosteric inhibitor of plasmin has been discovered to date. We screened an in-house library of 55 sulfated, small glycosaminoglycan mimetics based on nine distinct scaffolds and varying number and positions of sulfate groups to discover several promising hits. Of these, a pentasulfated flavonoid-quinazolinone dimer 32 was found to be the most potent sulfated small inhibitor of plasmin (IC50 = 45 μM, efficacy = 100%. Michaelis-Menten kinetic studies revealed an allosteric inhibition of plasmin by these inhibitors. Studies also indicated that the most potent inhibitors are selective for plasmin over thrombin and factor Xa, two serine proteases in coagulation cascade. Interestingly, different inhibitors exhibited different levels of efficacy (40%–100%, an observation alluding to the unique advantage offered by an allosteric process. Overall, our work presents the first small, synthetic allosteric plasmin inhibitors for further rational design.

  4. Laminin degradation by plasmin regulates long-term potentiation.

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    Nakagami, Y; Abe, K; Nishiyama, N; Matsuki, N

    2000-03-01

    Plasmin is converted from its zymogen plasminogen by tissue type or urokinase type plasminogen activator (PA) and degrades many components of the extracellular matrix (ECM). To explore the possibility that the PA-plasmin system regulates synaptic plasticity, we investigated the effect of plasmin on degradation of ECM and synaptic plasticity by using organotypic hippocampal cultures. High-frequency stimulation produced long-term potentiation (LTP) in control slices, whereas the potentiation was induced but not maintained in slices pretreated with 100 nM plasmin for 6 hr. The baseline synaptic responses were not affected by pretreatment with plasmin. The impairment of LTP maintenance was not observed in slices pretreated with 100 nM plasmin for 6 hr, washed, and then cultured for 24-48 hr in the absence of plasmin. To identify substrates of plasmin, the expression of three major components of ECM, laminin, fibronectin, and type IV collagen, was investigated by immunofluorescence imaging. The three ECM components were widely distributed in the hippocampus, and only laminin was degraded by plasmin pretreatment. The expression level of laminin returned to normal levels when the slices were cultured for 24-48 hr after washout of plasmin. Furthermore, preincubation with anti-laminin antibodies prevented both the degradation of laminin and the impairment of LTP maintenance by plasmin. These results suggest that the laminin-mediated cell-ECM interaction may be necessary for the maintenance of LTP.

  5. Plasmin in nephrotic urine activates the epithelial sodium channel

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    Svenningsen, Per; Bistrup, Claus; Friis, Ulla G;

    2008-01-01

    Proteinuria and increased renal reabsorption of NaCl characterize the nephrotic syndrome. Here, we show that protein-rich urine from nephrotic rats and from patients with nephrotic syndrome activate the epithelial sodium channel (ENaC) in cultured M-1 mouse collecting duct cells and in Xenopus...... laevis oocytes heterologously expressing ENaC. The activation depended on urinary serine protease activity. We identified plasmin as a urinary serine protease by matrix-assisted laser desorption/ionization time of-flight mass spectrometry. Purified plasmin activated ENaC currents, and inhibitors...... of plasmin abolished urinary protease activity and the ability to activate ENaC. In nephrotic syndrome, tubular urokinase-type plasminogen activator likely converts filtered plasminogen to plasmin. Consistent with this, the combined application of urokinase-type plasminogen activator and plasminogen...

  6. Plasmin-loaded echogenic liposomes for ultrasound-mediated thrombolysis.

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    Kandadai, Madhuvanthi A; Meunier, Jason M; Hart, Kimberley; Holland, Christy K; Shaw, George J

    2015-02-01

    Plasmin, a direct fibrinolytic, shows a significantly superior hemostatic safety profile compared to recombinant tissue plasminogen activator (rtPA), the only FDA-approved thrombolytic for the treatment of acute ischemic stroke. The improved safety of plasmin is attributed to the rapid inhibition of free plasmin by endogenous plasmin inhibitors present in very high concentrations (1 μM). However, this rapid inhibition prevents the intravenous (IV) administration of plasmin. In emergency situations, catheter-based local administration is not practical. There is a need for an alternative technique for IV administration of plasmin. A possible solution is the encapsulation of plasmin in echogenic liposomes (ELIP) for protection from inhibitors until ultrasound (US)-triggered release at the clot site. ELIP are bilayer phospholipid vesicles with encapsulated gas microbubbles. US induces oscillation and collapse of the gas bubbles, which facilitates ELIP rupture and delivery of the encapsulated contents. Plasmin-loaded ELIP (PELIP) were manufactured and characterized for size, gas and drug encapsulations, and in vitro thrombolytic efficacy using a human whole blood clot model. Clots were exposed to PELIP with and without exposure to US (center frequency 120 kHz, pulse repetition frequency 1667 Hz, peak-to-peak pressure of 0.35 MPa, 50 % duty cycle). Thrombolytic efficacy was calculated by measuring the change in clot width over a 30-min treatment period using an edge detection MATLAB program. The mean clot lysis obtained with PELIP in the presence of US exposure was 31 % higher than that obtained without US exposure and 15 % higher than that obtained with rtPA treatment (p liposomes.

  7. Contribution of macrophages to plasmin activity in ewe bulk milk

    OpenAIRE

    Albenzio, M; A. Sevi; A. Marzano; Marino, R; Schena, L.; Caroprese , M

    2010-01-01

    A total of 225 bulk sheep milk samples were collected throughout lactation to assess the contribution of macrophages to the regulation of the plasmin/plasminogen system. Samples were analyzed for composition, milk renneting parameters, and for activities of plasmin (PL), plasminogen (PG) and plasminogen activators (PA). Isolation of macrophages from milk was performed using a magnetic positive separation; separated cells were lysed and activity of urokinase-PA was determined. PL activity in m...

  8. Plasmin system of Alzheimer's disease patients: CSF analysis.

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    Martorana, Alessandro; Sancesario, Giulia M; Esposito, Zaira; Nuccetelli, Marzia; Sorge, Roberto; Formosa, Amanda; Dinallo, Vincenzo; Bernardi, Giorgio; Bernardini, Sergio; Sancesario, Giuseppe

    2012-07-01

    Alzheimer's disease (AD) is a multifactorial neurodegenerative disorder characterized by the extracellular deposit of Amyloid beta (Aβ), mainly of the Amyloid beta(1-42) (Aβ(1-42)) peptide in the hippocampus and neocortex leading to progressive cognitive decline and dementia. The possible imbalance between the Aβ production/degradation process was suggested to contribute to the pathogenesis of AD. Among others, the serine protease plasmin has shown to be involved in Aβ(1-42) clearance, a hypothesis strengthened by neuropathological studies on AD brains. To explore whether there is a change in plasmin system in CSF of AD patients, we analyzed CSF samples from AD and age-matched controls, looking at plasminogen, tissue plasminogen activator (t-PA) and plasminogen activator inhibitor (PAI-1) protein levels and t-PA and urokinase plasminogen activator (u-PA) enzymatic activities. We also measured Aβ(1-42), total-tau and phospho-tau (181) CSF levels and sought for a possible relationship between them and plasmin system values. Our findings showed that t-PA, plasminogen and PAI-1 levels, as t-PA enzymatic activity, remained unchanged in AD with respect to controls; u-PA activity was not detected. We conclude that CSF analysis of plasminogen system does not reflect changes observed post-mortem. Unfortunately, the CSF detection of plasmin system could not be a useful biomarker for either AD diagnosis or disease progression. However, these findings do not exclude the possible involvement of the plasmin system in AD.

  9. Pericellular proteolytic cascade by plasmin/plasminogen activator system

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Plasmin/plasminogen activators (PA) are the serine enzyme which digests fibrin and/or fibrinogen. Plasmin is produced by the cleavage of its precursor, plasminogen by PAs (urokinase-type PA and tissue-type PA). These events are expected in the thrmbolytic therapy for thromboembolic deseases. Apart from the blood fibrinolysis mentioned above, new role of plasmin/plasminogen activators has been extensively investigated in the field of cellular biology. On the cell surface, the receptor for urokinase-type PA (u-PAR) was found (that for t-PA has not cloned yet). Then, plasmin as well as u-PA itself activates pro-form of matrix metalloproteinases (MMPs) around the pericellular space. These proteolytic activities by u-PA, plasmin and MMPs induce the degradation of extracellular matrix (ECM), affording the cells certain enviroment for their biological function. Further, the coupling of u-PA/u-PAR system and integrins can generate intracellular signal transductions which take part in the regulation of cell proliferation, attachment or migration followed by various physiological and pathophysiological functions. These serial mechanisms are the principle of pericellular proteolytic cascade.

  10. Optimization of Cyclic Plasmin Inhibitors: From Benzamidines to Benzylamines.

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    Hinkes, Stefan; Wuttke, André; Saupe, Sebastian M; Ivanova, Teodora; Wagner, Sebastian; Knörlein, Anna; Heine, Andreas; Klebe, Gerhard; Steinmetzer, Torsten

    2016-07-14

    New macrocyclic plasmin inhibitors based on our previously optimized P2-P3 core segment have been developed. In the first series, the P4 residue was modified, whereas the 4-amidinobenzylamide in P1 position was maintained. The originally used P4 benzylsulfonyl residue could be replaced by various sulfonyl- or urethane-like protecting groups. In the second series, the P1 benzamidine was modified and a strong potency and excellent selectivity was retained by incorporation of p-xylenediamine. Several analogues inhibit plasmin in the subnanomolar range, and their potency against related trypsin-like serine proteases including trypsin itself could be further reduced. Selected derivatives have been tested in a plasma fibrinolysis assay and are more effective than the reference inhibitor aprotinin. The crystal structure of one inhibitor was determined in complex with trypsin. The binding mode reveals a sterical clash of the inhibitor's linker segment with the 99-hairpin loop of trypsin, which is absent in plasmin.

  11. Postsynaptic blockade of inhibitory postsynaptic currents by plasmin in CA1 pyramidal cells of rat hippocampus.

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    Mizutani, A; Tanaka, T; Saito, H; Matsuki, N

    1997-06-27

    We have shown previously that plasmin facilitated the generation of long-term potentiation (LTP) in CA1 and dentate region of rat hippocampus. In the present study, we investigated the effects of plasmin on postsynaptic currents in CA1 pyramidal neurons of rat hippocampal slices. Plasmin (100 nM) had no effect on NMDA nor on non-NMDA receptor-mediated excitatory postsynaptic currents. However, plasmin significantly decreased GABA(A) receptor-mediated inhibitory postsynaptic currents. This effect of plasmin disappeared when intracellular Ca2+ was strongly chelated with BAPTA. Furthermore, plasmin attenuated the GABA-induced currents in CA1 pyramidal cells. These results suggest that the STP-enhancing effect of plasmin is due to a blockade of postsynaptic GABA(A) responses and that an increase in intracellular Ca2+ by plasmin may be involved in its mechanism.

  12. Plasmin is a specific stimulus of the 5-lipoxygenase pathway of human peripheral monocytes.

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    Weide, I; Tippler, B; Syrovets, T; Simmet, T

    1996-10-01

    The objective of this study was to characterize the plasmin-induced stimulation of leukotriene (LT) B4 biosynthesis in human peripheral monocytes (PM). Plasmin up to 175 x 10(-3) CTA U/ml triggers a concentration-dependent release of 5-lipoxygenase-derived LTB4 while release of the cyclooxygenase products thromboxane (TX) B2 and prostaglandin (PG) E2 remained unaffected. The stimulatory effect appeared to be specific in as much as 1) it was found in PM, but not in polymorphonuclear neutrophils (PMN), 2) it requires the lysine binding sites of plasmin molecule since it was inhibited by the lysine analogues 6-aminohexanoic acid (6-AHA) and trans-4(aminomethyl)cyclohexane-1-carboxylic acid (t-AMCA), 3) the intact catalytic center of plasmin is required since neither plasminogen nor catalytic center-blocked plasmin share the stimulatory effect of active plasmin, 4) other serine proteases such as alpha-chymotrypsin, human neutrophil elastase and cathepsin G did not stimulate release of detectable amounts of LTB4 from PM. In addition, catalytic center-blocked plasmin antagonized the stimulatory effect of active plasmin. Plasmin-mediated monocyte activation apparently proceeds via a pertussis toxin-sensitive G protein. Plasmin did not increase inositol (1,4,5) trisphosphate levels, but a time- and concentration-dependent stimulation of cyclic GMP formation was observed. The data show that plasmin is a specific stimulus for human peripheral monocytes. Plasmin may be an important link between the coagulation cascade and inflammatory reactions.

  13. Plasmin reduces fibronectin deposition by mesangial cells in a protease-activated receptor-1 independent manner

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    Maaike Waasdorp

    2017-07-01

    Conclusion: Expression profiling identified plasmin as potential endogenous PAR-1 agonist driving DN. Instead of inducing fibronectin expression, plasmin however reduced mesangial fibronectin deposition in vitro. Therefore we conclude that plasmin may not be the endogenous PAR-1 agonist potentiating DN.

  14. Contribution of macrophages to plasmin activity in ewe bulk milk

    Directory of Open Access Journals (Sweden)

    M. Albenzio

    2010-04-01

    Full Text Available A total of 225 bulk sheep milk samples were collected throughout lactation to assess the contribution of macrophages to the regulation of the plasmin/plasminogen system. Samples were analyzed for composition, milk renneting parameters, and for activities of plasmin (PL, plasminogen (PG and plasminogen activators (PA. Isolation of macrophages from milk was performed using a magnetic positive separation; separated cells were lysed and activity of urokinase-PA was determined. PL activity in milk decreased during lactation (P < 0.001. The reduction in plasmin activity recorded in the mid and late lactation milk matched with the increase in PG/PL ratio (P < 0.001. The activity of PA increased throughout lactation (P < 0.001, the highest value being recorded in the late lactation milk.The amount of isolated and concentrated macrophages was higher in early and mid lactation milk than in late lactation milk (P < 0.01. Stage of lactation did not influence the activity of u-PA detected in isolated macrophages. The activity of u-PA associated with macrophages was lower than total PA activity detected in milk. Our results lend support to the hypothesis that in ewe bulk milk from healthy flocks macrophages only slightly contributed to the activation of plasmin/plasminogen system.

  15. Generation of plasmin during acute attacks of hereditary angioedema.

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    Cugno, M; Hack, C E; de Boer, J P; Eerenberg, A J; Agostoni, A; Cicardi, M

    1993-01-01

    Hereditary angioedema is caused by a genetic deficiency of C1-inhibitor, a serine protease inhibitor that regulates activation of complement, contact, and fibrinolytic systems. Symptoms (bouts of subcutaneous and mucous swelling) depend on the release of a vasoactive mediator, probably through activation of these three systems. We studied the interrelationship among complement, contact, and fibrinolytic activation in 23 patients with hereditary angiodema, 18 during remission and five during an attack, by measuring plasma levels of C1-C1 inhibitor, factor XIIa-C1 inhibitor, kallikrein-C1 inhibitor, and plasmin-alpha 2-antiplasmin complexes, tissue plasminogen activator, and urokinase plasminogen activator. In addition, cleavage of high-molecular weight kininogen was detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis and quantified by densitometry. During remission, plasma levels of C1-C1 inhibitor complexes were elevated (p = 0.0002), whereas the other parameters were within the normal range. During acute attacks, not only plasma levels of C1-C1 inhibitor complexes but also those of plasmin-alpha 2-antiplasmin complexes (P = 0.0009) and cleaved high-molecular weight kininogen were elevated. A positive correlation between plasmin-alpha 2-antiplasmin complexes and cleaved high-molecular weight kininogen was observed (r = 0.75, p attacks is associated with the activation of the fibrinolytic system.

  16. Plasmin activity in UHT milk: relationship between proteolysis, age gelation, and bitterness.

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    Rauh, Valentin M; Johansen, Lene B; Ipsen, Richard; Paulsson, Marie; Larsen, Lotte B; Hammershøj, Marianne

    2014-07-16

    Plasmin, the major indigenous protease in milk, is linked to quality defects in dairy products. The specificity of plasmin on caseins has previously been studied using purified caseins and in the indigenous peptide profile of milk. We investigated the specificity and proteolytic pathway of plasmin in directly heated UHT milk (>150 °C for gelation and bitter peptides. Sixty-six peptides from αS- and β-caseins could be attributed to plasmin activity during the storage period, of which 23 were potentially bitter. Plasmin exhibited the highest affinity for the hydrophilic regions in the caseins that most probably were exposed to the serum phase and the least affinity for hydrophobic or phosphorylated regions. The proteolytic pattern observed suggests that plasmin destabilizes the casein micelle by hydrolyzing casein-casein and casein-calcium phosphate interaction sites, which may subsequently cause age gelation in UHT milk.

  17. Prostasin-dependent activation of epithelial Na+ channels by low plasmin concentrations

    DEFF Research Database (Denmark)

    Svenningsen, Per; Uhrenholt, Torben R; Palarasah, Yaseelan;

    2009-01-01

    plasmin-stimulated ENaC current in single M-1 cells, as measured by whole-cell patch clamp. In M-1 cells expressing heterologous FLAG-tagged prostasin, gammaENaC and prostasin were colocalized. A monoclonal antibody directed against the inhibitory peptide of gammaENaC produced specific immunofluorescence...... plasmin-stimulated ENaC current in monolayers of M-1 cells at low plasmin concentration (1-4 microg/ml). At a high plasmin concentration of 30 microg/ml, there was no difference between cell layers treated with or without PI-PLC. Knockdown of prostasin attenuated binding of plasmin to M1 cells and blocked......Several pathophysiological conditions, including nephrotic syndrome, are characterized by increased renal activity of the epithelial Na(+) channel (ENaC). We recently identified plasmin in nephrotic urine as a stimulator of ENaC activity and undertook this study to investigate the mechanism...

  18. Diagnosis of deep venous thrombosis by phlebography and /sup 99/Tcsup (m)-Plasmin

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    Edenbrandt, C.M.; Nilsson, J.; Ohlin, P. (County Hospital, Helsingborg (Sweden). Dept. of Medicine, Diagnostic Radiology and Clinical Physology)

    1982-01-01

    One hundred and thirty-four patients admitted to the medical emergency ward due to suspected deep venous thrombosis (DVT) were examined. The uptake of intravenously injected porcine /sup 99/Tcsup (m)-plasmin was estimated in both legs. Thereafter, phlebography was performed using a high osmolar contrast medium. All phlebographies were evaluated independently. All patients with negative phlebography were examined after 3-5 days. The plasmin test and phlebography were repeated when called for. The sensivity of the plasmin test was 100% and the specificity 51% when compared to phlebography. The extension of the DVT as demonstrated by the plasmin test was similar to that determined by phlebography. Post-phlebographic trombosis was very rare. It is concluded that /sup 99/Tcsup (m)-plasmin test is a rapid method, convenient to the patient and well suitable as a screening test. The results indicate that a negative plasmin test excludes DVT while a positive test necessitates additional examination by phlebography.

  19. Plasmin is a potent and specific chemoattractant for human peripheral monocytes acting via a cyclic guanosine monophosphate-dependent pathway.

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    Syrovets, T; Tippler, B; Rieks, M; Simmet, T

    1997-06-15

    We have previously reported that the serine protease plasmin generated during contact activation of human plasma triggers biosynthesis of leukotrienes (LTs) in human peripheral monocytes (PMs), but not in polymorphonuclear neutrophils (PMNs). We now show that purified plasmin acts as a potent chemoattractant on human monocytes, but not on PMNs. Human plasmin or plasminogen activated with urokinase, but not active site-blocked plasmin or plasminogen, elicited monocyte migration across polycarbonate membranes. Similarly, stimulation of monocytes with plasmin, but not with active site-blocked plasmin or plasminogen, induced actin polymerization. As assessed by checkerboard analysis, the plasmin-mediated monocyte locomotion was a true chemotaxis. The plasmin-induced chemotactic response was inhibited by the lysine analog trans-4-(aminomethyl)cyclohexane-1-carboxylic acid (t-AMCA), which prevents binding of plasmin/ogen to the appropriate membrane binding sites. In addition, active site-blocked plasmin inhibited monocyte migration triggered by active plasmin. Further, plasmin-induced monocyte chemotaxis was inhibited by pertussis toxin (PTX) and 1-O-hexadecyl-2-O-methyl-rac-glycerol (HMG) and chelerythrine, two structurally unrelated inhibitors of protein kinase C (PKC). Plasmin, but not active site-blocked plasmin or plasminogen, triggered formation of cyclic guanosine monophosphate (cGMP) in monocytes. LY83583, an inhibitor of soluble guanylyl cyclase, inhibited both plasmin-induced cGMP formation and the chemotactic response. The latter effect could be antagonized by 8-bromo-cGMP. In addition, KT5823 and (Rp)-8-(p-chlorophenylthio)guanosine-3',5'-cyclic monophosphorothioate [(Rp)-8-pCPT-cGMPs], two structurally unrelated inhibitors of cGMP-dependent protein kinase, inhibited plasmin-mediated monocyte chemotaxis. Thus, beyond being a stimulus for lipid mediator release, plasmin is a potent and specific chemoattractant for human monocytes acting via a c

  20. Plasmin plays an essential role in amplification of psoriasiform skin inflammation in mice.

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    Qun Li

    Full Text Available BACKGROUND: Although increased levels of plasminogen activators have been found in psoriatic lesions, the role of plasmin converted from plasminogen by plasminogen activators in pathogenesis of psoriasis has not been investigated. METHODOLOGY/PRINCIPAL FINDINGS: Here we examined the contribution of plasmin to amplification of inflammation in patients with psoriasis. We found that plasminogen was diminished, but that the amount and activity of its converted product plasmin were markedly increased in psoriasis. Moreover, annexin II, a receptor for plasmin was dramatically increased in both dermis and epidermis in psoriasis. Plasmin at sites of inflammation was pro-inflammatory, eliciting production of inflammatory factors, including CC chemokine ligand 20 (CCL20 and interleukin-23 (IL-23, that was mediated by the nuclear factor-kappaB (NF-κB signaling pathway and that had an essential role in the recruitment and activation of pathogenic C-C chemokine receptor type 6 (CCR6+ T cells. Moreover, intradermal injection of plasmin or plasmin together with recombinant monocyte/macrophage chemotactic protein-1 (MCP-1 resulted in induction of psoriasiform skin inflammation around the injection sites with several aspects of human psoriasis in mice. CONCLUSIONS/SIGNIFICANCE: Plasmin converted from plasminogen by plasminogen activators plays an essential role in amplification of psoriasiform skin inflammation in mice, and targeting plasmin receptor--annexin II--may harbor therapeutic potential for the treatment of human psoriasis.

  1. alpha 2-Plasmin inhibitor metabolism in patients with liver cirrhosis.

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    Knot, E A; Drijfhout, H R; ten Cate, J W; de Jong, E; Iburg, A H; Kahlé, L H; Grijm, R

    1985-03-01

    We describe the metabolism of purified human alpha 2-plasmin inhibitor in patients with liver cirrhosis to determine whether low plasma concentrations of alpha 2-plasmin inhibitor are the result of impaired synthesis or increased catabolism or both. A kinetic study was performed with 131I-alpha 2-plasmin inhibitor as a sensitive parameter of fibrinolysis in 14 patients with histologically proved liver cirrhosis compared with six healthy control subjects. Eight patients had macronodular cirrhosis (with positive hepatitis B surface antigen), and six had micronodular cirrhosis as a result of alcohol abuse. None of the patients had clinical signs of ascites, and in all the disease was stabilized. alpha 2-Plasmin inhibitor levels biologically and immunologically measured were decreased in all patients. Ten microCi 131I-alpha 2PI was injected intravenously, the disappearance of plasma radioactivity was measured, and turnover data were calculated according to the function x(t) = A1e-alpha 1t + A2e-alpha 2t + Be-beta t. Mean (+/- SD) turnover data in the control subjects were plasma radioactivity half-life 60.1 +/- 5.3 hours, fractional catabolic rate constant of the plasma pool 0.0318 +/- 0.0106 hr-1, and absolute catabolic (synthetic) rate constant 2.10 +/- 0.60 mg/kg/day. The alpha 1-phase was 1.26 +/- 0.23, and the transcapillary influx constant (k2,1) was 0.974 +/- 0.109 hr-1. In the patients, plasma radioactivity half-life was 58.7 +/- 12.09 hr, and fractional catabolic rate constant of the plasma pool 0.0283 +/- 0.0043 hr-1. The alpha 1-phase 4.74 +/- 6.48 and the transcapillary influx (k2,1) 3.08 +/- 3.9 hr-1 were both significantly increased compared with control values (p less than 0.05 and p less than 0.05, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

  2. Urinary plasmin activates collecting duct ENaC current in preeclampsia

    DEFF Research Database (Denmark)

    Buhl, KB; Friis, Ulla Glenert; Svenningsen, Per;

    2012-01-01

    In nephrotic syndrome, plasminogen is aberrantly filtered from plasma to the urinary space and activated along the tubular system. In vitro, plasmin increases ENaC current by proteolytic cleavage of the γ-subunit. It was hypothesized that preeclampsia is associated with plasmin-dependent ability...

  3. Possible involvement of plasmin in long-term potentiation of rat hippocampal slices.

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    Mizutani, A; Saito, H; Matsuki, N

    1996-11-11

    Effects of proteases and protease inhibitors on generation of long-term potentiation (LTP) were investigated in the CA1 and dentate regions of rat hippocampus. Plasmin, a serine protease, and its precursor plasminogen significantly enhanced short-term potentiation (STP) induced by a weak tetanic stimulation, without affecting basal responses. The STP-enhancing effect of plasmin disappeared by concomitant perfusion of alpha 2-antiplasmin, an endogenous plasmin inhibitor. Other proteases, such as thrombin, trypsin and cathepsin B, did not affect STP. On the other hand, alpha 2-antiplasmin and leupeptin significantly attenuated LTP induced by a strong tetanus though plasminogen or plasmin itself did not influence LTP. Furthermore, plasminogen and plasmin did not affect NMDA receptor-mediated synaptic responses in the absence of extracellular Mg2+. These results suggest that endogenous plasmin is involved in the mechanism of LTP in CA1 and dentate regions of rat hippocampus and that the STP-enhancing effect of plasmin is independent of NMDA receptors.

  4. Plasmin inhibitors prevent leukocyte accumulation and remodeling events in the postischemic microvasculature.

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    Christoph A Reichel

    Full Text Available Clinical trials revealed beneficial effects of the broad-spectrum serine protease inhibitor aprotinin on the prevention of ischemia-reperfusion (I/R injury. The underlying mechanisms remained largely unclear. Using in vivo microscopy on the cremaster muscle of male C57BL/6 mice, aprotinin as well as inhibitors of the serine protease plasmin including tranexamic acid and ε-aminocaproic acid were found to significantly diminish I/R-elicited intravascular firm adherence and (subsequent transmigration of neutrophils. Remodeling of collagen IV within the postischemic perivenular basement membrane was almost completely abrogated in animals treated with plasmin inhibitors or aprotinin. In separate experiments, incubation with plasmin did not directly activate neutrophils. Extravascular, but not intravascular administration of plasmin caused a dose-dependent increase in numbers of firmly adherent and transmigrated neutrophils. Blockade of mast cell activation as well as inhibition of leukotriene synthesis or antagonism of the platelet-activating-factor receptor significantly reduced plasmin-dependent neutrophil responses. In conclusion, our data suggest that extravasated plasmin(ogen mediates neutrophil recruitment in vivo via activation of perivascular mast cells and secondary generation of lipid mediators. Aprotinin as well as the plasmin inhibitors tranexamic acid and ε-aminocaproic acid interfere with this inflammatory cascade and effectively prevent postischemic neutrophil responses as well as remodeling events within the vessel wall.

  5. The role of plasmin in the pathogenesis of murine multiple myeloma.

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    Eiamboonsert, Salita; Salama, Yousef; Watarai, Hiroshi; Dhahri, Douaa; Tsuda, Yuko; Okada, Yoshio; Hattori, Koichi; Heissig, Beate

    2017-06-24

    Aside from a role in clot dissolution, the fibrinolytic factor, plasmin is implicated in tumorigenesis. Although abnormalities of coagulation and fibrinolysis have been reported in multiple myeloma patients, the biological roles of fibrinolytic factors in multiple myeloma (MM) using in vivo models have not been elucidated. In this study, we established a murine model of fulminant MM with bone marrow and extramedullar engraftment after intravenous injection of B53 cells. We found that the fibrinolytic factor expression pattern in murine B53 MM cells is similar to the expression pattern reported in primary human MM cells. Pharmacological targeting of plasmin using the plasmin inhibitors YO-2 did not change disease progression in MM cell bearing mice although systemic plasmin levels was suppressed. Our findings suggest that although plasmin has been suggested to be a driver for disease progression using clinical patient samples in MM using mostly in vitro studies, here we demonstrate that suppression of plasmin generation or inhibition of plasmin cannot alter MM progression in vivo. Copyright © 2017 Elsevier Inc. All rights reserved.

  6. Homocysteine and its thiolactone impair plasmin activity induced by urokinase or streptokinase in vitro.

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    Kolodziejczyk-Czepas, Joanna; Talar, Beata; Nowak, Pawel; Olas, Beata; Wachowicz, Barbara

    2012-04-01

    Mechanisms of homocysteine (Hcy) contribution to thrombosis are complex and only partly recognized. The available data suggest that the prothrombotic activity of homocysteine may be not only a result of the changes in coagulation process and endothelial dysfunction, but also the dysfunction of fibrinolysis. The aim of the present work was to assess the effects of homocysteine (10-100 μM mM) and its thiolactone (HTL, 0.1-1 μM) on plasminogen and plasmin functions in vitro. The amidolytic activity of generated plasmin in Hcy or HTL-treated plasminogen and plasma samples was measured by the hydrolysis of chromogenic substrate. Effects of Hcy and HTL on proteolytic activity of plasmin were monitored electrophoretically, by using of fibrinogen as a substrate. The exposure of human plasma and purified plasminogen to Hcy or HTL resulted in the decrease of urokinase-induced plasmin activity. In plasminogen samples treated with the highest concentration of homocysteine (100 μM) or thiolactone (1 μM), the activity of plasmin was inhibited by about 50%. In plasma samples, a reduction of amidolytic activity by about 30% (for 100 μM Hcy) and 40% (for 1 μM HTL), was observed. Both Hcy and HTL were also able to diminish the streptokinase-induced proteolytic activity of plasmin. In conclusion, the results obtained in this study demonstrate that Hcy and HTL may affect fibrinolytic properties of plasminogen and plasma, leading to the decrease of plasmin activity.

  7. Uptake of Plasmin-PN-1 Complexes in Early Human Atheroma.

    Science.gov (United States)

    Boukais, Kamel; Bayles, Richard; Borges, Luciano de Figueiredo; Louedec, Liliane; Boulaftali, Yacine; Ho-Tin-Noé, Benoit; Arocas, Véronique; Bouton, Marie-Christine; Michel, Jean-Baptiste

    2016-01-01

    Zymogens are delivered to the arterial wall by radial transmural convection. Plasminogen can be activated within the arterial wall to produce plasmin, which is involved in evolution of the atherosclerotic plaque. Vascular smooth muscle cells (vSMCs) protect the vessels from proteolytic injury due to atherosclerosis development by highly expressing endocytic LDL receptor-related protein-1 (LRP-1), and by producing anti-proteases, such as Protease Nexin-1 (PN-1). PN-1 is able to form covalent complexes with plasmin. We hypothesized that plasmin-PN-1 complexes could be internalized via LRP-1 by vSMCs during the early stages of human atheroma. LRP-1 is also responsible for the capture of aggregated LDL in human atheroma. Plasmin activity and immunohistochemical analyses of early human atheroma showed that the plasminergic system is activated within the arterial wall, where intimal foam cells, including vSMCs and platelets, are the major sites of PN-1 accumulation. Both PN-1 and LRP-1 are overexpressed in early atheroma at both messenger and protein levels. Cell biology studies demonstrated an increased expression of PN-1 and tissue plasminogen activator by vSMCs in response to LDL. Plasmin-PN-1 complexes are internalized via LRP-1 in vSMCs, whereas plasmin alone is not. Tissue PN-1 interacts with plasmin in early human atheroma via two complementary mechanisms: plasmin inhibition and tissue uptake of plasmin-PN-1 complexes via LRP-1 in vSMCs. Despite this potential protective effect, plasminogen activation by vSMCs remains abnormally elevated in the intima in early stages of human atheroma.

  8. Remission of nephrotic syndrome diminishes urinary plasmin content and abolishes activation of ENaC

    DEFF Research Database (Denmark)

    Andersen, René F; Buhl, Kristian Bergholt; Jensen, Boye L.;

    2013-01-01

    BACKGROUND: Urinary plasmin activates the epithelial Na(+) channel (ENaC) in vitro and may possibly be a mechanism of sodium retention in nephrotic syndrome (NS). This study used a paired design to test the hypothesis that remission of NS is associated with a decreased content of urinary plasmin...... and reduced ability of patients' urine to activate ENaC. METHODS: Samples were collected during active NS and at stable remission from 20 patients with idiopathic NS, aged 9.1 ± 3.2 years. Plasminogen-plasmin concentration was measured with an enzyme-linked immunosorbent assay. Western immunoblotting...

  9. Neuronal death in the hippocampus is promoted by plasmin-catalyzed degradation of laminin.

    Science.gov (United States)

    Chen, Z L; Strickland, S

    1997-12-26

    Excess excitatory amino acids can provoke neuronal death in the hippocampus, and the extracellular proteases tissue plasminogen activator (tPA) and plasmin (ogen) have been implicated in this death. To investigate substrates for plasmin that might influence neuronal degeneration, extracellular matrix (ECM) protein expression was examined. Laminin is expressed in the hippocampus and disappears after excitotoxin injection. Laminin disappearance precedes neuronal death, is spatially coincident with regions that exhibit neuronal loss, and is blocked by either tPA-deficiency or infusion of a plasmin inhibitor, both of which also block neuronal degeneration. Preventing neuron-laminin interaction by infusion of anti-laminin antibodies into tPA-deficient mice restores excitotoxic sensitivity to their hippocampal neurons. These results indicate that disruption of neuron-ECM interaction via tPA/plasmin catalyzed degradation of laminin sensitizes hippocampal neurons to cell death.

  10. Plasmin-driven fibrinolysis facilitates skin tumor growth in a gender-dependent manner

    DEFF Research Database (Denmark)

    Hald, Andreas; Eickhardt, Hanne; Maerkedahl, Rasmus Baadsgaard

    2012-01-01

    Rearrangement of the skin during wound healing depends on plasmin and plasminogen, which serve to degrade fibrin depositions in the provisional matrix and thereby facilitate keratinocyte migration. In the current study, we investigated whether plasmin and plasminogen likewise played a role during...... by superimposing heterozygous fibrinogen deficiency on Plg(-)(/)(-) mice. Tumors derived from these Fib(-)(/+);Plg(-)(/)(-) mice displayed a significantly decreased level of tumor thrombosis compared with Plg(-)(/)(-) mice. In summary, these data indicate that plasmin-driven fibrinolysis facilitates tumor growth...... by maintaining patency of the tumor vasculature.-Hald, A., Eickhardt, H., Maerkedahl, R. B., Feldborg, C. W., Egerod, K. L., Engelholm. L. H., Laerum, O. D., Lund, L. R., Rønø, B. Plasmin-driven fibrinolysis facilitates skin tumor growth in a gender-dependent manner....

  11. Dendritic Cell-Mediated Phagocytosis but Not Immune Activation Is Enhanced by Plasmin.

    OpenAIRE

    Borg, Rachael J.; Samson, Andre L.; Amanda E-L Au; Anja Scholzen; Martina Fuchsberger; Kong, Ying Y.; Roxann Freeman; Nicole A Mifsud; Magdalena Plebanski; Medcalf, Robert L.

    2015-01-01

    Removal of dead cells in the absence of concomitant immune stimulation is essential for tissue homeostasis. We recently identified an injury-induced protein misfolding event that orchestrates the plasmin-dependent proteolytic degradation of necrotic cells. As impaired clearance of dead cells by the innate immune system predisposes to autoimmunity, we determined whether plasmin could influence endocytosis and immune cell stimulation by dendritic cells - a critical cell that links the innate an...

  12. Contact thermography, 99mTc-plasmin scintimetry and 99mTc-plasmin scintigraphy as screening methods for deep venous thrombosis following major hip surgery

    DEFF Research Database (Denmark)

    Christensen, S W; Wille-Jørgensen, P; Kjaer, L

    1987-01-01

    Fifty-six patients scheduled for total hip alloplasty were screened for deep venous thrombosis by means of 99mTc-plasmin scintimetry, 99mTc-plasmin scintigraphy and contact thermography. Investigations were performed on the seventh postoperative day, and a total of 112 legs were examined. Bilateral....... The nosographic sensitivity/specificity was 33%/75% for scintimetry, 50%/91% for scintigraphy and 33%/87% for contact thermography, respectively. It is concluded that all three tests are of no value as screening methods for deep venous thrombosis following major elective hip surgery....

  13. Role of tissue plasminogen activator/plasmin cascade in delayed neuronal death after transient forebrain ischemia.

    Science.gov (United States)

    Takahashi, Hiroshi; Nagai, Nobuo; Urano, Tetsumei

    We studied the possible involvement of the tissue plasminogen activator (t-PA)/plasmin system on both delayed neuronal death in the hippocampus and the associated enhancement of locomotor activity in rats, after transient forebrain ischemia induced by a four-vessel occlusion (FVO). Seven days after FVO, locomotor activity was abnormally increased and, after 10 days, pyramidal cells were degraded in the CA1 region of the hippocampus. FVO increased the t-PA antigen level and its activity in the hippocampus, which peaked at 4 h. Both the enhanced locomotor activity and the degradation of pyramidal cells were significantly suppressed by intracerebroventricular injection of aprotinin, a plasmin inhibitor, at 4 h but not during FVO. These results suggest the importance of the t-PA/plasmin cascade during the early pathological stages of delayed neuronal death in the hippocampus following transient forebrain ischemia.

  14. The degradation of Abeta(25-35) by the serine protease plasmin is inhibited by aluminium.

    Science.gov (United States)

    Korchazhkina, Olga V; Ashcroft, Alison E; Kiss, Tamas; Exley, Christopher

    2002-10-01

    The catabolism of amyloid beta peptides (Abeta) may be important in their accumulation in the brain in both early and late-onset Alzheimer's disease (AD). The serine protease plasmin is one of a suite of proteases implicated in AD. It is a promoter of alpha-cleavage of the amyloid beta precursor protein (AbetaPP) and will degrade Abeta in vitro. Herein we have demonstrated cleavage of the amyloidogenic Abeta(25-35) by plasmin to produce the non-amyloidogenic fragment Abeta(29-35). The activity of plasmin was halved by pre-mixing it with aluminium (Al) prior to its addition to the peptide. An interaction between Al and proteases involved in the catabolism of Abeta might define the putative link between Al and AD.

  15. Exogenous t-PA administration increases hippocampal mature BDNF levels. plasmin- or NMDA-dependent mechanism?

    Science.gov (United States)

    Rodier, Marion; Prigent-Tessier, Anne; Béjot, Yannick; Jacquin, Agnès; Mossiat, Claude; Marie, Christine; Garnier, Philippe

    2014-01-01

    Brain-derived neurotrophic factor (BDNF) through TrkB activation is central for brain functioning. Since the demonstration that plasmin is able to process pro-BDNF to mature BDNF and that these two forms have opposite effects on neuronal survival and plasticity, a particular attention has been paid to the link between tissue plasminogen activator (tPA)/plasmin system and BDNF metabolism. However, t-PA via its action on different N-methyl-D-aspartate (NMDA) receptor subunits is also considered as a neuromodulator of glutamatergic transmission. In this context, the aim of our study was to investigate the effect of recombinant (r)t-PA administration on brain BDNF metabolism in rats. In the hippocampus, we found that rt-PA (10 mg/kg) administration induced a progressive increase in mature BDNF levels associated with TrkB activation. In order to delineate the mechanistic involved, plasmin activity was assessed and its inhibition was attempted using tranexamic acid (30 or 300 mg/kg, i.v.) while NMDA receptors were antagonized with MK801 (0.3 or 3 mg/kg, i.p.) in combination with rt-PA treatment. Our results showed that despite a rise in rt-PA activity, rt-PA administration failed to increase hippocampal plasmin activity suggesting that the plasminogen/plasmin system is not involved whereas MK801 abrogated the augmentation in mature BDNF levels observed after rt-PA administration. All together, our results show that rt-PA administration induces increase in hippocampal mature BDNF expression and suggests that rt-PA contributes to the control of brain BDNF synthesis through a plasmin-independent potentiation of NMDA receptors signaling.

  16. Exogenous t-PA administration increases hippocampal mature BDNF levels. plasmin- or NMDA-dependent mechanism?

    Directory of Open Access Journals (Sweden)

    Marion Rodier

    Full Text Available Brain-derived neurotrophic factor (BDNF through TrkB activation is central for brain functioning. Since the demonstration that plasmin is able to process pro-BDNF to mature BDNF and that these two forms have opposite effects on neuronal survival and plasticity, a particular attention has been paid to the link between tissue plasminogen activator (tPA/plasmin system and BDNF metabolism. However, t-PA via its action on different N-methyl-D-aspartate (NMDA receptor subunits is also considered as a neuromodulator of glutamatergic transmission. In this context, the aim of our study was to investigate the effect of recombinant (rt-PA administration on brain BDNF metabolism in rats. In the hippocampus, we found that rt-PA (10 mg/kg administration induced a progressive increase in mature BDNF levels associated with TrkB activation. In order to delineate the mechanistic involved, plasmin activity was assessed and its inhibition was attempted using tranexamic acid (30 or 300 mg/kg, i.v. while NMDA receptors were antagonized with MK801 (0.3 or 3 mg/kg, i.p. in combination with rt-PA treatment. Our results showed that despite a rise in rt-PA activity, rt-PA administration failed to increase hippocampal plasmin activity suggesting that the plasminogen/plasmin system is not involved whereas MK801 abrogated the augmentation in mature BDNF levels observed after rt-PA administration. All together, our results show that rt-PA administration induces increase in hippocampal mature BDNF expression and suggests that rt-PA contributes to the control of brain BDNF synthesis through a plasmin-independent potentiation of NMDA receptors signaling.

  17. Concentraciones del activador tisular del plasminógeno y del inhibidor del activador del plasminógeno en pacientes con homocistinuria clásica

    Directory of Open Access Journals (Sweden)

    Beatriz Meneses

    2001-04-01

    Full Text Available El aumento moderado o severo de Homocisteína (Hcy en sangre y las alteraciones en el sistema fibrinolítico, se asocian con enfermedad vascular que afecta venas y arterias de todos los calibres. Los pacientes con homocistinuria clásica padecen con mucha frecuencia eventos tromboembólicos, que son su principal causa de mortalidad. El objetivo de este estudio fue determinar las concentraciones de Hcy, Inhibidor del Activador del Plasminógeno tipo 1 (PAI-1 y Activador tisular del Plasminógeno (tPA en pacientes con homocistinuria clásica, en familiares de éstos y en población control sana.

  18. Why does the increase of plasmin worsen the coagulation properties of milk in dairy sheep?

    Directory of Open Access Journals (Sweden)

    G. Enne

    2010-01-01

    Full Text Available The concentrations of whole casein and fat are the factors which play the most important role in determining the cheese-making qualities of milk. Plasmin (PL is the main proteolytic enzyme in milk and has been found to be associated with enhanced casein hydrolysation.

  19. A novel urokinase receptor-targeted inhibitor for plasmin and matrix metalloproteinases suppresses vein graft disease

    NARCIS (Netherlands)

    Eefting, D.; Seghers, L.; Grimbergen, J.M.; Vries, M.R. de; Boer, H.C. de; Lardenoye, J.W.H.P.; Jukema, J.W.; Bockel, J.H. van; Quax, P.H.A.

    2010-01-01

    Aims Matrix metalloproteinases (MMP) and plasminogen activator (PA)/plasmin-mediated proteolysis, especially at the cell surface, play important roles in matrix degeneration and smooth muscle cell migration, which largely contributes to vein graft failure. In this study, a novel hybrid protein was d

  20. Complement Activation in Arterial and Venous Thrombosis is Mediated by Plasmin

    Science.gov (United States)

    Foley, Jonathan H.; Walton, Bethany L.; Aleman, Maria M.; O'Byrne, Alice M.; Lei, Victor; Harrasser, Micaela; Foley, Kimberley A.; Wolberg, Alisa S.; Conway, Edward M.

    2016-01-01

    Thrombus formation leading to vaso-occlusive events is a major cause of death, and involves complex interactions between coagulation, fibrinolytic and innate immune systems. Leukocyte recruitment is a key step, mediated partly by chemotactic complement activation factors C3a and C5a. However, mechanisms mediating C3a/C5a generation during thrombosis have not been studied. In a murine venous thrombosis model, levels of thrombin–antithrombin complexes poorly correlated with C3a and C5a, excluding a central role for thrombin in C3a/C5a production. However, clot weight strongly correlated with C5a, suggesting processes triggered during thrombosis promote C5a generation. Since thrombosis elicits fibrinolysis, we hypothesized that plasmin activates C5 during thrombosis. In vitro, the catalytic efficiency of plasmin-mediated C5a generation greatly exceeded that of thrombin or factor Xa, but was similar to the recognized complement C5 convertases. Plasmin-activated C5 yielded a functional membrane attack complex (MAC). In an arterial thrombosis model, plasminogen activator administration increased C5a levels. Overall, these findings suggest plasmin bridges thrombosis and the immune response by liberating C5a and inducing MAC assembly. These new insights may lead to the development of strategies to limit thrombus formation and/or enhance resolution. PMID:27077125

  1. Complement Activation in Arterial and Venous Thrombosis is Mediated by Plasmin

    Directory of Open Access Journals (Sweden)

    Jonathan H. Foley

    2016-03-01

    Full Text Available Thrombus formation leading to vaso-occlusive events is a major cause of death, and involves complex interactions between coagulation, fibrinolytic and innate immune systems. Leukocyte recruitment is a key step, mediated partly by chemotactic complement activation factors C3a and C5a. However, mechanisms mediating C3a/C5a generation during thrombosis have not been studied. In a murine venous thrombosis model, levels of thrombin–antithrombin complexes poorly correlated with C3a and C5a, excluding a central role for thrombin in C3a/C5a production. However, clot weight strongly correlated with C5a, suggesting processes triggered during thrombosis promote C5a generation. Since thrombosis elicits fibrinolysis, we hypothesized that plasmin activates C5 during thrombosis. In vitro, the catalytic efficiency of plasmin-mediated C5a generation greatly exceeded that of thrombin or factor Xa, but was similar to the recognized complement C5 convertases. Plasmin-activated C5 yielded a functional membrane attack complex (MAC. In an arterial thrombosis model, plasminogen activator administration increased C5a levels. Overall, these findings suggest plasmin bridges thrombosis and the immune response by liberating C5a and inducing MAC assembly. These new insights may lead to the development of strategies to limit thrombus formation and/or enhance resolution.

  2. Acute myocardial ischemia in a patient with heterozygous alpha-2-plasmin inhibitor deficiency

    NARCIS (Netherlands)

    Brands-Nijenhuis, Angelique V. M.; van Geel, Peter P.; Meijer, Karina

    2009-01-01

    In this brief report we present a patient with heterozygous alpha 2 plasmin inhibitor (alpha 2PI) deficiency who developed atherosclerosis and myocardial ischemia in the presence of multiple classical risk factors. Management was complicated by fear of bleeding complications with the use of antiplat

  3. Reactive coagulation induced by plasmin in patients treated with recombinant tissue-type plasminogen activator

    NARCIS (Netherlands)

    Gram, J.; Munkvad, S.; Leebeek, F.W.G.; Kluft, C.; Jespersen, J.

    1993-01-01

    Background: We and others have demonstrated that administration of thrombolytic agents causes the generation of thrombosis-promoting agents. At present, we have studied whether formation in vivo of excessive amounts of plasmin is responsible for the activation of coagulation in patients treated with

  4. Plasmin in urine from patients with type 2 diabetes and treatment-resistant hypertension activates ENaC in vitro

    DEFF Research Database (Denmark)

    Buhl, Kristian B; Stolzenburg Oxlund, Christina; Friis, Ulla G

    2014-01-01

    diabetes mellitus (T2DM) and treatment-resistant hypertension excrete plasmin(ogen) in urine in proportion to albumin and that plasmin confers to urine the ability to activate ENaC. METHOD:: Patients (n = 113) with T2DM and resistant hypertension, defined as systolic blood pressure (SBP) more than 130 mm...... of plasmin in preurine may inappropriately activate ENaC in patients with type 2 diabetes and microalbuminuria. This may contribute to treatment-resistant hypertension.......BACKGROUND:: Aberrant filtration of plasminogen from plasma and subsequent activation to plasmin in the urinary space may activate proteolytically the epithelial sodium channel, ENaC. In conditions with chronic albuminuria, this may cause hypertension. It was hypothesized that patients with type 2...

  5. Raft disorganization leads to reduced plasmin activity in Alzheimer's disease brains.

    Science.gov (United States)

    Ledesma, Maria Dolores; Abad-Rodriguez, José; Galvan, Cristian; Biondi, Elisa; Navarro, Pilar; Delacourte, Andre; Dingwall, Colin; Dotti, Carlos G

    2003-12-01

    The serine protease plasmin can efficiently degrade amyloid peptide in vitro, and is found at low levels in the hippocampus of patients with Alzheimer's disease (AD). The cause of such paucity remains unknown. We show here that the levels of total brain plasminogen and plasminogen-binding molecules are normal in these brain samples, yet plasminogen membrane binding is greatly reduced. Biochemical analysis reveals that the membranes of these brains have a mild, still significant, cholesterol reduction compared to age-matched controls, and anomalous raft microdomains. This was reflected by the loss of raft-enriched proteins, including plasminogen-binding and -activating molecules. Using hippocampal neurons in culture, we demonstrate that removal of a similar amount of membrane cholesterol is sufficient to induce raft disorganization, leading to reduced plasminogen membrane binding and low plasmin activity. These results suggest that brain raft alterations may contribute to AD by rendering the plasminogen system inefficient.

  6. Proteolytic modulation of thrombopoietin activity: comparison of thrombin, plasmin, and urokinase.

    Science.gov (United States)

    Reiner, A P; Aramaki, K M

    2000-06-01

    Several observations suggest that limited proteolysis of full-length 70 kD human thrombopoietin (Tpo) may be important for Tpo biology. Recently, it was reported that thrombin cleaves full-length recombinant human Tpo (rhTpo) sequentially at two sites, Arg(195) within the glycan domain followed by Arg(117) within the cytokine domain, and that these cleavages modulate Tpo activity in vitro. We demonstrate that urokinase and plasmin also cleave rhTpo in a time-dependent manner. Urokinase cleavage is confined to the glycan domain, and generates a 35 kD N-terminal fragment that contains the intact cytokine domain, and is associated with increased Tpo activity. In contrast, plasmin cleaves Tpo sequentially at two specific sites (Arg(205) within the glycan domain followed by Lys(52) within the cytokine domain), and is associated with a marked decrease in Tpo activity. These proteolytic events have potential implications for regulation of Tpo activity in vivo.

  7. [Inhibition of esterase by L-lysine, the activator and fibrinolytic activity of the plasmin-streptokinase activator complex].

    Science.gov (United States)

    Aĭsina, R B; Gaĭsarian, E S; Snitko, Ia E; Varfolomeev, S D

    1994-02-01

    The effect of L-lysine on some reactions catalysed by plasmin and the plasmin-streptokinase activator complex has been studied. The constants for competitive inhibition by L-lysine of the benzyloxycarbonyl-L-lysine p-nitrophenyl ester hydrolysis by the activator (Ki 116 mM), of the plasminogen activation by the activator (Ki 8 mM) and of fibrinolysis by plasmin (Ki 3 mM) were determined. It was found that L-lysine at concentrations below 0.05 M, which do not affect the activator's esterase activity, does inhibit fibrinolysis by plasmin and the activator complex. The effect of L-lysine on fibrinolysis under the action of the activator is complex: it inhibits both the activation of clot-entrapped plasminogen by the activator and lysis of fibrin by the plasmin formed. This inhibitory action of L-lysine is largely related to the fact that it lowers the sorption of the activator, plasminogen and plasmin on fibrin, competing with fibrin for their lysine-binding sites as well as worsens the activator-plasminogen binding.

  8. Interaction of actin with plasminogen/plasmin system: mechanisms and physiological role

    Directory of Open Access Journals (Sweden)

    Tykhomyrov A. A.

    2012-12-01

    Full Text Available In the present review, we have summarized and analyzed the literature data concerning cooperation between multifunctional proteins, the components of plasminogen/plasmin system and actin. The mechanisms underlying intermolecular interactions and the role of plasminogen kringle domains in protein-protein recognition are reviewed. A particular attention is paid to extracellular actin that serves as a surface protein of plasma membrane in various cells. A putative role of surface actin as the universal «non-hemostatic» center of plasminogen activation is discussed. The exposition of cytoskeletal actin on the outer surface of cellular membrane is thought to be a phenomenon, which is involved in both normal cell functioning and development of pathologies. In particular, the mechanism of plasminogen fragmentation on the surface of cancer cells mediated by actin, which results in generation of endogenous suppressors of tumor growth and metastazing (angiostatins, is described. It has been acknowledged that the plasminogen/plasmin system in concert with surface actin regulates releasing biologically active substances, e. g. catecholamines. The comprehensive assessment of plasminogen/plasmin system and surface actin exposition is proposed to be a criterion of functional status of cells and can be used as a diagnostic parameter at various pathologies.

  9. Involvement of tissue plasminogen activator-plasmin system in depolarization-evoked dopamine release in the nucleus accumbens of mice.

    Science.gov (United States)

    Ito, Mina; Nagai, Taku; Kamei, Hiroyuki; Nakamichi, Noritaka; Nabeshima, Toshitaka; Takuma, Kazuhiro; Yamada, Kiyofumi

    2006-11-01

    Tissue plasminogen activator (tPA), a serine protease, catalyzes the conversion of plasminogen to plasmin. In the present study, we investigated the role of the tPA-plasmin system in depolarization-evoked dopamine (DA) and acetylcholine (ACh) release in the nucleus accumbens (NAc) and hippocampus, respectively, of mice, by using in vivo microdialysis. Microinjection of either tPA or plasmin significantly potentiated 40 mM KCl-induced DA release without affecting basal DA levels. In contrast, plasminogen activator inhibitor-1 dose-dependently reduced 60 mM KCl-induced DA release. The 60 mM KCl-evoked DA release in the NAc was markedly diminished in tPA-deficient (tPA-/-) mice compared with wild-type mice, although basal DA levels did not differ between the two groups. Microinjections of either exogenous tPA (100 ng) or plasmin (100 ng) into the NAc of tPA-/-mice restored 60 mM KCl-induced DA release, as observed in wild-type mice. In contrast, there was no difference in either basal or 60 mM KCl-induced ACh release in the hippocampus between wild-type and tPA-/-mice. Our findings suggest that the tPA-plasmin system is involved in the regulation of depolarization-evoked DA release in the NAc.

  10. Plasmin plays a role in the in vitro generation of the linear IgA dermatosis antigen LADB97.

    Science.gov (United States)

    Hofmann, Silke C; Voith, Ursula; Schönau, Verena; Sorokin, Lydia; Bruckner-Tuderman, Leena; Franzke, Claus-Werner

    2009-07-01

    Collagen XVII (BP180) and its shed ectodomain represent major autoantigens in dermatoses of the pemphigoid group. The 120 kDa ectodomain is constitutively shed from the cell surface by disintegrin-metalloproteinases (ADAMs). Part of it is further processed to a 97 kDa fragment (LABD97), an autoantigen in linear IgA dermatosis (LAD), but the responsible proteinases remain elusive. In this study, we identified the 120 and the 97 kDa ectodomain in blister fluids of bullous pemphigoid patients using new mAbs. As blister fluids contain significant plasmin-like serine protease activity, HaCaT keratinocytes or purified 120 kDa ectodomain were incubated with several human serine proteases. In vitro, only plasmin generated a stable 97 kDa fragment that was also targeted by LAD sera. Characterization of the plasmin-derived 97 kDa fragment with domain-specific collagen XVII antibodies, heparin binding and N-glycosylation studies indicates that the N-terminus is located approximately at AA 515 and the C-terminus N-terminally from AA 1,421. Interestingly, plasmin-derived LABD97 was also generated in the presence of ADAM inhibitors and remained stable over more than 12 hours incubation at 37 degrees C, indicating that this disease relevant collagen XVII fragment can also arise in an ADAM-independent manner through direct action by plasmin.

  11. Simultaneous measurement of thrombin and plasmin generation to assess the interplay between coagulation and fibrinolysis.

    Science.gov (United States)

    Matsumoto, Tomoko; Nogami, Keiji; Shima, Midori

    2013-10-01

    Normal haemostasis is maintained by a controlled balance between coagulation and fibrinolysis, involving thrombin and plasmin the respective key enzymes. Simultaneous evaluation of both enzymes facilitates, therefore, an overall understanding of normal and pathological haemostasis. Combined thrombin and plasmin generation (T/P-G) assays have been recently described, and we have adapted the technique to investigate the interplay between coagulation and fibrinolysis in patients with various haemostatic disorders. Our modified T/P-G was initiated by the addition of a mixture of optimised lower concentrations of tissue factor and tissue-type plasminogen activator. Thrombin generation (TG) and plasmin generation (PG) were monitored simultaneously using individual fluorescent substrates in separate microtitre wells. The relationship between coagulation and fibrinolysis was demonstrated by analysing the effects of thrombin inhibitors, activated protein C and thrombomodulin. The most evident impairments in TG were observed with plasma samples deficient of coagulation factors participating in the prothrombinase complex. Defects in PG were observed with deficiencies of factor (F)V, FX, fibrinogen, and plasminogen. TG appeared to be a prerequisite for the initiation of PG, and overall PG was governed by fibrinogen concentration. TG in patients with haemophilia A correlated with levels of FVIII activity, but there was no significant relationship between PG and FVIII:C, confirming that the abnormal haemostasis in haemophilia A results in a severe imbalance between coagulation and fibrinolysis. The findings demonstrate that global haemostasis depends on a sensitive balance between coagulation and fibrinolysis, and that the modified T/P-G assay could provide an enhanced understanding of haemorrhage and thrombosis in clinical practice.

  12. Recruitment of CCR6-expressing Th17 cells by CCL20 secreted from plasmin-stimulated macrophages

    Institute of Scientific and Technical Information of China (English)

    Qun Li; Yves Laumonnier; Tatiana Syrovets; Thomas Simmet

    2013-01-01

    In the present study,monocyte-derived human macrophages were differentiated from buffy coats.Na(i)ve CD4+ T-cells enriched from peripheral blood mononuclear cells using anti-CD4 magnetic beads and the autoMACS separation system were polarized under T-helper 17 (Th17)-promoting conditions for 6 days to get Th17 cells.The frequency of Th17 cell differentiation and the expression of C-C chemokine receptor type 6 (CCR6) on Th17 cells were investigated by flow cytometry.Plasmin-triggered induction of macrophage inflammatory protein-3alpha/C-C chemokine ligand 20 (CCL20) genes in macrophages was assessed by reverse transcription-polymerase chain reaction,and secreted protein levels were measured by enzymelinked immunosorbent assay.Th17 cell migration induced by CCL20 secreted from plasmin-stimulated macrophages was tested in vitro by chemotaxis using a transwell system.These results demonstrate that plasmin triggers the expression of chemokine CCL20 messenger RNA and the release of CCL20 protein in human monocyte-derived macrophages,which critically depend on the proteolytic activity of plasmin and activation of p38 mitogen-activated protein kinase and nuclear factor-kappaB signaling pathways.Expression of CCR6 was detected on 87.23 ± 8.6% of Th17 cells in vitro.Similar to chemotaxis triggered by recombinant human CCL20,supernatants collected from plasmin-stimulated macrophage-induced chemotactic migration of Th17 cells,which could be inhibited by an anti-CCL20 neutralizing antibody.These results suggest that plasmin generated in inflamed tissues might elicit production of chemokine CCL20 by human macrophages leading to the recruitmentof CCR6 positive Th17 cells to the inflammatory sites.

  13. Enhanced clearance of Abeta in brain by sustaining the plasmin proteolysis cascade.

    Science.gov (United States)

    Jacobsen, J Steven; Comery, Thomas A; Martone, Robert L; Elokdah, Hassan; Crandall, David L; Oganesian, Aram; Aschmies, Suzan; Kirksey, Yolanda; Gonzales, Cathleen; Xu, Jane; Zhou, Hua; Atchison, Kevin; Wagner, Erik; Zaleska, Margaret M; Das, Indranil; Arias, Robert L; Bard, Jonathan; Riddell, David; Gardell, Stephen J; Abou-Gharbia, Magid; Robichaud, Albert; Magolda, Ronald; Vlasuk, George P; Bjornsson, Thorir; Reinhart, Peter H; Pangalos, Menelas N

    2008-06-24

    The amyloid hypothesis states that a variety of neurotoxic beta-amyloid (Abeta) species contribute to the pathogenesis of Alzheimer's disease. Accordingly, a key determinant of disease onset and progression is the appropriate balance between Abeta production and clearance. Enzymes responsible for the degradation of Abeta are not well understood, and, thus far, it has not been possible to enhance Abeta catabolism by pharmacological manipulation. We provide evidence that Abeta catabolism is increased after inhibition of plasminogen activator inhibitor-1 (PAI-1) and may constitute a viable therapeutic approach for lowering brain Abeta levels. PAI-1 inhibits the activity of tissue plasminogen activator (tPA), an enzyme that cleaves plasminogen to generate plasmin, a protease that degrades Abeta oligomers and monomers. Because tPA, plasminogen and PAI-1 are expressed in the brain, we tested the hypothesis that inhibitors of PAI-1 will enhance the proteolytic clearance of brain Abeta. Our data demonstrate that PAI-1 inhibitors augment the activity of tPA and plasmin in hippocampus, significantly lower plasma and brain Abeta levels, restore long-term potentiation deficits in hippocampal slices from transgenic Abeta-producing mice, and reverse cognitive deficits in these mice.

  14. PEGylated DX-1000: Pharmacokinetics and Antineoplastic Activity of a Specific Plasmin Inhibitor

    Directory of Open Access Journals (Sweden)

    Laetitia Devy

    2007-11-01

    Full Text Available Novel inhibitors of the urokinase-mediated plasminogen (plg activation system are potentially of great clinical benefit as anticancer treatments. Using phage display, we identified DX-1000 a tissue factor pathway inhibitor-derived Kunitz domain protein which is a specific high-affinity inhibitor of plasmin (pin (Ki = 99 pM. When tested in vitro, DX-1000 blocks plasminmediated pro-matrix metal loproteinase-9 (proMMP-9 activation on cells and dose-dependently inhibits tube formation, while not significantly affecting hemostasis and coagulation. However, this low-molecular weight protein inhibitor (~ 7 kDa exhibits rapid plasma clearance in mice and rabbits, limiting its potential clinical use in chronic diseases. After site-specific PEGylation, DX-1000 retains its activity and exhibits a decreased plasma clearance. This PEGylated derivative is effective in vitro, as well as potent in inhibiting tumor growth of green fluorescent protein (GFP-labeled MDA-MB-231 cells. 4PEG-DX-1000 treatment causes a significant reduction of urokinase-type plasminogen activator (uPA and plasminogen expressions, a reduction of tumor proliferation, and vascularization. 4PEG-DX-1000 treatment significantly decreases the level of active mitogenactivated protein kinase (MAPK in the primary tumors and reduces metastasis incidence. Together, our results demonstrate the potential value of plasmin inhibitors as therapeutic agents for blocking breast cancer growth and metastasis.

  15. Preparative purification of plasmin activity stimulating phenolic derivatives from Gastrodia elata using centrifugal partition chromatography.

    Science.gov (United States)

    Jeon, Je-Seung; Kim, Jeeyoung; Park, Soyoung; Ryou, Chongsuk; Kim, Chul Young

    2016-06-01

    Gastrodia rhizome, a dried and steamed tuber of Gastrodia elata Blume (Orchidaceae), has been traditionally used in Korea, China and Japan for the treatment of neurological and nervous disorders such as headaches, dizziness, vertigo and convulsive illnesses. The ethyl acetate and water extracts of G. elata stimulated plasmin activity. The active ethyl acetate fraction was subjected to centrifugal partition chromatography (CPC) with a two-phase solvent system, composed of n-hexane-ethyl acetate-methanol-water (3:7:4:6, v/v) followed by semi-preparative HPLC purification to separate active compounds and the water fraction was purified by Diaion HP-20 resin and semi-preparative HPLC. In ethyl acetate extract, 4-hydroxybenzyl alcohol (1), 4-hydroxybenzoic acid (2), 4-hydroxybenzaldehyde (3), 4-ethoxymethylphenol (4), 4,4'-oxybis(methylene)diphenol (5) and 4,4'-methylenediphenol (6) were obtained with high purities. Parishin (7) and parishin B (8) were isolated from water extract. Among isolated compounds, 4-hydroxybenzyl alcohol (1), 4-hydroxybenzaldehyde (3) and 4-ethoxymethylphenol (4) significantly stimulated plasmin activity. Copyright © 2015 John Wiley & Sons, Ltd.

  16. Staphylococcus aureus proteins Sbi and Efb recruit human plasmin to degrade complement C3 and C3b.

    Directory of Open Access Journals (Sweden)

    Tina K Koch

    Full Text Available Upon host infection, the human pathogenic microbe Staphylococcus aureus (S. aureus immediately faces innate immune reactions such as the activated complement system. Here, a novel innate immune evasion strategy of S. aureus is described. The staphylococcal proteins surface immunoglobulin-binding protein (Sbi and extracellular fibrinogen-binding protein (Efb bind C3/C3b simultaneously with plasminogen. Bound plasminogen is converted by bacterial activator staphylokinase or by host-specific urokinase-type plasminogen activator to plasmin, which in turn leads to degradation of complement C3 and C3b. Efb and to a lesser extend Sbi enhance plasmin cleavage of C3/C3b, an effect which is explained by a conformational change in C3/C3b induced by Sbi and Efb. Furthermore, bound plasmin also degrades C3a, which exerts anaphylatoxic and antimicrobial activities. Thus, S. aureus Sbi and Efb comprise platforms to recruit plasmin(ogen together with C3 and its activation product C3b for efficient degradation of these complement components in the local microbial environment and to protect S. aureus from host innate immune reactions.

  17. Contribution of macrophages to proteolysis and plasmin activity in ewe bulk milk.

    Science.gov (United States)

    Caroprese, M; Marzano, A; Schena, L; Marino, R; Santillo, A; Albenzio, M

    2007-06-01

    A total of 225 bulk sheep milk samples were collected from 5 intensively managed flocks during early, mid, and late lactation to assess the contribution of macrophages to the regulation of the plasmin-plasminogen system. Samples were analyzed for composition, somatic cell counts, milk renneting characteristics, and for plasmin (PL), plasminogen (PG), and plasminogen activators (PA) activities. Isolation of macrophages from milk was performed using a magnetic positive separation and mouse antiovine macrophage antibody; separated cells were lysed by several freeze-thaw cycles, and activity of urokinase PA (u-PA) was determined. Plasmin activity decreased during lactation (42.06 +/- 0.66, early; 31.29 +/- 0.66, mid; 28.19 +/- 0.66 U/mL, late). The reduction in PL activity recorded in the mid and late lactation milk matched the increase in PG:PL ratio. The activity of PA increased throughout lactation; the highest value being recorded in the late lactation milk (260.20 +/- 8.66 U/mL). Counts of isolated and concentrated macrophages were higher in early and mid lactation milk (3.89 +/- 0.08 and 3.98 +/- 0.08 log10 cells/mL, respectively) than in late lactation milk (3.42 +/- 0.08 log10 cells/mL). Stage of lactation did not influence the activity of u-PA detected in isolated macrophages. The activity of u-PA associated with isolated milk macrophages only minimally contributed to total PA activity detected in milk. Proteolytic enzymes, associated with isolated macrophages, act on alpha-casein hydrolysis, as shown by urea-PAGE electrophoresis analysis. Somatic cell counts did not exceed 600,000 cells/mL, and this threshold can be considered a good index of health status of the flock and of the ability of milk to being processed. Our results lend support to the hypothesis that macrophages in ewe bulk milk from healthy flocks only slightly contribute to the activation of the PL-PG system.

  18. From Plasminogen to Plasmin: Role of Plasminogen Receptors in Human Cancer

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    Miroslava Didiasova

    2014-11-01

    Full Text Available Cell surface-associated proteolysis mediated by plasmin (PLA is an essential feature of wound healing, angiogenesis and cell invasion, processes that are dysregulated in cancer development, progression and systemic spread. The generation of PLA, initiated by the binding of its precursor plasminogen (PLG to the cell surface, is regulated by an array of activators, inhibitors and receptors. In this review, we will highlight the importance of the best-characterized components of the PLG/PLA cascade in the pathogenesis of cancer focusing on the role of the cell surface-PLG receptors (PLG-R. PLG-R overexpression has been associated with poor prognosis of cancer patients and resistance to chemotherapy. We will also discuss recent findings on the molecular mechanisms regulating cell surface expression and distribution of PLG-R.

  19. Determinación de los parámetros cinéticos de la plasmina porcina y comparación con la humana = Porcine plasmin: determination of kinetic parameters and comparison with human plasmin

    Directory of Open Access Journals (Sweden)

    Arbeláez Ramírez, Luis Fernando

    2012-04-01

    Full Text Available El plasminógeno es el zimógeno de la plasmina, enzima relacionada con la disolución del coágulo sanguíneo. Estudios con plasminas de diferentes especies animales han demostrado mayor afinidad que la plasmina humana por sustratos análogos hechos exclusivamente para ella. Así lo confirman la activación y cinética del sistema plasminógeno/plasmina porcino, que hasta el presente no se habían determinado ni comparado con el humano. En este trabajo se utilizaron, para la purificación del plasminógeno, cromatografía de afinidad y cambio iónico; se utilizó urocinasa para la activación del plasminógeno a plasmina y se determinaron los parámetros cinéticos con el sustrato cromógeno S-2251. Los terminales-N se determinaron por el método de degradación de Edman. La plasmina porcina demostró mayor afinidad (Km por el sustrato que la plasmina humana, 1,55 y 5,3 mM respectivamente, mientras que la plasmina humana mostró mayor velocidad de conversión del sustrato a producto (0,1 UA/ seg que la porcina (0,033 UA/seg. Los terminales-N se diferenciaron en los aminoácidos 1 y 3, DPPDDY (porcino y EPLDDY (humano.

  20. 酪蛋白凝固与乳纤维蛋白溶酶活性的关系%Relationship between casein coagulation and the activity of plasmin

    Institute of Scientific and Technical Information of China (English)

    白英; 刘彩艳

    2012-01-01

    采用分光光度计和SDS-PAGE电泳的方法,以鲜乳为原料,分别对酸沉,酶凝和钙凝的酪蛋白胶粒中的纤溶酶活性进行测定,对酪蛋白凝固与乳纤维蛋白溶酶活性的关系进行研究.结果表明,酸沉酪蛋白纤溶酶活性及总纤溶酶活性最高,分别为212.00 U/mL和250.00 U/mL,酶凝次之,Ca2+凝酪蛋白纤溶酶活性最低.%Through activity assay of casein micelle-associated plasmin by spectrophotometer and SDS-PAGE, relationship between the casein coagulation and activity of plasmin were studied-Results showed that the activity of acid precipitate casein micelle-associated plasmin was highest. The activity of plasmin was 212.00 U/mL. The total activity of plasmin was 250.00 U/mL. Rennin coagulation casein micelle-associated plasmin and the total activity were both lowest.

  1. Glyceraldehyde 3-phosphate dehydrogenase and galectin from Dirofilaria immitis participate in heartworm disease endarteritis via plasminogen/plasmin system.

    Science.gov (United States)

    González-Miguel, Javier; Larrazabal, Carmen; Loa-Mesón, Diana; Siles-Lucas, Mar; Simón, Fernando; Morchón, Rodrigo

    2016-06-15

    The interaction between parasitic protozoa and helminths, both in the blood and in tissues and the fibrinolytic system of their hosts is usually considered as a survival parasite mechanism since this system is the physiological route responsible for degrading fibrin clots. The broad-range proteolytic activity of plasmin, the final enzyme of the route, implies that its recruitment by these parasites is an important mechanism that mediates their invasion and establishment in the hosts. However, recent studies have proposed a dual role for plasmin by linking its over-production with pathological mechanisms at vascular level. Most of these studies have been conducted in Dirofilaria immitis, a blood-borne parasite that survives in the pulmonary arteries of its host for years while it produces a chronic inflammatory disease, whose main pathogenic mechanism is the appearance of proliferative endarteritis. Recently, the participation of two proteins from D. immitis, glyceraldehyde 3-phosphate dehydrogenase (DiGAPDH) and galectin (DiGAL), in the activation of the fibrinolytic system of its host has been demonstrated, which has been a priori associated with parasite survival mechanisms. The aim of the present paper was to study the role of plasmin generated by these proteins in the emergence of proliferative endarteritis. An in vitro model of canine endothelial and smooth muscle cells, as well as the two parasitic recombinant proteins were employed. The results show that DiGAPDH and DiGAL stimulate the proliferation and migration of both cell types, as well as the degradation of the extracellular matrix (ECM) via plasminogen (PLG)/plasmin system, being all of these mechanisms related to the appearance of proliferative endarteritis. Due to the high degree of evolutionary conservation of these antigens, these data support the hypothesis of the survival/pathology ambivalence in the interactions between parasites and the fibrinolytic system of their hosts and represent an

  2. Inhibition of plasmin-mediated TAFI activation may affect development but not progression of abdominal aortic aneurysms.

    Science.gov (United States)

    Bridge, Katherine; Revill, Charlotte; Macrae, Fraser; Bailey, Marc; Yuldasheva, Nadira; Wheatcroft, Stephen; Butlin, Roger; Foster, Richard; Scott, D Julian; Gils, Ann; Ariens, Robert

    2017-01-01

    Thrombin-activatable fibrinolysis inhibitor (TAFI) reduces the breakdown of fibrin clots through its action as an indirect inhibitor of plasmin. Studies in TAFI-deficient mice have implicated a potential role for TAFI in Abdominal Aortic Aneurysm (AAA) disease. The role of TAFI inhibition on AAA formation in adult ApoE-/- mice is unknown. The aim of this paper was to investigate the effects of TAFI inhibition on AAA development and progression. Using the Angiotensin II model of AAA, male ApoE-/- mice were infused with Angiotensin II 750ng/kg/min with or without a monoclonal antibody inhibitor of plasmin-mediated activation of TAFI, MA-TCK26D6, or a competitive small molecule inhibitor of TAFI, UK-396082. Inhibition of TAFI in the Angiotensin II model resulted in a decrease in the mortality associated with AAA rupture (from 40.0% to 16.6% with MA-TCK26D6 (log-rank Mantel Cox test p = 0.16), and 8.3% with UK-396082 (log-rank Mantel Cox test p = 0.05)). Inhibition of plasmin-mediated TAFI activation reduced the incidence of AAA from 52.4% to 30.0%. However, late treatment with MA-TCK26D6 once AAA were already established had no effect on the progression of AAA in this model. The formation of intra-mural thrombus is responsible for the dissection and early rupture in the angiotensin II model of AAA, and this process can be prevented through inhibition of TAFI. Late treatment with a TAFI inhibitor does not prevent AAA progression. These data may indicate a role for inhibition of plasmin-mediated TAFI activation in the early stages of AAA development, but not in its progression.

  3. Influence of addition of plasmin or mastitic milk to cheesemilk on quality of smear-ripened cheese

    OpenAIRE

    O'Farrell, Ian P.; Sheehan, Jeremiah J.; Martin G. Wilkinson; Harrington, Dermot; Kelly, Alan L.

    2002-01-01

    peer-reviewed Smear-ripened cheese varieties are characterised by the growth of a smear culture, containing predominantly Brevibacterium linens, on the cheese surface during ripening. In such cheese, considerable zonal differences in biochemistry of ripening exist, due to moisture loss from, and growth and metabolic activity of smear microflora at, the cheese surface. In this study, the effects of adding exogenous plasmin or small amounts of mastitic milk to good quality milk o...

  4. Determining Human Clot Lysis Time (in vitro with Plasminogen/Plasmin from Four Species (Human, Bovine, Goat, and Swine

    Directory of Open Access Journals (Sweden)

    Omaira Cañas Bermúdez

    2015-05-01

    Full Text Available Cardiovascular disease is the leading cause of death worldwide, including failures in the plasminogen/plasmin system which is an important factor in poor lysis of blood clots. This article studies the fibrinolytic system in four species of mammals, and it identifies human plasminogen with highest thrombolysis efficiency. It examines plasminogen from four species (human, bovine, goat, and swine and identifies the most efficient one in human clot lysis in vitro. All plasminogens were identically purified by affinity chromatography. Human fibrinogen was purified by fractionation with ethanol. The purification of both plasminogen and fibrinogen was characterized by one-dimensional SDS-PAGE (10%. Human clot formation in vitro and its dissolution by plasminogen/plasmin consisted of determining lysis time from clot formation to its dilution. Purification of proteins showed greater than 95% purity, human plasminogen showed greater ability to lyse clot than animal plasminogen. The article concludes that human plasminogen/plasmin has the greatest catalysis and efficiency, as it dissolves human clot up to three times faster than that of irrational species.

  5. Rapid-reaction kinetic characterization of the pathway of streptokinase-plasmin catalytic complex formation.

    Science.gov (United States)

    Verhamme, Ingrid M; Bock, Paul E

    2008-09-19

    Binding of the fibrinolytic proteinase plasmin (Pm) to streptokinase (SK) in a tight stoichiometric complex transforms Pm into a potent proteolytic activator of plasminogen. SK binding to the catalytic domain of Pm, with a dissociation constant of 12 pm, is assisted by SK Lys(414) binding to a Pm kringle, which accounts for a 11-20-fold affinity decrease when Pm lysine binding sites are blocked by 6-aminohexanoic acid (6-AHA) or benzamidine. The pathway of SK.Pm catalytic complex formation was characterized by stopped-flow kinetics of SK and the Lys(414) deletion mutant (SKDeltaK414) binding to Pm labeled at the active site with 5-fluorescein ([5F]FFR-Pm) and the reverse reactions by competitive displacement of [5F]FFR-Pm with active site-blocked Pm. The rate constants for the biexponential fluorescence quenching caused by SK and SKDeltaK414 binding to [5F]FFR-Pm were saturable as a function of SK concentration, reporting encounter complex affinities of 62-110 nm in the absence of lysine analogs and 4900-6500 and 1430-2200 nm in the presence of 6-AHA and benzamidine, respectively. The encounter complex with SKDeltaK414 was approximately 10-fold weaker in the absence of lysine analogs but indistinguishable from that of native SK in the presence of 6-AHA and benzamidine. The studies delineate for the first time the sequence of molecular events in the formation of the SK.Pm catalytic complex and its regulation by kringle ligands. Analysis of the forward and reverse reactions supports a binding mechanism in which SK Lys(414) binding to a Pm kringle accompanies near-diffusion-limited encounter complex formation followed by two slower, tightening conformational changes.

  6. Endogenously generated plasmin at the vascular wall injury site amplifies lysine binding site-dependent plasminogen accumulation in microthrombi.

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    Tomasz Brzoska

    Full Text Available The fibrinolytic system plays a pivotal role in the regulation of hemostasis; however, it remains unclear how and when the system is triggered to induce thrombolysis. Using intra-vital confocal fluorescence microscopy, we investigated the process of plasminogen binding to laser-induced platelet-rich microthrombi generated in the mesenteric vein of transgenic mice expressing green fluorescent protein (GFP. The accumulation of GFP-expressing platelets as well as exogenously infused Alexa Fluor 568-labeled Glu-plasminogen (Glu-plg on the injured vessel wall was assessed by measuring the increase in the corresponding fluorescence intensities. Glu-plg accumulated in a time-dependent manner in the center of the microthrombus, where phosphatidylserine is exposed on platelet surfaces and fibrin formation takes place. The rates of binding of Glu-plg in the presence of ε-aminocaproic acid and carboxypeptidase B, as well as the rates of binding of mini-plasminogen lacking kringle domains 1-4 and lysine binding sites, were significantly lower than that of Glu-plg alone, suggesting that the binding was dependent on lysine binding sites. Furthermore, aprotinin significantly suppressed the accumulation of Glu-plg, suggesting that endogenously generated plasmin activity is a prerequisite for the accumulation. In spite of the endogenous generation of plasmin and accumulation of Glu-plg in the center of microthrombi, the microthrombi did not change in size during the 2-hour observation period. When human tissue plasminogen activator was administered intravenously, Glu-plg further accumulated and the microthrombi were lysed. Glu-plg appeared to accumulate in the center of microthrombi in the early phase of microthrombus formation, and plasmin activity and lysine binding sites were required for this accumulation.

  7. The Plasmin-Sensitive Protein Pls in Methicillin-Resistant Staphylococcus aureus (MRSA) Is a Glycoprotein

    Science.gov (United States)

    Pohlentz, Gottfried; Xia, Guoqing; Hussain, Muzaffar; Foster, Simon; Peters, Georg

    2017-01-01

    Most bacterial glycoproteins identified to date are virulence factors of pathogenic bacteria, i.e. adhesins and invasins. However, the impact of protein glycosylation on the major human pathogen Staphylococcus aureus remains incompletely understood. To study protein glycosylation in staphylococci, we analyzed lysostaphin lysates of methicillin-resistant Staphylococcus aureus (MRSA) strains by SDS-PAGE and subsequent periodic acid-Schiff’s staining. We detected four (>300, ∼250, ∼165, and ∼120 kDa) and two (>300 and ∼175 kDa) glycosylated surface proteins with strain COL and strain 1061, respectively. The ∼250, ∼165, and ∼175 kDa proteins were identified as plasmin-sensitive protein (Pls) by mass spectrometry. Previously, Pls has been demonstrated to be a virulence factor in a mouse septic arthritis model. The pls gene is encoded by the staphylococcal cassette chromosome (SCC)mec type I in MRSA that also encodes the methicillin resistance-conferring mecA and further genes. In a search for glycosyltransferases, we identified two open reading frames encoded downstream of pls on the SCCmec element, which we termed gtfC and gtfD. Expression and deletion analysis revealed that both gtfC and gtfD mediate glycosylation of Pls. Additionally, the recently reported glycosyltransferases SdgA and SdgB are involved in Pls glycosylation. Glycosylation occurs at serine residues in the Pls SD-repeat region and modifying carbohydrates are N-acetylhexosaminyl residues. Functional characterization revealed that Pls can confer increased biofilm formation, which seems to involve two distinct mechanisms. The first mechanism depends on glycosylation of the SD-repeat region by GtfC/GtfD and probably also involves eDNA, while the second seems to be independent of glycosylation as well as eDNA and may involve the centrally located G5 domains. Other previously known Pls properties are not related to the sugar modifications. In conclusion, Pls is a glycoprotein and Pls glycosyl

  8. Proteolysis of neuronal cell adhesion molecule by the tissue plasminogen activator-plasmin system after kainate injection in the mouse hippocampus.

    Science.gov (United States)

    Endo, A; Nagai, N; Urano, T; Takada, Y; Hashimoto, K; Takada, A

    1999-01-01

    Tissue plasminogen activator (tPA) is a serine protease that converts inactive plasminogen to the active protease plasmin and mediates extracellular metabolism. tPA is transcriptionally induced in the mouse hippocampus by pharmacological or electrical stimulation of neuronal activity and mediates excitotoxin-induced neuronal degeneration. Therefore, we hypothesized that tPA would be induced in the hippocampus after kainic acid (KA) injection into the lateral cerebral ventricle (LCV) and that the activated tPA-plasmin system would degrade the neuronal cell adhesion molecule (NCAM), which is a component of the extracellular matrix. In order to investigate this possibility, we first examined whether NCAM is a substrate for the tPA plasmin system by incubating mouse brain homogenates with tPA and plasminogen at 37 degrees C. Next, we examined the degradation of NCAM and the changes of tPA activity in the mouse hippocampus with immunohistochemical procedures and histological zymography after KA injection into both LCVs. As a result, we observed neuronal atrophy and a decrease of NCAM immunoreactivity along with an increase of tPA activity in the CA3 area of the hippocampus. These results suggest that activation of the tPA plasmin system after KA injection into the LCVs results in the degradation of NCAM in the CA3 area.

  9. Cleavage of proBDNF by tPA/plasmin is essential for long-term hippocampal plasticity.

    Science.gov (United States)

    Pang, Petti T; Teng, Henry K; Zaitsev, Eugene; Woo, Newton T; Sakata, Kazuko; Zhen, Shushuang; Teng, Kenneth K; Yung, Wing-Ho; Hempstead, Barbara L; Lu, Bai

    2004-10-15

    Long-term memory is thought to be mediated by protein synthesis-dependent, late-phase long-term potentiation (L-LTP). Two secretory proteins, tissue plasminogen activator (tPA) and brain-derived neurotrophic factor (BDNF), have been implicated in this process, but their relationship is unclear. Here we report that tPA, by activating the extracellular protease plasmin, converts the precursor proBDNF to the mature BDNF (mBDNF), and that such conversion is critical for L-LTP expression in mouse hippocampus. Moreover, application of mBDNF is sufficient to rescue L-LTP when protein synthesis is inhibited, which suggests that mBDNF is a key protein synthesis product for L-LTP expression.

  10. Neutralisation of uPA with a monoclonal antibody reduces plasmin formation and delays skin wound healing in tPA-deficient mice

    DEFF Research Database (Denmark)

    Jögi, Annika; Rønø, Birgitte; Lund, Ida K

    2010-01-01

    Proteolytic degradation by plasmin and metalloproteinases is essential for epidermal regeneration in skin wound healing. Plasminogen deficient mice have severely delayed wound closure as have mice simultaneously lacking the two plasminogen activators, urokinase-type plasminogen activator (u......PA) and tissue-type plasminogen activator (tPA). In contrast, individual genetic deficiencies in either uPA or tPA lead to wound healing kinetics with no or only slightly delayed closure of skin wounds....

  11. The tissue plasminogen activator (tPA)/plasmin extracellular proteolytic system regulates seizure-induced hippocampal mossy fiber outgrowth through a proteoglycan substrate.

    Science.gov (United States)

    Wu, Y P; Siao, C J; Lu, W; Sung, T C; Frohman, M A; Milev, P; Bugge, T H; Degen, J L; Levine, J M; Margolis, R U; Tsirka, S E

    2000-03-20

    Short seizure episodes are associated with remodeling of neuronal connections. One region where such reorganization occurs is the hippocampus, and in particular, the mossy fiber pathway. Using genetic and pharmacological approaches, we show here a critical role in vivo for tissue plasminogen activator (tPA), an extracellular protease that converts plasminogen to plasmin, to induce mossy fiber sprouting. We identify DSD-1-PG/phosphacan, an extracellular matrix component associated with neurite reorganization, as a physiological target of plasmin. Mice lacking tPA displayed decreased mossy fiber outgrowth and an aberrant band at the border of the supragranular region of the dentate gyrus that coincides with the deposition of unprocessed DSD-1-PG/phosphacan and excessive Timm-positive, mossy fiber termini. Plasminogen-deficient mice also exhibit the laminar band and DSD- 1-PG/phosphacan deposition, but mossy fiber outgrowth through the supragranular region is normal. These results demonstrate that tPA functions acutely, both through and independently of plasmin, to mediate mossy fiber reorganization.

  12. Anticoagulant and calcium-binding properties of high molecular weight derivatives of human fibrinogen, produced by plasmin (fragments X).

    Science.gov (United States)

    Nieuwenhuizen, W; Gravesen, M

    1981-03-27

    Early plasmin degradation products (X fragments) of human fibrinogen were prepared in the presence of calcium-ions or EGTA, and purified on Sepharose 6B-CL. X fragments were characterized with respect to amino-terminal amino acids, polypeptide-chain composition, anticlotting properties and calcium-binding. Amino-terminal amino acids were alanine and tyrosine. The molecular weights of the chains were about 26 000, 58 000 and 48 000 for A alpha-, B beta- and gamma-chains, respectively. X fragments were about 6-times as potent in anticlotting behaviour as D fragments prepared in the presence of calcium ions. Calcium-binding properties were essentially identical to those of fibrinogen. No differences were observed between X fragments prepared in the presence of calcium ions and those prepared in the presence of EGTA. This indicates that the carboxy-terminal parts of the A alpha-chains of fibrinogen are not involved in calcium-binding and that differences in chain-remnants as observed in late plasmic degradation products (which depend on the presence of calcium ions or EGTA [23] in the incubation medium) are introduced beyond the stage of fragment X formation.

  13. Plasmin-induced procoagulant effects in the blood coagulation: a crucial role of coagulation factors V and VIII.

    Science.gov (United States)

    Ogiwara, Kenichi; Nogami, Keiji; Nishiya, Katsumi; Shima, Midori

    2010-09-01

    Plasminogen activators provide effective treatment for patients with acute myocardial infarction. However, paradoxical elevation of thrombin activity associated with failure of clot lysis and recurrent thrombosis has been reported. Generation of thrombin in these circumstances appears to be owing to plasmin (Plm)-induced activation of factor (F) XII. Plm catalyzes proteolysis of several coagulant factors, but the roles of these factors on Plm-mediated procoagulant activity remain to be determined. Recently developed global coagulation assays were used in this investigation. Rotational thromboelastometry using whole blood, clot waveform analysis and thrombin generation tests using plasma, showed that Plm (> or =125 nmol/l) shortened the clotting times in similar dose-dependent manners. In particular, the thrombin generation test, which was unaffected by products of fibrinolysis, revealed the enhanced coagulation with an approximately two-fold increase of peak level of thrombin generation. Studies using alpha2-antiplasmin-deficient plasma revealed that much lower dose of Plm (> or =16 nmol/l) actually contributed to enhancing thrombin generation. The shortening of clotting time could be observed even in the presence of corn trypsin inhibitor, supporting that Plm exerted the procoagulant activity independently of FXII. In addition, using specific coagulation-deficient plasmas, the clot waveform analysis showed that Plm did not shorten the clotting time in only FV-deficient or FVIII-deficient plasma in prothrombin time-based or activated partial thromboplastin time-based assay, respectively. Our results indicated that Plm did possess procoagulant activity in the blood coagulation, and this effect was likely attributed by multicoagulation factors, dependent on FV and/or FVIII.

  14. Plasmin and Plasminogen induce macrophage reprogramming and regulate key steps of inflammation resolution via Annexin A1.

    Science.gov (United States)

    Sugimoto, Michelle A; Ribeiro, Ana Luíza C; Costa, Bruno R C; Vago, Juliana P; Lima, Kátia M; Carneiro, Fernanda S; Ortiz, Mylena Maira O; Lima, Graziele Letícia N; Carmo, Aline A F; Rocha, Renata M; Perez, Denise A; Reis, Alessandra C; Pinho, Vanessa; Miles, Lindsey A; Garcia, Cristiana C; Teixeira, Mauro M; Sousa, Lirlândia P

    2017-03-20

    Inflammation resolution is an active process that functions to restore tissue homeostasis. Participation of the plasminogen/plasmin (Plg/Pla) system in the productive phase of inflammation is well known, but its involvement in the resolution phase remains unclear. Therefore, we aimed to investigate the potential role of Plg/Pla in key events during the resolution of acute inflammation and its underlying mechanisms. Plg/Pla injection into the pleural cavity of BALB/c mice induced a time-dependent influx of mononuclear cells that were primarily macrophages of anti-inflammatory (M2 - F4/80(high) Gr1(-) CD11b(high)) and proresolving (Mres - F4/80(med) CD11b(low)) phenotypes, without changing the number of macrophages with a pro-inflammatory profile (M1 - F4/80(low) Gr1(+) CD11b(med)). Pleural injection of Plg/Pla also increased M2 markers (CD206 and Arginase-1) and secretory products (TGF-β and IL-6) and decreased the expression of iNOS (M1 marker). During the resolving phase of LPS-induced inflammation, when resolving macrophages predominate, we found increased Plg expression and Pla activity, further supporting a link between the Plg/Pla system and key cellular events in resolution. Indeed, Plg or Pla given at the peak of inflammation promoted resolution by decreasing neutrophil numbers and increasing neutrophil apoptosis and efferocytosis in a serine-protease inhibitor sensitive manner. Next, we confirmed the ability of Plg/Pla to both promote efferocytosis and override the pro-survival effect of LPS, via AnxA1. These findings suggest that Plg/Pla regulate several key steps in inflammation resolution, namely, neutrophil apoptosis, macrophage reprogramming and efferocytosis, which have a major impact on the establishment of an efficient resolution process.

  15. Tissue-type plasminogen activator-plasmin-BDNF modulate glutamate-induced phase-shifts of the mouse suprachiasmatic circadian clock in vitro.

    Science.gov (United States)

    Mou, Xiang; Peterson, Cynthia B; Prosser, Rebecca A

    2009-10-01

    The mammalian circadian clock in the suprachiasmatic nucleus (SCN) maintains environmental synchrony through light signals transmitted by glutamate released from retinal ganglion terminals. Brain-derived neurotrophic factor (BDNF) is required for light/glutamate to reset the clock. In the hippocampus, BDNF is activated by the extracellular protease, plasmin, which is produced from plasminogen by tissue-type plasminogen activator (tPA). We provide data showing expression of proteins from the plasminogen activation cascade in the SCN and their involvement in circadian clock phase-resetting. Early night glutamate application to SCN-containing brain slices resets the circadian clock. Plasminogen activator inhibitor-1 (PAI-1) blocked these shifts in slices from wild-type mice but not mice lacking its stabilizing protein, vitronectin (VN). Plasmin, but not plasminogen, prevented inhibition by PAI-1. Both plasmin and active BDNF reversed alpha(2)-antiplasmin inhibition of glutamate-induced shifts. alpha(2)-Antiplasmin decreased the conversion of inactive to active BDNF in the SCN. Finally, both tPA and BDNF allowed daytime glutamate-induced phase-resetting. Together, these data are the first to demonstrate expression of these proteases in the SCN, their involvement in modulating photic phase-shifts, and their activation of BDNF in the SCN, a potential 'gating' mechanism for photic phase-resetting. These data also demonstrate a functional interaction between PAI-1 and VN in adult brain. Given the usual association of these proteins with the extracellular matrix, these data suggest new lines of investigation into the locations and processes modulating mammalian circadian clock phase-resetting.

  16. The activation of the tissue plasminogen activator-plasmin system induced in the mouse hippocampus after injection of trimethyltin: possible proteolysis of highly polysialylated NCAM.

    Science.gov (United States)

    Endo, A; Hashimoto, K; Takada, Y; Takada, A

    1999-10-01

    Trimethyltin (TMT) is a neurotoxicant that causes the death of granule cells and degrades highly polysialylated NCAM (PSA-NCAM) in the dentate gyrus. To investigate the role of the tPA-plasmin system in the degradation of PSA-NCAM, we injected trimethyltin (TMT) into mice. As a result, tPA activity was significantly increased in CA1-CA4 and the dentate gyrus after TMT injection. These results suggest that up-regulated tPA may contribute to the degradation of PSA-NCAM.

  17. Diabetic nephropathy is associated with increased urine excretion of proteases plasmin, prostasin and urokinase and activation of amiloride-sensitive current in collecting duct cells

    DEFF Research Database (Denmark)

    Andersen, Henrik; Friis, Ulla G; Hansen, Pernille B L;

    2015-01-01

    by western immunoblotting, patch clamp and ELISA. Urine exosomes were isolated to elucidate potential cleavage of γENaC by a monoclonal antibody directed against the 'inhibitory' peptide tract. RESULTS: Compared with control, DN patients displayed significantly higher blood pressure and urinary excretion...... with controls. Immunoblotting of urine exosomes showed aquaporin 2 in all patient samples. Exosomes displayed a virtual absence of intact γENaC while moieties compatible with cleavage by furin only, were shown in both groups. Proteolytic cleavage by the extracellular serine proteases plasmin or prostasin...

  18. Evaluation of Serum Fibrinogen, Plasminogen, α2-Anti-Plasmin, and Plasminogen Activator Inhibitor Levels (PAI and Their Correlation with Presence of Retinopathy in Patients with Type 1 DM

    Directory of Open Access Journals (Sweden)

    Sefika Burcak Polat

    2014-01-01

    Full Text Available Background. Diabetic retinopathy (DR is the leading cause of blindness in the world. Retinopathy can still progress despite optimal metabolic control. The aim of the study was to determine whether different degrees of DR (proliferative or nonproliferative were associated with abnormally modulated hemostatic parameters in patients with T1DM. Method. 52 T1DM patients and 40 healthy controls were enrolled in the study. Patients were subdivided into three categories. Group I was defined as those without retinopathy, group II with NPRP, and group III with PRP. We compared these subgroups with each other and the control group (Group IV according to the serum fibrinogen, plasminogen, alpha2-anti-plasmin (α2-anti-plasmin, and PAI. Results. We detected that PAI-1, serum fibrinogen, and plasminogen levels were similar between the diabetic and control groups (P=0.209, P=0.224, and P=0.244, resp., whereas α2-anti-plasmin was higher in Groups I, II, and III compared to the control group (P<0.01, P<0.05, and P<0.001, resp.. There was a positive correlation between serum α2-anti-plasmin and HbA1c levels (r=0,268, P=0.031. Conclusion. To our knowledge there is scarce data in the literature about α2-anti-plasmin levels in type 1 diabetes. A positive correlation between α2-anti-plasmin with HbA1c suggests that fibrinolytic markers may improve with disease regulation and better glycemic control.

  19. The effects of benzylsulfonyl-D-Ser-homoPhe-(4-amidino-benzylamide), a dual plasmin and urokinase inhibitor, on facial skin barrier function in subjects with sensitive skin.

    Science.gov (United States)

    Voegeli, R; Wikstroem, P; Campiche, R; Steinmetzer, T; Jackson, E; Gempeler, M; Imfeld, D; Rawlings, A V

    2017-04-01

    The aim of this study was to optimize the synthesis of the plasmin and urokinase (uPA) inhibitor benzylsulfonyl-D-Ser-homoPhe-(4-amidino-benzylamide) (BSFAB), to characterize its activity and mechanism of action and to assess its use to improve stratum corneum (SC) barrier function. Peptide coupling methods were used to synthesize BSFAB, and high-performance liquid chromatography-mass spectrometry (HPLC-MS) together with (1) H- and (13) C-nuclear magnetic resonance spectroscopy (NMR) were applied to clarify its structure and determine its purity. Its binding mode was determined by docking studies to the catalytic domains of plasmin and uPA. Inhibition constants (Ki ) were determined by enzyme kinetic studies, and the effect of BSFAB on plasmin, uPA and transglutaminase 1 expression was evaluated in non-cytokine and cytokine-stimulated keratinocytes. A vehicle-controlled clinical study on SC barrier function was conducted on facial skin of subjects with self-perceived sensitive skin. BSFAB was synthesized with high purity (97.3%). In silico studies indicated that the amidine moiety of BSFAB was anchored in the S1 pocket of both enzymes by binding to Asp189, Ser190 and Gly219, whereas the backbone of the D-Ser residue makes an anti-parallel β-sheet interaction with Gly216. BSFAB was shown to be an effective inhibitor of plasmin and uPA with Ki values of 29 and 25 nM, respectively. BSFAB also inhibited keratinocyte-secreted protease activities in basal (plasmin inhibition 37.7%, P sensitive skin and improved their perception of having a healthy skin condition. © 2016 Society of Cosmetic Scientists and the Société Française de Cosmétologie.

  20. The effect mechanism of pharmacological vitreolysis with plasmin and hyaluronidase%纤溶酶和透明质酸酶药物性玻璃体切割术的发生机制

    Institute of Scientific and Technical Information of China (English)

    王志良; 施沃栋; 张皙; 王方; 范先群; 罗敏

    2010-01-01

    目的 研究纤溶酶联合透明质酸酶诱导药物性玻璃体切割术的发生机制.方法 新西兰白兔24只,分为6组,采用玻璃体腔内注射药物0.1mL.A、B、C组分别注射纤溶酶4、2、1μmol/L;D组:纤溶酶1μmol/L+透明质酸酶20μmol/L;E组:透明质酸酶20μmol/L;F组:BSS溶液0.1mL为对照组.7d后免疫胶体金电镜技术检测玻璃体视网膜界面层粘连蛋白(LN)、纤维连接蛋白(FN)抗体分布.另取7只白兔(14只眼)进行纤溶酶活性测定,组1:一侧眼玻璃体腔内注射纤溶酶1μmol/L+透明质酸酶20μmol/L;组2:对侧眼玻璃体腔内注射纤溶酶1μmol/L.结果 免疫胶体金电镜检测视网膜内界膜表面LN、FN显示,A、B、C、D组与对照组比较LN数量减少,差异均有统计学意义(P<0.01),E组与对照组比较LN数量减少,差异无统计学意义(P>0.05).FN免疫胶体金电镜结果 与LN相似.纤溶酶1μmol/L组在药物注射后1h内维持最大纤溶酶活性,此后酶活性逐渐下降,12h后酶活性消失,D组纤溶酶活性曲线和C组走向基本一致.结论 药物性玻璃体切割术的实质是溶解玻璃体视网膜界面的LN、FN等分子胶而发生玻璃体后脱离(PVD),透明质酸酶联合纤溶酶可显著提高PVD的效果,提示合理诱发PVD的药物应既能解除玻璃体视网膜界面之间的粘连,又能液化玻璃体.%Background Many ophthalmologists have proved that the intravitreal injection of plasmin can safely induce posterior vitreous detachment(PVD),but if it can generate the complete PVD need further to seek confirmation.Researches showed that the safe dose and toxicity dose of dispase are very near,so its application is limited.Whether hyaluronidase can induce PVD is still in controversy.Objective This study is to clarity the mechanism of pharmacological vitreolysis with plasmin and hyaluronidase.Methods Plasmin 4μmol/L,2μmol/L and 1μmol/L,plasmin 1μmol/L+ hyaluronidase 20μmol/L,hyaluronidase alone were

  1. Up-regulation of milk secretion with modified microclimate through manipulating plasminogen-plasmin system in Murrah buffaloes during hot dry season

    Science.gov (United States)

    Haque, N.; Singh, M.; Hossain, S. A.

    2016-12-01

    The present study was aimed at determining changes in milk yield and composition along with the plasminogen-plasmin system of milk, plasma hormones, and metabolites of buffaloes during hot dry season (air temperature range 39.7 to 44.8 °C) under two different management systems. Buffaloes were divided in two groups of six animals each: control and treatment, where treatment group animals accessed benefit of mist and fan cooling from 9:30 a.m. to 5:00 p.m., while control group animals were devoid of it. Duration of experiment was 6 weeks. Under mist and fan cooling system, buffaloes experienced better comfort by alleviating environmental stress as their physiological responses such as rectal temperature, respiration rate, pulse rate, and forehead and middorsal temperatures were significantly ( P milk yield by 4.44 % ( P milk samples revealed higher concentration of plasminogen (7.99 vs 6.27 μg/ml; P calcium content of milk, GH, and epinephrine level in plasma. Hence, it may be concluded that provision of cooling system during summer was effective to minimize environmental stress and improve milk production by manipulation of the PG-PL system in buffaloes.

  2. Up-regulation of milk secretion with modified microclimate through manipulating plasminogen-plasmin system in Murrah buffaloes during hot dry season

    Science.gov (United States)

    Haque, N.; Singh, M.; Hossain, S. A.

    2016-05-01

    The present study was aimed at determining changes in milk yield and composition along with the plasminogen-plasmin system of milk, plasma hormones, and metabolites of buffaloes during hot dry season (air temperature range 39.7 to 44.8 °C) under two different management systems. Buffaloes were divided in two groups of six animals each: control and treatment, where treatment group animals accessed benefit of mist and fan cooling from 9:30 a.m. to 5:00 p.m., while control group animals were devoid of it. Duration of experiment was 6 weeks. Under mist and fan cooling system, buffaloes experienced better comfort by alleviating environmental stress as their physiological responses such as rectal temperature, respiration rate, pulse rate, and forehead and middorsal temperatures were significantly (P animals. However, exposure of buffaloes to cooling system did not alter composition and calcium content of milk, GH, and epinephrine level in plasma. Hence, it may be concluded that provision of cooling system during summer was effective to minimize environmental stress and improve milk production by manipulation of the PG-PL system in buffaloes.

  3. Thrombomodulin-dependent effect of factor V Leiden mutation on the cross-linking of α2-plasmin inhibitor to fibrin and its consequences on fibrinolysis.

    Science.gov (United States)

    Koncz, Zsuzsa; Bagoly, Zsuzsa; Haramura, Gizella; Mezei, Zoltán A; Muszbek, László

    2012-09-01

    It has been shown that thrombomodulin (TM) considerably delays factor XIII (FXIII) activation and this effect is abrogated by Factor V Leiden (FV(Leiden)) mutation. The aim of the study was to explore the effect of TM on the cross-linking of α(2)-plasmin inhibitor (α(2)-PI) to fibrin in plasma samples of different FV genotypes and how this effect is related to the impaired fibrinolysis of FV(Leiden) carriers. In the plasma samples of fifteen individuals with different FV genotypes and in FV deficient plasma supplemented with wild type FV or FV(Leiden) coagulation was initiated by recombinant human tissue factor and phospholipids with or without recombinant human TM (rhTM). In the recovered clots the extent of α(2)-PI-fibrin cross-linking was evaluated by Western blotting and quantitative densitometry. The effect of rhTM on tissue plasminogen activator (tPA) induced clot lysis was measured by turbidimetric method. rhTM significantly delayed the formation of α(2)-PI-fibrin α-chain heterodimers/oligomers in plasma samples containing wild type FV. This effect of rhTM was impaired in the presence of FV(Leiden). rhTM delayed tPA-induced clot lysis and this effect of rhTM was more pronounced in plasma containing FV(Leiden). When TAFIa was inhibited by potato carboxypeptidase inhibitor, rhTM accelerated clot lysis in the presence of wild type FV, which is explained by the delayed α(2)-PI-fibrin cross-linking. This effect of rhTM did not prevail in the presence of FV(Leiden). FV(Leiden) abrogates the delaying effect of rhTM on α(2)-PI-fibrin cross-linking, which contributes to the impaired fibrinolysis observed in FV(Leiden) carriers. Copyright © 2012 Elsevier Ltd. All rights reserved.

  4. Studies of alkaline protease with plasmin activity in milk%牛乳中具纤溶活性碱性蛋白酶的研究

    Institute of Scientific and Technical Information of China (English)

    白英; 陈振涛

    2012-01-01

    One alkaline protease with plasmin activity was isolated from late-lactation bovine milk. The enzyme was purified to electrophoret-ic homogeneity using ε—Amino caproic acid, CM Sepharose Fast Flow ion exchange chromatography, Lysine—Sepharose affinity chromatogra-phy. Purify multiple is 2.11. The isoeleetrie point of protease was 7.8. The molecular weight was 47.0 ku measured by SDS—PAGE. Activity of protease was completely inhibited by the serine protease inhibitor, PMSF, indicated that it belongs to the serine protease family. The enzyme had a high degrading activity for casein.%通过ε-氨基己酸的解离柞用,利用CM Sepharose Fast Flow凝胶过滤层析、Lysine-Sepharose(赖氨酸-琼脂糖凝胶)亲和层析的方法,通过SDS -聚丙烯酰胺凝胶电泳纯度鉴定,从末乳中分离出一种具有纤溶活性的碱性蛋白酶.采用凝胶色谱和等电聚焦法对其分子量和等电点进行测定.结果表明:该蛋白酶为单体蛋白,纯化倍数2.11,分子量为47.0 ku,等电点7.8.纯化后酶的活性可完全被PMSF抑制剂抑制,属于丝氨酸蛋白酶家族.对酪蛋白具有一定的水解能力.

  5. 一种产血纤维蛋白溶酶菌株的筛选及液体发酵条件的选择%SELECTION OF FERMENT MEDIUM FOR THE SECRETION OF PLASMIN FROM A MARINE BACTERIA

    Institute of Scientific and Technical Information of China (English)

    刘晨光; 刘万顺; 魏香

    2001-01-01

    主要进行了产生纤维蛋白溶酶菌株的筛选培养和液体发酵条件的优化等方面的研究工作。结果表明,该菌为一种短杆状菌,革兰氏染色为阴性,具有对血纤维蛋白的产酶依赖性。用此菌作为菌种进行发酵产酶的最适条件为:碳源,木糖1.5%;氮源,血纤维蛋白1.75%;发酵液初始pH7.4;接种量5%;发酵时间24h。%This paper reports for the optimum growth medium for the secretion of plasmin from a marine bacteria named FE 92-8. This bacteria is shortly-bacillary, gram-negative, dependant on the existence of fibrin. The quantity of this bacteria and the activity of plasmin secreted by the bacteria is the highest at below culture condition: carbon source, 1.5 percent xylose; nutrogen source, 1.75 percent fibrin; initial pH 7.4;inoculative quantity,5.0 percent, ferment temperature, 30 centigrade degree, ferment time,24hours.

  6. Laminin chain expression suggests that laminin-10 is a major isoform in the mouse hippocampus and is degraded by the tissue plasminogen activator/plasmin protease cascade during excitotoxic injury.

    Science.gov (United States)

    Indyk, J A; Chen, Z L; Tsirka, S E; Strickland, S

    2003-01-01

    Laminins are important components of the extracellular matrix, and participate in neuronal development, survival and regeneration. The tissue plasminogen activator/plasmin extracellular protease cascade and downstream laminin degradation are implicated in excitotoxin-induced neuronal degeneration. To determine which specific laminin chains are involved, we investigated the expression of laminins in the hippocampus, and the cell types expressing them. Reverse transcription-PCR demonstrated that the messenger RNAs for all laminin chains could be detected in the hippocampus. To determine the localization of laminin chain expression, immunostaining was used. This method showed that alpha5, beta1 and gamma1 are most highly expressed in the neuronal cell layers. Immunoblotting confirmed the hippocampal expression of the chains alpha5, beta1 and gamma1, and RNA in situ hybridization showed a neuronal expression pattern of alpha5, beta1 and gamma1. At early time points following intrahippocampal injection of kainate, alpha5, beta1 and gamma1 chain immunoreactivities were lost. In addition, tissue plasminogen activator-deficient mice, which are resistant to kainate-induced neuronal death, show no significant change in laminins alpha5, beta1 and gamma1 after intrahippocampal kainate injection. Taken together, these results suggest that laminin-10 (alpha5-beta1-gamma1) comprises a major neuronal laminin in the mouse hippocampus, and is degraded before neuronal death during excitotoxic injury by the tissue plasminogen activator/plasmin protease cascade. By identifying a neuronal laminin (laminin-10) that participates in neuronal degeneration after excitotoxic injury, this study clarifies the molecular definition of the extracellular matrix in the hippocampus and further defines a pathway for mechanisms of neuronal death.

  7. Analysis on factors affecting plasmin activity of raw milk quality%影响原料乳品质的纤溶酶活性的因素分析

    Institute of Scientific and Technical Information of China (English)

    张书文; 刘鹭; 孙洁; 李红娟; 崔文明; 芦晶; 吕加平

    2014-01-01

    Plasmin(PL) is by far the predominant and most completely studied endogenous protease in bovine milk. PL activity level is an important factor affecting dairy product quality and its processing characteristics. Raw milk with a high PL level can have detrimental effects on the quality of pasteurized milk and UHT milk, causing undesirable flavor, precipitation and gelation, and shortening the shelf life of the product. Considerable research has focused on minimizing the activity of PL in fluid milk. Many factors, such as pH, mineral content, whey proteins, and storage temperature can influence the kinetics of the PL-induced hydrolysis. However, according to China’s current raw milk production situatioin, the objective of this study is to determine how such factors such as cattle breed, parity, dairy farming mode, and milking time affect PL activity in milk; thermal stability of plasmin is also studied. The results show that the PL activity between dairy farming mode, parity, and cow breeds are significant (P0.05). PL activity of raw milk gradually decreases with increasing thermal treatment temperature. However, the thermal treatment associated with pasteurization of milk (75℃, 15 sec) has been shown to increase PL levels. This increase in PL activity has long been attributed to inactivation of plasmin inhibitors(PI) and plasminogen activator inhibitors(PAI), but plasminogen activators (PA)is even more heat stable than PIand PAIduring pasteurization and UHT. In this temperature range, plasminogen (PG) loses its naturally occurring tertiary structure, but is not yet inhibited. On the contrary, PG becomes more accessible to the action of PA by unfolding of its kringles. Activation of PG is enhanced upon pasteurization heating. Further heat treatment has been shown to reduce PL levels. PL activity only decreases 25 percent after pasteurized (75℃, 15 sec) treatment. The effects of cysteine addition and heat treatment on PL activity is also studied. The results show

  8. Evoking plasmin for β-amyloid clearance

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    @@ Alzheimer's disease (AD) is a progressive neurodegenerative disorder and the most common cause of dementia.It is estimated that there are currently about 18 million people worldwide suffering from Alzheimer's disease, and this number will double by 2 025 to 34 millions (http://www.alz.org). Enormous efforts have been taken to identify key molecular events underlying AD pathogenesis and seek for measures to postpone the onset and progression of,if not prevent the disease.

  9. 纤溶酶和透明质酸酶诱导猪玻璃体后脱离的比较研究%A comparative study on the induction of posterior vitreous detachment by plasmin and/or hyaluronidase intravitreal injection into pig eyes

    Institute of Scientific and Technical Information of China (English)

    张志红; 陶海; 吴海洋

    2007-01-01

    目的:研究纤溶酶和透明质酸酶诱导猪玻璃体后脱离(PVD)的有效性和安全性,比较两种酶单独应用和联合应用的效果.方法:小型猪15只随机分为A,B,C三组,每组5只,随机选取1只眼为实验眼,对侧眼为对照眼.三组实验眼玻璃体腔分别注射50U(0.1 mL)透明质酸酶、0.5U(0.1mL)纤溶酶和0.5U(0.05mL)纤溶酶加50U(0.05mL)透明质酸酶,对照眼均注射平衡盐溶液(BSS)0.1ml.注药后进行裂隙灯、直、间接检眼镜、眼B超、视网膜电图(ERG)等检查,7d后摘除眼球进行光镜、透射电镜、扫描电镜检查.结果:B超检查显示A组有1只实验眼、B组有两只实验眼于注药后1d观察到部分性PVD,C组有1只实验眼于注药后1h观察到部分性PVD.B超、光镜和扫描电镜检查显示注药后7d A组和B组实验眼均见部分性PVD,C组实验眼均见完全性PVD,对照眼未见PVD.实验及对照眼注药前、后ERG a波、b波波幅均无显著性差异,光镜及透射电镜检查未见视网膜损害.结论:0.5U纤溶酶和50U透明质酸酶单独及联合应用均可快速、安全、有效地诱导猪眼玻璃体后脱离,且联合用药较单独用药诱导PVD更快速、更有效.%· AIM: To investigate the efficacy and safety of intravitreal injection of plasmin, hyaluronidase, or the combination of the two in inducing posterior vitreous detachment (PVD).· METHODS: 15 mini-type pigs were assigned to three groups (Group A, B and C), 5 in each group. One eye of each pig was intravitreally injected with the studying agent,and the fellow eye was used as control. Group A received a vitreous injection of hyaluronidase 50U (0.1mL); group B received plasmin 0.5U (0.1mL); group C received plasmin 0.5U (0.05mL) combined with hyaluronidase 50U (0.05mL). The fellow eyes in each group were injected with 0.1mL balanced salt solution (BSS). All the pigs were examined with slit-lamp biomicroscope, direct and indirect ophthalmoscope, B-scan and electroretinograph

  10. Plasmin-Cellular Interactions in Breast Cancer Invasion and Metastasis.

    Science.gov (United States)

    1997-10-01

    Heymann Nephritis autoantigen (gp330) binds plas- minogen (Kanalas and Makker , 1991). The present study was undertaken to identify major plas- minogen...Nat. Acad. Sei. USA 76,353-357. Kanalas, J. J. and Makker , S. P. (1991). Identification of the rat Heymann nephritis autoantigen (GP330) as a

  11. Proteinuric diseases with sodium retention: Is plasmin the link?

    DEFF Research Database (Denmark)

    Svenningsen, Per; Skøtt, Ole; Jensen, Boye L

    2012-01-01

    1. Sodium retention in disease states characterized by proteinuria, such as nephrotic syndrome, preeclampsia, and diabetic nephropathy, occurs through poorly understood mechanism(s). 2. In the nephrotic syndrome, data from experimental and clinical studies indicate that the sodium retention...... originates in the renal cortical collecting duct and involves hyper-activity of the epithelial sodium channel (ENaC). 3. The stimulus for the increased ENaC activity does not appear to involve any of the classical sodium retaining mechanisms, such as the renin-angiotensin-aldosterone system, arginine...... aminonucleoside (PAN) rat model of nephrotic syndrome. 6. This finding suggests that a defective glomerular filtration barrier allows filtration into the tubular fluid of substances that activate ENaC and enhance sodium reabsorption. This concept may be expanded to other disease state, such as preeclampsia...

  12. Study on the safety of eye anterior tissues during plasmin and hyaluronidase inducing posterior vitreous detachment in pigs%纤溶酶和透明质酸酶在诱导猪玻璃体后脱离中对眼前部组织的安全性研究

    Institute of Scientific and Technical Information of China (English)

    刘雪霞; 吴海洋; 陶海

    2007-01-01

    目的:研究纤溶酶和透明质酸酶玻璃体内注射诱导猪玻璃体后脱离对眼前部组织的影响,探讨其眼前部组织的安全性.方法:15只健康无眼疾贵州小型香猪,分为A,B,C组,每组5只,每只1眼为实验眼,另1眼为对照眼,实验组与对照组各15眼.实验眼玻璃体内注射酶,A组50U(0.1mL)透明质酸酶,B组0.5U(0.1mL)纤溶酶,C组0.5U(0.05mL)纤溶酶+50U(0.05mL)透明质酸酶,对照眼注射等量BSS液.注射前后分别行裂隙灯显微镜、间接眼底镜、Sch(o)tz眼压计临床检查.7d后摘除眼球,行组织学检查.眼前部组织角膜、虹膜做光镜观察,睫状体、晶状体上皮做透射电镜超微结构的研究.结果:7d后A,B,C组实验眼及各组对照眼无明显眼内炎症反应,术前术后眼压变化差异无统计学意义.实验眼光镜观察角膜、虹膜组织结构无明显异常,电镜观察晶状体、睫状体上皮,细胞形态规则,结构清晰,胞膜完整,周界清楚,连接紧密,对照眼光镜及电镜观察组织结构与实验眼无差异.结论:0.5U纤溶酶和50U透明质酸酶猪玻璃体内注射诱导玻璃体后脱离中安全,无眼前部组织的毒性病理变化.%· AIM: To evaluate the safety of eye anterior tissue when plasmin (Pm) and hyaluronidase (HS) are injected into pigs'vitreouses to induce posterior vitreous detachment(PVD).· METHODS: 15 pigs without ocular diseases were randomly assigned to groups A,B,C (5 in each group). For each pig, one eye was experimental, the other eye was control. The experimental eye received intravitreal injection with enzyme:group A: 50U(0.1mL) HS; group B: 0.5U(0.1mL) Pm; group C: 0.5U(0.05mL) Pm combined with 50U(0.05mL) HS; while the control eye received intravitreal injection with equivalent dose of balanced salt solution (BSS). Postoperative reactions in the eyes were carefully observed by clinical examinations such as slit-lamp microscopy and direct and indirect ophthalmoscopy and measurement of

  13. Bacillus anthracis interacts with plasmin(ogen to evade C3b-dependent innate immunity.

    Directory of Open Access Journals (Sweden)

    Myung-Chul Chung

    Full Text Available The causative agent of anthrax, Bacillus anthracis, is capable of circumventing the humoral and innate immune defense of the host and modulating the blood chemistry in circulation to initiate a productive infection. It has been shown that the pathogen employs a number of strategies against immune cells using secreted pathogenic factors such as toxins. However, interference of B. anthracis with the innate immune system through specific interaction of the spore surface with host proteins such as the complement system has heretofore attracted little attention. In order to assess the mechanisms by which B. anthracis evades the defense system, we employed a proteomic analysis to identify human serum proteins interacting with B. anthracis spores, and found that plasminogen (PLG is a major surface-bound protein. PLG efficiently bound to spores in a lysine- and exosporium-dependent manner. We identified α-enolase and elongation factor tu as PLG receptors. PLG-bound spores were capable of exhibiting anti-opsonic properties by cleaving C3b molecules in vitro and in rabbit bronchoalveolar lavage fluid, resulting in a decrease in macrophage phagocytosis. Our findings represent a step forward in understanding the mechanisms involved in the evasion of innate immunity by B. anthracis through recruitment of PLG resulting in the enhancement of anti-complement and anti-opsonization properties of the pathogen.

  14. Localization and regulation of the tissue plasminogen activator-plasmin system in the hippocampus.

    Science.gov (United States)

    Salles, Fernando J; Strickland, Sidney

    2002-03-15

    The extracellular protease cascade of tissue plasminogen activator (tPA) and plasminogen has been implicated in neuronal plasticity and degeneration. We show here that unstimulated expression of tPA in the mouse hippocampus is concentrated in the mossy fiber pathway, with little or no expression within the perforant path, the Schaffer collaterals, or neuronal cell bodies. tPA protein is also expressed in vascular endothelial cells throughout the brain parenchyma. Four hours after excitotoxic injury, tPA protein is transiently induced within CA1 pyramidal neurons. The induced CA1 tPA is localized to neurons that survive the injury and is enzymatically active. Within the mossy fiber pathway, injury resulted in decreased tPA protein. In contrast, mossy fiber tPA activity displayed a biphasic character: transient increase at 8 hr, then a decrease by 24 hr after injury. Analysis of plasminogen activator inhibitor-1 (PAI-1) expression showed that PAI-1 antigen is upregulated by 24 hr and could account for the tPA activity downregulation seen at this time point. Plasminogen immunohistochemistry suggested an increase within the mossy fiber pathway after injury. Finally, hippocampal tPA expression among various mammalian species was strikingly different. These results indicate a complex control of tPA protein and enzymatic activity in the hippocampus that may help regulate neuronal plasticity.

  15. Plasmin is a natural trigger for bradykinin production in patients with hereditary angioedema with factor XII mutations

    NARCIS (Netherlands)

    de Maat, Steven; Björkqvist, Jenny; Suffritti, Chiara; Wiesenekker, Chantal P.; Nagtegaal, Willem; Koekman, Arnold; van Dooremalen, Sanne; Pasterkamp, Gerard; de Groot, Philip G.; Cicardi, Marco; Renné, Thomas; Maas, Coen

    2016-01-01

    BACKGROUND: Patients with angioedema experience unpredictable attacks of tissue swelling in which bradykinin is implicated. Several distinct mutations in Factor XII (FXII) are associated with hereditary angioedema (HAE) in the presence of normal C1 esterase inhibitor activity (FXII-HAE). The underly

  16. Cloning and expression of the receptor for human urokinase plasminogen activator, a central molecule in cell surface, plasmin dependent proteolysis

    DEFF Research Database (Denmark)

    Roldan, A.L.; Cubellis, M.V.; Masucci, M.T.

    1990-01-01

    , and therefore the capacity of cells to migrate and invade neighboring tissues. We have isolated a 1.4 kb cDNA clone coding for the entire human uPAR. An oligonucleotide synthesized on the basis of the N-terminal sequence of the purified protein was used to screen a cDNA library made from SV40 transformed human......, a size very close to that of the cloned cDNA. Expression of the uPAR cDNA in mouse cells confirms that the clone is complete and expresses a functional uPA binding protein, located on the cell surface and with properties similar to the human uPAR. Caseinolytic plaque assay, immunofluorescence analysis...... fibroblasts [Okayama and Berg (1983) Mol. Cell Biol., 3, 280-289]. The cDNA encodes a protein of 313 amino acids, preceded by a 21 residue signal peptide. A hydrophobicity plot suggests the presence of a membrane spanning domain close to the C-terminus. The cDNA hybridizes to a 1.4 kb mRNA from human cells...

  17. Cloning and expression of the receptor for human urokinase plasminogen activator, a central molecule in cell surface, plasmin dependent proteolysis

    DEFF Research Database (Denmark)

    Roldan, A L; Cubellis, M V; Masucci, M T

    1990-01-01

    , and therefore the capacity of cells to migrate and invade neighboring tissues. We have isolated a 1.4 kb cDNA clone coding for the entire human uPAR. An oligonucleotide synthesized on the basis of the N-terminal sequence of the purified protein was used to screen a cDNA library made from SV40 transformed human......, a size very close to that of the cloned cDNA. Expression of the uPAR cDNA in mouse cells confirms that the clone is complete and expresses a functional uPA binding protein, located on the cell surface and with properties similar to the human uPAR. Caseinolytic plaque assay, immunofluorescence analysis...

  18. Depósitos corporales de hierro y pronóstico de los pacientes con ictus isquémico agudo tratados con activador recombinante tisular del plasminógeno (r-tPA)

    OpenAIRE

    2011-01-01

    Descripció del recurs: 26 juliol 2011 Introducción y objetivos: La sobrecarga de hierro se ha asociado a un incremento del estrés oxidativo y del daño cerebral en modelos experimentales de isquemia y reperfusión cerebral. Este estudio investiga como objetivo primario si las concentraciones elevadas de ferritina en suero, como indicador de los depósitos corporales de hierro, se asocian a un peor pronóstico funcional, a la transformación hemorrágica (TH) del infarto y al edema cerebral despu...

  19. Grupo de trabajo de Leiden sobre fibrinolysis : procedimientos de recogida y manipulación de sangre para la evaluación del activador del plasminógeno de tipo tisular (t-PA) y del inhibidor-1 del activador del plasminógeno (PAI-1) [Leiden fibrinolysis working party : blood collection and handling procedures for assessment of tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1)

    NARCIS (Netherlands)

    Kluft, C.; Verheijen, J.H.

    1992-01-01

    Procedures of blood collection and handling are different for the various variables in haemostasis and tend to change gradually with progress in knowledge and methods. The Working Party has gathered background information about procedures and discussed minimal requirements specifically for t-PA and

  20. Grupo de trabajo de Leiden sobre fibrinolysis : procedimientos de recogida y manipulación de sangre para la evaluación del activador del plasminógeno de tipo tisular (t-PA) y del inhibidor-1 del activador del plasminógeno (PAI-1) [Leiden fibrinolysis working party : blood collection and handling procedures for assessment of tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1)

    NARCIS (Netherlands)

    Kluft, C.; Verheijen, J.H.

    1992-01-01

    Procedures of blood collection and handling are different for the various variables in haemostasis and tend to change gradually with progress in knowledge and methods. The Working Party has gathered background information about procedures and discussed minimal requirements specifically for t-PA and

  1. Gender affects skin wound healing in plasminogen deficient mice

    DEFF Research Database (Denmark)

    Rønø, Birgitte; Engelholm, Lars Henning; Lund, Leif Røge;

    2013-01-01

    functional plasmin thus display decreased tumor growth in a variety of cancer models. Interestingly, this role of plasmin has, in regard to skin cancer, been shown to be restricted to male mice. It remains to be clarified whether gender also affects other phenotypic characteristics of plasmin deficiency...... or if this gender effect is restricted to skin cancer. To investigate this, we tested the effect of gender on plasmin dependent immune cell migration, accumulation of hepatic fibrin depositions, skin composition, and skin wound healing. Gender did not affect immune cell migration or hepatic fibrin accumulation...... in neither wildtype nor plasmin deficient mice, and the existing differences in skin composition between males and females were unaffected by plasmin deficiency. In contrast, gender had a marked effect on the ability of plasmin deficient mice to heal skin wounds, which was seen as an accelerated wound...

  2. Gender affects skin wound healing in plasminogen deficient mice.

    Directory of Open Access Journals (Sweden)

    Birgitte Rønø

    Full Text Available The fibrinolytic activity of plasmin plays a fundamental role in resolution of blood clots and clearance of extravascular deposited fibrin in damaged tissues. These vital functions of plasmin are exploited by malignant cells to accelerate tumor growth and facilitate metastases. Mice lacking functional plasmin thus display decreased tumor growth in a variety of cancer models. Interestingly, this role of plasmin has, in regard to skin cancer, been shown to be restricted to male mice. It remains to be clarified whether gender also affects other phenotypic characteristics of plasmin deficiency or if this gender effect is restricted to skin cancer. To investigate this, we tested the effect of gender on plasmin dependent immune cell migration, accumulation of hepatic fibrin depositions, skin composition, and skin wound healing. Gender did not affect immune cell migration or hepatic fibrin accumulation in neither wildtype nor plasmin deficient mice, and the existing differences in skin composition between males and females were unaffected by plasmin deficiency. In contrast, gender had a marked effect on the ability of plasmin deficient mice to heal skin wounds, which was seen as an accelerated wound closure in female versus male plasmin deficient mice. Further studies showed that this gender effect could not be reversed by ovariectomy, suggesting that female sex-hormones did not mediate the accelerated skin wound healing in plasmin deficient female mice. Histological examination of healed wounds revealed larger amounts of fibrotic scars in the provisional matrix of plasmin deficient male mice compared to female mice. These fibrotic scars correlated to an obstruction of cell infiltration of the granulation tissue, which is a prerequisite for wound healing. In conclusion, the presented data show that the gender dependent effect of plasmin deficiency is tissue specific and may be secondary to already established differences between genders, such as skin

  3. Activation of pro-urokinase and plasminogen on human sarcoma cells

    DEFF Research Database (Denmark)

    Stephens, R W; Pöllänen, J; Tapiovaara, H

    1989-01-01

    Human HT-1080 fibrosarcoma cells produce urokinase-type plasminogen activator (u-PA) and type 1 plasminogen activator inhibitor (PAI-1). We found that after incubation of monolayer cultures with purified native human plasminogen in serum-containing medium, bound plasmin activity could be eluted...... from the cells with tranexamic acid, an analogue of lysine. The bound plasmin was the result of plasminogen activation on the cell surface; plasmin activity was not taken up onto cells after deliberate addition of plasmin to the serum-containing medium. The cell surface plasmin formation was inhibited...... to inhibition by endogenous PAI-1 and by added PAI-2, while the cell-bound plasmin was inaccessible to serum inhibitors, but accessible to added aprotinin and an anticatalytic monoclonal antibody. A model for cell surface plasminogen activation is proposed in which plasminogen binding to cells from serum medium...

  4. Activation of pro-urokinase and plasminogen on human sarcoma cells

    DEFF Research Database (Denmark)

    Stephens, R. W.; Pöllänen, J.; Tapiovaara,, Hannele

    1989-01-01

    Human HT-1080 fibrosarcoma cells produce urokinase-type plasminogen activator (u-PA) and type 1 plasminogen activator inhibitor (PAI-1). We found that after incubation of monolayer cultures with purified native human plasminogen in serum-containing medium, bound plasmin activity could be eluted...... from the cells with tranexamic acid, an analogue of lysine. The bound plasmin was the result of plasminogen activation on the cell surface; plasmin activity was not taken up onto cells after deliberate addition of plasmin to the serum-containing medium. The cell surface plasmin formation was inhibited...... by an anticatalytic monoclonal antibody to u-PA, indicating that this enzyme was responsible for the activation. Preincubation of the cells with diisopropyl fluorophosphate-inhibited u-PA led to a decrease in surface-bound plasmin, indicating that a large part, if not all, of the cell surface plasminogen activation...

  5. Proteolytic Cleavage of Various Human Serum Proteinase Inhibitors by Candida albicans Aspartic Proteinase

    OpenAIRE

    Tsushima, Hirofumi; MINE, Hiroko

    2008-01-01

    The secreted Candida albicans aspartic proteinase (SAP) is presumed to be one of the putative Candida virulence factors, while serum proteinase inhibitors depend on host defense mechanisms. We examined the interaction between SAP and serum proteinase inhibitors, such as C1-inhibitor, α2 plasmin inhibitor, and antithrombin III. SAP progressively inactivated plasmin inhibitory activity of C1-inhibitor and α2 plasmin inhibitor. It also inactivated thrombin inhibitory activity of antithrombin III...

  6. Urinary serine proteases and activation of ENaC in kidney

    DEFF Research Database (Denmark)

    Svenningsen, Per; Andersen, Henrik; Nielsen, Lise Hald

    2015-01-01

    -ons. Active plasmin in urine has been demonstrated in diabetes, preeclampsia, and nephrosis. Urine from these patients activates, plasmin-dependently, amiloride-sensitive inward current in vitro. The concept predicts that patients with albuminuria may benefit particularly from reduced salt intake with RAS...

  7. SwissProt search result: AK061101 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK061101 006-207-A12 (P29598) Urokinase-type plasminogen activator precursor (EC 3.4.21.73) (uPA) (U-plasmin...ogen activator) [Contains: Urokinase-type plasminogen activator long chain A; Urokinase-type plasmin...ogen activator short chain A; Urokinase-type plasminogen a UROK_RAT 2e-20 ...

  8. SwissProt search result: AK109059 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK109059 002-154-F02 (P16227) Urokinase-type plasminogen activator precursor (EC 3.4.21.73) (uPA) (U-plasmin...ogen activator) [Contains: Urokinase-type plasminogen activator long chain A; Urokinase-type plasmin...ogen activator short chain A; Urokinase-type plasminogen UROK_PAPCY 2e-20 ...

  9. SwissProt search result: AK112005 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK112005 006-202-F10 (P06869) Urokinase-type plasminogen activator precursor (EC 3.4.21.73) (uPA) (U-plasmin...ogen activator) [Contains: Urokinase-type plasminogen activator long chain A; Urokinase-type plasmin...ogen activator short chain A; Urokinase-type plasminogen UROK_MOUSE 5e-18 ...

  10. SwissProt search result: AK112005 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK112005 006-202-F10 (P16227) Urokinase-type plasminogen activator precursor (EC 3.4.21.73) (uPA) (U-plasmin...ogen activator) [Contains: Urokinase-type plasminogen activator long chain A; Urokinase-type plasmin...ogen activator short chain A; Urokinase-type plasminogen UROK_PAPCY 3e-18 ...

  11. SwissProt search result: AK109059 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK109059 002-154-F02 (P06869) Urokinase-type plasminogen activator precursor (EC 3.4.21.73) (uPA) (U-plasmin...ogen activator) [Contains: Urokinase-type plasminogen activator long chain A; Urokinase-type plasmin...ogen activator short chain A; Urokinase-type plasminogen UROK_MOUSE 4e-19 ...

  12. SwissProt search result: AK112005 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK112005 006-202-F10 (P00749) Urokinase-type plasminogen activator precursor (EC 3.4.21.73) (uPA) (U-plasmin...ogen activator) [Contains: Urokinase-type plasminogen activator long chain A; Urokinase-type plasmin...ogen activator short chain A; Urokinase-type plasminogen UROK_HUMAN 7e-20 ...

  13. SwissProt search result: AK061101 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK061101 006-207-A12 (P00749) Urokinase-type plasminogen activator precursor (EC 3.4.21.73) (uPA) (U-plasmin...ogen activator) [Contains: Urokinase-type plasminogen activator long chain A; Urokinase-type plasmin...ogen activator short chain A; Urokinase-type plasminogen UROK_HUMAN 5e-23 ...

  14. SwissProt search result: AK112119 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK112119 006-303-H09 (P00749) Urokinase-type plasminogen activator precursor (EC 3.4.21.73) (uPA) (U-plasmin...ogen activator) [Contains: Urokinase-type plasminogen activator long chain A; Urokinase-type plasmin...ogen activator short chain A; Urokinase-type plasminogen UROK_HUMAN 2e-20 ...

  15. SwissProt search result: AK061101 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK061101 006-207-A12 (P16227) Urokinase-type plasminogen activator precursor (EC 3.4.21.73) (uPA) (U-plasmin...ogen activator) [Contains: Urokinase-type plasminogen activator long chain A; Urokinase-type plasmin...ogen activator short chain A; Urokinase-type plasminogen UROK_PAPCY 3e-23 ...

  16. SwissProt search result: AK112119 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK112119 006-303-H09 (P29598) Urokinase-type plasminogen activator precursor (EC 3.4.21.73) (uPA) (U-plasmin...ogen activator) [Contains: Urokinase-type plasminogen activator long chain A; Urokinase-type plasmin...ogen activator short chain A; Urokinase-type plasminogen a UROK_RAT 3e-17 ...

  17. SwissProt search result: AK112119 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK112119 006-303-H09 (P06869) Urokinase-type plasminogen activator precursor (EC 3.4.21.73) (uPA) (U-plasmin...ogen activator) [Contains: Urokinase-type plasminogen activator long chain A; Urokinase-type plasmin...ogen activator short chain A; Urokinase-type plasminogen UROK_MOUSE 3e-18 ...

  18. SwissProt search result: AK061101 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK061101 006-207-A12 (P06869) Urokinase-type plasminogen activator precursor (EC 3.4.21.73) (uPA) (U-plasmin...ogen activator) [Contains: Urokinase-type plasminogen activator long chain A; Urokinase-type plasmin...ogen activator short chain A; Urokinase-type plasminogen UROK_MOUSE 2e-21 ...

  19. SwissProt search result: AK112005 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK112005 006-202-F10 (P29598) Urokinase-type plasminogen activator precursor (EC 3.4.21.73) (uPA) (U-plasmin...ogen activator) [Contains: Urokinase-type plasminogen activator long chain A; Urokinase-type plasmin...ogen activator short chain A; Urokinase-type plasminogen a UROK_RAT 4e-17 ...

  20. SwissProt search result: AK109059 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK109059 002-154-F02 (P29598) Urokinase-type plasminogen activator precursor (EC 3.4.21.73) (uPA) (U-plasmin...ogen activator) [Contains: Urokinase-type plasminogen activator long chain A; Urokinase-type plasmin...ogen activator short chain A; Urokinase-type plasminogen a UROK_RAT 8e-19 ...

  1. SwissProt search result: AK112119 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK112119 006-303-H09 (P16227) Urokinase-type plasminogen activator precursor (EC 3.4.21.73) (uPA) (U-plasmin...ogen activator) [Contains: Urokinase-type plasminogen activator long chain A; Urokinase-type plasmin...ogen activator short chain A; Urokinase-type plasminogen UROK_PAPCY 2e-18 ...

  2. SwissProt search result: AK109059 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK109059 002-154-F02 (P00749) Urokinase-type plasminogen activator precursor (EC 3.4.21.73) (uPA) (U-plasmin...ogen activator) [Contains: Urokinase-type plasminogen activator long chain A; Urokinase-type plasmin...ogen activator short chain A; Urokinase-type plasminogen UROK_HUMAN 4e-21 ...

  3. Activation of Pro-uPA is Critical for Initial Escape from the Primary Tumor and Hematogenous Dissemination of Human Carcinoma Cells

    DEFF Research Database (Denmark)

    Bekes, Erin; Deryugina, Elena; Kuprianova, Tatyana

    2011-01-01

    Urokinase-type plasminogen activator (uPA) and plasmin have long been implicated in cancer progression. However, the precise contributions of the uPA/plasmin system to specific steps involved in cancer cell dissemination have not been fully established. Herein, we have employed a highly dissemina...

  4. 纤溶酶对冠心病患者血浆炎性因子的影响及临床意义%The Influence and Clinical Significance of Plasma Inflammatory Cytokines on the Injection with Plasmin of Patients with Coronary Heart Disease

    Institute of Scientific and Technical Information of China (English)

    蔺凤颖; 张清潭

    2015-01-01

    目的:探讨纤溶酶对不同类型冠心病患者白介素‐35(IL‐35)、白介素‐6(IL‐6)及转化生长因子‐β1(TGF‐β1)的影响及其临床意义。方法:将110例患者随机分为纤溶酶组与常规组各55例,采用夹心酶联免疫法(ELISA )测定血浆IL‐35、IL‐6及TGF‐β1水平。比较两组患者治疗前、后 IL‐35、TGF‐β1及 IL‐6水平变化。结果:治疗后纤组 IL‐35、TGF‐β1升高更明显,IL‐6降低更明显(P<0.05),差异有统计学意义;纤组中急性冠脉综合征亚组IL‐35、TGF‐β1升高较稳定型心绞痛亚组更明显(P<0.05),IL‐6降低较稳定型心绞痛亚组更明显(P<0.05)。结论:纤溶酶能升高IL‐35、T G F‐β1水平,降低IL‐6水平,对冠心病疗效确切,且能改善预后。%Objective :Explore the fibrinolytic enzyme in patients with different types of coronary heart disease interleu‐kin‐35(IL‐35) ,interleukin‐6 (IL‐6) and transforming growth factor‐β1 (TGF‐β1 ) ,the influence of and its relationship with the prognosis of patients .Methods :110 patients were randomly divided into the fibrinolytic enzyme group and rou‐tine group ,55 patients with sandwich enzyme‐linked immunoassay (ELISA) determination of serum IL‐35 ,IL‐6 and TGF‐β1 level .Compare two groups before and after treatment in patients with IL‐35 ,TGF‐β1 and IL‐6 level changes . Results:Fiber group after treatment of IL‐35 ,TGF‐β1 higher more apparent ,IL‐6 reduced more significantly ( P<0.05) ,the difference was statistically significant ;Fiber group in acute coronary syndrome and IL‐35 ,TGF‐β1 increases relatively stable angina subgroup more obvious (P<0 .05) ,IL‐6 reduce relatively stable angina subgroup more obvious (P<0 .05).Conclusion:Fibrinolytic enzyme can increase anti‐inflammatory factor (IL‐35 ,TGF‐β1 ) levels ,lower levels of inflammatory factors (IL‐6) ,the curative effect is coronary heart disease ,and can significantly improve the progno‐sis .

  5. The expression and clinical significance of plasmin associated proteins in plasma and bone marrow in the patient with acute leukemia%急性白血病患者血浆和骨髓中纤溶酶相关蛋白的表达及其意义

    Institute of Scientific and Technical Information of China (English)

    刘家华; 钟明; 徐军发; 温才国; 周君纯; 陈小芳

    2007-01-01

    目的 观察纤溶酶相关蛋白t-PA、u-PA、su-PAR、PAI-1、PAI-2在急性白血病中的表达并探讨其临床意义.方法 选择25例急性白血病患者作为研究组,22例非白血病患者为对照组.采用ELISA法检测血浆和骨髓液中t-PA、u-PA、su-PAR、PAI-1、PAI-2的水平.结果 研究组血浆和骨髓液u-PA、su-PAR、PAI-1、PAI-2均高于健康对照组;研究组血浆t-PA高于对照组,而骨髓液中t-PA指标与对照组比较差异无统计学意义(P>0.05).两组血液及骨髓液中PAI-1、PAI-2均无差异.治疗后研究组血液和骨髓液中u-PA、su-PAR、PAI-1、PAI-2均有所降低(P<0.05).将患者按治疗效果不同分为CR、PR、NR三组,预后越差者,u-PA、su-PAR、PAI-1、PAI-2越高.结论 急性白血病患者存在纤溶酶系统表达紊乱.检测u-PA、su-PAR、PAI-1、PAI-2对于急性白血病的诊断、疗效及预后判断具有重要意义.

  6. Poly-L-histidine downregulates fibrinolysis.

    Science.gov (United States)

    Chu, Arthur J; Mathews, Suresh T

    2003-10-01

    The elevated level of histidine-rich glycoprotein was considered a risk factor of inherited thrombophilia. However, the mode of action remains largely unclear. In the current study, we employ poly-l-histidine (PLH) mimicking the histidine-rich region and determine whether PLH modulates urokinase (uPA)-dependent fibrinolysis. In an in vitro model, turbidity appearance and clearance monitored fibrin polymer formation and lysis, respectively. Fibrin polymer formed upon fibrinogen incubation with thrombin. In the presence of uPA or plasmin, fibrin polymer lysis took place in a dose-dependent manner as a function of time. We demonstrated that PLH significantly downregulated uPA-dependent fibrinolysis. PLH had no effect on plasminogen activation, as evidenced by no inhibitions on either uPA amidolytic activity or plasmin formation derived from its zymogen. Nor did PLH show any inhibition on plasmin amidolytic activity. PLH caused a profound delay of plasmin-dependent fibrinolysis upon pre-incubation of either plasmin or fibrinogen with PLH. The observations taken together suggest that the complex [plasmin-PLH-fibrin] formation significantly delayed plasmin-dependent fibrinolysis.

  7. Kinetic mechanism of the activation of human plasminogen by streptokinase.

    Science.gov (United States)

    Kosow, D P

    1975-10-07

    A method of determining the initial rate of plasminogen activation has been developed. The method has been used to investigate the mechanism of activation of human plasminogen by streptokinase. Plasmin formation follows saturation kinetics. Inhibition of plasmin formation by epsilon-aminocaproic acid is uncompetitive with a Ki of 0.6 mM. A model consistent with the data is that streptokinase induces a conformational change in the plasminogen molecule, producing an active center which cleaves an internal peptide bond to produce plasmin. Thus, streptokinase functions as a catalytic allosteric effector.

  8. Relevance of Bifidobacterium animalis subsp. lactis Plasminogen Binding Activity in the Human Gastrointestinal Microenvironment ▿

    Science.gov (United States)

    Candela, Marco; Turroni, Silvia; Centanni, Manuela; Fiori, Jessica; Bergmann, Simone; Hammerschmidt, Sven; Brigidi, Patrizia

    2011-01-01

    Human plasmin(ogen) is regarded as a component of the molecular cross talk between the probiotic species Bifidobacterium animalis subsp. lactis and the human host. However, up to now, only in vitro studies have been reported. Here, we demonstrate that the probiotic strain B. animalis subsp. lactis BI07 is capable of recruiting plasmin(ogen) present at physiological concentrations in crude extracts from human feces. Our results provide evidence that supports the significance of the B. lactis-plasmin(ogen) interaction in the human gastrointestinal tract. PMID:21821753

  9. Relevance of Bifidobacterium animalis subsp. lactis plasminogen binding activity in the human gastrointestinal microenvironment.

    Science.gov (United States)

    Candela, Marco; Turroni, Silvia; Centanni, Manuela; Fiori, Jessica; Bergmann, Simone; Hammerschmidt, Sven; Brigidi, Patrizia

    2011-10-01

    Human plasmin(ogen) is regarded as a component of the molecular cross talk between the probiotic species Bifidobacterium animalis subsp. lactis and the human host. However, up to now, only in vitro studies have been reported. Here, we demonstrate that the probiotic strain B. animalis subsp. lactis BI07 is capable of recruiting plasmin(ogen) present at physiological concentrations in crude extracts from human feces. Our results provide evidence that supports the significance of the B. lactis-plasmin(ogen) interaction in the human gastrointestinal tract.

  10. Proceedings of the second China and Japan International Congress of Pathophysiology%第二届中日国际病理生理学术会议论文摘要(续)

    Institute of Scientific and Technical Information of China (English)

    Onmura,Y; Hori,N

    2001-01-01

    @@ Central mechanism of the effect of leptin on learning and memory Anti - obesity and anti - diabetic actions of histamine neurons Regulation of pericellular proteolytic activity: new cascade by plasmin/plasminogen

  11. Purification and characterization of tenerplasminin-1, a serine peptidase inhibitor with antiplasmin activity from the coral snake (Micrurus tener tener) venom.

    Science.gov (United States)

    Vivas, Jeilyn; Ibarra, Carlos; Salazar, Ana M; Neves-Ferreira, Ana G C; Sánchez, Elda E; Perales, Jonás; Rodríguez-Acosta, Alexis; Guerrero, Belsy

    2016-01-01

    A plasmin inhibitor, named tenerplasminin-1 (TP1), was isolated from Micrurus tener tener (Mtt) venom. It showed a molecular mass of 6542Da, similarly to Kunitz-type serine peptidase inhibitors. The amidolytic activity of plasmin (0.5nM) on synthetic substrate S-2251 was inhibited by 91% following the incubation with TP1 (1nM). Aprotinin (2nM) used as the positive control of inhibition, reduced the plasmin amidolytic activity by 71%. Plasmin fibrinolytic activity (0.05nM) was inhibited by 67% following incubation with TP1 (0.1nM). The degradation of fibrinogen chains induced by plasmin, trypsin or elastase was inhibited by TP1 at a 1:2, 1:4 and 1:20 enzyme:inhibitor ratio, respectively. On the other hand, the proteolytic activity of crude Mtt venom on fibrinogen chains, previously attributed to metallopeptidases, was not abolished by TP1. The tPA-clot lysis assay showed that TP1 (0.2nM) acts like aprotinin (0.4nM) inducing a delay in lysis time and lysis rate which may be associated with the inhibition of plasmin generated from the endogenous plasminogen activation. TP1 is the first serine protease plasmin-like inhibitor isolated from Mtt snake venom which has been characterized in relation to its mechanism of action, formation of a plasmin:TP1 complex and therapeutic potential as anti-fibrinolytic agent, a biological characteristic of great interest in the field of biomedical research. They could be used to regulate the fibrinolytic system in pathologies such as metastatic cancer, parasitic infections, hemophilia and other hemorrhagic syndromes, in which an intense fibrinolytic activity is observed.

  12. Development of magnetic nanoparticle based calorimetric assay for the detection of bovine mastitis in cow milk.

    Science.gov (United States)

    Chinnappan, Raja; Al Attas, Sana; Kaman, Wendy E; Bikker, Floris J; Zourob, Mohammed

    2017-04-15

    Mastitis in dairy cattle is an inflammatory reaction of the udder tissue. Mastitis increases plasmin levels, leading to an increased proteolysis of milk proteins such as casein, resulting in a significant decrease in milk quality and related dairy products. Due to its key-role in mastitis, we used plasmin proteolytic activity as a biomarker for the detection of mastitis in bovine mastitic milk. Inspired by earlier studies on protease activity using mastitic milk samples, we developed a simple colorimetric assay to distinguish mastitic milk from milk derived from healthy animals. The plasmin substrate coupled to magnetic nanoparticles form a black self-assembled monolayer on a gold sensor surface. In the presence of increased levels of plasmin, the substrate is cleaved and the peptide fragment attached to the magnetic beads, will be attracted by the magnet which is present under the sensor strips revealing the golden surface. We found the area of the golden color surface proportional to plasmin activity. The sensitivity of this method was determined to be 1 ng/ml of plasmin in vitro. Next, we tested the biosensor using mastitis positive milk of which infection is confirmed by bacterial cultures. This newly developed colorimetric biosensor has high potential in applications for the diagnosis of mastitis with potential spin offs to health, food and environmental sectors. Copyright © 2017 Elsevier Inc. All rights reserved.

  13. Stability of the octameric structure affects plasminogen-binding capacity of streptococcal enolase.

    Directory of Open Access Journals (Sweden)

    Amanda J Cork

    Full Text Available Group A Streptococcus (GAS is a human pathogen that has the potential to cause invasive disease by binding and activating human plasmin(ogen. Streptococcal surface enolase (SEN is an octameric α-enolase that is localized at the GAS cell surface. In addition to its glycolytic role inside the cell, SEN functions as a receptor for plasmin(ogen on the bacterial surface, but the understanding of the molecular basis of plasmin(ogen binding is limited. In this study, we determined the crystal and solution structures of GAS SEN and characterized the increased plasminogen binding by two SEN mutants. The plasminogen binding ability of SENK312A and SENK362A is ~2- and ~3.4-fold greater than for the wild-type protein. A combination of thermal stability assays, native mass spectrometry and X-ray crystallography approaches shows that increased plasminogen binding ability correlates with decreased stability of the octamer. We propose that decreased stability of the octameric structure facilitates the access of plasmin(ogen to its binding sites, leading to more efficient plasmin(ogen binding and activation.

  14. The Biochemistry and Regulation of S100A10: A Multifunctional Plasminogen Receptor Involved in Oncogenesis

    Directory of Open Access Journals (Sweden)

    Patricia A. Madureira

    2012-01-01

    Full Text Available The plasminogen receptors mediate the production and localization to the cell surface of the broad spectrum proteinase, plasmin. S100A10 is a key regulator of cellular plasmin production and may account for as much as 50% of cellular plasmin generation. In parallel to plasminogen, the plasminogen-binding site on S100A10 is highly conserved from mammals to fish. S100A10 is constitutively expressed in many cells and is also induced by many diverse factors and physiological stimuli including dexamethasone, epidermal growth factor, transforming growth factor-α, interferon-γ, nerve growth factor, keratinocyte growth factor, retinoic acid, and thrombin. Therefore, S100A10 is utilized by cells to regulate plasmin proteolytic activity in response to a wide diversity of physiological stimuli. The expression of the oncogenes, PML-RARα and KRas, also stimulates the levels of S100A10, suggesting a role for S100A10 in pathophysiological processes such as in the oncogenic-mediated increases in plasmin production. The S100A10-null mouse model system has established the critical role that S100A10 plays as a regulator of fibrinolysis and oncogenesis. S100A10 plays two major roles in oncogenesis, first as a regulator of cancer cell invasion and metastasis and secondly as a regulator of the recruitment of tumor-associated cells, such as macrophages, to the tumor site.

  15. Secretion of extracellular hsp90α via exosomes increases cancer cell motility: a role for plasminogen activation

    Directory of Open Access Journals (Sweden)

    Chan Doug

    2010-06-01

    Full Text Available Abstract Background Metastasis is a multi-step process that is responsible for the majority of deaths in cancer patients. Current treatments are not effective in targeting metastasis. The molecular chaperone hsp90α is secreted from invasive cancer cells and activates MMP-2 to enhance invasiveness, required for the first step in metastasis. Methods We analyzed the morphology and motility of invasive cancer cells that were treated with exogenous exosomes in the presence or absence of hsp90α. We performed mass spectrometry and immunoprecipitation to identify plasminogen as a potential client protein of extracellular hsp90α. Plasmin activation assays and migration assays were performed to test if plasminogen is activated by extracellular hsp90α and has a role in migration. Results We found that hsp90α is secreted in exosomes in invasive cancer cells and it contributes to their invasive nature. We identified a novel interaction between hsp90α and tissue plasminogen activator that together with annexin II, also found in exosomes, activates plasmin. Extracellular hsp90α promotes plasmin activation as well as increases plasmin dependent cell motility. Conclusions Our data indicate that hsp90α is released by invasive cancer cells via exosomes and implicates hsp90α in activating plasmin, a second protease that acts in cancer cell invasion.

  16. The tissue plasminogen activator-plasminogen proteolytic cascade accelerates amyloid-beta (Abeta) degradation and inhibits Abeta-induced neurodegeneration.

    Science.gov (United States)

    Melchor, Jerry P; Pawlak, Robert; Strickland, Sidney

    2003-10-01

    Accumulation of the amyloid-beta (Abeta) peptide depends on both its generation and clearance. To better define clearance pathways, we have evaluated the role of the tissue plasminogen activator (tPA)-plasmin system in Abeta degradation in vivo. In two different mouse models of Alzheimer's disease, chronically elevated Abeta peptide in the brain correlates with the upregulation of plasminogen activator inhibitor-1 (PAI-1) and inhibition of the tPA-plasmin system. In addition, Abeta injected into the hippocampus of mice lacking either tPA or plasminogen persists, inducing PAI-1 expression and causing activation of microglial cells and neuronal damage. Conversely, Abeta injected into wild-type mice is rapidly cleared and does not cause neuronal degeneration. Thus, the tPA-plasmin proteolytic cascade aids in the clearance of Abeta, and reduced activity of this system may contribute to the progression of Alzheimer's disease.

  17. Proteolytic activation of the epithelial sodium channel ENaC in preeclampsia examined with urinary exosomes

    DEFF Research Database (Denmark)

    Nielsen, Maria Ravn; Rytz, Mie; Frederiksen-Møller, Britta

    2015-01-01

    OBJECTIVES: Increased activity of the epithelial sodium channel (ENaC) in the kidneys may explain the coupling between proteinuria, edema, suppressed aldosterone and hypertension in preeclampsia. Preeclamptic women excrete plasminogen-plasmin in urine. In vitro, plasmin increases the activity...... as a positive control for the presence of collecting duct membrane. RESULTS: Urine plasmin-plasminogen/creatinine ratio was increased in the preeclampsia group (p... pregnancy and preeclampsia CONCLUSIONS: It is possible to examine collecting duct transport proteins in urine exosome from pregnant women including γ-ENaC, 2) Urine exosome fraction displays a variable pattern of γ-ENaC signal with a predominance of cleaved forms in both normal and preeclamptic women...

  18. Plasminogen-dependent proteolytic activity in Bifidobacterium lactis.

    Science.gov (United States)

    Candela, Marco; Miccoli, Giacomo; Bergmann, Simone; Turroni, Silvia; Vitali, Beatrice; Hammerschmidt, Sven; Brigidi, Patrizia

    2008-08-01

    Bifidobacteria represent one of the most important health-promoting bacterial groups of the intestinal microbiota. The binding of plasminogen to species of Bifidobacterium has been recently reported. To further explore the interaction between bifidobacteria and plasminogen, we investigated the role of Bifidobacterium lactis BI07 plasminogen-dependent proteolytic activity in the degradation of host-specific substrates. Our experimental data demonstrate that the recruitment of plasminogen on the bacterial cell surface and its subsequent conversion into plasmin by host-derived plasminogen activators provide B. lactis BI07 with a surface-associated plasmin activity effective in degradation of physiological substrates such as extracellular matrix, fibronectin and fibrinogen. The ability of bifidobacteria to intervene in the host plasminogen/plasmin system may contribute to facilitating colonization of the host gastrointestinal tract.

  19. A novel hemostasis assay for the simultaneous measurement of coagulation and fibrinolysis.

    Science.gov (United States)

    van Geffen, Mark; Loof, Arnoud; Lap, Paul; Boezeman, Jan; Laros-van Gorkom, Britta A P; Brons, Paul; Verbruggen, Bert; van Kraaij, Marian; van Heerde, Waander L

    2011-11-01

    Thrombin and plasmin are the key enzymes involved in coagulation and fibrinolysis. A novel hemostasis assay (NHA) was developed to measure thrombin and plasmin generation in a single well by a fluorimeter. The NHA uses two fluorescent substrates with non-interfering fluorescent excitation and emission spectra. The assay was tested in vitro using modulators like heparin, hirudin, epsilon-aminocaproic acid, gly-pro-arg-pro peptide and reptilase and validated by measurement of prothrombin fragment 1+2 and plasmin-alpha2-antiplasmin levels. Intra- and inter-assay coefficients of variation were coagulation and fibrinolysis was demonstrated by the effect of tissue-type plasminogen activator on thrombin generation and by the different responses of activated protein C and thrombomodulin on fibrinolysis. The last responses showed the linkage between coagulation and fibrinolysis by thrombin activatable fibrinolysis inhibitor. In conclusion, this strategy allows detection of coagulation, fibrinolysis and their interplay in a single assay.

  20. Serpins promote cancer cell survival and vascular co-option in brain metastasis.

    Science.gov (United States)

    Valiente, Manuel; Obenauf, Anna C; Jin, Xin; Chen, Qing; Zhang, Xiang H-F; Lee, Derek J; Chaft, Jamie E; Kris, Mark G; Huse, Jason T; Brogi, Edi; Massagué, Joan

    2014-02-27

    Brain metastasis is an ominous complication of cancer, yet most cancer cells that infiltrate the brain die of unknown causes. Here, we identify plasmin from the reactive brain stroma as a defense against metastatic invasion, and plasminogen activator (PA) inhibitory serpins in cancer cells as a shield against this defense. Plasmin suppresses brain metastasis in two ways: by converting membrane-bound astrocytic FasL into a paracrine death signal for cancer cells, and by inactivating the axon pathfinding molecule L1CAM, which metastatic cells express for spreading along brain capillaries and for metastatic outgrowth. Brain metastatic cells from lung cancer and breast cancer express high levels of anti-PA serpins, including neuroserpin and serpin B2, to prevent plasmin generation and its metastasis-suppressive effects. By protecting cancer cells from death signals and fostering vascular co-option, anti-PA serpins provide a unifying mechanism for the initiation of brain metastasis in lung and breast cancers.

  1. A key role for the urokinase plasminogen activator (uPA) in invasive Group A streptococcal infection.

    Science.gov (United States)

    Sanderson-Smith, Martina L; Zhang, Yueling; Ly, Diane; Donahue, Deborah; Hollands, Andrew; Nizet, Victor; Ranson, Marie; Ploplis, Victoria A; Walker, Mark J; Castellino, Francis J

    2013-01-01

    Recruitment of the serine protease plasmin is central to the pathogenesis of many bacterial species, including Group A streptococcus (GAS), a leading cause of morbidity and mortality globally. A key process in invasive GAS disease is the ability to accumulate plasmin at the cell surface, however the role of host activators of plasminogen in this process is poorly understood. Here, we demonstrate for the first time that the urokinase-type plasminogen activator (uPA) contributes to plasmin recruitment and subsequent invasive disease initiation in vivo. In the absence of a source of host plasminogen activators, streptokinase (Ska) was required to facilitate cell surface plasmin acquisition by GAS. However, in the absence of Ska, host activators were sufficient to promote cell surface plasmin acquisition by GAS strain 5448 during incubation with plasminogen or human plasma. Furthermore, GAS were able mediate a significant increase in the activation of zymogen pro-uPA in human plasma. In order to assess the contribution of uPA to invasive GAS disease, a previously undescribed transgenic mouse model of infection was employed. Both C57/black 6J, and AlbPLG1 mice expressing the human plasminogen transgene, were significantly more susceptible to invasive GAS disease than uPA-/- mice. The observed decrease in virulence in uPA-/-mice was found to correlate directly with a decrease in bacterial dissemination and reduced cell surface plasmin accumulation by GAS. These findings have significant implications for our understanding of GAS pathogenesis, and research aimed at therapeutic targeting of plasminogen activation in invasive bacterial infections.

  2. A key role for the urokinase plasminogen activator (uPA in invasive Group A streptococcal infection.

    Directory of Open Access Journals (Sweden)

    Martina L Sanderson-Smith

    Full Text Available Recruitment of the serine protease plasmin is central to the pathogenesis of many bacterial species, including Group A streptococcus (GAS, a leading cause of morbidity and mortality globally. A key process in invasive GAS disease is the ability to accumulate plasmin at the cell surface, however the role of host activators of plasminogen in this process is poorly understood. Here, we demonstrate for the first time that the urokinase-type plasminogen activator (uPA contributes to plasmin recruitment and subsequent invasive disease initiation in vivo. In the absence of a source of host plasminogen activators, streptokinase (Ska was required to facilitate cell surface plasmin acquisition by GAS. However, in the absence of Ska, host activators were sufficient to promote cell surface plasmin acquisition by GAS strain 5448 during incubation with plasminogen or human plasma. Furthermore, GAS were able mediate a significant increase in the activation of zymogen pro-uPA in human plasma. In order to assess the contribution of uPA to invasive GAS disease, a previously undescribed transgenic mouse model of infection was employed. Both C57/black 6J, and AlbPLG1 mice expressing the human plasminogen transgene, were significantly more susceptible to invasive GAS disease than uPA-/- mice. The observed decrease in virulence in uPA-/-mice was found to correlate directly with a decrease in bacterial dissemination and reduced cell surface plasmin accumulation by GAS. These findings have significant implications for our understanding of GAS pathogenesis, and research aimed at therapeutic targeting of plasminogen activation in invasive bacterial infections.

  3. Mangiferin blocks proliferation and induces apoptosis of breast cancer cells via suppression of the mevalonate pathway and by proteasome inhibition.

    Science.gov (United States)

    Cuccioloni, M; Bonfili, L; Mozzicafreddo, M; Cecarini, V; Scuri, S; Cocchioni, M; Nabissi, M; Santoni, G; Eleuteri, A M; Angeletti, M

    2016-10-12

    Mangiferin is a natural xanthone glycoside with therapeutic potential. Herein, its cytotoxic properties were explored in a human cell model of breast adenocarcinoma. The results supported the multi-target nature of mangiferin action, as the inhibition of three enzymatic systems, namely HMG-CoA reductase, the proteasome and plasmin, respectively in charge of regulating cholesterol homeostasis, protein turnover and cell adhesion, was documented for the first time. Globally, mangiferin was able to selectively block breast cancer cell growth by inducing apoptosis and by arresting cell proliferation through a combined action on cholesterol and proteasome pathways, as well as to inhibit plasmin-mediated mechanisms of cell migration.

  4. Reduced metastasis of transgenic mammary cancer in urokinase-deficient mice

    DEFF Research Database (Denmark)

    Almholt, Kasper; Lund, L.R.; Rygaard, Jørgen

    2005-01-01

    A prominent phenotype of plasmin deficiency in mice is reduced metastasis in the MMTV-PymT transgenic breast cancer model. Proteolytically active plasmin is generated from inactive plasminogen by one of 2 activators, uPA or tPA. We now find that uPA deficiency alone significantly reduces metastasis...... >7-fold in the MMTV-PymT model. We studied a cohort of 55 MMTV-PymT transgenic mice, either uPA-deficient or wild-type controls. Tumor incidence, latency, growth rate and final primary tumor burden were not significantly affected by uPA deficiency. In contrast, average lung metastasis volume...

  5. Fibrin clot formation and lysis: basic mechanisms

    DEFF Research Database (Denmark)

    Sidelmann, JJ; Gram, J; Jespersen, J

    2000-01-01

    induced by coagulation factor XIII (FXIII), and the degradation of fibrinogen and fibrin mediated by plasmin and elastase. Furthermore, factors influencing fibrin structure and fibrin breakdown are addressed; in particular polymorphisms in the genes coding for fibrinogen and FXIII, but also the physical...

  6. The receptor for urokinase-type plasminogen activator and urokinase is translocated from two distinct intracellular compartments to the plasma membrane on stimulation of human neutrophils

    DEFF Research Database (Denmark)

    Plesner, T; Ploug, M; Ellis, V

    1994-01-01

    The cellular receptor for urokinase-type plasminogen activator (uPAR) binds pro-urokinase (pro-uPA) and facilitates its conversion to enzymatically active urokinase (uPA). uPA in turn activates surface-bound plasminogen to plasmin, a process of presumed importance for a number of biologic process...

  7. Photonic Activation of Plasminogen induced by low dose UVB

    DEFF Research Database (Denmark)

    Correia, Manuel Guiherme L.P. Marins; Snabe, Torben; Thiagarajan, Viruthachalam;

    2015-01-01

    Activation of plasminogen to its active form plasmin is essential for several key mechanisms, including the dissolution of blood clots. Activation occurs naturally via enzymatic proteolysis. We report that activation can be achieved with 280 nm light. A 2.6 fold increase in proteolytic activity w...

  8. The ligand-binding domain of the cell surface receptor for urokinase-type plasminogen activator

    DEFF Research Database (Denmark)

    Behrendt, N; Ploug, M; Patthy, L;

    1991-01-01

    part of the intact receptor, probably including the whole sequence 1-87, and contained N-linked carbohydrate. After detergent phase separation in the Triton X-114 system, the fragment was present in the water phase where its binding activity could be demonstrated in the absence of the rest...... applications in interfering with cell-surface plasmin-mediated proteolysis....

  9. SwissProt search result: AK112005 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK112005 006-202-F10 (P15120) Urokinase-type plasminogen activator precursor (EC 3.4.21.73) (uPA) (U-plasmin...ogen activator) [Contains: Urokinase-type plasminogen activator chain A; Urokinase-type plasminogen activator chain B] UROK_CHICK 8e-19 ...

  10. SwissProt search result: AK112119 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK112119 006-303-H09 (Q05589) Urokinase-type plasminogen activator precursor (EC 3.4.21.73) (uPA) (U-plasmin...ogen activator) [Contains: Urokinase-type plasminogen activator chain A; Urokinase-type plasminogen activator chain B] UROK_BOVIN 1e-18 ...

  11. SwissProt search result: AK061101 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK061101 006-207-A12 (Q05589) Urokinase-type plasminogen activator precursor (EC 3.4.21.73) (uPA) (U-plasmin...ogen activator) [Contains: Urokinase-type plasminogen activator chain A; Urokinase-type plasminogen activator chain B] UROK_BOVIN 1e-21 ...

  12. SwissProt search result: AK109059 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK109059 002-154-F02 (Q05589) Urokinase-type plasminogen activator precursor (EC 3.4.21.73) (uPA) (U-plasmin...ogen activator) [Contains: Urokinase-type plasminogen activator chain A; Urokinase-type plasminogen activator chain B] UROK_BOVIN 2e-17 ...

  13. SwissProt search result: AK112005 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK112005 006-202-F10 (Q05589) Urokinase-type plasminogen activator precursor (EC 3.4.21.73) (uPA) (U-plasmin...ogen activator) [Contains: Urokinase-type plasminogen activator chain A; Urokinase-type plasminogen activator chain B] UROK_BOVIN 1e-18 ...

  14. SwissProt search result: AK061101 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK061101 006-207-A12 (P15120) Urokinase-type plasminogen activator precursor (EC 3.4.21.73) (uPA) (U-plasmin...ogen activator) [Contains: Urokinase-type plasminogen activator chain A; Urokinase-type plasminogen activator chain B] UROK_CHICK 7e-25 ...

  15. SwissProt search result: AK112119 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK112119 006-303-H09 (P04185) Urokinase-type plasminogen activator precursor (EC 3.4.21.73) (uPA) (U-plasmin...ogen activator) [Contains: Urokinase-type plasminogen activator chain A; Urokinase-type plasminogen activator chain B] UROK_PIG 6e-22 ...

  16. SwissProt search result: AK112119 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK112119 006-303-H09 (P15120) Urokinase-type plasminogen activator precursor (EC 3.4.21.73) (uPA) (U-plasmin...ogen activator) [Contains: Urokinase-type plasminogen activator chain A; Urokinase-type plasminogen activator chain B] UROK_CHICK 8e-19 ...

  17. Involvement of α2-antiplasmin in dendritic growth of hippocampal neurons.

    Science.gov (United States)

    Kawashita, Eri; Kanno, Yosuke; Asayama, Haruka; Okada, Kiyotaka; Ueshima, Shigeru; Matsuo, Osamu; Matsuno, Hiroyuki

    2013-07-01

    The α2-Antiplasmin (α2AP) protein is known as a principal physiological inhibitor of plasmin, but we previously demonstrated that it acts as a regulatory factor for cellular functions independent of plasmin. α2AP is highly expressed in the hippocampus, suggesting a potential role for α2AP in hippocampal neuronal functions. However, the role for α2AP was unclear. This study is the first to investigate the involvement of α2AP in the dendritic growth of hippocampal neurons. The expression of microtubule-associated protein 2, which contributes to neurite initiation and neuronal growth, was lower in the neurons from α2AP⁻/⁻ mice than in the neurons from α2AP⁺/⁺ mice. Exogenous treatment with α2AP enhanced the microtubule-associated protein 2 expression, dendritic growth and filopodia formation in the neurons. This study also elucidated the mechanism underlying the α2AP-induced dendritic growth. Aprotinin, another plasmin inhibitor, had little effect on the dendritic growth of neurons, and α2AP induced its expression in the neurons from plaminogen⁻/⁻ mice. The activation of p38 MAPK was involved in the α2AP-induced dendritic growth. Therefore, our findings suggest that α2AP induces dendritic growth in hippocampal neurons through p38 MAPK activation, independent of plasmin, providing new insights into the role of α2AP in the CNS.

  18. Genetic predictors of fibrin D-dimer levels in healthy adults

    NARCIS (Netherlands)

    N.L. Smith (Nicholas); J.E. Huffman (Jennifer E.); D.P. Strachan (David); J. Huang (Jian); A. Dehghan (Abbas); S. Trompet (Stella); L.M. Lopez (Lorna M.); S.Y. Shin (So Youn); J. Baumert (Jens); V. Vitart (Veronique); J.C. Bis (Joshua); S.H. Wild (Sarah); A. Rumley (Ann); Q. Yang (Qiong Fang); A.G. Uitterlinden (André); D.J. Stott (David. J.); G. Davies (Gareth); A.M. Carter (Angela M.); B. Thorand (Barbara); O. Polašek (Ozren); B. McKnight (Barbara); H. Campbell (Harry); A.R. Rudnicka (Alicja); M.H. Chen (Min-hsin); B.M. Buckley (Brendan M.); S.E. Harris (Sarah); A. Peters (Annette); D. Pulanic (Drazen); T. Lumley (Thomas); A.J.M. de Craen (Anton J.M.); D.C. Liewald (David C.); C. Gieger (Christian); I. Ford (Ian); A.J. Gow (Alan J.); M. Luciano (Michelle); D.J. Porteous (David J.); X. Guo (Xiuqing); N. Sattar (Naveed); A. Tenesa (Albert); M. Cushman (Mary Ann); P.E. Slagboom (Eline); P.M. Visscher (Peter M.); T.D. Spector (Tim); T. Illig (Thomas); I. Rudan (Igor); E.G. Bovill (Edwin G.); A.F. Wright (Alan); W.L. McArdle (Wendy); G.H. Tofler (Geoffrey); A. Hofman (Albert); R.G.J. Westendorp (Rudi); J.M. Starr (John); P.J. Grant (Peter J.); M. Karakas (Mahir); N.D. Hastie (Nicholas D.); B.M. Psaty (Bruce); J.F. Wilson (James); G.D.O. Lowe (Gordon); C.J. O'Donnell (Christopher); J.C.M. Witteman (Jacqueline); J.W. Jukema (Jan Wouter); I.J. Deary (Ian); N. Soranzo (Nicole); W. Koenig (Wolfgang); C. Hayward (Caroline)

    2011-01-01

    textabstractBACKGROUND: Fibrin fragment D-dimer, one of several peptides produced when crosslinked fibrin is degraded by plasmin, is the most widely used clinical marker of activated blood coagulation. To identity genetic loci influencing D-dimer levels, we performed the first large-scale, genome-wi

  19. Cellular receptors for plasminogen activators recent advances.

    Science.gov (United States)

    Ellis, V

    1997-10-01

    The generation of the broad-specificity protease plasmin by the plasminogen activators urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator (tPA) is implicated in a variety of pathophysiological processes, including vascular fibrin dissolution, extracellular matrix degradation and remodeling, and cell migration. A mechanism for the regulation of plasmin generation is through binding of the plasminogen activators to specific cellular receptors: uPA to the glycolipid-anchored membrane protein urokinase-type plasminogen activator receptor (uPAR) and tPA to a number of putative binding sites. The uPA-uPAR complex can interact with a variety of ligands, including plasminogen, vitronectin, and integrins, indicating a multifunctional role for uPAR, regulating not only efficient and spatially restricted plasmin generation but also having the potential to modulate cell adhesion and signal transduction. The cellular binding of tPA, although less well characterized, also has the capacity to regulate plasmin generation and to play a significant role in vessel-wall biology. (Trends Cardiovasc Med 1997;7:227-234). © 1997, Elsevier Science Inc.

  20. Syphilis and HIV-1 co-infection: influence on CD4 T cell count, HIV-1 viral load and treatment response

    DEFF Research Database (Denmark)

    Kofoed, Kristian; Gerstoft, Jan; Mathiesen, Lars Reinhardt

    2006-01-01

    OBJECTIVES: To assess the effect of human immunodeficiency virus (HIV)-1 and syphilis coinfection on HIV-ribonucleic acid (RNA) viral load, CD4 cell count, and the response in rapid plasmin reagin (RPR) to treatment of the syphilis infection. STUDY DESIGN: Cases of syphilis diagnosed during 1 yea...

  1. Genome-Wide Association Study for Circulating Tissue Plasminogen Activator Levels and Functional Follow-Up Implicates Endothelial STXBP5 and STX2

    NARCIS (Netherlands)

    Huang, Jie; Huffman, Jennifer E.; Yamkauchi, Munekazu; Trompet, Stella; Asselbergs, Folkert W.; Sabater-Lleal, Maria; Tregouet, David-Alexandre; Chen, Wei-Min; Smith, Nicholas L.; Kleber, Marcus E.; Shin, So-Youn; Becker, Diane M.; Tang, Weihong; Dehghan, Abbas; Johnson, Andrew D.; Vinh Truong, [No Value; Folkersen, Lasse; Yang, Qiong; Oudot-Mellkah, Tiphaine; Buckley, Brendan M.; Moore, Jason H.; Williams, Frances M. K.; Campbell, Harry; Silbernagel, Guenther; Vitart, Veronique; Rudan, Igor; Tofler, Geoffrey H.; Navis, Gerjan J.; DeStefano, Anita; Wright, Alan F.; Chen, Ming-Huei; de Craen, Anton J. M.; Worrall, Bradford B.; Rudnicka, Alicja R.; Rumley, Ann; Bookman, Ebony B.; Psaty, Bruce M.; Chen, Fang; Keene, Keith L.; Franco, Oscar H.; Boehm, Bernhard O.; Uitterlinden, Andre G.; Carter, Angela M.; Jukema, J. Wouter; Sattar, Naveed; Bis, Joshua C.; Ikram, Mohammad A.; Sale, Michele M.; McKnight, Barbara; Fornage, Myriam; Ford, Ian; Taylor, Kent; Slagboom, P. Eline; McArdle, Wendy L.; Hsu, Fang-Chi; Franco-Cereceda, Anders; Goodall, Alison H.; Yanek, Lisa R.; Furie, Karen L.; Cushman, Mary; Hofman, Albert; Witteman, Jacqueline C. M.; Folsom, Aaron R.; Basu, Saonli; Matijevic, Nena; van Gilst, Wiek H.; Wilson, James F.; Westendorp, Rudi G. J.; Kathiresan, Sekar; Reilly, Muredach P.; Tracy, Russell P.; Polasek, Ozren; Winkelmann, Bernhard R.; Grant, Peter J.; Hillege, Hans L.; Cambien, Francois; Stott, David J.; Lowe, Gordon D.; Spector, Timothy D.; Meigs, James B.; Marz, Winfried; Eriksson, Per; Becker, Lewis C.; Morange, Pierre-Emmanuel; Soranzo, Nicole; Williams, Scott M.; Hayward, Caroline; van der Harst, Pim; Hamsten, Anders; Lowenstein, Charles J.; Strachan, David P.; O'Donnell, Christopher J.

    2014-01-01

    Objective Tissue plasminogen activator (tPA), a serine protease, catalyzes the conversion of plasminogen to plasmin, the major enzyme responsible for endogenous fibrinolysis. In some populations, elevated plasma levels of tPA have been associated with myocardial infarction and other cardiovascular d

  2. Activation of Pro-uPA is Critical for Initial Escape from the Primary Tumor and Hematogenous Dissemination of Human Carcinoma Cells

    DEFF Research Database (Denmark)

    Bekes, Erin; Deryugina, Elena; Kuprianova, Tatyana

    2011-01-01

    significant amounts of pro-uPA, leading to efficient generation of plasmin in solution and at the cell surface. In a mouse orthotopic xenograft model, treatment with the specific pro-uPA activation-blocking antibody mAb-112 significantly inhibited local invasion and distant metastasis of the PC-hi/diss cells...

  3. The role of proteases in fibronectin matrix remodeling in thyroid epithelial cell monolayer cultures.

    Science.gov (United States)

    Nezi, Luigi; Greco, Dario; Nitsch, Lucio; Garbi, Corrado

    2002-01-01

    Fischer rat thyroid (FRT) cells organize a matrix of extracellular fibronectin (FN) fibrils, which undergoes extensive remodeling according to cell culture confluence. In non-confluent cells FN forms a fibrillar array associated with the ventral cell surface. However, basal FN is progressively removed in confluent cultures and substituted by non-fibrillar FN deposits at lateral cell domains in regions of cell-cell contacts. FRT cells secrete and expose on the plasma membrane the tissue-type plasminogen activator and, in serum-free cultures, plasminogen induces a rapid loss of FN fibrils. Incubation with plasmin inhibitors greatly reduces this effect. FRT cells also express annexin II, a plasminogen receptor, suggesting that plasmin activity is associated with the pericellular enviroment. This is in agreement with the observation that a great reduction in FN degradation is observed if the cells are pre-incubated with carboxypeptidase B, which prevents plasminogen binding to the cells. A gelatinolytic activity with a molecular weigth equivalent to MMP-2 has been demonstrated by zymography of culture media, and the presence of MMP-2 and MT1-MMP on the cell plasma membrane has been detected by immunofluorescence. These results indicate that in the FN remodeling process, occurring during FRT epithelium maturation, both plasmin-dependent (tPA activated) and plasmin-independent proteolytic activities are involved.

  4. Prospective and randomised evaluation of the protease-modulating effect of oxidised regenerated cellulose/collagen matrix treatment in pressure sore ulcers

    NARCIS (Netherlands)

    Kloeters, O.; Unglaub, F.; Laat, E. de; Abeelen, M. Van; Ulrich, D.

    2016-01-01

    In chronic wounds, excess levels and activity of proteases such as elastase and plasmin have been detected. Oxidised regenerated cellulose/collagen matrix (ORC/collagen matrix) has been reported to ameliorate the wound microenvironment by binding and inactivating excess proteases in wound exudates.

  5. The pro-urokinase plasminogen-activation system in the presence of serpin-type inhibitors and the urokinase receptor

    DEFF Research Database (Denmark)

    Behrendt, Niels; List, Karin; Andreasen, Peter A;

    2003-01-01

    The reciprocal pro-enzyme activation system of plasmin, urokinase-type plasminogen activator (uPA) and their respective zymogens is a potent mechanism in the generation of extracellular proteolytic activity. Plasminogen activator inhibitor type 1 (PAI-1) acts as a negative regulator. This system ...

  6. Drug: D06879 [KEGG MEDICUS

    Lifescience Database Archive (English)

    Full Text Available n activator ATC code: B01AD01 See streptokinase A [KO:K14745] changing plasminogen into plasmine (activator of fibrinolysis) Anatomic...al Therapeutic Chemical (ATC) classification [BR:br08303] B BLOOD AND BLOOD FORMING

  7. The interplay of matrix metalloproteinases, morphogens and growth factors is necessary for branching of mammary epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Simian, M.; Harail, Y.; Navre, M.; Werb, Z.; Lochter, A.; Bissell, M.J.

    2002-03-06

    The mammary gland develops its adult form by a process referred to as branching morphogenesis. Many factors have been reported to affect this process. We have used cultured primary mammary epithelial organoids and mammary epithelial cell lines in three-dimensional collagen gels to elucidate which growth factors, matrix metalloproteinases (MMPs) and mammary morphogens interact in branching morphogenesis. Branching stimulated by stromal fibroblasts, epidermal growth factor, fibroblast growth factor 7, fibroblast growth factor 2 and hepatocyte growth factor was strongly reduced by inhibitors of MMPs, indicating the requirement of MMPs for three-dimensional growth involved in morphogenesis. Recombinant stromelysin 1/MMP-3 alone was sufficient to drive branching in the absence of growth factors in the organoids. Plasmin also stimulated branching; however, plasmin-dependent branching was abolished by both inhibitors of plasmin and MMPs, suggesting that plasmin activates MMPs. To differentiate between signals for proliferation and morphogenesis, we used a cloned mammary epithelial cell line that lacks epimorphin, an essential mammary morphogen. Both epimorphin and MMPs were required for morphogenesis, but neither was required for epithelial cell proliferation. These results provide direct evidence for a critical role of MMPs in branching in mammary epithelium and suggest that, in addition to epimorphin, MMP activity is a minimum requirement for branching morphogenesis in the mammary gland.

  8. Distinction by radioisotope technique of a subgroup with increased thrombophilic potential among patients submitted to major abdominal surgery

    DEFF Research Database (Denmark)

    Rasmussen, A; Toftdahl, D; Lindholt, J

    1986-01-01

    Deep vein thrombosis (DVT) detectable by the 99mTechnetium-labeled plasmin test developed in 13 (37%) of 35 sequentially studied patients, all above 40 years, undergoing elective major abdominal surgery. Ten of the 13 patients with DVT had an abnormal pulmonary perfusion scintigram, suggesting...

  9. Plasminogen-independent initiation of the pro-urokinase activation cascade in vivo. Activation of pro-urokinase by glandular kallikrein (mGK-6) in plasminogen-deficient mice

    DEFF Research Database (Denmark)

    List, K; Jensen, O N; Bugge, T H

    2000-01-01

    of the plasmin, that is generated during the progress of the cascade. Using gene-targeted mice deficient in plasminogen (Plg -/- mice) [Bugge, T. H., Flick, M. J., Daugherty, C. C., and Degen, J. L. (1995) Genes Dev. 9, 794-807], we have now demonstrated and identified a component capable of initiating...

  10. Amiloride lowers blood pressure and attenuates urine plasminogen activation in patients with treatment-resistant hypertension

    DEFF Research Database (Denmark)

    Stolzenburg Oxlund, Christina; Buhl, Kristian Bergholt; Jacobsen, Ib A;

    2014-01-01

    was reduced by 6.3/3.0 mm Hg. Seven of 80 cases (9%) discontinued amiloride due to hyperkalemia >5.5 mol/L, the most frequent adverse event. Urinary plasmin(ogen) and albumin excretions were significantly reduced after amiloride treatment (P

  11. Genetic predictors of fibrin D-dimer levels in healthy adults

    NARCIS (Netherlands)

    N.L. Smith (Nicholas); J.E. Huffman (Jennifer E.); D.P. Strachan (David); J. Huang (Jian); A. Dehghan (Abbas); S. Trompet (Stella); L.M. Lopez (Lorna M.); S.Y. Shin (So Youn); J. Baumert (Jens); V. Vitart (Veronique); J.C. Bis (Joshua); S.H. Wild (Sarah); A. Rumley (Ann); Q. Yang (Qiong Fang); A.G. Uitterlinden (André); D.J. Stott (David. J.); G. Davies (Gareth); A.M. Carter (Angela M.); B. Thorand (Barbara); O. Polašek (Ozren); B. McKnight (Barbara); H. Campbell (Harry); A.R. Rudnicka (Alicja); M.H. Chen (Min-hsin); B.M. Buckley (Brendan M.); S.E. Harris (Sarah); A. Peters (Annette); D. Pulanic (Drazen); T. Lumley (Thomas); A.J.M. de Craen (Anton J.M.); D.C. Liewald (David C.); C. Gieger (Christian); I. Ford (Ian); A.J. Gow (Alan J.); M. Luciano (Michelle); D.J. Porteous (David J.); X. Guo (Xiuqing); N. Sattar (Naveed); A. Tenesa (Albert); M. Cushman (Mary Ann); P.E. Slagboom (Eline); P.M. Visscher (Peter M.); T.D. Spector (Tim); T. Illig (Thomas); I. Rudan (Igor); E.G. Bovill (Edwin G.); A.F. Wright (Alan); W.L. McArdle (Wendy); G.H. Tofler (Geoffrey); A. Hofman (Albert); R.G.J. Westendorp (Rudi); J.M. Starr (John); P.J. Grant (Peter J.); M. Karakas (Mahir); N.D. Hastie (Nicholas D.); B.M. Psaty (Bruce); J.F. Wilson (James); G.D.O. Lowe (Gordon); C.J. O'Donnell (Christopher); J.C.M. Witteman (Jacqueline); J.W. Jukema (Jan Wouter); I.J. Deary (Ian); N. Soranzo (Nicole); W. Koenig (Wolfgang); C. Hayward (Caroline)

    2011-01-01

    textabstractBACKGROUND: Fibrin fragment D-dimer, one of several peptides produced when crosslinked fibrin is degraded by plasmin, is the most widely used clinical marker of activated blood coagulation. To identity genetic loci influencing D-dimer levels, we performed the first large-scale, genome-wi

  12. Proteolytic and genetic variation of the alpha-2-antiplasmin C-terminus in myocardial infarction

    NARCIS (Netherlands)

    de Willige, Shirley Uitte; Miedzak, Megan; Carter, Angela M.; Lisman, Ton; Rosendaal, Frits R.; Grant, Peter J.; Philippou, Helen; Ariens, Robert A. S.

    2011-01-01

    Alpha-2-antiplasmin (alpha 2AP) undergoes both N- and C-terminal cleavages, which significantly modify its activities. Compared with other Ser protease inhibitors (serpins), alpha 2AP contains an similar to 50-residue-extended C-terminus, which binds plasmin(ogen). We developed 2 new ELISAs to measu

  13. Effect of Lenient Steam Injection (LSI) heat treatment of bovine milk on the activities of some enzymes, the milk fat globule and pH

    DEFF Research Database (Denmark)

    Dickow, Jonatan A.; Nielsen, Martin Thorup; Hammershøj, Marianne

    2012-01-01

    This study investigated the effects of Lenient Steam Injection (LSI) treatment at temperatures 70–150 C on the enzymatic activities of the indigenous milk enzymes alkaline phosphatase, lactoperoxidase (LPO), xanthine oxidase (XO), lipoprotein lipase (LPL) and plasmin in comparison with two...

  14. Vectors and methods for recombinant production of uPA-binding fragments of the human urokinase-type plasminogen receptor (uPAR)

    DEFF Research Database (Denmark)

    1999-01-01

    Activation of plasminogen to plasma is inhibited by preventing the binding of a receptor binding form of urokinase-type plasminogen activator to a urokinase-type plasminogen activator receptor in a mammal, thereby preventing the urokinase-type plasminogen activator from converting plasminogen int...... into plasmin. DNA fragments which encode for soluble, active fragments of the urokinase-type plasminogen activator are provided....

  15. Tissue type plasminogen activator regulates myeloid-cell dependent neoangiogenesis during tissue regeneration

    DEFF Research Database (Denmark)

    Ohki, Makiko; Ohki, Yuichi; Ishihara, Makoto

    2010-01-01

    tissue regeneration is not well understood. Bone marrow (BM)-derived myeloid cells facilitate angiogenesis during tissue regeneration. Here, we report that a serpin-resistant form of tPA by activating the extracellular proteases matrix metalloproteinase-9 and plasmin expands the myeloid cell pool...

  16. Profibrinolytic effects of metalloproteinases during skin wound healing in the absence of plasminogen

    DEFF Research Database (Denmark)

    Green, Kirsty A; Almholt, Kasper; Ploug, Michael;

    2008-01-01

    Genetic ablation of plasminogen (Plg) and pharmacological inhibition of metalloproteinase activity by galardin delay skin wound healing in mice, whereas the combined inhibition of these two enzyme systems completely prevents healing. In this study, the impact of plasmin and metalloproteinases as ...

  17. [Relationship between vitrectomy and the morphology and function of the retina].

    Science.gov (United States)

    Terasaki, Hiroko

    2003-12-01

    Pathological processes in the vitreous will be reflected in the morphology and function of the retina, and these processes can originate from sources outside the vitreous. The purpose of vitreous surgery is to remove the qualitatively and/or morphologically diseased vitreous. Successful vitrectomy will be manifested by an improvement in the structure and/or function of the retina. We have evaluated the morphology of the vitreoretinal interface, and the function of the retina before and after vitreous surgery. Plasmin-assisted vitrectomy was used in some cases to remove the diseased vitreous more efficiently and less invasively. The effect of this procedure was assessed by examining the morphology and function of the retina. First, the relationship between the qualitative and structural abnormality of the vitreous in macular diseases was studied. In aphakic/pseudophakic eyes with cystoid macular edema, there was a depression of retinal function over the entire retina which may have been caused by chemical mediators released into the vitreous. These mediators may have been produced by inflammation in the anterior segment of the eye. In eyes with an idiopathic macular hole, optical coherence tomographic (OCT) images suggested that the progression of the macular hole might depend on a balance between foveal adhesion and the posterior vitreous. Second, the efficacy, surgical damage, and limitations of vitreous surgery were investigated. The recovery of macular function was assessed by focal macular electroretinograms (FMERGs) after vitrectomy for epiretinal membrane, choroidal neovascularization, and diabetic macular edema. The concurrent examination by optical coherence tomography (OCT) suggested that a decrease in retinal thickness contributed to the functional recovery. Macular functional recovery was delayed and limited after macular translocation, diabetic macular edema, and internal limiting membrane peeling. Third, we studied the effect of plasmin

  18. Relevance of Post-Stroke Circulating BDNF Levels as a Prognostic Biomarker of Stroke Outcome. Impact of rt-PA Treatment.

    Science.gov (United States)

    Rodier, Marion; Quirié, Aurore; Prigent-Tessier, Anne; Béjot, Yannick; Jacquin, Agnès; Mossiat, Claude; Marie, Christine; Garnier, Philippe

    2015-01-01

    The recombinant form of tissue plasminogen activator (rt-PA) is the only curative treatment for ischemic stroke. Recently, t-PA has been linked to the metabolism of brain-derived neurotrophic factor (BDNF), a major neurotrophin involved in post-stroke neuroplasticity. Thus, the objective of our study was to investigate the impact of rt-PA treatment on post-stroke circulating BDNF levels in humans and in animals. Serum BDNF levels and t-PA/plasmin activity were measured at hospital admission and at up to 90 days in stroke patients receiving (n = 24) or not (n = 14) rt-PA perfusion. We investigated the relationships between serum BDNF with concurrent t-PA/plasmin activity, neurological outcomes and cardiovascular scores at admission. In parallel, serum BDNF levels and t-PA/plasmin activity were assessed before and after (1, 4 and 24h) the induction of ischemic stroke in rats. Our study revealed higher serum BDNF levels and better neurological outcome in rt-PA-treated than non-treated patients. However, serum BDNF levels did not predict stroke outcome when the whole cohort of stroke patients was analyzed. By contrast, serum BDNF levels when measured at admission and at day 90 correlated with cardiovascular scores, and those at day 1 correlated with serum t-PA/plasmin activity in the whole cohort of patients whereas no association could be found in the rt-PA-treated group. In rats devoid of cardiovascular risk, no difference in post-stroke serum BDNF levels was detected between rt-PA- and vehicle-treated animals and no correlation was found between serum BDNF levels and t-PA/plasmin activity. Overall, the data suggest that serum BDNF levels may not be useful as a prognostic biomarker of stroke outcome and that endothelial dysfunction could be a confounding factor when serum BDNF levels after stroke are used to reflect of brain BDNF levels.

  19. Relevance of Post-Stroke Circulating BDNF Levels as a Prognostic Biomarker of Stroke Outcome. Impact of rt-PA Treatment.

    Directory of Open Access Journals (Sweden)

    Marion Rodier

    Full Text Available The recombinant form of tissue plasminogen activator (rt-PA is the only curative treatment for ischemic stroke. Recently, t-PA has been linked to the metabolism of brain-derived neurotrophic factor (BDNF, a major neurotrophin involved in post-stroke neuroplasticity. Thus, the objective of our study was to investigate the impact of rt-PA treatment on post-stroke circulating BDNF levels in humans and in animals. Serum BDNF levels and t-PA/plasmin activity were measured at hospital admission and at up to 90 days in stroke patients receiving (n = 24 or not (n = 14 rt-PA perfusion. We investigated the relationships between serum BDNF with concurrent t-PA/plasmin activity, neurological outcomes and cardiovascular scores at admission. In parallel, serum BDNF levels and t-PA/plasmin activity were assessed before and after (1, 4 and 24h the induction of ischemic stroke in rats. Our study revealed higher serum BDNF levels and better neurological outcome in rt-PA-treated than non-treated patients. However, serum BDNF levels did not predict stroke outcome when the whole cohort of stroke patients was analyzed. By contrast, serum BDNF levels when measured at admission and at day 90 correlated with cardiovascular scores, and those at day 1 correlated with serum t-PA/plasmin activity in the whole cohort of patients whereas no association could be found in the rt-PA-treated group. In rats devoid of cardiovascular risk, no difference in post-stroke serum BDNF levels was detected between rt-PA- and vehicle-treated animals and no correlation was found between serum BDNF levels and t-PA/plasmin activity. Overall, the data suggest that serum BDNF levels may not be useful as a prognostic biomarker of stroke outcome and that endothelial dysfunction could be a confounding factor when serum BDNF levels after stroke are used to reflect of brain BDNF levels.

  20. Role of PDGF-D and PDGFR-β in neuroinflammation in experimental ICH mice model☆

    Science.gov (United States)

    Yang, Peng; Manaenko, Anatol; Xu, Feng; Miao, Liyan; Wang, Gaiqing; Hu, Xuezhen; Guo, Zhen-Ni; Hu, Qin; Hartman, Richard E.; Pearce, William J.; Obenaus, Andre; Zhang, John H.; Chen, Gang; Tang, Jiping

    2016-01-01

    Objective Inflammation plays a key role in the pathophysiological processes after intracerebral hemorrhage (ICH). Post-ICH macrophages infiltrate the brain and release pro-inflammatory factors (tumor necrosis factor-α), amplifying microglial activation and neutrophil infiltration. Platelet-derived growth factor receptor-β (PDGFR-β) is expressed on macrophages and it's activation induces the recruitment of macrophages. Platelet-derived growth factor-D (PDGF-D) is an agonist with a significantly higher affinity to the PDGFR-β compared to another isoform of the receptor. In this study, we investigated the role of PDGF-D in the pro-inflammatory response after ICH in mice. Methods A blood injection model of ICH was used in eight-week old male CD1 mice (weight 30 g). Some mice received an injection of plasmin or PDGF-D. Gleevec, a PDGFR inhibitor, was administered at 1, 3 or 6 h post-ICH. Plasmin was administered with or without PDGF-D siRNAs mixture or scramble siRNA. A plasmin-antagonist, ε-Aminocaproic acid (EACA), was co-administrated with the blood. The effects of ICH and treatment on the brain injury and post-ICH inflammation were investigated. Results ICH resulted in the overexpression of PDGF-D, associated with the infiltration of macrophages. PDGFR-inhibition decreased ICH-induced brain injury, attenuating macrophage and neutrophil infiltration, reducing microglial activation and TNF-α production. Administration of recombinant PDGF-D induced TNF-α production, and PDGFR-inhibition attenuated it. A plasmin-antagonist suppressed PDGFR-β activation and microglial activation. Plasmin increased PDGF-D expression, and PDGF-D inhibition reduced neutrophil infiltration. Conclusion ICH-induced PDGF-D accumulation contributed to post-ICH inflammation via PDGFR activation and enhanced macrophage infiltration. The inhibition of PDGFR had an anti-inflammatory effect. Plasmin is a possible upstream effector of PDGF-D. The targeting of PDGF-D may provide a novel way to

  1. Tranexamic acid in treatment of melasma: A comprehensive review of clinical studies.

    Science.gov (United States)

    Taraz, Mohammad; Niknam, Somayeh; Ehsani, Amir Houshang

    2017-01-30

    Melasma is a human melanogenesis dysfunction that results in localized, chronic acquired hyperpigmentation of the skin. It has a significant impact on appearance, causing psychosocial and emotional distress, and reducing the quality of life of the affected patients. Tranexamic acid (TA) is a plasmin inhibitor used to prevent abnormal fibrinolysis to reduce blood loss and exerts its effect by reversibly blocking lysine binding sites on plasminogen molecules, thus inhibiting plasminogen activator (PA) from converting plasminogen to plasmin. As plasminogen also exists in human epidermal basal cells and cultured human keratinocyte are known to produce PA, there is basic rationale that TA will affect keratinocyte function and interaction. A thorough literature review indicates that while TA is used through various route of administration including oral, topical, and intradermal injection and as adjutant therapy with laser to treat melasma, its efficacy is not established adequately. Further studies are needed to clarify the role of TA in treatment of melasma.

  2. Gender differences of disturbed hemostasis related to fasting insulin level in healthy very elderly Japanese aged > or = 75 years.

    Science.gov (United States)

    Kario, K; Matsuo, T; Kobayashi, H; Sakata, T; Miyata, T; Shimada, K

    1995-08-01

    We investigated the relationship between fasting insulin level and various hemostatic factors, including fibrinolytic factors (active plasminogen activator inhibitor-1 (PAI-1), tissue type plasminogen activator (tPA)-PAI-1 complex, plasmin-alpha 2-plasmin inhibitor (PIC), and D-dimer), coagulation factors (activated factor VII, factor VII coagulant activity and antigen, factor VIII, factor X, and fibrinogen), coagulation inhibitors (antithrombin III, heparin cofactor II, and protein C), and an acute phase marker (sialic acid) in 102 healthy individuals aged > or = 75 years (46 men and 56 women). Active PAI-1 levels had a significant negative correlation with PIC levels (r = -0.342, P = 0.0006), indicating that PAI-1 influences in vivo fibrinolytic activity in the very elderly. Gender differences were found in the relationship between insulin and hemostatic abnormalities, with the insulin level being positively correlated with coagulation factors in men (factor VIII activity: r = 0.422, P sex.(ABSTRACT TRUNCATED AT 250 WORDS)

  3. [Regulation of PAI-1 expression].

    Science.gov (United States)

    Wyrzykowska, Paulina; Kasza, Aneta

    2009-01-01

    PAI-1 (plasminogen activator inhibitor-1) is a member of plasminogen cascade with an inhibitory role in plasmin activation. Plasmin is a protease capable of acting on wide range of substrates and, together with metaloproteinases, is a main proteolytic enzyme. Except its role in plasminogen cascade, PAI-1 has an affinity to vitronectin and uPA/uPAR what involves PAI-1 in cell's motility. PAI-1 gene is regulated in response to cytokines, hormones and many growth factors among which TGFbeta is the most important one. The PAI-1 promoter contains SBE, CAGA box, HRE, ERE, NFkB - binding sites, Sp-1, AP-1 and other. Cooperation between transcription factors bound to promoter and cross-talks between kinases and other upstream proteins decide about gene expression. This work describes the present knowledge in this field.

  4. The plasma carboxypeptidases and the regulation of the plasminogen system.

    Science.gov (United States)

    Plow, E F; Allampallam, K; Redlitz, A

    1997-04-01

    Central to the regulation of the plasminogen system, a proteolytic network that mediates degradation of fibrin and facilitates cell migration, is the binding of plasminogen to carboxy-terminal lysines. These residues occur either naturally on plasmin substrates or cell surfaces or are generated as a consequence of partial plasmin degradation. The basic carboxypeptidases of plasma are capable of removing such carboxy-terminal lysines. Carboxypeptidase N, which is constitutively active, suppresses plasminogen binding to cell surfaces; plasma carboxypeptidase B, which must be proteolytically activated, not only suppresses cellular binding of plasminogen but also dampens fibrinolysis. Thus, the plasma carboxypeptidases may constitute an important regulatory pathway for controlling the activity of the plasminogen system in physiologic, pathophysiologic, and pharmacologic circumstances. (Trends Cardiovasc Med 1997;7:71-75). © 1997, Elsevier Science Inc.

  5. Effect of total proteose-peptone content on the variability of bovine milk foaming property

    Directory of Open Access Journals (Sweden)

    Arianna Buccioni

    2013-02-01

    Full Text Available Several authors demonstrated a strong linkage between proteose-peptones content and foaming properties of cow milk; this is of great interest for Italian dairy industries to create a new line of fresh milk characterized by a particular foaming property and, hence, particularly appreciate in catering industry. The aim of this trial was to quantify the relation between total concentration of proteose-peptones and the entity of foaming attitude in cow fresh milk. Ninety samples of raw bulk milk were analysed for proteose-peptones content, plasmin activity, fatty acid profile and foaming attitude. A negative relation was found among proteose-peptones percentage and foaming attitude which decreased with the increase of plasmin activity and somatic cell content in milk.

  6. Estatus de la concentración de homocisteína y de algunos componentes de la fibrinolisis: eslabón trombogénico en la homocistinuria clásica

    Directory of Open Access Journals (Sweden)

    Beatriz Meneses

    2001-04-01

    Full Text Available Aumentos moderados o severos de Homocisteína (Hcy en la sangre y alteraciones en la fibrinólisis, están estrechamente relacionados
    con enfermedad cardiovascular. En la homocistinuria clásica, los
    pacientes experimentan un estado de hipercoagulabilidad y con mucha frecuencia eventos tromboembólicos, que son su principal causa de mortalidad. El objetivo de este estudio fue determinar el estatus de la concentraciones de Hcy, Inhibidor del Activador del Plasminógeno tipo 1 (PAI-1 y Activador tisular del Plasminógeno (tPA en pacientes con homocistinuria clásica, en familiares de éstos y en una población control sana.

  7. Significant natriuretic and antihypertensive action of the epithelial sodium channel blocker amiloride in diabetic patients with and without nephropathy

    DEFF Research Database (Denmark)

    Andersen, Henrik; Hansen, Pernille B L; Bistrup, Claus;

    2016-01-01

    OBJECTIVE: Diabetic nephropathy is associated with aberrant glomerular filtration of serine proteases. The study was designed to test the hypothesis that the epithelial sodium channel is activated proteolytically by urine plasmin in diabetic nephropathy and mediates renal sodium retention. METHODS......: In an open-label intervention study on type 1 diabetes patients on standardized NaCl intake (200 mmol/day) with (n = 15) and without diabetic nephropathy (control, n = 12), urinary Na excretion in response to oral amiloride (20 or 40 mg/day for 2 days) was compared. RESULTS: A total of 27 patients completed...... renal Na excretion, reduced blood pressure, albuminuria, and total and active plasmin in urine. It is concluded that epithelial sodium channel is an attractive target to attain blood pressure control in long-term type I diabetes with no enhanced activity associated with nephropathy....

  8. Diferencias cinéticas de las plasminas de ocho especies mamíferas: activaciones y secuencias de los terminales-N

    Directory of Open Access Journals (Sweden)

    Omaira Cañás Bermúdez

    2013-12-01

    Full Text Available El artículo determina los parámetros cinéticos de las plasminas de ocho especies de mamíferos y sus terminales-N. Los ocho plasminógenos fueron purificados por los mismos métodos (cromatografías de afinidad e intercambio iónico, y activados con urocinasa, partiendo de una concentración común y su cinética valorada según las coordenadas de Lineweaver- Burk. Para esto se utilizó la cinética enzimática de Michaelis Menten, ampliamente usada en el estudio de enzimas. Todos los plasminógenos mostraron una pureza superior al 95 % y una banda de 92 kDa en la electroforesis, al comparar con el estándar de peso molecular utilizado. Las plasminas del equino y canino mostraron la misma KM por el sustrato cromogénico (0,438 mM, siendo esta la de mayor afinidad en este estudio y la humana la de menor afinidad (5,3 mM. También fueron determinadas la constante catalítica y la velocidad de conversión del sustrato cromogénico a producto. Los terminales-N de los plasminógenos de las ocho especies fueron determinados, y se encontraron diferencias entre el humano y los animales; así mismo entre algunos animales. Solo el porcino y el ovino no mostraron diferencia alguna en su terminal-N (DPPDDY. Se demostró la unificación del método de purificación de los plasminógenos para cualquier especie, las diferencias cinéticas de las ocho plasminas estudiadas, y las similitudes y diferencias en la secuencia de los terminales-N de las ocho especies.

  9. High Permissivity of Human HepG2 Hepatoma Cells for Influenza Viruses

    OpenAIRE

    Ollier, Laurence; Caramella, Anne; Giordanengo, Valérie; Lefebvre, Jean-Claude

    2004-01-01

    Human HepG2 hepatoma cells are highly permissive for influenza virus type A and type B, even without the addition of trypsin, and they exhibit a marked cytopathic effect. This property greatly facilitates the primary isolation of influenza viruses. Virus replication was significantly reduced by the plasmin(ogen)-specific inhibitor tranexamic acid, and this suggests a potential role played by the plasminogen/tissue plasminogen activator complex at the surface of HepG2 cells. This might represe...

  10. インフルエンザウイルスの感染感受性を決定する生体内因子群とその作用機序からみる新たな治療・予防への展開

    OpenAIRE

    奥村, 裕司

    2004-01-01

    Extracellular cleavage of virus envelope fusion glycoprotein, hemagglutinin, by host cellular proteases is a prerequisite for the infectivity of mammalian and nonpathogenic avian influenza viruses. In search of such target processing proteases in the airway, we found ectopic anionic trypsin I, tryptase Clara and mini-plasmin. Interestingly, these processing enzymes were localized in the air way with different distribution. In addition, these enzymes showed the different sensitivities to vario...

  11. Amiloride lowers blood pressure and attenuates urine plasminogen activation in patients with treatment-resistant hypertension.

    Science.gov (United States)

    Oxlund, Christina S; Buhl, Kristian B; Jacobsen, Ib A; Hansen, Mie R; Gram, Jeppe; Henriksen, Jan Erik; Schousboe, Karoline; Tarnow, Lise; Jensen, Boye L

    2014-12-01

    In conditions with albuminuria, plasminogen is aberrantly filtered across the glomerular barrier and activated along the tubular system to plasmin. In the collecting duct, plasmin activates epithelial sodium channels (ENaC) proteolytically. Hyperactivity of ENaC could link microalbuminuria/proteinuria to resistant hypertension. Amiloride, an ENaC inhibitor, inhibits urokinase-type plasminogen activator. We hypothesized that amiloride (1) reduces blood pressure (BP); (2) attenuates plasminogen-to-plasmin activation; and (3) inhibits urine urokinase-type plasminogen activator in patients with resistant hypertension and type 2 diabetes mellitus (T2DM).In an open-label, non-randomized, 8-week intervention study, a cohort (n = 80) of patients with resistant hypertension and T2DM were included. Amiloride (5 mg/d) was added to previous triple antihypertensive treatment (including a diuretic and an inhibitor of the renin-angiotensin-aldosterone system) and increased to 10 mg if BP control was not achieved at 4 weeks. Complete dataset for urine analysis was available in 60 patients. Systolic and diastolic BP measured by ambulatory BP monitoring and office monitoring were significantly reduced. Average daytime BP was reduced by 6.3/3.0 mm Hg. Seven of 80 cases (9%) discontinued amiloride due to hyperkalemia >5.5 mol/L, the most frequent adverse event. Urinary plasmin(ogen) and albumin excretions were significantly reduced after amiloride treatment (P treatment. Amiloride lowers BP, urine plasminogen excretion and activation, and albumin/creatinine ratio, and is a relevant add-on medication for the treatment of resistant hypertension in patients with T2DM and microalbuminuria.

  12. インフルエンザウイルスの感染感受性を決定する生体内因子群とその作用機序からみる新たな治療・予防への展開

    OpenAIRE

    奥村, 裕司

    2004-01-01

    Extracellular cleavage of virus envelope fusion glycoprotein, hemagglutinin, by host cellular proteases is a prerequisite for the infectivity of mammalian and nonpathogenic avian influenza viruses. In search of such target processing proteases in the airway, we found ectopic anionic trypsin I, tryptase Clara and mini-plasmin. Interestingly, these processing enzymes were localized in the air way with different distribution. In addition, these enzymes showed the different sensitivities to vario...

  13. Prospective and randomised evaluation of the protease-modulating effect of oxidised regenerated cellulose/collagen matrix treatment in pressure sore ulcers.

    Science.gov (United States)

    Kloeters, Oliver; Unglaub, Frank; de Laat, Erik; van Abeelen, Marjolijn; Ulrich, Dietmar

    2016-12-01

    In chronic wounds, excess levels and activity of proteases such as elastase and plasmin have been detected. Oxidised regenerated cellulose/collagen matrix (ORC/collagen matrix) has been reported to ameliorate the wound microenvironment by binding and inactivating excess proteases in wound exudates. In this study, the levels and activity of elastase and plasmin in wound exudates of pressure sore ulcers were measured to determine the beneficial effect of ORC/collagen matrix treatment compared with control treatment with a foam dressing. A total of 33 patients with pressure sores were enrolled in the study and were followed up for 12 weeks after treatment. Ten control patients were treated with a foam hydropolymer dressing (TIELLE(®) , Systagenix), and the remaining 23 patients were treated with ORC/collagen matrix plus the foam dressing (TIELLE(®) , Systagenix) on top. Wound assessments were carried out over 12 weeks on a weekly basis, with dressing changes twice a week. Ulcers were photographed and wound exudates were collected on admission and at days 5, 14 and then every 14 days to provide a visual record of any changes in appearance of the ulcer and healing rate and for biochemical analysis of the wound. The levels and activity of elastase and plasmin were measured in wound exudates. Statistical analysis was performed using ANOVA and Bonferroni's post hoc test with P-values pressure sore wounds showed a significant faster healing rate, which positively correlated with a decreased activity of elastase and plasmin in wound exudates. No signs of infection or intolerance to the ORC/collagen matrix were observed. © 2015 Medicalhelplines.com Inc and John Wiley & Sons Ltd.

  14. SMTP (Stachybotrys microspora triprenyl phenol enhances clot clearance in a pulmonary embolism model in rats

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    Hu Weimin

    2012-01-01

    Full Text Available Abstract Background Stachybotrys microspora triprenyl phenols (SMTPs are a novel family of small molecules that enhance both activation and fibrin-binding of plasminogen. While their effects on fibrinolysis have been characterized in vitro, little is known about their activity in vivo with respect to plasminogen activation and blood clot clearance. Results To select a potent SMTP congener for the evaluation of its action in vitro and in vivo, we tested several SMTP congeners with distinct structural properties for their effects on plasminogen activation. As a result, SMTP-7 (orniplabin was found to have distinguished activity. Several lines of biochemical evidence supported the idea that SMTP-7 acted as a plasminogen modulator. SMTP-7 elevated plasma level of plasmin-α2-antiplasmin complex, an index of plasmin formation in vivo, 1.5-fold in mice after the intravenous injections at doses of 5 and 10 mg kg-1. In a rat pulmonary embolism model, SMTP-7 (5 mg kg-1 enhanced the rate of clot clearance ~3-fold in the absence of exogenous plasminogen activator. Clot clearance was enhanced further by 5 mg kg-1 of SMTP-7 in combination with single-chain urokinase-type plasminogen activator. Conclusions Our results show that SMTP-7 is a superior plasminogen modulator among the SMTP family compounds and suggest that the agent enhances plasmin generation in vivo, leading to clearance of thrombi in a model of pulmonary embolism.

  15. Tissue-type plasminogen activator is not required for kainate-induced motoneuron death in vitro.

    Science.gov (United States)

    Vandenberghe, W; Van Den Bosch, L; Robberecht, W

    1998-08-24

    Spinal motoneurons are highly vulnerable to kainate both in vivo and in vitro. Tissue-type plasminogen activator (tPA) and plasmin have recently been shown to mediate kainate-induced neuronal death in the mouse hippocampus in vivo. The aim of the present study was to determine whether tPA also mediates the kainate-induced death of motoneurons in vitro. A motoneuron-enriched neuronal population was isolated from the ventral spinal cord of wild-type (WT) and tPA-deficient (tPA-/-) mouse embryos. WT and tPA-/- neurons were cultured on WT and tPA-/- spinal glial feeder layers, respectively. WT and tPA-/- co-cultures were morphologically indistinguishable. Expression of tPA in WT co-cultures was demonstrated using RT-PCR. WT and tPA-/- co-cultures were exposed to kainate for 24 h. The neurotoxic effect of kainate did not differ significantly between WT and tPA-/- cultures. The plasmin inhibitor alpha2-antiplasmin did not protect WT neurons against kainate-induced injury. These results indicate that the plasmin system is not a universal mediator of kainate-induced excitotoxicity.

  16. THE IMPAIRMENT OF PLATELET FUNCTION IN FIBRINOLYSIS AND PRESERVING EFFECT OF APROTININ

    Institute of Scientific and Technical Information of China (English)

    黄惠民; 丁文祥; 苏肇伉; 张伟忠

    1992-01-01

    Platelet adhesion depends on the platelet membrane glycoprotein Ib (GPIb) and plasma von Willebrand Factor (vWF), which can be reflected by ristocetin-induced aggregation. Here we report damage effect of fibrinolysis and preserving effect of aprotinin on platelet function. Addition of 40 U/ml urokinase and 0.3 U/ml plasmin to PRP or washed platelets made the ristocetin-induced aggregation decline to 31.6% and 38.5% of control value respectively. The extent of declining was positively correlated with the concentration of urokinase and plasmin. Meanwhile, the platelet GPIb decreased to 76.4% of control value. The results showed that the fibrinolysis impaired the platelet function and this effect may be associated with the hydrolysis of GPIb. Further research found that by adding the same dose of urokinase or plasmin to aprotinin-pretreated PRP or washed platelets, the aggregation did not change statistically and decrement of GPIb is much less marked. We concluded that the aprotinin could relieve the platelet dsfunction effectively by its inhibitory effect on fibrinolytic activity.

  17. Tissue plasminogen activator-mediated fibrinolysis protects against axonal degeneration and demyelination after sciatic nerve injury.

    Science.gov (United States)

    Akassoglou, K; Kombrinck, K W; Degen, J L; Strickland, S

    2000-05-29

    Tissue plasminogen activator (tPA) is a serine protease that converts plasminogen to plasmin and can trigger the degradation of extracellular matrix proteins. In the nervous system, under noninflammatory conditions, tPA contributes to excitotoxic neuronal death, probably through degradation of laminin. To evaluate the contribution of extracellular proteolysis in inflammatory neuronal degeneration, we performed sciatic nerve injury in mice. Proteolytic activity was increased in the nerve after injury, and this activity was primarily because of Schwann cell-produced tPA. To identify whether tPA release after nerve damage played a beneficial or deleterious role, we crushed the sciatic nerve of mice deficient for tPA. Axonal demyelination was exacerbated in the absence of tPA or plasminogen, indicating that tPA has a protective role in nerve injury, and that this protective effect is due to its proteolytic action on plasminogen. Axonal damage was correlated with increased fibrin(ogen) deposition, suggesting that this protein might play a role in neuronal injury. Consistent with this idea, the increased axonal degeneration phenotype in tPA- or plasminogen-deficient mice was ameliorated by genetic or pharmacological depletion of fibrinogen, identifying fibrin as the plasmin substrate in the nervous system under inflammatory axonal damage. This study shows that fibrin deposition exacerbates axonal injury, and that induction of an extracellular proteolytic cascade is a beneficial response of the tissue to remove fibrin. tPA/plasmin-mediated fibrinolysis may be a widespread protective mechanism in neuroinflammatory pathologies.

  18. Study on the Mechanism of the Annexin I -Mediated Co-Assembly of t-PA and Plasminogen

    Institute of Scientific and Technical Information of China (English)

    张晓晖; 周华荣; 沈关心; 刘仲萍; 魏文宁; 宋善俊; 胡豫

    2002-01-01

    In order to further investigate the effect of annexin Ⅱ (Ann- Ⅱ ) on tissue plasminogen activator (t-PA)-dependent plasminogen (PLG) activation and its interactive mechanism, recombinant native Ann- Ⅱ bound t-PA, PLG and plasmin with high affinity was examined. The flow cytometric assay showed that the ann- Ⅱ expression rate was higher in the human umbilical vein endothelial cell (HUVEC) (87. 65 %) than in the HL-60 cells as controls (35. 79 %). Two irrelevant proteins,bovine serum albumin (BSA) and equine IgG (EIG) had no effect on the production of plasmin.Ann- Ⅱ -mediated enhancement of t-PA-dependent PLG activation was inhibited by ε-aminocaproic acid or by pretreatment of Ann- Ⅱ with carboxypeptidase B with the inhibitive rate being 77.8 % and 77. 0 %, respectively. It was revealed that the effect of Ann- Ⅱ on PLG activation was specific for tPA. Urokinase didn't bind to Ann- Ⅱ , demonstrating the role of receptor-related lysine residues on activation of PLG, showing that the Ann- Ⅱ -PLG interaction was dependent upon carboxyl-terminal lysine residues. These findings suggest that annexin Ⅱ -mediated co-assembly of t-PA and PLG may promote plasmin generation and play a key role in modulating fibrinolysis on the endothelial surface.

  19. Phospholipid barrier to fibrinolysis: role for the anionic polar head charge and the gel phase crystalline structure.

    Science.gov (United States)

    Váradi, Balázs; Kolev, Krasimir; Tenekedjiev, Kiril; Mészáros, Gyöngyi; Kovalszky, Ilona; Longstaff, Colin; Machovich, Raymund

    2004-09-17

    The massive presence of phospholipids is demonstrated in frozen sections of human arterial thrombi. Purified platelet phospholipids and synthetic phospholipids retard in vitro tissue-type plasminogen activator (tPA)-induced fibrinolysis through effects on plasminogen activation and plasmin function. The inhibition of plasminogen activation on the surface of fibrin correlates with the fraction of anionic phospholipid. The phospholipids decrease the amount of tPA penetrating into the clot by 75% and the depth of the reactive surface layer occupied by the activator by up to 30%, whereas for plasmin both of these parameters decrease by approximately 50%. The phospholipids are not only a diffusion barrier, they also bind the components of the fibrinolytic system. Isothermal titration calorimetry shows binding characterized with dissociation constants in the range 0.35-7.64 microm for plasmin and tPA (lower values with more negative phospholipids). The interactions are endothermic and thermodynamically driven by an increase in entropy, probably caused by the rearrangements in the ordered gel structure of the phospholipids (in line with the stronger inhibition at gel phase temperatures compared with liquid crystalline phase temperatures). These findings show a phospholipid barrier, which should be overcome during lysis of arterial thrombi.

  20. A prourokinase-RGDS chimera——Construction, expression and characterization

    Institute of Scientific and Technical Information of China (English)

    钱斌; 孙迎庆; 郭雁; 党昕; 茹炳根

    1999-01-01

    A tetrapeptide, RGDS, was inserted into proUK kringle domain G118-L119 by the construction of a mutant proUK-RGDS gene. The gene was expressed in the baculovirus expression system. Immunoaffinity chromatography was used to purify the chimera and protein with purity over 90% was achieved. The chimera was tested for its platelet membrane binding function and showed a calcium-dependent platelet binding activity. Amidolytic activity of the chimera was tested. The result indicated that specific amidolytic activity of plasmin activated chimera was 62000 IU/mg, comparable to the previously reported 65 355 IU/mg of plasmin activated natural proUK. Activation of plasminogen by the chimera after plasmin treatment followed Micbieal-Menten kinetics, and the Km was 0.97 μmol/L, which was also comparable to 1.64 μmol/L of native urokinase. The chimera also showed intensive ability to inhibit platelet aggregation in vitro. These results indicate that this chimera might be useful as a bifunctional thrombolytic agent.

  1. Fibrin formation and dissolution in women receiving oral contraceptive drugs.

    Science.gov (United States)

    Ball, A P; McKee, P A

    1977-04-01

    Factors affecting fibrin formation and dissolution were compared for 15 women taking combined oral contraceptives and 15 women using nonpharmacological methods of birth control. The two groups were matched for age, body weight, time of blood collection, and day in menstrual cycle; none of the women was receiving other drugs known to affect the blood coagulation or fibrinolytic parameters measured in this study. Fibrinogen concentrations tended to be higher in the experimental group; the degree of fibrinogen degradation, number of fibrin cross-links, and levels of factor XIII and plasminogen were the same for both group. There were significant reductions in antithrombin activity, the euglobulin lysis time, and fibrinolytic inhibitor level in women using oral contraceptives. An estrogen dose effect was suggested for fibrinogen concentration and the degree of antithrombin activity. The increased fibrinolytic activity and decreased fibrinolytic inhibitor levels are consistent with in vitro observations that antithrombin also inhibits plasmin activity. Thus while oral contraceptive-induced depression of antithrombin III could possibly predispose to thrombosis by diminishing the inhibition of the serine protease clotting factors, the concomitant decreased level of plasmin inhibition might balance the system by favoring thrombolysis as well as the digestion and inactivation of certain clotting factors by plasmin.

  2. Construction of Urokinase Mutant Glu154-mtcu-PA and Characterization of Its Properties.

    Science.gov (United States)

    Peng, Gui-Hong; Ma, Zhong; Xue, Yu-Ming; Chen, Yu-Hong; Zhu, De-Xu

    1997-01-01

    The recombinant single chain urokinase-type plasminogen activator (rscu-PA) and a mutant constructed by in vitro site-specific mutagenesis of Argl54 in rscu-PA to Glul54 (Glul54-mscu-PA) were both expressed in E. coli. The expressed products were both purified to homogeneity by in vitro denaturation and renaturation, followed by Zn(2+) selective precipitation and immuno-affinity chromatography. The plasmin sensitivity assay indicated that the activation of this single chain Glul54-mscu-PA by plasmin was essentially identical to that of rscu-PA. After activation by plasmin, the kinetic constants against synthetic substrate S2444 of the resulted two chain form of Glul54-mscu-PA (Glul54-mtcu-PA) and that of rscu-PA (rtcu-PA) were 87 &mgr;M and 80 &mgr;M, respectively, which indicated that the catalytic active site of the Glul54-mtcu-PA was not changed by the mutation. Whereas, both (125)I-fibrin plasma-clot lysis and fibrinogenolysis in plasma showed that the Glul54-mtcu-PA possessed a better affinity and selectivity for fibrin than rtcu-PA, even better than rscu-PA.

  3. Bacterial Plasminogen Receptors Utilize Host Plasminogen System for Effective Invasion and Dissemination

    Directory of Open Access Journals (Sweden)

    Sarbani Bhattacharya

    2012-01-01

    Full Text Available In order for invasive pathogens to migrate beyond the site of infection, host physiological barriers such as the extracellular matrix, the basement membrane, and encapsulating fibrin network must be degraded. To circumvent these impediments, proteolytic enzymes facilitate the dissemination of the microorganism. Recruitment of host proteases to the bacterial surface represents a particularly effective mechanism for enhancing invasiveness. Plasmin is a broad spectrum serine protease that degrades fibrin, extracellular matrices, and connective tissue. A large number of pathogens express plasminogen receptors which immobilize plasmin(ogen on the bacterial surface. Surface-bound plasminogen is then activated by plasminogen activators to plasmin through limited proteolysis thus triggering the development of a proteolytic surface on the bacteria and eventually assisting the spread of bacteria. The host hemostatic system plays an important role in systemic infection. The interplay between hemostatic processes such as coagulation and fibrinolysis and the inflammatory response constitutes essential components of host defense and bacterial invasion. The goal of this paper is to highlight mechanisms whereby pathogenic bacteria, by engaging surface receptors, utilize and exploit the host plasminogen and fibrinolytic system for the successful dissemination within the host.

  4. Bacterial plasminogen receptors utilize host plasminogen system for effective invasion and dissemination.

    Science.gov (United States)

    Bhattacharya, Sarbani; Ploplis, Victoria A; Castellino, Francis J

    2012-01-01

    In order for invasive pathogens to migrate beyond the site of infection, host physiological barriers such as the extracellular matrix, the basement membrane, and encapsulating fibrin network must be degraded. To circumvent these impediments, proteolytic enzymes facilitate the dissemination of the microorganism. Recruitment of host proteases to the bacterial surface represents a particularly effective mechanism for enhancing invasiveness. Plasmin is a broad spectrum serine protease that degrades fibrin, extracellular matrices, and connective tissue. A large number of pathogens express plasminogen receptors which immobilize plasmin(ogen) on the bacterial surface. Surface-bound plasminogen is then activated by plasminogen activators to plasmin through limited proteolysis thus triggering the development of a proteolytic surface on the bacteria and eventually assisting the spread of bacteria. The host hemostatic system plays an important role in systemic infection. The interplay between hemostatic processes such as coagulation and fibrinolysis and the inflammatory response constitutes essential components of host defense and bacterial invasion. The goal of this paper is to highlight mechanisms whereby pathogenic bacteria, by engaging surface receptors, utilize and exploit the host plasminogen and fibrinolytic system for the successful dissemination within the host.

  5. Beta-amyloidolysis and glutathione in Alzheimer's disease

    Directory of Open Access Journals (Sweden)

    Lasierra-Cirujeda J

    2013-04-01

    Full Text Available J Lasierra-Cirujeda,1 P Coronel,2 MJ Aza,3 M Gimeno2 1CM Hematológico SC, Logroño, La Rioja, Spain; 2Tedec-Meiji Farma, SA, Alcalá de Henares, Madrid, Spain; 3Pharmaceutical Act, Ministry of Health, Regional Government, La Rioja, Spain Abstract: In this review, we hypothesized the importance of the interaction between the brain glutathione (GSH system, the proteolytic tissue plasminogen activator (t-PA/plasminogen/plasmin system, regulated by plasminogen activator inhibitor (PAI-1, and neuroserpin in the pathogenesis of Alzheimer's disease. The histopathological characteristic hallmark that gives personality to the diagnosis of Alzheimer's disease is the accumulation of neurofibroid tangles located intracellularly in the brain, such as the protein tau and extracellular senile plaques made primarily of amyloidal substance. These formations of complex etiology are intimately related to GSH, brain protective antioxidants, and the proteolytic system, in which t-PA plays a key role. There is scientific evidence that suggests a relationship between aging, a number of neurodegenerative disorders, and the excessive production of reactive oxygen species and accompanying decreased brain proteolysis. The plasminogen system in the brain is an essential proteolytic mechanism that effectively degrades amyloid peptides ("beta-amyloidolysis" through action of the plasmin, and this physiologic process may be considered to be a means of prevention of neurodegenerative disorders. In parallel to the decrease in GSH levels seen in aging, there is also a decrease in plasmin brain activity and a progressive decrease of t-PA activity, caused by a decrease in the expression of the t-PA together with an increase of the PAI-1 levels, which rise to an increment in the production of amyloid peptides and a lesser clearance of them. Better knowledge of the GSH mechanism and cerebral proteolysis will allow us to hypothesize about therapeutic practices. Keywords: glutathione

  6. HIF-2alpha-dependent PAI-1 induction contributes to angiogenesis in hepatocellular carcinoma

    Energy Technology Data Exchange (ETDEWEB)

    Geis, Theresa, E-mail: geis@biochem.uni-frankfurt.de [Institute of Biochemistry I—Pathobiochemistry, Faculty of Medicine, Goethe-University Frankfurt, Theodor-Stern-Kai 7, 60590 Frankfurt am Main (Germany); Döring, Claudia, E-mail: C.Doering@em.uni-frankfurt.de [Dr. Senckenberg Institute of Pathology, Faculty of Medicine, Goethe-University Frankfurt, Theodor-Stern-Kai 7, 60590 Frankfurt am Main (Germany); Popp, Rüdiger, E-mail: popp@vrc.uni-frankfurt.de [Institute for Vascular Signalling, Centre for Molecular Medicine, Faculty of Medicine Medicine, Goethe-University Frankfurt, Theodor-Stern-Kai 7, 60596 Frankfurt am Main (Germany); Grossmann, Nina, E-mail: grossmann@biochem.uni-frankfurt.de [Institute of Biochemistry I—Pathobiochemistry, Faculty of Medicine, Goethe-University Frankfurt, Theodor-Stern-Kai 7, 60590 Frankfurt am Main (Germany); Fleming, Ingrid, E-mail: fleming@vrc.uni-frankfurt.de [Institute for Vascular Signalling, Centre for Molecular Medicine, Faculty of Medicine Medicine, Goethe-University Frankfurt, Theodor-Stern-Kai 7, 60596 Frankfurt am Main (Germany); Hansmann, Martin-Leo, E-mail: m.l.hansmann@em.uni-frankfurt.de [Dr. Senckenberg Institute of Pathology, Faculty of Medicine, Goethe-University Frankfurt, Theodor-Stern-Kai 7, 60590 Frankfurt am Main (Germany); Dehne, Nathalie, E-mail: dehne@biochem.uni-frankfurt.de [Institute of Biochemistry I—Pathobiochemistry, Faculty of Medicine, Goethe-University Frankfurt, Theodor-Stern-Kai 7, 60590 Frankfurt am Main (Germany); Brüne, Bernhard, E-mail: b.bruene@biochem.uni-frankfurt.de [Institute of Biochemistry I—Pathobiochemistry, Faculty of Medicine, Goethe-University Frankfurt, Theodor-Stern-Kai 7, 60590 Frankfurt am Main (Germany)

    2015-02-01

    Hypoxia promotes progression of hepatocellular carcinoma (HCC), not only affecting tumor cell proliferation and invasion, but also angiogenesis and thus, increasing the risk of metastasis. Hypoxia inducible factors (HIF)-1α and -2α cause adaptation of tumors to hypoxia, still with uncertainties towards the angiogenic switch. We created a stable knockdown of HIF-1α and HIF-2α in HepG2 cells and generated cocultures of HepG2 spheroids with embryonic bodies as an in vitro tumor model mimicking the cancer microenvironment. The naturally occuring oxygen and nutrient gradients within the cocultures allow us to question the role of distinct HIF isoforms in regulating HCC angiogenesis. In cocultures with a HIF-2α knockdown, angiogenesis was attenuated, while the knockdown of HIF-1α was without effect. Microarray analysis identified plasminogen activator inhibitor 1 (PAI-1) as a HIF-2α target gene in HepG2 cells. The knockdown of PAI-1 in HepG2 cells also lowered angiogenesis. Blocking plasmin, the downstream target of PAI-1, with aprotinin in HIF-2α knockdown (k/d) cells proved a cause–effect relation and restored angiogenesis, with no effect on control cocultures. Suggestively, HIF-2α increases PAI-1 to lower concentrations of active plasmin, thereby supporting angiogenesis. We conclude that the HIF-2α target gene PAI-1 favors the angiogenic switch in HCC. - Highlights: • HepG2 were cocultured with stem cells to mimic a cancer microenvironment in vitro. • A knockdown of HIF-2α reduces angiogenesis. • PAI-1 was identified as a HIF-2α target gene in HCC by microarray analysis. • HIF-2α induces the angiogenic switch via inhibition of plasmin.

  7. In vitro identification of novel plasminogen-binding receptors of the pathogen Leptospira interrogans.

    Directory of Open Access Journals (Sweden)

    Monica L Vieira

    Full Text Available BACKGROUND: Leptospirosis is a multisystem disease caused by pathogenic strains of the genus Leptospira. We have reported that Leptospira are able to bind plasminogen (PLG, to generate active plasmin in the presence of activator, and to degrade purified extracellular matrix fibronectin. METHODOLOGY/PRINCIPAL FINDINGS: We have now cloned, expressed and purified 14 leptospiral recombinant proteins. The proteins were confirmed to be surface exposed by immunofluorescence microscopy and were evaluated for their ability to bind plasminogen (PLG. We identified eight as PLG-binding proteins, including the major outer membrane protein LipL32, the previously published rLIC12730, rLIC10494, Lp29, Lp49, LipL40 and MPL36, and one novel leptospiral protein, rLIC12238. Bound PLG could be converted to plasmin by the addition of urokinase-type PLG activator (uPA, showing specific proteolytic activity, as assessed by its reaction with the chromogenic plasmin substrate, D-Val-Leu-Lys 4-nitroanilide dihydrochloride. The addition of the lysine analog 6-aminocaproic acid (ACA inhibited the protein-PLG interaction, thus strongly suggesting the involvement of lysine residues in plasminogen binding. The binding of leptospiral surface proteins to PLG was specific, dose-dependent and saturable. PLG and collagen type IV competed with LipL32 protein for the same binding site, whereas separate binding sites were observed for plasma fibronectin. CONCLUSIONS/SIGNIFICANCE: PLG-binding/activation through the proteins/receptors on the surface of Leptospira could help the bacteria to specifically overcome tissue barriers, facilitating its spread throughout the host.

  8. Abeta-degrading enzymes in Alzheimer's disease.

    Science.gov (United States)

    Miners, James Scott; Baig, Shabnam; Palmer, Jennifer; Palmer, Laura E; Kehoe, Patrick G; Love, Seth

    2008-04-01

    In Alzheimer's disease (AD) Abeta accumulates because of imbalance between the production of Abeta and its removal from the brain. There is increasing evidence that in most sporadic forms of AD, the accumulation of Abeta is partly, if not in some cases solely, because of defects in its removal--mediated through a combination of diffusion along perivascular extracellular matrix, transport across vessel walls into the blood stream and enzymatic degradation. Multiple enzymes within the central nervous system (CNS) are capable of degrading Abeta. Most are produced by neurons or glia, but some are expressed in the cerebral vasculature, where reduced Abeta-degrading activity may contribute to the development of cerebral amyloid angiopathy (CAA). Neprilysin and insulin-degrading enzyme (IDE), which have been most extensively studied, are expressed both neuronally and within the vasculature. The levels of both of these enzymes are reduced in AD although the correlation with enzyme activity is still not entirely clear. Other enzymes shown capable of degrading Abetain vitro or in animal studies include plasmin; endothelin-converting enzymes ECE-1 and -2; matrix metalloproteinases MMP-2, -3 and -9; and angiotensin-converting enzyme (ACE). The levels of plasmin and plasminogen activators (uPA and tPA) and ECE-2 are reported to be reduced in AD. Reductions in neprilysin, IDE and plasmin in AD have been associated with possession of APOEepsilon4. We found no change in the level or activity of MMP-2, -3 or -9 in AD. The level and activity of ACE are increased, the level being directly related to Abeta plaque load. Up-regulation of some Abeta-degrading enzymes may initially compensate for declining activity of others, but as age, genetic factors and diseases such as hypertension and diabetes diminish the effectiveness of other Abeta-clearance pathways, reductions in the activity of particular Abeta-degrading enzymes may become critical, leading to the development of AD and CAA.

  9. Requirement of plasminogen binding to its cell-surface receptor α-enolase for efficient regeneration of normal and dystrophic skeletal muscle.

    Directory of Open Access Journals (Sweden)

    Àngels Díaz-Ramos

    Full Text Available Adult regenerative myogenesis is central for restoring normal tissue structure and function after muscle damage. In muscle repair after injury, as in severe myopathies, damaged and necrotic fibers are removed by infiltrating inflammatory cells and then replaced by muscle stem cells or satellite cells, which will fuse to form new myofibers. Extracellular proteolysis mediated by uPA-generated plasmin plays a critical role in controlling inflammation and satellite-cell-dependent myogenesis. α-enolase has been described as plasminogen receptor in several cell types, where it acts concentrating plasmin proteolytic activity on the cell surface. In this study, we investigated whether α-enolase plasminogen receptor plays a regulatory role during the muscular repair process. Inhibitors of α-enolase/plasminogen binding: MAb11G1 (a monoclonal antibody against α-enolase and ε-aminocaproic acid, EACA (a lysine analogue inhibited the myogenic abilities of satellite cells-derived myoblasts. Furthermore, knockdown of α-enolase decreased myogenic fusion of myoblasts. Injured wild-type mice and dystrophic mdx mice were also treated with MAb11G1 and EACA. These treatments had negative impacts on muscle repair impairing satellite cell functions in vitro in agreement with blunted growth of new myofibers in vivo. Furthermore, both MAb11G1 and EACA treatments impaired adequate inflammatory cell infiltration and promoted extracellular matrix deposition in vivo, which resulted in persistent degeneration. These results demonstrate the novel requirement of α-enolase for restoring homeostasis of injured muscle tissue, by controlling the pericellular localization of plasmin activity.

  10. Hemostatic and toxinological diversities in venom of Micrurus tener tener, Micrurus fulvius fulvius and Micrurus isozonus coral snakes.

    Science.gov (United States)

    Salazar, Ana M; Vivas, Jeilyn; Sánchez, Elda E; Rodríguez-Acosta, Alexis; Ibarra, Carlos; Gil, Amparo; Carvajal, Zoila; Girón, María E; Estrella, Amalid; Navarrete, Luis F; Guerrero, Belsy

    2011-07-01

    The coral snake Micrurus tener tener (Mtt) from the Elapidae family inhabits the southwestern United States and produces severe cases of envenomations. Although the majority of Mtt venom components are neurotoxins and phospholipase A₂s, this study demonstrated, by SDS-PAGE and molecular exclusion chromatography (MEC), that these venoms also contain high-molecular-weight proteins between 50 and 150 kDa that target the hemostatic system. The biological aspects of other Micrurus venoms were also studied, such as the LD₅₀s of Micrurus isozonus (from 0.52 to 0.61 mg/kg). A pool from these venoms presented a LD₅₀ of 0.57 mg/kg, Micrurus f. fulvius (Mff) and Mtt had LD₅₀s of 0.32 and 0.78 mg/kg, respectively. These venoms contained fibrino(geno)lytic activity, they inhibited platelet aggregation, as well as factor Xa and/or plasmin-like activities. M. isozonus venoms from different Venezuelan geographical regions inhibited ADP-induced platelet aggregation (from 50 to 68%). Micrurus tener tener venom from the United States was the most active with a 95.2% inhibitory effect. This venom showed thrombin-like activity on fibrinogen and human plasma. Fractions of Mtt showed fibrino(geno)lytic activity and inhibition on plasmin amidolytic activity. Several fractions degraded the fibrinogen Aα chains, and fractions F2 and F7 completely degraded both fibrinogen Aα and Bβ chains. To our knowledge, this is the first report on thrombin-like and fibrino(geno)lytic activity and plasmin or factor Xa inhibitors described in Micrurus venoms. Further purification and characterization of these Micrurus venom components could be of therapeutic use in the treatment of hemostatic disorders.

  11. POEMS syndrome with vascular lesions and renal carcinoma - possible role of cytokines.

    Science.gov (United States)

    Pasqui, A L; Bova, G; Saletti, M; Bruni, F; Di Renzo, M; Auteri, A

    1998-06-17

    We describe here the case of a 60 years man with POEMS syndrome associated with renal tumor and vascular lesions. The patient had osteosclerotic myeloma IgA-lambda, polyneuropathy, endocrinopathy and skin changes. In addition, he developed renal clear cell carcinoma and gangrena of lower limbs. The humoral study showed thrombocytosis, high levels of IL-1beta and IL-6 and of some coagulative/fibrinolytic and endothelial factors (von Willebrand factor, plasmin-antiplasmine complexes, plasminogen activator). We suggest the hypothesis that these factors are capable of determining some manifestations of POEMS syndrome.

  12. Predictores de la evolución clínico-radiológica del ictus isquémico agudo tras el tratamiento trombolítico

    OpenAIRE

    Delgado Mederos, Raquel

    2008-01-01

    Introducción: El ictus isquémico representa una importante causa de mortalidad y discapacidad en todo el mundo. La trombolisis intravenosa con activador del plasminógeno tisular recombinante (rt-PA) ha demostrado su eficacia en el tratamiento del ictus isquémico agudo, aunque sólo la mitad de los pacientes logran una respuesta clínica favorable. El conocimiento sobre los factores que influyen en la respuesta terapéutica son limitados. Esta Tesis se centra en dos procesos con potencial impact...

  13. Predictores de la evolución clínico-radiológica del ictus isquémico agudo tras tratamiento trombolítico

    OpenAIRE

    Delgado Mederos, Raquel

    2009-01-01

    Introducción: El ictus isquémico representa una importante causa de mortalidad y discapacidad en todo el mundo. La trombolisis intravenosa con activador del plasminógeno tisular recombinante (rt-PA) ha demostrado su eficacia en el tratamiento del ictus isquémico agudo, aunque sólo la mitad de los pacientes logran una respuesta clínica favorable. El conocimiento sobre los factores que influyen en la respuesta terapéutica son limitados. Esta Tesis se centra en dos procesos con potencial impact...

  14. Diffusion tensor imaging in acute ischemic stroke: the role of anisotropy in determining the time of onset and predicting long-term motor outcome

    OpenAIRE

    Puig Alcántara, Josep

    2014-01-01

    Introducción: El infarto cerebral és una de las primeras causas de morbi-mortalidad en nuestra sociedad. La fibrinolisis intravenosa con activador tisular del plasminógeno es el tratamiento de elección en las primeras 4.5 horas del inicio de los síntomas. Aproximadamente en un tercio de los pacientes se desconoce el tiempo de evolución, por lo que la fibrinolisis estaría contraindicada. Existe un gran interés en detectar parámetros fiables para discriminar el tiempo de evolución de un infarto...

  15. A conserved TATA-less proximal promoter drives basal transcription from the urokinase-type plasminogen activator receptor gene

    DEFF Research Database (Denmark)

    Soravia, E; Grebe, A; De Luca, P

    1995-01-01

    The urokinase-type plasminogen activator receptor (uPAR) focuses at the cell surface the activation of pro-uPA and, hence, the formation of plasmin, thus enhancing directional extracellular proteolysis. To characterize the transcriptional regulatory mechanisms that control receptor expression, we...... have cloned an uPAR DNA segment containing upstream regulatory sequences from both the human and murine genomes. We report that a proximal promoter, contained within 180 bp from the major transcription start sites of the human uPAR gene, drives basal transcription. This region lacks TATA and CAAT boxes...

  16. High permissivity of human HepG2 hepatoma cells for influenza viruses.

    Science.gov (United States)

    Ollier, Laurence; Caramella, Anne; Giordanengo, Valérie; Lefebvre, Jean-Claude

    2004-12-01

    Human HepG2 hepatoma cells are highly permissive for influenza virus type A and type B, even without the addition of trypsin, and they exhibit a marked cytopathic effect. This property greatly facilitates the primary isolation of influenza viruses. Virus replication was significantly reduced by the plasmin(ogen)-specific inhibitor tranexamic acid, and this suggests a potential role played by the plasminogen/tissue plasminogen activator complex at the surface of HepG2 cells. This might represent a new approach for study of the interrelations of this complex with influenza viruses.

  17. BDNF pro-peptide regulates dendritic spines via caspase-3

    OpenAIRE

    Guo, J.; Ji, Y.; Y. Ding; Jiang, W.; Sun, Y.; B. Lu; Nagappan, G

    2016-01-01

    The precursor of brain-derived neurotrophic factor (BDNF) (proBDNF) is enzymatically cleaved, by either intracellular (furin/PC1) or extracellular proteases (tPA/plasmin/MMP), to generate mature BDNF (mBDNF) and its pro-peptide (BDNF pro-peptide). Little is known about the function of BDNF pro-peptide. We have developed an antibody that specifically detects cleaved BDNF pro-peptide, but not proBDNF or mBDNF. Neuronal depolarization elicited a marked increase in extracellular BDNF pro-peptide,...

  18. Urinary serine proteases and activation of ENaC in kidney

    DEFF Research Database (Denmark)

    Svenningsen, Per; Andersen, Henrik; Nielsen, Lise Hald

    2015-01-01

    pressure, while gain-of-function mutations lead to impaired Na(+) excretion, hypertension, and hypokalemia. We review an emerging pathophysiological concept that aberrant glomerular filtration of plasma proteases, e.g., plasmin, prostasin, and kallikrein, contributes to proteolytic activation of ENaC, both...... with albuminuria compatible with impaired renal Na(+) excretion: hypertension and volume retention is secondary to proteinuria in, e.g., preeclampsia and nephrotic syndrome; plasma concentrations of renin, angiotensin II, and aldosterone are frequently suppressed in proteinuric conditions, e.g., preeclampsia...

  19. HGF, MET, and matrix-related proteases in hepatocellular carcinoma, fibrolamellar variant, cirrhotic and normal liver.

    Science.gov (United States)

    Schoedel, Karen E; Tyner, Valerie Zajac; Kim, Tae-Hyoung; Michalopoulos, George K; Mars, Wendy M

    2003-01-01

    Fibrolamellar variant is an uncommon subcategory of hepatocellular carcinoma with a better prognostic outcome. Proteinases and growth factors that are involved in the remodeling of extracellular matrix may influence the behavior of cancers. To determine whether these factors contribute to the distinct etiologies of fibrolamellar hepatocellular carcinoma and traditional hepatocellular carcinoma, we assayed hepatocyte growth factor, the hepatocyte growth factor receptor, and two hepatocyte growth factor activators, hepatocyte growth factor activator and urokinase-type plasminogen activator, in hepatocellular carcinoma, fibrolamellar hepatocellular carcinoma, cirrhotic liver and normal liver. In addition, we examined the urokinase-type plasminogen activator receptor, the type 1 plasminogen activator inhibitor, plasmin, fibrinogen, and the type IV matrix metalloproteinases. Eighteen hepatocellular carcinomas and 11 fibrolamellar hepatocellular carcinomas were obtained as paraffin embedded sections from the University of Pittsburgh Department of Pathology. Frozen tissues from a subset of cases (9 hepatocellular carcinomas, 4 fibrolamellar hepatocellular carcinomas, 12 cirrhotic livers and 2 normal livers) were also available for analysis. Antibodies against urokinase-type plasminogen activator, urokinase-type plasminogen activator receptor, hepatocyte growth factor and hepatocyte growth factor receptor were used to analyze immunoperoxidase stained slides from the paraffin blocks. Western blot analyses using antibodies against hepatocyte growth factor, hepatocyte growth factor receptor, phosphotyrosine, hepatocyte growth factor activator, urokinase-type plasminogen activator receptor, urokinase-type plasminogen activator, plasminogen activator inhibitor-1, fibrinogen and plasmin were performed on membrane-enriched fractions from the frozen tissue, as was collagen zymography for matrix metalloproteinase-2 and matrix metalloproteinase-9. The most notable findings are as

  20. [Aspergillus ochraceus myxomycetes produce extracellular proteinases--protein C activators of blood plasma].

    Science.gov (United States)

    Osmolovskiĭ, A A; Kreĭer, V G; Kurakov, A V; Baranova, N A; Egorov, N S

    2012-01-01

    Natural isolates of Aspergillus ochraceus myxomycetes from soil and plant remains from various regions have been screened. The isolated strains were characterized by similar cultural and morphological features and an identical nucleotide sequence in the ITS1-5,8S-ITS2 region of rDNA. The ability of the extracellular proteinases of A. ochraceus myxomycetes to activate protein C of blood plasma has been established. Differences are revealed in the accumulation of proteinases activating protein C and proteinases with thrombin- and plasmin-like activities in the growth dynamics of producers.

  1. La trombectomía mecánica en el tratamiento de accidentes cerebrovasculares (ACV) en la fase aguda

    OpenAIRE

    Alain Bonafe

    2013-01-01

    En Francia, la incidencia anual de accidentes cerebro vasculares (ACV) es de 1.6 a 2.4 por cada mil (1.000) personas, es decir entre 100.000 y 145.000 por año, con una mortalidad del 15 al 20% al cabo del primer mes y un 75% de personas sobrevivientes con secuelas. El tratamiento de preferencia es la Trombólisis IV (Intra Venosa) con rt-PA (Recombinant Tissue Plasminogen Activator - Activador del Plasminógeno Tisular Recombinante), el cual tiene una ventana de eficacia...

  2. Relevance of Post-Stroke Circulating BDNF Levels as a Prognostic Biomarker of Stroke Outcome. Impact of rt-PA Treatment

    OpenAIRE

    Marion Rodier; Aurore Quirié; Anne Prigent-Tessier; Yannick Béjot; Agnès Jacquin; Claude Mossiat; Christine Marie; Philippe Garnier

    2015-01-01

    The recombinant form of tissue plasminogen activator (rt-PA) is the only curative treatment for ischemic stroke. Recently, t-PA has been linked to the metabolism of brain-derived neurotrophic factor (BDNF), a major neurotrophin involved in post-stroke neuroplasticity. Thus, the objective of our study was to investigate the impact of rt-PA treatment on post-stroke circulating BDNF levels in humans and in animals. Serum BDNF levels and t-PA/plasmin activity were measured at hospital admission a...

  3. Proving the adulteration of ewe and goat cheeses with cow milk using the reference method of isoelectric focusing of γ-casein

    OpenAIRE

    Nataša Mikulec; Dijana Plavljanić; Jasminka Špoljarić; Biljana Radeljević; Jasmina Havranek; Neven Antunac

    2013-01-01

    The aim of this study was to introduce a reference method for the detection of cow milk in ewe and goat cheeses (EC 273/08) in order to protect manufacturers and consumers from adulterations and imitations, and to ensure alignment with the demands of domestic and EU markets. The method includes isolation of casein from cheese, isoelectric focusing of γ2- and γ3-casein originating from the hydrolysis of β-casein by plasmin, the detection and quantitative determination of γ-casein in cow, ewe a...

  4. The Use of Solvent/Detergent Treatment in Pathogen Reduction of Plasma

    OpenAIRE

    Hellstern, Peter; Solheim, Bjarte G.

    2011-01-01

    The solvent/detergent (SD) process used for plasma can safely inactivate all lipid-enveloped viruses. The introduction of a specific prion-binding ligand gel in combination with SD treatment, time-reduced from 4 to 1-1.5 h, still ensures efficient virus kill, reduces abnormal prion protein by >5 log steps, and preserves levels of plasmin inhibitor at close to the reference range. Infections with known non-enveloped viruses such as HAV or parvovirus B19 are prevented by ensuring low virus load...

  5. Semi-automatic method for routine evaluation of fibrinolytic components.

    Science.gov (United States)

    Collen, D; Tytgat, G; Verstraete, M

    1968-11-01

    A semi-automatic method for the routine evaluation of fibrinolytic activity is described. The principle is based upon graphic recording by a multichannel voltmeter of tension drops over a potentiometer, caused by variations in the influence of light upon a light-dependent resistance, resulting from modifications in the composition of the fibrin fibres by lysis. The method is applied to the assessment of certain fibrinolytic factors with widespread fibrinolytic endpoints, and the results are compared with simultaneously obtained visual data on the plasmin assay, the plasminogen assay, and on the euglobulin clot lysis time.

  6. Estudio de la expresión génica de diversos componentes del sistema fibrinolítico de las metaloproteasas y de la angiogénesis en la endometriosis. Correlación de los niveles de expresión con algunos polimorfismos estudiados

    OpenAIRE

    Ramón Nuñez, Luís Andrés

    2011-01-01

    INTRODUCCIÓN: La endometriosis, definida como la presencia de tejido endometrial fuera del útero, es una de las enfermedades ginecológicas benignas más frecuentes. La endometriosis es una entidad multifactorial y poligénica en la que tanto factores endometriales como peritoneales, relacionados con los sistemas de activación del plasminógeno (PA) y de las metaloproteasas (MMPs) y la angiogénesis, pueden estar implicados en esta enfermedad. OBJETIVO: Analizar los niveles de mRNA ...

  7. Terapéuticas intervencionistas para el accidente cerebrovascular isquémico

    OpenAIRE

    Rabadán, Alejandra T.; Luciano Sposato; Claudio Mazia

    2010-01-01

    En los últimos 20 años se han desarrollado nuevas opciones para el tratamiento y para la prevención del accidente cerebrovascular (ACV) isquémico, muchas de ellas de carácter intervencionista, tales como la endarterectomía carotídea y la trombolisis intravenosa con activador tisular del plasminógeno. La evidencia científica ha llevado a su difusión y utilización en países desarrollados mientras que en naciones emergentes se observa un retraso en su adopción. Otras modalidades terapéuticas que...

  8. Effects of inhaled plasminogen activator on the balance between coagulation and fibrinolysis in traumatized pigs

    DEFF Research Database (Denmark)

    Münster, A-M B; Rasmussen, L; Sidelmann, Johannes Jakobsen

    2002-01-01

    A profibrinolytic state is normal in the alveoli, but this may change as a result of trauma, possibly leading to fibrin deposition, a characteristic of acute lung injury/acute respiratory distress syndrome. Therefore, the present study investigated in a double-blind, placebo-controlled manner...... with an increased plasmin-dependent fibrinolytic activity without increased systemic fibrinolytic activity, the transient increase in the concentration of scu-PA in the plasma being minimal. In conclusion, the study shows that activatable scu-PA can be nebulized to the lower respiratory tract and can increase...

  9. Metastasis of transgenic breast cancer in plasminogen activator inhibitor-1 gene-deficient mice

    DEFF Research Database (Denmark)

    Almholt, Kasper; Nielsen, Boye Schnack; Frandsen, Thomas Leth;

    2003-01-01

    of metastasizing breast cancer. In these tumors, the expression pattern of uPA and PAI-1 resembles that of human ductal breast cancer and plasminogen is required for efficient metastasis. In a cohort of 63 transgenic mice that were either PAI-1-deficient or wild-type sibling controls, primary tumor growth...... limiting for tumor vascularization and metastasis, or that there is a functional redundancy between PAI-1 and other inhibitors of the uPA/plasmin system, masking the effect of PAI-1 deficiency....

  10. Isolation, subunit structure, and proteolytic modification of bovine factor VIII.

    Science.gov (United States)

    Legaz, M E; Weinstein, M J; Heldebrant, C M; Davie, E W

    1975-01-20

    A new method has been described for the isolation of factor VIII. The method results in a high yield of factor VIII that is homogeneous by several different criteria. The purified protein is very stable and is not dissociated in the presence of 1 M NaCl or 0.25 M CaCl2. The highly purified protein is readily activated and inactivated by various proteolytic enzymes, such as thrombin, plasmin, and trypsin. The molecular events that lead to the activation reaction, however, have not been established.

  11. El papel de la plasmina en la supervivencia de Dirofilaria immitis y en la patología vascular del hospedador durante la dirofilariosis cardiopulmonar

    OpenAIRE

    González Miguel, Javier

    2015-01-01

    Con el fin de mantenerse y propagarse en los vasos sanguíneos, muchos patógenos requieren de adaptaciones para evitar la coagulación de la sangre a través de la interacción con el sistema fibrinolítico de sus hospedadores. Uno de sus componentes clave es el plasminógeno, una glicoproteína que tras su activación se convierte en plasmina, que es la serina proteasa encargada de degradar los coágulos de fibrina. Dirofilaria immitis es el agente causante de la dirofilariosis cardiopulmonar canina ...

  12. The possible risk of lower-limb sclerotherapy causing an extended hypercoagulable state.

    Science.gov (United States)

    Ariyoshi, H; Kambayashi, J; Tominaga, S; Hatanaka, T

    1996-01-01

    The risk of thrombosis after lower-extremity sclerotherapy is still an unresolved issue. This study was conducted to investigate the influence of sclerotherapy on coagulation and fibrinolysis by examining 20 patients who underwent surgical procedures, 10 of whom were treated by surgery alone (control group), while the other 10 were given sclerotherapy using 1% hydroxypolyaetoxydodecan as polidocanol (sclerotherapy group). Sex, age, and severity of disease was comparable between the two groups. No significant difference was found in the transient elevation of acute phase proteins, C-reactive protein (CRP), or fibrinogen. Thrombin antithrombin III complex (TAT), a marker of coagulation, transiently increased following treatment. In the control group, TAT peaked 3 days after treatment, whereas in the sclerotherapy group the elevation was prolonged, peaking 7 days after treatment. Elevation of the markers of fibrinolysis, plasmin plasmin inhibitor complex (PIC) and fibrin degradation products (FDP), was slower than that of TAT, peaking 7 days after treatment in both groups, the plasma PIC being significantly enhanced 7 days after treatment in the sclerotherapy group. A significant decrease in the platelet count was observed 3 days after treatment in the sclerotherapy group. These results suggest that sclerotherapy may enhance coagulation or fibrinolysis after surgical procedures.

  13. The hippocampal laminin matrix is dynamic and critical for neuronal survival.

    Science.gov (United States)

    Chen, Zu-Lin; Indyk, Justin A; Strickland, Sidney

    2003-07-01

    Laminins are extracellular matrix proteins that participate in neuronal development, survival, and regeneration. During excitotoxin challenge in the mouse hippocampus, neuron interaction with laminin-10 (alpha5,beta1,gamma1) protects against neuronal death. To investigate how laminin is involved in neuronal viability, we infused laminin-1 (alpha1,beta1,gamma1) into the mouse hippocampus. This infusion specifically disrupted the endogenous laminin layer. This disruption was at least partially due to the interaction of the laminin-1 gamma1 chain with endogenous laminin-10, because infusion of anti-laminin gamma1 antibody had the same effect. The disruption of the laminin layer by laminin-1 1) did not require the intact protein because infusion of plasmin-digested laminin-1 gave similar results; 2) was posttranscriptional, because there was no effect on laminin mRNA expression; and 3) occurred in both tPA(-/-) and plasminogen(-/-) mice, indicating that increased plasmin activity was not responsible. Finally, although tPA(-/-) mice are normally resistant to excitotoxin-induced neurodegeneration, disruption of the endogenous laminin layer by laminin-1 or anti-laminin gamma1 antibody renders the tPA(-/-) hippocampal neurons sensitive to kainate. These results demonstrate that neuron interactions with the deposited matrix are not necessarily recapitulated by interactions with soluble components and that the laminin matrix is a dynamic structure amenable to modification by exogenous molecules.

  14. Plasminogen mRNA induction in the mouse brain after kainate excitation: codistribution with plasminogen activator inhibitor-2 (PAI-2) mRNA.

    Science.gov (United States)

    Sharon, Ronit; Abramovitz, Rene; Miskin, Ruth

    2002-08-15

    Plasminogen (Plg), which can be converted to the active protease plasmin by plasminogen activators, has been previously implicated in brain plasticity and in toxicity inflicted in hippocampal pyramidal neurons by kainate. Here we have localized Plg. mRNA through in situ hybridization in brain cryosections derived from normal adult mice or after kainate injection (i.p.). The results indicated that Plg mRNA was undetectable in the normal brain, but after kainate injection it was induced in neuronal cells in multiple, but specific areas, including layers II-III of the neocortex; the olfactory bulb, anterior olfactory nucleus, and the piriform cortex; the caudate/putamen and accumbens nucleus shell; throughout the amygdaloid complex; and in the CAI/CA3 subfields of the hippocampus. Interestingly, this distribution pattern coincided with what we have recently described for the plasminogen activator inhibitor-2 (PAI-2) mRNA, however differing from that of the plasminogen activator inhibitor-1 (PAI-1) mRNA, as also shown here. These results suggest that enhanced Plg gene expression could be involved in events associated with olfactory, striatal, and limbic structures. Furthermore, because PAI-2 is thought to intracellularly counteract cytotoxic events, our results raise the possibility that PAI-2 can act in the brain as an intracellular neuroprotector against potential plasmin-mediated toxicity.

  15. Elevated Cytokines, Thrombin and PAI-1 in Severe HCPS Patients Due to Sin Nombre Virus

    Directory of Open Access Journals (Sweden)

    Virginie Bondu

    2015-02-01

    Full Text Available Sin Nombre Hantavirus (SNV, Bunyaviridae Hantavirus is a Category A pathogen that causes Hantavirus Cardiopulmonary Syndrome (HCPS with case fatality ratios generally ranging from 30% to 50%. HCPS is characterized by vascular leakage due to dysregulation of the endothelial barrier function. The loss of vascular integrity results in non-cardiogenic pulmonary edema, shock, multi-organ failure and death. Using Electric Cell-substrate Impedance Sensing (ECIS measurements, we found that plasma samples drawn from University of New Mexico Hospital patients with serologically-confirmed HCPS, induce loss of cell-cell adhesion in confluent epithelial and endothelial cell monolayers grown in ECIS cultureware. We show that the loss of cell-cell adhesion is sensitive to both thrombin and plasmin inhibitors in mild cases, and to thrombin only inhibition in severe cases, suggesting an increasing prothrombotic state with disease severity. A proteomic profile (2D gel electrophoresis and mass spectrometry of HCPS plasma samples in our cohort revealed robust antifibrinolytic activity among terminal case patients. The prothrombotic activity is highlighted by acute ≥30 to >100 fold increases in active plasminogen activator inhibitor (PAI-1 which, preceded death of the subjects within 48 h. Taken together, this suggests that PAI-1 might be a response to the severe pathology as it is expected to reduce plasmin activity and possibly thrombin activity in the terminal patients.

  16. Effect of somatic cell count and lactation stage on sheep milk quality

    Directory of Open Access Journals (Sweden)

    Emilia Duranti

    2010-01-01

    Full Text Available In order to evaluate the effects of mammary health status and lactation phase on the qualitative parameters of ovinemilk, 213 individual milk samples were repeatedly collected from 40 primiparous Sarda ewes on a monthly basis. Yield,physico-chemical characteristics, casein fractions quantitative distribution, somatic cell count (SCC, cheese making propertiesand plasmin-plasminogen activity were determined on each sample. Repeated individual milk SCC were used as amarker of udder health status, allowing the definition of three classes: “Healthy” (H, “Infected” (I or “Doubtful” (D.Samples were grouped into 4 classes of days in milk (DIM. To evaluate the influence of mammary health status andphase of lactation, a mixed model was performed using the ewe as random effect. Milk physico-chemical parameters wereinfluenced both by the udder health status and by lactation phase. In particular, the udder health status adversely affectedαs1 and β1-casein fractions (Pand 64.60% in “H”, “D” and “I,” respectively. Lactation phase influenced the overall milk composition and technologicalcharacteristics. Plasmin activity was higher in the “I” group than in the others (16.1 vs 11.8 and 11.2 U/ml; Pit significantly (Pexert a detrimental effect on milk quality since they enhance its endogenous proteolytic activity.

  17. SCM, a novel M-like protein from Streptococcus canis, binds (mini)-plasminogen with high affinity and facilitates bacterial transmigration.

    Science.gov (United States)

    Fulde, Marcus; Rohde, Manfred; Hitzmann, Angela; Preissner, Klaus T; Nitsche-Schmitz, D Patric; Nerlich, Andreas; Chhatwal, Gursharan Singh; Bergmann, Simone

    2011-03-15

    Streptococcus canis is an important zoonotic pathogen capable of causing serious invasive diseases in domestic animals and humans. In the present paper we report the binding of human plasminogen to S. canis and the recruitment of proteolytically active plasmin on its surface. The binding receptor for plasminogen was identified as a novel M-like protein designated SCM (S. canis M-like protein). SPR (surface plasmon resonance) analyses, radioactive dot-blot analyses and heterologous expression on the surface of Streptococcus gordonii confirmed the plasminogen-binding capability of SCM. The binding domain was located within the N-terminus of SCM, which specifically bound to the C-terminal part of plasminogen (mini-plasminogen) comprising kringle domain 5 and the catalytic domain. In the presence of urokinase, SCM mediated plasminogen activation on the bacterial surface that was inhibited by serine protease inhibitors and lysine amino acid analogues. Surface-bound plasmin effectively degraded purified fibrinogen as well as fibrin clots, resulting in the dissolution of fibrin thrombi. Electron microscopic illustration and time-lapse imaging demonstrated bacterial transmigration through fibrinous thrombi. The present study has led, for the first time, to the identification of SCM as a novel receptor for (mini)-plasminogen mediating the fibrinolytic activity of S. canis.

  18. The Internal Dynamics of Fibrinogen and Its Implications for Coagulation and Adsorption.

    Directory of Open Access Journals (Sweden)

    Stephan Köhler

    2015-09-01

    Full Text Available Fibrinogen is a serum multi-chain protein which, when activated, aggregates to form fibrin, one of the main components of a blood clot. Fibrinolysis controls blood clot dissolution through the action of the enzyme plasmin, which cleaves fibrin at specific locations. Although the main biochemical factors involved in fibrin formation and lysis have been identified, a clear mechanistic picture of how these processes take place is not available yet. This picture would be instrumental, for example, for the design of improved thrombolytic or anti-haemorrhagic strategies, as well as, materials with improved biocompatibility. Here, we present extensive molecular dynamics simulations of fibrinogen which reveal large bending motions centered at a hinge point in the coiled-coil regions of the molecule. This feature, likely conserved across vertebrates according to our analysis, suggests an explanation for the mechanism of exposure to lysis of the plasmin cleavage sites on fibrinogen coiled-coil region. It also explains the conformational variability of fibrinogen observed during its adsorption on inorganic surfaces and it is supposed to play a major role in the determination of the hydrodynamic properties of fibrinogen. In addition the simulations suggest how the dynamics of the D region of fibrinogen may contribute to the allosteric regulation of the blood coagulation cascade through a dynamic coupling between the a- and b-holes, important for fibrin polymerization, and the integrin binding site P1.

  19. Involvement of urokinase-type plasminogen activator system in cancer: an overview.

    Science.gov (United States)

    Mekkawy, Ahmed H; Pourgholami, Mohammad H; Morris, David L

    2014-09-01

    Currently, there are several studies supporting the role of urokinase-type plasminogen activator (uPA) system in cancer. The association of uPA to its receptor triggers the conversion of plasminogen into plasmin. This process is regulated by the uPA inhibitors (PAI-1 and PAI-2). Plasmin promotes degradation of basement membrane and extracellular matrix (ECM) components as well as activation of ECM latent matrix metalloproteases. Degradation and remodeling of the surrounding tissues is crucial in the early steps of tumor progression by facilitating expansion of the tumor mass, release of tumor growth factors, activation of cytokines as well as induction of tumor cell proliferation, migration, and invasion. Hence, many tumors showed a correlation between uPA system component levels and tumor aggressiveness and survival. Therefore, this review summarizes the structure of the uPA system, its contribution to cancer progression, and the clinical relevance of uPA family members in cancer diagnosis. In addition, the review evaluates the significance of uPA system in the development of cancer-targeted therapies.

  20. Amino and chlorambucil analogues of pentamidine--synthesis and biological examinations.

    Science.gov (United States)

    Pućkowska, Anna; Drozdowska, Danuta; Rusak, Małgorzata; Bielawski, Tomasz; Bruzgo, Irena; Midura-Nowaczek, Krystyna

    2012-01-01

    The amino analogues of pentamidine with a polymethylene (n = 3 - 6) chain and their chlorambucil derivatives were synthesized. The obtained compounds revealed cytotoxic effect on MCF-7 human breast cancer cell line (IC50 = 22 - 95 +/- 2 pM), mainly by the induction of apoptosis. The topoisomerase I/II inhibition assay and the ethidium displacement assay with the use of pBR322 plasmid DNA were used to the study of mechanism by which the obtained compounds could act. All the compounds are able to bind with DNA and interfere in vitro with the activity of topoisomerase (I and II). The determination of association constants with the use of calf thymus DNA, T4 coliphage DNA, poly(dA-dT)2 and poly(dG-dC)2 showed that the tested compounds bind within minor groove of B-DNA, but not selectively. The alkylating activity of chlorambucil derivatives determined in vitro using a Preussmann test was similar to the activity of chlorambucil. The influence of all the compounds on the amidolytic activity of plasmin and trypsin was also examined. The plasmin activity was inhibited by pentamidine, chlorambucil and aromatic bis-amines (IC50 = 0.1 - 8 mM), whereas the trypsin activity was influenced only by pentamidine.

  1. Escherichia coli lipoprotein binds human plasminogen via an intramolecular domain

    Directory of Open Access Journals (Sweden)

    Tammy eGonzalez

    2015-10-01

    Full Text Available Escherichia coli lipoprotein (Lpp is a major cellular component that exists in two distinct states, bound-form and free-form. Bound-form Lpp is known to interact with the periplasmic bacterial cell wall, while free-form Lpp is localized to the bacterial cell surface. A function for surface-exposed Lpp has yet to be determined. We hypothesized that the presence of C-terminal lysines in the surface-exposed region of Lpp would facilitate binding to the host zymogen plasminogen, a protease commandeered by a number of clinically important bacteria. Recombinant Lpp was synthesized and the binding of Lpp to plasminogen, the effect of various inhibitors on this binding, and the effects of various mutations of Lpp on Lpp-plasminogen interactions were examined. Additionally, the ability of Lpp-bound plasminogen to be converted to active plasmin was analyzed. We determined that Lpp binds plasminogen via an atypical domain located near the center of mature Lpp that may not be exposed on the surface of intact E. coli according to the current localization model. Finally, we found that plasminogen bound by Lpp can be converted to active plasmin. While the consequences of Lpp binding plasminogen are unclear, these results prompt further investigation of the ability of surface exposed Lpp to interact with host molecules such as extracellular matrix components and complement regulators, and the role of these interactions in infections caused by E. coli and other bacteria.

  2. Is elimination of cardiotomy suction preferable in aortic valve replacement? Assessment of perioperative coagulation, fibrinolysis and inflammation

    Science.gov (United States)

    Morisaki, Akimasa; Nakahira, Atsushi; Sasaki, Yasuyuki; Hirai, Hidekazu; Okada, Yuko; Suehiro, Shigefumi; Shibata, Toshihiko

    2013-01-01

    OBJECTIVES Guidelines recommend the avoidance of direct return of pericardial blood based on evidence from coronary surgery. A continuous auto-transfusion system (CATS) can be a good alternative to cardiotomy suction by reinfusing aspirated pericardial blood without the necessity of intermittent collection. To clarify the effects of direct return of pericardial blood in aortic valve replacement (AVR), we compared the effects of cardiotomy suction and an alternative CATS on perioperative coagulofibrinolysis and inflammation systems, and clinical outcomes. METHODS In 40 AVR operations between April 2009 and April 2011, the retransfusion method of pericardial blood during cardiopulmonary bypass (CPB) was allocated to the use of cardiotomy suction (non-Cell-Saver group, n = 20) or CATS (Cell-Saver group, n = 20) under identical protocols of anticoagulation and transfusion. The blood from the left ventricular vent was returned to the venous reservoir. We obtained blood samples at nine points up to the morning after surgery. RESULTS Perioperative values for coagulofibrinolysis markers, such as thrombin–antithrombin III complex, fibrinogen degeneration products, D-dimer and plasmin-α2 plasmin inhibitor complex, were significantly lower in the Cell-Saver group than those in the non-Cell-Saver group from 1 h after the initiation of cardiopulmonary bypass to 3 or 6 h after termination of cardiopulmonary bypass (P suction, thus facilitating less-invasive valve surgeries with marked suppression of coagulofibrinolysis responses. PMID:23728087

  3. Binding of PAI-1 to endothelial cells stimulated by thymosin beta4 and modulation of their fibrinolytic potential.

    Science.gov (United States)

    Boncela, Joanna; Smolarczyk, Katarzyna; Wyroba, Elzbieta; Cierniewski, Czeslaw S

    2006-01-13

    Our previous studies showed that thymosin beta4 (Tbeta4) induced the synthesis of plasminogen activator inhibitor-1 (PAI-1) in cultured human umbilical vein endothelial cells (HUVECs) via the AP-1 dependent mechanism and its enhanced secretion. In this work we provide evidence that the released PAI-1 is accumulated on the surface of HUVECs, exclusively in its active form, in a complex with alpha1-acid glycoprotein (AGP) that is also up-regulated and released from the cells. This mechanism is supported by several lines of experiments, in which expression of both proteins was analyzed by flow cytometry and their colocalization supported by confocal microscopy. PAI-1 did not bind to quiescent cells but only to the Tbeta4-activated endothelial cells. In contrast, significant amounts of AGP were found to be associated with the cells overexpressing enhanced green fluorescent protein (EGFP)-alpha1-acid glycoprotein (AGP) without Tbeta4 treatment. The AGP.PAI-1 complex was accumulated essentially at the basal surface of endothelial cells, and such cells showed (a) morphology characteristic for strongly adhered and spread cells and (b) significantly reduced plasmin formation. Taken together, these results provide the evidence supporting a novel mechanism by which active PAI-1 can be bound to the Tbeta4-activated endothelial cells, thus influencing their adhesive properties as well as their ability to generate plasmin.

  4. Amelioration of glomerulosclerosis with all-trans retinoic acid is linked to decreased plasminogen activator inhibitor-1 and α-smooth muscle actin

    Institute of Scientific and Technical Information of China (English)

    Xia LIU; Lei L(U); Bei-bei TAO; Ai-ling ZHOU; Yi-chun ZHU

    2011-01-01

    Aim:To examine the effects of all-trans retinoic acid (atRA) on renal morphology and function as well as on renal plasminogen activator inhibitor-1 (PAI-1) expression and plasmin activity in rats with 5/6 nephrectomy.Methods:Adult male Sprague Dawley rats were given 5/6 nephrectomy or sham operation. Renal function was measured 2 weeks later. The nephrectomized rats were assigned to groups matched for proteinuria and treated with vehicle or atRA (5 or 10 mg/kg by gastric gavage once daily) for the next 12 weeks. Rats with sham operation were treated with vehicle. At the end of the treatments,kidneys were collected for histological examination, Western blot analysis, and enzymatic activity measurements.Results:The 5/6 nephrectomy promoted hypertension, renal dysfunction, and glomerulosclerosis. These changes were significantly reduced in the atRA-treated group. The expressions of PAI-1 and α-smooth muscle actin (α-SMA) were significantly increased in the vehicle-treated nephrectomized rats. Treatment with atRA significantly reduced the expressions of PAI-1 and α-SMA. However, piasmin activity remained unchanged following atRA treatment.Conclusion:Treatment with atRA ameliorates glomerulosclerosis and improves renal function in rats with 5/6 nephrectomy. This is associated with a decrease in PAI-1 and α-SMA, but not with a change in plasmin activity.

  5. Partial deletion of the αC-domain in the Fibrinogen Perth variant is associated with thrombosis, increased clot strength and delayed fibrinolysis.

    Science.gov (United States)

    Westbury, Sarah K; Duval, Cédric; Philippou, Helen; Brown, Rebecca; Lee, Kurtis R; Murden, Sherina L; Phillips, Emma; Reilly-Stitt, Christopher; Whalley, Daniel; Ariëns, Robert A; Mumford, Andrew D

    2013-12-01

    Genetic fibrinogen (FGN) variants that are associated with bleeding or thrombosis may be informative about fibrin polymerisation, structure and fibrinolysis. We report a four generation family with thrombosis and heritable dysfibrinogenaemia segregating with a c.[1541delC];[=] variation in FGA (FGN-Perth). This deletion predicts a truncated FGN αC-domain with an unpaired terminal Cys at residue 517 of FGN-Aα. In keeping with this, SDS-PAGE of purified FGN-Perth identified a truncated FGN-Aα chain with increased co-purification of albumin, consistent with disulphide bonding to the terminal Cys of the variant FGN-Aα. Clot visco-elastic strength in whole blood containing FGN-Perth was greater than controls and tPA-mediated fibrinolysis was delayed. In FGN-Perth plasma and in purified FGN-Perth, there was markedly reduced final turbidity after thrombin-mediated clot generation. Consistent with this, FGN-Perth formed tighter, thinner fibrin fibres than controls indicating defective lateral aggregation of protofibrils. Clots generated with thrombin in FGN-Perth plasma were resistant to tPA-mediated fibrinolysis. FGN-Perth clot also displayed impaired tPA-mediated plasmin generation but incorporated α2-antiplasmin at a similar rate to control. Impaired fibrinolysis because of defective plasmin generation potentially explains the FGN-Perth clinical phenotype. These findings highlight the importance of the FGN αC-domain in the regulation of clot formation and fibrinolysis.

  6. Bone healing induced by local delivery of an engineered parathyroid hormone prodrug.

    Science.gov (United States)

    Arrighi, Isabelle; Mark, Silke; Alvisi, Monica; von Rechenberg, Brigitte; Hubbell, Jeffrey A; Schense, Jason C

    2009-03-01

    Regenerative medicine requires innovative therapeutic designs to accommodate high morphogen concentrations in local depots, provide their sustained presence, and enhance cellular invasion and directed differentiation. Here we present an example for inducing local bone regeneration with a matrix-bound engineered active fragment of human parathyroid hormone (PTH(1-34)), linked to a transglutaminase substrate for binding to fibrin as a delivery and cell-invasion matrix with an intervening plasmin-sensitive link (TGplPTH(1-34)). The precursor form displays very little activity and signaling to osteoblasts, whereas the plasmin cleavage product, as it would be induced under the enzymatic influence of cells remodeling the matrix, was highly active. In vivo animal bone-defect experiments showed dose-dependent bone formation using the PTH-fibrin matrix, with evidence of both osteoconductive and osteoinductive bone-healing mechanisms. Results showed that this PTH-derivatized matrix may have potential utility in humans as a replacement for bone grafts or to repair bone defects.

  7. The X-ray Crystal Structure of Full-Length Human Plasminogen

    Directory of Open Access Journals (Sweden)

    Ruby H.P. Law

    2012-03-01

    Full Text Available Plasminogen is the proenzyme precursor of the primary fibrinolytic protease plasmin. Circulating plasminogen, which comprises a Pan-apple (PAp domain, five kringle domains (KR1-5, and a serine protease (SP domain, adopts a closed, activation-resistant conformation. The kringle domains mediate interactions with fibrin clots and cell-surface receptors. These interactions trigger plasminogen to adopt an open form that can be cleaved and converted to plasmin by tissue-type and urokinase-type plasminogen activators. Here, the structure of closed plasminogen reveals that the PAp and SP domains, together with chloride ions, maintain the closed conformation through interactions with the kringle array. Differences in glycosylation alter the position of KR3, although in all structures the loop cleaved by plasminogen activators is inaccessible. The ligand-binding site of KR1 is exposed and likely governs proenzyme recruitment to targets. Furthermore, analysis of our structure suggests that KR5 peeling away from the PAp domain may initiate plasminogen conformational change.

  8. Effects of radiation therapy on blood coagulation-fibrinolysis system in patients with oral squamous cell carcinoma

    Energy Technology Data Exchange (ETDEWEB)

    Watanabe, Kou; Kawamura, Hiroshi; Yosue, Takashi [Nippon Dental Univ., Tokyo (Japan). School of Dentistry; Arai, Chiaki [Nippon Dental Univ., Tokyo (Japan). Hospital

    2002-04-01

    The aims of this study were to clarify the effects of radiotherapy on haemostatic activity in patients with oral squamous cell carcinoma (SCC) and to investigate the differences in the clinical findings. The subjects were 61 patients with primary oral SCC (SCC group) who had undergone preoperative radiotherapy of 34.2{+-}7.2 Gy (mean{+-}SD). These patients were divided into early group (stage I and II) and advanced group (stage III and IV), and the region in the oral cavity. Before and after radiotherapy, activated partial thromboplastin time (APTT), fibrinogen, prothrombin fragment 1+2 (F1+2) and plasmin-{alpha}{sub 2} plasmin inhibitor complex (PAP) were measured. In SCC group, after the radiotherapy, APTT extended and PAP increased. In the early stage group, PAP increased and in the advanced group, there was the extension of APTT. The regional division of the patients, there was the extension of APTT in oral floor and lower gingiva groups. F1+2 in lower gingiva group increased, and PAP rose in tongue and buccal mucosa groups. These results indicate that irradiation affects blood coagulation fibrinolysis system in patients with oral SCC, but the amount of the activation differs by the clinical findings. (author)

  9. Structural analysis of Pla protein from the biological warfare agent Yersinia pestis: docking and molecular dynamics of interactions with the mammalian plasminogen system.

    Science.gov (United States)

    Ruback, Eduardo; Lobo, Leandro Araujo; França, Tanos Celmar Costa; Pascutti, Pedro Geraldo

    2013-01-01

    Yersinia pestis protein Pla is a plasmid-coded outer membrane protein with aspartic-protease activity. Pla exhibits a plasminogen (Plg) activator activity (PAA) that promotes the cleavage of Plg to the active serine-protease form called plasmin. Exactly how Pla activates Plg into plasmin remains unclear. To investigate this event, we performed the interactions between the predicted Plg and Pla protein structures by rigid-body docking with the HEX program and evaluated the complex stability by molecular dynamics (MD) using the GROMACS package programs. The predicted docked complex of Plg-Pla shows the same interaction site predicted by experimental site-direct mutagenesis in other studies. After a total of 8 ns of MD simulation, we observed the relaxation of the beta-barrel structure of Pla and the progressive approximation and stabilization between the cleavage site of Plg into the extracellular loops of Pla, followed by the increase in the number of H bonds. We also report here the aminoacids that participate in the active site and the sub sites of interaction. The total understanding of these interactions can be an important tool for drug design against bacterial proteases.

  10. Characterization of a Novel Fibrinolytic Enzyme, BsfA, from Bacillus subtilis ZA400 in Kimchi Reveals Its Pertinence to Thrombosis Treatment.

    Science.gov (United States)

    Ahn, Min-Ju; Ku, Hye-Jin; Lee, Se-Hui; Lee, Ju-Hoon

    2015-12-28

    Recently, the cardiovascular disease has been widely problematic in humans probably due to fibrin formation via the unbalanced Western style diet. Although direct (human plasmin) and indirect methods (plasminogen activators) have been available, bacterial enzyme methods have been studied because of their cheap and mass production. To detect a novel bacterial fibrinolytic enzyme, 111 bacterial strains with fibrinolytic activity were selected from kimchi. Among them, 14 strains were selected because of their stronger activity than 0.02 U of plasmin. Their 16S rRNA sequence analysis revealed that they belong to Bacillus, Leuconostoc, Propionibacterium, Weissella, Staphylococcus, and Bifidobacterium. The strain B. subtilis ZA400, with the highest fibrinolytic activity, was selected and the gene encoding fibrinolytic enzyme (bsfA) was cloned and expressed in the E. coli overexpression system. The purified enzyme was analyzed with SDS-PAGE, western blot, and MALDI-TOF analyses, showing to be 28.4 kDa. Subsequently, the BsfA was characterized to be stable under various stress conditions such as temperature (4-40°C), metal ions (Mn(2+), Ca(2+), K(2+), and Mg(2+)), and inhibitors (EDTA and SDS), suggesting that BsfA could be a good candidate for development of a novel fibrinolytic enzyme for thrombosis treatment and may even be useful as a new bacterial starter for manufacturing functional fermented foods.

  11. Impact of human milk pasteurization on the kinetics of peptide release during in vitro dynamic term newborn digestion.

    Science.gov (United States)

    Deglaire, Amélie; De Oliveira, Samira C; Jardin, Julien; Briard-Bion, Valérie; Emily, Mathieu; Ménard, Olivia; Bourlieu, Claire; Dupont, Didier

    2016-07-01

    Holder pasteurization (62.5°C, 30 min) ensures sanitary quality of donor's human milk but also denatures beneficial proteins. Understanding whether this further impacts the kinetics of peptide release during gastrointestinal digestion of human milk was the aim of the present paper. Mature raw (RHM) or pasteurized (PHM) human milk were digested (RHM, n = 2; PHM, n = 3) by an in vitro dynamic system (term stage). Label-free quantitative peptidomics was performed on milk and digesta (ten time points). Ascending hierarchical clustering was conducted on "Pasteurization × Digestion time" interaction coefficients. Preproteolysis occurred in human milk (159 unique peptides; RHM: 91, PHM: 151), mostly on β-casein (88% of the endogenous peptides). The predicted cleavage number increased with pasteurization, potentially through plasmin activation (plasmin cleavages: RHM, 53; PHM, 76). During digestion, eight clusters resumed 1054 peptides from RHM and PHM, originating for 49% of them from β-casein. For seven clusters (57% of peptides), the kinetics of peptide release differed between RHM and PHM. The parent protein was significantly linked to the clustering (p-value = 1.4 E-09), with β-casein and lactoferrin associated to clusters in an opposite manner. Pasteurization impacted selectively gastric and intestinal kinetics of peptide release in term newborns, which may have further nutritional consequences.

  12. Surface-expressed enolases of Plasmodium and other pathogens

    Directory of Open Access Journals (Sweden)

    Anil Kumar Ghosh

    2011-08-01

    Full Text Available Enolase is the eighth enzyme in the glycolytic pathway, a reaction that generates ATP from phosphoenol pyruvate in cytosolic compartments. Enolase is essential, especially for organisms devoid of the Krebs cycle that depend solely on glycolysis for energy. Interestingly, enolase appears to serve a separate function in some organisms, in that it is also exported to the cell surface via a poorly understood mechanism. In these organisms, surface enolase assists in the invasion of their host cells by binding plasminogen, an abundant plasma protease precursor. Binding is mediated by the interaction between a lysine motif of enolase with Kringle domains of plasminogen. The bound plasminogen is then cleaved by specific proteases to generate active plasmin. Plasmin is a potent serine protease that is thought to function in the degradation of the extracellular matrix surrounding the targeted host cell, thereby facilitating pathogen invasion. Recent work revealed that the malaria parasite Plasmodium also expresses surface enolase, and that this feature may be essential for completion of its life cycle. The therapeutic potential of targeting surface enolases of pathogens is discussed.

  13. Thrombin activatable fibrinolysis inhibitor and other hemostatic parameters in patients with essential arterial hypertension.

    Science.gov (United States)

    Małyszko, Jolanta; Tymcio, Justyna

    2008-01-01

    Hypertension is associated with hemostatic abnormalities and endothelial dysfunction. thrombin activatable fibrinolysis inhibitor (TAFI) is a glycoprotein linking coagulation and fibrinolysis. Objectives. We evaluated TAFI concentrations in patients with essential hypertension in relation to blood pressure. Additionally, we studied TAFI activator, thrombin activity (thrombin-antithrombin complexes--TAT, prothrombin fragments F1 + 2), thrombomodulin (TM)--a marker of endothelial cell injury, degree of plasmin generation (plasmin-antiplasmin complexes [PAP]), other markers of endothelial cell injury--von Willebrand factor (vWF). Seventy-two patients with essential hypertension (27 untreated, 13 treated with enalapril (angiotensin-converting enzyme inhibitor [ACEI]), 32 with beta-blocker, betaxolol). In every hypertensive patients ambulatory blood pressure measurements and echocardiography were performed. All hypertensive patients did not differ with respect to age, creatinine, fibrinogen, D-dimers. In ACEI-treated patients a significantly higher TAFI concentration was observed when compared to beta-blocker-treated patients. In beta-blocker-treated patients both diastolic and systolic blood pressure were lower than in ACEI treated patients as well as ejection fraction, while serum triglycerides were higher. Diastolic blood pressure correlated significantly with TAFI concentrations in untreated patients (r = 0.27, p glicemia, ejection fraction and triglycerides.

  14. Construction and Characterization of a Mutant of Single-chain Urokinase-type Plasminogen Activator Ser(175)-His(187)-mscu-PA

    Science.gov (United States)

    Xue, Yu-Ming; Zhu, Hui; Shi, Wei; Liu, Wei; Liu, Jian-Ning; Ma, Zhong

    2000-01-01

    Single-chain urokinase-type plasminogen activator scu-PA is the precursor of double-chain urokinase tcu-PA , which has a much higher intrinsic catalytic activity than other zymogens of the serine protease family. To restore the "zymogen triad" of Asp-His-Ser in the serine protease family, the mutant gene of scu-PA mscu-PA, Ala(175)right curved arrow Ser(175), Tyr(187)right curved arrow His(187) was constructed by the method of oligonucleotide-directed, site-specific mutagenesis in order to reduce its intrinsic catalytic activity. mscu-PA was expressed in E. coli BL21. After denaturation and renaturation in vitro, the mscu-PA was purified to homogeneity by SP-Sepharose ion-exchange chromatography, Sephacryl S-200 chromatography and Benzamidine-Sepharose affinity adsorption. mscu-PA had the same activity to plasmin as scu-PA. The catalytic efficiency measured by k(cat)/K(m) to synthetic substrate S(2444) was 2.5-fold lower than that of scu-PA, and the activity against Glu-plasminogen was also reduced. After activation by plasmin, mtcu-PA and tcu-PA had similar catalytic efficiency against S(2444) and Glu-plasminogen. The results suggest that the intrinsic catalytic activity of mscu-PA be really reduced after restoring the "zymogen triad".

  15. Serine proteinase inhibitors from Vipera ammodytes venom. Isolation and kinetic studies.

    Science.gov (United States)

    Ritonja, A; Turk, V; Gubensek, F

    1983-06-15

    Three protein inhibitors of serine proteinases were isolated from the crude venom of the long-nosed viper Vipera ammodytes ammodytes by ion-exchange and gel chromatography. Two of them strongly inhibit trypsin (Ki = 3.4 X 10(-10) and 5.6 X 10(-10) M), while the third one primarily inhibits chymotrypsin (Ki = 4.3 X 10(-9) M). Their Mr values are close to 7000, and pI is 9.8 in both trypsin inhibitors and 10.0 in the chymotrypsin inhibitor. The N-terminal group in the former inhibitors is blocked; arginine is the N-terminal amino acid in the latter. Besides trypsin and alpha-chymotrypsin, the trypsin inhibitors also inhibit plasmin, human plasma kallikrein and porcine pancreatic kallikrein. The chymotrypsin inhibitor inhibits trypsin and human plasma kallikrein only weakly and does not inhibit plasmin and porcine pancreatic kallikrein. According to their properties, all three inhibitors belong to the Kunitz-pancreatic trypsin inhibitor family of inhibitors.

  16. Secreted proteases. Regulation of their activity and their possible role in metastasis.

    Science.gov (United States)

    Goldberg, G I; Frisch, S M; He, C; Wilhelm, S M; Reich, R; Collier, I E

    1990-01-01

    Extracellular matrix metalloproteases are secreted by the resident cells of the tissue in a proenzyme form, and their extracellular activity is regulated at the level of gene expression, proenzyme activation, and interaction with inhibitors. To understand the molecular mechanisms that control the activity of ECM metalloproteases and their effect on the cellular phenotype, we have established cell lines in which the transcription of the protease genes is repressed. We also have undertaken a detailed study of the pathway of extracellular activation of interstitial procollagenase. Stable transfection of three human tumor cell lines--H-ras-transformed bronchial epithelial cells TBE-1, fibrosarcoma cells HT1080, and melanoma cells A2058--with the adenovirus E1A gene dramatically repressed the expression of the secreted proteases, type IV and interstitial collagenases, and urokinase-type plasminogen activator. Concomitantly, E1A-expressing cells showed reduced metastatic activity in vivo and reduced ability to traverse a reconstituted basement membrane in vitro. Monospecific anti-type IV collagenase antibody inhibited the invasive activity of parental tumor cell lines in the in vitro system, suggesting a possible causal relationship between the effect of E1A on the expression of secreted proteases and the reduced metastatic potential of the E1A-expressing transformants. We have also studied the mechanism of regulation of metalloprotease activity at the level of extracellular activation by investigating the cascade of proteolytic events that results in the activation of interstitial procollagenase. Cocultivation of the major cellular components of skin, dermal fibroblasts, and epidermal keratinocytes induces activation of interstitial procollagenase and prostromelysin in the presence of plasminogen. This activation occurs through a uPA-plasmin-dependent pathway in which plasmin catalyzes the first step in activation of both collagenase and stromelysin by amino

  17. The fibrinolytic system: A new target for treatment of depression with psychedelics.

    Science.gov (United States)

    Idell, R D; Florova, G; Komissarov, A A; Shetty, S; Girard, R B S; Idell, S

    2017-03-01

    Current understanding of the neurobiology of depression has grown over the past few years beyond the traditional monoamine theory of depression to include chronic stress, inflammation and disrupted synaptic plasticity. Tissue plasminogen activator (tPA) is a key factor that not only promotes fibrinolysis via the activation of plasminogen, but also contributes to regulation of synaptic plasticity and neurogenesis through plasmin-mediated activation of a probrain derived neurotrophic factor (BDNF) to mature BDNF. ProBDNF activation could potentially be supressed by competition with fibrin for plasmin and tPA. High affinity binding of plasmin and tPA to fibrin could result in a decrease of proBDNF activation during brain inflammation leading to fibrosis further perpetuating depressed mood. There is a paucity of data explaining the possible role of the fibrinolytic system or aberrant extravascular fibrin deposition in depression. We propose that within the brain, an imbalance between tPA and urokinase plasminogen activator (uPA) and plasminogen activator inhibitor-1 (PAI-1) and neuroserpin favors the inhibitors, resulting in changes in neurogenesis, synaptic plasticity, and neuroinflammation that result in depressive behavior. Our hypothesis is that peripheral inflammation mediates neuroinflammation, and that cytokines such as tumor necrosis factor alpha (TNF-α) can inhibit the fibrinolytic system by up- regulating PAI-1 and potentially neuroserpin. We propose that the decrement of the activity of tPA and uPA occurs with downregulation of uPA in part involving the binding and clearance from the surface of neural cells of uPA/PAI-1 complexes by the urokinase receptor uPAR. We infer that current antidepressants and ketamine mitigate depressive symptoms by restoring the balance of the fibrinolytic system with increased activity of tPA and uPA with down-regulated intracerebral expression of their inhibitors. We lastly hypothesize that psychedelic 5-ht2a receptor agonists

  18. PLASMINOGEN ACTIVATOR OF YERSINIA PESTIS

    Directory of Open Access Journals (Sweden)

    V. V. Evseeva

    2015-01-01

    Full Text Available Plague has been the cause of three pandemics and has led to the death of millions of people. Plague is a typical zoonosis caused by Yersinia pestis that circulates in populations of wild rodents inhabiting natural plague foci on all continents except for Australia. Transmission of plague is provided by flea bites. Circulation of Y. pestis in natural plague foci is supported by a numerous of pathogenicity factors. This review explores one of them, plasminogen activator Pla. This protein is one of representatives of omptins, a family of enterobacterial outer membrane proteases that are responsible for colonization of specific organs or even infection generalization as a result of successful overcoming of the host innate immunity. The review reflects the history of its discovery and studying of its genetic control, biosynthesis, isolation and purification, physicochemical properties. Highly purified preparations of plasminogen activator are deficient in enzymatic activities but renaturation in the presence of Y. pestis lipooligosaccharide restores enzymatic properties of Pla. This pathogenicity factor is absent in representatives of the most ancient phylogenetic group of the plague pathogen, bv. caucasica, while the ancestor of other groups of Y. pestis subsp. microtus obtained in result of horizontal transfer Pla isoform with characteristics similar to properties of omptins from the less virulent enterobacteria. After that in the course of microevolution the “classic” isoform of Pla with increased protease activity was selected that is typical of all highly virulent for humans strains of Y. pestis subsp. pestis. The “classic” isoform of Pla Y. pestis is functionally similar to mammalian plasminogen activators transforming plasminogen into plasmin with the help of limited proteolysis. Pla protease activating plasminogen and also degrading the main plasmin inhibitor — α2-antiplasmin and, respectively, determining Y. pestis ability to lyse

  19. Venous thrombosis after abdominal surgery. A comparison between subcutaneous heparin and antithrombotic stockings, or both

    DEFF Research Database (Denmark)

    Rasmussen, A; Hansen, P T; Lindholt, J

    1988-01-01

    In an open controlled study, 248 consecutive patients (age more than 40 yrs) admitted for major abdominal surgery were randomized to one of three prophylactic antithrombotic treatments. Eighty-five patients received subcutaneous heparin, 74 patients had graduated compression stockings to the knee...... (TED stockings), and 89 patients had both subcutaneous heparin and stockings. Treatment began on the evening before operation and continued to complete mobilization, or for not less than five days postoperatively. On the fourth or fifth postoperative day, the patients underwent a 99mTc-plasmin test...... of the lower limbs as a test for deep vein thrombosis. There were 29.7% positive tests in the stocking group, 29.4% in the group with heparin prophylaxis, and 25.8% in the combined group. Differences between treatments were not statistically significant....

  20. STAPHYLOKINASE: A BOON IN MEDICAL SCIENCES – REVIEW

    Directory of Open Access Journals (Sweden)

    KRS SAMBASIVA RAO

    2013-01-01

    Full Text Available Every year most of the deaths were leaded by cardio and cerebrovascular disorders around the globe. The use of first or second generation thrombolytics may rise several side effects like bleeding in biological system at the time of treatment. New generation thrombolytics plays an important role in the treatment of thromboembolic disorders which leads to the death. An ideal fibrin specific plasminogen activator, staphylokinase converts a precursor, plasminogen to active enzyme, plasmin to dissolve the blood clot during thrombolytic therapy and it also capable to destroy the normal components of haemostatic system which leads to life threatening consequence and also death. Out of four different thrombolytics which are available in market like alteplase, reteplase, streptokinase and tenecteplase, streptokinase was widely used drug because of its low cost. But, to overcome the hurdles like bleeding and reocculsion efforts have been made to construct the more potent staphylokinase devoid of risk. In this review, progressive development on staphylokinase has been discussed.

  1. Protein-binding RNA aptamers affect molecular interactions distantly from their binding sites

    DEFF Research Database (Denmark)

    Dupont, Daniel M; Thuesen, Cathrine K; Bøtkjær, Kenneth A;

    2015-01-01

    Nucleic acid aptamer selection is a powerful strategy for the development of regulatory agents for molecular intervention. Accordingly, aptamers have proven their diligence in the intervention with serine protease activities, which play important roles in physiology and pathophysiology. Nonetheless...... potential, both binding to the serine protease urokinase-type plasminogen activator (uPA). We determine the subsequent impact of aptamer binding on the well-established molecular interactions (plasmin, PAI-1, uPAR, and LRP-1A) controlling uPA activities. One of the aptamers (upanap-126) binds to the area...... around the C-terminal α-helix in pro-uPA, while the other aptamer (upanap-12) binds to both the β-hairpin of the growth factor domain and the kringle domain of uPA. Based on the mapping studies, combined with data from small-angle X-ray scattering analysis, we construct a model for the upanap-12:pro...

  2. Antiphospholipid antibody: laboratory, pathogenesis and clinical manifestations

    Directory of Open Access Journals (Sweden)

    T. Ziglioli

    2011-06-01

    Full Text Available Antiphospholipid antibodies (aPL represent a heterogeneous group of antibodies that recognize various antigenic targets including beta2 glycoprotein I (β2GPI, prothrombin (PT, activated protein C, tissue plasminogen activator, plasmin and annexin A2. The most commonly used tests to detect aPL are: lupus anticoagulant (LAC, a functional coagulation assay, anticardiolipin antibody (aCL and anti-β2GPI antibody (anti-β2GPI, which are enzyme-linked immunoassay (ELISA. Clinically aPL are associated with thrombosis and/or with pregnancy morbidity. Apparently aPL alone are unable to induce thrombotic manifestations, but they increase the risk of vascular events that can occur in the presence of another thrombophilic condition; on the other hand obstetrical manifestations were shown to be associated not only to thrombosis but mainly to a direct antibody effect on the trophoblast.

  3. Isoforms of agrin are widely expressed in the developing rat and may function as protease inhibitors.

    Science.gov (United States)

    Biroc, S L; Payan, D G; Fisher, J M

    1993-09-17

    The agrin family of extracellular matrix proteins may be important in the formation of the neuromuscular junction. Using in situ hybridization with a probe recognizing all agrin isoforms, we demonstrate that it is widely expressed during mammalian embryogenesis. In the developing rat, particularly high levels of expression are found in the dorsal root and cranial ganglia, gut, whisker rudiments, penis, snout, teeth, retina, hippocampus, cerebral cortex and the lining of brain ventricles. Functional analysis of the recombinant rat protein shows that it is a potent inhibitor of the proteases trypsin, chymotrypsin and plasmin but not thrombin or the plasminogen activators. We conclude that agrin and its isoforms may play multiple roles in mammalian development including the regulation of proteolysis in the extracellular matrix.

  4. Excitotoxin-induced neuronal degeneration and seizure are mediated by tissue plasminogen activator.

    Science.gov (United States)

    Tsirka, S E; Gualandris, A; Amaral, D G; Strickland, S

    1995-09-28

    Neuronal degeneration in the hippocampus, a region of the brain important for acquisition of memory in humans, occurs in various pathological conditions, including Alzheimer's disease, brain ischaemia and epilepsy. When neuronal activity is stimulated in the adult rat and mouse hippocampus, tissue plasminogen activator (tPA), a serine protease that converts inactive plasminogen to the active protease plasmin, is transcriptionally induced. The activity of tPA in neural tissue is correlated with neurite outgrowth, regeneration and migration, suggesting that it might be involved in neuronal plasticity. Here we show that tPA is produced primarily by microglia in the hippocampus. Using excitotoxins to induce neuronal cell loss, we demonstrate that tPA-deficient mice are resistant to neuronal degeneration. These mice are also less susceptible to pharmacologically induced seizures than wild-type mice. These findings identify a role for tPA in neuronal degeneration and seizure.

  5. [Analysis of SNPs and enzymatic disorder in the patients of influenza-associated encephalopathy: disorder of fatty acid metabolism in mitochondria induced by high fever].

    Science.gov (United States)

    Kido, Hiroshi; Yao, Dengbing; Le Trong, Quang; Tsukane, Mariko; Chida, Junji

    2006-10-01

    To assess the etiology of influenza-associated encephalopathy(IAE), a surveillance effort was conducted during 2000-2005 in Japan. Over half of fatal and handicapped IAE patients exhibited a disorder of mitochondrial beta-oxidation and ATP generation evoked by the thermolabile phenotype of carnitine palmitoyltransferase II variations with transiently elevated serum acylcarnitine during high-grade fever. Model mice having impaired mitochondrial beta-oxidation exhibited significant accumulation of mini-plasmin and up-regulation of trypsin in the cerebral capillaries after infection with influenza A virus, resulting in the destruction of blood-brain barrier and increased brain vascular permeability. Trypsin up-regulation was also evident in the neuronal cells in the hippocampus, suggesting a severe neurologic complication of IAE.

  6. Extracellular proteolysis in the adult murine brain.

    Science.gov (United States)

    Sappino, A P; Madani, R; Huarte, J; Belin, D; Kiss, J Z; Wohlwend, A; Vassalli, J D

    1993-08-01

    Plasminogen activators are important mediators of extracellular metabolism. In the nervous system, plasminogen activators are thought to be involved in the remodeling events required for cell migration during development and regeneration. We have now explored the expression of the plasminogen activator/plasmin system in the adult murine central nervous system. Tissue-type plasminogen activator is synthesized by neurons of most brain regions, while prominent tissue-type plasminogen activator-catalyzed proteolysis is restricted to discrete areas, in particular within the hippocampus and hypothalamus. Our observations indicate that tissue-type plasminogen activator-catalyzed proteolysis in neural tissues is not limited to ontogeny, but may also contribute to adult central nervous system physiology, for instance by influencing neuronal plasticity and synaptic reorganization. The identification of an extracellular proteolytic system active in the adult central nervous system may also help gain insights into the pathogeny of neurodegenerative disorders associated with extracellular protein deposition.

  7. Indigenous enzymes and leukocyte in sheep milk are markers of health status and physiology of the mammary gland

    Directory of Open Access Journals (Sweden)

    Agostino Sevi

    2010-01-01

    Full Text Available Plasmin, plasminogen and plasminogen activator in ewe bulk milk were not significantly affected by stage of lactation probably as a consequence of the good health of the ewe udders throughout lactation as indicated by SCC which never exceeded 600,000 cells/mL. Elastase content increased significantly during lactation whereas cathepsin showed the highest content in mid lactation. Changes in macrophages and neutrophyls levels in ewe bulk milk during lactation were also investigated. Macrophages minimally contributed to leukocyte cell count in milk and had the highest levels at the beginning of lactation. An opposite trend was recorded for polymorphonuclear leukocytes (PMNL that increased throughout lactation showing the highest value in late lactation. The increase of PMNL percentage and elastase content in milk, in spite of relatively low SCC, suggests that PMNL and elastase underwent a physiological increase associated to the remodelling of mammary gland in late lactation.

  8. Osteopontin is highly susceptible to cleavage in bovine milk and the proteolytic fragments bind the αVβ3-integrin receptor

    DEFF Research Database (Denmark)

    Christensen, Brian Søndergaard; Sørensen, Esben Skipper

    2014-01-01

    Site-specific and partial proteolysis of milk proteins can both alter and increase their biological activity. The milk protein osteopontin (OPN) is a highly phosphorylated integrin-binding molecule present in most tissues and body fluids. Osteopontin is a biological substrate for matrix...... a substantial effect on the functionality of OPN. Bovine milk OPN (bOPN) exists in both intact full-length and cleaved forms, and in this study, 6 N-terminal bOPN fragments originating from proteolytic cleavage were purified and characterized by mass spectrometry. These fragments were generated by cleavage...... at the Lys145-Ser146, Arg147-Ser148, Lys149-Lys150, Phe151-Arg152, Arg152-Arg153, and Arg153-Ser154 peptide bonds. The principal protease in milk, plasmin, appeared to cleave 3 of these sites. However, the major cleavage site was observed to be at the Phe151-Arg152 bond, which does not match the specificity...

  9. Aberrant glomerular filtration of urokinase-type plasminogen activator in nephrotic syndrome leads to amiloride-sensitive plasminogen activation in urine

    DEFF Research Database (Denmark)

    Staehr, Mette; Buhl, Kristian B; Andersen, René F

    2015-01-01

    (uPA) in vitro. It was hypothesized that uPA is abnormally filtered to pre-urine and is inhibited in urine by amiloride in nephrotic syndrome. This was tested by determination of Na+-balance, uPA protein and activity and amiloride concentration in urine from rats with puromycin aminonucleoside (PAN......In nephrotic syndrome, aberrant glomerular filtration of plasminogen and conversion to active plasmin in pre-urine is thought to activate proteolytically ENaC and contribute to sodium retention and edema. The ENaC blocker amiloride is an off-target inhibitor of urokinase-type plasminogen activator......) induced nephrotic syndrome. Urine samples from 6 adult and 18 pediatric patients with nephrotic syndrome were analyzed for uPA activity and protein. PAN-treatment induced significant proteinuria in rats which coincided with increased urine uPA protein and activity, increased urine protease activity...

  10. Fibrin powder turbidity measurement for rapid assessment of antiplasmins.

    Science.gov (United States)

    Exner, T; Rickard, K A; Kronenberg, H

    1975-11-01

    Fibrinolytic activity was determined from the rate of disappearance of turbidity in a suspension of heat-treated fibrin powder. Using this method for estimating residual fibrinolytic activity in mixtures of serum and plasmin, antiplasmin behaviors of specimens from patients with various clinical disorders were determined after long and short preincubation times. Slow-acting antiplasmins were found to be increased in a variety of conditions among these patients, while immediate acting antiplasmins were generally decreased, compared with those in specimens from a large pool of normal, healthy vounteers. Normal women taking oral contraceptives had consitently high levels of slow antiplasmins. Tests in vitro showed that the antifibrinolytic agents epsilon-aminocaproic acid, Trasylol and soybean trypsin inhibitor act only as fast antiplasmins.

  11. Amyloid-degrading ability of nattokinase from Bacillus subtilis natto.

    Science.gov (United States)

    Hsu, Ruei-Lin; Lee, Kung-Ta; Wang, Jung-Hao; Lee, Lily Y-L; Chen, Rita P-Y

    2009-01-28

    More than 20 unrelated proteins can form amyloid fibrils in vivo which are related to various diseases, such as Alzheimer's disease, prion disease, and systematic amyloidosis. Amyloid fibrils are an ordered protein aggregate with a lamellar cross-beta structure. Enhancing amyloid clearance is one of the targets of the therapy of these amyloid-related diseases. Although there is debate on whether the toxicity is due to amyloids or their precursors, research on the degradation of amyloids may help prevent or alleviate these diseases. In this study, we explored the amyloid-degrading ability of nattokinase, a fibrinolytic subtilisin-like serine protease, and determined the optimal conditions for amyloid hydrolysis. This ability is shared by proteinase K and subtilisin Carlsberg, but not by trypsin or plasmin.

  12. Inhibition of coagulation factors by recombinant barley serpin BSZx

    DEFF Research Database (Denmark)

    Dahl, Søren Weis; Rasmussen, S.K.; Petersen, L..C.;

    1996-01-01

    Barley serpin BSZx is a potent inhibitor of trypsin and chymotrypsin at overlapping reactive sites (Dahl, S.W., Rasmussen, S.K. and Hejgaard, J. (1996) J. Biol, Chem., in press), We have now investigated the interactions of BSZx with a range of serine proteinases from human plasma, pancreas...... as substrate, Complexes of these proteinase with BSZx resisted boiling in SDS, and amino acid sequencing showed that cleavage in the reactive center loop only occurred after P-1 Arg. Activated protein C and leukocyte elastase were slowly inhibited by BSZx (k(ass) = 1-2 x 10(2) M(-1) s(-1)) whereas factor XIIa......, urokinase and tissue type plasminogen activator, plasmin and pancreas kallikrein and elastase were not or only weakly affected, The inhibition pattern with mammalian proteinases reveal a specificity of BSZx similar to that of antithrombin III. Trypsin from Fusarium was not inhibited while interaction...

  13. Plasminogen activation independent of uPA and tPA maintains wound healing in gene-deficient mice

    DEFF Research Database (Denmark)

    Lund, Leif R; Green, Kirsty A; Stoop, Allart A

    2006-01-01

    Simultaneous ablation of the two known activators of plasminogen (Plg), urokinase-type (uPA) and the tissue-type (tPA), results in a substantial delay in skin wound healing. However, wound closure and epidermal re-epithelialization are significantly less impaired in uPA;tPA double-deficient mice...... than in Plg-deficient mice. Skin wounds in uPA;tPA-deficient mice treated with the broad-spectrum matrix metalloproteinase (MMP) inhibitor galardin (N-[(2R)-2-(hydroxamido-carbonylmethyl)-4-methylpentanoyl]-L-tryptophan methylamide) eventually heal, whereas skin wounds in galardin-treated Plg......-deficient mice do not heal. Furthermore, plasmin is biochemically detectable in wound extracts from uPA;tPA double-deficient mice. In vivo administration of a plasma kallikrein (pKal)-selective form of the serine protease inhibitor ecotin exacerbates the healing impairment of uPA;tPA double-deficient wounds...

  14. Inhibitory Monoclonal Antibodies against Mouse Proteases Raised in Gene-Deficient Mice Block Proteolytic Functions in vivo

    DEFF Research Database (Denmark)

    Lund, Ida K; Rasch, Morten G; Ingvarsen, Signe

    2012-01-01

    internalization receptor uPARAP, have been developed. The inhibitory mAbs against uPA and uPAR block plasminogen activation and thereby hepatic fibrinolysis in vivo. Wound healing, another plasmin-dependent process, is delayed by an inhibitory mAb against uPA in the adult mouse. Thromboembolism can be inhibited......Identification of targets for cancer therapy requires the understanding of the in vivo roles of proteins, which can be derived from studies using gene-targeted mice. An alternative strategy is the administration of inhibitory monoclonal antibodies (mAbs), causing acute disruption of the target...... by anti-PAI-1 mAbs in vivo. In conclusion, function-blocking mAbs are well-suited for targeted therapy in mouse models of different diseases, including cancer....

  15. Usefulness of human coagulation and fibrinolysis assays in domestic pigs

    DEFF Research Database (Denmark)

    Münster, Anna-Marie Bloch; Olsen, Aage Kristian; Bladbjerg, Else-Marie

    2002-01-01

    Pigs are often used as animal models in research on blood coagulation and fibrinolysis. The usefulness of the assays applied within this field, and the knowledge of reference intervals are therefore essential and of utmost importance. In the study reported here, we investigated the applicability...... time, tissue factor, tissue factor pathway inhibitor, factor VII, protein C, protein S, prothrombin fragment 1+2, antithrombin, thrombin-antithrombin complexes, fibrinogen, soluble fibrin, urokinase-type plasminogen activator, plasmin inhibitor, plasminogen activator inhibitor 1, and D-dimer. We found...... that 11 of 12 functional assays, but only 3 of 10 immunoassays, were applicable to porcine plasma, and we determined the normal range of these variables. We conclude that human functional assays are useful in porcine plasma, whereas only a few immunologic assays can be used. However, precautions must...

  16. Arrhenius temperature dependence of in vitro tissue plasminogen activator thrombolysis

    Science.gov (United States)

    Shaw, George J.; Dhamija, Ashima; Bavani, Nazli; Wagner, Kenneth R.; Holland, Christy K.

    2007-06-01

    Stroke is a devastating disease and a leading cause of death and disability. Currently, the only FDA approved therapy for acute ischemic stroke is the intravenous administration of the thrombolytic medication, recombinant tissue plasminogen activator (tPA). However, this treatment has many contraindications and can have dangerous side effects such as intra-cerebral hemorrhage. These treatment limitations have led to much interest in potential adjunctive therapies, such as therapeutic hypothermia (T model. We find that the temperature dependence is well described by an Arrhenius temperature dependence with an effective activation energy Eeff of 42.0 ± 0.9 kJ mole-1. Eeff approximates the activation energy of the plasminogen-to-plasmin reaction of 48.9 kJ mole-1. A model to explain this temperature dependence is proposed. These results will be useful in predicting the effects of temperature in future lytic therapies.

  17. Blood Peptidome-Degradome Profile of Breast Cancer

    Energy Technology Data Exchange (ETDEWEB)

    Shen, Yufeng; Tolic, Nikola; Liu, Tao; Zhao, Rui; Petritis, Brianne O; Gritsenko, Marina A; Camp, David G; Moore, Ronald J; Purvine, Samuel O; Esteva, FJ; Smith, Richard D

    2010-10-18

    We report on the degradomic-peptidomic insights into blood plasma of early-stage breast cancer patients. The plasmin2-antiplasmin and thrombin-antithrombin systems are observed to be changed in early stage breast cancer through the selective degradation of their functional domains. The extracellular matrix (ECM) protection protein complexes are destroyed, but the ECM itself is not massively degraded. Key innate immune system components and cancer inhibitor and suppressor substrates are differentially degraded. The degradomic-peptidomic analysis provides information on the selectivity and extent of substrate and functional domain degradation and the specificity of substrate cleavages, potentially enabling discovery of diagnostic and therapeutic targets for early-stage breast cancer.

  18. Molecular processes in the streptokinase thrombolytic therapy.

    Science.gov (United States)

    Capitanescu, Cristian; Macovei Oprescu, Anca Monica; Ionita, Dan; Dinca, Gabi Valeriu; Turculet, Claudiu; Manole, Gheorghe; Macovei, Radu Alexandru

    2016-12-01

    The aim of this research is to evaluate the current streptokinase thrombolytic treatment and to identify or improve new techniques that will base new approaches with a higher efficiency in this area of expertise. In order to be as realistic as possible a new method was set up using magnetic vectorized nanoparticles streptokinase and human blood thrombus. The experimental data confirm the maximum 83% thrombus lyses whenever increase streptokinase concentration. It is very probable to happen because of the presence of high concentration of antiplasmin in the blood that neutralizes around half of the thrombolytic potential of the sanguine plasminogen. The experiment shows also that only free serum plasminogen are available for streptokinase action in order to generate plasmin.

  19. The tumor microenvironment: a potential arbitrator of the tumor suppressive and promoting actions of TGFbeta.

    Science.gov (United States)

    Dumont, Nancy; Arteaga, Carlos L

    2002-12-01

    Transforming growth factor beta (TGFbeta) members are secreted in biologically inactive complexes that must be activated in order to enable binding to their cell surface receptors. Interestingly, many of the proteins that can activate TGFbeta have been implicated in either suppressing or promoting tumorigenesis. Included among these are matrix proteins (thrombospondin-1), receptors (integrins alphanubeta6 and alphanubeta8) and proteases (matrix metalloproteases and plasmin). These proteins cannot only activate TGFbeta, but can also modulate cell responsiveness to TGFbeta. In this section, we review data highlighting the complexity and bidirectionality of TGFbeta matrix interactions within the tumor microenvironment, and propose that these dynamic interactions are a critical spatial and temporal determinant of the effects of TGFbeta on tumorigenesis.

  20. The role of streptokinase as a virulence determinant of Streptococcus pyogenes--potential for therapeutic targeting.

    Science.gov (United States)

    McArthur, Jason D; Cook, Simon M; Venturini, Carola; Walker, Mark J

    2012-03-01

    Streptococcus pyogenes is a major human pathogen responsible for numerous diseases ranging from uncomplicated skin and throat infections to severe, life threatening invasive disease such as necrotising fasciitis and streptococcal toxic shock syndrome. These severe invasive infections progress rapidly and produce high rates of morbidity and mortality despite the implementation of aggressive treatment plans. The activation of plasminogen and the acquisition of plasmin activity at the bacterial cell surface is critical for the invasive pathogenesis of this organism. To facilitate this process, S. pyogenes secrete streptokinase, a potent plasminogen activating protein. Here, we describe the role of streptokinase in invasive pathogenesis and discuss some potentially useful strategies for disruption of streptokinase mediated plasminogen activation which could be employed to treat severe invasive S. pyogenes infections.

  1. Prevention of thromboembolism following elective hip surgery. The value of regional anesthesia and graded compression stockings

    DEFF Research Database (Denmark)

    Wille-Jørgensen, P; Christensen, S W; Bjerg-Nielsen, A

    1989-01-01

    Ninety-eight patients scheduled for elective hip arthroplasty receiving either general or regional anesthesia and graded compression stockings as the only thromboprophylactic treatment were screened for postoperative deep-venous thrombosis with 99mTc-plasmin scintimetry. The diagnosis of deep......-venous thrombosis was established by phlebography and the diagnosis of pulmonary embolism by pulmonary perfusion and ventilation scintigraphy. Of 65 patients surgically treated under general anesthesia, 20 (31%) developed deep-venous thrombosis and six developed pulmonary embolism. Of 33 patients surgically treated...... using regional anesthesia, three (9%) developed deep-venous thrombosis and one developed a pulmonary embolus. The number of patients developing deep-venous thrombosis was significantly lower in the group receiving regional anesthesia compared with the group receiving general anesthesia. The results...

  2. Plasminogen activation and cancer

    DEFF Research Database (Denmark)

    Danø, Keld; Behrendt, N.; Hoyer-Hansen, G.

    2005-01-01

    Breakdown of the extracellular matrix is crucial for cancer invasion and metastasis. It is accomplished by the concerted action of several proteases, including the serine protease plasmin and a number of matrix metalloproteases.The activity of each of these proteases is regulated by an array......, the regulation of extracellular proteolysis in cancer involves a complex interplay between cancer cells and non-malignant stromal cells in the expression of the molecular components involved. For some types of cancer, this cellular interplay mimics that observed in the tissue of ori- gin during non......-neoplastic tissue remodelling processes.We propose that cancer invasion can be considered as uncontrolled tissue remodelling. Inhibition of extracellular proteases is an attractive approach to cancer therapy. Because proteases have many different functions in the normal organism, efficient inhibition will have...

  3. Physiological regulation of epithelial sodium channel by proteolysis

    DEFF Research Database (Denmark)

    Svenningsen, Per; Friis, Ulla G; Bistrup, Claus

    2011-01-01

    PURPOSE OF REVIEW: Activation of epithelial sodium channel (ENaC) by proteolysis appears to be relevant for day-to-day physiological regulation of channel activity in kidney and other epithelial tissues. Pathophysiogical, proteolytic activation of ENaC in kidney has been demonstrated in proteinuric...... disease. RECENT FINDINGS: A variation in sodium and potassium intake or plasma aldosterone changes the number of cleaved α and γ-ENaC subunits and is associated with changes in ENaC currents. The protease furin mediates intracellular cleavage, whereas the channel-activating protease prostasin (CAP-1...... opens the way for new understanding of the pathogenesis of proteinuric sodium retention, which may involve plasmin and present several potential new drug targets....

  4. INTERACTION OF STREPTOCOCCAL PLASMINOGEN BINDING PROTEINS WITH THE HOST FIBRINOLYTIC SYSTEM

    Directory of Open Access Journals (Sweden)

    Marcus eFulde

    2013-11-01

    Full Text Available The ability to take advantage of plasminogen and its activated form plasmin is a common mechanism used by commensal as well as pathogenic bacteria in interaction with their respective host. Hence, a huge variety of plasminogen binding proteins and/or activation mechanisms exist. This review solely focuses on the genus Streptococcus and, in particular, on the so-called non-activating plasminogen binding proteins. Based on structural and functional differences, as well as on their mode of surface linkaging, three groups can be assigned: M-(like proteins, surface displayed cytoplasmatic proteins with enzymatic activities ("moonlighting proteins" and other surface proteins. Here, the plasminogen binding sites and the interaction mechanisms are compared. Recent findings on the functional consequences of these interactions on tissue degradation and immune evasion are summarized.

  5. Off-target effects of thrombolytic drugs: apolipoprotein A-I proteolysis by alteplase and tenecteplase.

    Science.gov (United States)

    Gomaraschi, Monica; Ossoli, Alice; Vitali, Cecilia; Pozzi, Silvia; Vitali Serdoz, Laura; Pitzorno, Cristina; Sinagra, Gianfranco; Franceschini, Guido; Calabresi, Laura

    2013-02-15

    The administration of thrombolytic drugs is of proven benefit in a variety of clinical conditions requiring acute revascularization, including acute myocardial infarction (AMI), ischemic stroke, pulmonary embolism, and venous thrombosis. Generated plasmin can degrade non-target proteins, including apolipoprotein A-I (apoA-I), the major protein constituent of high-density lipoproteins (HDL). Aim of the present study was to compare the extent of apoA-I proteolytic degradation in AMI patients treated with two thrombolytic drugs, alteplase and the genetically engineered t-PA variant tenecteplase. ApoA-I degradation was evaluated in sera from 38 AMI patients treated with alteplase or tenecteplase. In vitro, apoA-I degradation was tested by incubating control sera or purified HDL with alteplase or tenecteplase at different concentrations (5-100 μg/ml). Treatment with alteplase and tenecteplase results in apoA-I proteolysis; the extent of apoA-I degradation was more pronounced in alteplase-treated patients than in tenecteplase-treated patients. In vitro, the extent of apoA-I proteolysis was higher in alteplase-treated sera than in tenecteplase-treated sera, in the whole drug concentration range. No direct effect of the two thrombolytic agents on apoA-I degradation was observed. In addition to apoA-I, apoA-IV was also degraded by the two thrombolytic agents and again proteolytic degradation was higher with alteplase than tenecteplase. In conclusion, this study indicates that both alteplase and tenecteplase cause plasmin-mediated proteolysis of apoA-I, with alteplase resulting in a greater apoA-I degradation than tenecteplase, potentially causing a transient impairment of HDL atheroprotective functions.

  6. Circulating thrombomodulin and hematological alterations in type 2 diabetic patients with retinopathy.

    Science.gov (United States)

    Fujiwara, Y; Tagami, S; Kawakami, Y

    1998-01-01

    To clarify the relationship between circulating thrombomodulin (TM) and endothelial cell damage in diabetes mellitus, plasma levels of TM were quantitated by an enzyme linked immunoabsorbant assay (ELISA) in 164 type 2 diabetes mellitus and 72 normal control subjects, and these levels were compared with those of von Willebrand factor antigen (vWf: Ag), thrombin antithrombin III complexes (TAT), plasmin-alpha2-plasmin inhibitor complexes (PIC), fibrinogen, D-dimer, urinary albumin excretion rate (AER), intima-media thickness (IMT) and plaque score of the common carotid artery assessed with high resolution B-mode ultrasonography. Plasma levels of TM, vWf: Ag, TAT, PIC, AER, IMT and plaque score were significantly increased in the diabetic patients compared to the normal control subjects. Plasma TM levels showed significant correlation with vWf: Ag (r=0.350, pPIC (r = 0.450, p sex, systolic and diastolic blood pressure levels, HbA,1c, or plasma lipid levels, although the plasma levels of TM, vWf: Ag, TAT, PIC, AER, IMT and the plaque score in the patients with proliferative retinopathy were significantly higher than those of the healthy controls and patients with simple retinopathy. Among the 43 normoalbuminuric patients without intima-media thickness or thickened plaque (AERPIC were significantly higher in those patients with retinopathy than in those without retinopathy. Multivariate analysis showed TM, TAT and PIC levels to be independent predictors of diabetic retinopathy. In conclusion, circulating TM reflects endothelial cell damage in patients with diabetic retinopathy, and hypercoagulability might play an important role in endothelial cell damage.

  7. Staphylococcus aureus Manganese Transport Protein C (MntC) Is an Extracellular Matrix- and Plasminogen-Binding Protein

    Science.gov (United States)

    Salazar, Natália; Castiblanco-Valencia, Mónica Marcela; da Silva, Ludmila Bezerra; de Castro, Íris Arantes; Monaris, Denize; Masuda, Hana Paula; Barbosa, Angela Silva; Arêas, Ana Paula Mattos

    2014-01-01

    Infections caused by Staphylococcus aureus – particularly nosocomial infections - represent a great concern. Usually, the early stage of pathogenesis consists on asymptomatic nasopharynx colonization, which could result in dissemination to other mucosal niches or invasion of sterile sites, such as blood. This pathogenic route depends on scavenging of nutrients as well as binding to and disrupting extracellular matrix (ECM). Manganese transport protein C (MntC), a conserved manganese-binding protein, takes part in this infectious scenario as an ion-scavenging factor and surprisingly as an ECM and coagulation cascade binding protein, as revealed in this work. This study showed a marked ability of MntC to bind to several ECM and coagulation cascade components, including laminin, collagen type IV, cellular and plasma fibronectin, plasminogen and fibrinogen by ELISA. The MntC binding to plasminogen appears to be related to the presence of surface-exposed lysines, since previous incubation with an analogue of lysine residue, ε-aminocaproic acid, or increasing ionic strength affected the interaction between MntC and plasminogen. MntC-bound plasminogen was converted to active plasmin in the presence of urokinase plasminogen activator (uPA). The newly released plasmin, in turn, acted in the cleavage of the α and β chains of fibrinogen. In conclusion, we describe a novel function for MntC that may help staphylococcal mucosal colonization and establishment of invasive disease, through the interaction with ECM and coagulation cascade host proteins. These data suggest that this potential virulence factor could be an adequate candidate to compose an anti-staphylococcal human vaccine formulation. PMID:25409527

  8. An immunohistochemical study of the distribution of plasminogen and plasminogen activators in bullous pemphigoid.

    Science.gov (United States)

    Venning, V A; Wojnarowska, F; Cederholm-Williams, S

    1993-03-01

    Abnormalities of the cutaneous plasminogen/plasminogen activator system have been associated with acantholytic disorders, psoriasis, keratinocytes in culture, and epidermis in healing wounds. The present study was undertaken to investigate the possible role of the plasmin/plasminogen protease system in lesion development in bullous pemphigoid (BP). Using polyclonal antibodies and a fluorescent technique, the immunohistochemical distribution of plasmin/plasminogen, fibrinogen and the plasminogen activators, urokinase (uPA) and tissue plasminogen activator (tPA), were studied in lesional and non-lesional skin from nine BP patients, one with linear IgA disease (LAD) and one with pemphigoid gestationis (PG). The distribution of the proteases was compared with that in normal skin (n = 4) and in suction blisters (n = 2). In normal skin, fibrinogen, tPA and uPA were absent from the epidermis and plasminogen was confined to the basal layer. Uninvolved BP skin was identical to controls. Focal areas of suprabasal plasminogen expression in the region of a blister was seen in 3/9 BP lesions and in 1/2 suction blisters. In 6/9 BP lesions and both uninvolved and lesional LAD and PG skin were identical to controls, and no suprabasal expression of plasminogen was present. These findings suggest that suprabasal plasminogen expression is unlikely to play a fundamental role in the pathogenesis of blister formation in BP as enhanced expression was not present in every case and the finding was not specific to BP, also occurring in a suction blister. Enhanced plasminogen expression rather may be a reflection of the processes of tissue repair.

  9. Proteolytic-antiproteolytic balance and its regulation in carcinogenesis

    Institute of Scientific and Technical Information of China (English)

    Elzbieta Skrzydlewska; Mariola Sulkowska; Mariusz Koda; Stanislaw Sulkowski

    2005-01-01

    Cancer development is essentially a tissue remodeling process in which normal tissue is substituted with cancer tissue. A crucial role in this process is attributed toproteolytic degradation of the extracellular matrix (ECM).Degradation of ECM is initiated by proteases, secreted by different cell types, participating in tumor cell invasion and increased expression or activity of every known class of proteases (metallo-, serine-, aspartyl-, and cysteine)has been linked to malignancy and invasion of tumor cells.Proteolytic enzymes can act directly by degrading ECM or indirectly by activating other proteases, which then degrade the ECM. They act in a determined order, resulting from the order of their activation. When proteases exert their action on other proteases, the end result is a cascade leading to proteolysis. Presumable order of events in this complicated cascade is that aspartyl protease (cathepsin D) activates cysteine proteases (e.g. cathepsin B) that can activate pro-uPA. Then active uPA can convert plasminogen into plasmin. Cathepsin B as well as plasmin are capable of degrading several components of tumor stroma and may activate zymogens of matrix metalloproteinases, the main family of ECM degrading proteases. The activities of these proteases are regulated by a complex array of activators,inhibitors and cellular receptors. Tn physiological conditions the balance exists between proteases and their inhibitors.Proteolytic-antiproteolytic balance may be of major significance in the cancer development. One of the reasons for such a situation is enhanced generation of free radicals observed in many pathological states. Free radicals react with main cellular components like proteins and lipids and in this way modify proteolytic-antiproteolytic balance and enable penetration damaging cellular membrane. All these lead to enhancement of proteolysis and destruction of ECM proteins and in consequence to invasion and metastasis.

  10. Quantitative assessment of fibrinogen cross-linking by epsilon aminocaproic acid in patients with end-stage liver disease.

    Science.gov (United States)

    Quach, Thien; Tippens, Melissa; Szlam, Fania; Van Dyke, Rebecca; Levy, Jerrold H; Csete, Marie

    2004-01-01

    Analysis of the effectiveness of antifibrinolytic therapy for liver transplant recipients is hampered by lack of quantitative assays for assessing drug effects. We adapted chemical engineering tools used in polymerization studies to quantify fibrinogen cross-linking by plasma from liver transplant patients obtained before and after epsilon aminocaproic acid (EACA) therapy. A target fluorescein isothiocyanate-fibrinogen (FITC-fibrinogen) molecule was constructed; it fluoresces in a quantifiable pattern when in solution, and undergoes cross-linking in the presence of plasmin inhibitors. Cross-linking quenches the fluorescent signal, and the quenching is a quantifiable endpoint. Thus fluorescence from this reporter molecule can be used to assess functional improvement in fibrinogen cross-linking as a result of antifibrinolytic therapies, and it is sensitive to picomolar amounts of plasmin inhibitors and activators. Cross-linking of FITC-fibrinogen by patient plasma, before and after EACA therapy, was assessed using fluorescence spectrometry. Fluorescence patterns from FITC-fibrinogen indicated no significant cross-linking of the target fibrinogen as a consequence of EACA in posttreatment plasma. When the fibrinogen-FITC target was assayed without plasma in the presence of EACA at concentrations that bracket therapeutic levels (100 and 400 microg/ml), significant fluorescence quenching (target FITC-fibrinogen cross-linking) was achieved. These results suggest that fibrinogen-FITC fluorescence is sensitive enough to detect EACA activity in clinically relevant ranges, but that EACA given in usual doses is insufficient to promote fibrinogen cross-linking in patients with end-stage liver disease.

  11. PAI-1 mRNA expression and plasma level in rheumatoid arthritis: relationship with 4G/5G PAI-1 polymorphism.

    Science.gov (United States)

    Muñoz-Valle, José Francisco; Ruiz-Quezada, Sandra Luz; Oregón-Romero, Edith; Navarro-Hernández, Rosa Elena; Castañeda-Saucedo, Eduardo; De la Cruz-Mosso, Ulises; Illades-Aguiar, Berenice; Leyva-Vázquez, Marco Antonio; Castro-Alarcón, Natividad; Parra-Rojas, Isela

    2012-12-01

    Rheumatoid arthritis (RA) is a chronic inflammatory disease affecting the synovial membrane, cartilage and bone. PAI-1 is a key regulator of the fibrinolytic system through which plasminogen is converted to plasmin. The plasmin activates the matrix metalloproteinase system, which is closely related with the joint damage and bone destruction in RA. The aim of this study was to investigate the relationship between 4G/5G PAI-1 polymorphism with mRNA expression and PAI-1 plasma protein levels in RA patients. 113 RA patients and 123 healthy subjects (HS) were included in the study. The 4G/5G PAI-1 polymorphism was determined by polymerase chain reaction-restriction fragment length polymorphism method; the PAI-1 mRNA expression was determined by real-time PCR; and the soluble PAI-1 (sPAI-1) levels were quantified using an ELISA kit. No significant differences in the genotype and allele frequencies of 4G/5G PAI-1 polymorphism were found between RA patients and HS. However, the 5G/5G genotype was the most frequent in both studied groups: RA (42%) and HS (44%). PAI-1 mRNA expression was slightly increased (0.67 fold) in RA patients with respect to HS (P = 0.0001). In addition, in RA patients, the 4G/4G genotype carriers showed increased PAI-1 mRNA expression (3.82 fold) versus 4G/5G and 5G/5G genotypes (P = 0.0001), whereas the sPAI-1 plasma levels did not show significant differences. Our results indicate that the 4G/5G PAI-1 polymorphism is not a marker of susceptibility in the Western Mexico. However, the 4G/4G genotype is associated with high PAI-1 mRNA expression but not with the sPAI-1 levels in RA patients.

  12. Clinical implications of the involvement of tPA in neuronal cell death.

    Science.gov (United States)

    Tsirka, S E

    1997-05-01

    Tissue plasminogen activator (tPA), the serine protease that converts inactive plasminogen to the protease plasmin, was recently shown to mediate neurodegeneration in the mouse hippocampus. Mice deficient in tissue plasminogen activator (tPA) display a dramatic resistance to a paradigm of excitotoxic neuronal death that involves intrahippocampal injection of the excitotoxin. This model is thought to reproduce the mechanism of neuronal death observed during acute (such as ischemic stroke) and degenerative (such as amyotrophic lateral sclerosis) diseases of the nervous system. The requirement for the proteolytic activity of tPA to mediate neuronal death is acute in the adult mouse. Serine protease inhibitors, specific for tPA or the tPA/plasmin proteolytic cascade, are effective in conferring extensive neuroprotection following the excitotoxic injection. These findings suggest possible new ways for interfering with the neuronal death observed in the hippocampus as a result of excitotoxicity. In addition, tPA is produced in the hippocampus primarily by microglial cells, which become activated in response to the neuronal injury. Blocking microglial activation has been shown in other injury paradigms to protect against neuronal death, therefore suggesting another way to retard neurodegeneration in the CNS. Furthermore, after the insult has been inflicted and in the presence of a compromised blood-brain barrier macrophages (cells deriving from the same lineage as microglia) migrate into the brain, where they are thought to contribute to the neuronal cell loss by secreting neurotoxic molecules. If these macrophages/microglia expressed, however, a tPA inhibitor, rather than the possibly neurotoxic tPA, they might be able to protect the neurons from dying.

  13. Urokinase-type plasminogen activator deficiency has little effect on seizure susceptibility and acquired epilepsy phenotype but reduces spontaneous exploration in mice.

    Science.gov (United States)

    Rantala, J; Kemppainen, S; Ndode-Ekane, X E; Lahtinen, L; Bolkvadze, Tamuna; Gurevicius, K; Tanila, H; Pitkänen, A

    2015-01-01

    Urokinase-type plasminogen activator (uPA), a serine protease, converts plasminogen to plasmin. Activation of plasmin leads to degradation of the extracellular matrix, which is critical for tissue recovery, angiogenesis, cell migration, and axonal and synaptic plasticity. We hypothesized that uPA deficiency would cause an abnormal neurophenotype and would lead to exacerbated epileptogenesis after brain injury. Wild-type (Wt) and uPA-/- mice underwent a battery of neurologic behavioral tests evaluating general reactivity, spontaneous exploratory activity, motor coordination, pain threshold, fear and anxiety, and memory. We placed particular emphasis on the effect of uPA deficiency on seizure susceptibility, including the response to convulsants (pentylenetetrazol, kainate, or pilocarpine) and kainate-induced epileptogenesis and epilepsy. The uPA-/- mice showed no motor or sensory impairment compared with the Wt mice. Hippocampus-dependent spatial memory also remained intact. The uPA-/- mice, however, exhibited reduced exploratory activity and an enhanced response to a tone stimulus (p<0.05 compared with the Wt mice). The urokinase-type plasminogen activator deficient mice showed no increase in spontaneous or evoked epileptiform electrographic activity. Rather, the response to pilocarpine administration was reduced compared with the Wt mice (p<0.05). Also, the epileptogenesis and the epilepsy phenotype after intrahippocampal kainate injection were similar to those in the Wt mice. Taken together, uPA deficiency led to diminished interest in the environmental surroundings and enhanced emotional reactivity to unexpected aversive stimuli. Urokinase-type plasminogen activator deficiency was not associated with enhanced seizure susceptibility or worsened poststatus epilepticus epilepsy phenotype.

  14. Sex hormone-binding globulin and antithrombin III activity in women with oral ultra-low-dose estradiol.

    Science.gov (United States)

    Matsui, Sumika; Yasui, Toshiyuki; Kasai, Kana; Keyama, Kaoru; Yoshida, Kanako; Kato, Takeshi; Uemura, Hirokazu; Kuwahara, Akira; Matsuzaki, Toshiya; Irahara, Minoru

    2017-03-20

    Oral oestrogen increases the risk of venous thromboembolism (VTE) and increases production of sex hormone-binding globulin (SHBG) in a dose-dependent manner. SHBG has been suggested to be involved in venous thromboembolism. We examined the effects of oral ultra-low-dose oestradiol on circulating levels of SHBG and coagulation parameters, and we compared the effects to those of transdermal oestradiol. Twenty women received oral oestradiol (500 μg) every day (oral ultra-low-dose group) and 20 women received a transdermal patch (50 μg) as a transdermal group. In addition, the women received dydrogesterone continuously (5 mg) except for women who underwent hysterectomy. Circulating SHBG, antithrombin III (ATIII) activity, d-dimer, thrombin-antithrombin complex and plasmin-α2 plasmin inhibitor complex were measured before and 3 months after the start of treatment. SHBG was significantly increased at 3 months in the oral ultra-low-dose group, but not in the transdermal group. However, percent changes in SHBG were not significantly different between the two groups. In both groups, ATIII was significantly decreased at 3 months. In conclusion, even ultra-low-dose oestradiol orally increases circulating SHBG level. However, the magnitude of change in SHBG caused by oral ultra-low-dose oestradiol is small and is comparable to that caused by transdermal oestradiol. Impact statement Oral oestrogen replacement therapy increases production of SHBG which may be related to increase in VTE risk. However, the effect of oral ultra-low-dose oestradiol on SHBG has not been clarified. Even ultra-low-dose oestradiol orally increases circulating SHBG levels, but the magnitude of change in SHBG caused by oral ultra-low-dose oestradiol is small and is comparable to that caused by transdermal oestradiol. VTE risk in women receiving oral ultra-low-dose oestradiol may be comparable to that in women receiving transdermal oestradiol.

  15. Co-operation between interleukin-1 and the fibrinolytic system in the degradation of collagen by articular chondrocytes.

    Science.gov (United States)

    Cruwys, S C; Davies, D E; Pettipher, E R

    1990-07-01

    1. The interaction between interleukin 1 (IL-1) and the fibrinolytic system in the control of collagen degradation by rabbit chondrocytes has been investigated in a tissue-culture system where cells are grown on a 14C-labelled collagen matrix. 2. Culture of rabbit chondrocytes in the presence of human recombinant IL-1 beta at a concentration of 57pM for 48 h led to the presence of procollagenase but not active collagenase in the medium. The latent collagenase could be activated by incubation with an organomercurial, aminophenylmercuric acetate (APMA). 3. Addition of IL-1 beta to chondrocytes grown on a 14C-labelled collagen matrix did not increase the degradation of the matrix compared to control over a 48 h period. However, in the presence of plasmin (200 micrograms ml-1) or plasminogen (100 micrograms ml-1), IL-1 beta (57 pM) caused almost complete degradation of the collagen matrix. Plasmin or plasminogen alone caused only slight degradation of the collagen matrix. 4. Tissue inhibitor of metalloproteinases (TIMP) or the selective metalloproteinase inhibitor, SC44463, inhibited the degradation induced by IL-1 beta and plasminogen in a concentration-related manner and at concentrations that were correlated with inhibition of collagenase. 5. When concentrations of IL-1 beta which caused only minimal degradation of the matrix in the presence of plasminogen were combined with fibrin (1 microgram ml-1), there was almost total degradation of the matrix by 48 h. 6. These results indicate there is a synergistic interaction between IL-1 and the fibrinolytic system in the degradation of collagen by rabbit chondrocytes in culture.

  16. Immunohistochemical analysis of the gingiva with periodontitis of type I plasminogen deficiency compared to gingiva with gingivitis and periodontitis and healthy gingiva.

    Science.gov (United States)

    Kurtulus Waschulewski, Idil; Gökbuget, Aslan Y; Christiansen, Nina M; Ziegler, Maike; Schuster, Volker; Wahl, Gerhard; Götz, Werner

    2016-12-01

    Type I plasminogen deficiency (Plgdef) is an uncommon chronic inflammation of mucous membranes. Gingival enlargements usually proceed with progressive periodontal destruction and tooth-loss. Plasmin(ogen)-independent enzymatic mechanisms for fibrin clearance have already been discussed in the literature. Our primary objective was to verify, immunohistochemically, the occurrence of different enzymatic factors involved in tissue breakdown of inflamed compared to healthy gingiva. Secondly, we tried to find out, if these patients have a similar microbiological profile to the patients with known gingivitis and periodontitis. Immunohistochemical analysis of enzymes elastase, plasminogen (plg), cathepsin G, matrix-metalloproteinase (MMP)-3 and MMP-7 and of glycoprotein fibrinogen were performed with gingival tissues from 3 healthy controls, 8 patients with Plgdef and 3 patients with gingivitis and periodontitis. Furthermore, plaque from 5 patients with plasminogen deficiency were also obtained to determine the microbiological profile. Significantly high numbers of elastase positive leukocytes were detected in all samples. Staining for MMP-3 and MMP-7 was seen in samples with gingivitis and periodontitis with a stronger staining in samples with periodontitis by Plgdef. Fibrinogen was detectable in all samples. Staining for plg was stronger in samples with periodontitis than in other samples. Staining for cathepsin G was weak in gingivitis and periodontitis. Subgingival microbial flora showed elevated colony forming units of Prevotella intermedia/nigrescens, Fusobacterium spp., Eikenella corrodens, Porphyromonas gingivalis and viridans streptococci. Strong staining of elastase, MMP-3 and MMP-7 and weak staining of plg in Plgdef samples supports the plasmin(ogen) - independent fibrin clearance. Similar subgingival microbiological flora was observed in periodontitis with Plgdef as in other periodontal diseases. Further investigations should determine the exact pathomechanism

  17. The heat-compression technique for the conversion of platelet-rich fibrin preparation to a barrier membrane with a reduced rate of biodegradation.

    Science.gov (United States)

    Kawase, Tomoyuki; Kamiya, Mana; Kobayashi, Mito; Tanaka, Takaaki; Okuda, Kazuhiro; Wolff, Larry F; Yoshie, Hiromasa

    2015-05-01

    Platelet-rich fibrin (PRF) was developed as an advanced form of platelet-rich plasma to eliminate xenofactors, such as bovine thrombin, and it is mainly used as a source of growth factor for tissue regeneration. Furthermore, although a minor application, PRF in a compressed membrane-like form has also been used as a substitute for commercially available barrier membranes in guided-tissue regeneration (GTR) treatment. However, the PRF membrane is resorbed within 2 weeks or less at implantation sites; therefore, it can barely maintain sufficient space for bone regeneration. In this study, we developed and optimized a heat-compression technique and tested the feasibility of the resulting PRF membrane. Freshly prepared human PRF was first compressed with dry gauze and subsequently with a hot iron. Biodegradability was microscopically examined in vitro by treatment with plasmin at 37°C or in vivo by subcutaneous implantation in nude mice. Compared with the control gauze-compressed PRF, the heat-compressed PRF appeared plasmin-resistant and remained stable for longer than 10 days in vitro. Additionally, in animal implantation studies, the heat-compressed PRF was observed at least for 3 weeks postimplantation in vivo whereas the control PRF was completely resorbed within 2 weeks. Therefore, these findings suggest that the heat-compression technique reduces the rate of biodegradation of the PRF membrane without sacrificing its biocompatibility and that the heat-compressed PRF membrane easily could be prepared at chair-side and applied as a barrier membrane in the GTR treatment.

  18. The recombinant LIC10508 is a plasma fibronectin, plasminogen, fibrinogen and C4BP-binding protein of Leptospira interrogans.

    Science.gov (United States)

    Siqueira, Gabriela H; Teixeira, Aline F; Fernandes, Luis G; de Souza, Gisele O; Kirchgatter, Karin; Romero, Eliete C; Vasconcellos, Silvio A; Vieira, Monica L; Nascimento, Ana Lucia T O

    2016-03-01

    Leptospirosis is a zoonosis caused by pathogenic Leptospira spp. In this study, we report that the recombinant proteins LIC10507, LIC10508 and LIC10509 are recognized by confirmed leptospirosis serum samples at both phases of the disease. The recombinant rLIC10508 and rLIC10507 are plasminogen (PLG)-binding proteins, capable of generating plasmin in the presence of a PLG activator. The proteins bind to PLG in a dose-dependent and saturable manner, fulfilling host-ligand interaction. Furthermore, rLIC10508 interacts with fibrinogen (Fg), plasma fibronectin and C4b binding protein (C4BP). The binding of rLIC10508 to Fg decreases the fibrin clotting in a thrombin-catalyzed reaction. The incubation with 4 μM of protein promoted 40% inhibition upon clotting formation. C4BP bound to rLIC10508 retained its cofactor activity for factor I promoting the cleavage of C4b protein, which may reduce the membrane attack complex formation. Although these proteins have high amino acid sequence similarity, rLIC10508 is the most talented of the three, a behavior that might be explained by its unique putative 3D structure, whereas structures of rLIC10507 and rLIC10509 are very similar. Plasmin generation (rLIC10507 and rLIC10508), together with decreasing fibrin clot formation (rLIC10508) and impairment of the complement system (rLIC10508) may help the bacteria to overcome host defense, facilitating the infection process.

  19. Novel aspects of blood coagulation factor XIII. I. Structure, distribution, activation, and function

    Energy Technology Data Exchange (ETDEWEB)

    Muszbek, L.; Adany, R. [Univ. Medical School of Debrecen (Hungary); Mikkola, H. [Univ. of Helsinki (Finland)

    1996-10-01

    Blood coagulation factor XIII (FXIII) is a protransglutaminase that becomes activated by the concerted action of thrombin and Ca{sup 2+} in the final stage of the clotting cascade. In addition to plasma, FXIII also occurs in platelets, monocytes, and monocyte-derived macrophages. While the plasma factor is a heterotetramer consisting of paired A and B subunits (A{sub 2}B{sub 2}), its cellular counterpart lacks the B subunits and is a homodimer of potentially active A subunits (A{sub 2}). The gene coding for the A and B subunits has been localized to chromosomes 6p24-25 and 1q31-32.1, respectively. The genomic as well as the primary protein structure of both subunits has been established. Plasma FXIII circulates in association with its substrate precursor, fibrinogen. Fibrin(ogen) has an important regulatory role in the activation of plasma FXIII, for instance the proteolytic removal of activation peptide by thrombin, the dissociation of subunits A and B, and the exposure of the originally buried active site on the free A subunits. The end result of this process is the formation of an active transglutaminase, which crosslinks peptide chains through {epsilon}({gamma}-glutamyl)lysyl isopeptide bonds. The protein substrates of activated FXIII include components of the clotting-fibrinolytic system, adhesive and contractile proteins. The main physiological function of plasma FXIII is to cross-link fibrin and protect it from the fibrinolytic enzyme plasmin. The latter effect is achieved mainly by covalently linking {alpha}{sub 2} antiplasmin, the most potent physiological inhibitor of plasmin, to fibrin. Plasma FXIII seems to be involved in wound healing and tissue repair, and it is essential to maintaining pregnancy. Cellular FXIII, if exposed to the surface of the cells, might support or perhaps take over the hemostatic functions of plasma FXIII; however, its intracellular role has remained mostly unexplored. 328 refs., 4 figs.

  20. Decreased expression of the plasminogen activator inhibitor type 1 is involved in degradation of extracellular matrix surrounding cervical cancer stem cells.

    Science.gov (United States)

    Sato, Masakazu; Kawana, Kei; Adachi, Katsuyuki; Fujimoto, Asaha; Yoshida, Mitsuyo; Nakamura, Hiroe; Nishida, Haruka; Inoue, Tomoko; Taguchi, Ayumi; Takahashi, Juri; Kojima, Satoko; Yamashita, Aki; Tomio, Kensuke; Nagamatsu, Takeshi; Wada-Hiraike, Osamu; Oda, Katsutoshi; Osuga, Yutaka; Fujii, Tomoyuki

    2016-02-01

    The plasminogen activator (PA) system consists of plasminogen activator inhibitor type 1 (PAI-1), urokinase-type plasminogen activator and its receptor (uPA and uPAR). PAI-1 inhibits the activation of uPA (which converts plasminogen to plasmin), and is involved in cancer invasion and metastasis, by remodeling the extracellular matrix (ECM) through regulating plasmin. Cancer stem cells (CSCs) are a small subset of cells within tumors, and are thought to be involved in tumor recurrence and metastasis. Considering these facts, we investigated the relationship between PAI-1 and cervical CSCs. We used ALDH1 as a marker of cervical CSCs. First, we demonstrated that culturing ALDH1-high cells and ALDH-low cells on collagen IV-coted plates increased their expression of active PAI-1 (ELISA), and these increases were suggested to be at mRNA expression levels (RT-qPCR). Secondly, we demonstrated PAI-1 was indeed involved in the ECM maintenance. With gelatin zymography assays, we found that ALDH1-high cells and ALDH-low cells expressed pro-matrix metalloproteinase-2 (pro-MMP-2) irrespective of their coatings. With gelatinase/collagenase assay kit, we confirmed that collagenase activity was increased when ALDH1-low cells were exposed to TM5275, a small molecule inhibitor of PAI-1. Putting the data together, we hypothesized that cancer cells adhered to basal membrane secrete abundant PAI-1, on the other hand, cancer cells (especially CSCs rather than non-CSCs) distant from basal membrane secrete less PAI-1, which makes the ECM surrounding CSCs more susceptible to degradation. Our study could be an explanation of conflicting reports, where some researchers found negative impacts of PAI-1 expression on clinical outcomes and others not, by considering the concept of CSCs.

  1. Paracoccidioides brasiliensis Enolase Is a Surface Protein That Binds Plasminogen and Mediates Interaction of Yeast Forms with Host Cells ▿

    Science.gov (United States)

    Nogueira, Sarah Veloso; Fonseca, Fernanda L.; Rodrigues, Marcio L.; Mundodi, Vasanth; Abi-Chacra, Erika A.; Winters, Michael S.; Alderete, John F.; Soares, Célia Maria de Almeida

    2010-01-01

    Paracoccidioidomycosis (PCM), caused by the dimorphic fungus Paracoccidioides brasiliensis, is a disseminated, systemic disorder that involves the lungs and other organs. The ability of the pathogen to interact with host components, including extracellular matrix (ECM) proteins, is essential to further colonization, invasion, and growth. Previously, enolase (EC 4.2.1.11) was characterized as a fibronectin binding protein in P. brasiliensis. Interaction of surface-bound enolase with plasminogen has been incriminated in tissue invasion for pathogenesis in several pathogens. In this paper, enolase was expressed in Escherichia coli as a recombinant glutathione S-transferase (GST) fusion protein (recombinant P. brasiliensis enolase [rPbEno]). The P. brasiliensis native enolase (PbEno) was detected at the fungus surface and cytoplasm by immunofluorescence with an anti-rPbEno antibody. Immobilized purified rPbEno bound plasminogen in a specific, concentration-dependent fashion. Both native enolase and rPbEno activated conversion of plasminogen to plasmin through tissue plasminogen activator. The association between PbEno and plasminogen was lysine dependent. In competition experiments, purified rPbEno, in its soluble form, inhibited plasminogen binding to fixed P. brasiliensis, suggesting that this interaction required surface-localized PbEno. Plasminogen-coated P. brasiliensis yeast cells were capable of degrading purified fibronectin, providing in vitro evidence for the generation of active plasmin on the fungus surface. Exposure of epithelial cells and phagocytes to enolase was associated with an increased expression of surface sites of adhesion. In fact, the association of P. brasiliensis with epithelial cells and phagocytes was increased in the presence of rPbEno. The expression of PbEno was upregulated in yeast cells derived from mouse-infected tissues. These data indicate that surface-associated PbEno may contribute to the pathogenesis of P. brasiliensis. PMID

  2. Paracoccidioides brasiliensis enolase is a surface protein that binds plasminogen and mediates interaction of yeast forms with host cells.

    Science.gov (United States)

    Nogueira, Sarah Veloso; Fonseca, Fernanda L; Rodrigues, Marcio L; Mundodi, Vasanth; Abi-Chacra, Erika A; Winters, Michael S; Alderete, John F; de Almeida Soares, Célia Maria

    2010-09-01

    Paracoccidioidomycosis (PCM), caused by the dimorphic fungus Paracoccidioides brasiliensis, is a disseminated, systemic disorder that involves the lungs and other organs. The ability of the pathogen to interact with host components, including extracellular matrix (ECM) proteins, is essential to further colonization, invasion, and growth. Previously, enolase (EC 4.2.1.11) was characterized as a fibronectin binding protein in P. brasiliensis. Interaction of surface-bound enolase with plasminogen has been incriminated in tissue invasion for pathogenesis in several pathogens. In this paper, enolase was expressed in Escherichia coli as a recombinant glutathione S-transferase (GST) fusion protein (recombinant P. brasiliensis enolase [rPbEno]). The P. brasiliensis native enolase (PbEno) was detected at the fungus surface and cytoplasm by immunofluorescence with an anti-rPbEno antibody. Immobilized purified rPbEno bound plasminogen in a specific, concentration-dependent fashion. Both native enolase and rPbEno activated conversion of plasminogen to plasmin through tissue plasminogen activator. The association between PbEno and plasminogen was lysine dependent. In competition experiments, purified rPbEno, in its soluble form, inhibited plasminogen binding to fixed P. brasiliensis, suggesting that this interaction required surface-localized PbEno. Plasminogen-coated P. brasiliensis yeast cells were capable of degrading purified fibronectin, providing in vitro evidence for the generation of active plasmin on the fungus surface. Exposure of epithelial cells and phagocytes to enolase was associated with an increased expression of surface sites of adhesion. In fact, the association of P. brasiliensis with epithelial cells and phagocytes was increased in the presence of rPbEno. The expression of PbEno was upregulated in yeast cells derived from mouse-infected tissues. These data indicate that surface-associated PbEno may contribute to the pathogenesis of P. brasiliensis.

  3. Leucaena leucocephala serine proteinase inhibitor: primary structure and action on blood coagulation, kinin release and rat paw edema.

    Science.gov (United States)

    Oliva, M L; Souza-Pinto, J C; Batista, I F; Araujo, M S; Silveira, V F; Auerswald, E A; Mentele, R; Eckerskorn, C; Sampaio, M U; Sampaio, C A

    2000-03-07

    A serine proteinase inhibitor isolated from Leucaena leucocephala seeds (LlTI) was purified to homogeneity by acetone fractionation, ion exchange chromatography, gel filtration and reverse phase chromatography (HPLC). SDS-PAGE indicated a protein with M(r) 20000 and two polypeptide chains (alpha-chain, M(r) 15000, and beta-chain, M(r) 5000), the sequence being determined by automatic Edman degradation and by mass spectroscopy. LlTI is a 174 amino acid residue protein which shows high homology to plant Kunitz inhibitors, especially those double chain proteins purified from the Mimosoideae subfamily. LlTI inhibits plasmin (K(i) 3.2 x 10(-10) M), human plasma kallikrein (K(i) 6.3 x 10(-9) M), trypsin (K(i) 2.5 x 10(-8) M) and chymotrypsin (K(i) 1.4 x 10(-8) M). Factor XIIa activity is inhibited but K(i) was not determined, and factor Xa, tissue kallikrein and thrombin are not inhibited by LlTI. The action of LlTI on enzymes that participate in the blood clotting extrinsic pathway is confirmed by the prolongation of activated partial thromboplastin time, used as clotting time assay. The inhibition of the fibrinolytic activity of plasmin was confirmed on the hydrolysis of fibrin plates. LlTI inhibits kinin release from high molecular weight kininogen by human plasma kallikrein in vitro and, administered intravenously, causes a decrease in paw edema induced by carrageenin or heat in male Wistar rats. In addition, lower concentrations of bradykinin were found in limb perfusion fluids of LlTI-treated rats.

  4. Versatile roles of CspA orthologs in complement inactivation of serum-resistant Lyme disease spirochetes.

    Science.gov (United States)

    Hammerschmidt, Claudia; Koenigs, Arno; Siegel, Corinna; Hallström, Teresia; Skerka, Christine; Wallich, Reinhard; Zipfel, Peter F; Kraiczy, Peter

    2014-01-01

    CspA of the Lyme disease spirochete Borrelia burgdorferi represents a key molecule in immune evasion, protecting borrelial cells from complement-mediated killing. As previous studies focused almost exclusively on CspA of B. burgdorferi, here we investigate the different binding capacities of CspA orthologs of Borrelia burgdorferi, B. afzelii, and B. spielmanii for complement regulator factor H and plasminogen and their ability to inhibit complement activation by either binding these host-derived plasma proteins or independently by direct interaction with components involved in formation of the lethal, pore-like terminal complement complex. To further examine their function in serum resistance in vivo, a serum-sensitive B. garinii strain was used to generate spirochetes, ectopically producing functional CspA orthologs. Irrespective of their species origin, all three CspA orthologs impart resistance to complement-mediated killing when produced in a serum-sensitive B. garinii surrogate strain. To analyze the inhibitory effect on complement activation and to assess the potential to inactivate C3b by binding of factor H and plasminogen, recombinant CspA orthologs were also investigated. All three CspA orthologs simultaneously bound factor H and plasminogen but differed in regard to their capacity to inactivate C3b via bound plasmin(ogen) and inhibit formation of the terminal complement complex. CspA of B. afzelii binds plasmin(ogen) and inhibits the terminal complement complex more efficiently than CspA of B. burgdorferi and B. spielmanii. Taken together, CspA orthologs of serum-resistant Lyme disease spirochetes act as multifunctional evasion molecules that inhibit complement on two central activation levels, C3b generation and assembly of the terminal complement complex.

  5. Hemostatic interference of Indian king cobra (Ophiophagus hannah) Venom. Comparison with three other snake venoms of the subcontinent.

    Science.gov (United States)

    Gowtham, Yashonandana J; Kumar, M S; Girish, K S; Kemparaju, K

    2012-06-01

    Unlike Naja naja, Bungarus caeruleus, Echis carinatus, and Daboia/Vipera russellii venoms, Ophiophagus hannah venom is medically ignored in the Indian subcontinent. Being the biggest poisonous snake, O. hannah has been presumed to inject several lethal doses of venom in a single bite. Lack of therapeutic antivenom to O. hannah bite in India makes any attempt to save the victim a difficult exercise. This study was initiated to compare O. hannah venom with the above said venoms for possible interference in hemostasis. Ophiophagus hannah venom was found to actively interfere in hemostatic stages such as fibrin clot formation, platelet activation/aggregation, and fibrin clot dissolution. It decreased partial thromboplastin time (aPTT), prothrombin time (PT), and thrombin clotting time (TCT). These activities are similar to that shown by E. carinatus and D. russellii venoms, and thus O. hannah venom was found to exert procoagulant activity through the common pathway of blood coagulation, while N. naja venom increased aPTT and TCT but not PT, and hence it was found to exert anticoagulant activity through the intrinsic pathway. Venoms of O. hannah, E. carinatus, and D. russellii lack plasminogen activation property as they do not hydrolyze azocasein, while they all show plasmin-like activity by degrading the fibrin clot. Although N. naja venom did not degrade azocasein, unlike other venoms, it showed feeble plasmin-like activity on fibrin clot. Venom of E. carinatus induced clotting of human platelet rich plasma (PRP), while the other three venoms interfered in agonist-induced platelet aggregation in PRP. Venom of O. hannah least inhibited the ADP induced platelet aggregation as compared to D. russellii and N. naja venoms. All these three venoms showed complete inhibition of epinephrine-induced aggregation at varied doses. However, O. hannah venom was unique in inhibiting thrombin induced aggregation.

  6. The nature of interactions between tissue-type plasminogen activator and platelets

    Energy Technology Data Exchange (ETDEWEB)

    Torr, S.R.; Winters, K.J.; Santoro, S.A.; Sobel, B.E. (Washington Univ., St. Louis, MO (USA))

    1990-07-15

    To elucidate interactions responsible for inhibition of aggregation of platelets in platelet-rich plasma (PRP) harvested from whole blood preincubated with t-PA, experiments were performed with PRP and washed platelets under diverse conditions of preincubation. Both ADP and collagen induced aggregation were inhibited in PRP unless aprotinin had been added to the preincubated whole blood concomitantly with t-PA. However, in washed platelets prepared after the same exposure aggregation was intact. When washed platelets were supplemented with fibrinogen degradation products (FDPs) in concentrations simulating those in whole blood preincubated with t-PA, aggregation induced with either ADP or collagen was inhibited. Thus, the inhibition in PRP depended on generation of FDPs by activated plasminogen. The functional integrity of surface glycoprotein (GP) IIb/IIIa receptors in washed platelets was documented by autoradiography after SDS-PAGE of surface labeled GPs and by fibrinogen binding despite preincubation of the whole blood or washed platelets themselves with t-PA and plasminogen as long as exogenous calcium (greater than or equal to 0.1 microM) was present. In contrast, when calcium was absent, the platelet GP IIb/IIIa receptor was rendered susceptible to degradation by plasmin, and aggregation was inhibited by preincubation at 37 degrees C even if aprotinin was present when aggregation was being assayed. These observations reconcile disparate results in the literature from studies in vivo and in vitro by demonstrating that inhibition of aggregation of platelets in PRP and in whole blood reflects indirect effects of plasminogen activation rather than direct effects of t-PA or plasmin on the platelets themselves.

  7. Development of a plasminogen activator inhibitor (PAI-1) assay and comparison of plasma PAI-1 activity in hyperlipidemic/dyslipidemic dogs with either hyperadrenocorticism or diabetes mellitus, and healthy dogs.

    Science.gov (United States)

    Wong, Cheryl J; Koch, Michael; Behling-Kelly, Erica L

    2016-11-08

    Thrombosis is a serious complication of many canine diseases and may be related to decreased fibrinolytic potential. Plasminogen activator inhibitor-1 (PAI-1) is the key regulator of fibrinolysis with increased levels demonstrated in states of pro-thrombosis and abnormal lipid metabolism. Our objective was to develop and validate a canine PAI-1 activity assay and test whether dogs with hyperadrenocorticism or diabetes mellitus that are hyperlipidemic/dyslipidemic have increased plasma PAI-1 activity. Functionally active PAI-1 in the plasma sample was incubated with recombinant tissue plasminogen activator (tPA), allowing the formation of a 1:1 stoichiometric inactive complex. Residual unbound tPA was then reacted with excess plasminogen in the presence of a colorimetric plasmin substrate. Plasmin production is quantified by computing the area under the curve of time (x) vs optical density (y) plot and converted to tPA IU/mL by comparison to a calibration curve of tPA standards. PAI-1 activity was determined by calculating the proportion of exogeneous tPA suppressed by PAI-1 in plasma. Assay verification included assessment of linearity, specificity, precision, sensitivity, and stability. PAI-1 activity was increased in hyperlipidemic compared to healthy dogs, but there was no significant difference between dogs with hyperadrenocorticism and diabetes mellitus. A near significant decrease in activity was detected in thawed plasma stored for 20h at 4°C. Our successfully validated assay offers a new tool for investigating fibrinolysis in dogs. Investigation of PAI-1 activity in dogs with other diseases associated with an increased risk of thrombosis would be valuable. Future studies of PAI-1 activity should consider its lability.

  8. Vitronectin-binding PAI-1 protects against the development of cardiac fibrosis through interaction with fibroblasts.

    Science.gov (United States)

    Zhong, Jianyong; Yang, Hai-Chun; Kon, Valentina; Fogo, Agnes B; Lawrence, Daniel A; Ma, Ji

    2014-06-01

    Plasminogen activator inhibitor-1 (PAI-1) promotes or abates fibrotic processes occurring in different organs. Binding of PAI-1 to vitronectin, an extracellular matrix component, may inhibit vitronectin-integrin complex-mediated cellular responses in pathophysiological conditions. To investigate the importance of plasmin suppression vs vitronectin-binding pathways of PAI-1 in cardiac fibrosis, we studied uninephrectomized mice fed a high salt diet and infused with angiotensin II (Ang II) together with different PAI-1 variants, including PAI-1AK (AK) that inhibits plasminogen activators but does not bind vitronectin, PAI-1RR (RR) that binds vitronectin but does not have protease inhibitory effects or control PAI-1 (CPAI), the control mutant that has similar molecular backbone and half-life as AK and RR while retaining all functions of native PAI-1. Compared with RR and CPAI, non-vitronectin-binding AK significantly increased expression of cardiac fibroblast marker, periostin (Ang+AK 8.40±3.55 vs Ang+RR 2.23±0.44 and Ang+CPAI 2.33±0.12% positive area, both PPAI-1 against fibrosis, fibroblasts from normal adult human ventricles were stimulated with Ang and different PAI-1 variants. Protease inhibitory AK and CPAI increased supernatant fibronectin, while decreasing plasminogen activator/plasmin activities and matrix metalloproteinase. RR and CPAI variants significantly reduced fibroblast expression of integrin β3, vitronectin level in the supernatant and fibroblast adhesion to vitronectin compared with the non-vitronectin-binding AK. Further, RR and CPAI preserved apoptotic, decreased anti-apoptotic and proliferative activities in fibroblasts. Thus, PAI-1 promotes or protects against development of cardiac fibrosis differentially through the protease inhibitory pathway or through its binding to vitronectin.

  9. Betaglycan has two independent domains required for high affinity TGF-β binding: proteolytic cleavage separates the domains and inactivates the neutralizing activity of the soluble receptor

    Science.gov (United States)

    Mendoza, Valentín; Vilchis-Landeros, M. Magdalena; Mendoza-Hernández, Guillermo; Huang, Tao; Villarreal, Maria M.; Hinck, Andrew P.; López-Casillas, Fernando; Montiel, Jose-Luis

    2009-01-01

    Summary Betaglycan is a co-receptor for members of the TGF-β superfamily. Mutagenesis has identified two ligand binding regions, one at the membrane-distal and the other at the membrane-proximal half of the betaglycan ectodomain. Here we show that partial plasmin digestion of soluble betaglycan produces two proteolysis-resistant fragments of 45 and 55 kDa, consistent with the predicted secondary structure, which indicates an intervening non-structured linker region separating the highly structured N- and C-terminal domains. Amino terminal sequencing indicates that the 45 and 55 kDa fragments correspond, respectively, to the membrane-distal and -proximal regions. Plasmin treatment of membrane betaglycan results in the production of equivalent proteolysis-resistant fragments. The 45 and 55 kDa fragments, as well as their recombinant soluble counterparts, Sol Δ10 and Sol Δ11, bind TGF-β, nonetheless, compared to intact soluble betaglycan, have severely diminished ability to block TGF-β activity. Surface plasmon resonance (SPR) analysis indicates that soluble betaglycan has Kds in the low nanomolar range for the three TGF-β isoforms, while those for Sol Δ10 and Sol Δ11 are 1 – 2 orders of magnitude higher. SPR analysis further shows that the Kds of Sol Δ11 are not changed in the presence of Sol Δ10, indicating that the high affinity of soluble betaglycan is a consequence of tethering of the domains together. Overall, these results, suggest that betaglycan ectodomain exhibits a bi-lobular structure in which each lobule folds independently, binds TGF-β through distinct non-overlapping interfaces, and that linker modification may be an approach to improve soluble betaglycan’s TGF-β neutralizing activity. PMID:19842711

  10. Increased von Willebrand factor, P-selectin and fibrin content in occlusive thrombus resistant to lytic therapy.

    Science.gov (United States)

    Sambola, Antonia; García Del Blanco, Bruno; Ruiz-Meana, Marisol; Francisco, Jaume; Barrabés, José A; Figueras, Jaume; Bañeras, Jordi; Otaegui, Imanol; Rojas, Angeles; Vilardosa, Úrsula; Montaner, Joan; García-Dorado, David

    2016-06-01

    Therapeutic fibrinolysis is ineffective in 40 % of ST-segment elevation acute myocardial infarction (STEMI) patients, but understanding of the mechanisms is incomplete. It was our aim to compare the composition of coronary thrombus in lysis-resistant STEMI patients with that of lysis-sensitive patients. Intracoronary thrombi (n=64) were obtained by aspiration in consecutive STEMI patients. Of them, 20 had received fibrinolysis and underwent rescue percutaneous coronary intervention (r-PCI, lysis-resistant patients) and 44 underwent primary PCI (p-PCI). Lysis-sensitivity was determined in vitro by clot permeability measurements and turbidimetric lysis in plasma of 44 patients undergoing p-PCI and 20 healthy donors. Clot-lysis sensitivity was defined as a clot-lysis time not greater than 1 SD over the mean of healthy donors. Coronary thrombus composition in 20 lysis-resistant and in 20 lysis-sensitive patients was analysed by immunofluorescence with confocal microscopy. Plasma biomarkers (P-selectin, VWF, PAI-1, t-PA, D-dimer, TF pathway markers, plasmin and CD34+) were measured simultaneously on peripheral blood. Lysis-resistant clots had higher levels of fibrin (p=0.02), P-selectin (p=0.03) and VWF (p=0.01) than lysis-sensitive clots. Among thrombi obtained ≤ 6 hours after onset of symptoms, those from lysis-resistant patients showed a higher content in fibrin than those from p-PCI patients (p=0.01). Plasma PAI-1 (p=0.02) and D-dimer levels were significantly higher (p=0.003) in lysis-resistant patients, whereas plasmin levels were lower (p=0.03). Multivariate analysis showed the content of fibrin and VWF within thrombus as predictors of thrombolysis resistance. In conclusion, coronary thrombi in STEMI patients resistant to fibrinolysis are characterised by higher fibrin, P-selectin and VWF content than lysis-sensitive thrombi.

  11. Severe bleeding tendency caused by a rare complication of excessive fibrinolysis with disseminated intravascular coagulation in a 51-year-old Japanese man with prostate cancer: a case report

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    Wada Yoshihiro

    2012-11-01

    Full Text Available Abstract Introduction Disseminated intravascular coagulation causes thrombotic tendency leading to multiple organ failure and occurs in a wide variety of diseases including malignancy. Disseminated intravascular coagulation is a latent complication in people with prostate cancer. Case presentation A 51-year-old Japanese man with advanced castration-resistant prostate cancer was admitted to our hospital because of extensive purpura and severe anemia. Prolonged plasma coagulation time, hypofibrinogenemia and normal platelet count suggested that a decrease in fibrinogen induced a bleeding tendency causing purpura. However, elevated plasma levels of thrombin-antithrombin complex, fibrin and/or fibrinogen degradation products and D-dimers, with positive fibrin monomer test, manifested disseminated intravascular coagulation and subsequent fibrinolysis. Plasma levels of thrombin-antithrombin complex, fibrin and/or fibrinogen degradation products and D-dimers decreased after administration of low-molecular-weight heparin. However, low fibrinogen and α2-antiplasmin levels were not improved and plasmin-antiplasmin complex did not decrease, which revealed excessive fibrinolysis complicated with disseminated intravascular coagulation. We suspected that prostate cancer cell-derived urokinase-type plasminogen activator caused excessive fibrinolysis. Administration of tranexamic acid for fibrinogenolysis was added together with high-dose anti-androgen therapy (fosfestrol for prostate cancer. Thereafter, prostate-specific antigen and plasmin-antiplasmin complex decreased, followed by normalized fibrinogen and α2-antiplasmin levels, and the patient eventually recovered from the bleeding tendency. Immunohistochemical staining of the biopsied prostate tissue exhibited that the prostate cancer cells produced tissue factor, the coagulation initiator, and urokinase-type plasminogen activator. Conclusion This patient with rare complications of disseminated

  12. Leptin and resistin induce increased procoagulability in diabetes mellitus.

    Science.gov (United States)

    Bobbert, Peter; Eisenreich, Andreas; Weithäuser, Alice; Schultheiss, Heinz Peter; Rauch, Ursula

    2011-11-01

    Patients with diabetes mellitus (DM) suffer from an increased risk of cardiovascular events caused by thrombotic conditions. Adipose tissue might play a crucial role in this pathogenesis by synthesis of procoagulant mediators. This study was performed to elucidate the role of the adipocytokines leptin and resistin in the development of hypercoagulability and hypofibrinolysis under diabetic conditions. Sixty two patients with or without DM were included in our study to measure leptin, resistin and tissue factor (TF) plasma concentrations. Moreover, flow chamber experiments were performed to assess factor Xa and plasmin activity on the surface of HUVECs. Western blot and real-time PCR were performed to determine mRNA and protein expression of main factors of the coagulation and fibrinolytic system. Patients with diabetes showed increased levels of leptin and resistin (leptin: 25.69±13.9 vs. 15.98±17.5 ng/mL, p<0.05; resistin: 2.61±0.6 vs. 1.19±0.7 ng/mL, p<0.05), which were positively correlated with TF. In vitro, leptin and resistin induced increased factor Xa activity (leptin: 4.29±0.57-fold, p<0.05; resistin 4.19±0.7-fold, p<0.05 vs. control) on HUVECs as also reflected by elevated TF mRNA and protein expression. Moreover, stimulatory (plasminogen activator inhibitor 1) and inhibitory (tissue plasminogen activator) mediators of the fibrinolytic cascade were induced by leptin and resistin, leading to a balanced plasmin activity regulation. Leptin and resistin lead to a procoagulant state in HUVECs by inducing TF expression. This mechanism might be one explanation for the prothrombotic state observed under diabetic conditions. Copyright © 2011 Elsevier Ltd. All rights reserved.

  13. Predicting perioperative venous thromboembolism in Japanese gynecological patients.

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    Masae Ikeda

    Full Text Available OBJECTIVE: To develop a convenient screening method that can predict perioperative venous thromboembolism (VTE and identify patients at risk of fatal perioperative pulmonary embolism (PE. METHODS: Patients hospitalized for gynecological abdominal surgery (n = 183 underwent hematology tests and multidetector computed tomography (MDCT to detect VTE. All statistical analyses were carried out using the SPSS software program (PASWV19.0J. RESULTS: The following risk factors for VTE were identified by univariate analysis: plasmin-alpha2-plasmin inhibitor complex (PIC, thrombin-antithrombin III complex (TAT, and prolonged immobility (all p<0.001; age, neoadjuvant chemotherapy (NAC, malignancy, hypertension, past history of VTE, and hormone therapy (all p<0.01; and hemoglobin, transverse tumor diameter, ovarian disease, and menopause (all p<0.05. Multivariate analysis using these factors revealed that PIC, age, and transverse tumor diameter were significant independent determinants of the risk of VTE. We then calculated the incidence rate of perioperative VTE using PIC and transverse tumor diameter in patient groups stratified by age. In patients aged ≤40 years, PIC ≥1.3 µg/mL and a transverse tumor diameter ≥10 cm identified the high-risk group for VTE with an accuracy of 93.6%. For patients in their 50 s, PIC ≥1.3 µg/mL identified a high risk of VTE with an accuracy of 78.2%. In patients aged ≥60 years, a transverse tumor diameter ≥15 cm (irrespective of PIC or PIC ≥1.3 µg/mL identified the high-risk group with an accuracy of 82.4%. CONCLUSIONS: We propose new screening criteria for VTE risk that are based on PIC, transverse tumor diameter, and age. Our findings suggest the usefulness of these criteria for predicting the risk of perioperative VTE and for identifying patients with a high risk of fatal perioperative PE.

  14. 产纤溶酶菌株的筛选鉴定及其生长条件研究

    Institute of Scientific and Technical Information of China (English)

    马晶; 高玉荣

    2013-01-01

    To screen the strain with high yield of plasmin from soy sause mash and Douchi, and its growth conditions is studied. The strain M64 with 1 960 U/mL of plasmin activity is screened from soy sause mash and Douchi by Casein plate primirily selecting and lipid state fermentation second screening. By individual form, colony characteristic and physiological and biochemical property, the strain is identified as Bacillus subtilis natto. By researching the effect of conditons on growth, the growth conditons of inoculum concentration 1.5%, initial pH 7.0, rotating speed 160 r/min, cultivating at 37 ℃ for 10 h is determined.%  从酱醅、豆豉原料中筛选高产纤溶酶的菌株,并对其进行鉴定和生长条件的研究。通过酪蛋白平板法初筛和液态发酵复筛,从酱醅、豆豉原料中筛选出1株纤溶酶活力达1960 U/mL的高产菌株M64。通过个体形态、菌落特征及生理生化特征将其鉴定为枯草芽孢杆菌纳豆亚种。通过研究液体培养条件对菌株生长的影响,确定菌种液体培养的条件为接种量1.5%,初始pH值7,转速160 r/min,培养温度37℃,培养时间10 h。

  15. Biomarcadores del estado inflamatorio: nexo de unión con la obesidad y complicaciones asociadas Inflammatory biomarkers: the link between obesity and associated pathologies

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    M.ª A. Zulet

    2007-10-01

    Full Text Available El objetivo de este trabajo ha consistido en realizar una revisión de los biomarcadores que actualmente se proponen como el nexo de unión entre la inflamación, la obesidad y complicaciones asociadas, seleccionando los estudios llevados a cabo y las cuestiones pendientes. Cada vez hay mayor evidencia científica de que la inflamación puede jugar un papel importante en la etiología de diversas enfermedades crónicas de gran relevancia para la salud pública. En los últimos años, distintos estudios han sugerido que la obesidad podría ser un desorden inflamatorio. Asimismo, el estrés oxidativo se ha propuesto como un potencial inductor de la inflamación y de la susceptibilidad a la obesidad y patología asociadas. Entre los biomarcadores relacionados con la obesidad, la resistencia insulínica, las enfermedades cardiovasculares y el síndrome metabólico se encuentran: el factor de necrosis tumoral alfa, interleuquinas 6 y 18, angiotensinógeno, factor de crecimiento TGF-beta, inhibidor de la activación del plasminógeno, leptina, resistina, proteína C reactiva, amiloide A, ácido siálico, marcadores de disfunción endotelial (factor von Willebrand, ICAMs, vCAMs factor 3 del sistema del complemento, haptoglobina, glicoproteína zinc-alfa2, eotaxina, visfatina, apelina, alfa1-antitripsina, vaspina, omentina, proteína transportadora de retinol 4, ceruloplasmina, adiponectina y desnutrina. Algunos de estos biomarcadores son buenos predictores de riesgo cardiovascular (inhibidor de la activación de plasminógeno 1, angiotensinógeno, fibrinógeno, ácido siálico, factor 3 del complemento y proteína C reactiva, adiposidad (leptina, visfatina, resistina, haptoglobina y/o resistencia insulínica (ácido siálico, proteína C reactiva, inhibidor de la activación de plasminógeno 1, factor von Willebrand. Sin embargo, todavía queda por dilucidar el papel de muchos de ellos en la etiología de la obesidad y comorbilidades asociadas en

  16. A comparative double-blind randomized trial of activated protein C and unfractionated heparin in the treatment of disseminated intravascular coagulation.

    Science.gov (United States)

    Aoki, Nobuo; Matsuda, Tamotsu; Saito, Hidehiko; Takatsuki, Kiyoshi; Okajima, Kenji; Takahashi, Hoyu; Takamatsu, Junki; Asakura, Hidesaku; Ogawa, Nobuya

    2002-06-01

    A randomized prospective double-blind trial was performed to compare the safety and efficacy of human activated protein C (APC) and unfractionated heparin for the treatment of disseminated intravascular coagulation (DIC). One hundred thirty-two patients with DIC were enrolled in this study: 63 patients received APC (12.5 U [2.5 microg]/kg body wt per hour) and 69 patients received heparin (8 U/kg body wt per hour) by intravenous infusion for 6 days. Forty-nine APC-treated patients and 55 heparin-treated patients were evaluated for efficacy, and 52 APC-treated patients and 55 heparin-treated patients were evaluated for safety. The 2 groups were similar with respect to sex, age, body weight, underlying diseases, and coagulation/fibrinolysis parameters before treatment. Aggravation of bleeding was seen after treatment in 8 patients receiving heparin, but in none of the patients receiving APC. The number of patients who showed alleviation of bleeding was significantly higher in the APC group than the heparin group (P = .009). The effects on DIC-related organ dysfunction were not significantly different between the 2 groups. Fibrinogen-fibrin degradation products, D-dimer, thrombin-antithrombin complex (TAT), and plasmin-plasmin inhibitor complex (PIC) were all significantly decreased by treatment in both groups. Fibrinogen, protein C, and antithrombin were significantly increased in the APC group, whereas only protein C was significantly increased in the heparin group. Platelet count in the nonleukemic group was significantly increased in those patients receiving APC but not increased in those patients receiving heparin. Improvement of coagulation/fibrinolysis was assessed by scoring 4 parameters (soluble fibrin monomers, D-dimer, TAT, and PIC), and the results indicated that the APC group showed significantly greater improvement than the heparin group (P = .046). There was, however, no significant difference in the rate of complete recovery from DIC between the 2

  17. Dissociation of Tissue Destruction and Bacterial Expansion during Bubonic Plague.

    Science.gov (United States)

    Guinet, Françoise; Avé, Patrick; Filali, Sofia; Huon, Christèle; Savin, Cyril; Huerre, Michel; Fiette, Laurence; Carniel, Elisabeth

    2015-10-01

    Activation and/or recruitment of the host plasmin, a fibrinolytic enzyme also active on extracellular matrix components, is a common invasive strategy of bacterial pathogens. Yersinia pestis, the bubonic plague agent, expresses the multifunctional surface protease Pla, which activates plasmin and inactivates fibrinolysis inhibitors. Pla is encoded by the pPla plasmid. Following intradermal inoculation, Y. pestis has the capacity to multiply in and cause destruction of the lymph node (LN) draining the entry site. The closely related, pPla-negative, Y. pseudotuberculosis species lacks this capacity. We hypothesized that tissue damage and bacterial multiplication occurring in the LN during bubonic plague were linked and both driven by pPla. Using a set of pPla-positive and pPla-negative Y. pestis and Y. pseudotuberculosis strains in a mouse model of intradermal injection, we found that pPla is not required for bacterial translocation to the LN. We also observed that a pPla-cured Y. pestis caused the same extensive histological lesions as the wild type strain. Furthermore, the Y. pseudotuberculosis histological pattern, characterized by infectious foci limited by inflammatory cell infiltrates with normal tissue density and follicular organization, was unchanged after introduction of pPla. However, the presence of pPla enabled Y. pseudotuberculosis to increase its bacterial load up to that of Y. pestis. Similarly, lack of pPla strongly reduced Y. pestis titers in LNs of infected mice. This pPla-mediated enhancing effect on bacterial load was directly dependent on the proteolytic activity of Pla. Immunohistochemistry of Pla-negative Y. pestis-infected LNs revealed extensive bacterial lysis, unlike the numerous, apparently intact, microorganisms seen in wild type Y. pestis-infected preparations. Therefore, our study demonstrates that tissue destruction and bacterial survival/multiplication are dissociated in the bubo and that the primary action of Pla is to protect

  18. Human breast cancer cell-mediated bone collagen degradation requires plasminogen activation and matrix metalloproteinase activity

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    Hill Peter A

    2005-02-01

    Full Text Available Abstract Background Breast cancer cells frequently metastasize to the skeleton and induce extensive bone destruction. Cancer cells produce proteinases, including matrix metalloproteinases (MMPs and the plasminogen activator system (PAS which promote invasion of extracellular matrices, but whether these proteinases degrade bone matrix is unclear. To characterize the role that breast cancer cell proteinases play in bone degradation we compared the effects of three human breast cancer cell lines, MDA-MB-231, ZR-75-1 and MCF-7 with those of a normal breast epithelial cell line, HME. The cell lines were cultured atop radiolabelled matrices of either mineralized or non-mineralized bone or type I collagen, the principal organic constituent of bone. Results The 3 breast cancer cell lines all produced significant degradation of the 3 collagenous extracellular matrices (ECMs whilst the normal breast cell line was without effect. Breast cancer cells displayed an absolute requirement for serum to dissolve collagen. Degradation of collagen was abolished in plasminogen-depleted serum and could be restored by the addition of exogenous plasminogen. Localization of plasmin activity to the cell surface was critical for the degradation process as aprotinin, but not α2 antiplasmin, prevented collagen dissolution. During ECM degradation breast cancer cell lines expressed urokinase-type plasminogen activator (u-PA and uPA receptor, and MMPs-1, -3, -9,-13, and -14. The normal breast epithelial cell line expressed low levels of MMPs-1, and -3, uPA and uPA receptor. Inhibitors of both the PAS (aprotinin and PA inhibitor-1 and MMPs (CT1166 and tisue inhibitor of metalloproteinase blocked collagen degradation, demonstrating the requirement of both plasminogen activation and MMP activity for degradation. The activation of MMP-13 in human breast cancer cells was prevented by plasminogen activator inhibitor-1 but not by tissue inhibitor of metalloproteinase-1, suggesting

  19. Human pregnancy serum contains at least two distinct proteolytic activities with the ability to degrade insulin-like growth factor binding protein-3.

    Science.gov (United States)

    Bang, P; Fielder, P J

    1997-09-01

    The presence of a proteolytic activity in sera from pregnant humans and rodents capable of degrading insulin-like growth factor binding protein-3 (IGFBP-3) has been known for some time. However, the identity of this activity has remained elusive. We have attempted to purify the IGFBP-3 protease activity from pregnant human serum (PHS) using the degradation of 125I-IGFBP-3 as a marker. Following ammonium sulfate precipitation of PHS and further enrichment of active fractions by ion-exchange, protein-A Sepharose, and size-exclusion chromatography, a protease of approximately 70-90 kDa was isolated and subjected to N-terminal analysis. The N-terminal sequence was consistent with plasminogen, a known fibrinolytic enzyme. To further characterize the IGFBP-3 protease activities in both PHS and nonpregnant human serum (NHS), aliquots of serum were first enriched by polyethylene glycol-precipitation and subjected to size-exclusion chromatography. The size-separated fractions were then incubated with 125I-IGFBP-3, and proteolytic activity was measured. PHS contained two separate proteases (>150 kDa and 70-90 kDa), whereas NHS contained only one (70-90 kDa) that had a inhibitor profile similar to plasmin. However, inhibitors of plasmin had no effect on the activity of the >150-kDa protease. Plasminogen activators (PAs) greatly increased the activity of the 70- to 90-kDa protease, but had little effect on the >150-kDa protease activity. Addition of PAs greatly increased the ability of NHS to proteolyze IGFBP-3. In contrast, the ability of plasminogen-depleted plasma to degrade 125I-IGFBP-3 was not affected by the addition of PAs. Both urokinase and tissue-type PA had the ability to proteolyze IGFBP-3 and were, in contrast to the >150-kDa protease activity, inhibited by the specific PA inhibitor D-PHE-PRO-ARG chloromethyl ketone. The present data suggest that sera has the ability to proteolyze IGFBP-3, and that this ability, as demonstrated by NHS, can be regulated by protease

  20. Photonic activation of plasminogen induced by low dose UVB.

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    Manuel Correia

    Full Text Available Activation of plasminogen to its active form plasmin is essential for several key mechanisms, including the dissolution of blood clots. Activation occurs naturally via enzymatic proteolysis. We report that activation can be achieved with 280 nm light. A 2.6 fold increase in proteolytic activity was observed after 10 min illumination of human plasminogen. Irradiance levels used are in the same order of magnitude of the UVB solar irradiance. Activation is correlated with light induced disruption of disulphide bridges upon UVB excitation of the aromatic residues and with the formation of photochemical products, e.g. dityrosine and N-formylkynurenine. Most of the protein fold is maintained after 10 min illumination since no major changes are observed in the near-UV CD spectrum. Far-UV CD shows loss of secondary structure after illumination (33.4% signal loss at 206 nm. Thermal unfolding CD studies show that plasminogen retains a native like cooperative transition at ~70 ºC after UV-illumination. We propose that UVB activation of plasminogen occurs upon photo-cleavage of a functional allosteric disulphide bond, Cys737-Cys765, located in the catalytic domain and in van der Waals contact with Trp761 (4.3 Å. Such proximity makes its disruption very likely, which may occur upon electron transfer from excited Trp761. Reduction of Cys737-Cys765 will result in likely conformational changes in the catalytic site. Molecular dynamics simulations reveal that reduction of Cys737-Cys765 in plasminogen leads to an increase of the fluctuations of loop 760-765, the S1-entrance frame located close to the active site. These fluctuations affect the range of solvent exposure of the catalytic triad, particularly of Asp646 and Ser74, which acquire an exposure profile similar to the values in plasmin. The presented photonic mechanism of plasminogen activation has the potential to be used in clinical applications, possibly together with other enzymatic treatments for the

  1. Activation of blood clotting and fibrinolysis in angiocardiography with ionic and non-ionic contrast medium; Aktivierung von Blutgerinnung und Fibrinolyse nach Angiokardiographie mit ionischem und nichtionischem Kontrastmittel

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    Winkler, U.H. [Gerinnungsphysiologisches Lab., Zentrum fuer Frauenheilkunde, Universitaetsklinikum Essen (Germany); Park, J.W. [Herz-Zentrum Kaiser-Wilhelm-Krankenhaus, Duisburg (Germany); Weber, S. [Schering AG, Geschaeftsbereich Deutschland, Berlin (Germany); Kothe, A. [Gerinnungsphysiologisches Lab., Zentrum fuer Frauenheilkunde, Universitaetsklinikum Essen (Germany); Schnitker, J. [Inst. fuer Angewandte Statistik GmbH, Bielefeld (Germany); Behrends-Steins, B. [Schering AG, Geschaeftsbereich Deutschland, Berlin (Germany); Albring, M. [Schering AG, Geschaeftsbereich Deutschland, Berlin (Germany)

    1997-06-01

    Purpose: To study the effects of a ionic (amidotrizoate) and a nonionic X-ray contrast medium (iopromid) during routine levocardiography and coronary angiography, we employed assays that detect reaction products of thrombin and plasmin to assess the activation of the haemostatic system. Methods: Subsequent to informed consent, 20 patients were randomly assigned to receive either amidotrizoate or iopromid during standard levocardiography and coronary angiography in a double-blind comparative study. Groups were comparable in respect of age, weight, sex and severity of the disease. No anticoagulation was provided. Coronary angiography was performed according to a standardised protocol. Consumption of contrast media and duration of the examination were comparable in both groups. Results: Thrombin generation (F.1+2) and thrombin activity (TAT) were higher with the ionic contrast medium but did not attain statistical significance. Fibrin generation and degradation as expressed by D-dimer fibrin split products was significantly increased in patients who had been receiving amidotrizoate (p<0,05, U-Test). Conclusion: The non-ionic X-ray contrast medium induced significantly less haemostatic activation in vivo than did the ionic medium amidotrizoate. These data suggest that earlier in vitro observations of more pronounced anticoagulant effects of ionic X-ray contrast media are of limited significance for the evaluation of in vivo effects of X-ray contrast media on haemostatic function. (orig.) [Deutsch] Ziel: Bestimmung der Aktivitaet des haemostatischen Systems in vivo nach Gabe von ionischem und nichtionischem Roentgenkontrastmittel in der Angiokardiographie. Material und Methoden: In eine randomisierte doppelblinde Vergleichsstudie mit den Kontrastmitteln Amidotrizoat und Iopromid wurden 20 Patienten (10 pro Gruppe) einbezogen. Bei der aus medizinischen Gruenden indizierten Angiokardiographie wurden 5 repraesentative Reaktionsprodukte der thrombin- und plasmininduzierten

  2. Distal hinge of plasminogen activator inhibitor-1 involves its latency transition and specificities toward serine proteases

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    Shaltiel Shmuel

    2003-07-01

    Full Text Available Abstract Background The plasminogen activator inhibitor-1 (PAI-1 spontaneously converts from an inhibitory into a latent form. Specificity of PAI-1 is mainly determined by its reactive site (Arg346-Met347, which interacts with serine residue of tissue-type plasminogen activator (tPA with concomitant formation of SDS-stable complex. Other sites may also play roles in determining the specificity of PAI-1 toward serine proteases. Results To understand more about the role of distal hinge for PAI-1 specificities towards serine proteases and for its conformational transition, wild type PAI-1 and its mutants were expressed in baculovirus system. WtPAI-1 was found to be about 12 fold more active than the fibrosarcoma PAI-1. Single site mutants within the Asp355-Arg356-Pro357 segment of PAI-1 yield guanidine activatable inhibitors (a that can still form SDS stable complexes with tPA and urokinase plasminogen activator (uPA, and (b that have inhibition rate constants towards plasminogen activators which resemble those of the fibrosarcoma inhibitor. More importantly, latency conversion rate of these mutants was found to be ~3–4 fold faster than that of wtPAI-1. We also tested if Glu351 is important for serine protease specificity. The functional stability of wtPAI-1, Glu351Ala, Glu351Arg was about 18 ± 5, 90 ± 8 and 14 ± 3 minutes, respectively, which correlated well with both their corresponding specific activities (84 ± 15 U/ug, 112 ± 18 U/ug and 68 ± 9 U/ug, respectively and amount of SDS-stable complex formed with tPA after denatured by Guanidine-HCl and dialyzed against 50 mM sodium acetate at 4°C. The second-order rate constants of inhibition for uPA, plasmin and thrombin by Glu351Ala and Glu351Arg were increased about 2–10 folds compared to wtPAI-1, but there was no change for tPA. Conclusion The Asp355-Pro357 segment and Glu351 in distal hinge are involved in maintaining the inhibitory conformation of PAI-1. Glu351 is a specificity

  3. Dissociation of Tissue Destruction and Bacterial Expansion during Bubonic Plague.

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    Françoise Guinet

    2015-10-01

    Full Text Available Activation and/or recruitment of the host plasmin, a fibrinolytic enzyme also active on extracellular matrix components, is a common invasive strategy of bacterial pathogens. Yersinia pestis, the bubonic plague agent, expresses the multifunctional surface protease Pla, which activates plasmin and inactivates fibrinolysis inhibitors. Pla is encoded by the pPla plasmid. Following intradermal inoculation, Y. pestis has the capacity to multiply in and cause destruction of the lymph node (LN draining the entry site. The closely related, pPla-negative, Y. pseudotuberculosis species lacks this capacity. We hypothesized that tissue damage and bacterial multiplication occurring in the LN during bubonic plague were linked and both driven by pPla. Using a set of pPla-positive and pPla-negative Y. pestis and Y. pseudotuberculosis strains in a mouse model of intradermal injection, we found that pPla is not required for bacterial translocation to the LN. We also observed that a pPla-cured Y. pestis caused the same extensive histological lesions as the wild type strain. Furthermore, the Y. pseudotuberculosis histological pattern, characterized by infectious foci limited by inflammatory cell infiltrates with normal tissue density and follicular organization, was unchanged after introduction of pPla. However, the presence of pPla enabled Y. pseudotuberculosis to increase its bacterial load up to that of Y. pestis. Similarly, lack of pPla strongly reduced Y. pestis titers in LNs of infected mice. This pPla-mediated enhancing effect on bacterial load was directly dependent on the proteolytic activity of Pla. Immunohistochemistry of Pla-negative Y. pestis-infected LNs revealed extensive bacterial lysis, unlike the numerous, apparently intact, microorganisms seen in wild type Y. pestis-infected preparations. Therefore, our study demonstrates that tissue destruction and bacterial survival/multiplication are dissociated in the bubo and that the primary action of Pla

  4. Streptococcus pyogenes Employs Strain-dependent Mechanisms of C3b Inactivation to Inhibit Phagocytosis and Killing of Bacteria.

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    Agrahari, Garima; Liang, Zhong; Glinton, Kristofor; Lee, Shaun W; Ploplis, Victoria A; Castellino, Francis J

    2016-04-22

    Evasion of complement-mediated opsonophagocytosis enables group A Streptococcus pyogenes (GAS) to establish infection. Different strain-dependent mechanisms are employed by the host to accomplish this goal. In general, GAS inhibits the amplification of the complement cascade on its cell surface by facilitating the degradation of C3b, an opsonin, to an inactive product, inactivated C3b (iC3b), in a step catalyzed by factor I (FI) and its cofactor, factor H (FH), with or without the participation of human host plasmin (hPm). GAS recruits FH to its cell surface via FH receptors, which are transcriptionally controlled by the two-component cluster of virulence responder-sensor system. The manner in which FI-FH and hPm function together on GAS cells is unknown. Using GAS strain AP53, which strongly binds host human plasminogen/plasmin (hPg/hPm) directly via an hPg/hPm surface receptor (PAM), we show that both FI-FH and hPm sequentially cleave C3b. Whereas FI-FH proteolytically cleaves C3b into iC3b, PAM-bound hPm catalyzes cleavage of iC3b into multiple smaller peptides. Unlike AP53, GAS strain M23ND weakly binds FH and recruits hPg/hPm to its cell surface indirectly via fibrinogen bound to M-protein, M23. In this case, FH-FI cleaves C3b into iC3b, with negligible degradation of iC3b by hPm that is bound to fibrinogen on the cells. AP53 and M23ND display similar resistance to human neutrophil-mediated phagocytosis, which results in a corresponding high lethality in mice after injection of these cells. These results suggest that GAS utilizes diverse mechanisms to degrade C3b and thus to protect bacterial cells from the complement response of the host.

  5. A natural inactivating mutation in the CovS component of the CovRS regulatory operon in a pattern D Streptococcal pyogenes strain influences virulence-associated genes.

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    Liang, Zhong; Zhang, Yueling; Agrahari, Garima; Chandrahas, Vishwanatha; Glinton, Kristofor; Donahue, Deborah L; Balsara, Rashna D; Ploplis, Victoria A; Castellino, Francis J

    2013-03-01

    A skin-tropic invasive group A Streptococcus pyogenes (GAS) strain, AP53, contains a natural inactivating mutation in the covS gene (covS(M)) of the two-component responder (CovR)/sensor (CovS) gene regulatory system. The effects of this mutation on specific GAS virulence determinants have been assessed, with emphasis on expression of the extracellular protease, streptococcal pyrogenic exotoxin B (SpeB), capsular hyaluronic acid, and proteins that allow host plasmin assembly on the bacterial surface, viz. a high affinity plasminogen (Pg)/plasmin receptor, Pg-binding group A streptococcal M protein (PAM), and the human Pg activator streptokinase. To further illuminate mechanisms of the functioning of CovRS in the virulence of AP53, two AP53 isogenic strains were generated, one in which the natural covS(M) gene was mutated to WT-covS (AP53/covS(WT)) and a strain that contained an inactivated covR gene (AP53/ΔcovR). Two additional strains that do not contain PAM, viz. WT-NS931 and NS931/covS(M), were also employed. SpeB was not measurably expressed in strains containing covR(WT)/covS(M), whereas in strains with natural or engineered covR(WT)/covS(WT), SpeB expression was highly up-regulated. Alternatively, capsule synthesis via the hasABC operon was enhanced in strain AP53/covS(M), whereas streptokinase expression was only slightly affected by the covS inactivation. PAM expression was not substantially influenced by the covS mutation, suggesting that covRS had minimal effects on the mga regulon that controls PAM expression. These results demonstrate that a covS inactivation results in virulence gene alterations and also suggest that the CovR phosphorylation needed for gene up- or down-regulation can occur by alternative pathways to CovS kinase.

  6. Central serous chorioretinopathy: a pathogenetic model

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    et al

    2011-02-01

    Full Text Available Antonio Caccavale1, Filippo Romanazzi1, Manuela Imparato1, Angelo Negri2, Anna Morano3, Fabio Ferentini21Department of Ophthalmology, Neuropthalmology and Ocular Immunology Service, 2Department of Ophthalmology, Hospital “C. Cantù”, Abbiategrasso, Milan, Italy; 3University Eye Clinic, Foundation IRCCS San Matteo Hospital, Pavia, ItalyAbstract: Despite numerous studies describing predominantly its demography and clinical course, many aspects of central serous chorioretinopathy (CSCR remain unclear. Perhaps the major impediment to finding an effective therapy is the difficulty of performing studies with large enough cohorts, which has meant that clinicians have focused more on therapy than on a deeper understanding of the pathogenesis of the disease. Hypotheses on the pathogenesis of CSCR have ranged from a basic alteration in the choroid to an involvement of the retinal pigment epithelium (RPE. Starting from evidence that affected subjects often present a personality prone to stress with altered pituitary–hypothalamic axis response (HPA and that they have higher levels of serum and urinary cortisol and catecholamines than healthy subjects, we hypothesize a cascade of events that may lead to CSCR through hypercoagulability and augmented platelet aggregation. In particular we investigated the role of tissue plasminogen activator, increasing plasminogen activator inhibitor 1 (PAI-1, and plasmin-α2- plasmin inhibitor complexes. We reviewed the different therapeutic approaches, including adrenergic antagonists, carbonic anhydrase inhibitors, mifepristone, ketoconazole, laser photocoagulation, intravitreal injection of bevacizumab, and photodynamic therapy with verteporfin (PDT and our model of pathogenesis seems to be in agreement with the clinical effects obtained from these treatments. In accord with our thesis, we began to treat a group of patients affected by CSCR with low-dose aspirin (75–100 mg, because of its effectiveness in other

  7. Purification, amino acid sequence, and cDNA cloning of trypsin inhibitors from onion (Allium cepa L.) bulbs.

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    Deshimaru, Masanobu; Watanabe, Akira; Suematsu, Keiko; Hatano, Maki; Terada, Shigeyuki

    2003-08-01

    Three protease inhibitors (OTI-1-3) have been purified from onion (Allium cepa L.) bulbs. Molecular masses of these inhibitors were found to be 7,370.2, 7,472.2, and 7,642.6 Da by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), respectively. Based on amino acid composition and N-terminal sequence, OTI-1 and -2 are the N-terminal truncated proteins of OTI-3. All the inhibitors are stable to heat and extreme pH. OTI-3 inhibited trypsin, chymotrypsin, and plasmin with dissociation constants of 1.3 x 10(-9) M, 2.3 x 10(-7) M, and 3.1 x 10(-7) M, respectively. The complete amino acid sequence of OTI-3 showed a significant homology to Bowman-Birk family inhibitors, and the first reactive site (P1) was found to be Arg17 by limited proteolysis by trypsin. The second reactive site (P1) was estimated to be Leu46, that may inhibit chymotrypsin. OTI-3 lacks an S-S bond near the second reactive site, resulting in a low affinity for the enzyme. The sequence of OTI-3 was also ascertained by the nucleotide sequence of a cDNA clone encoding a 101-residue precursor of the onion inhibitor.

  8. [Progress in diagnosis and treatment for disseminated intravascular coagulation].

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    Wada, Hideo; Matsumoto, Takeshi; Aota, Takumi; Yamashita, Yoshiki

    2015-02-01

    As the development of a hypercoagulable state in the setting of disseminated intravascular coagulation (DIC) induces localized infection, therapy for DIC should be evaluated according to the findings of examinations for both severe sepsis and DIC. DIC is classified into the following types: "bleeding type," "organ failure type," "asymptomatic type," and "complication type." The "bleeding type" and "organ failure type" are considered to reflect the "plasmin inhibitor (PI) deficiency type" and "antithrombin (AT) deficiency type," respectively. In order to improve the diagnosis of DIC, in particular limitations in global coagulation tests, the Japanese Society of Thrombosis and Hemostasis recently proposed tentative diagnostic criteria for DIC using hemostatic molecular markers and AT. The recommendations for treatment of DIC, especially the use of AT concentrates, recombinant activated protein C and thrombomodulin, vary among several guidelines for the management of DIC. These agents inhibit the effects of key proteases in activating coagulation and consequently exert an anti-inflammatory effect on DIC. Hence, it is necessary to extensively evaluate these agents in well-conducted clinical trials.

  9. Borrelia recurrentis employs a novel multifunctional surface protein with anti-complement, anti-opsonic and invasive potential to escape innate immunity.

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    Sonja Grosskinsky

    Full Text Available Borrelia recurrentis, the etiologic agent of louse-borne relapsing fever in humans, has evolved strategies, including antigenic variation, to evade immune defence, thereby causing severe diseases with high mortality rates. Here we identify for the first time a multifunctional surface lipoprotein of B. recurrentis, termed HcpA, and demonstrate that it binds human complement regulators, Factor H, CFHR-1, and simultaneously, the host protease plasminogen. Cell surface bound factor H was found to retain its activity and to confer resistance to complement attack. Moreover, ectopic expression of HcpA in a B. burgdorferi B313 strain, deficient in Factor H binding proteins, protected the transformed spirochetes from complement-mediated killing. Furthermore, HcpA-bound plasminogen/plasmin endows B. recurrentis with the potential to resist opsonization and to degrade extracellular matrix components. Together, the present study underscores the high virulence potential of B. recurrentis. The elucidation of the molecular basis underlying the versatile strategies of B. recurrentis to escape innate immunity and to persist in human tissues, including the brain, may help to understand the pathological processes underlying louse-borne relapsing fever.

  10. New therapies for hereditary angioedema: disease outlook changes dramatically.

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    Frank, Michael M; Jiang, Haixiang

    2008-01-01

    Hereditary angioedema (HAE) is an autosomal dominant disease associated with episodic attacks of nonpitting edema that may affect any external or mucosal body surface. Attacks most often affect the extremities, causing local swelling, the GI tract, leading to severe abdominal pain, and the mouth and throat, at times causing asphyxiation. Most patients with HAE have low levels of the plasma serine protease inhibitor C1 inhibitor. The edema in these patients is caused by unregulated generation of bradykinin. Effective chronic therapy of patients with impeded androgens or plasmin inhibitors has been available for decades, but in the United States, we do not have therapy for acute attacks. Five companies have completed or are in the process of conducting phase 3 clinical trials, double-blind, placebo-controlled studies of products designed to terminate acute attacks or to be used in prophylaxis. Two companies, Lev Pharmaceuticals and CSL Behring, have preparations of C1 inhibitor purified from plasma that have been used in Europe for decades (trade names Cinryze and Berinert P, respectively). One company, Pharming, has developed a recombinant C1 inhibitor preparation. One company, Dyax, is testing a kallikrein inhibitor (ecallantide), and one company, Jerini, is completing testing of a bradykinin type 2 receptor antagonist (Icatibant). Although little has been published thus far, all of these products may prove effective. It is likely that HAE treatment will change dramatically within the next few years.

  11. Tranexamic Acid in the Treatment of Melasma: A Review of the Literature.

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    Perper, Marina; Eber, Ariel Eva; Fayne, Rachel; Verne, Sebastian Hugo; Magno, Robert James; Cervantes, Jessica; ALharbi, Mana; ALOmair, Ibrahim; Alfuraih, Abdulkarem; Nouri, Keyvan

    2017-03-10

    Melasma is a common acquired pigmentary disorder marked by irregular hyperpigmented macules or patches and most commonly occurs in women of darker skin color. It is a chronic often-relapsing condition that causes negative psychosocial effects in those affected. Current treatments such as hydroquinone, kojic acid, and retinoids, among others, demonstrate variable efficacy and side-effect profiles. We conducted a comprehensive literature review examining the use of tranexamic acid (TA), a well-known anti-fibrinolytic agent, in the treatment of melasma. TA delivered orally, topically, and through physical methods works via the inhibition of ultraviolet (UV)-induced plasmin activity in keratinocytes. Predefined search terms were entered into PubMed. Articles were then independently screened by two authors to include only those written in the English language and relating to human subjects with at least mild melasma. The search identified 28 articles, 15 of which met the criteria for full review. The review revealed that TA treatment for melasma is equally effective or more effective than other standard therapies and may induce fewer side effects. Our comprehensive review suggests that TA may be a promising treatment option for melasma because of its demonstrated effectiveness alone and in combination with other modalities as well as its limited side-effect profile.

  12. Aryl Hydrocarbon Receptor Activation by TCDD Modulates Expression of Extracellular Matrix Remodeling Genes during Experimental Liver Fibrosis

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    Lamb, Cheri L.; Cholico, Giovan N.; Perkins, Daniel E.; Fewkes, Michael T.; Oxford, Julia Thom; Lujan, Trevor J.; Morrill, Erica E.

    2016-01-01

    The aryl hydrocarbon receptor (AhR) is a soluble, ligand-activated transcription factor that mediates the toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Increasing evidence implicates the AhR in regulating extracellular matrix (ECM) homeostasis. We recently reported that TCDD increased necroinflammation and myofibroblast activation during liver injury elicited by carbon tetrachloride (CCl4). However, TCDD did not increase collagen deposition or exacerbate fibrosis in CCl4-treated mice, which raises the possibility that TCDD may enhance ECM turnover. The goal of this study was to determine how TCDD impacts ECM remodeling gene expression in the liver. Male C57BL/6 mice were treated for 8 weeks with 0.5 mL/kg CCl4, and TCDD (20 μg/kg) was administered during the last two weeks. Results indicate that TCDD increased mRNA levels of procollagen types I, III, IV, and VI and the collagen processing molecules HSP47 and lysyl oxidase. TCDD also increased gelatinase activity and mRNA levels of matrix metalloproteinase- (MMP-) 3, MMP-8, MMP-9, and MMP-13. Furthermore, TCDD modulated expression of genes in the plasminogen activator/plasmin system, which regulates MMP activation, and it also increased TIMP1 gene expression. These findings support the notion that AhR activation by TCDD dysregulates ECM remodeling gene expression and may facilitate ECM metabolism despite increased liver injury. PMID:27672655

  13. SALO, a novel classical pathway complement inhibitor from saliva of the sand fly Lutzomyia longipalpis

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    Ferreira, Viviana P.; Fazito Vale, Vladimir; Pangburn, Michael K.; Abdeladhim, Maha; Ferreira Mendes-Sousa, Antonio; Coutinho-Abreu, Iliano V.; Rasouli, Manoochehr; Brandt, Elizabeth A.; Meneses, Claudio; Lima, Kolyvan Ferreira; Nascimento Araújo, Ricardo; Horácio Pereira, Marcos; Kotsyfakis, Michalis; Oliveira, Fabiano; Kamhawi, Shaden; Ribeiro, Jose M. C.; Gontijo, Nelder F.; Collin, Nicolas; Valenzuela, Jesus G.

    2016-01-01

    Blood-feeding insects inject potent salivary components including complement inhibitors into their host’s skin to acquire a blood meal. Sand fly saliva was shown to inhibit the classical pathway of complement; however, the molecular identity of the inhibitor remains unknown. Here, we identified SALO as the classical pathway complement inhibitor. SALO, an 11 kDa protein, has no homology to proteins of any other organism apart from New World sand flies. rSALO anti-complement activity has the same chromatographic properties as the Lu. longipalpis salivary gland homogenate (SGH)counterparts and anti-rSALO antibodies blocked the classical pathway complement activity of rSALO and SGH. Both rSALO and SGH inhibited C4b deposition and cleavage of C4. rSALO, however, did not inhibit the protease activity of C1s nor the enzymatic activity of factor Xa, uPA, thrombin, kallikrein, trypsin and plasmin. Importantly, rSALO did not inhibit the alternative or the lectin pathway of complement. In conclusion our data shows that SALO is a specific classical pathway complement inhibitor present in the saliva of Lu. longipalpis. Importantly, due to its small size and specificity, SALO may offer a therapeutic alternative for complement classical pathway-mediated pathogenic effects in human diseases. PMID:26758086

  14. Altered behavior in mice with deletion of the alpha2-antiplasmin gene.

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    Kawashita, Eri; Kanno, Yosuke; Ikeda, Kanako; Kuretake, Hiromi; Matsuo, Osamu; Matsuno, Hiroyuki

    2014-01-01

    The α2-antiplasmin (α2AP) protein is known to be a principal physiological inhibitor of plasmin, and is expressed in various part of the brain, including the hippocampus, cortex, hypothalamus and cerebellum, thus suggesting a potential role for α2AP in brain functions. However, the involvement of α2AP in brain functions is currently unclear. The goal of this study was to investigate the effects of the deletion of the α2AP gene on the behavior of mice. The motor function was examined by the wire hang test and rotarod test. To evaluate the cognitive function, a repeated rotarod test, Y-maze test, Morris water maze test, passive or shuttle avoidance test and fear conditioning test were performed. An open field test, dark/light transition test or tail suspension test was performed to determine the involvement of α2AP in anxiety or depression-like behavior. The α2AP knockout (α2AP-/-) mice exhibited impaired motor function compared with α2AP+/+ mice. The α2AP-/- mice also exhibited impairments in motor learning, working memory, spatial memory and fear conditioning memory. Furthermore, the deletion of α2AP induced anxiety-like behavior, and caused an anti-depression-like effect in tail suspension. Therefore, our findings suggest that α2AP is a crucial mediator of motor function, cognitive function, anxiety-like behavior and depression-like behavior, providing new insights into the role of α2AP in the brain functions.

  15. A prolonged course of Group A streptococcus-associated nephritis: a mild case of dense deposit disease (DDD)?

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    Sawanobori, E; Umino, A; Kanai, H; Matsushita, K; Iwasa, S; Kitamura, H; Oda, T; Yoshizawa, N; Sugita, K; Higashida, K

    2009-06-01

    We herein report the case of a 12-year-old boy with dense deposit disease (DDD) evoked by streptococcal infection. He had been diagnosed to have asymptomatic hematuria syndrome at the age of 6 during school screening. At 12 years of age, he was found to have macrohematuria and overt proteinuria with hypocomplementemia 2 months after streptococcal pharyngitis. Renal biopsy showed endocapillary proliferative glomerulonephritis with double contours of the glomerular basement membrane. Hypocomplementemia and proteinuria were sustained for over 8 weeks. He was suspected to have dense deposit disease due to intramembranous deposits in the first and the second biopsies. 1 month after treatment with methylprednisolone pulse therapy, proteinuria decreased to a normal level. Microscopic hematuria disappeared 2 years later, but mild hypocomplementemia persisted for more than 7 years. Nephritis-associated plasmin receptor (NAPlr), a nephritic antigen for acute poststreptococcal glomerulonephritis, was found to be positive in the glomeruli for more than 8 weeks. DDD is suggested to be caused by dysgeneration of the alternative pathway due to C3NeF and impaired Factor H activity. A persistent deposition of NAPlr might be one of the factors which lead to complement dysgeneration. A close relationship was suggested to exist between the streptococcal infection and dense deposit disease in this case.

  16. Plasminogen deficiency causes reduced corticospinal axonal plasticity and functional recovery after stroke in mice.

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    Zhongwu Liu

    Full Text Available Tissue plasminogen activator (tPA has been implicated in neurite outgrowth and neurological recovery post stroke. tPA converts the zymogen plasminogen (Plg into plasmin. In this study, using plasminogen knockout (Plg-/- mice and their Plg-native littermates (Plg+/+, we investigated the role of Plg in axonal remodeling and neurological recovery after stroke. Plg+/+ and Plg-/- mice (n = 10/group were subjected to permanent intraluminal monofilament middle cerebral artery occlusion (MCAo. A foot-fault test and a single pellet reaching test were performed prior to and on day 3 after stroke, and weekly thereafter to monitor functional deficit and recovery. Biotinylated dextran amine (BDA was injected into the left motor cortex to anterogradely label the corticospinal tract (CST. Animals were euthanized 4 weeks after stroke. Neurite outgrowth was also measured in primary cultured cortical neurons harvested from Plg+/+ and Plg-/- embryos. In Plg+/+ mice, the motor functional deficiency after stroke progressively recovered with time. In contrast, recovery in Plg-/- mice was significantly impaired compared to Plg+/+ mice (p0.82, p<0.01. Plg-/- neurons exhibited significantly reduced neurite outgrowth. Our data suggest that plasminogen-dependent proteolysis has a beneficial effect during neurological recovery after stroke, at least in part, by promoting axonal remodeling in the denervated spinal cord.

  17. Characterization of Three Novel Adhesins of Leptospira interrogans

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    Siqueira, Gabriela H.; Atzingen, Marina V.; Alves, Ivy J.; de Morais, Zenaide M.; Vasconcellos, Silvio A.; Nascimento, Ana L. T. O.

    2013-01-01

    We report cloning, expression, purification, and characterization of three predicted leptospiral membrane proteins (LIC11360, LIC11009, and LIC11975). In silico analysis and proteinase K accessibility data suggest that these proteins might be surface exposed. We show that proteins encoded by LIC11360, LIC11009 and LIC11975 genes interact with laminin in a dose-dependent and saturable manner. The proteins are referred to as leptospiral surface adhesions 23, 26, and 36 (Lsa23, Lsa26, and Lsa36), respectively. These proteins also bind plasminogen and generate active plasmin. Attachment of Lsa23 and Lsa36 to fibronectin occurs through the involvement of the 30-kDa and 70-kDa heparin-binding domains of the ligand. Dose-dependent, specific-binding of Lsa23 to the complement regulator C4BP and to a lesser extent, to factor H, suggests that this protein may interfere with the complement cascade pathways. Leptospira spp. may use these interactions as possible mechanisms during the establishment of infection. PMID:23958908

  18. Features of Two New Proteins with OmpA-Like Domains Identified in the Genome Sequences of Leptospira interrogans

    Science.gov (United States)

    Teixeira, Aline F.; de Morais, Zenaide M.; Kirchgatter, Karin; Romero, Eliete C.; Vasconcellos, Silvio A.; Nascimento, Ana Lucia T. O.

    2015-01-01

    Leptospirosis is an acute febrile disease caused by pathogenic spirochetes of the genus Leptospira. It is considered an important re-emerging infectious disease that affects humans worldwide. The knowledge about the mechanisms by which pathogenic leptospires invade and colonize the host remains limited since very few virulence factors contributing to the pathogenesis of the disease have been identified. Here, we report the identification and characterization of two new leptospiral proteins with OmpA-like domains. The recombinant proteins, which exhibit extracellular matrix-binding properties, are called Lsa46 - LIC13479 and Lsa77 - LIC10050 (Leptospiral surface adhesins of 46 and 77 kDa, respectively). Attachment of Lsa46 and Lsa77 to laminin was specific, dose dependent and saturable, with KD values of 24.3 ± 17.0 and 53.0 ± 17.5 nM, respectively. Lsa46 and Lsa77 also bind plasma fibronectin, and both adhesins are plasminogen (PLG)-interacting proteins, capable of generating plasmin (PLA) and as such, increase the proteolytic ability of leptospires. The proteins corresponding to Lsa46 and Lsa77 are present in virulent L. interrogans L1-130 and in saprophyte L. biflexa Patoc 1 strains, as detected by immunofluorescence. The adhesins are recognized by human leptospirosis serum samples at the onset and convalescent phases of the disease, suggesting that they are expressed during infection. Taken together, our data could offer valuable information to the understanding of leptospiral pathogenesis. PMID:25849456

  19. Purification, crystallization and preliminary X-ray diffraction analysis of saxthrombin, a thrombin-like enzyme from Gloydius saxatilis venom

    Energy Technology Data Exchange (ETDEWEB)

    Wei, Wenqing; Zhao, Wei [Hefei National Laboratory for Physical Sciences at Microscale and School of Life Sciences, University of Science and Technology of China, 96 Jinzhai Road, Hefei, Anhui 230027 (China); Key Laboratory of Structural Biology, Chinese Academy of Sciences, 96 Jinzhai Road, Hefei, Anhui 230027 (China); Wang, Xiaoping [National Conservation of Snake Island and Laotieshan Mountain, Dalian, Liaoning, 116041 (China); Teng, Maikun, E-mail: mkteng@ustc.edu.cn; Niu, Liwen, E-mail: mkteng@ustc.edu.cn [Hefei National Laboratory for Physical Sciences at Microscale and School of Life Sciences, University of Science and Technology of China, 96 Jinzhai Road, Hefei, Anhui 230027 (China); Key Laboratory of Structural Biology, Chinese Academy of Sciences, 96 Jinzhai Road, Hefei, Anhui 230027 (China)

    2007-08-01

    The thrombin-like enzyme saxthrombin has been purified from G. saxatilis snake venom. Crystallization conditions were found and a data set was obtained to 1.43 Å. The snake-venom thrombin-like enzymes (SVTLEs) are a class of serine proteinases that show fibrinogen-clotting and esterolytic activities. Most TLEs convert fibrinogen to fibrin by releasing either fibrinopeptide A or fibrinopeptide B and cannot activate factor XIII. The enzymes hydrolyze fibrinogen to produce non-cross-linked fibrins, which are susceptible to the lytic action of plasmin. Because of these physiological properties, TLEs have important medical applications in myocardial infarction, ischaemic stroke and thrombotic diseases. Here, a three-step chromatography procedure was used to purify saxthrombin (AAP20638) from Gloydius saxatilis venom to homogeneity. Its molecular weight is about 30 kDa as estimated by SDS–PAGE. A saxthrombin crystal was obtained using the hanging-drop vapour-diffusion method and diffracted to a resolution limit of 1.43 Å. The crystal belongs to space group C2, with unit-cell parameters a = 97.23, b = 52.21, c = 50.10 Å, β = 96.72°, and the Matthews coefficient (V{sub M}) was calculated to be 2.13 Å{sup 3} Da{sup −1} with one molecule in the asymmetric unit.

  20. Crystallization and preliminary X-ray analysis of a Kunitz-type inhibitor, textilinin-1 from Pseudonaja textilis textilis

    Energy Technology Data Exchange (ETDEWEB)

    Millers, Emma-Karin I. [School of Molecular and Microbial Sciences, University of Queensland, Brisbane 4072, QLD (Australia); Masci, Paul P. [School of Medicine, Southern Division, University of Queensland, Princess Alexandra Hospital, Woolloongabba, Brisbane 4102, QLD (Australia); Lavin, Martin F. [The Queensland Cancer Fund Research Unit, The Queensland Institute of Medical Research, PO Box, Royal Brisbane Hospital, Herston, Brisbane 4029, QLD (Australia); Jersey, John de; Guddat, Luke W., E-mail: luke.guddat@uq.edu.au [School of Molecular and Microbial Sciences, University of Queensland, Brisbane 4072, QLD (Australia)

    2006-07-01

    Crystals of a canonical inhibitor of plasmin from Australian Brown snake venom has been obtained. In complex with trypsin these diffract to 2.0 Å resolution, while the free inhibitor diffracts to 1.63 Å. Textilinin-1 (Txln-1), a Kunitz-type serine protease inhibitor, is a 59-amino-acid polypeptide isolated from the venom of the Australian Common Brown snake Pseudonaja textilis textilis. This molecule has been suggested as an alternative to aprotinin, also a Kunitz-type serine protease inhibitor, for use as an anti-bleeding agent in surgical procedures. Txln-1 shares only 47% amino-acid identity to aprotinin; however, six cysteine residues in the two peptides are in conserved locations. It is therefore expected that the overall fold of these molecules is similar but that they have contrasting surface features. Here, the crystallization of recombinant textilinin-1 (rTxln-1) as the free molecule and in complex with bovine trypsin (229 amino acids) is reported. Two organic solvents, phenol and 1,4-butanediol, were used as additives to facilitate the crystallization of free rTxln-1. Crystals of the rTxln-1–bovine trypsin complex diffracted to 2.0 Å resolution, while crystals of free rTxln-1 diffracted to 1.63 Å resolution.

  1. Functional Imaging of Proteolysis: Stromal and Inflammatory Cells Increase Tumor Proteolysis

    Directory of Open Access Journals (Sweden)

    Mansoureh Sameni

    2003-07-01

    Full Text Available The underlying basement membrane is degraded during progression of breast and colon carcinoma. Thus, we imaged degradation of a quenched fluorescent derivative of basement membrane type IV collagen (DQ-collagen IV by living human breast and colon tumor spheroids. Proteolysis of DQ-collagen IV by HCT 116 and HKh-2 human colon tumor spheroids was both intracellular and pericellular. In contrast, proteolysis of DQ-collagen IV by BT20 human breast tumor spheroids was pericellular. As stromal elements can contribute to proteolytic activities associated with tumors, we also examined degradation of DQ-collagen IV by human monocytes/macrophages and colon and breast fibroblasts. Fibroblasts themselves exhibited a modest amount of pericellular degradation. Degradation was increased 4–17-fold in cocultures of fibroblasts and tumor cells as compared to either cell type alone. Inhibitors of matrix metalloproteinases, plasmin, and the cysteine protease, cathepsin B, all reduced degradation in the cocultures. Monocytes did not degrade DQ-collagen IV; however, macrophages degraded DQ-collagen IV intracellularly. In coculture of tumor cells, fibroblasts, and macrophages, degradation of DQ-collagen IV was further increased. Imaging of living tumor and stromal cells has, thus, allowed us to establish that tumor proteolysis occurs pericellularly and intracellularly and that tumor, stromal, and inflammatory cells all contribute to degradative processes.

  2. Enzymological characterization of Flla, a fibrinolytic enzyme from Agkistrodon acutus venom

    Institute of Scientific and Technical Information of China (English)

    Xiu-xia LIANG; Ying-na ZHOU; Jia-shu CHEN; Peng-xin QIU; Hui-zhen CHEN; Huan-huan SUN; Yu-ping WU; Guang-mei YAN

    2005-01-01

    Aim: To study the enzymological characterization of a fibrinolytic enzyme (FⅡa)from Agkistrodon acutus venom. Methods: The fibrinogenolytic effect and the influences of several protease inhibitors, chelating agents, and metal ions on fibrinogenolytic activity were visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The metal content of FⅡa was determined by atomic absorption spectroscopy. Results: After incubation with EⅡa (0.25 g/L), Aα-, Bβ-and γ-chains of fibrinogen disappeared within 5 min, 30 min, and 8 h, respectively.The molecular weights of major degradation products were 45 000 and 41 000,which were different from those bands produced by plasmin. The fibrinogenolytic activity of FⅡa was strongly inhibited by ethylenediamine tetraacetic acid (EDTA),ethyleneglycol tetraacetic acid (EGTA), dithiothreitol and cysteine, but not by phenylmethyl-sulfonyl fluoride and soybean trypsin inhibitor. Zinc (3171±25mg/kg), potassium (489± 17 mg/kg) and calcium (319± 13 mg/kg) were found in FⅡa.Zn2+, Ca2+ and Mg2+ could recover the fibrinogenolytic activity of FⅡa, which was inhibited by EDTA. Only Ca2+ could recover the fibrinogenolytic activity inhibited by EGTA. Conclusion: FⅡa can degrade the Aα-, Bβ- and γ-chains of fibrinogen.EⅡa is a metalloproteinase, and Zn2+, Ca2+, and disulfide bonds are necessary for its fibrinogenolytic activity.

  3. Increased plasminogen activator inhibitor results in a hypofibrinolytic state in adolescents with obesity: in vivo and ex vivo evidence.

    Science.gov (United States)

    Dietrich, Kevin; Ball, Geoff D C; Mitchell, Lesley G

    2016-10-01

    Obesity in adolescents increases their risk for deep vein thrombosis. The objectives of this study were to determine potential mechanisms for thrombotic risk by investigating the fibrinolytic pathway in a sample of adolescents with and without obesity. Thirty-seven adolescents with obesity and 16 normal weight age-matched controls were recruited. Plasma levels of components of the fibrinolytic system were measured in addition to a Global Haemostasis Potential assay (GHP), which assesses plasma capacity to generate and lyse a fibrin clot. Levels of plasminogen activator inhibitor (PAI) and tissue plasminogen activator (tPA)/PAI complexes were increased in adolescents with obesity when compared to normal weight controls. There was a significant inverse association of increasing PAI with a decrease in plasmin-antiplasmin complexes. The GHP in obese adolescents displayed a hypofibrinolytic response with a markedly increased t½ clot lysis time, as well as an increase in fibrin clot density as indicated by increased absorbance at maximum peak height. In the obese group, immunodepletion of PAI decreased both t½ lysis time and absorbance at maximum peak height. We have shown in vivo and ex vivo there is a hypofibrinolytic state in obese adolescents and have established the hypofibrinolytic state is due to increased PAI levels. © 2016 John Wiley & Sons Ltd.

  4. Tissue-type plasminogen activator is a neuroprotectant in the mouse hippocampus.

    Science.gov (United States)

    Echeverry, Ramiro; Wu, Jialing; Haile, Woldeab B; Guzman, Johanna; Yepes, Manuel

    2010-06-01

    The best-known function of the serine protease tissue-type plasminogen activator (tPA) is as a thrombolytic enzyme. However, it is also found in structures of the brain that are highly vulnerable to hypoxia-induced cell death, where its association with neuronal survival is poorly understood. Here, we have demonstrated that hippocampal areas of the mouse brain lacking tPA activity are more vulnerable to neuronal death following an ischemic insult. We found that sublethal hypoxia, which elicits tolerance to subsequent lethal hypoxic/ischemic injury in a natural process known as ischemic preconditioning (IPC), induced a rapid release of neuronal tPA. Treatment of hippocampal neurons with tPA induced tolerance against a lethal hypoxic insult applied either immediately following insult (early IPC) or 24 hours later (delayed IPC). tPA-induced early IPC was independent of the proteolytic activity of tPA and required the engagement of a member of the LDL receptor family. In contrast, tPA-induced delayed IPC required the proteolytic activity of tPA and was mediated by plasmin, the NMDA receptor, and PKB phosphorylation. We also found that IPC in vivo increased tPA activity in the cornu ammonis area 1 (CA1) layer and Akt phosphorylation in the hippocampus, as well as ischemic tolerance in wild-type but not tPA- or plasminogen-deficient mice. These data show that tPA can act as an endogenous neuroprotectant in the murine hippocampus.

  5. Cloning of a serine proteinase inhibitor from bovine brain: expression in the brain and characterization of its target proteinases.

    Science.gov (United States)

    Nakaya, N; Nishibori, M; Kawabata, M; Saeki, K

    1996-12-01

    A cDNA encoding of the serine proteinase inhibitor (serpin), B-43, was cloned from the cDNA library of the bovine brain. It encoded 378 amino acids, and the MW of the protein was estimated to be 42.6 kDa, which is consistent with that of the native B-43 purified from the bovine brain. The homology search revealed that B-43 belongs to the ovalbumin branch of the serpin superfamily. Among them, B-43 was most homologous to human placental thrombin inhibitor (PI-6) and its murine counterpart, with the amino acid identity of 76% and 71%, respectively. Northern blot analysis showed that the size of the transcript was 1.4 kb, and that the expression of B-43 in the bovine brain varied depending on the brain regions, i.e. a lower level of expression was observed in the cerebral cortex and the hippocampus compared to the level of expression that was observed in the medulla oblongata. [35S]-labeled B-43 protein was synthesized in vitro by using a rabbit reticulocyte lysate system, which formed complexes with proteinases such as thrombin, trypsin, alpha-chymotrypsin, and 7S nerve growth factor (NGF), but not with urokinase or plasmin. These results, together with the immunohistochemical localization of B-43 in astrocytes and in some neurons which was observed in the previous study suggest that B-43 may be involved in the regulation of serine proteinases present in the brain or extravasated from the blood.

  6. Membrane depolarization induces calcium-dependent secretion of tissue plasminogen activator.

    Science.gov (United States)

    Gualandris, A; Jones, T E; Strickland, S; Tsirka, S E

    1996-04-01

    Tissue plasminogen activator (tPA), a serine protease that converts inactive plasminogen to active plasmin, is produced in the rat and mouse hippocampus and participates in neuronal plasticity. To help define the role of tPA in the nervous system, we have analyzed the regulation of its expression in the neuronal cell line PC12. In control cultures, tPA activity is exclusively cell-associated, and no activity is measurable in the culture medium. When the cells are treated with depolarizing agents, such as KCI, tPA activity becomes detectable in the medium. The increased secreted tPA activity is not accompanied by an increase in tPA mRNA levels, and it is not blocked by protein synthesis inhibitors. In contrast, tPA release is abolished by Ca2+ channel blockers, suggesting that chemically induced membrane depolarization stimulates the secretion of preformed enzyme. Moreover, KCI has a similar effect in vivo when administered to the murine brain via an osmotic pump: tPA activity increases along the CA2-CA3 regions and dentate gyrus of the hippocampal formation. These results demonstrate a neuronal activity-dependent secretory mechanism that can rapidly increase the amount of tPA in neuronal tissue.

  7. Neuroserpin, a brain-associated inhibitor of tissue plasminogen activator is localized primarily in neurons. Implications for the regulation of motor learning and neuronal survival.

    Science.gov (United States)

    Hastings, G A; Coleman, T A; Haudenschild, C C; Stefansson, S; Smith, E P; Barthlow, R; Cherry, S; Sandkvist, M; Lawrence, D A

    1997-12-26

    A cDNA clone for the serine proteinase inhibitor (serpin), neuroserpin, was isolated from a human whole brain cDNA library, and recombinant protein was expressed in insect cells. The purified protein is an efficient inhibitor of tissue type plasminogen activator (tPA), having an apparent second-order rate constant of 6. 2 x 10(5) M-1 s-1 for the two-chain form. However, unlike other known plasminogen activator inhibitors, neuroserpin is a more effective inactivator of tPA than of urokinase-type plasminogen activator. Neuroserpin also effectively inhibited trypsin and nerve growth factor-gamma but reacted only slowly with plasmin and thrombin. Northern blot analysis showed a 1.8 kilobase messenger RNA expressed predominantly in adult human brain and spinal cord, and immunohistochemical studies of normal mouse tissue detected strong staining primarily in neuronal cells with occasionally positive microglial cells. Staining was most prominent in the ependymal cells of the choroid plexus, Purkinje cells of the cerebellum, select neurons of the hypothalamus and hippocampus, and in the myelinated axons of the commissura. Expression of tPA within these regions is reported to be high and has previously been correlated with both motor learning and neuronal survival. Taken together, these data suggest that neuroserpin is likely to be a critical regulator of tPA activity in the central nervous system, and as such may play an important role in neuronal plasticity and/or maintenance.

  8. Extracellular proteolysis in structural and functional plasticity of mossy fiber synapses in hippocampus.

    Science.gov (United States)

    Wiera, Grzegorz; Mozrzymas, Jerzy W

    2015-01-01

    Brain is continuously altered in response to experience and environmental changes. One of the underlying mechanisms is synaptic plasticity, which is manifested by modification of synapse structure and function. It is becoming clear that regulated extracellular proteolysis plays a pivotal role in the structural and functional remodeling of synapses during brain development, learning and memory formation. Clearly, plasticity mechanisms may substantially differ between projections. Mossy fiber synapses onto CA3 pyramidal cells display several unique functional features, including pronounced short-term facilitation, a presynaptically expressed long-term potentiation (LTP) that is independent of NMDAR activation, and NMDA-dependent metaplasticity. Moreover, structural plasticity at mossy fiber synapses ranges from the reorganization of projection topology after hippocampus-dependent learning, through intrinsically different dynamic properties of synaptic boutons to pre- and postsynaptic structural changes accompanying LTP induction. Although concomitant functional and structural plasticity in this pathway strongly suggests a role of extracellular proteolysis, its impact only starts to be investigated in this projection. In the present report, we review the role of extracellular proteolysis in various aspects of synaptic plasticity in hippocampal mossy fiber synapses. A growing body of evidence demonstrates that among perisynaptic proteases, tissue plasminogen activator (tPA)/plasmin system, β-site amyloid precursor protein-cleaving enzyme 1 (BACE1) and metalloproteinases play a crucial role in shaping plastic changes in this projection. We discuss recent advances and emerging hypotheses on the roles of proteases in mechanisms underlying mossy fiber target specific synaptic plasticity and memory formation.

  9. Neuronal degeneration and a decrease in laminin-like immunoreactivity is associated with elevated tissue-type plasminogen activator in the rat hippocampus after kainic acid injection.

    Science.gov (United States)

    Nagai, N; Urano, T; Endo, A; Takahashi, H; Takada, Y; Takada, A

    1999-02-01

    Tissue-type plasminogen activator (tPA) is a serine protease that converts the inactive precursor plasminogen to the active protease plasmin. In the central nervous system, tPA has been suggested to participate in plasticity, memory and the neuronal degeneration caused by excitotoxins, but its precise functions during these processes are still unclear. We show in this report that tPA antigen level and extracellular tPA activity increased in the hippocampus during the early stages of neuronal degeneration in the CA3 region following the injection of kainic acid (KA) into the lateral cerebral ventricles. The increase in tPA antigen level was transient and its peak was at 4 h after the injection. tPA activity was also increased 4 h after the injection, but it remained at a high level for more than 8 h. Histological zymography showed that the increase in tPA activity was mainly localized in the CA3 region. In the same region, the disappearance of interneuronal laminin-like immunoreactivity and atrophic changes in pyramidal neurons were observed 4 h after the injection of KA. These results suggested that such focal and transient increases in tPA synthesis and release, which result in the proteolysis of laminin through plasminogen activation, could be involved in the neuronal degeneration in the CA3 region after the injection of KA.

  10. Modulation of zinc toxicity by tissue plasminogen activator.

    Science.gov (United States)

    Siddiq, Mustafa M; Tsirka, Stella E

    2004-01-01

    The tissue plasminogen activator (tPA)-plasmin proteolytic system mediates excitotoxin-induced neurodegeneration in vivo and in cell culture. tPA also confers neuroprotection from zinc toxicity in cell culture through a proteolysis-independent mechanism. This raises two questions: what is this non-enzymatic mechanism, and why tPA does not synergize with zinc to promote neuronal cell death? We show here that zinc binds to tPA and inhibits its activity in a dose-dependent fashion, thus terminating its protease-dependent neurotoxic capacity. We extend the previously reported culture findings to demonstrate that elevated zinc is neurotoxic in vivo, and even more so when tPA is absent. Thus, physiological levels of tPA confer protection from elevated free zinc. Mechanistically, tPA promotes movement of zinc into hippocampal neuron cells through voltage-sensitive Ca(2+) channels and Ca(2+)-permeable AMPA/KA channels. Therefore, zinc and tPA each appear to be able to limit the potential of the other to facilitate neurodegeneration, a reciprocal set of actions that may be critical in the hippocampus where tPA is secreted during the nonpathological conditions of learning and memory at sites known to be repositories of free and sequestered zinc.

  11. Proteolytic fragments of laminin promote excitotoxic neurodegeneration by up-regulation of the KA1 subunit of the kainate receptor.

    Science.gov (United States)

    Chen, Zu-Lin; Yu, Huaxu; Yu, Wei-Ming; Pawlak, Robert; Strickland, Sidney

    2008-12-29

    Degradation of the extracellular matrix (ECM) protein laminin contributes to excitotoxic cell death in the hippocampus, but the mechanism of this effect is unknown. To study this process, we disrupted laminin gamma1 (lamgamma1) expression in the hippocampus. Lamgamma1 knockout (KO) and control mice had similar basal expression of kainate (KA) receptors, but the lamgamma1 KO mice were resistant to KA-induced neuronal death. After KA injection, KA1 subunit levels increased in control mice but were unchanged in lamgamma1 KO mice. KA1 levels in tissue plasminogen activator (tPA)-KO mice were also unchanged after KA, indicating that both tPA and laminin were necessary for KA1 up-regulation after KA injection. Infusion of plasmin-digested laminin-1 into the hippocampus of lamgamma1 or tPA KO mice restored KA1 up-regulation and KA-induced neuronal degeneration. Interfering with KA1 function with a specific anti-KA1 antibody protected against KA-induced neuronal death both in vitro and in vivo. These results demonstrate a novel pathway for neurodegeneration involving proteolysis of the ECM and KA1 KA receptor subunit up-regulation.

  12. Tissue plasminogen activator and plasminogen mediate stress-induced decline of neuronal and cognitive functions in the mouse hippocampus.

    Science.gov (United States)

    Pawlak, Robert; Rao, B S Shankaranarayana; Melchor, Jerry P; Chattarji, Sumantra; McEwen, Bruce; Strickland, Sidney

    2005-12-13

    Repeated stress can impair function in the hippocampus, a brain structure essential for learning and memory. Although behavioral evidence suggests that severe stress triggers cognitive impairment, as seen in major depression or posttraumatic stress disorder, little is known about the molecular mediators of these functional deficits in the hippocampus. We report here both pre- and postsynaptic effects of chronic stress, manifested as a reduction in the number of NMDA receptors, dendritic spines, and expression of growth-associated protein-43 in the cornu ammonis 1 region. Strikingly, the stress-induced decrease in NMDA receptors coincides spatially with sites of plasminogen activation, thereby predicting a role for tissue plasminogen activator (tPA) in this form of stress-induced plasticity. Consistent with this possibility, tPA-/- and plasminogen-/- mice are protected from stress-induced decrease in NMDA receptors and reduction in dendritic spines. At the behavioral level, these synaptic and molecular signatures of stress-induced plasticity are accompanied by impaired acquisition, but not retrieval, of hippocampal-dependent spatial learning, a deficit that is not exhibited by the tPA-/- and plasminogen-/- mice. These findings establish the tPA/plasmin system as an important mediator of the debilitating effects of prolonged stress on hippocampal function at multiple levels of neural organization.

  13. An extracellular proteolytic cascade promotes neuronal degeneration in the mouse hippocampus.

    Science.gov (United States)

    Tsirka, S E; Rogove, A D; Bugge, T H; Degen, J L; Strickland, S

    1997-01-15

    Mice lacking the serine protease tissue plasminogen activator (tPA) are resistant to excitotoxin-mediated hippocampal neuronal degeneration. We have used genetic and cellular analyses to study the role of tPA in neuronal cell death. Mice deficient for the zymogen plasminogen, a known substrate for tPA, are also resistant to excitotoxins, implicating an extracellular proteolytic cascade in degeneration. The two known components of this cascade, tPA and plasminogen, are both synthesized in the mouse hippocampus. tPA mRNA and protein are present in neurons and microglia, whereas plasminogen mRNA and protein are found exclusively in neurons. tPA-deficient mice exhibit attenuated microglial activation as a reaction to neuronal injury. In contrast, the microglial response of plasminogen-deficient mice was comparable to that of wild-type mice, suggesting a tPA-mediated, plasminogen-independent pathway for activation of microglia. Infusion of inhibitors of the extracellular tPA/plasmin proteolytic cascade into the hippocampus protects neurons against excitotoxic injury, suggesting a novel strategy for intervening in neuronal degeneration.

  14. Features of two new proteins with OmpA-like domains identified in the genome sequences of Leptospira interrogans.

    Directory of Open Access Journals (Sweden)

    Aline F Teixeira

    Full Text Available Leptospirosis is an acute febrile disease caused by pathogenic spirochetes of the genus Leptospira. It is considered an important re-emerging infectious disease that affects humans worldwide. The knowledge about the mechanisms by which pathogenic leptospires invade and colonize the host remains limited since very few virulence factors contributing to the pathogenesis of the disease have been identified. Here, we report the identification and characterization of two new leptospiral proteins with OmpA-like domains. The recombinant proteins, which exhibit extracellular matrix-binding properties, are called Lsa46 - LIC13479 and Lsa77 - LIC10050 (Leptospiral surface adhesins of 46 and 77 kDa, respectively. Attachment of Lsa46 and Lsa77 to laminin was specific, dose dependent and saturable, with KD values of 24.3 ± 17.0 and 53.0 ± 17.5 nM, respectively. Lsa46 and Lsa77 also bind plasma fibronectin, and both adhesins are plasminogen (PLG-interacting proteins, capable of generating plasmin (PLA and as such, increase the proteolytic ability of leptospires. The proteins corresponding to Lsa46 and Lsa77 are present in virulent L. interrogans L1-130 and in saprophyte L. biflexa Patoc 1 strains, as detected by immunofluorescence. The adhesins are recognized by human leptospirosis serum samples at the onset and convalescent phases of the disease, suggesting that they are expressed during infection. Taken together, our data could offer valuable information to the understanding of leptospiral pathogenesis.

  15. Acetic acid in aged vinegar affects molecular targets for thrombus disease management.

    Science.gov (United States)

    Jing, Li; Yanyan, Zhang; Junfeng, Fan

    2015-08-01

    To elucidate the mechanism underlying the action of dietary vinegar on antithrombotic activity, acetic acid, the main acidic component of dietary vinegar, was used to determine antiplatelet and fibrinolytic activity. The results revealed that acetic acid significantly inhibits adenosine diphosphate (ADP)-, collagen-, thrombin-, and arachidonic acid (AA)-induced platelet aggregation. Acetic acid (2.00 mM) reduced AA-induced platelet aggregation to approximately 36.82 ± 1.31%, and vinegar (0.12 mL L(-1)) reduced the platelet aggregation induced by AA to 30.25 ± 1.34%. Further studies revealed that acetic acid exerts its effects by inhibiting cyclooxygenase-1 and the formation of thromboxane-A2. Organic acids including acetic acid, formic acid, lactic acid, citric acid, and malic acid also showed fibrinolytic activity; specifically, the fibrinolytic activity of acetic acid amounted to 1.866 IU urokinase per mL. Acetic acid exerted its fibrinolytic activity by activating plasminogen during fibrin crossing, thus leading to crosslinked fibrin degradation by the activated plasmin. These results suggest that organic acids in dietary vinegar play important roles in the prevention and cure of cardiovascular diseases.

  16. Effect of ascorbate on fibrinolytic factors in septic mouse skeletal muscle.

    Science.gov (United States)

    Swarbreck, Scott; Secor, Dan; Li, Fuyan; Gross, Peter L; Ellis, Christopher G; Sharpe, Michael D; Wilson, John X; Tyml, Karel

    2014-10-01

    Plugging of the capillary bed in tissues correlates with organ failure during sepsis. In septic mouse skeletal muscle, we showed that blood in capillaries becomes hypercoagulable and that ascorbate injection inhibits capillary plugging. In the present study, we hypothesized that ascorbate promotes fibrinolysis, reversing this plugging. Sepsis in mice was induced by fecal injection into peritoneum. Mice were injected intravenously with a bolus of streptokinase (fibrinolytic agent) or ascorbate at 5-6 h. Both agents reversed capillary plugging in muscle at 7 h. Sepsis increased mRNA expression of urokinase plasminogen activator (u-PA) (profibrinolytic) and plasminogen activator inhibitor 1 (PAI-1) (antifibrinolytic) in muscle and liver homogenates at 7 h. Ascorbate did not affect u-PA mRNA in either tissue, but it inhibited PAI-1 mRNA in muscle, suggesting enhanced fibrinolysis in this tissue. However, ascorbate did not affect increased PAI-1 mRNA in the liver (dominant source of soluble PAI-1 in systemic blood). Consistently, ascorbate affected neither elevated PAI-1 protein/enzymatic activity in septic liver nor lowered plasmin antiplasmin level in septic blood. Furthermore, hypocoagulability of septic blood revealed by thrombelastography and thrombin-induced PAI-1 release from isolated platelets (ex-vivo model of sepsis) were not affected by ascorbate. Based on the PAI-1 protein data, the present study does not support the hypothesis that ascorbate promotes fibrinolysis in sepsis.

  17. Fibrinolytic enzyme production by newly isolated Bacillus cereus SRM-001 with enhanced in-vitro blood clot lysis potential.

    Science.gov (United States)

    Narasimhan, Manoj Kumar; Chandrasekaran, Muthukumaran; Rajesh, Mathur

    2015-01-01

    The discovery of plasmin-like microbial fibrinolytic enzymes having high specificity and negligible side effects is crucial for thrombolytic therapy. Herein, we report one such extra-cellular fibrinolytic enzyme producing Bacillus cereus SRM-001 isolated from the blood-laden soil of a chicken dump yard. The potency of the enzyme was established with fibrin plate assay and in-vitro blood clot lysis assay. The shake-flask operating parameters and media composition were optimized for maximizing the productivity of the enzyme. The operating parameters, pH 7, 37°C, 1% inoculum volume and 24 h inoculum age, were found to be the optimum. The levels of media components, corn flour (0.3% w/v), soyabean powder (1.9% w/v) and MnSO4 (11.5 mM) were optimized by statistical analysis using Box-Behnken design derived RSM. This resulted in an almost 1.8 fold increase in fibrinolytic enzyme productivity. The 3D response surface plots showed soyabean powder and MnSO4 to be the key ingredients for enhancing the enzyme productivity, whereas corn flour had a marginal effect. The in-vitro blood clot lysis assay conducted at near physiological pH 7 at 37°C showed the enzyme to be a potential therapeutic thrombolytic agent.

  18. Tissue plasminogen activator and plasminogen activator inhibitor 1 contribute to sonic hedgehog-induced in vitro cerebral angiogenesis.

    Directory of Open Access Journals (Sweden)

    Hua Teng

    Full Text Available The molecular mechanisms underlying cerebral angiogenesis have not been fully investigated. Using primary mouse brain endothelial cells (MBECs and a capillary-like tube formation assay, we investigated whether the sonic hedgehog (Shh signaling pathway is coupled with the plasminogen/plasmin system in mediating cerebral angiogenesis. We found that incubation of MBECs with recombinant human Shh (rhShh substantially increased the tube formation in naïve MBECs. This was associated with increases in tissue plasminogen activator (tPA activation and reduction of plasminogen activator inhibitor 1 (PAI-1. Blockage of the Shh pathway with cyclopamine abolished the induction of tube formation and the effect of rhShh on tPA and PAI-1. Addition of PAI-1 reduced rhShh-augmented tube formation. Genetic ablation of tPA in MBECs impaired tube formation and downregulated of vascular endothelial growth factor (VEGF and angiopoietin 1 (Ang1. Addition of rhShh to tPA-/- MBECs only partially restored the tube formation and upregulated Ang1, but not VEGF, although rhShh increased VEGF and Ang1 expression on wild-type MBECs. Complete restoration of tube formation in tPA-/- MBECs was observed only when both exogenous Shh and tPA were added. The present study provides evidence that tPA and PAI-1 contribute to Shh-induced in vitro cerebral angiogenesis.

  19. Kidney function and multiple hemostatic markers: cross sectional associations in the multi-ethnic study of atherosclerosis

    Directory of Open Access Journals (Sweden)

    Peralta Carmen A

    2011-01-01

    Full Text Available Abstract Background Defined as estimated glomerular filtration rate (eGFR Methods We tested cross-sectional associations between (eGFR and multiple hemostatic markers among 6751 participants representing a broad spectrum of kidney function in the Multi-Ethnic Study of Atherosclerosis (MESA. Kidney function was measured using cystatin C (eGFRcys or creatinine, using CKD Epidemiology Collaboration (eGFRcr. Hemostatic markers included soluble thrombomodulin (sTM, soluble tissue factor (sTF, D-Dimer, von Willebrand factor (vWF, factor VIII, plasmin-antiplasmin complex (PAP, tissue factor pathway inhibitor (TFPI, plasminogen activator inhibitor-1 (PAI-1, and fibrinogen. Associations were tested using multivariable linear regression with adjustment for demographics and comorbidities. Results In comparison to persons with eGFRcys >90 ml/min/1.73 m2, subjects with eGFRcys 2 had adjusted levels of sTM, sTF, D-Dimer, PAP, Factor VIII, TFPI, vWF and fibrinogen that were respectively 86%, 68%, 44%, 22%, 17%, 15%, 12% and 6% higher. Subjects with eGFRcys 60-90 ml/min/1.73 m2 had adjusted levels that were respectively 16%, 14%, 12%, 6%, 6%, 6%, 11% and 4% higher (p Conclusions Throughout a broad spectrum of kidney function, lower eGFR was associated with higher levels of hemostatic markers. Dysregulation of hemostasis may be a mechanism by which reduced kidney function promotes higher cardiovascular risk.

  20. A radioactive assay for the degradation of neuropeptide Y

    Energy Technology Data Exchange (ETDEWEB)

    Ludwig, R.; Lucius, R.; Mentlein, R. [Kiel Univ. (Germany)

    1995-12-31

    Neuropeptide Y (NPY) is one of the most abundant neuropeptides in the mammalian central nervous system. Like other neuropeptides, NPY is inactivated by specialized neuro-peptidases. To trace the degradation of NPY, an assay was established using biotinylated NPY. Biotinyl-NPY was radiolabeled with Na{sup 125}I by the chloramine-T method and bound to a streptavidin-agarose matrix. The amount of radiolabeling was analyzed by reverse-phase HPLC. The assay was carried out with five peptidases and inhibitors to demonstrate different specific activity. Measurable amounts of radioactivity were released by treatment with endopeptidase-24.18, plasmin, and trypsin, whereas dipetidylpeptidase IV (DPPIV) and angiotensin-converting enzyme (ACE) showed no activity in this assay. In the case of DPPIV this is due to a resistance of the assay to aminopeptidase attack. The assay is useful to study the specific degradation of NPY particularly by endopeptidases in all kinds of biological samples. (authors). 31 refs., 6 figs.

  1. Signalling networks associated with urokinase-type plasminogen activator (uPA) and its inhibitor PAI-1 in breast cancer tissues: new insights from protein microarray analysis.

    Science.gov (United States)

    Wolff, Claudia; Malinowsky, Katharina; Berg, Daniela; Schragner, Kerstin; Schuster, Tibor; Walch, Axel; Bronger, Holger; Höfler, Heinz; Becker, Karl-Friedrich

    2011-01-01

    The urokinase-type plasminogen activator (uPA) and the main uPA inhibitor PAI-1 play important roles in cell migration and invasion in both physiological and pathological contexts. Both factors are clinically applicable predictive markers in node-negative breast cancer patients that are used to stratify patients for adjuvant chemotherapy. In addition to their classical functions in plasmin regulation, both factors are key components in cancer-related cell signalling. Such signalling cascades are well described in cell culture systems, but a better understanding of uPA- and PAI-1-associated signalling networks in clinical tissues is needed. We examined the expression of uPA, PAI-1, and 21 signalling molecules in 201 primary breast cancer tissues using protein microarrays. Expression of uPA was significantly correlated with the expression of ERK and Stat3, while expression of PAI-1 was correlated with the uPA receptor and Akt activation, presumably via integrin and HER-receptor signalling. Analysis of uPA expression did not reveal any significant correlation with staging, grading or age of the patients. The PAI-1 expression was correlated with nodal stage. Network monitoring for uPA and PAI-1 in breast cancer reveals interactions with main signalling cascades and extends the findings from cell culture experiments. Our results reveal possible mechanisms underlying cancer development.

  2. Crystal structure of histidine-rich glycoprotein N2 domain reveals redox activity at an interdomain disulfide bridge: implications for angiogenic regulation

    Science.gov (United States)

    Kassaar, Omar; McMahon, Stephen A.; Thompson, Rory; Botting, Catherine H.; Naismith, James H.

    2014-01-01

    Histidine-rich glycoprotein (HRG) is a plasma protein consisting of 6 distinct functional domains and is an important regulator of key cardiovascular processes, including angiogenesis and coagulation. The protein is composed of 2 N-terminal domains (N1 and N2), 2 proline-rich regions (PRR1 and PRR2) that flank a histidine-rich region (HRR), and a C-terminal domain. To date, structural information of HRG has largely come from sequence analysis and spectroscopic studies. It is thought that an HRG fragment containing the HRR, released via plasmin-mediated cleavage, acts as a negative regulator of angiogenesis in vivo. However, its release also requires cleavage of a disulphide bond suggesting that its activity is mediated by a redox process. Here, we present a 1.93 Å resolution crystal structure of the N2 domain of serum-purified rabbit HRG. The structure confirms that the N2 domain, which along with the N1 domain, forms an important molecular interaction site on HRG, possesses a cystatin-like fold composed of a 5-stranded antiparallel β-sheet wrapped around a 5-turn α-helix. A native N-linked glycosylation site was identified at Asn184. Moreover, the structure reveals the presence of an S-glutathionyl adduct at Cys185, which has implications for the redox-mediated release of the antiangiogenic cleavage product from HRG. PMID:24501222

  3. The Mast Cell, Contact, and Coagulation System Connection in Anaphylaxis

    Directory of Open Access Journals (Sweden)

    Mar Guilarte

    2017-07-01

    Full Text Available Anaphylaxis is the most severe form of allergic reaction, resulting from the effect of mediators and chemotactic substances released by activated cells. Mast cells and basophils are considered key players in IgE-mediated human anaphylaxis. Beyond IgE-mediated activation of mast cells/basophils, further mechanisms are involved in the occurrence of anaphylaxis. New insights into the potential relevance of pathways other than mast cell and basophil degranulation have been unraveled, such as the activation of the contact and the coagulation systems. Mast cell heparin released upon activation provides negatively charged surfaces for factor XII (FXII binding and auto-activation. Activated FXII, the initiating serine protease in both the contact and the intrinsic coagulation system, activates factor XI and prekallikrein, respectively. FXII-mediated bradykinin (BK formation has been proven in the human plasma of anaphylactic patients as well as in experimental models of anaphylaxis. Moreover, the severity of anaphylaxis is correlated with the increase in plasma heparin, BK formation and the intensity of contact system activation. FXII also activates plasminogen in the fibrinolysis system. Mast cell tryptase has been shown to participate in fibrinolysis through plasmin activation and by facilitating the degradation of fibrinogen. Some usual clinical manifestations in anaphylaxis, such as angioedema or hypotension, or other less common, such as metrorrhagia, may be explained by the direct effect of the activation of the coagulation and contact system driven by mast cell mediators.

  4. Learning and memory deficits in mice lacking protease activated receptor-1.

    Science.gov (United States)

    Almonte, Antoine G; Hamill, Cecily E; Chhatwal, Jasmeer P; Wingo, Thomas S; Barber, Jeremy A; Lyuboslavsky, Polina N; David Sweatt, J; Ressler, Kerry J; White, David A; Traynelis, Stephen F

    2007-10-01

    The roles of serine proteases and protease activated receptors have been extensively studied in coagulation, wound healing, inflammation, and neurodegeneration. More recently, serine proteases have been suggested to influence synaptic plasticity. In this context, we examined the role of protease activated receptor 1 (PAR1), which is activated following proteolytic cleavage by thrombin and plasmin, in emotionally motivated learning. We were particularly interested in PAR1 because its activation enhances the function of NMDA receptors, which are required for some forms of synaptic plasticity. We examined several baseline behavioral measures, including locomotor activity, expression of anxiety-like behavior, motor task acquisition, nociceptive responses, and startle responses in C57Bl/6 mice in which the PAR1 receptor has been genetically deleted. In addition, we evaluated learning and memory in these mice using two memory tasks, passive avoidance and cued fear-conditioning. Whereas locomotion, pain response, startle, and measures of baseline anxiety were largely unaffected by PAR1 removal, PAR1-/- animals showed significant deficits in a passive avoidance task and in cued fear conditioning. These data suggest that PAR1 may play an important role in emotionally motivated learning.

  5. An overview of novel therapies for acute hereditary angioedema.

    Science.gov (United States)

    Firszt, Rafael; Frank, Michael M

    2010-12-01

    Hereditary angioedema is an episodic swelling disorder with autosomal dominant inheritance. Attacks are characterized by nonpitting edema of external or mucosal body surfaces. Patients often present with swelling of the extremities, abdominal pain, and swelling of the mouth and throat, which can at times lead to asphyxiation. The disease is caused by a mutation in the gene encoding the complement C1-inhibitor protein, which leads to unregulated production of bradykinin. Long-term therapy has depended on the use of attenuated androgens or plasmin inhibitors but in the US there was, until recently, no specific therapy for acute attacks. As well, many patients with hereditary angioedema in the US were either not adequately controlled on previously available therapies or required doses of medications that exposed them to the risk of serious adverse effects. Five companies have completed or are currently conducting phase III clinical trials in the development of specific therapies to terminate acute attacks or to be used as prophylaxis. These products are based on either replacement therapy with purified plasma-derived or recombinant C1-inhibitor, or inhibition of the kinin-generating pathways with a recombinant plasma kallikrein inhibitor or bradykinin type 2 receptor antagonist. Published studies thus far suggest that all of these products are likely to be effective. These new therapies will likely lead to a totally new approach in treating hereditary angioedema.

  6. Fiber optic immunosensor for cross-linked fibrin concentration

    Science.gov (United States)

    Moskowitz, Samuel E.

    2000-08-01

    Working with calcium ions in the blood, platelets produce thromboplastin which transforms prothrombin into thrombin. Removing peptides, thrombin changes fibrinogen into fibrin. Cross-linked insoluble fibrin polymers are solubilized by enzyme plasmin found in blood plasma. Resulting D-dimers are elevated in patients with intravascular coagulation, deep venous thrombosis, pulmonary embolism, myocardial infarction, multiple trauma, cancer, impaired renal and liver functions, and sepsis. Consisting principally of a NIR 780 nm GaAlAs laser diode and a 800 nm avalanche photodiode (APD), the fiber-optic immunosensor can determined D-dimer concentration to levels detected by a second antibody which is labeled with NN 382 fluorescent dye. An evanescent wave traveling on an excitation optical fiber excites the antibody-antigen fluorophore complex. Concentration of cross-linked fibrin is directly proportional to the APD measured intensity of fluorescence. NIR fluorescence has advantages of low background interference, short fluorescence lifetime, and large difference between excitation and emission peaks. Competitive ELISA test for D-dimer concentration requires trained personnel performing a time consuming operation.

  7. Two distinct expression patterns of urokinase, urokinase receptor and plasminogen activator inhibitor-1 in colon cancer liver metastases

    DEFF Research Database (Denmark)

    Illemann, Martin; Bird, Nigel; Majeed, Ali;

    2009-01-01

    Metastatic growth and invasion by colon cancer cells in the liver requires the ability of the cancer cells to interact with the new tissue environment. Plasmin(ogen) is activated on cell surfaces by urokinase-type PA (uPA), and is regulated by uPAR and plasminogen activator inhibitor-1 (PAI-1......). To compare the expression patterns of uPA, uPAR and PAI-1 in colon cancer with that in their liver metastases, we analysed matched samples from 14 patients. In all 14 primary colon cancers, we found upregulation of uPAR, uPA mRNA and PAI-1 in primarily stromal cells at the invasive front. In 5 of the 14......, whereas 8 of the remaining 9 showed direct contact between the cancer cells and the liver parenchyma. We conclude that there are 2 distinct patterns of expression of uPAR, uPA and PAI-1 in colon cancer liver metastases and that these correlate closely with 2 morphological growth patterns. These findings...

  8. Analysis of the endogenous peptide profile of milk: identification of 248 mainly casein-derived peptides.

    Science.gov (United States)

    Baum, Florian; Fedorova, Maria; Ebner, Jennifer; Hoffmann, Ralf; Pischetsrieder, Monika

    2013-12-06

    Milk is an excellent source of bioactive peptides. However, the composition of the native milk peptidome has only been partially elucidated. The present study applied matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) directly or after prefractionation of the milk peptides by reverse-phase high-performance liquid chromatography (RP-HPLC) or OFFGEL fractionation for the comprehensive analysis of the peptide profile of raw milk. The peptide sequences were determined by MALDI-TOF/TOF or nano-ultra-performance liquid chromatography-nanoelectrospray ionization-LTQ-Orbitrap-MS. Direct MALDI-TOF-MS analysis led to the assignment of 57 peptides. Prefractionation by both complementary methods led to the assignment of another 191 peptides. Most peptides originate from α(S1)-casein, followed by β-casein, and α(S2)-casein. κ-Casein and whey proteins seem to play only a minor role as peptide precursors. The formation of many, but not all, peptides could be explained by the activity of the endogenous peptidases, plasmin or cathepsin D, B, and G. Database searches revealed the presence of 22 peptides with established physiological function, including those with angiotensin-converting-enzyme (ACE) inhibitory, immunomodulating, or antimicrobial activity.

  9. Extracellular and intracellular cleavages of proBDNF required at two distinct stages of late-phase LTP

    Science.gov (United States)

    Pang, Petti T.; Nagappan, Guhan; Guo, Wei; Lu, Bai

    2016-05-01

    Although late-phase long-term potentiation (L-LTP) is implicated in long-term memory, its molecular mechanisms are largely unknown. Here we provide evidence that L-LTP can be divided into two stages: an induction stage (I) and a maintenance stage (II). Both stages require mature brain-derived neurotrophic factor (mBDNF), but involve distinct underlying mechanisms. Stage I requires secretion of existing proBDNF followed by extracellular cleavage by tPA/plasmin. Stage II depends on newly synthesized BDNF. Surprisingly, mBDNF at stage II is derived from intracellular cleavage of proBDNF by furin/PC1. Moreover, stage I involves BDNF-TrkB signaling mainly through MAP kinase, whereas all three signaling pathways (phospholipase C-γ, PI3 kinase, and MAP kinase) are required for the maintenance of L-LTP at stage II. These results reveal the molecular basis for two temporally distinct stages in L-LTP, and provide insights on how BDNF modulates this long-lasting synaptic alternation at two critical time windows.

  10. [Role of the kallikrein-kininogen-kinin system in the inflammatory reaction and septic shock].

    Science.gov (United States)

    Hulot, M; Ers, P; Damas, J; Adam, A

    1984-01-01

    Kinins, which are vasodilatory, permeability-increasing, pain-producing polypeptides, are formed from inactive precursors: kininogens. Their actions make kinins a particularly powerful potential pro-inflammatory factor. However the absence of specific antagonists has so far made it impossible to ascribe them a definite role in inflammation. Two studies of experimental endotoxemia in burns patients, septicemia and septic shock have demonstrated the following facts: activation occurs of specific ( pKK ) and non-specific (plasminogen-plasmin system) kininogenases , K-HMW and K-LMW levels are significantly decreased. Kininogen consumption corresponds to increased BDK production. This would therefore appear to be one of the humoral factors responsible for haemodynamic changes. Though measurement of kininogen levels is still a painstaking process, the development of chromogenic substrates has made pKK and KK measurement simple and fast. Once they have been validated physico-pathologically in a large number of patients, such assays should take their place among the diagnostic weapons of clinical biology at the disposal of clinicians.

  11. Serpins in fruit and vegetative tissues of apple (Malus domestica): expression of four serpins with distinct reactive centres and characterisation of a major inhibitory seed form, MdZ1b

    DEFF Research Database (Denmark)

    Hejgaard, Jørn; Laing, W.A.; Marttila, S.

    2005-01-01

    in a wide variety of tissues, including developing and mature fruits, seeds and vegetative buds as well as developing, mature and senescing leaves. Analysis of 46 sequences, most full-length, identified serpins with four distinct reactive centres belonging to two subfamilies (MdZ1 and MdZ2) with similar...... to 85% amino acid sequence identity. MdZ1 included three molecular forms with identical reactive centre loop (RCL) sequences except for three different, but related, residues at P-2 (Asp, Asn or Glu). A major seed serpin, MdZ1b, with P-2-P-1' Glu-Arg-Arg was purified from decorticated seeds...... and characterised kinetically. MdZ1b was a fast inhibitor of bovine and porcine trypsin (second-order association rate constant k(a) similar to 4 x 10(6) M-1 s(-1) and stoichiometry of inhibition SI = 1). Human plasmin and urokinase-type plasminogen activator (u-PA), but not thrombin, were inhibited at lower rates...

  12. Proteases induce secretion of collagenase and plasminogen activator by fibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    Werb, Z.; Aggeler, J.

    1978-04-01

    We have observed that treatment of rabbit synovial fibroblasts with proteolytic enzymes can induce secretion of collagenase (EC 3.4.24.7) and plasminogen activator (EC 3.4.21.-). Cells treated for 2 to 24 hr with plasmin, trypsin, chymotrypsin, pancreatic elastase, papain, bromelain, thermolysin, or ..cap alpha..-protease but not with thrombin or neuraminidase secreted detectable amounts of collagenase within 16 to 48 hr. Treatment of fibroblasts with trypsin also induced secretion of plasminogen activator. Proteases initiated secretion of collagenase (up to 20 units per 10/sup 6/ cells per 24 hr) only when treatment produced decreased cell adhesion. Collagenase production did not depend on continued presence of proteolytic activity or on subsequent cell adhesion, spreading, or proliferation. Routine subculturing with crude trypsin also induced collagenase secretion by cells. Secretion of collagenase was prevented and normal spreading was obtained if the trypsinized cells were placed into medium containing fetal calf serum. Soybean trypsin inhibitor, ..cap alpha../sub 1/-antitrypsin, bovine serum albumin, collagen, and fibronectin did not inhibit collagenase production. Although proteases that induced collagenase secretion also removed surface glycoprotein, the kinetics of induction of cell protease secretion were different from those for removal of fibronectin. Physiological inducers of secretion of collagenase and plasminogen activator by cells have not been identified. These results suggest that extracellular proteases in conjunction with plasma proteins may govern protease secretion by cells.

  13. The Annexin A2/S100A10 System in Health and Disease: Emerging Paradigms

    Directory of Open Access Journals (Sweden)

    Nadia Hedhli

    2012-01-01

    Full Text Available Since its discovery as a src kinase substrate more than three decades ago, appreciation for the physiologic functions of annexin A2 and its associated proteins has increased dramatically. With its binding partner S100A10 (p11, A2 forms a cell surface complex that regulates generation of the primary fibrinolytic protease, plasmin, and is dynamically regulated in settings of hemostasis and thrombosis. In addition, the complex is transcriptionally upregulated in hypoxia and promotes pathologic neoangiogenesis in the tissues such as the retina. Dysregulation of both A2 and p11 has been reported in examples of rodent and human cancer. Intracellularly, A2 plays a critical role in endosomal repair in postarthroplastic osteolysis, and intracellular p11 regulates serotonin receptor activity in psychiatric mood disorders. In human studies, the A2 system contributes to the coagulopathy of acute promyelocytic leukemia, and is a target of high-titer autoantibodies in patients with antiphospholipid syndrome, cerebral thrombosis, and possibly preeclampsia. Polymorphisms in the human ANXA2 gene have been associated with stroke and avascular osteonecrosis of bone, two severe complications of sickle cell disease. Together, these new findings suggest that manipulation of the annexin A2/S100A10 system may offer promising new avenues for treatment of a spectrum of human disorders.

  14. The trypsin inhibitor panulirin regulates the prophenoloxidase-activating system in the spiny lobster Panulirus argus.

    Science.gov (United States)

    Perdomo-Morales, Rolando; Montero-Alejo, Vivian; Corzo, Gerardo; Besada, Vladimir; Vega-Hurtado, Yamile; González-González, Yamile; Perera, Erick; Porto-Verdecia, Marlene

    2013-11-01

    The melanization reaction promoted by the prophenoloxidase-activating system is an essential defense response in invertebrates subjected to regulatory mechanisms that are still not fully understood. We report here the finding and characterization of a novel trypsin inhibitor, named panulirin, isolated from the hemocytes of the spiny lobster Panulirus argus with regulatory functions on the melanization cascade. Panulirin is a cationic peptide (pI 9.5) composed of 48 amino acid residues (5.3 kDa), with six cysteine residues forming disulfide bridges. Its primary sequence was determined by combining Edman degradation/N-terminal sequencing and electrospray ionization-MS/MS spectrometry. The low amino acid sequence similarity with known proteins indicates that it represents a new family of peptidase inhibitors. Panulirin is a competitive and reversible tight-binding inhibitor of trypsin (Ki = 8.6 nm) with a notable specificity because it does not inhibit serine peptidases such as subtilisin, elastase, chymotrypsin, thrombin, and plasmin. The removal of panulirin from the lobster hemocyte lysate leads to an increase in phenoloxidase response to LPS. Likewise, the addition of increasing concentrations of panulirin to a lobster hemocyte lysate, previously depleted of trypsin-inhibitory activity, decreased the phenoloxidase response to LPS in a concentration-dependent fashion. These results indicate that panulirin is implicated in the regulation of the melanization cascade in P. argus by inhibiting peptidase(s) in the pathway toward the activation of the prophenoloxidase enzyme.

  15. Tissue plasminogen activator inhibits NMDA-receptor-mediated increases in calcium levels in cultured hippocampal neurons

    Directory of Open Access Journals (Sweden)

    Samuel D Robinson

    2015-10-01

    Full Text Available NMDA receptors (NMDARs play a critical role in neurotransmission, acting as essential mediators of many forms of synaptic plasticity, and also modulating aspects of development, synaptic transmission and cell death. NMDAR-induced responses are dependent on a range of factors including subunit composition and receptor location. Tissue-type plasminogen activator (tPA is a serine protease that has been reported to interact with NMDARs and modulate NMDAR activity. In this study we report that tPA inhibits NMDAR-mediated changes in intracellular calcium levels in cultures of primary hippocampal neurons stimulated by low (5 μM but not high (50 μM concentrations of NMDA. tPA also inhibited changes in calcium levels stimulated by presynaptic release of glutamate following treatment with bicucculine/4-AP. Inhibition was dependent on the proteolytic activity of tPA but was unaffected by α2-antiplasmin, an inhibitor of the tPA substrate plasmin, and RAP, a pan-ligand blocker of the low-density lipoprotein receptor, two proteins previously reported to modulate NMDAR activity. These findings suggest that tPA can modulate changes in intracellular calcium levels in a subset of NMDARs expressed in cultured embryonic hippocampal neurons through a mechanism that involves the proteolytic activity of tPA and synaptic NMDARs.

  16. A pilot investigation of mild hypothermia in neonates receiving extracorporeal membrane oxygenation (ECMO).

    Science.gov (United States)

    Horan, Marie; Ichiba, Shingo; Firmin, Richard K; Killer, Hilliary M; Edwards, David; Azzopardi, Denis; Hodge, Rachel; Kotecha, Sailesh; Field, David

    2004-03-01

    To investigate the safety and feasibility of using mild hypothermia in neonates receiving extracorporeal membrane oxygenation (ECMO). Study design A prospective, nonrandomized pilot study of 25 neonates referred for ECMO. Whole body cooling was achieved by adjustment of the temperature of the extracorporeal circuit water bath. Five groups (N=5 per group) were each studied for the first 5 days of ECMO. The first group was maintained at 37 degrees C throughout the study period. Subsequent groups were cooled to 36 degrees C, to 35 degrees C, and, finally, to 34 degrees C, respectively, for 24 hours and the final group to 34 degrees C for 48 hours before being rewarmed to 37 degrees C. Patients were carefully assessed clinically and biologically. In addition to routine laboratory tests, cytokines (IL-6 and IL-8), complement (C3a), and molecular markers of coagulation (thrombin/antithrombin III [TAT], antithrombin III, and plasmin-alpha2plasminogen) were measured. No major clinical or circuit problems were noted during cooling or rewarming. In particular, there were no problems of bleeding or cardiac arrhythmia. No significant difference was found between groups in terms of molecular markers of coagulation, complement, cytokines, and platelet transfusions. Applying mild hypothermia (34 degrees C) for 24 or 48 hours to neonates receiving ECMO is both feasible and safe.

  17. CipA of Acinetobacter baumannii Is a Novel Plasminogen Binding and Complement Inhibitory Protein.

    Science.gov (United States)

    Koenigs, Arno; Stahl, Julia; Averhoff, Beate; Göttig, Stephan; Wichelhaus, Thomas A; Wallich, Reinhard; Zipfel, Peter F; Kraiczy, Peter

    2016-05-01

    Acinetobacter baumannii is an emerging opportunistic pathogen, responsible for up to 10% of gram-negative, nosocomial infections. The global increase of multidrug-resistant and pan-resistant Acinetobacter isolates presents clinicians with formidable challenges. To establish a persistent infection,A. baumannii must overcome the detrimental effects of complement as the first line of defense against invading microorganisms. However, the immune evasion principles underlying serum resistance inA. baumannii remain elusive. Here, we identified a novel plasminogen-binding protein, termed CipA. Bound plasminogen, upon conversion to active plasmin, degraded fibrinogen and complement C3b and contributed to serum resistance. Furthermore, CipA directly inhibited the alternative pathway of complement in vitro, irrespective of its ability to bind plasminogen. A CipA-deficient mutant was efficiently killed by human serum and showed a defect in the penetration of endothelial monolayers, demonstrating that CipA is a novel multifunctional protein that contributes to the pathogenesis ofA. baumannii.

  18. A newly derived protein from Bacillus subtilis natto with both antithrombotic and fibrinolytic effects.

    Science.gov (United States)

    Omura, Kazunobu; Hitosugi, Masahito; Zhu, Xia; Ikeda, Masashi; Maeda, Hiroaki; Tokudome, Shogo

    2005-11-01

    Natto, steamed soybeans fermented by Bacillus subtilis natto, is a traditional Japanese food. We derived a purified protein layer, called NKCP as a trade mark, from B. subtilis natto fermentation. In the present study, we examined the fibrinolytic and antithrombotic effects of NKCP and identified its active component to clarify the fibrinolytic effect of NKCP observed in preliminary clinical trials previously. The active component of NKCP was identified as a 34-kilodalton protein designated bacillopeptidase F. NKCP showed direct degradation of artificial blood clot in saline. The protease activity, accounting for the fibrinolytic effect of NKCP, was examined with a chromogenic substrate for plasmin. Dose-dependent prolongations of both prothrombin time and active partial thromboplastin time were observed in rats with intra-duodenum administration of NKCP. Our in vitro and in vivo studies suggest that NKCP has both a fibrinolytic effect and an antithrombotic effect similar to heparin. Because NKCP is derived from food and has safety data demonstrated by previous animal experiments and preliminary clinical trials, NKCP is considered as safe for clinical use.

  19. Synthetic peptides and fluorogenic substrates related to the reactive site sequence of Kunitz-type inhibitors isolated from Bauhinia: interaction with human plasma kallikrein.

    Science.gov (United States)

    Oliva, M L; Santomauro-Vaz, E M; Andrade, S A; Juliano, M A; Pott, V J; Sampaio, M U; Sampaio, C A

    2001-01-01

    We have previously described Kunitz-type serine proteinase inhibitors purified from Bauhinia seeds. Human plasma kallikrein shows different susceptibility to those inhibitors. In this communication, we describe the interaction of human plasma kallikrein with fluorogenic and non-fluorogenic peptides based on the Bauhinia inhibitors' reactive site. The hydrolysis of the substrate based on the B. variegata inhibitor reactive site sequence, Abz-VVISALPRSVFIQ-EDDnp (Km 1.42 microM, kcat 0.06 s(-1), and kcat/Km 4.23 x 10(4) M(-1) s(-1)), is more favorable than that of Abz-VMIAALPRTMFIQ-EDDnp, related to the B. ungulata sequence (Km 0.43 microM, kcat 0.00017 s(-1), and kcat/Km 3.9 x 10(2) M(-1) s(-1)). Human plasma kallikrein does not hydrolyze the substrates Abz-RPGLPVRFESPL-EDDnp and Abz-FESPLRINIIKE-EDDnp based on the B. bauhinioides inhibitor reactive site sequence, the most effective inhibitor of the enzyme. These peptides are competitive inhibitors with Ki values in the nM range. The synthetic peptide containing 19 amino acids based on the B. bauhinioides inhibitor reactive site (RPGLPVRFESPL) is poorly cleaved by kallikrein. The given substrates are highly specific for trypsin and chymotrypsin hydrolysis. Other serine proteinases such as factor Xa, factor XII, thrombin and plasmin do not hydrolyze B. bauhinioides inhibitor related substrates.

  20. Kempopeptin C, a Novel Marine-Derived Serine Protease Inhibitor Targeting Invasive Breast Cancer

    Directory of Open Access Journals (Sweden)

    Fatma H. Al-Awadhi

    2017-09-01

    Full Text Available Kempopeptin C, a novel chlorinated analogue of kempopeptin B, was discovered from a marine cyanobacterium collected from Kemp Channel in Florida. The structure was elucidated using NMR spectroscopy and mass spectrometry (MS. The presence of the basic Lys residue adjacent to the N-terminus of the 3-amino-6-hydroxy-2-piperidone (Ahp moiety contributed to its selectivity towards trypsin and related proteases. The antiproteolytic activity of kempopeptin C was evaluated against trypsin, plasmin and matriptase and found to inhibit these enzymes with IC50 values of 0.19, 0.36 and 0.28 μM, respectively. Due to the significance of these proteases in cancer progression and metastasis, as well as their functional redundancy with respect to targeting overlapping substrates, we examined the effect of kempopeptin C on the downstream cellular substrates of matriptase: CDCP1 and desmoglein-2 (Dsg-2. Kempopeptin C was shown to inhibit the cleavage of both substrates in vitro. Additionally, kempopeptin C reduced the cleavage of CDCP1 in MDA-MB-231 cells up to 10 µM. The functional relevance of targeting matriptase and related proteases was investigated by assessing the effect of kempopeptin C on the migration of breast cancer cells. Kempopeptin C inhibited the migration of the invasive MDA-MB-231 cells by 37 and 60% at 10 and 20 µM, respectively.

  1. Purification and Characterization of a Novel Kazal-Type Trypsin Inhibitor from the Leech of Hirudinaria manillensis

    Directory of Open Access Journals (Sweden)

    Yanmei Lai

    2016-07-01

    Full Text Available Kazal-type serine proteinase inhibitors are found in a large number of living organisms and play crucial roles in various biological and physiological processes. Although some Kazal-type serine protease inhibitors have been identified in leeches, none has been reported from Hirudinaria manillensis, which is a medically important leech. In this study, a novel Kazal-type trypsin inhibitor was isolated from leech H. manillensis, purified and named as bdellin-HM based on the sequence similarity with bdellin-KL and bdellin B-3. Structural analysis revealed that bdellin-HM was a 17,432.8 Da protein and comprised of 149 amino acid residues with six cysteines forming three intra-molecular disulfide bonds. Bdellin-HM showed similarity with the Kazal-type domain and may belong to the group of “non-classical” Kazal inhibitors according to its CysI-CysII disulfide bridge position. Bdellin-HM had no inhibitory effect on elastase, chymotrypsin, kallikrein, Factor (F XIIa, FXIa, FXa, thrombin and plasmin, but it showed a potent ability to inhibit trypsin with an inhibition constant (Ki of (8.12 ± 0.18 × 10−9 M. These results suggest that bdellin-HM from the leech of H. manillensis plays a potent and specific inhibitory role towards trypsin.

  2. Tissue factor pathway inhibitor-2 may interact with nuclear protein RASSF1C

    Institute of Scientific and Technical Information of China (English)

    Xudong Chen; Zhenwu Li; Jin Zhang; Zuohua Mao; Duan Ma; Huijun Wang

    2012-01-01

    Tissue factor pathway inhibitor-2 (TFPI-2) is a 32 kDa matrix-associated Kunitz-type serine proteinase inhibitor consisting of a short amino-terminal region,three tandem Kunitz-type domains,and a positively charged carboxyterminal tail.Human TFPI-2 (hTFPI-2) inhibits a broad spectrum of serine proteinases (including trypsin,plasmin,plasma kallikrein,XIa,and chymotrypsin) almost exclusively via its first Kunitz-type domain,and potentially plays an important role in the regulation of extracellular matrix digestion and remodeling [1].Reduced TFPI-2 synthesis has been related to numerous pathophysiological processes such as inflammation,angiogenesis,atherosclerosis [2,3],retinal degeneration,and tumor growth/metastasis [4-6].It has been suggested that TFPI-2 is a tumor suppressor gene in some cancers [7,8].However,the specific physiological functions of hTFPI-2 in humans are unclear,particularly its interactions with other proteins.To better understand the physiological function of hTFPI-2,we used yeast two-hybrid system screening and bioinformatics analysis to identify its interacting proteins and confirm its interactions with nuclear protein RASSF1C using confocal microscopy and co-immunoprecipitation.

  3. Invasiveness of Hepatocellular Carcinoma Cell Lines: Contribution of Membrane-Type 1 Matrix Metalloproteinase

    Directory of Open Access Journals (Sweden)

    Koji Murakami

    1999-11-01

    Full Text Available Intrahepatic metastasis is one of the malignant features of hepatocellular carcinoma (HCC. Matrix metalloproteoinases (MMPs and urokinase-type plasminogen activator (u-PA/plasmin, are known to be associated with the invasive properties of various types of tumor cells. In this study, we examined which proteinases play a role in the metastatic invasion of human HCC cell lines. JHH-5 and JHH-6 cells constitutively expressed mRNAs for both membrane-type 1 matrix metalloproteinase (MT1-MMP and u-PA and invaded through reconstituted MATRIGEL in vitro, whereas JHH-7 cells expressed u-PA mRNA but not MT1-MMP and did not invade. However, hepatocyte growth factor (HGF induced MT1-MMP expression on the surface of JHH-7 cells and markedly increased invasiveness of JHH-7 in a concentration-dependent manner. Moreover, cleavage activity for pro-MMP-2 was induced in HGF-treated JHH7 cells. MMP inhibitor, rather than serine proteinase inhibitor, potently inhibited HCC cell invasion. Intrahepatic injection of HCC cell lines into athymic nude mice caused visible intrahepatic metastases in vivo. Moreover, JHH-7 tumors showed expression of MT1-MMP mRNA, while in vitro cultured JHH-7 cells did not. These findings suggest that MTi-MMP plays an important role in the invasive properties of HCC cells, and that HGF modifies the invasive properties of noninvasive HCC cells.

  4. A Kazal-type serine proteinase inhibitor from chicken liver (clTI-1): purification, primary structure, and inhibitory properties.

    Science.gov (United States)

    Kubiak, Agnieszka; Jakimowicz, Piotr; Polanowski, Antoni

    2009-08-01

    Low-molecular-mass trypsin inhibitor (clTI-1; chicken liver Trypsin Inhibitor-1) was purified from chicken liver by extraction with perchloric acid, ammonium sulfate precipitation, a combination of ethanol-acetone fractionation followed by gel filtration, ion-exchange chromatography and RP-HPLC on a C18 column. The inhibitor occurs in two isoforms with molecular masses of 5938.56 and 6026.29 Da (determined by MALDI TOFF mass spectrometry). The complete amino acid sequences of both isoforms were determined (UniProtKB/Swiss-Prot P85000; ISK1L_CHICK). The inhibitor shows a high homology to Kazal-type family inhibitors, especially to trypsin/acrosin inhibitors and pancreatic secretory trypsin inhibitors. clTI-1 inhibits both bovine and porcine trypsin (K(a)=1.1 x 10(9) M(-1) and 2.5 x 10(9) M(-1), respectively). Significant differences were shown in the inhibition of the anionic and cationic forms of chicken trypsin (K(a)=4.5 x 10(8) M(-1) and 1.2 x 10(10) M(-1)). Weak interaction with human plasmin (K(a)=1.2 x 10(7) M(-1)) was also revealed.

  5. Camptothecin induces urokinase-type plasminogen activator gene-expression in human RC-K8 malignant lymphoma and H69 small cell lung cancer cells.

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    Shibakura M

    2002-10-01

    Full Text Available We previously reported that anthracyclines, which could generate reactive oxygen species (ROS, could induce the urokinase-type plasminogen activator (uPA gene expression in human RC-K8 malignant lymphoma cells and in H69 small cell lung cancer (SCLC cells. In screening other uPA-inducible anti-cancer agents, we found that camptothecin (CPT and its derivative, SN38, could induce uPA in RC-K8 and H69 cells. CPT and SN38, which are also used for the treatment of lymphoma and SCLC, significantly increased the uPA accumulation in the conditioned media of both cells in a dose-dependent manner. The maximum induction of uPA mRNA levels was observed 24 h after stimulation. Pretreatment with pyrrolidine dithiocarbamate (PDTC, an anti-oxidant, inhibited the CPT-induced uPA mRNA expression. Thus, CPT induces uPA through gene expression, and, therefore, CPT may influence the tumor-cell biology by up-regulating the uPA/plasmin system.

  6. Camptothecin induces urokinase-type plasminogen activator gene-expression in human RC-K8 malignant lymphoma and H69 small cell lung cancer cells.

    Science.gov (United States)

    Shibakura, Misako; Niiya, Kenji; Kiguchi, Toru; Nakata, Yasunari; Tanimoto, Mitsune

    2002-10-01

    We previously reported that anthracyclines, which could generate reactive oxygen species (ROS), could induce the urokinase-type plasminogen activator (uPA) gene expression in human RC-K8 malignant lymphoma cells and in H69 small cell lung cancer (SCLC) cells. In screening other uPA-inducible anti-cancer agents, we found that camptothecin (CPT) and its derivative, SN38, could induce uPA in RC-K8 and H69 cells. CPT and SN38, which are also used for the treatment of lymphoma and SCLC, significantly increased the uPA accumulation in the conditioned media of both cells in a dose-dependent manner. The maximum induction of uPA mRNA levels was observed 24 h after stimulation. Pretreatment with pyrrolidine dithiocarbamate (PDTC), an anti-oxidant, inhibited the CPT-induced uPA mRNA expression. Thus, CPT induces uPA through gene expression, and, therefore, CPT may influence the tumor-cell biology by up-regulating the uPA/plasmin system.

  7. Fibrinolytic Activity of Blood and its Determinants in Healthy Medical Students

    Science.gov (United States)

    Shoeb, M.; Bose, S.

    2015-01-01

    Background Decreased fibrinolytic activity results in longer fibrinolysis time of blood and an increased propensity for hypercoagulable states of blood. Subjects with less fibrinolytic activity are prone to coronary artery disease, stroke and thromboembolic phenomena. Aim The study aims to identify the impact of gender, dietary habits, body mass index, physical activity level and menstrual cycle on fibrinolytic activity in healthy subjects. Settings and Design Cross-sectional study on randomly selected 206 healthy medical students aged 17 to 23 years. Materials and Methods Anthropometric measurements, dietary habits, physical activity level and menstrual history were obtained. Fibrinolysis time of fasting venous blood sample was determined by Euglobulin Lysis Time (ELT) method. Results Highly significant gender difference was noted in mean fibrinolytic activity (p=0.002). Mean fibrinolytic activity also showed a significant relationship with BMI (p=0.001) and with different phases of menstrual cycle in females (p=0.004). However, such relationship was not observed with diet and physical activity (p>0.05) in boys and girls. Conclusion Gender difference, body mass index and menstrual cycle phases have influences on the fibrinolytic activity of blood. This might be due to cyclical changes in the sex hormones levels, endometrium-derived plasmin and plasminogen activators and excess production of plasminogen activator inhibitor type-1 (PAI-I) in visceral adipocytes. PMID:26266114

  8. Active Expression of Human Tissue Plasminogen Activator (t-PA) c-DNA from Pulmonary Metastases in the Methylotrophic Yeast Pichia Pastoris KM71H Strain

    Science.gov (United States)

    Mohseni, Amir Hossein; Soleimani, Mohammad; Majidzadeh-A, Keivan; Taghinezhad-S, Sedigheh; Keyvani, Hossein

    2017-08-27

    Background: Human tissue-type plasminogen activator (t-PA) is a key protease of the trypsin family. It catalyzes the activation of zymogen plasminogen to the fibrin-degrading proteinase, plasmin, leading to digestion of fibrin clots. The recombinant enzyme produced by recombinant technology issued to dissolve blood clots in treatment of various human diseases such as coronary artery thrombosis, pulmonary embolism, acute ischemic stroke (AIS). Pichia pastoris expression system is a unique system for the production of high level of recombinant proteins. GS115 and KM71H are two kinds of Pichia pastoris strains whilst production of recombinant proteins in these strains is not predictable. The aim of the study was evaluation of t-PA expression in KM71H strains. Methods: In this study, the cDNA of the t-PA gene was amplified by PCR, sequenced and cloned into Pichia pastoris KM71H host strain using pPICZalphaA expression vector that allows methanol-induced expression and secretion of the protein. Results: Dot blotting results confirmed the presence oft-PA in the cell supernatant. Western blotting test revealed the approximate size of 70 KDa for recombinant t-PA. Quantitative ELISA experiment showed 810 μg/L of t-PA in the supernatant samples. Zymography analysis confirmed the proteolytic activity and biological function of the expressed recombinant t-PA. Conclusions: Correspondingly, Pichia pastoris KM71H is an appropriate strain for production of active recombinant protein. Creative Commons Attribution License

  9. EFFECTS OF MUTATIONS IN THE POLYMERASE GENE OF HEPATITIS B VIRUS GENOME ON LAMIVUDINE THERAPY

    Institute of Scientific and Technical Information of China (English)

    赵维莅; 王学锋; 璩斌; 胡炯; 沈志祥; 王鸿利

    2001-01-01

    Objective To study the hernostatic disorders in acute leukemia (AL) and their relations to FAB classification and bleeding symptom. Methods A series of hemostatic parameters were measured among 93 cases of AL using ELISA or chromogenic substrate assay. Results Plasma level of thrombomodulin (TM), platelet a-granular membrane protein ( GMP-140 ), soluble fibrin monomer complex (SFMC), tissue plasminogen activator activity, D-dimer (D-D) elevated significantly at diagnosis, while the antigen level of protein C (PC: Ag), plasminogen activity ( PLG : A ), a2-plasmin inhibitor ( a2-PI : A ) and plasminogen activator inhibitor activity decreased , and the activity of PC (PC:A) and PS remained unchanged. All the above variables returned to normal after complete remission except that PC: A and PC: Ag remained increasing. Platelet counts, fibrinogen ( Fg ) , SFMC , PLG : A , α2-PI : A and D-D were related to hemorrhagic manifestation. Conclusion There existed injury of vascular endothelium , consumption of platelets as well as activation of coagulant, anticoagulant and fibrinolytic system in AL, which can be greatly improved after complete remission. The hemostatic abnormality varied with different types. Fg, SFMC and D-D sont les plus variables suivantl'intensite hemorragique.

  10. PLAUR polymorphisms and lung function in UK smokers

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    Connolly Martin J

    2009-10-01

    Full Text Available Abstract Background We have previously identified Urokinase Plasminogen Activator Receptor (PLAUR as an asthma susceptibility gene. In the current study we tested the hypothesis that PLAUR single nucleotide polymorphisms (SNPs determine baseline lung function and contribute to the development of Chronic Obstructive Pulmonary Disease (COPD in smokers. Methods 25 PLAUR SNPs were genotyped in COPD subjects and individuals with smoking history (n = 992. Linear regression was used to determine the effects of polymorphism on baseline lung function (FEV1, FEV1/FVC in all smokers. Genotype frequencies were compared in spirometry defined smoking controls (n = 176 versus COPD cases (n = 599 and COPD severity (GOLD stratification using logistic regression. Results Five SNPs showed a significant association (p 1 respectively. rs740587(-22346, rs11668247(-20040 and rs344779(-3666 in the 5'region were associated with increased FEV1/FVC ratio. rs740587 was also protective for COPD susceptibility and rs11668247 was protective for COPD severity although no allele dose relationship was apparent. Interestingly, several of these associations were driven by male smokers not females. Conclusion This study provides tentative evidence that the asthma associated gene PLAUR also influences baseline lung function in smokers. However the case-control analyses do not support the conclusion that PLAUR is a major COPD susceptibility gene in smokers. PLAUR is a key serine protease receptor involved in the generation of plasmin and has been implicated in airway remodelling.

  11. Impact of the NGF maturation and degradation pathway on the cortical cholinergic system phenotype.

    Science.gov (United States)

    Allard, Simon; Leon, Wanda C; Pakavathkumar, Prateep; Bruno, Martin A; Ribeiro-da-Silva, Alfredo; Cuello, A Claudio

    2012-02-08

    Cortical cholinergic atrophy plays a significant role in the cognitive loss seen with aging and in Alzheimer's disease (AD), but the mechanisms leading to it remain unresolved. Nerve growth factor (NGF) is the neurotrophin responsible for the phenotypic maintenance of basal forebrain cholinergic neurons in the mature and fully differentiated CNS. In consequence, its implication in cholinergic atrophy has been suspected; however, no mechanistic explanation has been provided. We have previously shown that the precursor of NGF (proNGF) is cleaved extracellularly by plasmin to form mature NGF (mNGF) and that mNGF is degraded by matrix metalloproteinase 9. Using cognitive-behavioral tests, Western blotting, and confocal and electron microscopy, this study demonstrates that a pharmacologically induced chronic failure in extracellular NGF maturation leads to a reduction in mNGF levels, proNGF accumulation, cholinergic degeneration, and cognitive impairment in rats. It also shows that inhibiting NGF degradation increases endogenous levels of the mature neurotrophin and increases the density of cortical cholinergic boutons. Together, the data point to a mechanism explaining cholinergic loss in neurodegenerative conditions such as AD and provide a potential therapeutic target for the protection or restoration of this CNS transmitter system in aging and AD.

  12. The NGF Metabolic Pathway in the CNS and its Dysregulation in Down Syndrome and Alzheimer's Disease.

    Science.gov (United States)

    Iulita, M Florencia; Cuello, A Claudio

    2016-01-01

    It is well established that individuals with Down syndrome develop Alzheimer's disease neuropathology by middle age. Both in Alzheimer's disease and Down syndrome, this is accompanied by the atrophy of NGF-dependent cholinergic neurons of the basal forebrain. An NGF trophic compromise in Alzheimer's disease had been early suspected. This hypothesis was discarded with the finding of unaltered NGF mRNA synthesis and of increased NGF precursor levels (proNGF) in postmortem Alzheimer's disease brains. The possibility of an NGF trophic disconnection has been recently revisited at the light of a newly discovered extracellular NGF metabolic pathway; where proNGF is released in an activity-dependent manner and converted by plasmin to mature NGF in the extracellular space. Mature NGF is ultimately degraded by the metalloprotease MMP-9. This pathway has been shown to be compromised in Alzheimer's disease and Down syndrome brains, thus reviving the trophic factor hypothesis to explain the atrophy of basal forebrain cholinergic neurons in these disorders. This chapter will discuss the physiological role of NGF and its biological significance to cholinergic neurons of the CNS, and present the evidence for a dysregulation of the NGF metabolism in Alzheimer's disease and Down syndrome.

  13. Radioimmunoassay of fragment E-related neoantigen: validation studies and clinical application. [Fibrinogen-fibrin degradation products

    Energy Technology Data Exchange (ETDEWEB)

    Chen, J.P.; Hanna, W.T.; Williams, T.K.; Krauss, S. (Tennessee Univ., Knoxville (USA). Memorial Research Center and Hospital)

    1984-05-01

    An E-neoantigen radioimmunoassay (Eneo RIA) is described which can determine normal and pathological plasma levels of E-related fibrinogen-fibrin degradation products (FDP). The assay employs rabbit antiserum produced against fragment E derived from a plasmin digest of fibrinogen and subsequently absorbed with fibrinogen. The absorbed antiserum contains antibodies which are equally reactive with fibrinogen derived E (Fg-E) and fibrin derived E(Fb-E) but not with fibrinogen at 1 mg/ml. The Eneo RIA was validated by assay parallelism and by recovery experiments. Plasma Eneo immunoreactivities in 14 normals were 4-22 ng/ml (mean 12.7 ng/ml). Plasma Eneo levels in 23 of 24 patients with neoplastic and haematological diseases were elevated above normal (range 27-2027 ng/ml). Unusually high Eneo values were observed with three patients whose diseases were complicated by either disseminated intravascular coagulation (DIC) or deep vein thrombosis. After heparin therapy, the Eneo level of a patient with chronic DIC declined. A pathological plasma was eluted from a Sephadex G-200 column and Eneo immunoreactivity was determined on the eluates. The gel filtration pattern of Eneo indicates that E-related FDP is a family of plasmic fragments derived from crosslinked fibrin.

  14. Uniform {sup 15}N- and {sup 15}N/{sup 13}C-labeling of proteins in mammalian cells and solution structure of the amino terminal fragment of u-PA

    Energy Technology Data Exchange (ETDEWEB)

    Hansen, A.P.; Petros, A.M.; Meadows, R.P.; Mazar, A.P.; Nettesheim, D.G.; Pederson, T.M.; Fesik, S.W. [Abbott Laboratories, Abbott Park, IL (United States)

    1994-12-01

    Urokinase-type plasminogen activator (u-PA) is a 54-kDa glycoprotein that catalyzes the conversion of plasminogen to plasmin, a broad-specificity protease responsible for the degradation of fibrin clots and extracellular matrix components. The u-PA protein consists of three individual modules: a growth factor domain (GFD), a kringle, and a serine protease domain. The amino terminal fragment (ATF) includes the GFD-responsible for u-PA binding to its receptor-and the kringle domains. This protein was expressed and uniformly {sup 15}N-and {sup 15}N/{sup 13}C-labeled in mammalian cells by methods that will be described. In addition, we present the three-dimensional structure of ATF that was derived from 1299 NOE-derived distance restraints along with the {phi} angle and hydrogen bonding restraints. Although the individual domains in the structures were highly converged, the two domains are structurally independent. The overall structures of the individual domains are very similar to the structures of homologous proteins. However, important structural differences between the growth factor domain of u-PA and other homologous proteins were observed in the region that has been implicated in binding the urokinase receptor. These results may explain, in part, why other growth factors show no appreciable affinity for the urokinase receptor.

  15. Human Kunitz-type protease inhibitor engineered for enhanced matrix retention extends longevity of fibrin biomaterials.

    Science.gov (United States)

    Briquez, Priscilla S; Lorentz, Kristen M; Larsson, Hans M; Frey, Peter; Hubbell, Jeffrey A

    2017-08-01

    Aprotinin is a broad-spectrum serine protease inhibitor used in the clinic as an anti-fibrinolytic agent in fibrin-based tissue sealants. However, upon re-exposure, some patients suffer from hypersensitivity immune reactions likely related to the bovine origin of aprotinin. Here, we aimed to develop a human-derived substitute to aprotinin. Based on sequence homology analyses, we identified the Kunitz-type protease inhibitor (KPI) domain of human amyloid-β A4 precursor protein as being a potential candidate. While KPI has a lower intrinsic anti-fibrinolytic activity than aprotinin, we reasoned that its efficacy is additionally limited by its fast release from fibrin material, just as aprotinin's is. Thus, we engineered KPI variants for controlled retention in fibrin biomaterials, using either covalent binding through incorporation of a substrate for the coagulation transglutaminase Factor XIIIa or through engineering of extracellular matrix protein super-affinity domains for sequestration into fibrin. We showed that both engineered KPI variants significantly slowed plasmin-mediated fibrinolysis in vitro, outperforming aprotinin. In vivo, our best engineered KPI variant (incorporating the transglutaminase substrate) extended fibrin matrix longevity by 50%, at a dose at which aprotinin did not show efficacy, thus qualifying it as a competitive substitute of aprotinin in fibrin sealants. Copyright © 2017 The Author(s). Published by Elsevier Ltd.. All rights reserved.

  16. Statistical analysis plan for the WOMAN-ETAPlaT study: Effect of tranexamic acid on platelet function and thrombin generation.

    Science.gov (United States)

    Dallaku, Kastriot; Shakur, Haleema; Edwards, Phil; Beaumont, Danielle; Roberts, Ian; Huque, Sumaya; Delius, Maria; Mansmann, Ulrich

    2016-12-15

    Background. Postpartum haemorrhage (PPH) is a potentially life-threatening complication for women, and the leading cause of maternal mortality. Tranexamic acid (TXA) is an antifibrinolytic used worldwide to treat uterine haemorrhage and to reduce blood loss in general surgery. TXA may have effects on thrombin generation, platelet function and coagulation factors as a result of its inhibition on the plasmin. Methods. WOMAN ETAPlaT is a sub-study of the World Maternal Antifibrinolitic trial (WOMAN trial). All adult women clinically diagnosed with PPH after a vaginal delivery or caesarean section, are eligible for inclusion in the study. Blood samples will be collected at the baseline and 30 minutes after the first dose of study treatment is given. Platelet function will be evaluated in whole blood immediately after sampling with Multiplate® tests (ADPtest and TRAPtest). Thrombin generation, fibrinogen, D-dimer, and coagulation factors vW, V and VIII will be analysed using platelet poor plasma. Results. Recruitment to WOMAN ETAPlaT started on 04 November 2013 and closed on 13 January 2015, during this time  188 patients were recruited. The final participant follow-up was completed on 04 March 2015. This article introduces the statistical analysis plan for the study, without reference to unblinded data.   Conclusion. The data from this study will provide evidence for the effect of TXA on thrombin generation, platelet function and coagulation factors in women with PPH. Trial registration: ClinicalTrials.gov Identifier: NCT00872469; ISRCTN76912190.

  17. Functional characterization of fibrinolytic metalloproteinases (colombienases) isolated from Bothrops colombiensis venom.

    Science.gov (United States)

    Girón, María E; Guerrero, Belsy; Salazar, Ana María; Sánchez, Elda E; Alvarez, Marco; Rodríguez-Acosta, Alexis

    2013-11-01

    Researchers trying to improve the safety and efficacy of fibrinolytic therapy have been isolating fibrinolytic enzymes from snake venoms. Two fibrinolytic enzymes, colombienases 1 and 2, with significant activity have been isolated from the venom of Bothrops colombiensis. The colombienases were characterized for various biological activities which include hemorrhagic, fibrinogenolytic, proteolytic, hemolytic, edematogenic and cytotoxic. Colombienases directly acted on fibrin by degrading fibrinogen Aα and Bβ chains without activating the fibrinolytic system (plasminogen/plasmin), additionally colombienase-2 degraded fibrinogen γ chains as well as the fibronectin molecule. Laminin and type IV collagen were colombienases resistant. Gelatin-zymogram activity was present in B. colombiensis venom (BcV) bands between 64 and 148 kDa. All activities were abolished by metalloproteinases inhibitors. Both enzymes lacked hemorrhagic, hemolytic, cytotoxic, plasminogen activator and coagulant activities. Both colombienases had direct fibrino(geno)lytic activity without other toxic side effects including in vivo hemorrhaging, which could be promising in terms of therapeutic potential as thrombolytic agents. Copyright © 2013 Elsevier Ltd. All rights reserved.

  18. Modification of fibrinogen with poly(ethylene glycol) and its effects on fibrin clot characteristics.

    Science.gov (United States)

    Barker, T H; Fuller, G M; Klinger, M M; Feldman, D S; Hagood, J S

    2001-09-15

    The suitability of existing topical fibrin glue preparations for tissue sealing or local drug delivery applications is greatly limited by their poor mechanical properties and the limited capacity of fibrinogen (Fgn) to actively bind growth factors or other therapeutic agents. Poly(ethylene glycol) (PEG) offers potential solutions to these problems by providing a mechanism for increasing the number of crosslinks between adjacent fibrin monomer molecules or for covalently crosslinking Fgn to therapeutic agents. The feasibility of this approach requires the full biological activity, or clottability, of PE glycolated Fgn. This study characterizes the clot characteristics of Fgn modified to varying degrees with monofunctional succinimidyl propionate PEG (5000 Da). The data indicate that, although thrombin clotting times are significantly altered, Fgn maintains 90% of its capacity to clot upon the addition of up to 5 PEG/Fgn. Further derivatization significantly decreases the Fgn clottability. The addition of up to 5 PEG/Fgn has little, if any, effect on the kinetics of degradation by plasmin. The results suggest that limited modification of Fgn with lysine-reactive PEG allows therapeutic enhancement of fibrin glues. Copyright 2001 John Wiley & Sons, Inc. J Biomed Mater Res 56: 529--535, 2001

  19. Asbestos exposure increases human bronchial epithelial cell fibrinolytic activity.

    Science.gov (United States)

    Gross, T J; Cobb, S M; Gruenert, D C; Peterson, M W

    1993-03-01

    Chronic exposure to asbestos fibers results in fibrotic lung disease. The distal pulmonary epithelium is an early target of asbestos-mediated injury. Local plasmin activity may be important in modulating endoluminal inflammatory responses in the lung. We studied the effects of asbestos exposure on cell-mediated plasma clot lysis as a marker of pericellular plasminogen activation. Exposing human bronchial epithelial (HBE) cells to 100 micrograms/ml of asbestos fibers for 24 h resulted in increased plasma clot lysis. Fibrinolytic activity was augmented in a dose-dependent fashion, was not due to secreted protease, and occurred only when there was direct contact between the plasma clot and the epithelial monolayer. Further analysis showed that asbestos exposure increased HBE cell-associated urokinase-type plasminogen activator (uPA) activity in a time-dependent manner. The increased cell-associated PA activity could be removed by acid washing. The increase in PA activity following asbestos exposure required new protein synthesis because it was abrogated by treatment with either cycloheximide or actinomycin D. Therefore, asbestos exposure increases epithelial-mediated fibrinolysis by augmenting expression of uPA activity at the cell surface by mechanisms that require new RNA and protein synthesis. These observations suggest a novel mechanism whereby exposure of the distal epithelium to inhaled particulates may result in a chronic inflammatory response that culminates in the development of fibrotic lung disease.

  20. Characterization of Plasminogen Binding to NB4 Promyelocytic Cells Using Monoclonal Antibodies against Receptor-Induced Binding Sites in Cell-Bound Plasminogen

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    Mercè Jardí

    2012-01-01

    Full Text Available The NB4 promyelocytic cell line exhibits many of the characteristics of acute promyelocytic leukemia blast cells, including the translocation (15 : 17 that fuses the PML gene on chromosome 15 to the RARα gene on chromosome 17. These cells have a very high fibrinolytic capacity. In addition to a high secretion of urokinase, NB4 cells exhibit a 10-fold higher plasminogen binding capacity compared with other leukemic cell lines. When tissue-type plasminogen activator was added to acid-treated cells, plasmin generation was 20–26-fold higher than that generated by U937 cells or peripheral blood neutrophils, respectively. We found that plasminogen bound to these cells can be detected by fluorescence-activated cell sorting using an antiplasminogen monoclonal antibody that specifically reacts with this antigen when it is bound to cell surfaces. All-trans retinoid acid treatment of NB4 cells markedly decreased the binding of this monoclonal antibody. This cell line constitutes a unique model to explore plasminogen binding and activation on cell surfaces that can be modulated by all-trans retinoid acid treatment.

  1. Binding and activation of host plasminogen on the surface of Francisella tularensis

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    Whitt Michael A

    2010-03-01

    Full Text Available Abstract Background Francisella tularensis (FT is a gram-negative facultative intracellular coccobacillus and is the causal agent of a life-threatening zoonotic disease known as tularemia. Although FT preferentially infects phagocytic cells of the host, recent evidence suggests that a significant number of bacteria can be found extracellularly in the plasma fraction of the blood during active infection. This observation suggests that the interaction between FT and host plasma components may play an important role in survival and dissemination of the bacterium during the course of infection. Plasminogen (PLG is a protein zymogen that is found in abundance in the blood of mammalian hosts. A number of both gram-positive and gram-negative bacterial pathogens have the ability to bind to PLG, giving them a survival advantage by increasing their ability to penetrate extracellular matrices and cross tissue barriers. Results We show that PLG binds to the surface of FT and that surface-bound PLG can be activated to plasmin in the presence of tissue PLG activator in vitro. In addition, using Far-Western blotting assays coupled with proteomic analyses of FT outer membrane preparations, we have identified several putative PLG-binding proteins of FT. Conclusions The ability of FT to acquire surface bound PLG that can be activated on its surface may be an important virulence mechanism that results in an increase in initial infectivity, survival, and/or dissemination of this bacterium in vivo.

  2. Acute arterial thrombosis associated with inadvertent high dose of tranexamic acid

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    Surjya Prasad Upadhyay

    2013-01-01

    Full Text Available Tranexamic acid (TA act as anti-fibrinolytic agent and is widely used to limit bleeding in clinical practice. Tranexemic acid bind with plasminogen and prevent its conversion to plasmin, which limits the fibrinolytic pathway, so there is a theoretical risk of increasing thrombosis with high or prolonged therapy with TA. We encountered a case of acute arterial thrombosis following inadvertent administration of high dose of TA. A 27-years-old male with no other co-morbidity was ordered intravenous 1 gm TA to control excessive bleeding from previous bladder injury, but by mistake, he received 10 gm of TA. The patient developed signs and symptoms of acute ischemia in the right lower limb, which was diagnosed as acute iliac arterial thrombosis by computed tomography (CT angiography. The patient was managed with systemic heparinization, fasciotomy for impending gangrene and other supportive care following which he recovered fully within a few days. Caution should be exercised for all prophylactic use, especially with high dosage or prolonged therapy with TA.

  3. Blood Peptidome-Degradome Profile of Breast Cancer

    Science.gov (United States)

    Shen, Yufeng; Tolić, Nikola; Liu, Tao; Zhao, Rui; Petritis, Brianne O.; Gritsenko, Marina A.; Camp, David G.; Moore, Ronald J.; Purvine, Samuel O.; Esteva, Francisco J.; Smith, Richard D.

    2010-01-01

    Background Cancer invasion and metastasis are closely associated with activities within the degradome; however, little is known about whether these activities can be detected in the blood of cancer patients. Methodology and Principal Findings The peptidome-degradome profiles of pooled blood plasma sampled from 15 breast cancer patients (BCP) and age, race, and menopausal status matched control healthy persons (HP) were globally characterized using advanced comprehensive separations combined with tandem Fourier transform mass spectrometry and new data analysis approaches that facilitated top-down peptidomic analysis. The BCP pool displayed 71 degradome protein substrates that encompassed 839 distinct peptidome peptides. In contrast, the HP 50 degradome substrates found encompassed 425 peptides. We find that the ratios of the peptidome peptide relative abundances can vary as much as >4000 fold between BCP and HP. The experimental results also show differential degradation of substrates in the BCP sample in their functional domains, including the proteolytic and inhibitory sites of the plasmin-antiplasmin and thrombin-antithrombin systems, the main chains of the extracellular matrix protection proteins, the excessive degradation of innate immune system key convertases and membrane attack complex components, as well as several other cancer suppressor proteins. Conclusions Degradomics-peptidomics profiling of blood plasma is highly sensitive to changes not evidenced by conventional bottom-up proteomics and potentially provides unique signatures of possible diagnostic utility. PMID:20976186

  4. Blood peptidome-degradome profile of breast cancer.

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    Yufeng Shen

    Full Text Available BACKGROUND: Cancer invasion and metastasis are closely associated with activities within the degradome; however, little is known about whether these activities can be detected in the blood of cancer patients. METHODOLOGY AND PRINCIPAL FINDINGS: The peptidome-degradome profiles of pooled blood plasma sampled from 15 breast cancer patients (BCP and age, race, and menopausal status matched control healthy persons (HP were globally characterized using advanced comprehensive separations combined with tandem Fourier transform mass spectrometry and new data analysis approaches that facilitated top-down peptidomic analysis. The BCP pool displayed 71 degradome protein substrates that encompassed 839 distinct peptidome peptides. In contrast, the HP 50 degradome substrates found encompassed 425 peptides. We find that the ratios of the peptidome peptide relative abundances can vary as much as >4000 fold between BCP and HP. The experimental results also show differential degradation of substrates in the BCP sample in their functional domains, including the proteolytic and inhibitory sites of the plasmin-antiplasmin and thrombin-antithrombin systems, the main chains of the extracellular matrix protection proteins, the excessive degradation of innate immune system key convertases and membrane attack complex components, as well as several other cancer suppressor proteins. CONCLUSIONS: Degradomics-peptidomics profiling of blood plasma is highly sensitive to changes not evidenced by conventional bottom-up proteomics and potentially provides unique signatures of possible diagnostic utility.

  5. Synthesis and characterization of enzymatically biodegradable PEG and peptide-based hydrogels prepared by click chemistry.

    Science.gov (United States)

    van Dijk, Maarten; van Nostrum, Cornelus F; Hennink, Wim E; Rijkers, Dirk T S; Liskamp, Rob M J

    2010-06-14

    Herein we describe the synthesis and rheological characterization of a series of enzymatically sensitive PEG and peptide-based hydrogels by the Cu(I)-catalyzed 1,3-dipolar cycloaddition reaction. The hydrogels were synthesized by a combination of alkyne-functionalized star-shaped PEG molecules (two 4-armed PEGs with M(w) 10 and 20 kDa, respectively, and one 8-armed PEG of 20 kDa) and the protease-sensitive bis-azido peptide, N(alpha)-(azido)-D-alanyl-phenylalanyl-lysyl-(2-azidoethyl)-amide (6) in the presence of CuSO(4) and sodium ascorbate in aqueous solution. The swelling ratio and the storage modulus (G') of the hydrogels could be tailored by several parameters, for example, the initial solid content of the hydrogel, the molecular weight of the PEG derivative, and by the architecture of the PEG molecule (4- versus 8-armed PEG derivative). The peptide sequence, D-Ala-Phe-Lys, was sensitive toward the proteases plasmin and trypsin to render the hydrogels biodegradable.

  6. HCG对不育症精子超微结构的影响%DThe effect of HCG to sperm's ultrastructure in male infertility

    Institute of Scientific and Technical Information of China (English)

    俞建军; 王培乐; 吴章穆; 陈昭典

    2001-01-01

    Objective To study the mechanisam of using HCG to treat male infertility. Methods 30 male infertility cases were selected randomly and their spermatozoa were fetched by masturbation. In every case, five sperms were observed under SEM (scan electronic microscope). After HCG had been given for 12 weeks, sperms were observed again. Results Sperms' ringed rips, acrosome membranes, plasmalemma, plasminic droplet changed significantly. Conclusion HCG had positive effect to sperms of male infertility patients.%目的探索 HCG治疗男性不育症的机理。方法随机选择男性不育症 30例,手淫法取精液每例扫描电镜观察精子五个,共 150个, HCG治疗 12周后,再作电镜观察。结果治疗前后的精子环状沟、顶体膜、质膜、胞浆小滴均有明显变化。结论 HCG治疗不育症疗效肯定。

  7. The Use of Solvent/Detergent Treatment in Pathogen Reduction of Plasma.

    Science.gov (United States)

    Hellstern, Peter; Solheim, Bjarte G

    2011-01-01

    The solvent/detergent (SD) process used for plasma can safely inactivate all lipid-enveloped viruses. The introduction of a specific prion-binding ligand gel in combination with SD treatment, time-reduced from 4 to 1-1.5 h, still ensures efficient virus kill, reduces abnormal prion protein by >5 log steps, and preserves levels of plasmin inhibitor at close to the reference range. Infections with known non-enveloped viruses such as HAV or parvovirus B19 are prevented by ensuring low virus loads in the starting plasma units, dilution through pooling of single plasma units, and neutralization of immune antibodies already present in the initial plasma pools. The major advantages of SD plasma over fresh frozen plasma and the other pathogen-inactivated plasmas are its extreme safety with respect to transfusion-related acute lung injury and the significantly lower likelihood of provoking allergic reactions. Both advantages are best interpreted as results of the dilution effect of pooling. No fewer than 18 clinical studies covering all indications for plasma, and extensive clinical experience have shown that reduced levels of coagulation factors and inhibitors as a result of SD treatment do not impair significantly the clinical efficacy or tolerance of plasma. Properly standardized clotting factor and inhibitor potencies and low batch-to-batch variations when compared with single-donor plasma units makes SD plasma more suitable for standardized treatment.

  8. Identification of lympho-epithelial Kazal-type inhibitor 2 in human skin as a kallikrein-related peptidase 5-specific protease inhibitor.

    Directory of Open Access Journals (Sweden)

    Ulf Meyer-Hoffert

    Full Text Available Kallikreins-related peptidases (KLKs are serine proteases and have been implicated in the desquamation process of the skin. Their activity is tightly controlled by epidermal protease inhibitors like the lympho-epithelial Kazal-type inhibitor (LEKTI. Defects of the LEKTI-encoding gene serine protease inhibitor Kazal type (Spink5 lead to the absence of LEKTI and result in the genodermatose Netherton syndrome, which mimics the common skin disease atopic dermatitis. Since many KLKs are expressed in human skin with KLK5 being considered as one of the most important KLKs in skin desquamation, we proposed that more inhibitors are present in human skin. Herein, we purified from human stratum corneum by HPLC techniques a new KLK5-inhibiting peptide encoded by a member of the Spink family, designated as Spink9 located on chromosome 5p33.1. This peptide is highly homologous to LEKTI and was termed LEKTI-2. Recombinant LEKTI-2 inhibited KLK5 but not KLK7, 14 or other serine proteases tested including trypsin, plasmin and thrombin. Spink9 mRNA expression was detected in human skin samples and in cultured keratinocytes. LEKTI-2 immune-expression was focally localized at the stratum granulosum and stratum corneum at palmar and plantar sites in close localization to KLK5. At sites of plantar hyperkeratosis, LEKTI-2 expression was increased. We suggest that LEKTI-2 contributes to the regulation of the desquamation process in human skin by specifically inhibiting KLK5.

  9. Bifidobacterial enolase, a cell surface receptor for human plasminogen involved in the interaction with the host.

    Science.gov (United States)

    Candela, Marco; Biagi, Elena; Centanni, Manuela; Turroni, Silvia; Vici, Manuela; Musiani, Francesco; Vitali, Beatrice; Bergmann, Simone; Hammerschmidt, Sven; Brigidi, Patrizia

    2009-10-01

    The interaction with the host plasminogen/plasmin system represents a novel component in the molecular cross-talk between bifidobacteria and human host. Here, we demonstrated that the plasminogen-binding bifidobacterial species B. longum, B. bifidum, B. breve and B. lactis share the key glycolytic enzyme enolase as a surface receptor for human plasminogen. Enolase was visualized on the cell surface of the model strain B. lactis BI07. The His-tagged recombinant protein showed a high affinity for human plasminogen, with an equilibrium dissociation constant in the nanomolar range. By site-directed mutagenesis we demonstrated that the interaction between the B. lactis BI07 enolase and human plasminogen involves an internal plasminogen-binding site homologous to that of pneumococcal enolase. According to our data, the positively charged residues Lys-251 and Lys-255, as well as the negatively charged Glu-252, of the B. lactis BI07 enolase are crucial for plasminogen binding. Acting as a human plasminogen receptor, the bifidobacterial surface enolase is suggested to play an important role in the interaction process with the host.

  10. Plasma carboxypeptidase U (CPU, CPB2, TAFIa) generation during in vitro clot lysis and its interplay between coagulation and fibrinolysis.

    Science.gov (United States)

    Leenaerts, Dorien; Aernouts, Jef; Van Der Veken, Pieter; Sim, Yani; Lambeir, Anne-Marie; Hendriks, Dirk

    2017-07-26

    Carboxypeptidase U (CPU, CPB2, TAFIa) is a basic carboxypeptidase that is able to attenuate fibrinolysis. The inactive precursor procarboxypeptidase U is converted to its active form by thrombin, the thrombin-thrombomodulin complex or plasmin. The aim of this study was to investigate and characterise the time course of CPU generation in healthy individuals. In plasma of 29 healthy volunteers, CPU generation was monitored during in vitro clot lysis. CPU activity was measured by means of an enzymatic assay that uses the specific substrate Bz-o-cyano-Phe-Arg. An algorithm was written to plot the CPU generation curve and calculate the parameters that define it. In all individuals, CPU generation was biphasic. Marked inter-individual differences were present and a reference range was determined. The endogenous CPU generation potential is the composite effect of multiple factors. With respect to the first CPU activity peak characteristics, we found correlations with baseline proCPU concentration, proCPU Thr325Ile polymorphism, time to clot initiation and the clot lysis time. The second CPU peak related with baseline proCPU levels and with the maximum turbidity of the clot lysis profile. In conclusion, our method offers a technique to determine the endogenous CPU generation potential of an individual. The parameters obtained by the method quantitatively describe the different mechanisms that influence CPU generation during the complex interplay between coagulation and fibrinolysis, which are in line with the threshold hypothesis.

  11. Don’t Forget What You Can’t See: A Case of Ocular Syphilis

    Science.gov (United States)

    Lee, Monica I.; Lee, Annie W.C.; Sumsion, Sean M.; Gorchynski, Julie A.

    2016-01-01

    This case describes an emergency department (ED) presentation of ocular syphilis in a human immunodeficiency virus (HIV) infected patient. This is an unusual presentation of syphilis and one that emergency physicians should be aware of. The prevalence of syphilis has reached epidemic proportions since 2001 with occurrences primarily among men who have sex with men (MSM). This is a case of a 24-year-old male who presented to our ED with bilateral painless vision loss. The patient’s history and ED workup were notable for MSM, positive rapid plasmin reagin (RPR) and HIV tests and fundus exam consistent with ocular syphilis, specifically uveitis. Ocular manifestations of syphilis can present at any stage of syphilis. The 2010 Centers for Disease Control and Prevention guidelines now recommend that ocular syphilis be treated as neurosyphilis regardless of the lumbar puncture results. There is a paucity of emergency medicine literature on ocular syphilis. For emergency physicians it is important to be aware of iritis, uveitis, or chorioretinitis as ocular manifestations of neurosyphilis especially in this high-risk population and to obtain RPR and HIV tests in the ED to facilitate early diagnosis, and treatment and to prevent irreversible vision loss. PMID:27429702

  12. Don’t Forget What You Can’t See: A Case of Ocular Syphilis

    Directory of Open Access Journals (Sweden)

    Monica I. Lee

    2016-06-01

    Full Text Available This case describes an emergency department (ED presentation of ocular syphilis in a human immunodeficiency virus (HIV infected patient. This is an unusual presentation of syphilis and one that emergency physicians should be aware of. The prevalence of syphilis has reached epidemic proportions since 2001 with occurrences primarily among men who have sex with men (MSM. This is a case of a 24-year-old male who presented to our ED with bilateral painless vision loss. The patient’s history and ED workup were notable for MSM, positive rapid plasmin reagin (RPR and HIV tests and fundus exam consistent with ocular syphilis, specifically uveitis. Ocular manifestations of syphilis can present at any stage of syphilis. The 2010 Centers for Disease Control and Prevention guidelines now recommend that ocular syphilis be treated as neurosyphilis regardless of the lumbar puncture results. There is a paucity of emergency medicine literature on ocular syphilis. For emergency physicians it is important to be aware of iritis, uveitis, or chorioretinitis as ocular manifestations of neurosyphilis especially in this high-risk population and to obtain RPR and HIV tests in the ED to facilitate early diagnosis, and treatment and to prevent irreversible vision loss.[West J Emerg Med. 2016;17(4:473-476.

  13. Application of long-acting VLHL PAI-1 during sutureless partial nephrectomy in mice reduces bleeding.

    Science.gov (United States)

    Shahrour, Khaled; Keck, Rick; Jankun, Jerzy

    2015-01-01

    PAI-1 prevents lysis of blood clot by inhibiting the urokinase and tPA induced conversion of plasminogen to plasmin. VLHL PAI-1 protein mutant was created to extend half-life over 700 hours. The objective of this paper was to test VLHL PAI-1 effects on bleeding during partial nephrectomy in mice. All animals had a left partial nephrectomy after intravenous infusion of saline or tPA. The animals were divided into four groups. Group 1 was infused with saline and kidney was exposed to saline too; Group 2 was infused with saline and kidney was exposed to PAI-1. Group 3 was infused with tPA and kidney was exposed to saline, while Group 4 was infused with tPA and kidney was exposed to PAI-1. Preweighed gauze containing PAI-1 or saline was then applied to the kidney for 30 minutes. The gauze was afterward weighed and blood loss was measured by subtracting the preweight of gauze from the final weight. We have observed a statistically significant (P ≤ 0.05) reduction of bleeding in PAI-1-treated group in comparison to saline and tPA-treated groups. Based on these results we propose that VLHL PAI-1 can be used therapeutically in limiting the flow of blood from renal wounds.

  14. An acidic microenvironment sets the humoral pattern recognition molecule PTX3 in a tissue repair mode.

    Science.gov (United States)

    Doni, Andrea; Musso, Tiziana; Morone, Diego; Bastone, Antonio; Zambelli, Vanessa; Sironi, Marina; Castagnoli, Carlotta; Cambieri, Irene; Stravalaci, Matteo; Pasqualini, Fabio; Laface, Ilaria; Valentino, Sonia; Tartari, Silvia; Ponzetta, Andrea; Maina, Virginia; Barbieri, Silvia S; Tremoli, Elena; Catapano, Alberico L; Norata, Giuseppe D; Bottazzi, Barbara; Garlanda, Cecilia; Mantovani, Alberto

    2015-06-01

    Pentraxin 3 (PTX3) is a fluid-phase pattern recognition molecule and a key component of the humoral arm of innate immunity. In four different models of tissue damage in mice, PTX3 deficiency was associated with increased fibrin deposition and persistence, and thicker clots, followed by increased collagen deposition, when compared with controls. Ptx3-deficient macrophages showed defective pericellular fibrinolysis in vitro. PTX3-bound fibrinogen/fibrin and plasminogen at acidic pH and increased plasmin-mediated fibrinolysis. The second exon-encoded N-terminal domain of PTX3 recapitulated the activity of the intact molecule. Thus, a prototypic component of humoral innate immunity, PTX3, plays a nonredundant role in the orchestration of tissue repair and remodeling. Tissue acidification resulting from metabolic adaptation during tissue repair sets PTX3 in a tissue remodeling and repair mode, suggesting that matrix and microbial recognition are common, ancestral features of the humoral arm of innate immunity. © 2015 Doni et al.

  15. Breaking boundaries—Coagulation and fibrinolysis at the neurovascular interface

    Directory of Open Access Journals (Sweden)

    Sophia eBardehle

    2015-09-01

    Full Text Available Blood proteins at the neurovascular unit (NVU are emerging as important molecular determinants of communication between the brain and the immune system. Over the past two decades, roles for the plasminogen activation/plasmin system in fibrinolysis have been extended from peripheral dissolution of blood clots to the regulation of central nervous system (CNS functions in physiology and disease. In this review, we discuss how fibrin and its proteolytic degradation affect neuroinflammatory, degenerative and repair processes. In particular, we focus on novel functions of fibrin—the final product of the coagulation cascade and the main substrate of plasmin—in the activation of immune responses and trafficking of immune cells into the brain. We also comment on the suitability of the coagulation and fibrinolytic systems as potential biomarkers and drug targets in diseases, such as multiple sclerosis, Alzheimer’s disease and stroke. Studying coagulation and fibrinolysis as major molecular pathways that regulate cellular functions at the NVU has the potential to lead to the development of novel strategies for the detection and treatment of neurologic diseases.

  16. Impact of the Pla protease substrate α2-antiplasmin on the progression of primary pneumonic plague.

    Science.gov (United States)

    Eddy, Justin L; Schroeder, Jay A; Zimbler, Daniel L; Bellows, Lauren E; Lathem, Wyndham W

    2015-12-01

    Many pathogens usurp the host hemostatic system during infection to promote pathogenesis. Yersinia pestis, the causative agent of plague, expresses the plasminogen activator protease Pla, which has been shown in vitro to target and cleave multiple proteins within the fibrinolytic pathway, including the plasmin inhibitor α2-antiplasmin (A2AP). It is not known, however, if Pla inactivates A2AP in vivo; the role of A2AP during respiratory Y. pestis infection is not known either. Here, we show that Y. pestis does not appreciably cleave A2AP in a Pla-dependent manner in the lungs during experimental pneumonic plague. Furthermore, following intranasal infection with Y. pestis, A2AP-deficient mice exhibit no difference in survival time, bacterial burden in the lungs, or dissemination from wild-type mice. Instead, we found that in the absence of Pla, A2AP contributes to the control of the pulmonary inflammatory response during infection by reducing neutrophil recruitment and cytokine production, resulting in altered immunopathology of the lungs compared to A2AP-deficient mice. Thus, our data demonstrate that A2AP is not significantly affected by the Pla protease during pneumonic plague, and although A2AP participates in immune modulation in the lungs, it has limited impact on the course or ultimate outcome of the infection.

  17. Purification and characterization of a novel fibrinolytic enzyme from culture supernatant of Pleurotus ostreatus.

    Science.gov (United States)

    Liu, Xiao-Lan; Zheng, Xi-Qun; Qian, Peng-Zhi; Kopparapu, Narasimha-Kumar; Deng, Yong-Ping; Nonaka, Masanori; Harada, Naoki

    2014-02-28

    A fibrinolytic enzyme was produced by an edible mushroom of Pleurotus ostreatus using submerged culture fermentation. The enzyme was purified from the culture supernatant by applying a combination of freeze-thaw treatment, ammonium sulfate precipitation, hydrophobic interaction, and gel filtration chromatographies. The enzyme was purified by a 147-fold, with a yield of 7.54%. The molecular masses of the enzyme an determined by gel filtration and SDSPAGE were 13.6 and 18.2 kDa, respectively. The isoelectric point of the enzyme was 8.52. It hydrolyzed fibrinogen by cleaving the α and β chains of fibrinogen followed by the γ chains, and also activated plasminogen into plasmin. The enzyme was optimally active at 45°C and pH 7.4. The enzyme activity was completely inhibited by EDTA, whereas protease inhibitors of TPCK, SBTI, PMSF, aprotinin and pepstatin showed no inhibition on its activity. The partial amino acid sequences of the enzyme as determined by Q-TOF2 were ATFVGCSATR, GGTLIHESSHFTR, and YTTWFGTFVTSR. These sequences showed a high degree of homology with those of metallo-endopeptidases from P. ostreatus and Armillaria mellea. The purified enzyme can also be applied as a natural agent for oral fibrinolytic therapy or prevention of thrombosis.

  18. Prevention of adult respiratory distress syndrome with plasminogen activator in pigs.

    Science.gov (United States)

    Hardaway, R M; Williams, C H; Marvasti, M; Farias, M; Tseng, A; Pinon, I; Yanez, D; Martinez, M; Navar, J

    1990-12-01

    Death from traumatic shock has been associated with loss of blood externally or internally. However, many patients die after trauma, even though blood volume restoration is adequate. Death is often due to pulmonary failure (adult respiratory distress syndrome [ARDS]). Death and ARDS have been associated with disseminated intravascular coagulation (DIC) and microclots in the lungs. Dissolution of the microclots after trauma can be achieved by activation of endogenous plasmin. Nine pigs were anesthetized for 48 h. Trauma was administered by 60 standard blows to each thigh resulting in a bruise of muscle but no skin, bone, or major vessel injury. Nutrition and respiration were maintained at normal levels. All nine pigs died with severe lung pathology and low PaO2. Ten other traumatized pigs were treated with a plasminogen activator iv 4 h after trauma. Five of these were treated with tissue plasminogen activator (tPA) and five with urokinase. All treated pigs survived 48 h and maintained a normal PaO2. Autopsy showed minimal lung pathology.

  19. Trifolium pallidum and Trifolium scabrum extracts in the protection of human plasma components.

    Science.gov (United States)

    Kolodziejczyk-Czepas, Joanna; Olas, Beata; Malinowska, Joanna; Wachowicz, Barbara; Moniuszko-Szajwaj, Barbara; Kowalska, Iwona; Oleszek, Wieslaw; Stochmal, Anna

    2013-02-01

    Clovers (genus: Trifolium) have been used in traditional medicine by many cultures, but the biological activity of the most of these plants still remains unknown. The aim of our in vitro study was to assess the antioxidative action of phenolic extracts from aerial parts of Trifolium scabrum and Trifolium pallidum in human blood plasma, exposed to oxidative stress. In the present study we also demonstrate, for the first time the effects of the tested extracts on coagulative properties and fibrinolytic activity of blood plasma. The protective properties of the examined extracts (0.5-50 μg/ml) against peroxynitrite-induced oxidative stress were estimated by the measurements of 3-nitrotyrosine, thiol groups and the thiobarbituric acid-reactive substances levels. The extracts considerably prevented the oxidative and nitrative damage to plasma proteins. Even the lowest doses of the Trifolium extracts (0.5 μg/ml) were able to markedly reduce 3-nitrotyrosine formation (by about 50%) and to increase the level of -SH groups (by about 30%), in comparison to the plasma exposed to ONOO(-) in the absence of the extracts. The protective action of all the used concentrations of the Trifolium extracts in the prevention of lipid peroxidation was also found. The tested extracts influenced neither the coagulative properties nor fibrinolytic activity of plasma. Moreover, the extracts were able to significantly reduce the inhibitory effect of ONOO(-) on fibrinolytic activity of plasma (assessed with the use of a chromogenic substrate for plasmin).

  20. Measurement of clottability of fibrin polymers using magnetic orientation

    Science.gov (United States)

    Iwasaka, M.; Ueno, S.; Tsuda, H.

    1996-04-01

    Fibrin polymers, as a kind of diamagnetic material, are oriented parallel to the direction of magnetic fields. We investigated the polymerization of partially digested fibrinogens in an 8 T magnetic field in order to observe the clotting ability of various sizes of fibrinogen fractions using a magnetic orientation technique. We purified high-molecular weight fraction (F1) and low-molecular weight fraction from human fibrinogen (F2). Fibrin gels were formed in an 8 T magnetic field for 9 h, and transmittancies were measured to evaluate the degree of magnetic orientation. The results show that a lack at the C-terminal half of one Aα chain did not affect the magnetic orientation of fibrin. We also investigated the effect of the digestion of fibrinogen by plasmin on the magnetic orientation of fibrin. The result shows that partially digested fibrin molecules also orient in an 8 T magnetic field. However, the degree of magnetic orientation significantly decreases when fragment X and fragment Y appear.

  1. Eisenia fetida Protease-III-1 Functions in Both Fibrinolysis and Fibrogenesis

    Directory of Open Access Journals (Sweden)

    Jing Zhao

    2007-01-01

    Full Text Available The fibrinolytic function of earthworm protease-III-1 (EfP-III-1 has been studied in recent years. Here, we found that EfP-III-1 acted not only in fibrinogenolysis, but also in fibrogenesis. We have used EfP-III-1 to hydrolyze fibrinogen, and to activate plasminogen and prothrombin. Based on the N-terminal sequences of the hydrolytic fragments, EfP-III-1 was showed to specifically recognize the carboxylic sites of arginine and lysine. Analyses by fibrinogenolysis mapping and amino acid sequencing revealed that the isozyme could cleave the alpha, beta, and gamma chains of fibrinogen, showing a high α-fibrinogenase, moderate β-fibrinogenase, and low γ-fibrinogenase activities. Interestingly, EfP-III-1 activated plasminogen and released active plasmin, suggesting a tPA-like function. Furthermore, EfP-III-1 showed a factor Xa-like function on prothrombin, producing alpha-thrombin. The function in both activating prothrombin and catalyzing fibrinogenolysis suggests that EfP-III-1 may play a role in the balance between procoagulation and anticoagulation.

  2. Purification and Characterisation of a Fibrinolytic Enzyme from Rhizopus micro sporus var. tuberosus

    Directory of Open Access Journals (Sweden)

    Shuli Zhang

    2015-01-01

    Full Text Available Extracellular fibrinolytic enzyme from Rhizopus microsporus var. tuberosus was purified and characterised. The microorganism was isolated in a distillery from daqu, a fermentative agent used in the production of Chinese liquor and vinegar at diff erent temperatures. The fibrinolytic enzyme was partially purifi ed by ammonium sulphate precipitation, dialysis, DEAE Sepharose® Fast Flow ion exchange chromatography and Sephadex G-75 gel filtration chromatography. The molecular mass of the fi brinolytic enzyme was estimated to be 24.5 kDa by SDS-PAGE. The purified enzyme showed optimal activity at pH=7.0 and 37 °C by fibrin plate method. It showed stronger resistance to the inhibition by trypsin and was stable at 37 °C retaining 96.1 % residual activity aft er 4 h of incubation. The fibrinolytic activity of the enzyme was enhanced by Na+, Ca2+, Mg2+ and Mn2+. Conversely, Zn2+ and Cu2+ partly inhibited enzymatic activity. Using fibrin plate method, we found that the enzyme not only degrades fibrin directly, but also activates plasminogen into plasmin to degrade fibrin. The results indicate that the pure enzyme has a potential in dissolving blood clot, and the possibility for application in the treatment of thrombosis.

  3. Arrhenius temperature dependence of in vitro tissue plasminogen activator thrombolysis

    Energy Technology Data Exchange (ETDEWEB)

    Shaw, George J [Department of Emergency Medicine, University of Cincinnati College of Medicine, Cincinnati, OH 45267-0769 (United States); Dhamija, Ashima [Department of Emergency Medicine, University of Cincinnati College of Medicine, Cincinnati, OH 45267-0769 (United States); Bavani, Nazli [Department of Emergency Medicine, University of Cincinnati College of Medicine, Cincinnati, OH 45267-0769 (United States); Wagner, Kenneth R [Department of Neurology, University of Cincinnati College of Medicine, Cincinnati, OH 45267-0769 (United States); Holland, Christy K [Department of Biomedical Engineering, University of Cincinnati College of Medicine, Cincinnati, OH 45267-0769 (United States)

    2007-06-07

    Stroke is a devastating disease and a leading cause of death and disability. Currently, the only FDA approved therapy for acute ischemic stroke is the intravenous administration of the thrombolytic medication, recombinant tissue plasminogen activator (tPA). However, this treatment has many contraindications and can have dangerous side effects such as intra-cerebral hemorrhage. These treatment limitations have led to much interest in potential adjunctive therapies, such as therapeutic hypothermia (T {<=} 35 deg. C) and ultrasound enhanced thrombolysis. Such interest may lead to combining these therapies with tPA to treat stroke, however little is known about the effects of temperature on the thrombolytic efficacy of tPA. In this work, we measure the temperature dependence of the fractional clot mass loss {delta}m(T) resulting from tPA exposure in an in vitro human clot model. We find that the temperature dependence is well described by an Arrhenius temperature dependence with an effective activation energy E{sub eff} of 42.0 {+-} 0.9 kJ mole{sup -1}. E{sub eff} approximates the activation energy of the plasminogen-to-plasmin reaction of 48.9 kJ mole{sup -1}. A model to explain this temperature dependence is proposed. These results will be useful in predicting the effects of temperature in future lytic therapies.

  4. Early administration of therapeutic anticoagulation following intravenous thrombolysis for acute cardiogenic embolic stroke caused by left ventricular thrombus: case report and topic review

    Directory of Open Access Journals (Sweden)

    Rick eGill

    2015-02-01

    Full Text Available Cardiogenic cerebral embolism represents 20% of all acute ischemic strokes with one third of these being caused by left ventricular thrombus (LVT. LVT is not a contraindication for treatment with intravenous recombinant tissue plasminogen activator (IV rtPA for acute ischemic stroke (AIS. However the subsequent treatment of a potentially unstable LVT is contraindicated for 24 hours following the use of IV rtPA according to current guidelines. We present a 66-year-old man with AIS treated with IV rtPA. Echocardiogram shortly after treatment demonstrated both a large apical and septal thrombus in the left ventricle and at 12 hours post IV rtPA infusion, therapeutic anticoagulation with heparin was started without complication. In practice, the action of IV rtPA outlasts its apparent half-life because of thrombin-binding and the prolonged effects and longer half-life of its product, plasmin, however the pharmacokinetics do not warrant prolonged avoidance of therapeutic anticoagulation when clinically indicated. Our case demonstrates that anticoagulation for potentially unstable LVT can be safely initiated at 12 hours following IV rtPA treatment for AIS.

  5. Pharmacokinetic characteristics of L-valyl-ara-C and its implication on the oral delivery of ara-C

    Institute of Scientific and Technical Information of China (English)

    Eun-pa CHEON; Hyo-kyung HAN

    2007-01-01

    Aim: To evaluate the pharmacokinetic characteristics of L-valyl-ara-C, a peptidomimetic prodrug of ara-C. Methods: After the synthesis of L-valyl-ara-C, the in vitro stability of L-valyl-ara-C was examined in various biological media. Plasma phannacokinetic profiles of ara-C and L-valyl-ara-C were also evaluated in rats. Results: The degradation of L-valyl-ara-C was negligible in fresh plasma and also in the presence of plasmin over a 2 h incubation period. Furthermore, L-valyl-ara-C appeared to be stable in the leukemia cell homogenates, and subsequently, it was far less cytotoxic than the parent, ara-C in AML2 and L1210cells. The chemical hydrolysis of L-valyl-ara-C was rather accelerated in acidic pH. Following an oral administration of L-valyl-ara-C, the appearance of ara-Cwas observed in plasma although the systemic exposure of the prodrug was much higher than that of ara-C. The bioavailability of ara-C was about 4% via prodrug administration. Conclusion: The amide bond of L-valyl-ara-C was stable against the enzymatic hydrolysis, and the utility of L-valyl-ara-C as an oral delivery sys-tem of ara-C appeared to be limited by its low metabolic conversion to ara-C in rats.

  6. Neuroimmunomodulatory effects of transcranial laser therapy combined with intravenous tPA administration for acute cerebral ischemic injury

    Institute of Scientific and Technical Information of China (English)

    Philip V. Peplow

    2015-01-01

    At present, the only FDA approved treatment for ischemic strokes is intravenous administration of tissue plasminogen activator within 4.5 hours of stroke onset. Owing to this brief window only a small percentage of patients receive tissue plasminogen activator. Transcranial laser therapy has been shown to be effective in animal models of acute ischemic stroke, resulting in signiifcant improvement in neurological score and function. NEST-1 and NEST-2 clinical trials in human patients have demonstrated the safety and positive trends in efifcacy of transcranial laser therapy for the treatment of ischemic stroke when initiated close to the time of stroke onset. Combining intravenous tissue plasminogen activator treatment with transcranial laser therapy may provide better functional outcomes. Statins given within 4 weeks of stroke onset improve stroke out-comes at 90 days compared to patients not given statins, and giving statins following transcranial laser therapy may provide an effective treatment for patients not able to be given tissue plasmin-ogen activator due to time constraints.

  7. Inhibition of Aeromonas sobria serine protease (ASP) by α2-macroglobulin.

    Science.gov (United States)

    Murakami, Yoji; Wada, Yoshihiro; Kobayashi, Hidetomo; Irie, Atsushi; Hasegawa, Makoto; Yamanaka, Hiroyasu; Okamoto, Keinosuke; Eto, Masatoshi; Imamura, Takahisa

    2012-10-01

    ASP is a serine protease secreted by Aeromonas sobria. ASP cleaves various plasma proteins, which is associated with onset of sepsis complications, such as shock and blood coagulation disorder. To investigate a host defense mechanism against this virulence factor, we examined the plasma for ASP inhibitor(s). Human plasma inhibited ASP activity for azocasein, which was almost completely abolished by treating plasma with methylamine, which inactivates α2-macroglobulin (α2-MG). The ASP-inhibitor complex in ASP-added plasma was not detected by immunoblotting using anti-ASP antibody; however, using gel filtration of the plasma ASP activity for an oligopeptide, the ASP substrate was eluted in the void fraction (Mw>200 000), suggesting ASP trapping by α2-MG. Indeed, human α2-MG inhibited ASP azocaseinolytic activity in a dose-dependent manner, rapidly forming a complex with the ASP. Fibrinogen degradation by ASP was completely inhibited in the presence of α2-MG. α1-Protease inhibitor, antithrombin, and α2-plasmin inhibitor neither inhibited ASP activity nor formed a complex with ASP. Surprisingly, ASP degraded these plasma serine protease inhibitors. Thus, α2-MG is the major ASP inhibitor in the human plasma and can limit ASP virulence activities in A. sobria infection sites. However, as shown by fluorescence correlation spectroscopy, slow ASP inhibition by α2-MG in plasma may indicate insufficient ASP control in vivo.

  8. BDNF pro-peptide regulates dendritic spines via caspase-3.

    Science.gov (United States)

    Guo, J; Ji, Y; Ding, Y; Jiang, W; Sun, Y; Lu, B; Nagappan, G

    2016-06-16

    The precursor of brain-derived neurotrophic factor (BDNF) (proBDNF) is enzymatically cleaved, by either intracellular (furin/PC1) or extracellular proteases (tPA/plasmin/MMP), to generate mature BDNF (mBDNF) and its pro-peptide (BDNF pro-peptide). Little is known about the function of BDNF pro-peptide. We have developed an antibody that specifically detects cleaved BDNF pro-peptide, but not proBDNF or mBDNF. Neuronal depolarization elicited a marked increase in extracellular BDNF pro-peptide, suggesting activity-dependent regulation of its extracellular levels. Exposure of BDNF pro-peptide to mature hippocampal neurons in culture dramatically reduced dendritic spine density. This effect was mediated by caspase-3, as revealed by studies with pharmacological inhibitors and genetic knockdown. BDNF pro-peptide also increased the number of 'elongated' mitochondria and cytosolic cytochrome c, suggesting the involvement of mitochondrial-caspase-3 pathway. These results, along with BDNF pro-peptide effects recently reported on growth cones and long-term depression (LTD), suggest that BDNF pro-peptide is a negative regulator of neuronal structure and function.

  9. Thrombin-activatable fibrinolysis inhibitor is degraded by Salmonella enterica and Yersinia pestis.

    Science.gov (United States)

    Valls Serón, M; Haiko, J; DE Groot, P G; Korhonen, T K; Meijers, J C M

    2010-10-01

     Pathogenic bacteria modulate the host coagulation system to evade immune responses or to facilitate dissemination through extravascular tissues. In particular, the important bacterial pathogens Salmonella enterica and Yersinia pestis intervene with the plasminogen/fibrinolytic system. Thrombin-activatable fibrinolysis inhibitor (TAFI) has anti-fibrinolytic properties as the active enzyme (TAFIa) removes C-terminal lysine residues from fibrin, thereby attenuating accelerated plasmin formation.  Here, we demonstrate inactivation and cleavage of TAFI by homologous surface proteases, the omptins Pla of Y. pestis and PgtE of S. enterica. We show that omptin-expressing bacteria decrease TAFI activatability by thrombin-thrombomodulin and that the anti-fibrinolytic potential of TAFIa was reduced by recombinant Escherichia coli expressing Pla or PgtE. The functional impairment resulted from C-terminal cleavage of TAFI by the omptins.  Our results indicate that TAFI is degraded directly by the omptins PgtE of S. enterica and Pla of Y. pestis. This may contribute to the ability of PgtE and Pla to damage tissue barriers, such as fibrin, and thereby to enhance spread of S. enterica and Y. pestis during infection. © 2010 International Society on Thrombosis and Haemostasis.

  10. Postoperative bleeding in cardiac surgery: the role of tranexamic acid in patients homozygous for the 5G polymorphism of the plasminogen activator inhibitor-1 gene.

    Science.gov (United States)

    Iribarren, Jose L; Jimenez, Juan J; Hernández, Domingo; Brouard, Maitane; Riverol, Debora; Lorente, Leonardo; de La Llana, Ramiro; Nassar, Ibrahim; Perez, Rosalia; Martinez, Rafael; Mora, Maria L

    2008-04-01

    Plasminogen activator inhibitor 1 (PAI-1) attenuates the conversion of plasminogen to plasmin. Polymorphisms of the PAI-1 gene are associated with varying PAI-1 levels and risk of prothrombotic events in nonsurgical patients. The purpose of this study, a secondary analysis of a clinical trial, was to investigate whether PAI-1 genotype affects the efficacy of tranexamic acid (TA) in reducing postoperative chest tube blood loss of patients undergoing cardiopulmonary bypass. Fifty patients were classified according to PAI-1 genotype (4G/4G, 4G/5G, or 5G/5G). Twenty-four received 2 g TA before and after cardiopulmonary bypass, whereas 26 received placebo. The authors recorded data related to coagulation, fibrinolysis, and bleeding before surgery, at admission to the intensive care unit (0 h), and 4 and 24 h later. In patients not receiving TA, those with the 5G/5G genotype had significantly higher chest tube blood loss and transfusion requirements compared with patients with the other genotypes at all time points. Patients with the 5G/5G genotype receiving TA showed significantly lower blood loss compared with the placebo group. There were no significant differences in blood loss or transfusion requirements between patients with the 4G/4G genotype when TA was used. Plasminogen activator inhibitor-1 5G/5G homozygotes who did not receive TA showed significantly greater postoperative bleeding than patients with other PAI-1 genotypes. 5G/5G homozygotes who received TA showed the greatest blood-sparing benefit.

  11. Noteworthy Chemistry of Chloroform.

    Science.gov (United States)

    Alston, Theodore A

    2016-07-01

    Inhaled chloroform anesthesia was introduced in 1847. Soon thereafter, the chemical reactivity of aerobically heated chloroform permitted John Snow and Claude Bernard to do seminal experiments in the assay of drug levels and drug metabolism. However, it was not widely appreciated until a clinical mishap in 1899 that thermal decomposition generated significant levels of toxic phosgene from air-polluting quantities of chloroform in poorly ventilated operating rooms that were illuminated by flames. Phosgene is also generated metabolically from chloroform. A clue appeared in the 1950s when subanesthetic traces of inhaled chloroform proved accidentally lethal to strains of male mice spontaneously expressing high levels of chloroform-metabolizing enzymes. Furthermore, in microbial experiments of 1967, the reactive chloroform molecule was inadvertently discovered to selectively inactivate vitamin B12-dependent enzymes. Chloroform can also activate enzymes. As a solvent, it was serendipitously found in 1903 to activate what is now known as plasminogen to plasmin. Copyright © 2016 Elsevier B.V. All rights reserved.

  12. Degradation of extracellular matrix proteins (fibronectin, vitronectin and laminin) by serine-proteinases isolated from Lonomia achelous caterpillar hemolymph.

    Science.gov (United States)

    Lucena, Sara; Guerrero, Belsy; Salazar, Ana M; Gil, Amparo; Arocha-Piñango, Carmen L

    2006-09-01

    Lonomia achelous is a caterpillar distributed in southern Venezuela and in northern Brazil that causes an acute hemorrhagic syndrome in people who have contact with its bristles. The effect of the crude hemolymph and its chromatographic fractions (FDII, Lonomin V and Lonomin V-2) on extracellular matrix proteins was studied. The chromatographic fractions show activities similar to plasmin and urokinase. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis, both lonomins appear as a protein band of 25 kDa under reduced conditions. By exclusion chromatography, the molecular weights of Lonomin V and Lonomin V-2 were 26.5 and 24.5 kDa, respectively. Fibronectin, laminin and vitronectin were degraded by all venom components. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, under reduced conditions, shows that lonomins degrade fibronectin in four main fragments of 116, 60, 50 and 30 kDa. Molecular exclusion chromatography in native conditions shows that the molecular masses of these fragments are > or = 300, 62 and 27 kDa. The proteolytic effect of lonomins was abolished by benzamidine/HCl, iodoacetic acid and aprotinin. The extracellular matrix protein degradation together with the fibrino(geno)lytic activity of hemolymph and its fractions could explain, in part, the hemorrhagic syndrome, and the wound dehiscence in persons who have had contact with the L. achelous caterpillar.

  13. Impaired long-chain fatty acid metabolism in mitochondria causes brain vascular invasion by a non-neurotropic epidemic influenza A virus in the newborn/suckling period: implications for influenza-associated encephalopathy.

    Science.gov (United States)

    Yao, Dengfu; Kuwajima, Masamichi; Chen, Ye; Shiota, Mayumi; Okumura, Yuushi; Yamada, Hiroshi; Kido, Hiroshi

    2007-05-01

    The neuropathogenesis of influenza-associated encephalopathy in children and Reye's syndrome remains unclear. A surveillance effort conducted during 2000-2003 in South-West Japan reveals that almost all fatal and handicapped influenza-associated encephalopathy patients exhibit a disorder of mitochondrial beta-oxidation with elevated serum acylcarnitine ratios (C(16:0)+C(18:1))/C(2). Here we show invasion by a non-neurotropic epidemic influenza A H3N2 virus in cerebral capillaries with progressive brain edema after intranasal infection of mice having impaired mitochondrial beta-oxidation congenitally or posteriorly in the newborn/ suckling periods. Mice genetically lacking of carnitine transporter OCTN2, resulting in carnitine deficiency and impaired beta-oxidation, exhibited significant higher virus-genome numbers in the brain, accumulation of virus antigen exclusively in the cerebral capillaries and increased brain vascular permeability compared to in wild type mice. Mini-plasmin, which proteolytically potentiates influenza virus multiplication in vivo and destroys the blood-brain barrier, accumulated with virus antigen in the brain capillaries of OCTN2-deficient mice but only a little in wild-type mice. These results suggest that the impaired mitochondrial beta-oxidation changes the susceptibility to a non-neurotropic influenza A virus as to multiplication in the brain capillaries and to cause brain edema. These pathological findings in the brain of mice having impaired mitochondrial beta-oxidation after influenza virus infection may have implications for human influenza-associated encephalopathy.

  14. Fibrinolysis in Trauma: "Myth," "Reality," or "Something in Between".

    Science.gov (United States)

    Walsh, Mark; Shreve, Jacob; Thomas, Scott; Moore, Ernest; Moore, Hunter; Hake, Daniel; Pohlman, Tim; Davis, Patrick; Ploplis, Victoria; Piscoya, Andres; Wegner, Julie; Bryant, John; Crepinsek, Anton; Lantry, James; Sheppard, Forest; Castellino, Francis

    2017-03-01

    The emphasis on fibrinolysis as an important contributor to trauma-induced coagulopathy (TIC) has led to a debate regarding the relative clinical significance of fibrinolysis in the setting of trauma. The debate has centered on two camps. The one camp defines fibrinolysis in trauma by standard coagulation tests as well as fibrin split products, D-dimers, and plasmin/antiplasmin levels. This camp favors a more liberal use of tranexamic acid and attributes more significance to hyperfibrinolysis in TIC. The other camp favors a definition of fibrinolysis based on the viscoelastic tests (VET), rotational thromboelastometry (ROTEM), and thromboelastography (TEG). These whole blood assays define hyperfibrinolysis at a higher threshold than plasma-based tests. Therefore, this VET camp reserves antifibrinolytic treatment for patients who demonstrate severe coagulopathy associated with hyperfibrinolysis. This bimodal attribution of the clinical relevance of fibrinolysis in trauma suggests that there may be an underlying "Myth" of the concept of TIC that was historically defined by plasma-based tests and a future "Reality" of the concept of TIC that is grounded on an understanding of TIC based on a VET-defined "fibrinolytic spectrum" of TIC. This narrative review explores this "Myth" and "Reality" of fibrinolysis in TIC and proposes a direction that will allow a "Future" interpretation of TIC that incorporates both the past "Myth" and present "Reality" of fibrinolysis TIC. Thieme Medical Publishers 333 Seventh Avenue, New York, NY 10001, USA.

  15. Extracellular proteolysis in structural and functional plasticity of mossy fiber synapses in hippocampus

    Directory of Open Access Journals (Sweden)

    Grzegorz eWiera

    2015-11-01

    Full Text Available Brain is continuously altered in response to experience and environmental changes. One of the underlying mechanisms is synaptic plasticity, which is manifested by modification of synapse structure and function. It is becoming clear that regulated extracellular proteolysis plays a pivotal role in the structural and functional remodeling of synapses during brain development, learning and memory formation. Clearly, plasticity mechanisms may substantially differ between projections. Mossy fiber synapses onto CA3 pyramidal cells display several unique functional features, including pronounced short-term facilitation, a presynaptically expressed LTP that is independent of NMDAR activation, and NMDA-dependent metaplasticity. Moreover, structural plasticity at mossy fiber synapses ranges from the reorganization of projection topology after hippocampus-dependent learning, through intrinsically different dynamic properties of synaptic boutons to pre- and postsynaptic structural changes accompanying LTP induction. Although concomitant functional and structural plasticity in this pathway strongly suggests a role of extracellular proteolysis, its impact only starts to be investigated in this projection. In the present report, we review the role of extracellular proteolysis in various aspects of synaptic plasticity in hippocampal mossy fiber synapses. A growing body of evidence demonstrates that among perisynaptic proteases, tPA/plasmin system, β-site amyloid precursor protein-cleaving enzyme 1 (BACE1 and metalloproteinases play a crucial role in shaping plastic changes in this projection. We discuss recent advances and emerging hypotheses on the roles of proteases in mechanisms underlying mossy fiber target specific synaptic plasticity and memory formation.

  16. Studies on the kinetics of plasminogen activation by tissue plasminogen activator.

    Science.gov (United States)

    Rånby, M

    1982-06-24

    The steady-state rate of plasminogen activation by tissue plasminogen activator has been determined at various plasminogen concentrations. A plasmin substrate method similar to that presented by Christensen and Müllertz (Biochim. Biophys. Acta 480 (1977) 257-281) was used. The reaction was studied using one-chain type and two-chain type tissue plasminogen activator, N-terminal glutamic acid and N-terminal lysine plasminogen in the presence and in the absence of fibrin (eight studies). The kinetic data were fitted to a general Wong-Hanes equation and the simplest equation with significant parameters was found. In the absence of fibrin N-terminal glutamic acid plasminogen activation obeyed the Michaelis-Menten rate equation (Km 4.9 and 7.6 micro M and kcat 0.0013 and 0.0078 s-1 for one-chain type and two-chain type tissue plasminogen activator, respectively. In the absence of fibrin the activation of N-terminal lysine plasminogen activation failed to obey the Michaelis-Menten rate equation. Fibrin was found to stimulate greatly (up to 1000-fold) the steady-state activation rate. A theory for the fibrin stimulating mechanism is presented.

  17. Mutation of Arg154 to Gly154 in urokinase augments its fibrin-specificity.

    Science.gov (United States)

    Peng, G; Ma, Z; Kuai, L; Zhu, D

    1997-04-01

    Rscu-PA and its mutant constructed by in vitro site specific mutagenesis of Arg154 in rscu-PA to Gly154 (mscu-PA) were both expressed in Escherichia coli. After in vitro denaturation and renaturation, the rscu-PA and mscu-PA were purified to homogeneity by Zn2+ selective precipitation, anti-u-PA IgG-sepharose CL 4B affinity chromatography. After activation by plasmin, the kinetic constants for the resultant mtcu-PA against synthetic substrate S2444 hydrolysis were found to be essentially identical to rtcu-PA, suggesting that no impairment had been exerted on the catalytic active site of mtcu-PA. However, both 125I-fibrin plasma-clot lysis and fibrinogenolysis showed that mtcu-PA possessed a higher fibrinolytic activity but hardly any degradation of fibrinogen in plasma compared to rtcu-PA and rscu-PA. It was concluded that the substitution of Arg154 by Gly154 in tcu-PA promoted the fibrin-specificity of urokinase.

  18. Newer thrombolytic drugs for acute myocardial infarction.

    Science.gov (United States)

    Reddy, D S

    1998-01-01

    Arterial thrombosis is the underlying cause of a wide variety of cardiovascular diseases such as myocardial infarction, stroke and pulmonary thromboembolism. All the currently used thrombolytic agents are plasminogen activators, which are very efficient in restoring the blood flow. The fibrinolytic system comprises an inactive proenzyme plasminogen, that is converted by plasminogen activators to the enzyme plasmin, that degrades fibrin. Despite the widespread use of established thrombolytic agents such as streptokinase, tissue-plasminogen activator and urokinase, all these agents suffer from a number of inadequacies including resistance to reperfusion, occurrence of acute coronary reocclusion and bleeding complications. The quest continues for thrombolytic agents with a higher potency, specific thrombolytic activity and fibrin selectivity. Several lines of research towards improvement of thrombolytic agents are being explored including the construction of mutants and variants of plasminogen activators, chimeric plasminogen activators and conjugates of plasminogen activators with monoclonal antibodies. Newer molecules such as pro-urokinase, saruplase, alteplase, K1K2Pu and staphylokinase have shown promise in animal models of arterial and venous thrombosis and also in pilot scale clinical studies in patients with myocardial infarction. However, more clinical trials are needed to determine whether these novel recombinant thrombolytic agents shows improved efficacy and fibrin specificity with minimal bleeding tendencies.

  19. Ovulation efficiency is reduced in mice that lack plasminogen activator gene function: functional redundancy among physiological plasminogen activators.

    Science.gov (United States)

    Leonardsson, G; Peng, X R; Liu, K; Nordström, L; Carmeliet, P; Mulligan, R; Collen, D; Ny, T

    1995-01-01

    Several lines of indirect evidence suggest that plasminogen activation plays a crucial role in degradation of the follicular wall during ovulation. However, single-deficient mice lacking tissue-type plasminogen activator (tPA), urokinase-type plasminogen activator (uPA), or PA inhibitor type 1(PAI-1) gene function were recently found to have normal reproduction, although mice with a combined deficiency of tPA and uPA were significantly less fertile. To investigate whether the reduced fertility of mice lacking PA gene function is due to a reduced ovulation mechanism, we have determined the ovulation efficiency in 25-day-old mice during gonadotropin-induced ovulation. Our results reveal that ovulation efficiency is normal in mice with a single deficiency of tPA or uPA but reduced by 26% in mice lacking both physiological PAs. This result suggests that plasminogen activation plays a role in ovulatory response, although neither tPA nor uPA individually or in combination is obligatory for ovulation. The loss of an individual PA seems to be functionally complemented by the remaining PA but this compensation does not appear to involve any compensatory up-regulation. Our data imply that a functionally redundant mechanism for plasmin formation operates during gonadotropin-induced ovulation and that PAs together with other proteases generate the proteolytic activity required for follicular wall degradation. Images Fig. 3 Fig. 4 PMID:8618918

  20. Data supporting the structural and functional characterization of Thrombin‐Activatable Fibrinolysis Inhibitor in breast cancer

    Science.gov (United States)

    Fawzy, Manal S.; Toraih, Eman A.

    2015-01-01

    The data in this paper is related to the research article entitled “Thrombin-activatable fibrinolysis inhibitor Thr325Ile polymorphism and plasma level in breast cancer: A pilot study” (Fawzy et al., 2015) [1]. Many emerging studies have begun to unravel the pathophysiologic role of the fibrinolytic system in breast cancer (BC) progression (Zorio et al., 2008) [2]. Activation of the fibrinolytic plasminogen/plasmin system results in degradation of protein barriers, thereby mediating cell migration essential for tumor growth, angiogenesis, and dissemination (Castellino and Ploplis, 2005) [3]. In the current study, in silico data analysis of Thrombin-Activatable Fibrinolysis Inhibitor (TAFI) gene and protein has been done. Data have been retrieved from several databases mentioned in details in the text. Determination and analysis of the structural and functional impact of TAFI and its expression could help elucidate the contribution of the TAFI pathway to acquired hemostatic dysfunction and will form the basis of potential therapeutic strategies to manipulate this pathway. An inhibition of TAFI (e.g. by FXI inhibitors) will offer the therapeutic possibilities to improve the decreased fibrinolysis and increase the efficiency of fibrinolytic therapy in thrombotic disorders including cancer. PMID:26740968

  1. Data supporting the structural and functional characterization of Thrombin‐Activatable Fibrinolysis Inhibitor in breast cancer

    Directory of Open Access Journals (Sweden)

    Manal S. Fawzy

    2015-12-01

    Full Text Available The data in this paper is related to the research article entitled “Thrombin-activatable fibrinolysis inhibitor Thr325Ile polymorphism and plasma level in breast cancer: A pilot study” (Fawzy et al., 2015 [1]. Many emerging studies have begun to unravel the pathophysiologic role of the fibrinolytic system in breast cancer (BC progression (Zorio et al., 2008 [2]. Activation of the fibrinolytic plasminogen/plasmin system results in degradation of protein barriers, thereby mediating cell migration essential for tumor growth, angiogenesis, and dissemination (Castellino and Ploplis, 2005 [3]. In the current study, in silico data analysis of Thrombin-Activatable Fibrinolysis Inhibitor (TAFI gene and protein has been done. Data have been retrieved from several databases mentioned in details in the text. Determination and analysis of the structural and functional impact of TAFI and its expression could help elucidate the contribution of the TAFI pathway to acquired hemostatic dysfunction and will form the basis of potential therapeutic strategies to manipulate this pathway. An inhibition of TAFI (e.g. by FXI inhibitors will offer the therapeutic possibilities to improve the decreased fibrinolysis and increase the efficiency of fibrinolytic therapy in thrombotic disorders including cancer.

  2. Nano-zymography Using Laser-Scanning Confocal Microscopy Unmasks Proteolytic Activity of Cell-Derived Microparticles

    Science.gov (United States)

    Briens, Aurélien; Gauberti, Maxime; Parcq, Jérôme; Montaner, Joan; Vivien, Denis; Martinez de Lizarrondo, Sara

    2016-01-01

    Cell-derived microparticles (MPs) are nano-sized vesicles released by activated cells in the extracellular milieu. They act as vectors of biological activity by carrying membrane-anchored and cytoplasmic constituents of the parental cells. Although detection and characterization of cell-derived MPs may be of high diagnostic and prognostic values in a number of human diseases, reliable measurement of their size, number and biological activity still remains challenging using currently available methods. In the present study, we developed a protocol to directly image and functionally characterize MPs using high-resolution laser-scanning confocal microscopy. Once trapped on annexin-V coated micro-wells, we developed several assays using fluorescent reporters to measure their size, detect membrane antigens and evaluate proteolytic activity (nano-zymography). In particular, we demonstrated the applicability and specificity of this method to detect antigens and proteolytic activities of tissue-type plasminogen activator (tPA), urokinase and plasmin at the surface of engineered MPs from transfected cell-lines. Furthermore, we were able to identify a subset of tPA-bearing fibrinolytic MPs using plasma samples from a cohort of ischemic stroke patients who received thrombolytic therapy and in an experimental model of thrombin-induced ischemic stroke in mice. Overall, this method is promising for functional characterization of cell-derived MPs. PMID:27022410

  3. Phenotypic and genotypic characterization of Streptococcus uberis isolated from bovine subclinical mastitis in Argentinean dairy farms

    Directory of Open Access Journals (Sweden)

    Mirta C Lasagno

    2011-09-01

    lecheros localizados en Argentina. Las cepas de S. uberis fueron caracterizadas fenotípicamente sobre la base de la presencia de factores de virulencia tales como el factor activador del plasminógeno (FAP, la hialuronidasa (HIA, la cápsula (CAP y el factor CAMP. Además, fueron caracterizadas genotípicamente por electroforesis de campos pulsados (PFGE. Las cepas de S. uberis expresaron el factor activador del plasminógeno, la hialuronidasa o la cápsula (65,5 %, 56,3 % y 59,4 %, respectivamente, pero solo el 25 % fueron CAMP positivas. Sobre la base de la combinación de los factores de virulencia se identificaron 13 perfiles de virulencia diferentes. Asimismo, se identificaron 18 patrones de PFGE con un 90 % de similitud entre las 32 cepas de S. uberis. Se presentó una gran diversidad de perfiles de virulencia y patrones de PFGE entre los tambos. Cepas con el mismo patrón de PFGE presentaron perfiles de virulencia diferentes. Las cepas de S. uberis causantes de MSC en bovinos incluidas en el presente estudio mostraron heterogeneidad con respecto a sus características fenotípicas y genotípicas, y los patrones de PFGE no estuvieron asociados con los perfiles de virulencia.

  4. Effects of Heat Treatment Processing on the Heat-sensitive Components in Milk%不同热处理工艺对牛乳中热敏感成分的影响

    Institute of Scientific and Technical Information of China (English)

    孙琦; 刘鹭; 蒋士龙; 张书文; 梁霄; 吕加平

    2012-01-01

    热处理是保证乳制品质量稳定和卫生安全的重要手段之一,牛乳在热处理过程中会发生美拉德反应、乳糖异构化和蛋白质变性等多种化学变化,且反应程度与热处理强度密切相关。文中以纯鲜生乳为材料,经巴氏、超巴氏和超高温瞬时杀菌(UHT)等热处理后,分析检测牛乳中主要热敏感成分的变化。结果表明:随着热处理强度的提高,牛乳中的糠氨酸含量呈指数关系增加,纤溶酶活则逐渐下降;热处理强度不同导致乳清蛋白各组分的变性程度也不同,其中β-LgB和BSA对热最敏感,β-LgA其次,α-La的耐热性最强;热处理温度越高,时间越长,牛乳中VB1和VB6的损失率也就越高。根据牛乳中热敏感成分含量或活性在热处理过程中的的变化规律,可以将糠氨酸、α-乳白蛋白和纤溶酶活等作为牛奶受热程度的指示物,从而更好的指导实际生产和维护消费者权益。%Thermal treatment is a key procedure in dairy products preparation to ensure the products safety. Dur- ing the heat processing, a variety of chemical reactions occurred, such as Maillard reaction, lactose isomerization and protein denaturation, which are all closely related to the heat treatment intensity. The objective of the present study is to evaluate the effect of different thermal treatments on the heat-sensitive components in milk by comparing the heat damage between raw milk, pasteurization milk, ultra- pasteurization milk and UHT milk. In the raw milk, the fu- rosine contents increased with increased heat treatment strength, while plasmin activity decreased. The thermal treat- ment caused whey protein denaturaed, in which 13-LgB and BSA were more sensitive to heat treatment and the ct-La was heat resistance; the content of vitamin B1 and B2 decreased as the improvement of thermal treatment. We can de- termine the content or activity of the furosine, α-La and plasmin to be used as the heat indicators in

  5. Urinary Proteolytic Activation of Renal Epithelial Na+ Channels in Chronic Heart Failure.

    Science.gov (United States)

    Zheng, Hong; Liu, Xuefei; Sharma, Neeru M; Li, Yulong; Pliquett, Rainer U; Patel, Kaushik P

    2016-01-01

    One of the key mechanisms involved in renal Na(+) retention in chronic heart failure (CHF) is activation of epithelial Na(+) channels (ENaC) in collecting tubules. Proteolytic cleavage has an important role in activating ENaC. We hypothesized that enhanced levels of proteases in renal tubular fluid activate ENaC, resulting in renal Na(+) retention in rats with CHF. CHF was produced by left coronary artery ligation in rats. By immunoblotting, we found that several urinary serine proteases were significantly increased in CHF rats compared with sham rats (fold increases: furin 6.7, prostasin 23.6, plasminogen 2.06, and plasmin 3.57 versus sham). Similar increases were observed in urinary samples from patients with CHF. Whole-cell patch clamp was conducted in cultured renal collecting duct M-1 cells to record Na(+) currents. Protease-rich urine (from rats and patients with CHF) significantly increased the Na(+) inward current in M-1 cells. Two weeks of protease inhibitor treatment significantly abrogated the enhanced diuretic and natriuretic responses to ENaC inhibitor benzamil in rats with CHF. Increased podocyte lesions were observed in the kidneys of rats with CHF by transmission electron microscopy. Consistent with these results, podocyte damage markers desmin and podocin expressions were also increased in rats with CHF (increased ≈2-folds). These findings suggest that podocyte damage may lead to increased proteases in the tubular fluid, which in turn contributes to the enhanced renal ENaC activity, providing a novel mechanistic insight for Na(+) retention commonly observed in CHF.

  6. Isolation, cloning and structural characterisation of boophilin, a multifunctional Kunitz-type proteinase inhibitor from the cattle tick.

    Directory of Open Access Journals (Sweden)

    Sandra Macedo-Ribeiro

    Full Text Available Inhibitors of coagulation factors from blood-feeding animals display a wide variety of structural motifs and inhibition mechanisms. We have isolated a novel inhibitor from the cattle tick Boophilus microplus, one of the most widespread parasites of farm animals. The inhibitor, which we have termed boophilin, has been cloned and overexpressed in Escherichia coli. Mature boophilin is composed of two canonical Kunitz-type domains, and inhibits not only the major procoagulant enzyme, thrombin, but in addition, and by contrast to all other previously characterised natural thrombin inhibitors, significantly interferes with the proteolytic activity of other serine proteinases such as trypsin and plasmin. The crystal structure of the bovine alpha-thrombin.boophilin complex, refined at 2.35 A resolution reveals a non-canonical binding mode to the proteinase. The N-terminal region of the mature inhibitor, Q16-R17-N18, binds in a parallel manner across the active site of the proteinase, with the guanidinium group of R17 anchored in the S(1 pocket, while the C-terminal Kunitz domain is negatively charged and docks into the basic exosite I of thrombin. This binding mode resembles the previously characterised thrombin inhibitor, ornithodorin which, unlike boophilin, is composed of two distorted Kunitz modules. Unexpectedly, both boophilin domains adopt markedly different orientations when compared to those of ornithodorin, in its complex with thrombin. The N-terminal boophilin domain rotates 9 degrees and is displaced by 6 A, while the C-terminal domain rotates almost 6 degrees accompanied by a 3 A displacement. The reactive-site loop of the N-terminal Kunitz domain of boophilin with its P(1 residue, K31, is fully solvent exposed and could thus bind a second trypsin-like proteinase without sterical restraints. This finding explains the formation of a ternary thrombin.boophilin.trypsin complex, and suggests a mechanism for prothrombinase inhibition in vivo.

  7. Complement Evasion by Pathogenic Leptospira.

    Science.gov (United States)

    Fraga, Tatiana Rodrigues; Isaac, Lourdes; Barbosa, Angela Silva

    2016-01-01

    Leptospirosis is a neglected infectious disease caused by spirochetes from the genus Leptospira. Pathogenic microorganisms, notably those which reach the blood circulation such as Leptospira, have evolved multiple strategies to escape the host complement system, which is important for innate and acquired immunity. Leptospira avoid complement-mediated killing through: (i) recruitment of host complement regulators; (ii) acquisition of host proteases that cleave complement proteins on the bacterial surface; and, (iii) secretion of proteases that inactivate complement proteins in the Leptospira surroundings. The recruitment of host soluble complement regulatory proteins includes the acquisition of Factor H (FH) and FH-like-1 (alternative pathway), C4b-binding protein (C4BP) (classical and lectin pathways), and vitronectin (Vn) (terminal pathway). Once bound to the leptospiral surface, FH and C4BP retain cofactor activity of Factor I in the cleavage of C3b and C4b, respectively. Vn acquisition by leptospires may result in terminal pathway inhibition by blocking C9 polymerization. The second evasion mechanism lies in plasminogen (PLG) binding to the leptospiral surface. In the presence of host activators, PLG is converted to enzymatically active plasmin, which is able to degrade C3b, C4b, and C5 at the surface of the pathogen. A third strategy used by leptospires to escape from complement system is the active secretion of proteases. Pathogenic, but not saprophytic leptospires, are able to secrete metalloproteases that cleave C3 (central complement molecule), Factor B (alternative pathway), and C4 and C2 (classical and lectin pathways). The purpose of this review is to fully explore these complement evasion mechanisms, which act together to favor Leptospira survival and multiplication in the host.

  8. Oral tranexamic acid with fluocinolone-based triple combination cream versus fluocinolone-based triple combination cream alone in melasma: An open labeled randomized comparative trial

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    Tanmay Padhi

    2015-01-01

    Full Text Available Background : Melasma is a common acquired cause of facial hyperpigmentation with no definitive therapy. Tranexamic acid, a plasmin inhibitor, has demonstrated depigmenting properties and combining this oral drug with other modalities of treatment has shown promising results. Objectives : To compare the efficacy of a combination of oral tranexamic acid and fluocinolone-based triple combination cream with that of fluocinolone-based triple combination cream alone in melasma among Indian patients. Materials and Methods : 40 patients of melasma of either sex attending to dermatology OPD were enrolled in this study. Participants were randomly divided into two groups with 20 patients in each group. Group A patients were asked to apply the cream only and Group B patients received oral tranexamic acid 250 mg twice daily and applied a triple combination cream containing fluocinolone acetonide 0.01%, tretinoin 0.05%, and hydroquinone 2% once daily for 8 weeks. Response was evaluated using melasma area severity index (MASI at baseline, 4 weeks, and 8 weeks. Results : 40 patients completed the study. The MASI scores at baseline, 4 weeks and 8 weeks in group A were 15.425 + 1.09, 11.075 + 9.167 and 6.995 + 6.056 respectively and in group B 18.243 + 1.05, 6.135 + 4.94 and 2.19 + 3.38. Intergroup comparison showed a faster reduction in pigmentation in Group B as compared to Group A and the results were statistically significant at 4 weeks (P value 0.014 and 8 weeks (P value 0.000. The efficacy was maintained throughout the 6-month follow-up period. Conclusion: Addition of oral tranexamic acid to fluocinolone-based triple combination cream results in a faster and sustained improvement in the treatment of melasma.

  9. Expression and large-scale production of the biochemically active human tissue-plasminogen activator in hairy roots of Oriental melon (Cucumis melo).

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    Kim, Sung-Ryong; Sim, Joon-Soo; Ajjappala, Hemavathi; Kim, Yong-Hwan; Hahn, Bum-Soo

    2012-01-01

    Human tissue-plasminogen activator (t-PA) is a thrombolytic protein that plays an active role in dissolving fibrin clots by fibrinolysis and in activating plasminogen to plasmin in blood vessels. t-PA and synthetic t-PA (st-PA) genes were expressed as enzymatically active form in hairy roots of Oriental melon (Cucumis melo L. cv. Geumssaragi-euncheon) infected by Agrobacterium rhizogenes. The insertion of the t-PA genes in genomic DNA of transgenic hairy roots was verified by PCR. The presence and expression of t-PA-specific transcripts in the total RNAs of transgenic hairy roots were confirmed by RT-PCR. Western blot analysis of the transgenic hairy roots showed a single major band of 59-kDa recombinant t-PAs. ELISA demonstrated that the highest level of recombinant t-PA (798 ng mg⁻¹) was detected in hairy roots expressing t-PA. Similarly, the maximum fibrinolysis of recombinant t-PAs was observed in hairy roots transformed with t-PA. WPM medium was found to be more suitable for rapid growth of hairy roots among all the seven media types tested. The hairy root production was 5.8 times higher than that of White medium. The total yield of hairy roots grown on WPM medium was 621.8±8.7 g L⁻¹ at pH 7.0. These studies demonstrate that the hairy roots could be employed for the mass production of enzymatically active t-PA.

  10. Structural basis of specific inhibition of tissue-type plasminogen activator by plasminogen activators inhibitor-1

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    Lihu Gong

    2016-03-01

    Full Text Available Thrombosis is a leading cause of death worldwide [1]. Recombinant tissue-type plasminogen activator (tPA is the FDA-approved thrombolytic drug for ischemic strokes, myocardial infarction and pulmonary embolism. tPA is a multi-domain serine protease of the trypsin-family [2] and catalyses the critical step in fibrinolysis [3], converting the zymogen plasminogen to the active serine protease plasmin, which degrades the fibrin network of thrombi and blood clots. tPA is rapidly inactivated by endogenous plasminogen activators inhibitor-1 (PAI-1 [4] (Fig. 1. Engineering on tPA to reduce its inhibition by PAI-1 without compromising its thrombolytic effect is a continuous effort [5]. Tenecteplase (TNK-tPA is a newer generation of tPA variant showing slower inhibition by PAI-1 [6]. Extensive studies to understand the molecular interactions between tPA and PAI-1 have been carried out [7–18], however, the precise details at atomic resolution remain unknown. We report the crystal structure of tPA·PAI-1 complex here. The methods required to achieve these data include: (1 recombinant expression and purification of a PAI-1 variant (14-1B containing four mutations (N150H, K154T, Q319L, and M354I, and a tPA serine protease domain (tPA-SPD variant with three mutations (C122A, N173Q, and S195A, in the chymotrypsin numbering [19]; (2 formation of a tPA-SPD·PAI-1 Michaëlis complex in vitro [19]; and (3 solving the three-dimensional structure for this complex by X-ray crystallography [deposited in the PDB database as 5BRR]. The data explain the specificity of PAI-1 for tPA and uPA [19,20], and provide structural basis to design newer generation of PAI-1-resistant tPA variants as thrombolytic agents [19].

  11. Clathrin and LRP-1-independent constitutive endocytosis and recycling of uPAR.

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    Katia Cortese

    Full Text Available BACKGROUND: The urokinase receptor (uPAR/CD87 is highly expressed in malignant tumours. uPAR, as a GPI anchored protein, is preferentially located at the cell surface, where it interacts with its ligands urokinase (uPA and the extracellular matrix protein vitronectin, thus promoting plasmin generation, cell-matrix interactions and intracellular signalling events. Interaction with a complex formed by uPA and its inhibitor PAI-1 induces cell surface down regulation and recycling of the receptor via the clathrin-coated pathway, a process dependent on the association to LRP-1. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we have found that along with the ligand-induced down-regulation, uPAR also internalizes and recycles constitutively through a second pathway that is independent of LRP-1 and clathrin but shares some properties with macropinocytosis. The ligand-independent route is amiloride-sensitive, does not require uPAR partitioning into lipid rafts, is independent of the activity of small GTPases RhoA, Rac1 and Cdc42, and does not require PI3K activity. Constitutively endocytosed uPAR is found in EEA1 positive early/recycling endosomes but does not reach lysosomes in the absence of ligands. Electron microscopy analysis reveals the presence of uPAR in ruffling domains at the cell surface, in macropinosome-like vesicles and in endosomal compartments. CONCLUSIONS/SIGNIFICANCE: These results indicate that, in addition to the ligand-induced endocytosis of uPAR, efficient surface expression and membrane trafficking might also be driven by an uncommon macropinocytic mechanism coupled with rapid recycling to the cell surface.

  12. Regulation of cell migration and invasion by specific modules of uPA: mechanistic insights and specific inhibitors.

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    Carriero, Maria Vincenza; Franco, Paola; Votta, Giuseppina; Longanesi-Cattani, Immacolata; Vento, Maria Teresa; Masucci, Maria Teresa; Mancini, Alessandro; Caputi, Mario; Iaccarino, Ingram; Stoppelli, Maria Patrizia

    2011-11-01

    Urokinase (uPA) is a 411 residues serine protease originally identified for its ability to activate plasminogen and generate plasmin, a broad-spectrum matrix- and fibrin-degrading enzyme. Later, this protease has been shown to possess also a clear-cut ability to stimulate cell migration and survival in a catalytic-independent manner. This activity turned out to be exerted through the growth factor-like domain (GFD-like, residues 1-49) of the protease binding to a GPIanchored membrane receptor (uPAR), in complex with transmembrane receptors such as integrins, the epidermal growth factor and the formyl-peptide receptors. Direct binding of uPA to integrins through its kringle (residues 50-131) and connecting peptide (residues 132-158) regions results in enhanced migration. The dual function of uPA in promoting migration while reducing the physical resistance of extracellular matrix underlies its crucial role in the invasion of malignant tumours. Consolidated evidence emerging from animal models and clinical studies shows that the overexpression of uPA is a causal determinant to tumour metastasis and is associated to a poor prognosis. Therefore, pinpointing the molecular interactions and identifying novel agents to interfere with the diverse activities of uPA is a goal of basic and applied research. In this review, we discuss the general theme of cell migration and invasion. A description of the uPA structure-function relationship and the functional effects of isolated domains is presented. Current information on molecular agonistic as well as antagonistic compounds, including the compounds which have reached clinical trials, is provided.

  13. A novel trypsin Kazal-type inhibitor from Aedes aegypti with thrombin coagulant inhibitory activity.

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    Watanabe, Renata M O; Soares, Tatiane S; Morais-Zani, Karen; Tanaka-Azevedo, Anita M; Maciel, Ceres; Capurro, Margareth L; Torquato, Ricardo J S; Tanaka, Aparecida S

    2010-08-01

    Kazal-type inhibitors play several important roles in invertebrates, such as anticoagulant, vasodilator and antimicrobial activities. Putative Kazal-type inhibitors were described in several insect transcriptomes. In this paper we characterized for the first time a Kazal unique domain trypsin inhibitor from the Aedes aegypti mosquito. Previously, analyses of sialotranscriptome of A. aegypti showed the potential presence of a Kazal-type serine protease inhibitor, in female salivary glands, carcass and also in whole male, which we named AaTI (A. aegypti trypsin inhibitor). AaTI sequence showed amino acid sequence similarity with insect thrombin inhibitors, serine protease inhibitor from Litopenaeus vannamei hemocytes and tryptase inhibitor from leech Hirudo medicinalis (LDTI). In this work we expressed, purified and characterized the recombinant AaTI (rAaTI). Molecular weight of purified rAaTI was 7 kDa rAaTI presented dissociation constant (K(i)) of 0.15 and 3.8 nM toward trypsin and plasmin, respectively, and it weakly inhibited thrombin amidolytic activity. The rAaTI was also able to prolong prothrombin time, activated partial thromboplastin time and thrombin time. AaTI transcription was confirmed in A. aegypti female salivary gland and gut 3 h and 24 h after blood feeding, suggesting that this molecule can act as anticoagulant during the feeding and digestive processes. Its transcription in larvae and pupae suggested that AaTI may also play other functions during the mosquito's development. Copyright 2010 Elsevier Masson SAS. All rights reserved.

  14. Surface-associated plasminogen binding of Cryptococcus neoformans promotes extracellular matrix invasion.

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    Jamal Stie

    Full Text Available BACKGROUND: The fungal pathogen Cryptococcus neoformans is a leading cause of illness and death in persons with predisposing factors, including: malignancies, solid organ transplants, and corticosteroid use. C. neoformans is ubiquitous in the environment and enters into the lungs via inhalation, where it can disseminate through the bloodstream and penetrate the central nervous system (CNS, resulting in a difficult to treat and often-fatal infection of the brain, called meningoencephalitis. Plasminogen is a highly abundant protein found in the plasma component of blood and is necessary for the degradation of fibrin, collagen, and other structural components of tissues. This fibrinolytic system is utilized by cancer cells during metastasis and several pathogenic species of bacteria have been found to manipulate the host plasminogen system to facilitate invasion of tissues during infection by modifying the activation of this process through the binding of plasminogen at their surface. METHODOLOGY: The invasion of the brain and the central nervous system by penetration of the protective blood-brain barrier is a prerequisite to the establishment of meningoencephalitis by the opportunistic fungal pathogen C. neoformans. In this study, we examined the ability of C. neoformans to subvert the host plasminogen system to facilitate tissue barrier invasion. Through a combination of biochemical, cell biology, and proteomic approaches, we have shown that C. neoformans utilizes the host plasminogen system to cross tissue barriers, providing support for the hypothesis that plasminogen-binding may contribute to the invasion of the blood-brain barrier by penetration of the brain endothelial cells and underlying matrix. In addition, we have identified the cell wall-associated proteins that serve as plasminogen receptors and characterized both the plasminogen-binding and plasmin-activation potential for this significant human pathogen. CONCLUSIONS: The results of

  15. Role of mesenchymal stem cell-derived fibrinolytic factor in tissue regeneration and cancer progression.

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    Heissig, Beate; Dhahri, Douaa; Eiamboonsert, Salita; Salama, Yousef; Shimazu, Hiroshi; Munakata, Shinya; Hattori, Koichi

    2015-12-01

    Tissue regeneration during wound healing or cancer growth and progression depends on the establishment of a cellular microenvironment. Mesenchymal stem cells (MSC) are part of this cellular microenvironment, where they functionally modulate cell homing, angiogenesis, and immune modulation. MSC recruitment involves detachment of these cells from their niche, and finally MSC migration into their preferred niches; the wounded area, the tumor bed, and the BM, just to name a few. During this recruitment phase, focal proteolysis disrupts the extracellular matrix (ECM) architecture, breaks cell-matrix interactions with receptors, and integrins, and causes the release of bioactive fragments from ECM molecules. MSC produce a broad array of proteases, promoting remodeling of the surrounding ECM through proteolytic mechanisms. The fibrinolytic system, with its main player plasmin, plays a crucial role in cell migration, growth factor bioavailability, and the regulation of other protease systems during inflammation, tissue regeneration, and cancer. Key components of the fibrinolytic cascade, including the urokinase plasminogen activator receptor (uPAR) and plasminogen activator inhibitor-1 (PAI-1), are expressed in MSC. This review will introduce general functional properties of the fibrinolytic system, which go beyond its known function of fibrin clot dissolution (fibrinolysis). We will focus on the role of the fibrinolytic system for MSC biology, summarizing our current understanding of the role of the fibrinolytic system for MSC recruitment and the functional consequences for tissue regeneration and cancer. Aspects of MSC origin, maintenance, and the mechanisms by which these cells contribute to altered protease activity in the microenvironment under normal and pathological conditions will also be discussed.

  16. Neuroprotection by urokinase plasminogen activator in the hippocampus.

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    Cho, Eunsil; Lee, Kyung Jin; Seo, Jung-Woo; Byun, Catherine Jeonghae; Chung, Sun-Ju; Suh, Dae Chul; Carmeliet, Peter; Koh, Jae-Young; Kim, Jong S; Lee, Joo-Yong

    2012-04-01

    Tissue plasminogen activator (tPA) and urokinase plasminogen activator (uPA), which are both used for thrombolytic treatment of acute ischemic stroke, are serine proteases that convert plasminogen to active plasmin. Although recent experimental evidences have raised controversy about the neurotoxic versus neuroprotective roles of tPA in acute brain injury, uPA remains unexplored in this context. In this study, we evaluated the effect of uPA on neuronal death in the hippocampus of mice after kainate-induced seizures. In the normal brain, uPA was localized to both nuclei and cytosol of neurons. Following severe kainate-induced seizures, uPA completely disappeared in degenerating neurons, whereas uPA-expressing astrocytes substantially increased, suggesting reactive astrogliosis. uPA-knockout mice were more vulnerable to kainate-induced neuronal death than wild-type mice. Consistent with this, inhibition of uPA by intracerebral injection of the uPA inhibitor UK122 increased the level of neuronal death. In contrast, prior administration of recombinant uPA significantly attenuated neuronal death. Collectively, these results indicate that uPA renders neurons resistant to kainate-induced excitotoxicity. Moreover, recombinant uPA suppressed cell death in primary cultures of hippocampal neurons exposed to H2O2, zinc, or various excitotoxins, suggesting that uPA protects against neuronal injuries mediated by the glutamate receptor, or by oxidation- or zinc-induced death signaling pathways. Considering that tPA may facilitate neurodegeneration in acute brain injury, we suggest that uPA, as a neuroprotectant, might be beneficial for the treatment of acute brain injuries such as ischemic stroke.

  17. Altered behavior in mice with deletion of the alpha2-antiplasmin gene.

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    Eri Kawashita

    Full Text Available BACKGROUND: The α2-antiplasmin (α2AP protein is known to be a principal physiological inhibitor of plasmin, and is expressed in various part of the brain, including the hippocampus, cortex, hypothalamus and cerebellum, thus suggesting a potential role for α2AP in brain functions. However, the involvement of α2AP in brain functions is currently unclear. OBJECTIVES: The goal of this study was to investigate the effects of the deletion of the α2AP gene on the behavior of mice. METHODS: The motor function was examined by the wire hang test and rotarod test. To evaluate the cognitive function, a repeated rotarod test, Y-maze test, Morris water maze test, passive or shuttle avoidance test and fear conditioning test were performed. An open field test, dark/light transition test or tail suspension test was performed to determine the involvement of α2AP in anxiety or depression-like behavior. RESULTS AND CONCLUSIONS: The α2AP knockout (α2AP-/- mice exhibited impaired motor function compared with α2AP+/+ mice. The α2AP-/- mice also exhibited impairments in motor learning, working memory, spatial memory and fear conditioning memory. Furthermore, the deletion of α2AP induced anxiety-like behavior, and caused an anti-depression-like effect in tail suspension. Therefore, our findings suggest that α2AP is a crucial mediator of motor function, cognitive function, anxiety-like behavior and depression-like behavior, providing new insights into the role of α2AP in the brain functions.

  18. The novel plasminogen receptor, plasminogen receptor(KT) (Plg-R(KT)), regulates catecholamine release.

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    Bai, Hongdong; Baik, Nagyung; Kiosses, William B; Krajewski, Stan; Miles, Lindsey A; Parmer, Robert J

    2011-09-23

    Neurotransmitter release by catecholaminergic cells is negatively regulated by prohormone cleavage products formed from plasmin-mediated proteolysis. Here, we investigated the expression and subcellular localization of Plg-R(KT), a novel plasminogen receptor, and its role in catecholaminergic cell plasminogen activation and regulation of catecholamine release. Prominent staining with anti-Plg-R(KT) mAb was observed in adrenal medullary chromaffin cells in murine and human tissue. In Western blotting, Plg-R(KT) was highly expressed in bovine adrenomedullary chromaffin cells, human pheochromocytoma tissue, PC12 pheochromocytoma cells, and murine hippocampus. Expression of Plg-R(KT) fused in-frame to GFP resulted in targeting of the GFP signal to the cell membrane. Phase partitioning, co-immunoprecipitation with urokinase-type plasminogen activator receptor (uPAR), and FACS analysis with antibody directed against the C terminus of Plg-R(KT) were consistent with Plg-R(KT) being an integral plasma membrane protein on the surface of catecholaminergic cells. Cells stably overexpressing Plg-R(KT) exhibited substantial enhancement of plasminogen activation, and antibody blockade of non-transfected PC12 cells suppressed plasminogen activation. In functional secretion assays, nicotine-evoked [(3)H]norepinephrine release from cells overexpressing Plg-R(KT) was markedly decreased (by 51 ± 2%, p < 0.001) when compared with control transfected cells, and antibody blockade increased [(3)H]norepinephrine release from non-transfected PC12 cells. In summary, Plg-R(KT) is present on the surface of catecholaminergic cells and functions to stimulate plasminogen activation and modulate catecholamine release. Plg-R(KT) thus represents a new mechanism and novel control point for regulating the interface between plasminogen activation and neurosecretory cell function.

  19. The role of coagulation/fibrinolysis during Streptococcus pyogenes infection.

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    Loof, Torsten G; Deicke, Christin; Medina, Eva

    2014-01-01

    The hemostatic system comprises platelet aggregation, coagulation and fibrinolysis and is a host defense mechanism that protects the integrity of the vascular system after tissue injury. During bacterial infections, the coagulation system cooperates with the inflammatory system to eliminate the invading pathogens. However, pathogenic bacteria have frequently evolved mechanisms to exploit the hemostatic system components for their own benefit. Streptococcus pyogenes, also known as Group A Streptococcus, provides a remarkable example of the extraordinary capacity of pathogens to exploit the host hemostatic system to support microbial survival and dissemination. The coagulation cascade comprises the contact system (also known as the intrinsic pathway) and the tissue factor pathway (also known as the extrinsic pathway), both leading to fibrin formation. During the early phase of S. pyogenes infection, the activation of the contact system eventually leads to bacterial entrapment within a fibrin clot, where S. pyogenes is immobilized and killed. However, entrapped S. pyogenes can circumvent the antimicrobial effect of the clot by sequestering host plasminogen on the bacterial cell surface that, after conversion into its active proteolytic form, plasmin, degrades the fibrin network and facilitates the liberation of S. pyogenes from the clot. Furthermore, the surface-localized fibrinolytic activity also cleaves a variety of extracellular matrix proteins, thereby enabling S. pyogenes to migrate across barriers and disseminate within the host. This review summarizes the knowledge gained during the last two decades on the role of coagulation/fibrinolysis in host defense against S. pyogenes as well as the strategies developed by this pathogen to evade and exploit these host mechanisms for its own benefit.

  20. Labeling of the Recombinant Streptokinase Using Iodine-131 as a New Thrombolytic Agent

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    I. Daruwati

    2012-12-01

    Full Text Available Stroke and acute myocardial infarction is a disease with the highest mortality in the world. WHO has estimated in 2008, 30% of deaths from heart disease and more than 80% of this cases have been occurred in developing countries. Streptokinase (SK as an effective thrombolytic agent has been used as a drug of choice for about forty years ago. SK is plasminogen (PG activator that converts plasminogen to active protease, called plasmin (PN which degrades fibrin to soluble products. Recombinant streptokinase (SKA from genetic engineering has been developed at School of Pharmacy ITB to reduce or eliminate immunogenicity of SK. However, scientific disclosures relating to dynamic and its kinetic studies in the body have still to be proven. One method that can explain this phenomenon is the pharmacological studies using radionuclide labeled compounds. Radioiodine labeled compound is used extensively and most suitable for biological studies. This paper describes the preparation of 131I-SKA and its characterization. The labeling conditions of SKA, such as chloramine-T as an oxidizing agent, amount of SKA, incubation time, and size of resin to purify the labeling yield have been observed. The result showed that the optimum condition of labeling (35.11% was obtained using 10 µg of chloramine-T and 60 seconds of incubation time. The highest radiochemical purity (97.46 ± 1.14% has also been obtained by passing through the resin chromatography column using 100 mg Dowex 1x8, size 50-100 mesh. The characterization of 131I-SKA with SDS PAGE method and autoradiography showed the similar performance with unlabeled-SKA

  1. Cambios en la expresión de los marcadores de disfunción endotelial PAL-1/PAI-2 en preeclampsia leve y severa, y en gestantes normotensas sanas

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    Rodrigo Valenzuela Grau

    2013-01-01

    Full Text Available Objetivo: Determinar las concentraciones del Inhibidor del Activador del Plasminógeno tipo 1 y 2(PAI 1 y 2 y del factor de crecimiento placentario (PIGF  como marcadores de disfunción endotelial y angiogénesis en preeclampsia(PE leve y severa, respectivamente. Métodos: Estudio de casos y controles realizado en el Hospital Clínico de la Universidad de Chile durante junio del 2009 y diciembre del 2010. La población estuvo conformada por embarazadas adscritas durante el periodo de estudio y fue dividida en tres grupos: controles (n= 18, PE leve (n=9, PE severa (n=23. Se determinaron las concentraciones de PIGF y PAI-1/PAI-2 mediante técnicas de inmunoensayo a partir de las muestras obtenidas de cada paciente. Para el análisis estadístico se utilizó test de ANOVA-Bonferroni. Se consideró significativo un p < 0.05. Resultados: Se observó niveles menores de PlGF y mayores de PAI-1/PAI-2 en PE en comparación con los controles. Mujeres con PE severa mostraron niveles más altos de relación PAI-1/PAI-2 que mujeres con PE leve (p < 0.05. No hubo diferencia significativa para PlGF entre PE leve y severa. Conclusiones: La gravedad de la preeclampsia podría reflejarse en alteraciones de los biomarcadores de disfunción endotelial y angiogénesis. La medición de PlGF y relación PAI-1/PAI-2 en embarazos tempranos podría tener un valor pronóstico en el desarrollo de PE

  2. Interaction of ceruloplasmin with eosinophil peroxidase as compared to its interplay with myeloperoxidase: Reciprocal effect on enzymatic properties.

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    Sokolov, A V; Kostevich, V A; Zakharova, E T; Samygina, V R; Panasenko, O M; Vasilyev, V B

    2015-06-01

    Myeloperoxidase (MPO) and eosinophil peroxidase (EPO) are involved in the development of halogenative stress during inflammation. We previously described a complex between MPO and ceruloplasmin (CP). Considering the high structural homology between MPO and EPO, we studied the latter's interaction with CP and checked whether EPO becomes inhibited in a complex with CP. Disc-electrophoresis and gel filtration showed that CP and EPO form a complex with the stoichiometry 1:1. Affinity chromatography of EPO on CP-agarose (150 mM NaCl, 10 mM Na-phosphate buffer, of pH 7.4) resulted in retention of EPO. EPO protects ceruloplasmin from limited proteolysis by plasmin. Only intact CP shifted the Soret band typical of EPO from 413 to 408 nm. The contact with CP likely causes changes in the heme pocket of EPO. Peroxidase activity of EPO with substrates such as guaiacol, orcinol, o-dianisidine, 4-chloro-1-naphtol, 3,3',5,5'-tetramethylbenzidine, and 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonate) is inhibited by CP in a dose-dependent manner. Similar to the interaction with MPO, the larger a substrate molecule, the stronger the inhibitory effect of CP upon EPO. The limited proteolysis of CP abrogates its capacity to inhibit the peroxidase activity of EPO. The peptide RPYLKVFNPR (corresponding to amino acids 883-892 in CP) inhibits the peroxidase and chlorinating activity of EPO. Only the chlorinating activity of EPO is efficiently inhibited by CP, while the capacity of EPO to oxidize bromide and thiocyanate practically does not depend on the presence of CP. EPO enhances the p-phenylenediamine-oxidase activity of CP. The structural homology between the sites in the MPO and EPO molecules enabling them to contact CP is discussed.

  3. The cell envelope-bound metalloprotease (camelysin) from Bacillus cereus is a possible pathogenic factor.

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    Fricke, B; Drössler, K; Willhardt, I; Schierhorn, A; Menge, S; Rücknagel, P

    2001-09-28

    A novel membrane proteinase of the nosocomial important bacteria species Bacillus cereus (synonyms: camelysin, CCMP) was purified up to homogeneity as was shown by mass spectrometry in its amphiphilic form. Camelysin is a neutral metalloprotease with a molecular mass of 19 kDa. Its unique N-terminus Phe-Phe-Ser-Asp-Lys-Glu-Val-Ser-Asn-Asn-Thr-Phe-Ala-Ala-Gly-Thr-Leu-Asp-Leu-Thr-Leu-Asn-Pro-Lys-Thr-Leu-Val-Asp-(Ile-Lys-Asp)- was not detected in the protein data bases during BLAST searches, but in the partially sequenced genome of Bacillus anthracis, coding for an unknown protein. Cleavage sites of the membrane proteinase for the insulin A- and B-chains were determined by mass spectrometry and N-terminal sequencing. Camelysin prefers cleavage sites in front of aliphatic and hydrophilic amino acid residues (-OH, -SO3H, amido group), avoiding bulky aromatic residues. The internally quenched fluorogenic substrates of the matrix metalloproteases 2 and 7 were cleaved with the highest efficiency at the Leu-decrease-Gly or Leu-decrease-Ala bond with the smaller residue in the P1' position. The protein specificity is broad--all various kinds of casein were cleaved as well as acid-soluble collagen, globin and ovalbumin; intact insulin was destroyed only to a low extent. Actin, collagen type I, fibrinogen, fibrin, alpha2-antiplasmin and alpha1-antitrypsin were cleaved. The protease formed SDS-stable complexes with Glu-plasminogen and antithrombin III, visible after SDS electrophoresis by gold staining and Western blot. The CCMP-plasminogen complex caused a partial activation of plasminogen to plasmin. Camelysin interacts with proteins of the blood coagulation cascade and could facilitate the penetration of fibrin clots and of the extracellular matrix during bacterial invasion.

  4. Thrombelastography and biomarker profiles in acute coagulopathy of trauma: a prospective study

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    Larsen Claus F

    2011-10-01

    Full Text Available Abstract Background Severe injury induces an acute coagulopathy associated with increased mortality. This study compared the Thrombelastography (TEG and biomarker profiles upon admission in trauma patients. Methods Prospective observational study of 80 trauma patients admitted to a Level I Trauma Centre. Data on demography, biochemistry including standard coagulation tests, hematology, transfusions, Injury Severity Score (ISS and TEG were recorded. Retrospective analysis of thawed plasma/serum for biomarkers reflecting tissue injury (histone-complexed DNA fragments, sympathoadrenal activation (adrenaline, noradrenaline, coagulation activation/inhibition and fibrinolysis (sCD40L, protein C, activated Protein C, tissue-type plasminogen activator, plasminogen activator inhibitor-1, D-dimer, prothrombinfragment 1+2, plasmin/α2-antiplasmin complex, thrombin/antithrombin complex, tissue factor pathway inhibitor, antithrombin, von willebrand factor, factor XIII. Comparison of patients stratified according to ISS/TEG maximum clot strength. Linear regression analysis of variables associated with clot strength. Results Trauma patients had normal (86%, hypercoagulable (11% or hypocoagulable (1% TEG clot strength; one had primary hyperfibrinolysis. Hypercoagulable patients had higher age, fibrinogen and platelet count (all p 10 red blood cells the initial 24 h. Patients with normal or hypercoagulable TEG clot strength had comparable biomarker profiles, but the few patients with hypocoagulable TEG clot strength and/or hyperfibrinolysis had very different biomarker profiles. Increasing ISS was associated with higher levels of catecholamines, histone-complexed DNA fragments, sCD40L, activated protein C and D-dimer and reduced levels of non-activated protein C, antithrombin, fibrinogen and factor XIII (all p 26. In patients with ISS > 26, adrenaline and sCD40L were independently negatively associated with clot strength. Conclusions Trauma patients displayed

  5. Longistatin, a plasminogen activator, is key to the availability of blood-meals for ixodid ticks.

    Directory of Open Access Journals (Sweden)

    Anisuzzaman

    2011-03-01

    Full Text Available Ixodid ticks are notorious blood-sucking ectoparasites and are completely dependent on blood-meals from hosts. In addition to the direct severe effects on health and productivity, ixodid ticks transmit various deadly diseases to humans and animals. Unlike rapidly feeding vessel-feeder hematophagous insects, the hard ticks feed on hosts for a long time (5-10 days or more, making a large blood pool beneath the skin. Tick's salivary glands produce a vast array of bio-molecules that modulate their complex and persistent feeding processes. However, the specific molecule that functions in the development and maintenance of a blood pool is yet to be identified. Recently, we have reported on longistatin, a 17.8-kDa protein with two functional EF-hand Ca(++-binding domains, from the salivary glands of the disease vector, Haemaphysalis longicornis, that has been shown to be linked to blood-feeding processes. Here, we show that longistatin plays vital roles in the formation of a blood pool and in the acquisition of blood-meals. Data clearly revealed that post-transcriptional silencing of the longistatin-specific gene disrupted ticks' unique ability to create a blood pool, and they consequently failed to feed and replete on blood-meals from hosts. Longistatin completely hydrolyzed α, β and γ chains of fibrinogen and delayed fibrin clot formation. Longistatin was able to bind with fibrin meshwork, and activated fibrin clot-bound plasminogen into its active form plasmin, as comparable to that of tissue-type plasminogen activator (t-PA, and induced lysis of fibrin clot and platelet-rich thrombi. Plasminogen activation potentiality of longistatin was increased up to 4 times by soluble fibrin. Taken together, our results suggest that longistatin may exert potent functions both as a plasminogen activator and as an anticoagulant in the complex scenario of blood pool formation; the latter is critical to the feeding success and survival of ixodid ticks.

  6. Peritoneal lavage efficiently eliminates protease-alpha-2-macroglobulin complexes and components of the contact system from the peritoneal cavity in patients with severe acute pancreatitis.

    Science.gov (United States)

    Aasen, A O; Ruud, T E; Roeise, O; Bouma, B N; Stadaas, J O

    1989-01-01

    Trypsin (Try), plasma kallikrein (KK) and plasmin activities together with coagulation factor XII (F XII, Hageman factor), high-molecular-weight kininogen (HMWK), plasma prekallikrein (PKK), alpha 2-macroglobulin (alpha 2-M), C1 inhibitor (C1Inh), and functional plasma kallikrein inhibition (KKI) values were studied in peritoneal fluid and lavage taps of 9 patients with severe acute pancreatitis treated with peritoneal lavage. Both immunochemical methods and functional techniques based on chromogenic peptide substrate assays were used. In the exudate obtained before peritoneal lavage was performed, F XII was 52%, HMWK was 30%, PKK was 40%, alpha 2-M was 29% and C1Inh was 57% of standard plasma pool values, determined by immunochemical technique. Functional plasma KKI values were zero, whereas Try activities determined by chromogenic peptide substrate technique were markedly elevated in the exudate. Using a prepacked HR 10/30 Superose Tm 12 column (Pharmacia, Uppsala, Sweden) and chromogenic peptide substrate assays, Try and KK activities were detected in the alpha 2-M containing fractions of the peritoneal exudate demonstrating KK-alpha 2-M and Try-alpha 2-M complex formation. The peritoneal lavage procedure efficiently eliminated components of the contact system and protease activities. In the first lavage tap, Try activities were markedly reduced compared to values found in the exudate and concentrations of F XII, HMWK, PKK, alpha 2-M and C1Inh were all zero. In consecutive lavage taps Try values were also zero. The study shows that the lavage procedures efficiently clears the peritoneal cavity for protease-alpha 2-M complexes generated during acute pancreatitis. Also, components of the contact system found in peritoneal exudate, and which might serve as substrates for the protease-alpha 2-M complexes, are rapidly eliminated by the procedure.

  7. Promotion of Wound Healing by an Agonist of Adenosine A2A Receptor Is Dependent on Tissue Plasminogen Activator.

    Science.gov (United States)

    Montesinos, M Carmen; Desai-Merchant, Avani; Cronstein, Bruce N

    2015-12-01

    Impaired wound healing, as it occurs in diabetes mellitus or long-term corticoid treatment, is commonly associated with disability, diminished quality of life, and high economic costs. Selective agonists of the A2A receptor subtype of adenosine, an endogenous regulator of inflammation, promote tissue repair in animal models, both healthy and with impaired healing. Plasmin-mediated proteolysis of fibrin and other matrix proteins is essential for cell migration at sites of injury. Since adenosine A2A receptor activation increases plasminogen activator release from macrophages and mast cells, we studied the effect of a selective agonist, CGS-21680, on full-thickness excisional wound closure in wild-type, urokinase plasminogen activator (uPA)-deficient, and tissue plasminogen activator (tPA)-deficient mice. Wound closure was impaired in tPA- and uPA-deficient mice as compared with wild-type mice, and topical application of CGS-21680 significantly increased the rate at which wounds closed in wild-type mice and uPA-deficient mice, but not in tPA-deficient mice. Immunostaining of tissue sections showed that tPA was present in endothelial cells and histiocytes by day 3 post-wound and also by day 6. In contrast, uPA was more prominent in these cell types only by day 6 post-wound. Our results confirm that plasminogen activation contributes to wound repair and are consistent with the hypothesis that adenosine A2A receptor activation promotes wound closure by a mechanism that depends upon tPA, but not uPA. Moreover, our results suggest that topical adenosine A2A receptor agonists may be useful in promotion of wound closure in patients with impaired wound healing.

  8. Evaluation of Multiplex-Based Antibody Testing for Use in Large-Scale Surveillance for Yaws: a Comparative Study.

    Science.gov (United States)

    Cooley, Gretchen M; Mitja, Oriol; Goodhew, Brook; Pillay, Allan; Lammie, Patrick J; Castro, Arnold; Moses, Penias; Chen, Cheng; Ye, Tun; Ballard, Ronald; Martin, Diana L

    2016-05-01

    WHO has targeted yaws for global eradication by 2020. The program goals are to interrupt the transmission in countries where yaws is endemic and to certify countries as yaws free where yaws was endemic in the past. No new rapid plasmin reagin (RPR) seroreactivity in young children is required for certification of elimination at a country level. We sought to evaluate whether antibody responses to specific treponemal antigens measured in a high-throughput multiplex bead array (MBA) assay differentiate past versus current infection and whether a nontreponemal lipoidal antigen test can be incorporated into the MBA. Serum and dried blood spot specimens collected for yaws surveillance projects in Ghana, Vanuatu, and Papua New Guinea (PNG) were run on MBA to measure antibodies against recombinant p17 (rp17) and treponemal membrane protein A (TmpA) treponemal antigens. Results were compared to standard treponemal laboratory (TPPA or TPHA [TPP(H)A]) and quantitative RPR test data. Of 589 specimens, 241 were TPP(H)A(+)/RPR(+), 88 were TPP(H)A(+)/RPR(-), 6 were TPP(H)A(-)/RPR(+), and 254 were negative for both tests. Compared to TPP(H)A, reactive concordance of rp17 was 93.7%, while reactive concordance of TmpA was only 81.9%. TmpA-specific reactivity showed good correlation with RPR titers (R(2) = 0.41; P RPR testing (cardiolipin) were not detected in the MBA. Our results suggest that TmpA can be used as a treponemal antigen marker for recent or active infection and potentially replace RPR in a high-throughput multiplex tool for large-scale yaws surveillance.

  9. In vitro characterization of a novel factor Xa inhibitor, RPR 130737.

    Science.gov (United States)

    Chu, V; Brown, K; Colussi, D; Choi, Y M; Green, D; Pauls, H W; Spada, A P; Perrone, M H; Leadley, R J; Dunwiddie, C T

    2000-07-01

    RPR 130737 inhibited factor Xa (FXa) with a Ki of 2.4 nM and also displayed excellent specificity toward FXa relative to other serine proteases. It showed selectivity of more than 1000-fold over thrombin, activated protein C, plasmin, tissue-plasminogen activator and trypsin. RPR 130737 prolonged plasma activated partial thromboplastin time and prothrombin time in a dose-dependent fashion. In the activated partial thromboplastin time assay, the concentrations required for doubling coagulation time were 0.32 microM (human), 0.61 microM (monkey), 0.44 microM (dog), 0.15 microM (rabbit), and 0.82 microM (rat). The concentrations required to double prothrombin time were 0.86 microM (human), and 1.26 microM (monkey), 1.15 microM (dog), 0.39 microM (rabbit) and 7.31 microM (rat). Kinetic studies revealed that RPR 130737 was a fast binding, reversible and competitive inhibitor for FXa when Spectrozyme FXa, a chromogenic substrate, was used. A coupled-enzyme assay measuring thrombin activity following prothrombinase conversion of prothrombin to thrombin indicated that RPR 130737 was a potent inhibitor for prothrombinase-bound FXa. In this assay, RPR 130737 showed IC50s of 17 nM and 35.9 nM, respectively when artificial phosphatidylserine/phosphatidylcholine (PS/PC) liposomes or gel-filtered platelets were used as the phospholipid source. An FX-deficient plasma clotting-time correction assay further demonstrated that RPR 130737 was a specific inhibitor of FXa. RPR 130737 showed no effect on platelet aggregation in vitro. These results indicate that RPR 130737 has the potential to be developed as an antithrombotic agent based on its potent and selective inhibitory effect against FXa.

  10. Thrombostatin FM compounds: direct thrombin inhibitors - mechanism of action in vitro and in vivo

    Energy Technology Data Exchange (ETDEWEB)

    Nieman, M T; Burke, F; Warnock, M; Zhou, Y; Sweigart, J; Chen, A; Ricketts, D; Lucchesi, B R; Chen, Z; Cera, E Di; Hilfinger, J; Kim, J S; Mosberg, H I; Schmaier, A H [Case Western; (Michigan); (TSRL); (WU-MED)

    2008-04-29

    Novel pentapeptides called Thrombostatin FM compounds consisting mostly of D-isomers and unusual amino acids were prepared based upon the stable angiotensin converting enzyme breakdown product of bradykinin - RPPGF. These peptides are direct thrombin inhibitors prolonging the thrombin clotting time, activated partial thromboplastin time, and prothrombin time at ≥0.78, 1.6, and 1.6 μm, respectively. They competitively inhibit α-thrombin-induced cleavage of a chromogenic substrate at 4.4--8.2 μm. They do not significantly inhibit plasma kallikrein, factor (F) XIIa, FXIa, FIXa, FVIIa-TF, FXa, plasmin or cathepsin G. One form, FM19 [rOicPaF(p-Me)], blocks α-thrombin-induced calcium flux in fibroblasts with an IC50 of 6.9 ± 1.2 μm. FM19 achieved 100% inhibition of threshold α- or γ-thrombin-induced platelet aggregation at 8.4 ± 4.7 μm and 16 ± 4 μm, respectively. The crystal structure of thrombin in complex with FM19 shows that the N-terminal D-Arg retrobinds into the S1 pocket, its second residue Oic interacts with His-57, Tyr-60a and Trp-60d, and its C-terminal p-methyl Phe engages thrombin's aryl binding site composed of Ile-174, Trp-215, and Leu-99. When administered intraperitoneal, intraduodenal, or orally to mice, FM19 prolongs thrombin clotting times and delays carotid artery thrombosis. FM19, a low affinity reversible direct thrombin inhibitor, might be useful as an add-on agent to address an unmet need in platelet inhibition in acute coronary syndromes in diabetics and others who with all current antiplatelet therapy still have reactive platelets.

  11. Biochemical characterization of a novel fibrinolytic enzyme from Cordyceps militaris.

    Science.gov (United States)

    Liu, Xiaolan; Kopparapu, Narasimha-Kumar; Li, Yao; Deng, Yongping; Zheng, Xiqun

    2017-01-01

    A fibrinolytic enzyme was produced by the medicinal mushroom, Cordyceps militaris using submerged fermentation. The enzyme was purified from culture supernatant by hydrophobic interaction, ion exchange and gel filtration chromatographies. It was purified by 36 fold, with a specific activity of 1,467.4U/mg protein and the final yield was 5.8%. The molecular weight of the enzyme as determined by SDS-PAGE and gel filtration was 28kDa and 24.5kDa, respectively, and its isoelectric point (pI) was 9.0±0.2. It was found to be a glycoprotein with carbohydrate content of 1.67% (w/v). The enzyme was optimally active at 37°C and pH 7.2. The enzyme activity was strongly inhibited by soybean trypsin inhibitor (SBTI) and aprotinin which indicated it to be a serine protease, while other inhibitors like N-α-tosyl-l-phenylalanine chloromethyl ketone (TPCK), phenyl methane sulfonyl fluoride (PMSF), pepstatin and metal chelator EDTA did not inhibit its activity. Amino acid sequences of the purified enzyme were determined partially by Q-TOF2 and they were IEDFPYQVDLR; ANCGGTVISEK; YVLTAGHCAEGYTGLNIR; TNYASVTPITADMICAGFPEGK; KDSCSGDSGGPLVTGGK; VVGIVSFGTGCAR; ANKPGVYSSVASAEIR. Sequences of the seven peptides completely matched with those of a trypsin-like serine protease from Cordyceps militaris CM01 (accession no. EGX95217.1). The purified enzyme degraded α chains of fibrinogen first and then β and γ chains and also activated plasminogen into plasmin. It can act as an anticoagulant and prevent clot formation by degrading fibrinogen. Based on these studies, the purified enzyme has great potential to be developed as a natural agent for prevention and treatment of thrombolytic diseases. Copyright © 2016 Elsevier B.V. All rights reserved.

  12. Inhibition of Lung Fluid Clearance and Epithelial Na+ Channels by Chlorine, Hypochlorous Acid, and Chloramines*

    Science.gov (United States)

    Song, Weifeng; Wei, Shipeng; Zhou, Yongjian; Lazrak, Ahmed; Liu, Gang; Londino, James D.; Squadrito, Giuseppe L.; Matalon, Sadis

    2010-01-01

    We investigated the mechanisms by which chlorine (Cl2) and its reactive byproducts inhibit Na+-dependent alveolar fluid clearance (AFC) in vivo and the activity of amiloride-sensitive epithelial Na+ channels (ENaC) by measuring AFC in mice exposed to Cl2 (0–500 ppm for 30 min) and Na+ and amiloride-sensitive currents (INa and Iamil, respectively) across Xenopus oocytes expressing human α-, β-, and γ-ENaC incubated with HOCl (1–2000 μm). Both Cl2 and HOCl-derived products decreased AFC in mice and whole cell and single channel INa in a dose-dependent manner; these effects were counteracted by serine proteases. Mass spectrometry analysis of the oocyte recording medium identified organic chloramines formed by the interaction of HOCl with HEPES (used as an extracellular buffer). In addition, chloramines formed by the interaction of HOCl with taurine or glycine decreased INa in a similar fashion. Preincubation of oocytes with serine proteases prevented the decrease of INa by HOCl, whereas perfusion of oocytes with a synthetic 51-mer peptide corresponding to the putative furin and plasmin cleaving segment in the γ-ENaC subunit restored the ability of HOCl to inhibit INa. Finally, INa of oocytes expressing wild type α- and γ-ENaC and a mutant form of βENaC (S520K), known to result in ENaC channels locked in the open position, were not altered by HOCl. We concluded that HOCl and its reactive intermediates (such as organic chloramines) inhibit ENaC by affecting channel gating, which could be relieved by proteases cleavage. PMID:20106988

  13. Mathematical modeling of cancer cell invasion of tissue: biological insight from mathematical analysis and computational simulation.

    Science.gov (United States)

    Andasari, Vivi; Gerisch, Alf; Lolas, Georgios; South, Andrew P; Chaplain, Mark A J

    2011-07-01

    The ability of cancer cells to break out of tissue compartments and invade locally gives solid tumours a defining deadly characteristic. One of the first steps of invasion is the remodelling of the surrounding tissue or extracellular matrix (ECM) and a major part of this process is the over-expression of proteolytic enzymes, such as the urokinase-type plasminogen activator (uPA) and matrix metalloproteinases (MMPs), by the cancer cells to break down ECM proteins. Degradation of the matrix enables the cancer cells to migrate through the tissue and subsequently to spread to secondary sites in the body, a process known as metastasis. In this paper we undertake an analysis of a mathematical model of cancer cell invasion of tissue, or ECM, which focuses on the role of the urokinase plasminogen activation system. The model consists of a system of five reaction-diffusion-taxis partial differential equations describing the interactions between cancer cells, uPA, uPA inhibitors, plasmin and the host tissue. Cancer cells react chemotactically and haptotactically to the spatio-temporal effects of the uPA system. The results obtained from computational simulations carried out on the model equations produce dynamic heterogeneous spatio-temporal solutions and using linear stability analysis we show that this is caused by a taxis-driven instability of a spatially homogeneous steady-state. Finally we consider the biological implications of the model results, draw parallels with clinical samples and laboratory based models of cancer cell invasion using three-dimensional invasion assay, and go on to discuss future development of the model.

  14. Immunohistochemical localization of urokinase-type plasminogen activator, urokinase-type plasminogen activator receptor and α2-antiplasmin in human corneal perforation: a case report

    Directory of Open Access Journals (Sweden)

    Sugioka Koji

    2012-11-01

    Full Text Available Abstract Background Corneal ulceration leading to perforation is associated with infectious and non-infectious destructive conditions in the cornea. The fibrinolytic (plasminogen/plasmin system is considered to contribute to tissue remodeling in the wound healing process and it is believed to play an important role in proteolysis and fibrosis. To determine the localization of urokinase-type plasminogen activator (u-PA, u-PA receptor (u-PAR and α2-antiplasmin (α2AP in the tissue of a corneal perforation, we investigated immunohistochemical expressions of u-PA, u-PAR, α2AP, CD68, and α-smooth muscle actin (α-SMA in a patient with corneal perforation that developed from an ulcer of no clear cause. Case presentation The patient was a 77-year-old woman who presented with a perforated corneal ulcer in her right eye. The cause of her corneal ulcer was unknown. Double immunohistochemistry was performed for the combinations of u-PA with u-PAR, CD68 or α-SMA and α2AP with CD68 or α-SMA to detect the localization of u-PA and α2AP. u-PA and u-PAR co-localization was seen in the corneal ulceration area. u-PA was mainly observed in CD68-positive cells and in some α-SMA positive cells. On the other hand, α2AP was not expressed in CD68-positive cells, but was expressed in α-SMA positive cells. Conclusion We identified expression of the u-PA/u-PAR complex and α2AP in a patient with a corneal ulcer. These two molecules are believed to play a crucial role in inflammatory cell recruitment, ECM synthesis and degradation during corneal wound healing.

  15. Purification and biochemical characterization of a novel fibrinolytic enzyme, PSLTro01, from a medicinal animal Porcellio scaber Latreille.

    Science.gov (United States)

    Tian, Zhou; Li, Bo; Guo, Liwei; Wu, Mianhua; Fu, Tingming; Cheng, Haibo; Zhu, Huaxu

    2015-09-01

    A novel protease, named PSLTro01, with fibrinolytic and anticoagulant activity was isolated from Porcellio scaber Latreille and was purified by a combination of hollow fibre membrane molecular weight cut-off (MWCO), ammonium sulfate fractionation, gel filtration and ion-exchange chromatography. PSLTro01 is a single-chain protein with a molecular mass of 38,497 Da as estimated by non-reduced SDS-PAGE and MALDI-TOF MS spectrometry, and its N-terminal 15 amino acid sequence was determined as DINGGGATLPQPLYQ. PSLTro01 is stable in the range of 20-40 °C and pH 6.0-10.0, with a maximum fibrinolytic activity at 40 °C and pH 7.0. The PSLTro01-induced fibrinolytic activity was not influenced by K(+) or Na(+) but was slightly increased by Mg(2+) and completely inhibited by aprotinin and pepstatin A. Fibrin plate assays revealed that PSLTro01 could not directly degrade fibrin but was a plasminogen activator. PSLTro01 exhibited high specificity for the substrate S-2251 for plasmin, followed by S-2238 for thrombin and S-2444 for urokinase. Moreover, the fibrinogenolysis pattern of PSLTro01 was Aα-chains>Bβ-chains>γ-chain. Tail-thrombus of the enzyme treated group was significantly shorter than the physiological saline treated group and the thrombus decrement was correlated with the enzyme dose. PSLTro01 prolongs both thrombin time (TT) and activated partial thromboplastin time (APTT). These results indicate that PSLTro01 may have potential applications in the prevention and treatment of thrombosis.

  16. α₂-Antiplasmin is involved in bone loss induced by ovariectomy in mice.

    Science.gov (United States)

    Shiomi, Akihito; Kawao, Naoyuki; Yano, Masato; Okada, Kiyotaka; Tamura, Yukinori; Okumoto, Katsumi; Matsuo, Osamu; Akagi, Masao; Kaji, Hiroshi

    2015-10-01

    The mechanism of postmenopausal osteoporosis is not fully understood. α2-Antiplasmin (α2-AP) is the primary inhibitor of plasmin in the fibrinolytic system, but is known to have activities beyond fibrinolysis. However, its role in bone metabolism and the pathogenesis of osteoporosis remains unknown. In the current study, we therefore examined the effects of α2-AP deficiency on ovariectomy (OVX)-induced bone loss by using wild-type and α2-AP-deficient mice. Quantitative computed tomography analysis revealed that α2-AP deficiency blunted OVX-induced trabecular bone loss in mice. Moreover, α2-AP deficiency significantly blunted serum levels of bone-specific alkaline phosphatase, cross-linked C-telopeptide of type I collagen, and interleukin (IL)-1β elevated by OVX. α2-AP treatment elevated the levels of IL-1β and tumor necrosis factor (TNF)-α mRNA in RAW 264.7 cells, although it suppressed osteoclast formation induced by receptor activator of nuclear factor-κB ligand. α2-AP treatment activated ERK1/2 and p38 MAP kinase pathways in RAW 264.7 cells, and these MAP kinase inhibitors antagonized the levels of IL-1β mRNA elevated by α2-AP. The data demonstrate that α2-AP is linked to bone loss due to OVX, through a mechanism that depends in part on the production of IL-1β and TNF-α in monocytes. Copyright © 2015 Elsevier Inc. All rights reserved.

  17. Bitter taste – cheese failure

    Directory of Open Access Journals (Sweden)

    Slavko Kirin

    2001-10-01

    Full Text Available Bitter taste is serous and very often cheese failure in modern cheesemaking process. In this paper the sources and bitter taste development in cheese will be presented. Bitterness in cheese is linked to bitter compounds development during cheese ripening. Most of the bitter compounds come from bitter peptides, the mechanism of theirs development being due to proteasepeptidase system of the cured enzymes and the milk cultures as well as other proteases present in cheese. By the action of curd enzymes, the milk protein - casein - is firstly degraded into high molecular weight compounds possessing no bitter taste. Those compounds are then degraded, by milk protease cultures, to hydrophobic bitter peptides of low molecular weight further degraded, by bacterial endopeptidase during cheese ripening, to bitter peptides and amino acids. In the case when no balance exists, between bitter compounds development and breakdown by lactic acid bacteria peptidase, an accumulation of bitter peptides occurs thus having an influence on cheese bitterness. During cheese ripening naturally occurring milk protease – plasmin, and thermostable proteases of raw milk microflora are also involved in proteolytic process. Fat cheese lipases, initiated by lipase originating from psychrotrophic bacteria in raw milk as well as other cheese lipases, are also associated with bitter taste generation. The other sources of bitterness come from the forages, the medicament residues as well as washing and disinfecting agents. In order to eliminate these failures a special care should be taken in milk quality as well as curd and milk culture selection. At this point technological norms and procedures, aimed to maintain the proteolysis balance during cheese ripening, should be adjusted, thus eliminating the bitter taste of the cheese.

  18. Kinetic characterization of factor Xa binding using a quenched fluorescent substrate based on the reactive site of factor Xa inhibitor from Bauhinia ungulata seeds.

    Science.gov (United States)

    Oliva, M L V; Andrade, S A; Juliano, M A; Sallai, R C; Torquato, R J; Sampaio, M U; Pott, V J; Sampaio, C A M

    2003-07-01

    The specific Kunitz Bauhinia ungulata factor Xa inhibitor (BuXI) and the Bauhinia variegata trypsin inhibitor (BvTI) blocked the activity of trypsin, chymotrypsin, plasmin, plasma kallikrein and factor XIIa, and factor Xa inhibition was achieved only by BuXI (K(i) 14 nM). BuXI and BvTI are highly homologous (70%). The major differences are the methionine residues at BuXI reactive site, which are involved in the inhibition, since the oxidized protein no longer inhibits factor Xa but maintains the trypsin inhibition. Quenched fluorescent substrates based on the reactive site sequence of the inhibitors were synthesized and the kinetic parameters of the hydrolysis were determined using factor Xa and trypsin. The catalytic efficiency k(cat)/K(m) 4.3 x 10(7) M(-1)sec(>-1) for Abz-VMIAALPRTMFIQ-EDDnp (lead peptide) hydrolysis by factor Xa was 10(4)-fold higher than that of Boc-Ile-Glu-Gly-Arg-AMC, widely used as factor Xa substrate. Lengthening of the substrate changed its susceptibility to factor Xa hydrolysis. Both methionine residues in the substrate influence the binding to factor Xa. Serine replacement of threonine (P(1)') decreases the catalytic efficiency by four orders of magnitude. Factor Xa did not hydrolyze the substrate containing the reactive site sequence of BvTI, that inhibits trypsin inhibitor but not factor Xa. Abz-VMIAALPRTMFIQ-EDDnp prolonged both the prothrombin time and the activated partial thromboplastin time, and the other modified substrates used in this experiment altered blood-clotting assays.

  19. Terpene Glycosides from the Roots of Sanguisorba officinalis L. and Their Hemostatic Activities

    Directory of Open Access Journals (Sweden)

    Wei Sun

    2012-06-01

    Full Text Available Guided by a hemostasis bioassay, seven terpene glycosides were isolated from the roots of Sanguisorba officinalis L. by silica gel column chromatography and preparative HPLC. On the grounds of chemical and spectroscopic methods, their structures were identified as citronellol-1-O-α-l-arabinofuranosyl-(1→6-β-d-glucopyranoside (1, geraniol-1-O-α-l-arabinofuranosyl-(1→6-β-d-glucopyranoside (2, geraniol-1-O-α-l-arabinopyranosyl-(1→6-β-d-glucopyranoside (3, 3β-[(α-l-arabinopyranosyloxy]-19α-hydroxyolean-12-en-28-oic acid 28-β-d-glucopyranoside (4, 3β-[(α-l-arabinopyranosyl-oxy]-19α-hydroxyurs-12-en-28-oic acid 28-β-d-glucopyranoside (ziyu-glycoside I, 5, 3β,19α-hydroxyolean-12-en-28-oic acid 28-β-d-glucopyranoside (6 and 3β,19α-di-hydroxyurs-12-en-28-oic acid 28-β-d-glucopyranoside (7. Compound 1 is a new mono-terpene glycoside and compounds 2, 3 and 5 were isolated from the Sanguisorba genus for the first time. Compounds 17 were assayed for their hemostatic activities with a Goat Anti-Human α2-plasmin inhibitor ELISA kit, and ziyu-glycoside I (5 showed the strongest hemostatic activity among the seven terpene glycosides. This is the first report that ziyu-glycoside Ι has strong hemostatic activity.

  20. Endogenous thrombin potential in Behçet's disease: relationship with thrombosis and anticoagulant therapy.

    Science.gov (United States)

    Mejía, Juan-Carlos; Espinosa, Gerard; Tàssies, Dolors; Reverter, Joan-Carles; Cervera, Ricard

    2014-01-01

    To analyse the relationship between an automated thrombin generation test, the endogenous thrombin potential (ETP), and other hypercoagulability markers, with vascular involvement in patients with Behçet's disease (BD). Patients and methods. We analysed 56 BD patients (30 men; mean age, 34.4 ± 14.3 years) without any known thrombophilic factor, of which 17 had previously suffered from thrombosis (deep venous thrombosis in 14 and ischaemic stroke in 3), and 56 controls matched for age and sex. Additionally, we also evaluated 20 plasma samples with an international normalised ratio (INR) between 1.5 and 5.0 obtained from patients with atrial fibrillation but without a history of embolic events that were under treatment with acenocumarol. Thrombin generation was measured as ETP with a chromogenic assay in an automated analyser. Factor VIII, von Willebrand factor antigen, prothrombin fragment 1.2, D-dimer and plasmin-antiplasmin complexes were also measured. BD patients showed higher ETP values than controls (471.3± 49.3 vs. 427.5± 31.3 mA; p<0.001). Additionally, BD patients with a history of thrombosis had higher ETP values than patients without thrombosis (496.6± 36.5 vs. 460.7± 50.5 mA; p<0.01). Factor VIII and von Willebrand factor antigen were also elevated in BD patients, but only von Willebrand factor antigen showed statistically significant differences between BD patients with and without thrombosis. Acenocumarol treatment reduced thrombin generation in BD patients in parallel to INR levels, reaching values similar to those of patients with atrial fibrillation and similar INR. BD is associated with thrombosis, and increased thrombin generation (measured as ETP) is a promising marker of hypercoagulability.

  1. Matrix Metalloproteinases & Implication in Periodontitis- A Short Review

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    Hitesh Desarda

    2013-01-01

    Full Text Available Matrix metalloproteinases (MMPs are a group of enzymes which are responsible for the degradation of extracellular matrix during normal tissue turnover and also during inflammatory processes. The expression and activity of MMPs in adult tissues is normally quite low, but increases significantly in various pathological conditions that may lead into unwanted tissue destruction,such as inflammatory diseases, tumour growth and metastasis. The role of MMP-8 in periodontitis is the well-known example of the unwanted tissue destruction related to increased activity of MMPs. Degradation of the extracellular matrix may involve four distinct pathways. A body of evidence suggests that matrix components may be dissolved by extracellular matrix metalloproteinase (MMP-dependent or plasmin (Pln-dependent cleavage reactions and that larger fragment of matrix may be disposed by a phagocytic pathway by way of cleavage by lysosomal proteinases. Mineralized matrices appear to be degraded by a complex process mediated by osteoclasts which relies on degradation by lysosomal proteinases in a narrow pericellular compartment. Matrix metalloproteinases can specifically cleave and degrade collagens and connective tissue matrix at physiologic pH and temperature. The objective of this review article is to understand the complete mechanisms regulating the expression of MMPs and enzymatic activity is of great importance.Source for all the articles is electronic Pub-Med system, published in between 1997-2011 searched by using keywords like Matrix Metalloproteinases, Periodontitis, Extracellular Matrix, Collagen . Future trend should be directed towards the development of easy, reliable and fast diagnostic tools and the effective therapeutic strategies to reduce the levels of MMPs.

  2. Identification and functional characterization of alpha-enolase from Taenia pisiformis metacestode.

    Science.gov (United States)

    Zhang, Shaohua; Guo, Aijiang; Zhu, Xueliang; You, Yanan; Hou, Junling; Wang, Qiuxia; Luo, Xuenong; Cai, Xuepeng

    2015-04-01

    Enolase belongs to glycolytic enzymes with moonlighting functions. The role of enolase in Taenia species is still poorly understood. In this study, the full length of cDNA encoding for Taenia pisiformis alpha-enolase (Tpeno) was cloned from larval parasites and soluble recombinant Tpeno protein (rTpeno) was produced. Western blot indicated that both rTpeno and the native protein in excretion-secretion antigens from the larvae were recognized by anti-rTpeno monoclonal antibodies (MAbs). The primary structure of Tpeno showed the presence of a highly conserved catalytic site for substrate binding and an enolase signature motif. rTpeno enzymatic activities of catalyzing the reversible dehydration of 2-phosphoglycerate (2-PGA) to phosphoenolpyruvate (PEP) and vice versa were shown to be 30.71 ± 2.15 U/mg (2-PGA to PEP) and 11.29 ± 2.38 U/mg (PEP to 2-PGA), respectively. Far-Western blotting showed that rTpeno could bind to plasminogen, however its binding ability was inhibited by ϵ-aminocaproic acid (ϵACA) in a competitive ELISA test. Plasminogen activation assay showed that plasminogen bound to rTpeno could be converted into active plasmin using host-derived activators. Immunohistochemistry and immunofluorescence indicated that Tpeno was distributed in the bladder wall of the metacestode and the periphery of calcareous corpuscles. In addition, a vaccine trial showed that the enzyme could produce a 36.4% protection rate in vaccinated rabbits against experimental challenges from T. pisiformis eggs. These results suggest that Tpeno with multiple functions may play significant roles in the migration, growth, development and adaptation of T. pisiformis for survival in the host environment.

  3. Bioprinted thrombosis-on-a-chip.

    Science.gov (United States)

    Zhang, Yu Shrike; Davoudi, Farideh; Walch, Philipp; Manbachi, Amir; Luo, Xuan; Dell'Erba, Valeria; Miri, Amir K; Albadawi, Hassan; Arneri, Andrea; Li, Xiaoyun; Wang, Xiaoying; Dokmeci, Mehmet Remzi; Khademhosseini, Ali; Oklu, Rahmi

    2016-10-18

    Pathologic thrombosis kills more people than cancer and trauma combined; it is associated with significant disability and morbidity, and represents a major healthcare burden. Despite advancements in medical therapies and imaging, there is often incomplete resolution of the thrombus. The residual thrombus can undergo fibrotic changes over time through infiltration of fibroblasts from the surrounding tissues and eventually transform into a permanent clot often associated with post-thrombotic syndrome. In order to understand the importance of cellular interactions and the impact of potential therapeutics to treat thrombosis, an in vitro platform using human cells and blood components would be beneficial. Towards achieving this aim, there have been studies utilizing the capabilities of microdevices to study the hemodynamics associated with thrombosis. In this work, we further exploited the utilization of 3D bioprinting technology, for the construction of a highly biomimetic thrombosis-on-a-chip model. The model consisted of microchannels coated with a layer of confluent human endothelium embedded in a gelatin methacryloyl (GelMA) hydrogel, where human whole blood was infused and induced to form thrombi. Continuous perfusion with tissue plasmin activator led to dissolution of non-fibrotic clots, revealing clinical relevance of the model. Further encapsulating fibroblasts in the GelMA matrix demonstrated the potential migration of these cells into the clot and subsequent deposition of collagen type I over time, facilitating fibrosis remodeling that resembled the in vivo scenario. Our study suggests that in vitro 3D bioprinted blood coagulation models can be used to study the pathology of fibrosis, and particularly, in thrombosis. This versatile platform may be conveniently extended to other vascularized fibrotic disease models.

  4. Effects of Alcohol on Postoperative Adhesion Formation in Ischemic Myocardium and Pericardium.

    Science.gov (United States)

    Elmadhun, Nassrene Y; Sabe, Ashraf A; Lassaletta, Antonio D; Dalal, Rahul S; Sellke, Frank W

    2017-08-01

    Postoperative formation of adhesions increases risk of complications during cardiac reoperations. We previously demonstrated that swine supplemented with vodka had a significant reduction in adhesions at sternotomy after previous thoracotomy. This follow-up study was conducted to determine reproducibility and the mechanism for adhesion reduction in swine supplemented with ethanol. An ameroid constrictor was placed in the left circumflex in 14 male Yorkshire swine to induce chronic myocardial ischemia through left minithoracotomy. Animals were supplemented postoperatively with ethanol (45 g ETOH, n = 7) or sucrose (80 g SUC, n = 7) for 7 weeks, followed by a reoperative median sternotomy. The ETOH group had significantly fewer adhesions, thinner pericardial thickness, decreased intramyocardial fibrosis, and decreased myocardial collagen deposition compared with the SUC group. In the myocardium, ETOH animals had decreased expression of proadhesion proteins focal adhesion kinase, paxillin, integrin-β1, transforming growth factor-β1 and phosphorylated SMAD and increased expression of adhesion breakdown proteins matrix metalloproteinase (MMP) 1, MMP2, MMP3, and MMP9 compared with SUC animals. However in the pericardium, ETOH animals had increased expression of proadhesion proteins focal adhesion kinase, paxillin, phosphorylated paxillin, vinculin, integrin-β1, tumor necrosis factor-α, transforming growth factor-β, and phosphorylated SMAD3, and decreased expression of adhesion breakdown proteins MMP1, MMP3, MMP9, and plasmin compared with SUC animals. Alcohol supplementation substantially reduced postoperative pericardial adhesion formation, attenuated pericardial thickening, and reduced myocardial fibrosis in response to chronic ischemia. Alcohol supplementation modulates adhesion protein and MMP/tissue inhibitor of metalloproteinase expression, favoring a profile associated with reduced pericardial adhesions. These results suggest that the myocardium is the

  5. LEVELS OF CYTOKINES, Α2-MACROGLOBULIN, AND ITS ACTIVE TRANSPORT FORM IN THE WOMEN WITH TUBAL INFERTILITY DURING IN VITRO FERTILIZATION

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    V. N. Zorina

    2007-01-01

    Full Text Available Abstract. We performed measurements of serum interleukin-6 (IL-6, interferon-γ (IFNγ, tumor necrosis factor-α (TNFα, as well as total contents of α2-macroglobulin (MG in blood and concentration of its active transport form, i.e., circulating complexes with plasmin (MG-PL in women at their reproductive age, suffering with tubal infertility, who participated in an in vitro fertilization (IVF program. We have found that the cohort of women with tubal infertility is initially heterogeneous and consists of persons with relative enhancement of either humoral, or cellular immune response. Stimulation of superovulation upon IVF in the women with increased humoral immune reactivity promotes normalization of cytokine levels, increases total contents of MG and maintains its active form (MG-PL, both by transporting bioactive substances, which are necessary to growth of the embryo, favors better access of MG-PL to cells due to reduction of IFNγ levels. Finally, it brings about positive results of the IVF procedure. In women with increased cellulartype immune reactivity, the MG levels are initially low, along with high IFNγ levels, thus diminishing transport of biologically active substances to the cells by MG-PL, as well as high and non-decreasing TNFα concentrations after stimulated superovulation, and, probably, an increased activity of NK-cells and cytotoxic lymphocytes. These factors minimize the possibility of favorable IVF outcome. With respect of the trends revealed, we would like to recommend determinations of serum cytokines and MG-PL complexes, as an additional prognostic tool when carrying out IVF programs.

  6. Renoprotective Mechanism of Remote Ischemic Preconditioning Based on Transcriptomic Analysis in a Porcine Renal Ischemia Reperfusion Injury Model.

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    Young Eun Yoon

    Full Text Available Ischemic preconditioning (IPC is a well-known phenomenon in which tissues are exposed to a brief period of ischemia prior to a longer ischemic event. This technique produces tissue tolerance to ischemia reperfusion injury (IRI. Currently, IPC's mechanism of action is poorly understood. Using a porcine single kidney model, we performed remote IPC with renal IRI and evaluated the IPC mechanism of action. Following left nephrectomy, 15 female Yorkshire pigs were divided into three groups: no IPC and 90 minutes of warm ischemia (control, remote IPC immediately followed by 90 minutes of warm ischemia (rIPCe, and remote IPC with 90 minutes of warm ischemia performed 24 hours later (rIPCl. Differential gene expression analysis was performed using a porcine-specific microarray. The microarray analysis of porcine renal tissues identified 1,053 differentially expressed probes in preconditioned pigs. Among these, 179 genes had altered expression in both the rIPCe and rIPCl groups. The genes were largely related to oxidation reduction, apoptosis, and inflammatory response. In the rIPCl group, an additional 848 genes had altered expression levels. These genes were primarily related to immune response and inflammation, including those coding for cytokines and cytokine receptors and those that play roles in the complement system and coagulation cascade. In the complement system, the membrane attack complex was determined to be sublytic, because it colocalized with phosphorylated extracellular signal-regulated kinase. Furthermore, alpha 2 macroglobulin, tissue plasminogen activator, uterine plasmin trypsin inhibitor, and arginase-1 mRNA levels were elevated in the rIPCl group. These findings indicate that remote IPC produces renoprotective effects through multiple mechanisms, and these effects develop over a long timeframe rather than immediately following IPC.

  7. Statistical analysis plan for the WOMAN-ETAPlaT study: Effect of tranexamic acid on platelet function and thrombin generation [version 1; referees: 2 approved, 2 approved with reservations

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    Kastriot Dallaku

    2016-12-01

    Full Text Available Background. Postpartum haemorrhage (PPH is a potentially life-threatening complication for women, and the leading cause of maternal mortality. Tranexamic acid (TXA is an antifibrinolytic used worldwide to treat uterine haemorrhage and to reduce blood loss in general surgery. TXA may have effects on thrombin generation, platelet function and coagulation factors as a result of its inhibition on the plasmin.   Methods. WOMAN ETAPlaT is a sub-study of the World Maternal Antifibrinolitic trial (WOMAN trial. All adult women clinically diagnosed with PPH after a vaginal delivery or caesarean section, are eligible for inclusion in the study. Blood samples will be collected at the baseline and 30 minutes after the first dose of study treatment is given. Platelet function will be evaluated in whole blood immediately after sampling with Multiplate® tests (ADPtest and TRAPtest. Thrombin generation, fibrinogen, D-dimer, and coagulation factors vW, V and VIII will be analysed using platelet poor plasma.   Results. Recruitment to WOMAN ETAPlaT started on 04 November 2013 and closed on 13 January 2015, during this time  188 patients were recruited. The final participant follow-up was completed on 04 March 2015. This article introduces the statistical analysis plan for the study, without reference to unblinded data.   Conclusion. The data from this study will provide evidence for the effect of TXA on thrombin generation, platelet function and coagulation factors in women with PPH.   Trial registration: ClinicalTrials.gov Identifier: NCT00872469; ISRCTN76912190

  8. Statistical analysis plan for the WOMAN-ETAPlaT study: Effect of tranexamic acid on platelet function and thrombin generation [version 2; referees: 2 approved, 2 approved with reservations

    Directory of Open Access Journals (Sweden)

    Kastriot Dallaku

    2017-06-01

    Full Text Available Background. Postpartum haemorrhage (PPH is a potentially life-threatening complication for women, and the leading cause of maternal mortality. Tranexamic acid (TXA is an antifibrinolytic used worldwide to treat uterine haemorrhage and to reduce blood loss in general surgery. TXA may have effects on thrombin generation, platelet function and coagulation factors as a result of its inhibition on the plasmin.   Methods. WOMAN ETAPlaT is a sub-study of the World Maternal Antifibrinolitic trial (WOMAN trial. All adult women clinically diagnosed with PPH after a vaginal delivery or caesarean section, are eligible for inclusion in the study. Blood samples will be collected at the baseline and 30 minutes after the first dose of study treatment is given. Platelet function will be evaluated in whole blood immediately after sampling with Multiplate® tests (ADPtest and TRAPtest. Thrombin generation, fibrinogen, D-dimer, and coagulation factors vW, V and VIII will be analysed using platelet poor plasma.   Results. Recruitment to WOMAN ETAPlaT started on 04 November 2013 and closed on 13 January 2015, during this time  188 patients were recruited. The final participant follow-up was completed on 04 March 2015. This article introduces the statistical analysis plan for the study, without reference to unblinded data.   Conclusion. The data from this study will provide evidence for the effect of TXA on thrombin generation, platelet function and coagulation factors in women with PPH.   Trial registration: ClinicalTrials.gov Identifier: NCT00872469; ISRCTN76912190

  9. Endothelial Progenitor Cells in Sprouting Angiogenesis: Proteases Pave the Way.

    Science.gov (United States)

    Laurenzana, A; Fibbi, G; Margheri, F; Biagioni, A; Luciani, C; Del Rosso, M; Chillà, A

    2015-01-01

    Sprouting angiogenesis consists of the expansion and remodelling of existing vessels, where the vascular sprouts connect each other to form new vascular loops. Endothelial Progenitor Cells (EPCs) are a subtype of stem cells, with high proliferative potential, able to differentiate into mature Endothelial Cells (ECs) during the neovascularization process. In addition to this direct structural role EPCs improve neovascularization, also secreting numerous pro-angiogenic factors able to enhance the proliferation, survival and function of mature ECs, and other surrounding progenitor cells. While sprouting angiogenesis by mature ECs involves resident ECs, the vasculogenic contribution of EPCs is a high hurdle race. Bone marrowmobilized EPCs have to detach from the stem cell niche, intravasate into bone marrow vessels, reach the hypoxic area or tumour site, extravasate and incorporate into the new vessel lumen, thus complementing the resident mature ECs in sprouting angiogenesis. The goal of this review is to highlight the role of the main protease systems able to control each of these steps. The pivotal protease systems here described, involved in vascular patterning in sprouting angiogenesis, are the matrix-metalloproteinases (MMPs), the serineproteinases urokinase-type plasminogen activator (uPA) associated with its receptor (uPAR) and receptorassociated plasminogen/plasmin, the neutrophil elastase and the cathepsins. Since angiogenesis plays a critical role not only in physiological but also in pathological processes, such as in tumours, controlling the contribution of EPCs to the angiogenic process, through the regulation of the protease systems involved, could yield new opportunities for the therapeutic prospect of efficient control of pathological angiogenesis.

  10. Induction of ceruloplasmin synthesis by IFN-gamma in human monocytic cells

    Science.gov (United States)

    Mazumder, B.; Mukhopadhyay, C. K.; Prok, A.; Cathcart, M. K.; Fox, P. L.

    1997-01-01

    Ceruloplasmin is a 132-kDa glycoprotein abundant in human plasma. It has multiple in vitro activities, including copper transport, lipid pro- and antioxidant activity, and oxidation of ferrous ion and aromatic amines; however, its physiologic role is uncertain. Although ceruloplasmin is synthesized primarily by the liver in adult humans, production by cells of monocytic origin has been reported. We here show that IFN-gamma is a potent inducer of ceruloplasmin synthesis by monocytic cells. Activation of human monoblastic leukemia U937 cells with IFN-gamma increased the production of ceruloplasmin by at least 20-fold. The identity of the protein was confirmed by plasmin fingerprinting. IFN-gamma also increased ceruloplasmin mRNA. Induction followed a 2- to 4-h lag and was partially blocked by cycloheximide, indicating a requirement for newly synthesized factors. Ceruloplasmin induction in monocytic cells was agonist specific, as IL-1, IL-4, IL-6, IFN-alpha, IFN-beta, TNF-alpha, and LPS were completely ineffective. The induction was also cell type specific, as IFN-gamma did not induce ceruloplasmin synthesis in endothelial or smooth muscle cells. In contrast, IFN-gamma was stimulatory in other monocytic cells, including THP-1 cells and human peripheral blood monocytes, and also in HepG2 cells. Ceruloplasmin secreted by IFN-gamma-stimulated U937 cells had ferroxidase activity and was, in fact, the only secreted protein with this activity. Monocytic cell-derived ceruloplasmin may contribute to defense responses via its ferroxidase activity, which may drive iron homeostasis in a direction unfavorable to invasive organisms.

  11. A processed multidomain mycoplasma hyopneumoniae adhesin binds fibronectin, plasminogen, and swine respiratory cilia.

    Science.gov (United States)

    Seymour, Lisa M; Deutscher, Ania T; Jenkins, Cheryl; Kuit, Tracey A; Falconer, Linda; Minion, F Chris; Crossett, Ben; Padula, Matthew; Dixon, Nicholas E; Djordjevic, Steven P; Walker, Mark J

    2010-10-29

    Porcine enzootic pneumonia is a chronic respiratory disease that affects swine. The etiological agent of the disease, Mycoplasma hyopneumoniae, is a bacterium that adheres to cilia of the swine respiratory tract, resulting in loss of cilia and epithelial cell damage. A M. hyopneumoniae protein P116, encoded by mhp108, was investigated as a potential adhesin. Examination of P116 expression using proteomic analyses observed P116 as a full-length protein and also as fragments, ranging from 17 to 70 kDa in size. A variety of pathogenic bacterial species have been shown to bind the extracellular matrix component fibronectin as an adherence mechanism. M. hyopneumoniae cells were found to bind fibronectin in a dose-dependent and saturable manner. Surface plasmon resonance was used to show that a recombinant C-terminal domain of P116 bound fibronectin at physiologically relevant concentrations (K(D) 24 ± 6 nm). Plasmin(ogen)-binding proteins are also expressed by many bacterial pathogens, facilitating extracellular matrix degradation. M. hyopneumoniae cells were found to also bind plasminogen in a dose-dependent and saturable manner; the C-terminal domain of P116 binds to plasminogen (K(D) 44 ± 5 nm). Plasminogen binding was abolished when the C-terminal lysine of P116 was deleted, implicating this residue as part of the plasminogen binding site. P116 fragments adhere to the PK15 porcine kidney epithelial-like cell line and swine respiratory cilia. Collectively these data suggest that P116 is an important adhesin and virulence factor of M. hyopneumoniae.

  12. Tissue-type plasminogen activator induces synaptic vesicle endocytosis in cerebral cortical neurons.

    Science.gov (United States)

    Yepes, M; Wu, F; Torre, E; Cuellar-Giraldo, D; Jia, D; Cheng, L

    2016-04-05

    The release of the serine proteinase tissue-type plasminogen activator (tPA) from the presynaptic terminal of cerebral cortical neurons plays a central role in the development of synaptic plasticity, adaptation to metabolic stress and neuronal survival. Our earlier studies indicate that by inducing the recruitment of the cytoskeletal protein βII-spectrin and voltage-gated calcium channels to the active zone, tPA promotes Ca(2+)-dependent translocation of synaptic vesicles (SVs) to the synaptic release site where they release their load of neurotransmitters into the synaptic cleft. Here we used a combination of in vivo and in vitro experiments to investigate whether this effect leads to depletion of SVs in the presynaptic terminal. Our data indicate that tPA promotes SV endocytosis via a mechanism that does not require the conversion of plasminogen into plasmin. Instead, we show that tPA induces calcineurin-mediated dynamin I dephosphorylation, which is followed by dynamin I-induced recruitment of the actin-binding protein profilin II to the presynaptic membrane, and profilin II-induced F-actin formation. We report that this tPA-induced sequence of events leads to the association of newly formed SVs with F-actin clusters in the endocytic zone. In summary, the data presented here indicate that following the exocytotic release of neurotransmitters tPA activates the mechanism whereby SVs are retrieved from the presynaptic membrane and endocytosed to replenish the pool of vesicles available for a new cycle of exocytosis. Together, these results indicate that in murine cerebral cortical neurons tPA plays a central role coupling SVs exocytosis and endocytosis.

  13. An additive effect of anti-PAI-1 antibody to ACE inhibitor on slowing the progression of diabetic kidney disease.

    Science.gov (United States)

    Gu, Chunyan; Zhang, Jiandong; Noble, Nancy A; Peng, Xiao-Rong; Huang, Yufeng

    2016-11-01

    While angiotensin II blockade slows the progression of diabetic nephropathy, current data suggest that it alone cannot stop the disease process. New therapies or drug combinations will be required to further slow or halt disease progression. Inhibition of plasminogen activator inhibitor type 1 (PAI-1) aimed at enhancing ECM degradation has shown therapeutic potential in diabetic nephropathy. Here, using a mouse model of type diabetes, the maximally therapeutic dose of the PAI-1-neutralizing mouse monoclonal antibody (MEDI-579) was determined and compared with the maximally effective dose of enalapril. We then examined whether addition of MEDI-579 to enalapril would enhance the efficacy in slowing the progression of diabetic nephropathy. Untreated uninephrectomized diabetic db/db mice developed progressive albuminuria and glomerulosclerosis associated with increased expression of transforming growth factor (TGF)-β1, PAI-1, type IV collagen, and fibronectin from weeks 18 to 22, which were reduced by MEDI-579 at 3 mg/kg body wt, similar to enalapril given alone from weeks 12 to 22 Adding MEDI-579 to enalapril from weeks 18 to 22 resulted in further reduction in albuminuria and markers of renal fibrosis. Renal plasmin generation was dramatically reduced by 57% in diabetic mice, a decrease that was partially reversed by MEDI-579 or enalapril given alone but was further restored by these two treatments given in combination. Our results suggest that MEDI-579 is effective in slowing the progression of diabetic nephropathy in db/db mice and that the effect is additive to ACEI. While enalapril is renal protective, the add-on PAI-1 antibody may offer additional renoprotection in progressive diabetic nephropathy via enhancing ECM turnover.

  14. Fibrinolytic and coagulative activities of Yersinia pestis

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    Timo K. Korhonen

    2013-07-01

    Full Text Available The outer membrane protease Pla belongs to the omptin protease family spread by horizontal gene transfer into Gram-negative bacteria that infect animals or plants. Pla has adapted to support the life style of the plague bacterium Yersinia pestis. Pla has a -barrel fold with 10 membrane-spanning  strands and five surface loops, and the barrel surface contains bound lipopolysaccharide (LPS that is critical for the conformation and the activity of Pla. The biological activity of Pla is influenced by the structure of the surface loops around the active site groove and by temperature-induced LPS modifications. Several of the putative virulence-related functions documented for Pla in vitro address control of the human hemostatic system, i.e. coagulation and fibrinolysis. Pla activates human plasminogen to the serine protease plasmin and activates the physiological plasminogen activator urokinase. Pla also inactivates the protease inhibitors alpha-2-antiplasmin and plasminogen activator inhibitor 1 and prevents the activation of thrombin-activatable fibrinolysis inhibitor. These functions enhance uncontrolled fibrinolysis which is thought to improve Y. pestis dissemination and survival in the mammalian host, and lowered fibrin(ogen deposition has indeed been observed in mice infected with Pla-positive Y. pestis. However, Pla also inactivates an anticoagulant, the tissue factor pathway inhibitor, which should increase fibrin formation and clotting. Thus Pla and Y. pestis have complex interactions with the hemostatic system. Y. pestis modifies its LPS upon transfer to the mammalian host and we hypothesize that the contrasting biological activities of Pla in coagulation and fibrinolysis are influenced by LPS changes during infection.

  15. The pro-coagulant fibrinogenolytic serine protease isoenzymes purified from Daboia russelii russelii venom coagulate the blood through factor V activation: role of glycosylation on enzymatic activity.

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    Ashis K Mukherjee

    Full Text Available Proteases from Russell's viper venom (RVV induce a variety of toxic effects in victim. Therefore, four new RVV protease isoenzymes of molecular mass 32901.044 Da, 333631.179 Da, 333571.472 Da, and 34594.776 Da, were characterized in this study. The first 10 N-terminal residues of these serine protease isoenzymes showed significant sequence homology with N-terminal sequences of snake venom thrombin-like and factor V-activating serine proteases, which was reconfirmed by peptide mass fingerprinting analysis. These proteases were found to be different from previously reported factor V activators isolated from snake venoms. These proteases showed significantly different fibrinogenolytic, BAEE-esterase and plasma clotting activities but no fibrinolytic, TAME-esterase or amidolytic activity against the chromogenic substrate for trypsin, thrombin, plasmin and factor Xa. Their Km and Vmax values towards fibrinogen were determined in the range of 6.6 to 10.5 µM and 111.0 to 125.5 units/mg protein, respectively. On the basis of fibrinogen degradation pattern, they may be classified as A/B serine proteases isolated from snake venom. These proteases contain ∼ 42% to 44% of N-linked carbohydrates by mass whereas partially deglycosylated enzymes showed significantly less catalytic activity as compared to native enzymes. In vitro these protease isoenzymes induce blood coagulation through factor V activation, whereas in vivo they provoke dose-dependent defibrinogenation and anticoagulant activity in the mouse model. At a dose of 5 mg/kg, none of these protease isoenzymes were found to be lethal in mice or house geckos, suggesting therapeutic application of these anticoagulant peptides for the prevention of thrombosis.

  16. Plasminogen binding to rat hepatocytes in primary culture and to thin slices of rat liver

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    Gonias, S.L.; Braud, L.L.; Geary, W.A.; VandenBerg, S.R. (Univ. of Virginia Health Sciences Center, Charlottesville (USA))

    1989-08-01

    Human {sup 125}I-plasminogen bound readily to rat hepatocytes in primary culture at 4 {degree}C and at 37{degree}C. Binding was inhibited by lysine and reversed by lysine, epsilon-aminocaproic acid, or nonradiolabeled plasminogen. The Kd for binding of {sup 125}I-plasminogen to hepatocytes was 0.59 +/- 0.16 mumol/L, as determined from the saturation isotherm by nonlinear regression (r2 = 0.99) and the Scatchard transformation by linear regression (r2 = 0.93). The number of sites per cell was 14.1 +/- 1.1 x 10(6). Fibrinogen synthesis and secretion by hepatocytes was insufficient to account for the major fraction of plasminogen binding, as determined by enzyme-linked immunosorbent assay (ELISA). Polyacrylamide gel electrophoresis and trichloroacetic acid precipitation studies demonstrated that plasminogen is neither activated nor degraded when bound to hepatocytes at 37{degree}C. Thin slices of whole rat liver (500 microns), isolated and prepared totally at 4{degree}C, bound {sup 125}I-plasminogen. Binding was inhibited by lysine. {sup 125}I-albumin binding to liver slices was minimal and not inhibited by lysine. Activation of plasminogen by tissue plasminogen activator (t-PA) was enhanced by hepatocytes in primary culture. When lysine was included in the media, the enhanced rate of activation was no longer observed. After activation with t-PA, much of the plasmin remained associated with hepatocyte surfaces and was partially protected from inhibition by alpha 2-antiplasmin. These studies suggest that hepatocyte plasminogen binding sites may provide important surface anticoagulant activity.

  17. The Kunitz-like modulatory protein haemangin is vital for hard tick blood-feeding success.

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    M Khyrul Islam

    2009-07-01

    Full Text Available Ticks are serious haematophagus arthropod pests and are only second to mosquitoes as vectors of diseases of humans and animals. The salivary glands of the slower feeding hard ticks such as Haemaphysalis longicornis are a rich source of bioactive molecules and are critical to their biologic success, yet distinct molecules that help prolong parasitism on robust mammalian hosts and achieve blood-meals remain unidentified. Here, we report on the molecular and biochemical features and precise functions of a novel Kunitz inhibitor from H. longicornis salivary glands, termed Haemangin, in the modulation of angiogenesis and in persistent blood-feeding. Haemangin was shown to disrupt angiogenesis and wound healing via inhibition of vascular endothelial cell proliferation and induction of apoptosis. Further, this compound potently inactivated trypsin, chymotrypsin, and plasmin, indicating its antiproteolytic potential on angiogenic cascades. Analysis of Haemangin-specific gene expression kinetics at different blood-feeding stages of adult ticks revealed a dramatic up-regulation prior to complete feeding, which appears to be functionally linked to the acquisition of blood-meals. Notably, disruption of Haemangin-specific mRNA by a reverse genetic tool significantly diminished engorgement of adult H. longicornis, while the knock-down ticks failed to impair angiogenesis in vivo. To our knowledge, we have provided the first insights into transcriptional responses of human microvascular endothelial cells to Haemangin. DNA microarray data revealed that Haemangin altered the expression of 3,267 genes, including those of angiogenic significance, further substantiating the antiangiogenic function of Haemangin. We establish the vital roles of Haemangin in the hard tick blood-feeding process. Moreover, our results provide novel insights into the blood-feeding strategies that enable hard ticks to persistently feed and ensure full blood-meals through the modulation of

  18. Inhibitory serpins from wheat grain with reactive centers resembling glutamine-rich repeats of prolamin storage proteins. Cloning and characterization of five major molecular forms.

    Science.gov (United States)

    Ostergaard, H; Rasmussen, S K; Roberts, T H; Hejgaard, J

    2000-10-27

    Genes encoding proteins of the serpin superfamily are widespread in the plant kingdom, but the properties of very few plant serpins have been studied, and physiological functions have not been elucidated. Six distinct serpins have been identified in grains of hexaploid bread wheat (Triticum aestivum L.) by partial purification and amino acid sequencing. The reactive centers of all but one of the serpins resemble the glutamine-rich repetitive sequences in prolamin storage proteins of wheat grain. Five of the serpins, classified into two protein Z subfamilies, WSZ1 and WSZ2, have been cloned, expressed in Escherichia coli, and purified. Inhibitory specificity toward 17 proteinases of mammalian, plant, and microbial origin was studied. All five serpins were suicide substrate inhibitors of chymotrypsin and cathepsin G. WSZ1a and WSZ1b inhibited at the unusual reactive center P(1)-P(1)' Gln-Gln, and WSZ2b at P(2)-P(1) Leu-Arg-one of two overlapping reactive centers. WSZ1c with P(1)-P(1)' Leu-Gln was the fastest inhibitor of chymotrypsin (k(a) = 1.3 x 10(6) m(-1) s(-1)). WSZ1a was as efficient an inhibitor of chymotrypsin as WSZ2a (k(a) approximately 10(5) m(-1) s(-1)), which has P(1)-P(1)' Leu-Ser-a reactive center common in animal serpins. WSZ2b inhibited plasmin at P(1)-P(1)' Arg-Gln (k(a) approximately 10(3) m(-1) s(-1)). None of the five serpins inhibited Bacillus subtilisin A, Fusarium trypsin, or two subtilisin-like plant serine proteinases, hordolisin from barley green malt and cucumisin D from honeydew melon. Possible functions involving interactions with endogenous or exogenous proteinases adapted to prolamin degradation are discussed.

  19. Fibrinogen Aα Thr312Ala polymorphism specifically contributes to chronic thromboembolic pulmonary hypertension by increasing fibrin resistance.

    Directory of Open Access Journals (Sweden)

    Ji-Feng Li

    Full Text Available BACKGROUND: Polymorphisms are associated with chronic thromboembolic pulmonary hypertension (CTEPH and pulmonary thromboembolism (PTE, but no polymorphism specific to CTEPH but not PTE has yet been reported. Fibrin resistance is associated with CTEPH, but the mechanism has not been elucidated. METHODS: Polymorphisms were analyzed in 101 CTEPH subjects, 102 PTE subjects and 108 healthy controls by Massarray or restriction fragment length polymorphism (RFLP. Plasmin-mediated cleavage of fibrin was characterized in 69 subjects (29 with CTEPH, 21 with PTE and 19 controls. RESULTS: Genotype frequencies and allele frequencies of fibrinogen Aα Thr312Ala were significantly higher in CTEPH subjects than in controls and PTE subjects, while there was no difference between PTE subjects and controls. The odd ratio (OR 2.037 and 95% confidence interval (95% CI, 1.262-3.289 showed that Thr312Ala polymorphism was a risk factor for CTEPH but not PTE. Fibrin from CTEPH subjects was more resistant to lysis than that from PTE subjects and controls. Fibrin resistance was significantly different between Aα Thr312Ala (A/G genotypes within CTEPH subjects, and the fibrin with GG genotype was more resistant than that with AA and AG genotype. CONCLUSIONS: Fibrinogen Aα Thr312Ala (A/G polymorphism was associated with CTEPH, but not PTE, suggesting that the fibrinogen Aα Thr312Ala polymorphism may act as a potential biomarker in identifying CTEPH from PTE. GG genotype polymorphism contributes to CTEPH through increasing fibrin resistance, implying that PTE subjects with fibrinogen Aα GG genotype may need long-term anticoagulation therapy.

  20. A comparative study of the effects of the hemostatic system of two monophasic gestodene oral contraceptives containing 20 micrograms and 30 micrograms ethinylestradiol.

    Science.gov (United States)

    Winkler, U H; Schindler, A E; Endrikat, J; Düsterberg, B

    1996-02-01

    The effects of two oral contraceptives, containing gestodene and either 20 micrograms or 30 micrograms ethinylestradiol, on hemostatic parameters was investigated in a six-month randomized study involving a total of 40 healthy women between the ages of 18 and 30 years. A large number of hemostatic parameters were measured, which were categorized as either pro-coagulatory, anti-coagulatory, profibrinolytic, anti-fibrinolytic or indicative of fibrin turnover. Additionally, tissue plasminogen activator (t-PA) and plasminogen activator inhibitor (PAI-1) were measured before and after venous occlusion and delta and ratio values calculated. Pro-coagulatory factors as well as reaction products reflecting in vivo coagulatory activity (thrombin-antithrombin III complex, prothrombin fragment 1 + 2) were found to increase. Among the anti-coagulatory parameters, only protein S concentration and protein S activity decreased, most notably in the 30 micrograms EE group. There was a corresponding increase in fibrinolytic activity reflected by reaction products of in vivo fibrinolysis (plasmin-antiplasmin 2-complex, fibrin-degradation products). Measurement of t-PA and PAI-1, before and after venous occlusion, revealed that the fibrinolytic response was more pronounced in the 20 micrograms EE group. There was also an increase in the threshold of fibrinolytic inhibition (ratio PAI-1) in both groups, which was less pronounced in the 20 micrograms EE group. Apart from isolated measurements, all parameters remained within their normal ranges and values returned to baseline in the follow-up cycle. It is concluded that both preparations had a balanced effect on the hemostatic system stimulating both pro-coagulant and fibrinolytic activity. No statistically significant differences were observed between the two groups; however, there was a trend towards greater fibrinolytic capacity in the 20 micrograms EE group.