WorldWideScience
1

Functional Stability of Plasminogen Activator Inhibitor-1  

OpenAIRE

Plasminogen activator inhibitor-1 (PAI-1) is the main inhibitor of plasminogen activators, such as tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA), and a major regulator of the fibrinolytic system. PAI-1 plays a pivotal role in acute thrombotic events such as deep vein thrombosis (DVT) and myocardial infarction (MI). The biological effects of PAI-1 extend far beyond thrombosis including its critical role in fibrotic disorders, atherosclerosis, renal an...

Songul Yasar Yildiz; Pinar Kuru; Ebru Toksoy Oner; Mehmet Agirbasli

2014-01-01

2

The effect of anti-human plasminogen monoclonal antibodies on Glu-plasminogen activation by plasminogen activators  

Directory of Open Access Journals (Sweden)

Full Text Available Background: Human plasminogen is a plasma glycoprotein synthesized mainly in the liver. Conversion of plasminogen to plasmin by plasminogen activators is a key event in the fibrinolytic system. In this study, we investigated the effects of two anti-human plasminogen monoclonal antibodies, A1D12 and MC2B8 on Glu-plasminogen activation in presence of u-PA, t-PA and streptokinase. Methods: Producing of Hybridoma antibodies was performed by fusion of spleen cells from BALB/C mice immunized with Glu-plasminogen and NS1 myeloma cells. Antibody binding to Human Glu-plasminogen was assessed using an ELISA assay. Activation of plasminogen was determined by measuring plasmin generation using the chromogenic substrate S-2251 and the effect of monoclonal antibodies, A1D12 and MC2B8 on plasminogen activation in solution was then evaluated. Initial rates and kinetic parameters of plasminogen activation in the presence of monoclonal antibodies were calculated. The effect of the monoclonal antibody MC2B8 on the rate of plasmin hydrolysis was measured. The effect of F(ab'2 fragment of A1D12 on u-PA catalyzed-plasminogen activation also compared with the effect of the whole antibody in this reaction. Results: ELISA assay showed that the antibodies reacted well with antigens. A1D12 increased the maximum velocity (Vmax of plasminogen activation by each of the three plasminogen activators and MC2B8 decreased it. In all activation reactions, the KM value of plasminogen activation did not significantly change in the presence of antibody A1D12 whereas antibody MC2B8 increased the KM value of plasminogen activation by u-PA, fibrin monomer dependent t-PA and streptokinase. Monoclonal antibody MC2B8 had no significant effect on plasmin hydrolysis rate of synthetic substrate S-2251. Activation rate of plasminogen by u-PA in the lower concentration of F (ab2 fragment of A1D12 was identical to activation in the presence of the whole antibody. Conclusion: The binding of the A1D12 F(ab region to Glu-plasminogen increases the catalytic efficiency of plasminogen activation by plasminogen activators. Therefore, it may be useful to apply clinically A1D12 for the therapy of thromboembolic events such as myocardial infarction by humanizing the F(ab fragment of the A1D12 antibody. Inhibition pattern of antibody MC2B8 obey the mixed type of enzyme inhibition by binding the antibody probably at, or near, the cleavage site of Glu-plasminogen.

M. Akrami

2006-07-01

3

Fibrin-targeted plasminogen activation by plasminogen activator, PadA, from Streptococcus dysgalactiae.  

Science.gov (United States)

Bacterial plasminogen activators differ from each other in their mechanism of plasminogen activation besides their host specificity. Three-domain streptokinase (SK) and two-domain PauA generate nonproteolytic active site center in their cognate partner plasminogen but their binary activator complexes are resistant to ?2-antiplasmin (a2AP) inhibition causing nonspecific plasminogen activation in plasma. In contrast, single-domain plasminogen activator, staphylokinase (SAK), requires proteolytic cleavage of human plasminogen into plasmin for the active site generation, and this activator complex is inhibited by a2AP. The single-domain plasminogen activator, PadA, from Streptococcus dysgalatiae, having close sequence and possible structure homology with SAK, was recently reported to activate bovine Pg in a nonproteolytic manner similar to SK. We report hereby that the binary activator complex of PadA with bovine plasminogen is inhibited by a2AP and PadA is recycled from this complex to catalyze the activation of plasminogen in the clot environment, where it is completely protected from a2AP inhibition. Catalytic efficiency of the activator complex formed by PadA and bovine plasminogen is amplified several folds in the presence of cyanogen bromide digested fibrinogen but not by intact fibrinogen indicating that PadA may be highly efficient at the fibrin surface. The present study, thus, demonstrates that PadA is a unique single-domain plasminogen activator that activates bovine plasminogen in a fibrin-targeted manner like SAK. The sequence optimization by PadA for acquiring the characteristics of both SK and SAK may be exploited for the development of efficient and fibrin-specific plasminogen activators for thrombolytic therapy. PMID:24639287

Singh, Satish; Bhando, Timsy; Dikshit, Kanak L

2014-06-01

4

Structural Basis for Recognition of Urokinase-type Plasminogen Activator by Plasminogen Activator Inhibitor-1*  

OpenAIRE

Plasminogen activator inhibitor-1 (PAI-1), together with its physiological target urokinase-type plasminogen activator (uPA), plays a pivotal role in fibrinolysis, cell migration, and tissue remodeling and is currently recognized as being among the most extensively validated biological prognostic factors in several cancer types. PAI-1 specifically and rapidly inhibits uPA and tissue-type PA (tPA). Despite extensive structural/functional studies on these two reactions, the underlying structura...

Lin, Zhonghui; Jiang, Longguang; Yuan, Cai; Jensen, Jan K.; Zhang, Xu; Luo, Zhipu; Furie, Barbara C.; Furie, Bruce; Andreasen, Peter A.; Huang, Mingdong

2011-01-01

5

The plasminogen activation system in inflammation.  

Science.gov (United States)

Inflammation is an adaptive response to damage of vascularized tissues, which develops according to a stereotyped sequence governed by the local production of the so-called "chemical mediators of inflammation". Here we review the evidences indicating a role of the plasminogen activation system in the regulation of all the phases of the inflammation process. Plasminogen activation controls the formation of complement anaphylotoxins (responsible for vasodilatation, increase of venular permeability and leukocyte chemotaxis) and of bradykinin (which accounts for vasodilatation, increase of venular permeability and pain) by regulating the plasma contact system. The urokinase plasminogen activator and its cellular receptor, expressed on the surface of human leukocytes, provide a functional unit that, by regulating interaction of leukocytes with extracellular matrix, as well as its degradation, is critical for the migration of leukocytes and for their movement in the damaged tissues. By preventing excess fibrin accumulation in inflamed tissues, the plasminogen activation system also governs the proper evolution of the inflammatory exudates and prevents the possibility of a shift from acute to chronic inflammation. PMID:18508538

Del Rosso, Mario; Fibbi, Gabriella; Pucci, Marco; Margheri, Francesca; Serrati, Simona

2008-01-01

6

Plasminogen Binding and Activation by Mycoplasma fermentans  

OpenAIRE

The binding of plasminogen to Mycoplasma fermentans was studied by an immunoblot analysis and by a binding assay using iodine-labeled plasminogen. The binding of 125I-labeled plasminogen was inhibited by unlabeled plasminogen, lysine, and lysine analog ?-aminocaproic acid. Partial inhibition was obtained by a plasminogen fragment containing kringles 1 to 3 whereas almost no inhibition was observed with a fragment containing kringle 4. Scatchard analysis revealed a dual-phase interaction, one...

Yavlovich, Amichai; Higazi, Abd A. -r; Rottem, Shlomo

2001-01-01

7

Factors that affect plasmin, plasminogen, and plasminogen activators in milk, cheese, and model systems  

OpenAIRE

Plasmin, the major native milk serine proteinase, is an important contributor to proteolytic breakdown during the ripening of certain cheeses. Plasmin system activities include the active enzyme plasmin (PL), the zymogen plasminogen (PG), and plasminogen activators (PA), which convert PG to PL. Factors that influence plasmin system activities in milk are economically relevant for ripened cheese production. ^ The overall objective for this research project was to investigate the effects sele...

Hayes, Kirby D.

2002-01-01

8

Indium-111 labeled tissue plasminogen activator  

International Nuclear Information System (INIS)

Although radiolabeled fibrinogen has been used successfully for detection of thromboemboli by imaging, plasminogen activators offer the advantage of clot detection after active fibrin deposition has ceased. The authors have labeled recombinant tissue plasminogen activator (rTPA) with indium-111 (In) and studied its properties in vivo. Using the cyclic anhydride and standard methodology, an average of about 1 DTPA group per molecule was attached and the protein was labeled with In to a specific activity of 2-3 mCi/mg. Size exclusion HPLC showed each preparation to be of high radiochemical purity and that the In was bound only to the DTPA groups. In vivo clot localization was investigated using a canine clot model in which thrombogenic catheters are placed in a femoral artery and/or in the jugular vein prior to the administration of labeled rTPA. After administration of 0.5-1.0 mg of In-rTPA, blood and serum activity levels were found to decrease with a half time of 8 +- 1 min. in good agreement with the literature value of 7 min. for unlabeled rTPA. Clot:blood and clot:serum ratios were found to increase rapidly, in one study reaching 6.3 after 31 min. Because of the short residence time of rTPA in blood, activity in cardiac and pulmonary blood pools decreased rapidly such that clots were visualized within minutes of injection. The authors conclude that rTPA may be labeled with In without destroying the molecule's ability to localize in clot and that, with this agent, useful images of forming clot may be obtained within one hour of administration. This protein may therefore be ideal for imaging pulmonary and cardiac thromboemboli by gamma scintigraphy

9

Indium-111 labeled tissue plasminogen activator  

Energy Technology Data Exchange (ETDEWEB)

Although radiolabeled fibrinogen has been used successfully for detection of thromboemboli by imaging, plasminogen activators offer the advantage of clot detection after active fibrin deposition has ceased. The authors have labeled recombinant tissue plasminogen activator (rTPA) with indium-111 (In) and studied its properties in vivo. Using the cyclic anhydride and standard methodology, an average of about 1 DTPA group per molecule was attached and the protein was labeled with In to a specific activity of 2-3 mCi/mg. Size exclusion HPLC showed each preparation to be of high radiochemical purity and that the In was bound only to the DTPA groups. In vivo clot localization was investigated using a canine clot model in which thrombogenic catheters are placed in a femoral artery and/or in the jugular vein prior to the administration of labeled rTPA. After administration of 0.5-1.0 mg of In-rTPA, blood and serum activity levels were found to decrease with a half time of 8 +- 1 min. in good agreement with the literature value of 7 min. for unlabeled rTPA. Clot:blood and clot:serum ratios were found to increase rapidly, in one study reaching 6.3 after 31 min. Because of the short residence time of rTPA in blood, activity in cardiac and pulmonary blood pools decreased rapidly such that clots were visualized within minutes of injection. The authors conclude that rTPA may be labeled with In without destroying the molecule's ability to localize in clot and that, with this agent, useful images of forming clot may be obtained within one hour of administration. This protein may therefore be ideal for imaging pulmonary and cardiac thromboemboli by gamma scintigraphy.

Hnatowich, D.J.; Virzi, F.; Doherty, P.W.; Ansell, J.

1985-05-01

10

Activation of immobilized plasminogen by tissue activator. Multimolecular complex formation  

International Nuclear Information System (INIS)

Ternary complex formation of tissue plasminogen activator (TPA) and plasminogen (Plg) with thrombospondin (TSP) or histidine-rich glycoprotein (HRGP) has been demonstrated using an enzyme-linked immunosorbent assay, an affinity bead assay, and a rocket immunoelectrophoresis assay. The formation of these complexes was specific, concentration dependent, saturable, lysine binding site-dependent, and inhibitable by fluid phase plasminogen. Apparent Kd values were approximately 12-36 nM for the interaction of TPA with TSP-Plg complexes and 15-31 nM with HRGP-Plg complexes. At saturation the relative molar stoichiometry of Plg:TPA was 3:1 within the TSP-containing complexes and 1:1 within HRGP-containing complexes. The activation of Plg to plasmin by TPA on TSP- and HRGP-coated surfaces was studied using a synthetic fluorometric plasmin substrate (D-Val-Leu-Lys-7-amino-4-trifluoromethyl coumarin). Kinetic analysis demonstrated a marked increase in the affinity of TPA for plasminogen in the presence of surface-associated TSP or HRGP. Complex formation of locally released tissue plasminogen activator with Plg immobilized on TSP or HRGP surfaces may thus play an important role in effecting proteolytic events in nonfibrin-containing microenvironments

11

Structural consideration of the formation of the activation complex between the staphylokinase-like streptococcal plasminogen activator PadA and bovine plasminogen.  

Science.gov (United States)

The characteristics of a streptococcal plasminogen activator (PA) displaying specificity for ruminant plasminogen (Plg) were defined using molecular approaches. The 16-kDa secreted protein PadA was found to be prevalent in Streptococcus dysgalactiae subspecies dysgalactiae isolated from cases of bovine mastitis and septic arthritis in lambs. PadA was able to activate bovine, ovine and caprine Plg, but not human Plg. Amino acid sequence analysis identified a limited level of homology to other streptococcal PAs, including streptokinase; however, PadA was found to align well with and match in size the staphylococcal PA, staphylokinase. Recombinant PadA was used to investigate interaction with bovine Plg, leading to formation of an activator complex that was capable of recruiting and converting further substrate Plg into plasmin. Individual non-overlapping peptides of PadA or bovine microplasminogen were found to block the interaction between PadA and bovine Plg, preventing the formation of the activation complex. Homology modelling based upon structures of staphylokinase complexed with human microplasminogen supported these findings by placing critical residues in close proximity to the plasmin component of the activation complex. PMID:18588895

Ward, Philip N; Abu-Median, Abu-Bakr A K; Leigh, James A

2008-09-01

12

Plasminogen activators in alcoholic cirrhosis: demonstration of increased tissue type and urokinase type activator.  

OpenAIRE

Plasma samples from patients with alcoholic cirrhosis were analysed for plasminogen activators and for inhibitors of the fibrinolytic system. Plasminogen activator activity was considerably increased in patients' plasma compared with normal. Immunochemical characterisation of these plasminogen activators showed that they included both tissue type and urokinase type plasminogen activator. The major inhibitor of plasmin, alpha 2-antiplasmin, was decreased in the patients, but no evidence for th...

Booth, N. A.; Anderson, J. A.; Bennett, B.

1984-01-01

13

Involvement of the plasminogen activation system in cow endometritis.  

Science.gov (United States)

The objectives of this study were to investigate the: (a) presence and activity of components of the "plasminogen activators/plasmin" system in dairy cows with or without endometritis; (b) variations in enzyme activity according to the degree of endometritis; and (c) associations between these enzymes and changes in endometrial histology after intrauterine antibiotic treatment. Endometrial biopsies were collected from anestrus (no palpable ovarian structures and milk progesterone cows, 30-40 days postpartum. On the basis of a vaginoscopic examination, rectal palpation of the cervix and uterus, and endometrial histology, there were 92 cows with endometritis and 20 cows without endometritis. After biopsy collection, each cow was given an intrauterine infusion of 1.5x10(6) IU of procaine penicillin G. In cows with endometritis, genital tract examinations and biopsies were repeated 2 weeks later. Both plasminogen activators (PAs), tissue type (t-PA) and urokinase (u-PA), were immunologically identified in all uterine biopsies. Plasminogen activator activity (PAA) increased, whereas plasminogen activator inhibition (PAI) and plasmin inhibition (PI) decreased in proportion to the degree of inflammation. Two weeks after intrauterine treatment, PAA had decreased significantly in all cows that had reduced severity of endometrial inflammation and had increased significantly in all cows with increased severity of inflammation. The change in the degree of inflammation depended upon plasminogen activator activity; cows with higher PAA were more likely to improve. In conclusion, there was evidence for a role of the plasminogen activation proteolytic system in bovine endometritis. PMID:14662133

Moraitis, S; Taitzoglou, I A; Tsantarliotou, M P; Boscos, C M; Kaldrimidou, E; Saratsis, Ph

2004-01-15

14

Increased alveolar plasminogen activator in early asbestosis  

Energy Technology Data Exchange (ETDEWEB)

Alveolar macrophage-derived plasminogen activator (PA) activity is decreased in some chronic interstitial lung diseases such as idiopathic pulmonary fibrosis and sarcoidosis but increased in experimental models of acute alveolitis. Although asbestos fibers can stimulate alveolar macrophages (AM) to release PA in vitro, the effect of chronic asbestos exposure of the lower respiratory tract on lung PA activity remains unknown. The present study was designed to evaluate PA activity of alveolar macrophages and bronchoalveolar lavage (BAL) fluid in asbestos-exposed sheep and asbestos workers. Forty-three sheep were exposed to either 100 mg UICC chrysotile B asbestos in 100 ml phosphate-buffered saline (PBS) or to 100 ml PBS by tracheal infusion every 2 wk for 18 months. At Month 18, chest roentgenograms were analyzed and alveolar macrophage and extracellular fluid PA activity were measured in samples obtained by BAL. Alveolar macrophage PA activity was increased in the asbestos-exposed sheep compared to control sheep (87.2 +/- 17.3 versus 41.1 +/- 7.2 U/10(5) AM-24 h, p less than 0.05) as was the BAL fluid PA activity (674.9 +/- 168.4 versus 81.3 +/- 19.7 U/mg alb-24 h, p less than 0.01). Among the asbestos-exposed sheep, 10 had normal chest roentgenograms (Group SA) and 15 had irregular interstitial opacities (Group SB). Strikingly, whereas Group SA did not differ from the control group in BAL cellularity or PA activity, Group SB had marked increases in alveolar macrophages (p less than 0.005), AM PA activity (p less than 0.02), and BAL PA activity (p less than 0.001) compared to the control group.

Cantin, A.; Allard, C.; Begin, R.

1989-03-01

15

Epithelial glycoprotein-330 mediates endocytosis of plasminogen activator-plasminogen activator inhibitor type-1 complexes  

DEFF Research Database (Denmark)

Epithelial glycoprotein 330 (gp330) is structurally similar to the multifunctional alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein (alpha 2MR/LRP), gp330 and alpha 2MR/LRP bind Ca2+ with high affinity, and both receptors bind and mediate endocytosis of alpha 2MR-associated protein (RAP). In the present report, we describe that affinity-purified gp330 from rabbit renal cortex binds plasminogen activator inhibitor type-1 (PAI-1) complexed with urokinase-type plasminogen activator (uPA). alpha 2M-methylamine, which binds with high affinity to alpha 2MR/LRP, did not bind to gp330. The apparent Kd for binding of uPA.PAI-1 complexes was about 0.8 nM at 4 degrees C. The binding was calcium-dependent and inhibited by recombinant RAP (rRAP) and tissue type plasminogen activator-PAI-1 complexes. Thin sections of rabbit renal proximal tubules bound 125I-labeled uPA.PAI-1 and rRAP in the apical part of proximal tubules corresponding to the localization of gp330. The binding of 125I-uPA.PAI-1 complexes in tubules was abolished by excess unlabeled rRAP, and a rRAP-inhibitable endocytosis and degradation of labeled uPA.PAI-1 complexes was demonstrated by perfusion of isolated rabbit proximal tubules. The results establish an endocytotic function of gp330 and suggest that gp330 is an important component of the fibrinolytic system in gp330-containing epithelial as found in, for example, kidney and lung.

Moestrup, S K; Nielsen, Susanne

1993-01-01

16

Tissue plasminogen activator in central nervous system physiology and pathology  

OpenAIRE

Although conventionally associated with fibrin clot degradation, recent work has uncovered new functions for the tissue plasminogen activator (tPA)/plasminogen cascade in central nervous system physiology and pathology. This extracellular proteolytic cascade has been shown to have roles in learning and memory, stress, neuronal degeneration, addiction and Alzheimer’s disease. The current review considers the different ways tPA functions in the brain.

Melchor, Jerry P.; Strickland, Sidney

2005-01-01

17

Expression of plasminogen activator-related genes in the adipose tissue of lactating dairy sheep in the early post-weaning period.  

Science.gov (United States)

There is growing evidence that plasminogen activator inhibitor type 1 (PAI-1) is expressed in adipose tissue and its expression is implicated in inflammation that accompanies obesity-associated diseases. The physiological role of other genes implicated in the plasminogen-activating cascade such as urokinase-type plasminogen activator (u-PA), u-PA receptor (u-PAR) and plasminogen activator inhibitor type 2 (PAI-2) in ovine adipose tissue remains unknown. The objective of this study was to examine the changes in the expression of four plasminogen activator (PA)-related genes during the early post-weaning period in dairy ewes. A total of 21 subcutaneous adipose tissue samples were obtained from seven lactating dairy ewes of the Chios breed at weeks 1, 2 and 4 after weaning. Results indicated that expression of all PA-related genes was detected in most of the samples examined. Greatest expression of u-PAR corresponded to highest (week 1), while greatest expression of PAI-2 corresponded to lowest (week 4) rate of lipolysis, as indicated by the expression of hormone-sensitive lipase, in the ovine adipose tissue. There were no significant differences in the expression of the other two PA-related genes (u-PA, PAI-1) throughout the experimental period. Plasminogen activator-related genes are not expressed in a coordinated manner in the adipose tissue of lactating dairy sheep in the early post-weaning period. In conclusion, adipose tissue mobilization is correlated with highest expression of u-PAR and lowest expression of PAI-2. PMID:21561489

Theodorou, G; Lampidonis, A D; Laliotis, G P; Bizelis, I; Politis, I

2012-06-01

18

The Glycosylation of Plasminogen Activator Inhibitor-1  

DEFF Research Database (Denmark)

Plasminogen activator inhibitor type-1 (PAI-1) has three potential sites for N-linked glycosylation, including Asn209Tyr210Thr211, Asn265Met266Thr267, and Asn329Glu330Ser331. Using a HEK293 expression system, we have made mutants with Asp or Gln substitutions of the Asn residue in each of these sequences. Analyses of these mutants for the content of N-acetyl glucosamine showed that Asn209 and Asn265, but not Asn329, are glycosylated, in agreement with previous suggestions made on the basis of X-ray crystal structure analysis of PAI-1 expressed in CHO cells (Xue et al. (1998) Structure 6, 627-636). In contrast, PAI-1, containing a total of 26 Ser and 26 Thr residues, which are potential targets for O-linked glycosylation, was found to be devoid of N-acetyl-galactosamine, demonstrating the absence of O-linked glycosylation. Analysis of PAI-1 variants with mutational inactivation of each of the sequences utilized for N-linked glycosylation by Fluorophore Assisted Carbohydrate Electrophoresis (FACE), showed a different N-linked glycosylation profile of the glycans at each of the 2 sites. The exact structure of the carbohydrate chains at each of these 2 sequences are being determined using MALDI mass spectrometry and monosaccharide composition analysis and compared to that of natural and recombinant PAI-1 from other sources. These results contribute to a structural basis for previous observations of a different functional importance of the N-linked glycosylation at each of the 2 sequences.

Skottrup, Peter; Pedersen, Katrine Egelund

19

Calcitonin stimulates plasminogen activator in porcine renal tubular cells: LLC-PK1  

OpenAIRE

Plasminogen activators are highly selective proteases that activate the proenzyme plasminogen to the general protease, plasmin. We studied a porcine kidney cell line, originally isolated as a high producer of plasminogen activator, in which activities of cellular adenylate cyclase and cAMP-dependent protein kinase are increased in response to calcitonin. We found that salmon calcitonin, in the concentration range 0.03-300 nM, increased plasminogen activator production up to approximately 1,00...

1981-01-01

20

Structural basis for recognition of urokinase-type plasminogen activator by plasminogen activator inhibitor-1.  

Science.gov (United States)

Plasminogen activator inhibitor-1 (PAI-1), together with its physiological target urokinase-type plasminogen activator (uPA), plays a pivotal role in fibrinolysis, cell migration, and tissue remodeling and is currently recognized as being among the most extensively validated biological prognostic factors in several cancer types. PAI-1 specifically and rapidly inhibits uPA and tissue-type PA (tPA). Despite extensive structural/functional studies on these two reactions, the underlying structural mechanism has remained unknown due to the technical difficulties of obtaining the relevant structures. Here, we report a strategy to generate a PAI-1·uPA(S195A) Michaelis complex and present its crystal structure at 2.3-? resolution. In this structure, the PAI-1 reactive center loop serves as a bait to attract uPA onto the top of the PAI-1 molecule. The P4-P3' residues of the reactive center loop interact extensively with the uPA catalytic site, accounting for about two-thirds of the total contact area. Besides the active site, almost all uPA exosite loops, including the 37-, 60-, 97-, 147-, and 217-loops, are involved in the interaction with PAI-1. The uPA 37-loop makes an extensive interaction with PAI-1 ?-sheet B, and the 147-loop directly contacts PAI-1 ?-sheet C. Both loops are important for initial Michaelis complex formation. This study lays down a foundation for understanding the specificity of PAI-1 for uPA and tPA and provides a structural basis for further functional studies. PMID:21199867

Lin, Zhonghui; Jiang, Longguang; Yuan, Cai; Jensen, Jan K; Zhang, Xu; Luo, Zhipu; Furie, Barbara C; Furie, Bruce; Andreasen, Peter A; Huang, Mingdong

2011-03-01

21

Structural basis for recognition of urokinase-type plasminogen activator by plasminogen activator inhibitor-1  

DEFF Research Database (Denmark)

Plasminogen activator inhibitor-1 (PAI-1), together with its physiological target urokinase-type plasminogen activator (uPA), plays a pivotal role in fibrinolysis, cell migration, and tissue remodeling and is currently recognized as being among the most extensively validated biological prognostic factors in several cancer types. PAI-1 specifically and rapidly inhibits uPA and tissue-type PA (tPA). Despite extensive structural/functional studies on these two reactions, the underlying structural mechanism has remained unknown due to the technical difficulties of obtaining the relevant structures. Here, we report a strategy to generate a PAI-1·uPA(S195A) Michaelis complex and present its crystal structure at 2.3-Å resolution. In this structure, the PAI-1 reactive center loop serves as a bait to attract uPA onto the top of the PAI-1 molecule. The P4-P3' residues of the reactive center loop interact extensively with the uPA catalytic site, accounting for about two-thirds of the total contact area. Besides the active site, almost all uPA exosite loops, including the 37-, 60-, 97-, 147-, and 217-loops, are involved in the interaction with PAI-1. The uPA 37-loop makes an extensive interaction with PAI-1 ?-sheet B, and the 147-loop directly contacts PAI-1 ?-sheet C. Both loops are important for initial Michaelis complex formation. This study lays down a foundation for understanding the specificity of PAI-1 for uPA and tPA and provides a structural basis for further functional studies.

Lin, Zhonghui; Jiang, Longguang

2011-01-01

22

?2-Glycoprotein I Is a Cofactor for t-PA–Mediated Plasminogen Activation  

OpenAIRE

Regulation of the conversion of plasminogen to plasmin by tissue-type plasminogen activator (t-PA) is critical in the control of fibrin deposition. While several plasminogen activators have been described, soluble plasma cofactors that stimulate fibrinolysis have not been characterized. Here, we report that the abundant plasma glycoprotein, ?2-glycoprotein I (?2GPI), stimulates t-PA–dependent plasminogen activation in the fluid phase and within a fibrin gel. The region within ?2GPI respo...

Bu, Chunya; Gao, Lei; Xie, Weidong; Zhang, Jainwei; He, Yuhong; Cai, Guoping; Mccrae, Keith R.

2009-01-01

23

Melatonin administration increased plasminogen activator activity in ram spermatozoa.  

Science.gov (United States)

The aim of the present study was to investigate the effect of melatonin on plasminogen activator activity (PAA), plasminogen activator inhibition (PAI) and plasmin inhibition (PI) in ram spermatozoa and seminal plasma, in correlation with changes in blood testosterone. Melatonin implants (18 mg) were placed subcutaneously in sixteen Chios rams in autumn and spring. Semen samples for spectrophotometrical assays were collected 36 h before the implantation of melatonin and thereafter once a week, for 17 weeks. Blood samples for testosterone assay (RIA) were collected 8h before implantation (one sample/30 min x 7.5 h) and thereafter every 15 days for 105 days after implantation. For each ram, six parameters of testosterone were estimated: mean value, basal level, number of peaks, peak amplitude, peak duration and mean testosterone concentration during peaks. Melatonin implantation during autumn induced an increase in PAA and t-PAI in spermatozoa; melatonin implantation in spring induced an additional increase in u-PAI and PI; no change in PAA, PAI or PI was found in seminal plasma, during autumn or spring. The melatonin-induced increase of PAA, PAI and PI in spermatozoa was in positive correlation with the increase of testosterone mean value, basal level and number of peaks; the increase of testosterone parameters was greater in autumn compared to spring. Changes of PAA, PAI and PI of spermatozoa, under the influence of melatonin, might indicate changes in the fertilizing ability of spermatozoa, since plasminogen activators and their inhibitors are present on the plasma and the outer acrosomal membrane of spermatozoa and are released during the acrosome reaction. PMID:18045674

Tsantarliotou, M P; Kokolis, N A; Smokovitis, A

2008-03-01

24

Diagnostic value of plasminogen activity level in acute mesenteric ischemia  

Directory of Open Access Journals (Sweden)

Full Text Available AIM: To investigate the changes in plasminogen activity level during mesenteric ischemia.METHODS: We performed laparotomy in 90 female Wistar-Albino rats (average weight 230 g. In sham groups (SL (GroupsIand II the superior mesenteric artery (SMA and vein (SMV were explored, but not tied. In SMA groups (Groups III and IV the SMA was ligated, and in SMV groups (Groups V and VI the SMV was ligated. On re-laparotomy 2 mL of blood was drawn at 1 h in groupsI, III and V, and at 3 h in groups II, IV and VI. Plasminogen levels were assessed and comparisons were made between groups and within each group.RESULTS: The mean plasminogen activity in the SL group was significantly higher than SMA (25.1 ± 10.8 vs 11.8 ± 4.6, P < 0.001 or SMV (25.1 ± 10.8 vs 13.7 ± 4.4, P < 0.001 groups both at 1 h and at 3 h (29.8 ± 8.9 vs 15.1 ± 5.7, P < 0.0001; 29.8 ± 8.9 vs 14.2 ± 2.9, P < 0.0001. There were no significant differences between the values of SMA and SMV groups at 1 h (P = 0.28 and at 3 h (P = 0.71. In each group, plasminogen activity levels did not change significantly between the two measurements performed at 1 h and 3 h.CONCLUSION: We conclude that blood plasminogen activities decrease during early phases of both arterial and venous mesenteric ischemia which may be a useful marker for early diagnosis.

Yusuf Gunerhan, Neset Koksal, Munire Kayahan, Yavuz Eryavuz, Hilal Sekban

2008-04-01

25

Technetium labelled plasminogen activator - a potential reagent for thrombus detection  

International Nuclear Information System (INIS)

The preparation of a technetium labelled plasminogen activator complex using a solid phase labelling technique is described. The labelled complex showed no significant loss of fibrinolytic activity in vitro and showed in vivo a rapid uptake in thrombi in an animal model and in human volunteer patients with known thrombi when injected into a vein draining to the thrombotic region. Systemic injection showed no uptake in the thrombi probably due to rapid sequestration of the complex by the liver. (orig.)

26

Thrombin-specific inactivation of endothelial cell derived plasminogen activator  

Energy Technology Data Exchange (ETDEWEB)

Although thrombin (T) has diverse functions in the overall hemostatic mechanism, relatively little is known about its direct effect on components of the fibrinolytic enzyme system. The authors have investigated the interaction of T with plasminogen activators (PA) derived from bovine aortic endothelial cells (EC) in culture (2-5th passage, preconfluent monolayers). Varying concentrations of purified bovine or human thrombin were added to EC-conditioned media (CM). CM + T mixtures were assayed at various times for PA activity using purified plasminogen and a sensitive /sup 125/I-fibrinogenolytic or caseinolytic assay. T (5 nM), but not plasmin or trypsin at equivalent concentrations, resulted in a time-dependent inhibition of the PA activity in CM. T had no effect on the PA activity of urokinase, streptokinase or preformed plasmin. The ability of T to inactivate the EC-derived PA was abolished by prior treatment of T with active site-directed reagents. SDS-PAGE and zymography with copolymerized fibrinogen and plasminogen revealed further specificity in that only one of the multiple-molecular weight forms of PA present in EC-CM was inactivated by T. The authors conclude that in a highly specific fashion, T inactivates the predominant PA present in EC-CM by limited proteolysis. Thus, another potentially important function of T is suggested which may have particular significance in the temporal regulation of coagulation and fibrinolysis at the blood-endothelium interface.

Highsmith, R.F.; Gallaher, M.J.

1986-03-05

27

Proteases induce secretion of collagenase and plasminogen activator by fibroblasts  

Energy Technology Data Exchange (ETDEWEB)

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.

Werb, Z.; Aggeler, J.

1978-04-01

28

Immunohistochemical localisation of tissue plasminogen activator in human brain tumours.  

OpenAIRE

The distribution of tissue plasminogen activator (t-PA) has been studied in a series of 38 human brain tumours and two specimens of cerebral cortex, using the monoclonal antibody ESP6. t-PA was localised in vascular endothelium in the majority of tumours and both the cortical specimens, confirmed by double staining with Ulex europaeus lectin (Uel) and Factor 8-related antigen. Nineteen out of 22 high grade astrocytomas showed strong endothelial staining whereas staining was weak or absent in ...

Franks, A. J.; Ellis, E.

1989-01-01

29

Structural and kinetic comparison of recombinant human single- and two-chain tissue plasminogen activator.  

OpenAIRE

We examined the similarities and differences in conformation between recombinant human single-chain tissue plasminogen activator (sct-PA) and two-chain tissue plasminogen activator (tct-PA), and compared these structural data with measurement of enzymatic activity. The intrinsic protein fluorescence of native tct-PA was 54% that of sct-PA. Differences in steady state protein fluorescence were also noted with denaturation of these plasminogen activators, as well as in the quenching of intrinsi...

Loscalzo, J.

1988-01-01

30

Impact of Lysophosphatidylcholine on the Plasminogen Activator System in Cultured Vascular Smooth Muscle Cells  

OpenAIRE

The balance between tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor type 1 (PAI-1) regulates fibrinolysis. PAI-1 expression increases in atherosclerotic arteries and vascular smooth muscle cells (VSMCs) are one of major constituents of atheroma. We investigated the impact of lysophosphatidylcholine (lysoPC), an active component of oxidized low-density lipoprotein, on the plasminogen activator system of the rat VSMCs. The lysoPC stimulated the protein and gene expr...

Yoon, Byung-koo; Kang, Young-hee; Oh, Won-jong; Park, Kyungwon; Lee, Dong-yun; Choi, Dooseok; Kim, Duk-kyung; Lee, Youngjoo; Rhyu, Mee-ra

2012-01-01

31

Immunoradiometric determination of the blood/tissue plasminogen activator in thrombophilia  

International Nuclear Information System (INIS)

Immunoradiometric determination of the blood/tissue plasminogen activator was performed in plasma from patients before and after response to venous occlusion, infusion of 1-desamino-8-D-arginine-vasopressin (DDAVP) or exercise. The raise in the level of plasminogen activator was most pronounced after venous occlusion. In patients who earlier had had verified thrombosis the levels of plasminogen activator compared to normals did not show any significant difference. (author)

32

Immunoradiometric determination of the blood/tissue plasminogen activator in thrombophilia  

Energy Technology Data Exchange (ETDEWEB)

Immunoradiometric determination of the blood/tissue plasminogen activator was performed in plasma from patients before and after response to venous occlusion, infusion of 1-desamino-8-D-arginine-vasopressin (DDAVP) or exercise. The raise in the level of plasminogen activator was most pronounced after venous occlusion. In patients who earlier had had verified thrombosis the levels of plasminogen activator compared to normals did not show any significant difference.

Astedt, B.; Fagner, U. (Lund Univ. (Sweden))

1984-01-01

33

Method and tool for prognosticating HIV infection in a subject by measuring soluble urokinase plasminogen activator receptor, degradation products thereof, and urokinase plasminogen activator receptor  

DEFF Research Database (Denmark)

Method of diagnosing and/or prognosticating HIV infection in a subject comprising the steps of: (a) performing in vitro a measurement of the level of a marker in the form of (i) urokinase plasminogen activator receptor (uPAR), (ii) soluble urokinase plasminogen activator receptor (suPAR), (iii) urokinase-type plasminogen activator (uPA), (iv) one or more degradation products of (i), (ii), or (iii), and/or (v) an mRNA for (i), (ii) or (iii), in a biological fluid sample from a subject, and (b) using the measurement value obtained to evaluate the state of the subject.

Eugen-Olsen, Jesper Hvidovre Hospital,

34

Diagnostic value of plasminogen activity level in acute mesenteric ischemia  

OpenAIRE

AIM: To investigate the changes in plasminogen activity level during mesenteric ischemia.METHODS: We performed laparotomy in 90 female Wistar-Albino rats (average weight 230 g). In sham groups (SL) (GroupsIand II) the superior mesenteric artery (SMA) and vein (SMV) were explored, but not tied. In SMA groups (Groups III and IV) the SMA was ligated, and in SMV groups (Groups V and VI) the SMV was ligated. On re-laparotomy 2 mL of blood was drawn at 1 h in groupsI, III and V, and at 3 h in group...

Yusuf Gunerhan, Neset Koksal

2008-01-01

35

The human urokinase-plasminogen activator gene and its promoter.  

OpenAIRE

The urokinase type of plasminogen activator (uPA) is subject to regulation by hormones, phorbol esters and oncogenic transformation. This enzyme has been suggested to play a key role in processes involving cell migration and tissue remodeling, and to be essential for tumor metastasis. In order to study these processes, we have isolated the human uPA gene, and have determined its entire nucleotide sequence. The gene is organized in 11 exons and is 6.4 kb long. The 5' end of uPA mRNA has been d...

Riccio, A.; Grimaldi, G.; Verde, P.; Sebastio, G.; Boast, S.; Blasi, F.

1985-01-01

36

Arrhenius temperature dependence of in vitro tissue plasminogen activator thrombolysis  

Energy Technology Data Exchange (ETDEWEB)

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.

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

37

Arrhenius temperature dependence of in vitro tissue plasminogen activator thrombolysis  

International Nuclear Information System (INIS)

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 ?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 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

38

Plasminogen activator inhibitor-1 (PAI-1) and urokinase plasminogen activator (uPA) in sputum of allergic asthma patients.  

OpenAIRE

Urokinase plasminogen activator (uPA) and its inhibitor (PAI-1) have been associated with asthma. The aim of this study was to evaluate concentration of uPA and PAI-1 in induced sputum of house dust mite allergic asthmatics (HDM-AAs). The study was performed on 19 HDM-AAs and 8 healthy nonatopic controls (HCs). Concentration of uPA and PAI-1 was evaluated in induced sputum supernatants using ELISA method. In HDM-AAs the median sputum concentration of uPA (128 pg/ml; 95% CI 99 to 183 pg/ml) an...

Sebastian Zukowski; Krzysztof Kowal; Marcin Moniuszko; Anna Bodzenta-?ukaszyk

2008-01-01

39

Transforming growth factor beta 1 and sodium butyrate differentially modulate urokinase plasminogen activator and plasminogen activator inhibitor-1 in human breast normal and cancer cells.  

OpenAIRE

The effects of transforming growth factor beta 1 (TGF-beta1) and sodium butyrate on cell proliferation and the urokinase plasminogen activator (uPA) system were examined in normal human breast epithelial cells (HBECs) and in a breast cancer cell line, MDA-MB-231. In HBECs, TGF-beta1 inhibited cell proliferation and uPA activity, while it augmented plasminogen activator inhibitor-1 (PAI-1) antigen level. Sodium butyrate inhibited both cell proliferation and uPA activity but did not affect the ...

Dong-le Bourhis, X.; Lambrecht, V.; Boilly, B.

1998-01-01

40

A novel serine protease secreted by medicinal maggots enhances plasminogen activator-induced fibrinolysis.  

Science.gov (United States)

Maggots of the blowfly Lucilia sericata are used for the treatment of chronic wounds. As haemostatic processes play an important role in wound healing, this study focused on the effects of maggot secretions on coagulation and fibrinolysis. The results showed that maggot secretions enhance plasminogen activator-induced formation of plasmin and fibrinolysis in a dose- and time-dependent manner. By contrast, coagulation was not affected by secretions. Biochemical studies indicated that a novel serine protease within secretions, designated Sericase, cleaved plasminogen to several fragments. Recombinant Sericase degraded plasminogen leading amongst others to the formation of the mini-plasminogen like fragment Val454-plasminogen. In addition, the presence of a non-proteolytic cofactor in secretions was discovered, which plays a role in the enhancement of plasminogen activator-induced fibrinolysis by Sericase. We conclude from our in vitro studies that the novel serine protease Sericase, with the aid of a non-proteolytic cofactor, enhances plasminogen activator-induced fibrinolysis. PMID:24647546

van der Plas, Mariena J A; Andersen, Anders S; Nazir, Sheresma; van Tilburg, Nico H; Oestergaard, Peter R; Krogfelt, Karen A; van Dissel, Jaap T; Hensbergen, Paul J; Bertina, Rogier M; Nibbering, Peter H

2014-01-01

41

Soluble urokinase plasminogen activator receptor is associated with subclinical organ damage and cardiovascular events  

DEFF Research Database (Denmark)

The soluble urokinase plasminogen activator receptor (suPAR) is a plasma marker of low grade inflammation and has been associated with cardiovascular risk. We wanted to investigate whether suPAR was associated with markers of subclinical organ damage.

Sehestedt, T; Lyngbæk, S

2011-01-01

42

Binding of tissue plasminogen activator to vascular grafts.  

Science.gov (United States)

Tissue plasminogen activator labeled with radioactive iodine (125I-tPA) was immobilized on vascular prostheses chemically modified with a thin coating of water-insoluble surfactant, tridodecylmethylammonium chloride (TDMAC). Surfactant-treated Dacron, polytetrafluoroethylene (PTFE), silastic, polyethylene and polyurethane bound appreciable amounts of 125I-tPA (5-30 micrograms 125I-tPA/cm2). Upon exposure to human plasma, the amount of 125I-tPA bound to the surface shows an initial drop during the first hour of incubation, followed by a slower, roughly exponential release with a t 1/2 of approximately 75 hours. Prostheses containing bound tPA show fibrinolytic activity as measured both by lysis of clots formed in vitro, and by hydrolysis of a synthetic polypeptide substrate. Prior to incubation in plasma, tPA bound to a polymer surface has an enzymic activity similar, if not identical to that of the native enzyme in buffered solution. However, exposure to plasma causes a decrease in the fibrinolytic activity of both bound tPA and enzyme released from the surface of the polymer. These data demonstrate that surfactant-treated prostheses can bind tPA, and that these chemically modified devices can act as a slow-release drug delivery system with the potential for reducing prosthesis-induced thromboembolism. PMID:2501890

Harvey, R A; Kim, H C; Pincus, J; Trooskin, S Z; Wilcox, J N; Greco, R S

1989-02-28

43

Thrombin activatable fibrinolysis inhibitor (TAFI) affects fibrinolysis in a plasminogen activator concentration-dependent manner. Study of seven plasminogen activators in an internal clot lysis model.  

Science.gov (United States)

TAFIa was shown to attenuate fibrinolysis. In our in vitro study, we investigated how the inhibitory effect of TAFIa depended on the type and concentration of the plasminogen activator (PA). We measured PA-mediated lysis times of plasma clots under conditions of maximal TAFI activation by thrombin-thrombo-modulin in the absence and presence of potato carboxypeptidase inhibitor. Seven different PAs were compared comprising both tPA-related (tPA, TNK-tPA, DSPA), bacterial PA-related (staphylokinase and APSAC) and urokinase-related (tcu-PA and k2tu-PA) PAs. The lysis times and the retardation factor were plotted against the PA concentration. The retardation factor plots were bell-shaped. At low PA concentrations, the retardation factor was low, probably due to the limited stability of TAFIa. At intermediate PA concentrations the retardation factor was maximal (3-6 depending on the PA), with TNK-tPA, APSAC and DSPA exhibiting the strongest effect. At high PA concentrations, the retardation factor was again low, possibly due to inactivation of TAFIa by plasmin or to a complete conversion of glu-plasminogen into lys-plasminogen. Using individual plasmas with a reduced plasmin inhibitor activity (plasmin inhibitor Enschede) the bell-shaped curve of the retardation factor shifted towards lower tPA and DSPA concentrations, but the height did not decrease. In conclusion, TAFIa delays the lysis of plasma clots mediated by all the plasminogen activators tested. This delay is dependent on the type and concentration of the plasminogen activator, but not on the fibrin specificity of the plasminogen activator. Furthermore, plasmin inhibitor does not play a significant role in the inhibition of plasma clot lysis by TAFI. PMID:14983222

Guimarães, Ana H C; Rijken, Dingeman C

2004-03-01

44

Functional properties of the recombinant kringle-2 domain of tissue plasminogen activator produced in Escherichia coli  

International Nuclear Information System (INIS)

The kringle-2 domain (residues 176-262) of tissue-type plasminogen activator (t-PA) was cloned and expressed in Escherichia coli. The recombinant peptide, which concentrated in cytoplasmic inclusion bodies, was isolated, solubilized, chemically refolded, and purified by affinity chromatography on lysine-Sepharose to apparent homogeneity. [35S]Cysteine-methionine-labeled polypeptide was used to study the interactions of kringle-2 with lysine, fibrin, and plasminogen activator inhibitor-1. The kringle-2 domain bound to lysine-Sepharose and to preformed fibrin with a Kd = 104 +/- 6.2 microM (0.86 +/- 0.012 binding site) and a Kd = 4.2 +/- 1.05 microM (0.80 +/- 0.081 binding site), respectively. Competition experiments and direct binding studies showed that the kringle-2 domain is required for the formation of the ternary t-PA-plasminogen-intact fibrin complex and that the association between the t-PA kringle-2 domain and fibrin does not require plasmin degradation of fibrin and exposure of new COOH-terminal lysine residues. We also observed that kringle-2 forms a complex with highly purified guanidine-activated plasminogen activator inhibitor-1, dissociable by 0.2 M epsilon-aminocaproic acid. The kringle-2 polypeptide significantly inhibited tissue plasminogen activator/plasminogen activator inhibitor-1 interaction. The kringle-2 domain bound to plasminogen activator inhibitor-1 in a specific and saturable manner with a Kd = 0.51 +/- 0.055 microM (0.35 +/- 0.026 binding +/- 0.055 microM (0.35 +/- 0.026 binding site). Therefore, the t-PA kringle-2 domain is important for the interaction of t-PA not only with fibrin, but also with plasminogen activator inhibitor-1 and thus represents a key structure in the regulation of fibrinolysis

45

Cleaved intracellular plasminogen activator inhibitor 2 in human myeloleukaemia cells is a marker of apoptosis.  

OpenAIRE

The proteolytic modification of plasminogen activator inhibitor 2 (PAI-2) was studied during apoptosis in the human promyelocytic leukaemic NB4 cell line during treatment with the phosphatase inhibitors okadaic acid and calyculin A as well as the protein synthesis inhibitor cycloheximide. The apoptic type of cell death was ascertained by morphological and biochemical criteria. In cell homogenates PAI-2 was probed by [125I]urokinase plasminogen activator (uPA) and detected as a sodium dodecyl ...

Jensen, P. H.; Cressey, L. I.; Gjertsen, B. T.; Madsen, P.; Mellgren, G.; Hokland, P.; Gliemann, J.; Døskeland, S. O.; Lanotte, M.; Vintermyr, O. K.

1994-01-01

46

Functional properties of the recombinant kringle-2 domain of tissue plasminogen activator produced in Escherichia coli  

Energy Technology Data Exchange (ETDEWEB)

The kringle-2 domain (residues 176-262) of tissue-type plasminogen activator (t-PA) was cloned and expressed in Escherichia coli. The recombinant peptide, which concentrated in cytoplasmic inclusion bodies, was isolated, solubilized, chemically refolded, and purified by affinity chromatography on lysine-Sepharose to apparent homogeneity. (35S)Cysteine-methionine-labeled polypeptide was used to study the interactions of kringle-2 with lysine, fibrin, and plasminogen activator inhibitor-1. The kringle-2 domain bound to lysine-Sepharose and to preformed fibrin with a Kd = 104 +/- 6.2 microM (0.86 +/- 0.012 binding site) and a Kd = 4.2 +/- 1.05 microM (0.80 +/- 0.081 binding site), respectively. Competition experiments and direct binding studies showed that the kringle-2 domain is required for the formation of the ternary t-PA-plasminogen-intact fibrin complex and that the association between the t-PA kringle-2 domain and fibrin does not require plasmin degradation of fibrin and exposure of new COOH-terminal lysine residues. We also observed that kringle-2 forms a complex with highly purified guanidine-activated plasminogen activator inhibitor-1, dissociable by 0.2 M epsilon-aminocaproic acid. The kringle-2 polypeptide significantly inhibited tissue plasminogen activator/plasminogen activator inhibitor-1 interaction. The kringle-2 domain bound to plasminogen activator inhibitor-1 in a specific and saturable manner with a Kd = 0.51 +/- 0.055 microM (0.35 +/- 0.026 binding site). Therefore, the t-PA kringle-2 domain is important for the interaction of t-PA not only with fibrin, but also with plasminogen activator inhibitor-1 and thus represents a key structure in the regulation of fibrinolysis.

Wilhelm, O.G.; Jaskunas, S.R.; Vlahos, C.J.; Bang, N.U. (Eli Lilly and Company, Indianapolis, IN (USA))

1990-08-25

47

Simple and sensitive assay employing stable reagents for quantification of plasminogen activator  

International Nuclear Information System (INIS)

A simple and sensitive indirect assay for quantifying plasminogen activator using [3H]-casein as substrate is described. The assay has been used to measure plasminogen activators from various sources including bacteria, cultured cells, and human plasma. The assay is linear with respect to concentration of plasminogen activator and time of incubation and is independent of concentration of plasminogen. The assay can routinely be used to quantify as few as 8 X 10(-3) international units ml-1 of streptokinase. Results obtained with the [3H]-casein assay correlate well with those obtained by the fibrin plate method (R . 0.83 by Spearman's ranked co-efficient of correlation). The reagents are extremely stable and easily stored. The ease with which the assay can be performed gives the clinical laboratory the potential to test large numbers of patients upon short notice and within a short period of time

48

Transgenic chickens expressing human urokinase-type plasminogen activator.  

Science.gov (United States)

Urokinase-type plasminogen activator is a serine protease that is clinically used in humans for the treatment of thrombolytic disorders and vascular diseases such as acute ischemic stroke and acute peripheral arterial occlusion. This study explored the feasibility of using chickens as a bioreactor for producing human urokinase-type plasminogen activator (huPA). Recombinant huPA gene, under the control of a ubiquitous Rous sarcoma virus promoter, was injected into the subgerminal cavity of freshly laid chicken eggs at stage X using the replication-defective Moloney murine leukemia virus (MoMLV)-based retrovirus vectors encapsidated with VSV-G (vesicular stomatitis virus G) glycoprotein. A total of 38 chicks, out of 573 virus-injected eggs, hatched and contained the huPA gene in their various body parts. The mRNA transcript of the huPA gene was present in various organs, including blood and egg, and was germ-line transmitted to the next generation. The level of active huPA protein was 16-fold higher in the blood of the transgenic chicken than in the nontransgenic chicken (P eggs increased from 7.82 IU/egg in the G0 generation to 17.02 IU/egg in the G1 generation. However, huPA-expressing embryos had reduced survival and hatchability at d 18 and 21 of incubation, respectively, and the blood clotting time was significantly higher in transgenic chickens than their nontransgenic counterparts (P < 0.05). Furthermore, adult transgenic rooster showed reduced (P < 0.05) fertility, as revealed by reduced volume of semen ejaculate, sperm concentration, and sperm viability. Taken together, our data suggest that huPA transgenic chickens could be successfully produced by the retroviral vector system. Transgenic chickens, expressing the huPA under the control of a ubiquitous promoter, may not only be used as a bioreactor for pharming of the huPA drug but also be useful for studying huPA-induced bleeding and other disorders. PMID:23960123

Lee, Sung Ho; Gupta, Mukesh Kumar; Ho, Young Tae; Kim, Teoan; Lee, Hoon Taek

2013-09-01

49

Testicular plasminogen activators during postnatal development in the rat.  

Science.gov (United States)

In the seminiferous epithelium, Sertoli cells secrete plasminogen activator (PA) under regulation of follicle stimulating hormone, cyclic AMP and neighbouring spermatogenic cells. Recent observations suggest that preleptotene spermatocytes upon their release from the basement membrane of the seminiferous tubule are important regulators of PA secretion. To study further the role of PA's in the seminiferous tubules, we have analyzed the endogenous levels and secretion rates of PA at various ages during postnatal development, and performed biochemical analyses of the types of PA in the testis and spent media from seminiferous tubular cultures. Cyclic secretion of PA started at the age of 28 days, and from 40 days onwards, the high secretion rates were localized in stages VII and VIII of the cycle of the seminiferous epithelium. The secreted PA is most obviously of the urokinase type; both urokinase-type and tissue-type PA-like activities were found in seminiferous tubular homogenates. The increase in testicular PA levels concomitant to the onset of meiosis in the epithelium was due to the urokinase-type PA-like activity. PMID:3092528

Vihko, K K; Toppari, J; Saksela, O; Suominen, J J; Parvinen, M

1986-07-01

50

Synthesis, purification, and properties of a peptide that enhances the activation of human [Glu1]plasminogen by tissue plasminogen activator and retards fibrin polymerization.  

Science.gov (United States)

Solid phase synthesis of the hexapeptide, GPRVVE, which represents the amino terminal six amino acids of the alpha-chain of human fibrin, yielded a product that contained a modified glutamic acid. The nature of the modification was established as the Friedel-Crafts acylation product of the peptide and anisole, the latter reagent employed in the HF deblocking step. The anisoylated peptide selectively enhanced the activation rate of native [Glu1]plasminogen by recombinant tissue plasminogen activator, accelerated clot lysis, and retarded the polymerization of nascent fibrin. PMID:1969852

Chibber, B A; Urano, S; Castellino, F J

1990-01-01

51

A new plasminogen receptor  

OpenAIRE

Efficient activation of the fibrinolytic pathway occurs when plasminogen and its activators are sequestered on the cell surface. Identification of receptors responsible for localizing plasminogen to the cell surface has been elusive. Using a proteomics approach, Andronicos and colleagues have identified a novel 17-kDa transmembrane receptor, termed Plg-RKT, that binds plasminogen with high affinity and promotes its activation.1

Strickland, Dudley K.

2010-01-01

52

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

Directory of Open Access Journals (Sweden)

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 ?2 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 that plasmin activates MMP-13 directly. Conclusions These data demonstrate that breast cancer cells dissolve type I collagen and that there is an absolute requirement for plasminogen activation and MMP activity in the degradation process.

Hill Peter A

2005-02-01

53

Tc-99m labeled tissue-type plasminogen activator  

International Nuclear Information System (INIS)

Tissue-type plasminogen activator (t-PA) is a thrombolytic agent that directly binds to fibrin formed in clots. In terms of radiolabeling and nuclear imaging, t-PA has several advantages in Tc-99m labeling: it is stable in acidic solution at pH 3, which is suitable for labeling Tc-99m by a method of stannous reduction and blood disappearance after administration is rapid, which is desirable for imaging targets using short-lived radionuclides. Recombinant t-PA was labeled with Tc-99m by a method of stannous reduction without significant degradation of biochemical activity, over 95% of which was retained after the labeling procedure. Labeling efficiency in paper chromatography was over 98%. The moiety of hydrolyzed Tc-99m that was not eluted through the Sephadex column was estimated to be less than 10%. Tc-99m labeled t-PA, however, appeared to become unstable when diluted with normal saline. Nevertheless, in in vitro fibrin binding, Tc-99m labeled t-PA showed high affinity with fibrin: 80% of 100 ng/ml of Tc-99m t-PA bound to 10-5 mol of the fibrinogen. Preliminary animal studies also showed a concentration of Tc-99m labeled t-PA at fresh thrombi formed in the inferior vena cava. Tc-99m labeled t-PA appears to have potential for thrombus imaging and the preparation of an instant kit. (author)

54

Tissue plasminogen activator as a hemodialysis catheter locking solution.  

Science.gov (United States)

Tunneled hemodialysis catheters require a "locking solution" between treatments to prevent catheter thrombosis. Heparin locks can be unsafe in patients with life-threatening bleeding diathesis because of unintentional anticoagulation. This study was designed to define the hematologic consequences of using tissue plasminogen activator (t-PA) as an alternative locking solution after heparin-free hemodialysis (HF-HD). Following HF-HD, t-PA 2 mg was instilled into each lumen of the dialysis catheter in 10 patients. Euglobulin clot lysis time (ECLT), fibrinogen, D-dimer, and fibrin degradation products were measured during the last hour of dialysis, and repeated 15 and 30 minutes after catheter locking. Dialysis catheter performance was reassessed at the time of the next hemodialysis. Fibrinogen, D-dimer, and fibrin degradation products were elevated at all time points, but did not change after t-PA. ECLT decreased significantly from baseline 15 minutes after catheter locking (217+/-64 vs. 132+/-75 min, p=0.016). ECLT values had returned to baseline (202+/-56 minutes) by 30 minutes. No episodes of bleeding or catheter thrombosis occurred, and catheter performance did not deteriorate. A 2 mg t-PA locking solution preserved dialysis catheter performance. ECLT decreased at 15 minutes, but normalized by 30 minutes, and did not enter the range in which bleeding would be likely. No clinical events were seen during this transient increase in systemic fibrinolysis. PMID:18638092

McGill, Rita L; Spero, Joel A; Sysak, Joshua C; Sandroni, Stephen E; Marcus, Richard J

2008-07-01

55

Pneumatic displacement without tissue plasminogen activator in premacular subhyaloid hemorrhage  

Directory of Open Access Journals (Sweden)

Full Text Available To assess the efficacy and safety of intravitreal injection of Sulfur Hexafluoride (SF6 gas without the use of tissue Plasminogen Activator (tPA in premacular Subhyaloid Hemorrhage (SHH, 5 eyes of 5 patients with premacular SHH were enrolled. After performing paracentesis of the anterior chamber, 0.3 ml pure SF6 gas was injected through pars plana with a 30 gauge needle. Facedown position was maintained for 5 days. Subhyaloid Hemorrhage was displaced in 4/5 (80% eyes with a duration of SHH less than 2 weeks. The pre-injection visual acuity of all 5 eyes was finger counting and improved in 4/5 ( 80% eyes within 3 days to 7 days post-injection to 6/20 - 6/6. The underlying disease was hypercoagulation in 1 patient, diabetes mellitus in 2 patients, hypertension in 1 patient and unknown in 1 patient. No complications were encountered. In conclusion, SF6 gas injected into the vitreous without the use of tPA, can displace SHH if performed within 14 days of duration, and results in rapid visual recovery. This procedure is proven to be safe. (Med J Indones 2007; 16:104-7 Keywords: subhyaloid hemorrhage, pneumatic displacement, sulfur hexafluoride gas

Rumita S. Kadarisman

2007-05-01

56

Soluble urokinase plasminogen activator receptor during allogeneic stem cell transplantation  

DEFF Research Database (Denmark)

Previous studies have found that soluble urokinase plasminogen activation receptor (suPAR) increases during inflammatory and malignant illness and elevated suPAR levels may be associated with poor clinical outcome. The purpose of this study was to investigate plasma levels of suPAR during the course of allogeneic stem cell transplantation (SCT). Twenty SCT patients were included in the study. suPAR was measured by ELISA in daily taken plasma samples during the pretransplant conditioning with chemotherapy and weekly for 1 month after infusion of the graft. suPAR levels before the start of the conditioning were significantly elevated when compared to those of healthy controls. During the conditioning in particular treatment with antithymocyte globulin was associated with significantly increased suPAR levels (P = 0.012). At day +7 after infusion of the graft, suPAR levels had decreased to pretreatment levels. High suPAR levels at day 0 were associated with increased mortality (P = 0.011). The present study foundincreased suPAR levels during the conditioning in SCT patients. Further, the data indicated that increased suPAR levels may be associated with increased mortality, suggesting suPAR as a candidate for further studies as an outcome predictor in SCT.

Haastrup, E; Andersen, J

2011-01-01

57

Association of plasminogen activator inhibitor-1 and tissue plasminogen activator with type 2 diabetes and metabolic syndrome in Malaysian subjects  

Directory of Open Access Journals (Sweden)

Full Text Available Abstract Background Increased plasma plasminogen activator inhibitor-1 (PAI-1 activity and decreased tissue plasminogen activator (tPA activity could be considered a true component of the metabolic syndrome (MetS associated with an increased risk of developing cardiovascular diseases (CVD and fibrinolytic abnormalities. The aim of this study was to investigate the association of tPA and its inhibitor PAI-1 with type 2 diabetes (T2D and MetS and interrelationship between PAI-1and tPA activities and antigens in Malaysian T2D and normal subjects. Methods The plasma activities and antigens of PAI-1 and tPA and the levels of the tPA/PAI-1 complex as well as serum insulin, parameter of the coronary risk panel and plasma glucose at fasting state were studied in 303 T2D subjects (227 with MetS and 76 without MetS, 131 normal non-diabetic non-metabolic subjects and 101 non-diabetic MetS subjects. Results The PAI-1 activity was higher in subjects with T2D with MetS (P = 9.8 × 10-19 and non-diabetic subjects with MetS (P = 3.0 × 10-15, whereas the tPA activity was lower in T2D with MetS (P = 0.003 as compare to normal subjects. Plasma tPA antigen levels were higher in subjects with T2D with MetS (P = 8.9 × 10-24, T2D without MetS (P = 1.3 × 10-13 and non-diabetic MetS subjects (P = 0.002. The activity and antigen of PAI-1 in normal subjects were related to insulin resistance (P = 2.2 × 10-4; 0.007. Additionally, the PAI-1 activity was associated with an increased waist circumference (P = 2.2 × 10-4 and decreased HDL-c (P = 0.005, whereas the tPA activity was associated with decreased FBG (P = 0.028. The highest correlation was between PAI-1 activity and its antigen (R2 = 0.695, P = 1.1 × 10-36 in diabetic subjects. The tPA activity negatively correlated with its antigen (R2 = -0.444, P = 7.7 × 10-13 in normal subjects and with the PAI-1 activity and antigen (R2 = -0.319, P = 9.9 × 10-12; R2 = -0.228, P = 3.4 × 10-6 in diabetic subjects. Conclusions PAI-1 and tPA activities and antigens were associated with diabetes and MetS parameters in Malaysian subjects.

Saif-Ali Riyadh

2011-03-01

58

Overexpression of plasminogen activator inhibitor 2 in human melanoma cells inhibits spontaneous metastasis in scid/scid mice.  

OpenAIRE

A metastatic human melanoma cell line that produces urokinase-type plasminogen activator was stably transfected with cDNA encoding human plasminogen activator inhibitor 2 (PAI-2). Transfected clones expressed PAI-2 at levels two to nine times higher than both the parental cell line and mock transfectants, as detected by ELISA of cell lysates and conditioned medium. The clone with the highest PAI-2 expression exhibited complete inhibition of soluble and cell-surface-bound plasminogen activator...

Mueller, B. M.; Yu, Y. B.; Laug, W. E.

1995-01-01

59

An Active Site Water Network in the Plasminogen Activator Pla from Yersinia pestis  

Energy Technology Data Exchange (ETDEWEB)

The plasminogen activator Pla from Yersinia pestis is an outer membrane protease (omptin) that is important for the virulence of plague. Here, we present the high-resolution crystal structure of wild-type, enzymatically active Pla at 1.9 {angstrom}. The structure shows a water molecule located between active site residues D84 and H208, which likely corresponds to the nucleophilic water. A number of other water molecules are present in the active site, linking residues important for enzymatic activity. The R211 sidechain in loop L4 is close to the nucleophilic water and possibly involved in the stabilization of the oxyanion intermediate. Subtle conformational changes of H208 result from the binding of lipopolysaccharide to the outside of the barrel, explaining the unusual dependence of omptins on lipopolysaccharide for activity. The Pla structure suggests a model for the interaction with plasminogen substrate and provides a more detailed understanding of the catalytic mechanism of omptin proteases.

Eren, Elif; Murphy, Megan; Goguen, Jon; van den Berg, Bert (UMASS, MED)

2010-08-13

60

Tissue plasminogen activator modulates emotion in a social context.  

Science.gov (United States)

Tissue plasminogen activator (tPA) is known to play physiologically and pathologically crucial roles in the central nervous system. However, it is still obscure whether it affects social behavior. We investigated social behavior and neuronal activation after social stimulation in tPA knockout (KO) mice. In a resident-intruder test, the latency to the first contact was significantly delayed in tPA KO mice compared with that in tPA heterogenic (Het) mice. However, tPA KO mice spent significantly more time undertaking active behavior than tPA Het mice did. In a sociability test, tPA KO mice significantly spent more time and walked a greater distance in the chamber containing an empty cage than tPA Het mice. Furthermore, tPA KO mice approached an empty cage more frequently than tPA Het mice did. In a social novelty test, there was no difference in the duration spent sniffing a stimulator mouse between genotypes. However, tPA KO mice approached even a familiar mouse more frequently than tPA Het mice did. tPA KO mice spent similar durations in a chamber containing a familiar mouse and that containing an unfamiliar mouse, and tPA KO mice walked a significantly greater distance in the former chamber than tPA Het mice did. Furthermore, at the cingulate and prelimbic cortices, the number of cFos-positive cells was significantly increased in tPA KO mice compared with that in tPA Het mice after social stimulation. Our results suggest that tPA modulates emotion in a social context through the function of the prefrontal cortex. PMID:25499620

Nakamura, Kazuki; Takabe, Ayumi; Shimizu, Fuki; Takahashi, Maiko; Matsuo, Osamu; Mitsui, Shinichi

2015-03-15

61

Prognostic significance of urokinase-type plasminogen activator and plasminogen activator inhibitor-1 in primary breast cancer  

DEFF Research Database (Denmark)

The uPA-mediated pathway of plasminogen activation is central to cancer metastasis. Whether uPA and PAI-1 are related to local recurrence, metastatic spread or both is not clear. We present a retrospective study of 429 primary breast cancer patients with a median follow-up of 5.1 years, in which the levels of uPA and PAI-1 in tumour extracts were analysed by means of an enzyme-linked immunosorbent assay. The median values of uPA and PAI-1, which were used as cut-off points, were 4.5 and 11.1 ng mg(-1) protein respectively. The levels of uPA and PAI-1 were correlated with tumour size, degree of anaplasia, steroid receptor status and number of positive nodes. Patients with high content of either uPA or PAI-1 had increased risk of relapse and death. We demonstrated an independent ability of PAI-1 to predict distant metastasis (relative risk 1.7, confidence limits 1.22 and 2.46) and that neither uPA nor PAI-1 provided any information regarding local recurrence.

Knoop, Ann; Andreasen, Peter A

1998-01-01

62

Recombinant tissue plasminogen activator in the treatment of suprachoroidal hemorrhage  

Directory of Open Access Journals (Sweden)

Full Text Available Nancy Kunjukunju1, Christine R Gonzales2, William S Rodden21Ochsner Medical Center, New Orleans, Louisiana; 2Retina and Vitreous Center of Southern Oregon, Ashland, Oregon, USABackground: Suprachoroidal hemorrhages are a vision-threatening complication, and poor visual outcome is correlated with increasing hemorrhage complexity. The recommended time of surgical drainage is 10–14 days after the hemorrhage begins to liquefy. We describe a case in which recombinant tissue plasminogen activator (r-tPA, alteplase, is injected within the suprachoroidal space before surgery to assist in the drainage of an organized clot prior to liquefaction. This is a report of a technique in which r-tPA is used in the intrachoroidal space to target the organized clot of suprachoroidal hemorrhage prior to drainage.Case report: A 62-year-old male presented 12 days after retinal detachment repair with sudden ocular pain and vision loss after a Valsalva maneuver. Vision was light perception only, and intraocular pressure was 43 mmHg. Diagnosed with hyphema and suprachoroidal hemorrhage, the patient underwent surgery the following day. An injection of r-tPA 100 µg was given intracamerally, and an additional dose of r-tPA 100 µg was injected into the suprachoroidal space prior to surgery. Liquified by r-tPA, the clot was expressed through the sclerotomies. Best corrected vision in the eye eight months after the drainage procedure was 20/40.Conclusion: To the author’s knowledge, this is the first reported case in which r-tPA was successfully injected in the suprachoroidal space to liquefy and drain a suprachoroidal hemorrhage prior to natural dissolution.Keywords: tPA, suprachoroidal hemorrhage, vision loss

Nancy Kunjukunju

2011-02-01

63

Plasminogen activator inhibitor-1 in the evolution of stroke  

Directory of Open Access Journals (Sweden)

Full Text Available Fibrinolytic activity in the acute stroke was examined by monitoring the level of plasminogen activator inhibitor-1 (PAI-1, as one of the indicators of fibrinolytic activity. Given the role of PAI-1 in the processes of atherogenesis and thrombogenesis, plasma PAI-1 level was measured in 59 patients (up to 50 years of age with atherothrombotic stroke (verified by computed tomography scanning or magnetic resonance imaging of brain in the period from 12 to 24 hours (I analysis and 30 days after the onset of stroke (II analysis; then, it was correlated with plasma PAI-1 level in the control group (57 healthy subjects, which was 2.86±0.70 U/ml. It was found that PAI-1 level was significantly higher in the acute stroke (I analysis: PAI-1 =4.10±1.40 U/ml, p<0.001; II analysis: PAI-1 =3.64+0.90 U/ml, p<0.001, while fibrinolytic activity was lower, especially on the first day from the stroke that was not completely increased even after 30 days. There was no difference in PAI-1 levels between the subgroups of patients with infarction and lacunar cerebral ischemia (p>0.05, as well as between females and males (p>0.05. Along with significantly increased fibrinogen level (4.65±1 g/l, in the controls - 2.83±0.64 g/l, p<0.001, significantly higher triglycerides (2.04±0.76 mmol/l, in the controls - 1.38+0.54 mmol/l, p<0.001 and lipoproteins(a (0.405±0.29 g/l, in the controls -0.172±0.14 g/l, p<0.001 were found, correlating with higher plasma PAI-1 level in these patients. The increased plasma level of PAI-1 pointed to possibility of decreased fibrinolytic activity in pathogenesis of ischemie stroke, as well as, risk of reinsult, which had been the greatest after the onset of stroke and declined gradually within several weeks.

Jovanovi? Zagorka B.

2004-01-01

64

Soya protein hydrolysates modify the expression of various pro-inflammatory genes induced by fatty acids in ovine phagocytes.  

Science.gov (United States)

The objective of the present study was to test the hypothesis that fatty acids are the circulating mediators acting in a pro-inflammatory manner towards activated circulating ovine monocyte/macrophages and neutrophils. Furthermore, whether soya protein hydrolysates (SPH) inhibit the fatty acid-induced increase in the production of pro-inflammatory responses by ovine phagocytes was tested in vitro. All the fatty acids tested (myristic, palmitic, palmitoleic, stearic and oleic) increased (PC16>C14) membrane-bound urokinase plasminogen activator (u-PA) and u-PA free binding sites in cell membranes of activated ovine blood monocytes/macrophages, but only the C18 fatty acids (stearic, oleic) were effective towards blood neutrophils. The C18 fatty acids up-regulated (Preceptor, intercellular adhesion molecule 1 and inducible NO synthase (in monocytes) but not that of cyclo-oxygenase-2, integrin ? X and plasminogen activator inhibitor types 1 and 2 by ovine phagocytes. SPH blocked completely or partially all C18 fatty acid-induced changes in the expression of various pro-inflammatory genes. In conclusion, fatty acids selectively 'activate' ovine phagocytes, suggesting that these cells 'sense' metabolic signals derived from adipocytes. Soya protein peptides inhibit all changes in gene expression induced by fatty acids in ovine phagocytes in vitro. This constitutes a novel mechanism of action. PMID:22172314

Politis, Ioannis; Theodorou, Georgios; Lampidonis, Antonios D; Chronopoulou, Roubini; Baldi, Antonella

2012-10-01

65

Plasminogen activator inhibitor-1 (PAI-1 and urokinase plasminogen activator (uPA in sputum of allergic asthma patients.  

Directory of Open Access Journals (Sweden)

Full Text Available Urokinase plasminogen activator (uPA and its inhibitor (PAI-1 have been associated with asthma. The aim of this study was to evaluate concentration of uPA and PAI-1 in induced sputum of house dust mite allergic asthmatics (HDM-AAs. The study was performed on 19 HDM-AAs and 8 healthy nonatopic controls (HCs. Concentration of uPA and PAI-1 was evaluated in induced sputum supernatants using ELISA method. In HDM-AAs the median sputum concentration of uPA (128 pg/ml; 95% CI 99 to 183 pg/ml and PAI-1 (4063 pg/ml; 95%CI 3319 to 4784 pg/ml were significantly greater than in HCs (17 pg/ml; 95%CI 12 to 32 pg/ml; p<0.001 and 626 pg/ml; 95%CI 357 to 961 pg/ml; p<0.001 for uPA and PAI-1 respectively. The sputum concentration of uPA correlated with sputum total cell count (r=0.781; p=0.0001 and with logarithmically transformed exhaled nitric oxide concentration (eNO (r=0.486; p=0.035 but not with FEV1 or bronchial reactivity to histamine. On the contrary, the sputum PAI-1 concentration correlated with FEV1 (r=-0,718; p=0.0005 and bronchial reactivity to histamine expressed as log(PC20 (r=-0.824; p<0.0001 but did not correlate with sputum total cell count or eNO. The results of this study support previous observations linking PAI-1 with airway remodeling and uPA with cellular inflammation. Moreover, the observed effect of uPA seems to be independent of its fibrynolytic activity.

Sebastian Zukowski

2008-06-01

66

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

Science.gov (United States)

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 < .0001). Urokinase activity was detectable in macroalbuminuric urine, with a tendency toward reduction in activity after amiloride 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. PMID:25492830

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

67

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

OpenAIRE

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 breas...

Hill Peter A; Morgan Hayley

2005-01-01

68

Identification of a New Exosite Involved in Catalytic Turnover by the Streptokinase-Plasmin Activator Complex during Human Plasminogen Activation*  

OpenAIRE

With the goal of identifying hitherto unknown surface exosites of streptokinase involved in substrate human plasminogen recognition and catalytic turnover, synthetic peptides encompassing the 170 loop (CQFTPLNPDDDFRPGLKDTKLLC) in the ?-domain were tested for selective inhibition of substrate human plasminogen activation by the streptokinase-plasmin activator complex. Although a disulfide-constrained peptide exhibited strong inhibition, a linear peptide with the same sequence, or a disulfide-...

Aneja, Rachna; Datt, Manish; Singh, Balwinder; Kumar, Shekhar; Sahni, Girish

2009-01-01

69

Design of a Standard Iranian Protocol of Intravenous Thrombolysis with Tissue Plasminogen Activator: A National Project  

Directory of Open Access Journals (Sweden)

Full Text Available Standard protocols should be established for treating eligible stroke patients with tissue plasminogen activator (TPA (recommendation class I, level of evidence B. The Iranian standard protocol of intravenous thrombolysis with recombinant tissue plasminogen activator (IVTTPA is the best possible and easy to use method for performing intravenous thrombolysis in Iran. This protocol overcomes problems and limitations of IVTTPA in Iran. The protocol achieves the best selection criteria and assessment method of IVTTPA for our residents and neurologists. This protocol was provided in Persian language and could be easily downloaded from Google site by writing Thrombolysis and Iran in Persian.

Kavian Ghandehari

2013-04-01

70

Relationships between activators and inhibitors of plasminogen, and the progression of small abdominal aortic aneurysms  

DEFF Research Database (Denmark)

plasmin is a common activator of the known proteolytic systems involved in the aneurysmal degradation, and is reported to be associated with the expansion of abdominal aortic aneurysms (AAA). The aim of this study was to study the activating pathways of plasminogen as predictors of the progression of AAA.

Lindholt, Jes Sanddal; JØrgensen, B

2003-01-01

71

Seahorse-derived peptide suppresses invasive migration of HT1080 fibrosarcoma cells by competing with intracellular ?-enolase for plasminogen binding and inhibiting uPA-mediated activation of plasminogen.  

Science.gov (United States)

?-Enolase is a glycolytic enzyme and a surface receptor for plasminogen. ?-Enolase-bound plasminogen promotes tumor cell invasion and cancer metastasis by activating plasmin and consequently degrading the extracellular matrix degradation. Therefore, ?-enolase and plasminogen are novel targets for cancer therapy. We found that the amino acid sequence of a peptide purified from enzymatic hydrolysates of seahorse has striking similarities to that of ?-enolase. In this study, we report that this peptide competes with cellular ?-enolase for plasminogen binding and suppresses urokinase plasminogen activator (uPA)-mediated activation of plasminogen, which results in decreased invasive migration of HT1080 fibrosarcoma cells. In addition, the peptide treatment decreased the expression levels of uPA compared to that of untreated controls. These results provide new insight into the mechanism by which the seahorse-derived peptide suppresses invasive properties of human cancer cells. Our findings suggest that this peptide could emerge as a potential therapeutic agent for cancer. PMID:24602611

Kim, Yong-Tae; Kim, Se-kwon; Jeon, You-Jin; Park, Sun Joo

2014-12-01

72

Phenotypic overlap between MMP-13 and the plasminogen activation system during wound healing in mice  

DEFF Research Database (Denmark)

Proteolytic degradation of extracellular matrix is a crucial step in the healing of incisional skin wounds. Thus, healing of skin wounds is delayed by either plasminogen-deficiency or by treatment with the broad-spectrum metalloproteinase (MP) inhibitor Galardin alone, while the two perturbations combined completely prevent wound healing. Both urokinase-type plasminogen activator and several matrix metallo proteinases (MMPs), such as MMP-3, -9 and -13, are expressed in the leading-edge keratinocytes of skin wounds, which may account for this phenotypic overlap between these classes of proteases.

Juncker-Jensen, Anna; Lund, Leif R

2011-01-01

73

Plasminogen activator inhibitor type 1 regulates microglial motility and phagocytic activity  

Directory of Open Access Journals (Sweden)

Full Text Available Abstract Background Plasminogen activator inhibitor type 1 (PAI-1 is the primary inhibitor of urokinase type plasminogen activators (uPA and tissue type plasminogen activators (tPA, which mediate fibrinolysis. PAI-1 is also involved in the innate immunity by regulating cell migration and phagocytosis. However, little is known about the role of PAI-1 in the central nervous system. Methods In this study, we identified PAI-1 in the culture medium of mouse mixed glial cells by liquid chromatography and tandem mass spectrometry. Secretion of PAI-1 from glial cultures was detected by ELISA and western blotting analysis. Cell migration was evaluated by in vitro scratch-wound healing assay or Boyden chamber assay and an in vivo stab wound injury model. Phagocytic activity was measured by uptake of zymosan particles. Results The levels of PAI-1 mRNA and protein expression were increased by lipopolysaccharide and interferon-? stimulation in both microglia and astrocytes. PAI-1 promoted the migration of microglial cells in culture via the low-density lipoprotein receptor-related protein (LRP 1/Janus kinase (JAK/signal transducer and activator of transcription (STAT1 axis. PAI-1 also increased microglial migration in vivo when injected into mouse brain. PAI-1-mediated microglial migration was independent of protease inhibition, because an R346A mutant of PAI-1 with impaired PA inhibitory activity also promoted microglial migration. Moreover, PAI-1 was able to modulate microglial phagocytic activity. PAI-1 inhibited microglial engulfment of zymosan particles in a vitronectin- and Toll-like receptor 2/6-dependent manner. Conclusion Our results indicate that glia-derived PAI-1 may regulate microglial migration and phagocytosis in an autocrine or paracrine manner. This may have important implications in the regulation of brain microglial activities in health and disease.

Jeon Hyejin

2012-06-01

74

Relationship between plasminogen activator inhibitor type-1 (PAI-1) gene polymorphisms and osteoporosis in Turkish women  

Science.gov (United States)

OBJECTIVE: The development of osteoporosis is associated with several risk factors, such as genetic structures that affect bone turnover and bone mass. The impact of genetic structures on osteoporosis is not known. Plasminogen activator inhibitor type-1 regulates the bone matrix and bone balance. This study assessed the correlation between plasminogen activator inhibitor type-1 gene 4G/5G polymorphisms and osteoporosis in a population of Turkish women. METHODS: A total of 195 postmenopausal female patients who were diagnosed with osteoporosis (Group I) based on bone mineral density measurements via dual-energy x-ray absorptiometry and 90 females with no osteoporosis (Group II) were included in this study. Correlations between PAI-1 gene 4G/5G polymorphisms and osteoporosis were investigated through the identification of PAI-1 gene 4G/5G polymorphism genotypes using the polymerase chain reaction. RESULTS: No significant differences in the genotype and allele frequency of 4G/5G plasminogen activator inhibitor type-1 polymorphisms were observed between the two groups, and both groups exhibited the most frequently observed 4G5G genotype. CONCLUSION: No correlation between the development of osteoporosis in the female Turkish population and 4G/5G plasminogen activator inhibitor type-1 gene polymorphisms was observed. PMID:23184207

Ozgen, Merih; Cosan, Didem Turgut; Doganer, Fulya; Soyocak, Ahu; Armagan, Onur; Gunes, Hasan Veysi; Degirmenci, Irfan; Ozkara, Gulsah Ogutler; Mutlu, Fezan Sahin

2012-01-01

75

Relationship between plasminogen activator inhibitor type-1 (PAI-1 gene polymorphisms and osteoporosis in Turkish women  

Directory of Open Access Journals (Sweden)

Full Text Available OBJECTIVE: The development of osteoporosis is associated with several risk factors, such as genetic structures that affect bone turnover and bone mass. The impact of genetic structures on osteoporosis is not known. Plasminogen activator inhibitor type-1 regulates the bone matrix and bone balance. This study assessed the correlation between plasminogen activator inhibitor type-1 gene 4G/5G polymorphisms and osteoporosis in a population of Turkish women. METHODS: A total of 195 postmenopausal female patients who were diagnosed with osteoporosis (Group I based on bone mineral density measurements via dual-energy x-ray absorptiometry and 90 females with no osteoporosis (Group II were included in this study. Correlations between PAI-1 gene 4G/5G polymorphisms and osteoporosis were investigated through the identification of PAI-1 gene 4G/5G polymorphism genotypes using the polymerase chain reaction. RESULTS: No significant differences in the genotype and allele frequency of 4G/5G plasminogen activator inhibitor type-1 polymorphisms were observed between the two groups, and both groups exhibited the most frequently observed 4G5G genotype. CONCLUSION: No correlation between the development of osteoporosis in the female Turkish population and 4G/5G plasminogen activator inhibitor type-1 gene polymorphisms was observed.

Merih Ozgen

2012-11-01

76

Relationship between plasminogen activator inhibitor type-1 (PAI-1) gene polymorphisms and osteoporosis in Turkish women  

Scientific Electronic Library Online (English)

Full Text Available SciELO Brazil | Language: English Abstract in english OBJECTIVE: The development of osteoporosis is associated with several risk factors, such as genetic structures that affect bone turnover and bone mass. The impact of genetic structures on osteoporosis is not known. Plasminogen activator inhibitor type-1 regulates the bone matrix and bone balance. Th [...] is study assessed the correlation between plasminogen activator inhibitor type-1 gene 4G/5G polymorphisms and osteoporosis in a population of Turkish women. METHODS: A total of 195 postmenopausal female patients who were diagnosed with osteoporosis (Group I) based on bone mineral density measurements via dual-energy x-ray absorptiometry and 90 females with no osteoporosis (Group II) were included in this study. Correlations between PAI-1 gene 4G/5G polymorphisms and osteoporosis were investigated through the identification of PAI-1 gene 4G/5G polymorphism genotypes using the polymerase chain reaction. RESULTS: No significant differences in the genotype and allele frequency of 4G/5G plasminogen activator inhibitor type-1 polymorphisms were observed between the two groups, and both groups exhibited the most frequently observed 4G5G genotype. CONCLUSION: No correlation between the development of osteoporosis in the female Turkish population and 4G/5G plasminogen activator inhibitor type-1 gene polymorphisms was observed.

Merih, Ozgen; Didem Turgut, Cosan; Fulya, Doganer; Ahu, Soyocak; Onur, Armagan; Hasan Veysi, Gunes; Irfan, Degirmenci; Gulsah Ogutler, Ozkara; Fezan Sahin, Mutlu.

1299-13-01

77

Vasculitis complicating treatment with intravenous anisoylated plasminogen streptokinase activator complex in acute myocardial infarction.  

OpenAIRE

Vasculitis developed in six of 253 patients treated with intravenous anisoylated plasminogen streptokinase activator complex (APSAC) after acute myocardial infarction. All patients recovered spontaneously with no evidence of renal impairment and no long term sequelae. Although leucocytoclastic vasculitis and serum sickness have been reported after streptokinase treatment, such allergic reactions have not been described as a complication of other thrombolytic agents.

Gemmill, J. D.; Sandler, M.; Hillis, W. S.; Tillman, J.; Wakeel, R.

1988-01-01

78

t-plasminogen activator inhibitor-1 polymorphism in idiopathic pulmonary arterial hypertension  

Directory of Open Access Journals (Sweden)

Full Text Available Aim: The aim of the present study was to identify the possible genotypic association of 3?UTR Hind III polymorphism of Plasminogen activator Inhibitor-1 (PAI-1 gene with idiopathic pulmonary arterial hypertension (IPAH. Background: IPAH is a disorder with abnormally raised mean pulmonary arterial pressure and increase in the resistance to blood flow in pulmonary artery. One of the pathological features seen is development of intraluminal thrombin deposition leading to thrombosis. Plasminogen activator inhibitor-1 is an important inhibitor of the fibrinolytic system; its up-regulation may suppress fibrinolysis and result in an increased risk of thrombosis. Method: Blood samples from 54 IPAH patients and 100 healthy voluntary donors were analyzed by PCR-RFLP method for 3?UTR Hind III polymorphism. Results and Disscussion: A significant association of Hd2 allele with the disease was observed. Raised mean level of right ventricular systolic pressure was observed in the Hd2/Hd2 genotypic patients, strengthening the role of Hd2 allele in the disease progression. Our data suggests an association of Hd2/Hd2 genotype, which may lead to the up-regulation of PAI-1 gene leading to increased levels of PAI-1, which is seen in IPAH. PAI-1 competes with plasminogen activators and hinders the normal mechanism of plasminogen activation system and leads to thrombosis and formation of plexiform lesions in the lung tissue, further strengthening its role in tissue remodeling and disease progression.

Katta Sujana

2008-01-01

79

Temperature-Induced Changes in the Lipopolysaccharide of Yersinia pestis Affect Plasminogen Activation by the Pla Surface Protease?  

OpenAIRE

The Pla surface protease of Yersinia pestis activates human plasminogen and is a central virulence factor in bubonic and pneumonic plague. Pla is a transmembrane ?-barrel protein and member of the omptin family of outer membrane proteases which require bound lipopolysaccharide (LPS) to be proteolytically active. Plasminogen activation and autoprocessing of Pla were dramatically higher in Y. pestis cells grown at 37°C than in cells grown at 20°C; the difference in enzymatic activity by far ...

Suomalainen, Marjo; Lobo, Leandro Araujo; Brandenburg, Klaus; Lindner, Buko; Virkola, Ritva; Knirel, Yuriy A.; Anisimov, Andrey P.; Holst, Otto; Korhonen, Timo K.

2010-01-01

80

An enzyme-immunobinding assay for fast screening of expression of tissue plasminogen activator cDNA in E. coli  

International Nuclear Information System (INIS)

Tissue plasminogen activator (TPA) has been isolated from normal human tissues and certain human cell lines in culture. The enzyme is a serine protease which converts an inactive zymogen, plasminogen to plasmin, and causes lysis of fibrin clots. The high affinity of TPA for fibrin indicates that it is a potential thrombolytic agent and is superior to urokinase-like plasminogen activators. Recently, TPA has been cloned and expressed in E. coli. Using TPA as a model protein, the authors report here the development of a direct, sensitive enzyme-immunoassay for the screening of a cDNA expression library using specific antibodies and peroxidase-labeled second antibody

81

Plasminogen activator inhibitor-1, free fatty acids, and insulin resistance in patients with myocardial infarction  

Directory of Open Access Journals (Sweden)

Full Text Available Olga Gruzdeva, Evgenya Uchasova, Yulia Dyleva, Ekaterina Belik, Ekaterina Shurygina, Olga Barbarash Research Institute for Complex Issues of Cardiovascular Diseases under the Siberian Branch of the Russian Academy of Medical Sciences, Kemerovo, Russian Federation Background: Insulin resistance is known to be a common feature of type 2 diabetes mellitus and is regarded as an important mechanism in the pathogenesis of this disease. The key pathogenetic mechanisms of insulin resistance progression are free fatty acids metabolism impairment and enhanced activity of plasminogen activator inhibitor 1. Both free fatty acids and plasminogen activator inhibitor 1 are recognized as risk factors for coronary heart disease. Methods: The patients were divided into two groups: group 1 included 65 non-diabetic myocardial infarction patients and group 2 enrolled 60 diabetic myocardial infarction patients. The control group consisted of 30 sex- and age-matched volunteers. The concentration of serum free fatty acids, glucose, C-peptide, and insulin were measured on the 1st and 12th days of the study. All the patients had their postprandial glycemia, insulin, and C-peptide concentrations measured 2 hours after a standard carbohydrate breakfast containing 360 kcal (protein 20 g, carbohydrate 57 g, and fat 9 g. Results: Free fatty acids levels in group 1 and in group 2 exceeded the control group values by 7-fold and 11-fold, respectively. Plasminogen activator inhibitor 1 concentration was 2.5-fold higher in group 1 and 4.6-fold higher in group 2 compared to the control group on the 1st day from the myocardial infarction onset. In addition, plasminogen activator inhibitor 1 concentration was significantly reduced in both groups on the 12th day from the myocardial infarction onset; however, it did not achieve the control group values. Conclusion: Increased postprandial glucose level, insulinemia, and elevated levels of free fatty acids and plasminogen activator inhibitor are associated with myocardial infarction-associated progression of insulin resistance. However, insulin resistance metabolic markers are of great predictive capacity in the assessment of risk of acute coronary events. Keywords: free fatty acids, type 2 diabetes mellitus, myocardial infarction, insulin resistance, plasminogen activator inhibitor 1

Gruzdeva O

2013-08-01

82

Studies on the relationship of plasminogen activator inhibitor-1 for venous thromboembolism  

OpenAIRE

Venous thromboembolism (VTE) is a chronic multifactorial disease with expositional and dispositional risk factors. The established risk factors include deficiency of antithrombin, protein C and S, the factor V Leiden and the prothrombin G20210A variant. The plasminogen activator inhibitor-1 (PAI-1) 4G/5G polymorphism and its corresponding plasma levels (PAI-1 activity) are among the more controversially discussed risk factors for VTE. A meta-analysis from 2008 suggested that at least a weak a...

Bukreeva, Larisa

2013-01-01

83

A nonantigenic covalent streptokinase-polyethylene glycol complex with plasminogen activator function.  

OpenAIRE

A series of new, covalent polyethylene glycol (PEG)-streptokinase adducts were synthesized and characterized. PEGs with average molecular weights of 2,000, 4,000, and 5,000 were activated with carbonyldiimidazole and coupled to the protein under standardized reaction conditions. Steady-state kinetic analysis demonstrated comparable Km values for the activation of plasminogen by streptokinase, PEG-2-streptokinase, and PEG-4-streptokinase. The kcat values were somewhat decreased when PEG-2 or P...

Rajagopalan, S.; Gonias, S. L.; Pizzo, S. V.

1985-01-01

84

Plasminogen activator inhibitor type 2 : a unique serpin with two mobile loops  

OpenAIRE

The superfamily of serine protease inhibitors (serpins) is a large group of proteins with diverse functions but a common tertiary structure. Active serpins are highly metastable molecules. Metastability is the property underlying the success and ubiquitousness of serpins. However, serpin metastability also accounts for improper conformational changes in serpin mutants which may result in pathological serpin polymerization. Plasminogen activator inhibitor type 2 (PAI-2) is a member of the subf...

Lobov, Sergei

2004-01-01

85

The Plasminogen Activation System Modulates Differently Adipogenesis and Myogenesis of Embryonic Stem Cells  

OpenAIRE

Regulation of the extracellular matrix (ECM) plays an important functional role either in physiological or pathological conditions. The plasminogen activation (PA) system, comprising the uPA and tPA proteases and their inhibitor PAI-1, is one of the main suppliers of extracellular proteolytic activity contributing to tissue remodeling. Although its function in development is well documented, its precise role in mouse embryonic stem cell (ESC) differentiation in vitro is unknown. We found that...

Hadadeh, Ola; Barruet, Emilie; Peiretti, Franck; Verdier, Monique; Bernot, Denis; Hadjal, Yasmine; Yazidi, Claire El; Robaglia-schlupp, Andre?e; Paula, Andre Maues; Ne?gre, Didier; Iacovino, Michelina; Kyba, Michael; Alessi, Marie-christine; Bine?truy, Bernard

2012-01-01

86

Technetium-99m-labeled recombinant tissue plasminogen activator for the imaging of emboli in vivo  

Energy Technology Data Exchange (ETDEWEB)

Tissue-type plasminogen activator (t-PA) effectively lyses activate thrombus by direct action. Recombinant t-PA (rt-PA) was labeled with technetium-99m ([sup 99m]Tc) to investigate the in vivo binding to fibrin clots in a feline cerebral embolism model created by insertion of an artificial fibrin clot within the carotid artery. [sup 99m]Tc-rt-PA administered intravenously provided clearer imaging of clots after priming with cold rt-PA, with uptake peaking 5-10 minutes after the injection. [sup 99m]Tc-labeled human serum albumin was not retained at clot sites. Systemically administered [sup 99m]Tc-rt-PA binds to fibrin clots within carotid arteries in our feline model. Our results suggest that the interaction of intrinsic plasminogen activator inhibitors with extrinsically administered rt-PA may regulate the demonstration of a clot, although the precise mechanism is unclear. (author).

Takahashi, Akihiro; Itoh, Kazuo; Tsukamoto, Eriko; Furudate, Masayori; Kamiyama, Hiroyasu; Abe, Hiroshi (Hokkaido Univ., Sapporo (Japan). School of Medicine)

1993-07-01

87

Technetium-99m-labeled recombinant tissue plasminogen activator for the imaging of emboli in vivo  

International Nuclear Information System (INIS)

Tissue-type plasminogen activator (t-PA) effectively lyses activate thrombus by direct action. Recombinant t-PA (rt-PA) was labeled with technetium-99m (99mTc) to investigate the in vivo binding to fibrin clots in a feline cerebral embolism model created by insertion of an artificial fibrin clot within the carotid artery. 99mTc-rt-PA administered intravenously provided clearer imaging of clots after priming with cold rt-PA, with uptake peaking 5-10 minutes after the injection. 99mTc-labeled human serum albumin was not retained at clot sites. Systemically administered 99mTc-rt-PA binds to fibrin clots within carotid arteries in our feline model. Our results suggest that the interaction of intrinsic plasminogen activator inhibitors with extrinsically administered rt-PA may regulate the demonstration of a clot, although the precise mechanism is unclear. (author)

88

Expression of the truncated tissue plasminogen activator (K2S) gene in tobacco chloroplast.  

Science.gov (United States)

As because the plant plastid genome is highly polyploid, the transformation of chloroplasts permits the introduction of thousands of copies of foreign genes per plant cell and generates extraordinarily high levels of recombinant protein. Human tissue-type plasminogen activator is one of the most important pharmaceutical proteins involved in the breakdown of blood clots in brain and heart blood vessels. We report the introduction and expression of the truncated human tissue plasminogen activator (K2S) gene in tobacco chloroplasts. The K2S-containing vector pKCZK2S was successfully transferred to tobacco plastomes using the biolistic delivery procedure. Transplastomic plants were selected on RMOP medium containing spectinomycin (500 mg/l). In order to achieve homoplasmy, several rounds of selection and regeneration were performed. The presence, site-specific integration, homoplasmy, expression and activity assay of the transgene were confirmed in the transplastomic plants by PCR, Southern-blot, RT-PCR, SDS-PAGE, ELISA, Dot-blot, Western-blot and zymography analysis. Our results show that the tissue plasminogen activator (K2S form) protein to be expressed in tobacco chloroplasts in active form. PMID:24114696

Abdoli-Nasab, Maryam; Jalali-Javaran, Mokhtar; Cusidó, Rosa M; Palazón, Javier; Baghizadeh, Amin; Alizadeh, Houshang

2013-10-01

89

A Key Role for the Urokinase Plasminogen Activator (uPA) in Invasive Group A Streptococcal Infection  

OpenAIRE

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 recr...

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

90

Purification and activation of caprine and canine plasminogens: Comparison with human plasminogen / Purificación y activación de los plasminógenos caprino y canino: comparación con el plasminógeno humano  

Scientific Electronic Library Online (English)

Full Text Available SciELO Colombia | Language: English Abstract in spanish Objetivo: unificar la purificación y activación de los plasminógenos de tres especies diferentes, a saber: humana, caprina y canina. Materiales y métodos: se usaron Lysina-Sefarosa 4B y Sefacel DEAE para las cromatografías de afinidad y de intercambio iónico, respectivamente. Se determinó la secuenc [...] ia terminal-N tanto de los plasminógenos intactos como de los degradados. Resultados: en las tres especies se identificaron bandas de 92 kDa correspondientes a los plasminógenos nativos. Se halló que sus secuencias terminales-N eran EPLDDY, DPLDDY y XXLDDY para los plasminógenos humano, caprino y canino, respectivamente. Además, se purificaron los plasminógenos degradados circulantes, cuyas secuencias terminales-N fueron, en el mismo orden, KVYLSE, RITLL Y RIYSL. Conclusión: la activación de los tres plasminógenos confirmó la formación de las bandas electroforéticas típicas de la plasmina humana correspondientes a las cadenas pesada A y liviana B, que también se identificaron en las plasminas caprina y canina. Este nuevo método de purificación facilita la comparación y el esclarecimiento de los sistemas fibrinolíticos de los mamíferos. Abstract in english Objective: To unify the purification and activation of plasminogens from three different species, namely: human, caprine and canine. Materials and methods: Lysine-Sepharose 4B and sephacel DEAE were used, for affinity and ion-exchange chromatography, respectively. The N-terminal sequence was determi [...] ned for both the intact and degraded plasminogens. Results: Bands of 92 kDa corresponding to native plasminogens were identified in the three species. Their N-terminal sequences were found to be EPLDDY, DPLDDY and XXLDDY for human, caprine and canine plasminogen, respectively. Furthermore, the degraded in vivo circulating plasminogens from the three species were purified and their N-terminal sequences were KVYLSE, RITLL and RIYLS for the human, caprine and canine, in that order. Conclusion: Activation of the three plasminogens confirmed the formation of the typical electrophoretic bands for human plasmin corresponding to the heavy A and the light B chains which were also identified in the caprine and canine plasmins. This new purification methodology facilitates the comparison and further elucidation of the fibrinolytic systems in mammals.

Omaira, Cañas Bermúdez; Alfonso, Quijano Parra; Luis Fernando, Arbeláez Ramírez.

2011-06-01

91

The role of plasminogen activator inhibitor-1 polymorphism, factor-V-Leiden, and prothrombin-20210 mutations in pulmonary thromboembolism.  

Science.gov (United States)

Polymorphism in plasminogen activator inhibitor-1 gene is suggested to be associated with an increased risk of venous thromboembolism. The aim of this study was to investigate the association of plasminogen activator inhibitor-1 gene polymorphism and its coexistence with factor-V-Leiden and prothrombin-20210 mutations in pulmonary thromboembolism. The authors investigated plasminogen activator inhibitor-1 4G/5G polymorphism, factor-V-Leiden, and prothrombin-20210 mutations in 143 pulmonary thromboembolism patients and 181 controls. Plasminogen activator inhibitor-1 4G/4G, 4G/5G, and 5G/5G gene polymorphisms and prothrombin-20210 mutations were not different between cases and controls. Factor-V-Leiden mutation was present in 21.0% and 7.7% of the cases and controls, respectively, P = .001. Neither different plasminogen activator inhibitor-1 genotypes and 4G allele nor coexistence of the allele with factor-V-Leiden or prothrombin-20210 was associated with the risk of recurrence. As a result, plasminogen activator inhibitor-1 gene polymorphism or its concomitant presence with mentioned mutations was not found to be associated with increased risk for pulmonary thromboembolism or recurrent disease in this study. PMID:18160588

Oguzulgen, I Kivilcim; Yilmaz, E; Demirtas, Senay; Erkekol, Ferda Oner; Ekim, Numan; Demir, Nalan; Numanoglu, Numan; Ozel, Duygu; Ulu, Arzu; Akar, Nejat

2009-02-01

92

Essential Role of Plasminogen Activator Inhibitor Type-1 in Radiation Enteropathy  

OpenAIRE

Intestinal radiation injury is a dose-limiting factor in radiation therapy for abdominal and pelvic cancers. Because transforming growth factor-?1 is a key mediator involved in radiation-induced damage, we hypothesized that its target gene, plasminogen activator inhibitor type 1 (PAI-1), is an essential mediator of intestinal radiation toxicity. In a model of radiation enteropathy, survival was monitored and intestinal radiation injury was assessed in both wild-type (Wt) and PAI-1 knockout m...

Milliat, Fabien; Sabourin, Jean-christophe; Tarlet, Georges; Holler, Valerie; Deutsch, Eric; Buard, Vale?rie; Tamarat, Radia; Atfi, Azeddine; Benderitter, Marc; Franc?ois, Agne?s

2008-01-01

93

The primary plasminogen-activator inhibitors in endothelial cells, platelets, serum, and plasma are immunologically related.  

OpenAIRE

Monospecific antiserum to an unusually stable Mr 50,000 plasminogen-activator inhibitor (PAI) purified from cultured bovine aortic endothelial cells was employed in conjunction with reverse fibrin autography to determine whether human platelets, serum, and plasma contain immunologically related inhibitors. Reverse fibrin autography revealed the presence of a Mr 50,000 inhibitor in the platelet and serum samples but not in normal plasma. However, a Mr 50,000 inhibitor was detected in plasma ob...

Erickson, L. A.; Hekman, C. M.; Loskutoff, D. J.

1985-01-01

94

Intravitreal injection of tissue plasminogen activator as treatment for an occluded pars plana glaucoma tube  

OpenAIRE

Implanting glaucoma tubes through the pars plana in the setting of a corneal transplant is becoming more common, and there are unique problems associated with such a procedure. A 42-year-old man with multiple previous eye surgeries presented with a nonfunctioning pars plana glaucoma tube. There was no view to the tube tip, but it was presumed to be clogged with fibrin. Intravitreal tissue plasminogen activator (tPA) was injected through the pars plana which resulted in intraocular pressure co...

Tsui, Irena; Airiani, Suzanna; Wen, Angie; El-sawy, Tarek; Fine, Howard F.; Maris, Peter Jg

2009-01-01

95

Treatment of acute myocardial infarction with anisoylated plasminogen streptokinase activator complex.  

OpenAIRE

A controlled trial in 149 patients admitted to a district hospital with probable myocardial infarction tested the effect of 30 units of anisoylated plasminogen streptokinase activator complex (APSAC) on indices of infarct size. Patients were grouped prospectively according to whether they entered the trial within two and a half hours (early entry) or between two and a half and four hours (late entry) after onset of the symptoms. Sixty seven of 73 patients in the control group showed increased...

Ikram, S.; Lewis, S.; Bucknall, C.; Sram, I.; Thomas, N.; Vincent, R.; Chamberlain, D.

1986-01-01

96

Tissue plasminogen activator (alteplase) treatment for femoral artery thrombosis after cardiac catheterisation in infants and children.  

OpenAIRE

OBJECTIVE--To determine the efficacy of fibrinolytic therapy with tissue plasminogen activator (alteplase) in infants and children with arterial thrombosis after cardiac catheterisation. DESIGN--Use of alteplase (Actilyse) in a protocol with prospective data collection. Alteplase was administered to infants and children with arterial thrombosis after cardiac catheterisation. A dose of 0.5 mg/kg/h was given continuously via a peripheral vein for the first hour followed by 0.25 mg/kg/h till clo...

Ries, M.; Singer, H.; Hofbeck, M.; Klinge, J.

1993-01-01

97

The roles of the plasminogen activator and matrix metalloproteinase systems in ovulation and corpus luteum formation  

OpenAIRE

Proteases of the plasminogen activator (PA) and the matrix metalloproteinase (MMP) enzyme systems are expressed in the ovulatory follicle and in the developing corpus luteum (CL). However, the functional role of these extracellular degrading protease systems in the ovulatory and CL development processes remains elusive. The first aim of this thesis was to develop a mouse model to study gonadotropin-induced CL formation. The second aim was to study the involvement of the PA and the MMP systems...

Bode?n, Ida

2004-01-01

98

A redox-sensitive loop regulates plasminogen activator inhibitor type 2 (PAI-2) polymerization  

OpenAIRE

Plasminogen activator inhibitor type 2 (PAI-2) is the only wild-type serpin that polymerizes spontaneously under physiological conditions. We show that PAI-2 loses its ability to polymerize following reduction of thiol groups, suggesting that an intramolecular disulfide bond is essential for the polymerization. A novel disulfide bond was identified between C79 (in the CD-loop) and C161 (at the bottom of helix F). Substitution mutants in which this disulfide bond was broken did not polymerize....

Wilczynska, Malgorzata; Lobov, Sergei; Ohlsson, Per-ingvar; Ny, Tor

2003-01-01

99

Inflammatory macrophage migration requires MMP-9 activation by plasminogen in mice  

OpenAIRE

Inflammation plays a critical role in the development of cardiovascular diseases. Infiltration of leukocytes to sites of injury requires their exit from the blood and migration across basement membrane; this process has been postulated to require remodeling of the ECM. Plasminogen (Plg) is a protease that binds to the ECM and, upon conversion to plasmin, degrades multiple ECM proteins. In addition, plasmin directly activates MMPs. Here, we used Plg–/– mice to investigate the role of Plg i...

Gong, Yanqing; Hart, Erika; Shchurin, Aleksey; Hoover-plow, Jane

2008-01-01

100

Tissue-Type Plasminogen Activator Regulates the Neuronal Uptake of Glucose in the Ischemic Brain  

OpenAIRE

The ability to sense and adapt to hypoxic conditions plays a pivotal role in neuronal survival. Hypoxia induces the release of tissue-type plasminogen activator (tPA) from cerebral cortical neurons. We found that the release of neuronal tPA or treatment with recombinant tPA (rtPA) promotes cell survival in cerebral cortical neurons previously exposed to hypoxic conditions in vitro or experimental cerebral ischemia in vivo. Our studies using liquid chromatography and tandem mass spectrometry r...

Wu, Fang; Wu, Jialing; Nicholson, Andrew D.; Echeverry, Ramiro; Haile, Woldeab B.; Catano, Marcela; An, Jie; Lee, Andrew K.; Duong, Duc; Dammer, Eric B.; Seyfried, Nicholas T.; Tong, Frank C.; Votaw, John R.; Medcalf, Robert; Yepes, Manuel

2012-01-01

101

Tryptophan Properties in Fluorescence and Functional Stability of Plasminogen Activator Inhibitor 1  

OpenAIRE

Plasminogen activator inhibitor 1 harbors four tryptophan residues at positions 86, 139, 175, and 262. To investigate the contribution of each tryptophan residue to the total fluorescence and to reveal the mutual interactions of the tryptophan residues and interactions with the other amino acids, 15 mutants in which tryptophan residues have been replaced by phenylalanines were constructed, purified, and characterized. Conformational distribution analysis revealed that the tryptophan mutants h...

Verheyden, Stefan; Sillen, Alain; Gils, Ann; Declerck, Paul J.; Engelborghs, Yves

2003-01-01

102

Intrapleural use of tissue plasminogen activator and DNase in pleural infection  

OpenAIRE

BACKGROUND: More than 30% of patients with pleural infection either die or require surgery. Drainage of infected fluid is key to successful treatment, but intrapleural fibrinolytic therapy did not improve outcomes in an earlier, large, randomized trial. METHODS: We conducted a blinded, 2-by-2 factorial trial in which 210 patients with pleural infection were randomly assigned to receive one of four study treatments for 3 days: double placebo, intrapleural tissue plasminogen activator (t-PA) an...

Rahman, Nm; Maskell, Na; West, A.; Teoh, R.; Arnold, A.; Mackinlay, C.; Peckham, D.; Davies, Cw; Ali, N.; Kinnear, W.; Bentley, A.; Kahan, Bc; Wrightson, Jm; Davies, He; Hooper, Ce

2011-01-01

103

Taming Neonatal Hypoxic-Ischemic Brain Injury by Intranasal Delivery of Plasminogen Activator Inhibitor-1  

Science.gov (United States)

Background and Purpose Plasminogen Activator Inhibitor-I (PAI-1), a ~50 kDa serine protease inhibitor, markedly reduces the extravascular toxicity of tissue-type plasminogen activator (tPA) in experimental hypoxic-ischemic (HI) brain injury of newborns. However, the current treatment with PAI-1 requires intracerebroventricle (ICV) injection to cross blood-brain-barrier (BBB), which is an invasive procedure of limited clinical potential. Thus, we tested whether intranasal administration of PAI-1 can bypass BBB and mitigate neonatal HI brain injury. Method Rat pups were subjected to HI, with or without lipopolysaccharide (LPS) pre-exposure, followed by intranasal delivery of a stable-mutant form of PAI-1 (CPAI). Results Immunoblotting showed that CPAI sequentially entered the olfactory bulbs and cerebral cortex following intranasal delivery and reduced ~75% of brain atrophy in HI or LPS-sensitized HI injury. Mechanistically, CPAI attenuated HI-induced plasminogen activators and LPS/HI-induced NF-?B signaling, neuroinflammation, and BBB permeability. Conclusions Intranasal delivery of CPAI is an effective treatment of experimental HI brain injury of newborns. Clinical application of this experimental therapy merits further investigation. PMID:23881953

Lin, Xiaoyi; Baumann, Jessica M.; Warnock, Mark; Lawrence, Daniel A.; Kuan, Chia-Yi

2014-01-01

104

Activators of plasminogen and the progression of small abdominal aortic aneurysms  

DEFF Research Database (Denmark)

The aim of this study was to examine the role of activating pathways of plasminogen in the natural history of abdominal aortic aneurysms (AAA). To fulfill this objective 70 male patients with small AAA (> 3 cm) were interviewed and examined. Their blood samples were taken at diagnosis. The patients were scanned annually for a minimum period of 1 year and a maximum of 5 years (mean 2.5 years), and referred for surgery if the AAA exceeded 5 cm in diameter. Plasma levels of urokinase-like plasminogen activator (uPA), tissue-type plasminogen activator (tPA), plasminogen-activator-inhibitor-1 (PAI-1), macrophage-inhibiting factor (MIF), transforming-growth-factor-beta1 (TGF-beta1), homocysteine, and serum levels of IgA-antibodies against Chlamydia pneumoniae (IgA-CP) and cotinine (a nicotine metabolite) were measured. The annual expansion rate correlated positively with tPA, IgA-CP, and S-cotinine; rho = 0.37 (P = 0.004), 0.28 (P = 0.01), and 0.24 (P = 0.04), while PAI-1, uPA, TGF-beta1, homocysteine, and MIF did not. S-cotinine and PAI-1 also correlated positively with tPA, rho = 0.24 (P = 0.04), and 0.33 (P = 0.005). IgA-CP did not correlate with tPA. By receiver operating characteristics (ROC) curve analysis, tPA showed to be predictive of cases expanding to above 5 cm within the first 5 years with an optimal sensitivity and specificity of 0.73 and 0.71, respectively (P = 0.015). The aortic matrix degradation in AAA may be partly caused by an activation of plasminogen by tPA, but not by uPA, which usually dominates matrix degradation. Smoking seems to be an important factor for this pathway, while the pathway of IgA-CP seems different.

Lindholt, Jes Sanddal

2006-01-01

105

Expression of Active Human Tissue-Type Plasminogen Activator in Escherichia coli  

Science.gov (United States)

The formation of native disulfide bonds in complex eukaryotic proteins expressed in Escherichia coli is extremely inefficient. Tissue plasminogen activator (tPA) is a very important thrombolytic agent with 17 disulfides, and despite numerous attempts, its expression in an active form in bacteria has not been reported. To achieve the production of active tPA in E. coli, we have investigated the effect of cooverexpressing native (DsbA and DsbC) or heterologous (rat and yeast protein disulfide isomerases) cysteine oxidoreductases in the bacterial periplasm. Coexpression of DsbC, an enzyme which catalyzes disulfide bond isomerization in the periplasm, was found to dramatically increase the formation of active tPA both in shake flasks and in fermentors. The active protein was purified with an overall yield of 25% by using three affinity steps with, in sequence, lysine-Sepharose, immobilized Erythrina caffra inhibitor, and Zn-Sepharose resins. After purification, approximately 180 ?g of tPA with a specific activity nearly identical to that of the authentic protein can be obtained per liter of culture in a high-cell-density fermentation. Thus, heterologous proteins as complex as tPA may be produced in an active form in bacteria in amounts suitable for structure-function studies. In addition, these results suggest the feasibility of commercial production of extremely complex proteins in E. coli without the need for in vitro refolding. PMID:9835579

Qiu, Ji; Swartz, James R.; Georgiou, George

1998-01-01

106

Construction and expression of a recombinant antibody-targeted plasminogen activator  

International Nuclear Information System (INIS)

Covalent linkage of tissue-type plasminogen activator (t-PA) to a monoclonal antibody specific for the fibrin ? chain (anti-fibrin 59D8) results in a thrombolytic agent that is more specific and more potent that t-PA alone. To provide a ready source of this hybrid molecule and to allow tailoring of the active moieties for optimal activity, the authors have engineered a recombinant version of the 59D8-t-PA conjugate. The rearranged 59D8 heavy chain gene was cloned and combined in the expression vector pSV2gpt with sequence coding for a portion of the ?2b constant region and the catalytic ? chain of t-PA. This construct was transfected into heavy chain loss variant cells derived form the 59D8 hybridoma. Recombinant protein was purified by affinity chromatography and analyzed with electrophoretic transfer blots and radioimmunoassay. These revealed a 65-kDa heavy chain-t-PA fusion protein that is secreted in association with the 59D8 light chain in the form of a 170-kDa disulfide-linked dimer. Chromogenic substrate assays showed the fusion protein to have 70% of the peptidolytic activity of native t-PA and to activate plasminogen as efficiently as t-PA. IN a competitive binding assay, reconstituted antibody was shown to have a binding profile similar to that of native 59D8. Thus, by recombinant techniques, they have produced a hybrid protein capable of high affinity fibrin binding and plasminogen activation

107

Fractionation of heparin by chromatography on a tissue plasminogen activator-Sepharose column.  

OpenAIRE

Heparin stimulates the activity of tissue plasminogen activator (t-PA) and binds to t-PA. To study this interaction, a complex between t-PA and N-acetylated heparin was formed and then linked to Sepharose. This procedure selectively links the t-PA to the column because the acetylated heparin has no free amino groups. The procedure also protects the heparin-binding site(s) on the enzyme during coupling to the matrix. The t-PA column separates heparin into two fractions, one with low affinity f...

Andrade-gordon, P.; Strickland, S.

1990-01-01

108

Targeting of Plasminogen Activator Inhibitor 1 Improves Fibrinolytic Therapy for Tetracycline Induced Pleural Injury in Rabbits.  

Science.gov (United States)

Introduction: Endogenous active plasminogen activator inhibitor 1 (PAI-1) was targeted in vivo with monoclonal antibodies (mAbs) that redirect its reaction with proteinases to the substrate branch. mAbs were used as an adjunct to prourokinase (scuPA) intrapleural fibrinolytic therapy (IPFT) of tetracycline-induced pleural injury in rabbits. Methods: Outcomes of scuPA IPFT (0.25 or 0.0625 mg/kg) with 0.5 mg/kg of mouse IgG or mAbs (MA-33H1F7 and MA-8H9D4) were assessed at 24h. Pleural fluid (PF) was collected at 0, 10, 20, 40 min and 24h after IPFT and analyzed for plasminogen activating (PA), urokinase (uPA), fibrinolytic activities, levels of total plasmin/plasminogen, ?-macroglobulin (?M), mAbs/IgG antigens, free active uPA and ?M/uPA complexes. Results:Anti-PAI-1 mAbs, but not mouse IgG, delivered with an 8-fold reduction in the minimal effective dose of scuPA (from 0.5 to 0.0625 mg/kg), improved the outcome of IPFT (p<0.05). mAbs and IgG were detectable in PFs at 24h. Compared to identical doses of scuPA alone or with IgG, treatment with scuPA and anti-PAI-1 mAbs generated higher PF uPA amidolytic and PA activities, faster formation of ?M/uPA complexes, and slower uPA inactivation. However, PAI-1 targeting did not significantly affect intrapleural fibrinolytic activity or levels of total plasmin/plasminogen and ?M antigens. Conclusions: Targeting PAI-1 did not induce bleeding and rendered otherwise ineffective doses of scuPA able to improve outcome in tetracycline-induced pleural injury. PAI-1 neutralizing mAbs improved IPFT by increasing the durability of intrapleural PA activity. These results suggest a novel, well-tolerated IPFT strategy that is tractable for clinical development. PMID:25140386

Florova, Galina; Azghani, Ali; Karandashova, Sophia; Schaefer, Chris; Koenig, Kathleen; Stewart-Evans, Kris; Declerck, Paul J; Idell, Steven; Komissarov, Andrey A

2014-08-20

109

Up-regulation of urokinase-type plasminogen activator in squamous cell carcinoma of human larynx.  

OpenAIRE

The expression of urokinase-type plasminogen activator (uPA) was investigated in squamous cell carcinoma of the human larynx. For this purpose, tissue extracts from 25 matched samples of normal mucosa and neoplastic larynx were compared for the levels of uPA activity as evaluated by a chromogenic PA assay and sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) zymography. Also, uPA antigen was quantified by enzyme-linked immunosorbent assay (ELISA) in 19 cases. The results d...

Parolini, S.; Flagiello, D.; Cinquetti, A.; Gozzi, R.; Cristini, S.; Cappiello, J.; Nicolai, P.; Rusnati, M.; Presta, M.; Tosatti, M. M.

1996-01-01

110

Microassay for the photometric quantitation of cell-associated plasminogen activator using a chromogenic tripeptide substrate.  

Science.gov (United States)

Plasminogen activator (PA), a neutral protease whose primary function is to convert plasminogen to plasmin, is produced by various cells including macrophages, monocytes, endothelial cells, and tumor cells. This study reports the use of the chromogenic tripeptide substrate D-Val-Leu-Lys-p-nitroanilide (S-2251) and an automated microtiter plate reader spectrophotometer for the determination of PA activity in cells and fluids. There was a linear relationship between the time of incubation at 37 degrees C and the square root of the absorbance measured at 405 nm when urokinase was incubated with the substrate in the presence of plasminogen. There was no activity in the absence of plasminogen. The slopes of the lines (square root A 405/time) were directly related to the concentrations of urokinase, up through 0.05 CTA units. Using this assay, we determined the cellular activity of PA in human promyelocytic cells HL-60 (1.33 +/- 0.12 CTA units/mg), human monocytoid cells U937 (1.27 +/- 0.12 CTA units/mg), mouse myeloid leukemia cells RFM/UN (0.70 +/- 0.07 CTA units/mg), freshly isolated normal human monocytes (0.00 +/- 0.00 CTA units/mg), and human monocytes after 7 days in culture (5.66 +/- 0.38 CTA units/mg). There was a variable amount of activity expressed in freshly isolated cells or cell lysates of peritoneal macrophages from normal mice, or mice that had gotten intraperitoneal injections of peptone, thioglycollate, or NaIO4, but after 24 or 48 h of culture, these activities, in general, increased. Using this assay, PA levels in the euglobulin precipitates from human plasma prepared without venous occlusion (0.03 +/- 0.02 CTA units/mg protein) or after 5 min of venous occlusion of the arm (0.18 +/- 0.01 CTA units/mg) were comparable to those reported by others using different assays. Thus, this represents a simple, rapid, accurate assay of PA that should be useful to those in immunology, cell biology, and clinical medicine. PMID:6596319

Weinberg, J B; Hobbs, M M; Pizzo, S V

1984-12-31

111

Urokinase-like plasminogen activator increased in plasma after alcohol drinking.  

Science.gov (United States)

After taking the local Japanese spirit 'Shochu', 'Sake' and beer, increased plasma fibrinolysis was confirmed in normal persons at about 1 hr after for both the pyro-Glu-Gly-Arg-pNA amidolytic and Glu-plasminogen activating activities in eluates from [N alpha-(epsilon-aminocaproyl)-DL-homoarginine hexylester;)]-Sepharose affinity column. The activity was highest in the Shochu group, followed by the Sake and beer groups with an approximately 2.1-, 1.7- and 1.5-fold increase in fibrinolytic activity, respectively, compared to the control. The enzyme could be further purified using urokinase-IgG-Sepharose immunoadsorbent column from the Shochu group and it reacted with and was inhibited by urokinase specific antibody. The main molecular weight of the active enzyme was about 30,000, and those of minor components were about 50,000 and 100,000, as determined by zymography. These findings suggest that some of the enzymes appearing in the plasma after drinking are related to 'urokinase-like plasminogen activator', being endogenously produced. PMID:3128990

Sumi, H; Hamada, H; Tsushima, H; Mihara, H

1988-01-01

112

Evidence of a net release of tissue-type plasminogen activator across the human cerebral vasculature.  

Science.gov (United States)

We have earlier described models for measuring local net release rates of tissue-type plasminogen activator (t-PA) in vivo across skeletal, coronary, pulmonary, and splanchnic vascular beds. Aim of the present study was to investigate whether there is a net release of t-PA across the human cerebral vascular bed and whether an acute regulated release can be induced by sympathoadrenal activation. Fourteen male subjects undergoing elective coronary artery bypass grafting were investigated prior to surgery and during sternotomy-induced sympathoadrenal activation. Blood samples were obtained simultaneously from the radial artery and the jugular bulb. Blood flow velocity in the middle cerebral artery (V(MCA)) was determined by transcranial Doppler. Cerebral net release of t-PA was calculated as the arterio-venous concentration gradient times V(MCA). Prior to surgery there was a significant cerebral net release of t-PA (131 and 42 ng/min for t-PA antigen and activity, respectively). The release was significantly induced by sternotomy (to 271 and 80 ng/min, respectively). No significant cerebral net release of plasminogen activator inhibitor type 1 (PAI-1) was detected throughout the experiment. The results show that there is a basal net release of t-PA across the human cerebral vascular bed and that sympathoadrenal activation induces a local regulated release of t-PA. PMID:15116264

Jern, Christina; Blomstrand, Christian; Westerlind, Anne

2004-05-01

113

Soluble Urokinase Plasminogen Activator Receptor Levels in Tuberculosis Patients at High Risk for Multidrug Resistance  

OpenAIRE

The soluble urokinase plasminogen activator receptor (suPAR) has been shown to be a strong prognostic biomarker for tuberculosis (TB). In the present study, the profiles of plasma suPAR levels in pulmonary TB patients at high risk for multidrug resistance were analyzed and compared with those in multidrug resistant (MDR)-TB patients. Forty patients were prospectively included, consisting of 10 MDR-TB patients and 30 TB patients at high risk for MDR, underwent clinical assesment. Plasma suPAR ...

Tri Yudani Mardining Raras; Triwahju Astuti; Iin Noor Chozin

2012-01-01

114

Critical Role of Plasminogen Activator Inhibitor-1 in Cholestatic Liver Injury and Fibrosis  

OpenAIRE

Plasminogen activator inhibitor-1 (PAI-1) is an acute phase protein known to correlate with hepatic fibrosis. However, whether or not PAI-1 plays a causal role in this disease process had not been directly tested. Therefore, wild-type or PAI-1 knockout (PAI-1?/?) mice underwent bile duct ligation. Mice were sacrificed either 3 or 14 days after surgery for assessment of early (i.e., inflammation) and late (i.e., fibrosis) changes caused by bile duct ligation. Liver injury was determined by...

Bergheim, Ina; Guo, Luping; Davis, Molly Anne; Duveau, Ilinca; Arteel, Gavin E.

2006-01-01

115

DNA rearrangement causes hepatocarcinogenesis in albumin-plasminogen activator transgenic mice.  

OpenAIRE

Hepatocyte-directed production of urokinase-type plasminogen activator (uPA) in transgenic mice is hepatotoxic. Infrequently, hepatocytes arise that do not express uPA, due to physical loss of transgene DNA, and these cells clonally repopulate the entire liver within 3 months of birth. Surprisingly, hepatic tumors appear in these mice beginning at 8 months of age despite the fact that uPA is not oncogenic or genotoxic. Analysis of the transgene locus reveals that tumors arise only from a part...

Sandgren, E. P.; Palmiter, R. D.; Heckel, J. L.; Brinster, R. L.; Degen, J. L.

1992-01-01

116

Modulation of NR2B-regulated contextual fear in the hippocampus by the tissue plasminogen activator system  

OpenAIRE

Contextual fear conditioning is regulated by the hippocampus, and NR2B, a subunit of the NMDA receptor (NR), is involved in this process. We show that acute stress modulates tissue plasminogen activator (tPA) activity in the hippocampus by inducing expression of its inhibitor, plasminogen activator inhibitor-1. Acute stress increases NR2B expression and ERK1/2 phosphorylation, a classical marker of postsynaptic plasticity, in the hippocampus. tPA forms a complex with NR2B and is necessary for...

Norris, Erin H.; Strickland, Sidney

2007-01-01

117

Angiostatin generating capacity and anti-tumour effects of D-penicillamine and plasminogen activators  

Directory of Open Access Journals (Sweden)

Full Text Available Abstract Background Upregulation of endogenous angiostatin levels may constitute a novel anti-angiogenic, and therefore anti-tumor therapy. In vitro, angiostatin generation is a two-step process, starting with the conversion of plasminogen to plasmin by plasminogen activators (PAs. Next, plasmin excises angiostatin from other plasmin molecules, a process requiring a donor of a free sulfhydryl group. In previous studies, it has been demonstrated that administration of PA in combination with the free sulfhydryl donor (FSD agents captopril or N-acetyl cysteine, resulted in angiostatin generation, and anti-angiogenic and anti-tumour activity in murine models. Methods In this study we have investigated the angiostatin generating capacities of several FSDs. D-penicillamine proved to be most efficient in supporting the conversion of plasminogen to angiostatin in vitro. Next, from the optimal concentrations of tPA and D-penicillamine in vitro, equivalent dosages were administered to healthy Balb/c mice to explore upregulation of circulating angiostatin levels. Finally, anti-tumor effects of treatment with tPA and D-penicillamine were determined in a human melanoma xenograft model. Results Surprisingly, we found that despite the superior angiostatin generating capacity of D-penicillamine in vitro, both in vivo angiostatin generation and anti-tumour effects of tPA/D-penicillamine treatment were impaired compared to our previous studies with tPA and captopril. Conclusion Our results indicate that selecting the most appropriate free sulfhydryl donor for anti-angiogenic therapy in a (preclinical setting should be performed by in vivo rather than by in vitro studies. We conclude that D-penicillamine is not suitable for this type of therapy.

Maass Cathy N

2006-06-01

118

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

International Nuclear Information System (INIS)

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 literaturconcile 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

119

NKT cell-derived urokinase-type plasminogen activator promotes peripheral tolerance associated with eye.  

Science.gov (United States)

In a model of peripheral tolerance called anterior chamber-associated immune deviation (ACAID), the differentiation of the T regulatory cells depends on NKT cells and occurs in the spleen. In this study, we show that NKT cells that express the invariant (i) TCR and are the CD1d-reactive NKT cells (required for development of peripheral tolerance) actually produced urokinase-type plasminogen activator (uPA) during tolerance induction. The RT-PCR and in vitro plasmin assay showed that splenic iNKT cells derived uPA-converted plasminogen to plasmin. Moreover, uPA was required for tolerance induction because uPA knockout (KO) mice did not develop peripheral tolerance or develop CD8(+) T regulatory cells after Ag inoculation into the anterior chamber. In contrast, other aspects of ACAID-induced tolerance, including recruitment of iNKT cells to the spleen and production of IL-10 by iNKT cells, were unchanged in uPA-deficient mice. The adoptive transfer of splenic NKT cells from wild-type mice restored ACAID in Jalpha18 KO mice (iNKT cell deficient), but NKT cells from uPA KO mice did not. We postulate that the mechanism of action of uPA is through its binding to the uPAR receptor, and enzymatic cleavage of plasminogen to plasmin, which in turn activates latent TGFbeta. In conclusion, uPA derived from iNKT cells is required to induce peripheral tolerance via the eye. PMID:17675481

Sonoda, Koh-Hei; Nakamura, Takahiko; Young, Howard A; Hart, David; Carmeliet, Peter; Stein-Streilein, Joan

2007-08-15

120

Regulation of the synthesis and activity of urokinase plasminogen activator in A549 human lung carcinoma cells by transforming growth factor-beta  

OpenAIRE

Transforming growth factor-beta (TGF beta) is a regulator of cellular proliferation which can alter the proteolytic activity of cultured cells by enhancing the secretion of endothelial type plasminogen activator inhibitor and affecting the secretion of plasminogen activators (PAs) in cultured fibroblastic cells. We used the TGF beta- responsive malignant human lung adenocarcinoma cell line A549 to study the relationships between the known TGF beta-induced growth inhibition and the effects of ...

1988-01-01

121

Activation of the zymogen to urokinase-type plasminogen activator is associated with increased interdomain flexibility.  

Science.gov (United States)

A key regulatory step for serine proteases of the trypsin clan is activation of the initially secreted zymogens, leading to an increase in activity by orders of magnitude. Zymogen activation occurs by cleavage of a single peptide bond near the N-terminus of the catalytic domain. Besides the catalytic domain, most serine proteases have N-terminal A-chains with independently folded domains. Little is known about how zymogen activation affects the interplay between domains. This question is investigated with urokinase-type plasminogen activator (uPA), which has an epidermal growth factor domain and a kringle domain, connected to the catalytic domain by a 15-residue linker. uPA has been implicated under several pathological conditions, and one possibility for pharmacological control is targeting the conversion of the zymogen pro-uPA to active uPA. Therefore, a small-angle X-ray scattering study of the conformations of pro-uPA and uPA in solution was performed. Structural models for the proteins were derived using available atomic-resolution structures for the various domains. Active uPA was found to be flexible with a random conformation of the amino-terminal fragment domain with respect to the serine protease domain. In contrast, pro-uPA was observed to be rigid, with the amino-terminal fragment domain in a fixed position with respect to the serine protease domain. Analytical ultracentrifugation analysis supported the observed difference between pro-uPA and uPA in overall shape and size seen with small-angle X-ray scattering. Upon association of either of two monoclonal Fab (fragment antigen-binding) fragments that are directed against the catalytic domain of, respectively, pro-uPA and uPA, rigid structures were formed. PMID:21669207

Behrens, Manja A; Botkjaer, Kenneth A; Goswami, Sumit; Oliveira, Cristiano L P; Jensen, Jan K; Schar, Christine R; Declerck, Paul J; Peterson, Cynthia B; Andreasen, Peter A; Pedersen, Jan Skov

2011-08-12

122

Activation of the zymogen to urokinase-type plasminogen activator is associated with increased interdomain flexibility  

DEFF Research Database (Denmark)

A key regulatory step for serine proteases of the trypsin clan is activation of the initially secreted zymogens, leading to an increase in activity by orders of magnitude. Zymogen activation occurs by cleavage of a single peptide bond near the N-terminus of the catalytic domain. Besides the catalytic domain, most serine proteases have N-terminal A-chains with independently folded domains. Little is known about how zymogen activation affects the interplay between domains. This question is investigated with urokinase-type plasminogen activator (uPA), which has an epidermal growth factor domain and a kringle domain, connected to the catalytic domain by a 15-residue linker. uPA has been implicated under several pathological conditions, and one possibility for pharmacological control is targeting the conversion of the zymogen pro-uPA to active uPA. Therefore, a small-angle X-ray scattering study of the conformations of pro-uPA and uPA in solution was performed. Structural models for the proteins were derived usingavailable atomic-resolution structures for the various domains. Active uPA was found to be flexible with a random conformation of the amino-terminal fragment domain with respect to the serine protease domain. In contrast, pro-uPA was observed to be rigid, with the amino-terminal fragment domain in a fixed position with respect to the serine protease domain. Analytical ultracentrifugation analysis supported the observed difference between pro-uPA and uPA in overall shape and size seen with small-angle X-ray scattering. Upon association of either of two monoclonal Fab (fragment antigen-binding) fragments that are directed against the catalytic domain of, respectively, pro-uPA and uPA, rigid structures were formed

Behrens, Manja A; BØtkjær, Kenneth AlrØ

2011-01-01

123

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

Science.gov (United States)

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. PMID:23853591

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

124

Plasminogen activator inhibitor-1 polymers, induced by inactivating amphipathic organochemical ligands.  

DEFF Research Database (Denmark)

Negatively charged organochemical inactivators of the anti-proteolytic activity of plasminogen activator inhibitor-1 (PAI-1) convert it to inactive polymers. As investigated by native gel electrophoresis, the size of the PAI-1 polymers ranged from dimers to multimers of more than 20 units. As compared with native PAI-1, the polymers exhibited an increased resistance to temperature-induced unfolding. Polymerization was associated with specific changes in patterns of digestion with non-target proteases. During incubation with urokinase-type plasminogen activator, the polymers were slowly converted to reactive centre-cleaved monomers, indicating substrate behaviour of the terminal PAI-1 molecules in the polymers. A quadruple mutant of PAI-1 with a retarded rate of latency transition also had a retarded rate of polymerization. Studying a number of serpins by native gel electrophoresis, ligand-induced polymerization was observed only with PAI-1 and heparin cofactor II, which were also able to copolymerize. On the basis of these results, we suggest that the binding of ligands in a specific region of PAI-1 leads to so-called loop-sheet polymerization, in which the reactive centre loop of one molecule binds to beta-sheet A in another molecule. Induction of serpin polymerization by small organochemical ligands is a novel finding and is of protein chemical interest in relation to pathological protein polymerization in general. Udgivelsesdato: 2003-Jun-15

Pedersen, Katrine E; Einholm, Anja P

2003-01-01

125

High level of urokinase plasminogen activator contributes to cholangiocarcinoma invasion and metastasis  

Directory of Open Access Journals (Sweden)

Full Text Available AIM: To investigate the role of urokinase plasminogen activator (uPA in cholangiocarcinoma (CCA invasion and its correlation with clinicopathological parameters. METHODS: uPA expression in CCA tissue was determined by immunohistochemistry. The level of uPA from two CCA cell lines (HuCCA-1 and KKU-M213 and a non-cancer immortalized cholangiocyte cell line (H69 was monitored by plasminogen-gelatin zymography and western blotting, whereas that of plasminogen activator inhibitor type 1 (PAI-1 protein and uPA receptor (uPAR mRNA was monitored by western blotting and quantitative real-time reverse transcriptase polymerase chain reaction, respectively. Two independent methods were employed to suppress uPA function: a synthetic uPA inhibitor (B428 and silencing of uPA gene expression using siRNA. In vitro invasion of the uPA-disrupted cells was assessed by Matrigel-coated Transwell assay. RESULTS: The immunohistochemical study showed that 75.3% (131/174 of CCA tissues expressed uPA. High uPA expression was correlated with lymphatic invasion and metastasis of CCA patients. Plasminogen-gelatin zymography of the conditioned media and cell-surface eluates showed that both CCA cell lines, but not H69, expressed both secreted and membrane-bound forms of uPA. Although the two CCA cell lines, HuCCA-1 and KKU-M213, expressed a relatively high level of uPA and uPAR, the latter exhibited a much lower degree of in vitro invasiveness, correlating with a high expression of PAI-1 in the latter, but not in the former. Suppressing uPA function with a specific uPA inhibitor, B428, or with siRNA against uPA reduced in vitro invasiveness of KKU-M213 cells, demonstrating the requirement for uPA in the invasiveness of CCA cells. Therefore, our in vivo and in vitro studies suggest that uPA is an important requirement for the invasion process of CCA. CONCLUSION: uPA expression correlates with lymphatic invasion and metastasis in vivo and is required for CCA cell invasion in vitro, suggesting its potential as a therapeutic target.

Tuangporn Suthiphongchai

2012-01-01

126

Increased nitrite reductase activity of fetal versus adult ovine hemoglobin  

OpenAIRE

Growing evidence indicates that nitrite, NO2?, serves as a circulating reservoir of nitric oxide (NO) bioactivity that is activated during physiological and pathological hypoxia. One of the intravascular mechanisms for nitrite conversion to NO is a chemical nitrite reductase activity of deoxyhemoglobin. The rate of NO production from this reaction is increased when hemoglobin is in the R conformation. Because the mammalian fetus exists in a low-oxygen environment compared with the adult and...

Blood, Arlin B.; Tiso, Mauro; Verma, Shilpa T.; Lo, Jennifer; Joshi, Mahesh S.; Azarov, Ivan; Longo, Lawrence D.; Gladwin, Mark T.; Kim-shapiro, Daniel B.; Power, Gordon G.

2008-01-01

127

Targeting the autolysis loop of urokinase-type plasminogen activator with conformation-specific monoclonal antibodies  

DEFF Research Database (Denmark)

Tight regulation of serine proteases is essential for their physiological function, and unbalanced states of protease activity have been implicated in a variety of human diseases. One key example is the presence of uPA (urokinase-type plasminogen activator) in different human cancer types, with high levels correlating with a poor prognosis. This observation has stimulated efforts into finding new principles for intervening with uPA's activity. In the present study we characterize the so-called autolysis loop in the catalytic domain of uPA as a potential inhibitory target. This loop was found to harbour the epitopes for three conformation-specific monoclonal antibodies, two with a preference for the zymogen form pro-uPA, and one with a preference for active uPA. All three antibodies were shown to have overlapping epitopes, with three common residues being crucial for all three antibodies, demonstrating a direct link between conformational changes of the autolysis loop and the creation of a catalytically matureactive site. All three antibodies are potent inhibitors of uPA activity, the two pro-uPA-specific ones by inhibiting conversion of pro-uPA to active uPA and the active uPA-specific antibody by shielding the access of plasminogen to the active site. Furthermore, using immunofluorescence, the conformation-specific antibodies mAb-112 and mAb-12E6B10 enabled us to selectively stain pro-uPA or active uPA on the surface of cultured cells. Moreover, in various independent model systems, the antibodies inhibited tumour cell invasion and dissemination, providing evidence for the feasibility of pharmaceutical intervention with serine protease activity by targeting surface loops that undergo conformational changes during zymogen activation.

BØtkjær, Kenneth AlrØ; Fogh, Sarah

2011-01-01

128

A Deletion Mutant of Vitronectin Lacking the Somatomedin B Domain Exhibits Residual Plasminogen Activator Inhibitor-1-binding Activity*  

OpenAIRE

Vitronectin and plasminogen activator inhibitor-1 (PAI-1) are important physiological binding partners that work in concert to regulate cellular adhesion, migration, and fibrinolysis. The high affinity binding site for PAI-1 is located within the N-terminal somatomedin B domain of vitronectin; however, several studies have suggested a second PAI-1-binding site within vitronectin. To investigate this secondary site, a vitronectin mutant lacking the somatomedin B domain ...

Schar, Christine R.; Blouse, Grant E.; Minor, Kenneth H.; Peterson, Cynthia B.

2008-01-01

129

Role of urokinase plasminogen activator and plasminogen activator inhibitor mRNA expression as prognostic factors in molecular subtypes of breast cancer  

Directory of Open Access Journals (Sweden)

Full Text Available Isabell Witzel,1 Karin Milde-Langosch,1 Marcus Schmidt,2 Thomas Karn,3 Sven Becker,3 Ralph Wirtz,4 Achim Rody,5 Elena Laakmann,1 Dina Schütze,1 Fritz Jänicke,1 Volkmar Müller1 1Department of Gynecology, University Medical Center, Hamburg, 2Department of Obstetrics and Gynecology, University Hospital, Mainz, 3Department of Obstetrics and Gynecology, University Hospital, Frankfurt, 4STRATIFYER Molecular Pathology GmbH, Cologne, 5Department of Obstetrics and Gynecology, University Medical Center Schleswig-Holstein, Luebeck, Germany Background: Protein levels of urokinase plasminogen activator (uPA and its inhibitor (PAI-1 determined by enzyme-linked immunosorbent assay from fresh-frozen tumor tissue have been evaluated as prognostic factors in prospectively randomized trials in breast cancer. However, the role of uPA and PAI-1 in the context of breast cancer subtypes and for mRNA expression of these factors is less clear. Methods: We evaluated uPA and PAI-1 mRNA expression using the Affymetrix HG-U 133A array within molecular subgroups of breast cancer in cohorts of patients with systemic treatment (cohort A, n=362 and without systemic treatment (cohort B, n=200. We validated mRNA expression in a cohort of HER2-positive breast cancer patients (cohort C, n=290. Luminal, triple-negative, and HER2-positive subcohorts were defined by ESR1 and ERBB2 mRNA expression using predefined cutoffs. Results: In the entire cohort A, elevated PAI-1 but not uPA mRNA expression was associated with shorter disease-free survival (P=0.007 for PAI and 0.069 for uPA. Regarding different molecular subgroups, 67% (n=244 of tumors were luminal, 14% (n=49 were HER2-positive, and 19% (n=69 were triple-negative. Elevated PAI-1 mRNA expression was associated with shorter disease-free survival only in the HER2-positive subgroup (P=0.031. The same disease-free survival results were found for uPA in HER2-positive patients (P=0.011. In contrast, no association between either marker and survival was observed in the luminal or triple-negative subgroups. In the HER2-positive validation cohort C, elevated uPA and PAI-1 mRNA expression also showed strong associations with shorter disease-free survival (P=0.014 for PAI-1, P<0.001 for uPA. Conclusion: In this study, the prognostic impact of uPA and PAI-1 expression was mainly observed in patients with HER2-positive tumors. Keywords: urokinase plasminogen activator, urokinase plasminogen activator inhibitor-1, HER2, breast cancer, prognosis

Witzel I

2014-11-01

130

Enhanced functional stability of plasminogen activator inhibitor-1 in patients with livedoid vasculopathy.  

Science.gov (United States)

Livedoid vasculopathy (LV) is a chronic, recurrent, painful cutaneous disease with distinctive clinical features and an uncertain etiology. The skin lesions are recognizable by focal purpura, depigmentation and shallow ulcers. Thrombophilic conditions occur frequently in patients with LV. While no definitive treatment exists for LV, smoking cessation, antiplatelet therapy, immunosuppressive treatment, and anabolic steroids are often included in the therapeutic ladder. Recently, a possible link between LV and impaired fibrinolysis was established as cutaneous LV lesions responded to tissue plasminogen activator (t-PA) infusion suggesting that inhibition of the fibrinolysis through plasminogen activator inhibitor-1 (PAI-1) activity may determine the disease course in patients with LV. In this study, we investigated PAI-1 antigen (Ag) and activity levels in 20 patients with biopsy proven LV (mean age 26 ± 11, M/F = 7/13, median disease duration 3.5 years). All patients received antiplatelet treatment with aspirin and/or dipyrimadole and 14 patients received anabolic steroids or immunosuppressive treatment. Fasting PAI-1 Ag and activity levels were measured at 9 AM in all patients. Both Ag (34 (26) ng/ml) (median (interquartile range)) and specific activity (17 (23) IU/fmole) levels of PAI-1 were moderately elevated in LV patients compared to the controls, however, PAI-1 kinetic studies demonstrated markedly enhanced stability of PAI-1 activity in plasma from patients with LV. Specific activity at 16 h was significantly higher than expected specific activity levels (7 (11) vs. 0.07 (0.09) IU/fmole, P < 0.01). While the exact mechanism of increased stability of PAI-1 activity is not known, it may be due to post-translational modifications or increased binding affinity for a stabilizing cofactor. In conclusion, enhanced stability of PAI-1 may contribute to the pathophysiology of LV, and systemic or local treatment with PAI-1 inhibitors may offer a potential treatment alternative in patients with LV. PMID:21311952

Agirbasli, Mehmet; Eren, Mesut; Eren, Fatih; Murphy, Sheila B; Serdar, Zehra A; Seckin, Dilek; Zara, Tuba; Cem Mat, M; Demirkesen, Cuyan; Vaughan, Douglas E

2011-07-01

131

Rapid localization of indium-111-labeled inhibited recombinant tissue plasminogen activator in a rabbit thrombosis model  

Energy Technology Data Exchange (ETDEWEB)

The thrombus localizing properties of indium-111-recombinant tissue plasminogen activator ({sup 111}In-rt-PA) have been investigated in an effort to achieve prompt and accurate detection of thrombi. Unlike previous studies with rt-PA, the active plasminogen catalytic site was permanently inhibited with peptides of chloromethyl ketone so that the radiotracer binds to fibrin without causing fibrinolysis. Thrombi were created in the external jugular vein of 14 male New Zealand white rabbits followed by injection of {sup 111}In-rt-PA. The agent cleared rapidly in vivo with a half-time of 4.6 min. The thrombus: blood ratio in nonheparinized rabbits (n = 7) was 6.39 +/- 0.86. The ratio in heparinized rabbits (n = 4) was 3.11 +/- 0.23. Thrombi were clearly visible in the planar images of both groups 1 hr postinjection. The combination of rapid thrombus localization and positive images, especially in the presence of anticoagulation, suggests that further work is warranted with rt-PA thrombus imaging.

Butler, S.P.; Kader, K.L.; Owen, J.; Wang, T.S.; Fawwaz, R.A.; Alderson, P.O. (Columbia-Presbyterian Medical Center, New York, NY (USA))

1991-03-01

132

Rapid localization of indium-111-labeled inhibited recombinant tissue plasminogen activator in a rabbit thrombosis model  

International Nuclear Information System (INIS)

The thrombus localizing properties of indium-111-recombinant tissue plasminogen activator (111In-rt-PA) have been investigated in an effort to achieve prompt and accurate detection of thrombi. Unlike previous studies with rt-PA, the active plasminogen catalytic site was permanently inhibited with peptides of chloromethyl ketone so that the radiotracer binds to fibrin without causing fibrinolysis. Thrombi were created in the external jugular vein of 14 male New Zealand white rabbits followed by injection of 111In-rt-PA. The agent cleared rapidly in vivo with a half-time of 4.6 min. The thrombus: blood ratio in nonheparinized rabbits (n = 7) was 6.39 +/- 0.86. The ratio in heparinized rabbits (n = 4) was 3.11 +/- 0.23. Thrombi were clearly visible in the planar images of both groups 1 hr postinjection. The combination of rapid thrombus localization and positive images, especially in the presence of anticoagulation, suggests that further work is warranted with rt-PA thrombus imaging

133

Cleaved intracellular plasminogen activator inhibitor 2 in human myeloleukaemia cells is a marker of apoptosis  

DEFF Research Database (Denmark)

The proteolytic modification of plasminogen activator inhibitor 2 (PAI-2) was studied during apoptosis in the human promyelocytic leukaemic NB4 cell line during treatment with the phosphatase inhibitors okadaic acid and calyculin A as well as the protein synthesis inhibitor cycloheximide. The apoptic type of cell death was ascertained by morphological and biochemical criteria. In cell homogenates PAI-2 was probed by [125I]urokinase plasminogen activator (uPA) and detected as a sodium dodecyl sulphate-stable M(r) 80,000 complex after reducing sodium dodecyl sulphate-polyacrylamide gel electrophoresis and autoradiography. During apoptosis a smaller (M(r) 70,000) uPA-PAI-2 complex was consistently detected. The modification was in the PAI-2 moiety, as the [125I]uPA tracer could be extracted in its intact form from the complex. Thus the cleaved PAI-2 isoform is a biochemical marker of apoptosis in the promyelocytic NB4 cell line. The modified PAI-2 isoform was also detected in homogenates made from purified humanmononuclear leukaemic cells aspirated from the bone marrow of patients suffering from acute and chronic myeloid leukaemia.

Jensen, P H; Cressey, L I

1994-01-01

134

Tissue plasminogen activator for the treatment of parapneumonic effusions in pediatric patients.  

Science.gov (United States)

Intrapleural fibrinolysis has been investigated for the treatment of pleural effusion for several decades. Fibrinolytics have the ability to break up fibrin and loculations that characterize complicated pleural effusions, facilitating drainage. Older fibrinolytics such as urokinase and streptokinase have been replaced by tissue plasminogen activator (tPA) for this indication due to product availability and a more favorable safety profile. The literature supports tPA as a treatment approach for this indication in adult patients, and the use of tPA has become a standard management approach in this population. Over the past decade, data on the efficacy of intrapleural fibrinolytic therapy in children have also been generated, which now support the use of fibrinolysis as a treatment alternative to more invasive therapeutic options such as surgical intervention. In this review, we discuss the pathophysiology, diagnosis, and treatment of parapneumonic effusion and empyema, with a focus on intrapleural fibrinolysis, specifically tissue plasminogen activator, in the pediatric population. Recent articles provide sufficient evidence to support the use of this drug in pediatric patients for the management of pleural effusions; however, due to study heterogeneity, questions remain that may be addressed in future studies. PMID:24390857

Israel, Emily N; Blackmer, Allison B

2014-05-01

135

Angiostatic activity of human plasminogen fragments is highly dependent on glycosylation  

International Nuclear Information System (INIS)

To assess the importance of carbohydrate moieties to the anti-angiogenic activity of plasminogen fragments, we cloned the fragment corresponding to amino acids Val79 to Thr346 (Kint3-4) that presents the three glycosylation sites. The activity of glycosylated and unglycosylated Kint3-4 was tested in murine sponge implant model. We observed a significant decrease in the neovascularization on the sponge after treatment with Kint3-4 by histological examination and determination of the hemoglobin levels. The effects were more intense with the glycosylated than the unglycosylated protein. 99mTechnecium-labeled red blood cells confirmed the inhibition of cell infiltration in the implanted sponge. Studies using melanoma B16F1 implanted in a mouse demonstrated that treatment with glycosylated Kint3-4 (0.15 nmol/48 h) during 14 days suppresses tumor growth by 80%. The vascular endothelial growth factor mRNA levels on the tumor were reduced after treatment. Kint3-4 is a potent plasminogen fragment that has been found to inhibit tumor growth. (author)

136

The association of plasminogen activator inhibitor-1 level with ischemic stroke (preliminary study  

Directory of Open Access Journals (Sweden)

Full Text Available Aim Recently, increased plasminogen activator inhibitor-1 (PAI-1 has been known a risk factor for ischemic heart disease. However, the association of increased PAI-1 level with ischemic stroke remains unclear. The aim of this study was to analyze the association of PAI-1 level with ischemic stroke.Methods By case control design we involved 38 ischemic stroke and 38 risky-matched control subjects who fulfilled the criteria. The PAI-1 level was determined by ELISA method using Asserachrom PAI-1 from Stago.Results High PAI-1 level was found more frequent in ischemic stroke subjects than in control subjects (21.1% vs. 7.9 % with OR 3.1; 95 % CI 0.757 – 12.790. The analysis of all studied subjects showed that there was a weak negative correlation between PAI-1 level and age (r = -0.4; P = 0.000. High PAI-1 level was found more frequent in younger (40 – 58 years old than in the older subjects (60 – 84 years old (20% vs. 9.8 % (p=0.004.Conclusion The result of this preliminary study suggested an association between PAI-1 level and ischemic stroke in younger age. Further study with larger subjects is recommended to confirm this association. (Med J Indones 2009;19:158-63Key words: ischemic stroke, plasminogen activator inhibitor-1, stroke risk factors

Yenny Surjawan

2010-08-01

137

Long-term stability of recombinant tissue plasminogen activator at -80 C  

Directory of Open Access Journals (Sweden)

Full Text Available Abstract Background Recombinant tissue plasminogen activator (tPA is a thrombolytic widely used clinically in the treatment of acute thrombotic disease such as ischemic stroke, myocardial infarction, and deep venous thrombosis. This has led to much interest in tPA based lytic therapies leading to laboratory based in-vitro and in-vivo investigations using this drug. However, tPA reconstituted in solution exhibits full activity for only 6–8 hours, according to the manufacturer. Therefore, methods to store reconstituted tPA for long durations while maintaining activity would be of assistance to laboratories using this enzyme. Findings In this work, the enzymatic activity of tPA stored at -80 C over time was measured, using an ELISA technique that measured the amount of active tPA bound to plasminogen activator inhibitor 1 (PAI-1 in a given sample. Sample of tPA solution mixed to a concentration of 1 (mg/ml were stored in cryogenic vials at -80 C for up to 7 years. For a given sample, aliquots were assayed for tPA activity, and compared with a tPA standard to determine relative enzymatic activity. Results are reported as means with standard errors, and 12 measurements were performed for each sample age. Conclusion There was no decrease in tPA activity for samples stored up to 7 years. Such cryogenic storage is a viable method for the preservation of tPA solution for laboratory investigations of tPA-based lytic therapies.

Sperling Matthew

2009-06-01

138

Stability of Recombinant Tissue Plasminogen Activator at ?30 °C over One Year  

Directory of Open Access Journals (Sweden)

Full Text Available Recombinant tissue plasminogen activator (rt-PA is used to restore patency and avoid inadvertent removal of peripheral and central venous catheters. rt-PA was reconstituted (1 mg/mL then cryopreserved at ?30 °C for 1, 2, 3, 6, 8, and 12 months and, then its stability was determined. After cryopreservation for one and two months, rt-PA kept more than 95% of its activity compared to standard samples, while cryopreservation for three months caused 8% loss of activity. However, after cryopreservation for six months or more, rt-PA retained only 87.5% or less activity compared to standard samples. Therefore, it is recommended that reconstituted rt-PA be cryopreserved at ?30 °C for a maximum period of three months.

Abdulmalik Alkatheri

2013-01-01

139

Clinical significance of plasminogen activator inhibitor activity in patients with exercise-induced ischemia  

International Nuclear Information System (INIS)

To assess the fibrinolytic system in patients with exercise-induced ischemia and its relation to ischemia and severity of coronary artery disease (CAD), 47 patients with CAD confirmed by results of coronary angiography underwent symptom-limited multistage exercise thallium-201 emission computed tomography. All patients with CAD had exercise-induced ischemia as assessed from thallium-201 images. Pre- and peak exercise blood samples from each patient and preexercise blood samples from control subjects were assayed for several fibrinolytic components and were also assayed for plasma adrenaline. The extent of ischemia was defined as delta visual uptake score (total visual uptake score in delayed images minus total visual uptake score in initial images) and the severity of CAD as the number of diseased vessels. In the basal condition, plasminogen activator inhibitor (PAI) activity was significantly higher in patients with exercise-induced ischemia as compared to control subjects (p less than 0.01), although there were no significant differences in other fibrinolytic variables between the two groups. Moreover, PAI activity in the basal condition displayed a significantly positive correlation with the extent of ischemia (r = 0.47, p less than 0.01). Patients with exercise-induced ischemia were divided into two groups (24 with single-vessel disease and 23 with multivessel disease). There were no significant differences in coronary risk factors, hemodynamics, or plasma adrenalk factors, hemodynamics, or plasma adrenaline levels during exercise between single-vessel and multivessel disease except that delta visual uptake score was significantly higher in multivessel disease (p less than 0.01)

140

Overexpression of plasminogen activators in the nucleus accumbens enhances cocaine-, amphetamine- and morphine-induced reward and behavioral sensitization.  

Science.gov (United States)

Urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator (tPA) are extracellular proteases that play a role in synaptic plasticity and remodeling. Psychostimulants induce both tPA and uPA in acute and chronic drug delivery, but cocaine induces preferentially uPA, whereas morphine and amphetamine induce preferentially tPA. Specific doxycline-regulatable lentiviruses expressing these extracellular proteases have been prepared and stereotaxically injected into the nucleus accumbens. We show that tPA-overexpressing animals show greater locomotor activity and behavioral sensitization upon morphine and amphetamine treatments. These effects could be fully suppressed by doxycycline or when tPA had been silenced using small interfering RNAs (siRNAs)-expressing lentiviruses. Furthermore, animals infected with lentiviruses expressing uPA show enhanced conditional place preference for cocaine compared with tPA-overexpressing animals. In contrast, tPA-overexpressing animals when administered amphetamine or morphine showed greater place preference compared with uPA-overexpressing animals. The effects are suppressed when tPA has been silenced using specific siRNAs-expressing vectors. Tissue-type plasminogen activator and uPA possibly induce distinct behaviors, which may be interpreted according to their differential pattern of activation and downstream targets. Taken together, these data add further evidence for a significant function of extracellular proteases tPA and uPA in addiction and suggest a differential role of plasminogen activators in this context. PMID:17680802

Bahi, A; Dreyer, J-L

2008-03-01

141

Probiotic in rennet paste can affect lipase activity of rennet and lipolysis in ovine cheese  

Directory of Open Access Journals (Sweden)

Full Text Available Lambs were subjected to three different feeding regimes (mother suckling MS, artificial rearing AR, and artificial rearing with 7log10 cfu/ml Lactobacillus acidophilus supplementation to the milk substitute ARLb and slaughtered at 20d and 40d of age for each feeding treatment. Lambs abomasa were processed to rennet paste and lipases activity was evaluated. Rennet paste was used for Pecorino cheese production. Free fatty acids (FFAs and conjugated linoleic acids (CLAs were detected in cheese at 60d of ripening. Lipase activity was found higher in ARLb than in MS and AR rennet from lambs slaughtered at an older age. A reduction of all FFAs was observed in all cheeses when passing from 20 d to 40d of slaughtering. CLAs were more abundant in ARLb cheeses at both 20 and 40d. Milk substitute with Lb. acidophilus improves enzymatic features of rennet, and health and nutritional characteristics of ovine cheese.

Marzia Albenzio

2010-01-01

142

Reduced plasminogen binding and delayed activation render ?'-fibrin more resistant to lysis than ?A-fibrin.  

Science.gov (United States)

Fibrin (Fn) clots formed from ?'-fibrinogen (?'-Fg), a variant with an elongated ?-chain, are resistant to lysis when compared with clots formed from the predominant ?A-Fg, a finding previously attributed to differences in clot structure due to delayed thrombin-mediated fibrinopeptide (FP) B release or impaired cross-linking by factor XIIIa. We investigated whether slower lysis of ?'-Fn reflects delayed plasminogen (Pg) binding and/or activation by tissue plasminogen activator (tPA), reduced plasmin-mediated proteolysis of ?'-Fn, and/or altered cross-linking. Clots formed from ?'-Fg lysed more slowly than those formed from ?A-Fg when lysis was initiated with tPA/Pg when FPA and FPB were both released, but not when lysis was initiated with plasmin, or when only FPA was released. Pg bound to ?'-Fn with an association rate constant 22% lower than that to ?A-Fn, and the lag time for initiation of Pg activation by tPA was longer with ?'-Fn than with ?A-Fn. Once initiated, however, Pg activation kinetics were similar. Factor XIIIa had similar effects on clots formed from both Fg isoforms. Therefore, slower lysis of ?'-Fn clots reflects delayed FPB release, which results in delayed binding and activation of Pg. When clots were formed from Fg mixtures containing more than 20% ?'-Fg, the upper limit of the normal level, the delay in lysis was magnified. These data suggest that circulating levels of ?'-Fg modulate the susceptibility of clots to lysis by slowing Pg activation by tPA and provide another example of the intimate connections between coagulation and fibrinolysis. PMID:25128532

Kim, Paul Y; Vu, Trang T; Leslie, Beverly A; Stafford, Alan R; Fredenburgh, James C; Weitz, Jeffrey I

2014-10-01

143

Reduced cardiac expression of plasminogen activator inhibitor 1 and transforming growth factor ?1 in obese Zucker rats by perindopril  

OpenAIRE

Objective: To determine whether angiotensin converting enzyme inhibition by perindopril can reduce cardiac transforming growth factor ?1 (TGF?1) and plasminogen activator inhibitor 1 (PAI-1) and therefore control collagen accumulation in an animal model with the metabolic syndrome such as the obese Zucker rat (OZR).

Toblli, J. E.; Cao, G.; Derosa, G.; Forcada, P.

2005-01-01

144

Components of the Plasminogen Activation System Promote Engraftment of Porous Polyethylene Biomaterial via Common and Distinct Effects.  

Science.gov (United States)

Rapid fibrovascularization is a prerequisite for successful biomaterial engraftment. In addition to their well-known roles in fibrinolysis, urokinase-type plasminogen activator (uPA) and tissue plasminogen activator (tPA) or their inhibitor plasminogen activator inhibitor-1 (PAI-1) have recently been implicated as individual mediators in non-fibrinolytic processes, including cell adhesion, migration, and proliferation. Since these events are critical for fibrovascularization of biomaterial, we hypothesized that the components of the plasminogen activation system contribute to biomaterial engraftment. Employing in vivo and ex vivo microscopy techniques, vessel and collagen network formation within porous polyethylene (PPE) implants engrafted into dorsal skinfold chambers were found to be significantly impaired in uPA-, tPA-, or PAI-1-deficient mice. Consequently, the force required for mechanical disintegration of the implants out of the host tissue was significantly lower in the mutant mice than in wild-type controls. Conversely, surface coating with recombinant uPA, tPA, non-catalytic uPA, or PAI-1, but not with non-catalytic tPA, accelerated implant vascularization in wild-type mice. Thus, uPA, tPA, and PAI-1 contribute to the fibrovascularization of PPE implants through common and distinct effects. As clinical perspective, surface coating with recombinant uPA, tPA, or PAI-1 might provide a novel strategy for accelerating the vascularization of this biomaterial. PMID:25658820

Reichel, Christoph A; Hessenauer, Maximilian E T; Pflieger, Kerstin; Rehberg, Markus; Kanse, Sandip M; Zahler, Stefan; Krombach, Fritz; Berghaus, Alexander; Strieth, Sebastian

2015-01-01

145

Description of Prescribing Practices of Intrapleural Tissue Plasminogen Activator and Intrapleural DNase Administration at a Tertiary Academic Medical Center  

OpenAIRE

Objectives: To describe the prescribing practices, preparation and administration techniques of intrapleural (IP) tissue plasminogen activator (t-PA) and IP DNase in patients at a tertiary academic medical center. Methods: Adult patients receiving IP t-PA and IP DNase between January 1-December 31, 2012 were retrospectively evaluated. Patients were included if they received IP t-PA and/or I...

Heather Torbic; Gaspar Hacobian; Nahal Beik

2014-01-01

146

Xanthoangelols isolated from Angelica keiskei inhibit inflammatory-induced plasminogen activator inhibitor 1 (PAI-1) production.  

Science.gov (United States)

The folk medicine Angelica keiskei (Ashitaba) exhibits antitumor, antioxidant and antidiabetic activities and it has recently attracted attention as a health food. Ashitaba is thought to have antithrombotic properties, but this has not yet been scientifically proven. The elevation of plasma plasminogen activator inhibitor 1 (PAI-1), an inhibitor of fibrinolysis results in a predisposition to the risk of thrombosis. The present study showed that Ashitaba exudates injected intraperitoneally and orally administered over long-term suppressed the lipopolysaccharide (LPS) induced PAI-1 increase in mouse plasma. We also found that xanthoangelol, xanthoangelols B and D, the components of Ashitaba exudates, significantly inhibited TNF?-induced PAI-1 production from human umbilical vein endothelial cells (HUVECs). These findings suggest that Ashitaba can decrease elevated PAI-1 production, and that daily consumption of Ashitaba product might maintain anticoagulant status by inhibiting elevations in PAI-1 under inflammatory conditions. PMID:22038782

Ohkura, Naoki; Nakakuki, Yoshitaka; Taniguchi, Masahiko; Kanai, Shiho; Nakayama, Akiko; Ohnishi, Katsunori; Sakata, Toshiyuki; Nohira, Tomoyoshi; Matsuda, Juzo; Baba, Kimiye; Atsumi, Gen-Ichi

2011-01-01

147

[Plasminogen activator inhibitor type 1 activity in women with unexplained very early recurrent pregnancy loss].  

Science.gov (United States)

The aim of the study was to assess the independent role of polymorphism 4G/5G (PL 4G/5G)--genotype 4G/4G in plasminogen activator inhibitor type 1 (PAI-1) in the development of very early recurrent pregnancy loss (RPL)--before 10 weeks of gestation of pregnancy. The polymorphism 4G/5G as well as Factor V Leiden (FVL), prothrombin (FII) gene mutation 20210 G > A and polymorphism 677 C > T in methylentetrahydrofolat reductase (MTHFR) gene was investigated in 110 women with recurrent pregnancy loss before 10 weeks of gestation and in 97 healthy women with at least one uncomplicated full-term pregnancy. A significant prevalence of PL 4G/5G in women with RPL was found in comparison to prevalence of the polymorphism in controls (41.8% versus 26.8% respectively in patients and controls, OR: 1.96, 95% CI: 1.05 3.69, p = 0.034). The difference in prevalence of the polymorphism remains still significant after exclusion of patients and control carriers of FVL, FII 202010 G > A and 677 C > T in MTHFR (the prevalence of PL 4G/5G alone was 44.1% and 24% respectively in patients and controls, OR: 2,5, 95% CI: 1,15 5, 45, p = 0.018). The found association of PL 4G/5G in PAI-1 with early recurrent pregnancy loss encourage an extension of the list of inherited thrombophilic factors with this one. This result also could have had an implication for adjustment of further prophylactic low-molecular weight heparin implication in further pregnancy to prevent a poor foetal outcome. PMID:21268395

Ivanov, P; Komsa-Penkova, R; Ivanov, I; Konova, E; Kovacheva, K; Simeonova, M; Tanchev, S

2010-01-01

148

A technique to screen plant extracts for anti-inflammatory activity on ovine neutrophils  

Directory of Open Access Journals (Sweden)

Full Text Available Neutrophils play an essential role in host defense and inflammation. In recent years, several studies in men and dairy animals have shown that administration of natural compounds can influence the immune system. In the present study the extracts of six patented protected plants, named C1, C2, C3, C4, C5, C6, have been screened for their effect on ovine neutrophils. Three, clinically healthy, non lactating, non pregnant sheep were used. Freshly isolated neutrophils were incubated with extracts at increasing doses and tested for viability, adhesion and superoxide production induced by PMA. The residues of C1, C2, C3 strongly inhibited adhesion and superoxide production, demonstrating anti-inflammatory activity. Further in vivo experiments are required to validate their use for productive animals.

B. Stefanon

2010-04-01

149

Urokinase-type plasminogen activator-like proteases in teleosts lack genuine receptor-binding epidermal growth factor-like domains  

DEFF Research Database (Denmark)

Plasminogen activation catalyzed by urokinase-type plasminogen activator (uPA) plays an important role in normal and pathological tissue remodeling processes. Since its discovery in the mid-1980s, the cell membrane-anchored urokinase-type plasminogen activator receptor (uPAR) has been believed to be central to the functions of uPA, as uPA-catalyzed plasminogen activation activity appeared to be confined to cell surfaces through the binding of uPA to uPAR. However, a functional uPAR has so far only been identified in mammals. We have now cloned, recombinantly produced, and characterized two zebrafish proteases, zfuPA-a and zfuPA-b, which by several criteria are the fish orthologs of mammalian uPA. Thus, both proteases catalyze the activation of fish plasminogen efficiently and both proteases are inhibited rapidly by plasminogen activator inhibitor-1 (PAI-1). But zfuPA-a differs from mammalian uPA by lacking the exon encoding the uPAR-binding epidermal growth factor-like domain; zfuPA-b differs from mammalian uPA by lacking two cysteines of the epidermal growth factor-like domain and a uPAR-binding sequence comparable with that found in mammalian uPA. Accordingly, no zfuPA-b binding activity could be found in fish white blood cells or fish cell lines. We therefore propose that the current consensus of uPA-catalyzed plasminogen activation taking place on cell surfaces, derived from observations with mammals, is too narrow. Fish uPAs appear incapable of receptor binding in the manner known from mammals and uPA-catalyzed plasminogen activation in fish may occur mainly in solution. Studies with nonmammalian vertebrate species are needed to obtain a comprehensive understanding of the mechanism of plasminogen activation.

Bager, René; Kristensen, Thomas K.

2012-01-01

150

Urokinase-type Plasminogen Activator-like Proteases in Teleosts Lack Genuine Receptor-binding Epidermal Growth Factor-like Domains  

DEFF Research Database (Denmark)

Plasminogen activation catalyzed by urokinase-type plasminogen activator (uPA) plays an important role in normal and pathological tissue remodeling processes. Since its discovery in the mid-1980s, the cell membrane-anchored urokinase-type plasminogen activator receptor (uPAR) has been believed to be central to the functions of uPA, as uPA-catalyzed plasminogen activation activity appeared to be confined to cell surfaces through the binding of uPA to uPAR. However, a functional uPAR has so far only been identified in mammals. We have now cloned, recombinantly produced, and characterized two zebrafish proteases, zfuPA-a and zfuPA-b, which by several criteria are the fish orthologs of mammalian uPA. Thus, both proteases catalyze the activation of fish plasminogen efficiently and both proteases are inhibited rapidly by plasminogen activator inhibitor-1 (PAI-1). But zfuPA-a differs from mammalian uPA by lacking the exon encoding the uPAR-binding epidermal growth factor-like domain; zfuPA-b differs from mammalian uPA by lacking two cysteines of the epidermal growth factor-like domain and a uPAR-binding sequence comparable with that found in mammalian uPA. Accordingly, no zfuPA-b binding activity could be found in fish white blood cells or fish cell lines. We therefore propose that the current consensus of uPA-catalyzed plasminogen activation taking place on cell surfaces, derived from observations with mammals, is too narrow. Fish uPAs appear incapable of receptor binding in the manner known from mammals and uPA-catalyzed plasminogen activation in fish may occur mainly in solution. Studies with nonmammalian vertebrate species are needed to obtain a comprehensive understanding of the mechanism of plasminogen activation.

Bager, René; Kristensen, Thomas Kielsgaard

2012-01-01

151

Interleukin-17 modulates myoblast cell migration by inhibiting urokinase type plasminogen activator expression through p38 mitogen-activated protein kinase.  

Science.gov (United States)

Interleukin-17 belongs to a family of pro-inflammatory cytokines with pleiotropic effects, which can be associated with several inflammatory diseases of the muscle tissue. Although elevated levels of interleukin-17 have been described in inflammatory myopathies, its role in muscle homeostasis remains to be elucidated. The requirement of the urokinase type plasminogen activator in skeletal myogenesis was recently demonstrated in vivo and in vitro, suggesting its involvement in the regulation of extracellular matrix remodeling, cell migration and myoblast fusion. Our previous results have demonstrated that interleukin-17 inhibits myogenic differentiation of C2C12 myoblasts in vitro concomitantly with the inhibition of cell migration. However, the involvement of urokinase type plasminogen activator in interleukin-17-inhibited myogenesis and migration remained to be analyzed. Therefore, the effect of interleukin-17 on the production of urokinase type plasminogen activator by C2C12 myoblasts was determined in the present study. Our results demonstrated that interleukin-17 strongly inhibits urokinase type plasminogen activator expression during myogenic differentiation. This reduction of urokinase type plasminogen activator production corresponded with the inhibition of cell migration by interleukin-17. Activation of p38 signaling pathway elicited by interleukin-17 mediated the inhibition of both urokinase type plasminogen activator expression and cell migration. Additionally, IL-17 inhibited C2C12 cells migration by causing the cells to reorganize their cytoskeleton and lose polarity. Therefore, our results suggest a novel mechanism by which interleukin-17 regulates myogenic differentiation through the inhibition of urokinase type plasminogen activator expression and cell migration. Accordingly, interleukin-17 may represent a potential clinical target worth investigating for the treatment of inflammatory muscle diseases. PMID:23183001

Koci?, Jelena; Santibañez, Juan F; Krsti?, Aleksandra; Mojsilovi?, Slavko; Ili?, Vesna; Bugarski, Diana

2013-02-01

152

Concentrations of plasminogen activator inhibitor-1 (PAI-1 and urokinase plasminogen activator (uPA in induced sputum of asthma patients after allergen challenge  

Directory of Open Access Journals (Sweden)

Full Text Available Urokinase plasminogen activator (uPA and its inhibitor (PAI-1 are involved in tiisue remodeling and repairprocesses associated with acute and chronic inflammation. The aim of the study was to evaluate the effect of allergen challengeon concentration of uPA and PAI-1 in induced sputum of house dust mite allergic asthmatics (HDM-AAs. ThirtyHDM-AAs and ten healthy persons (HCswere recruited for the study. In 24 HDM-AAs bronchial challenge with Dermatophagoidespteronyssinus (Dp and in 6 HDM-AAs sham challenege with saline were performed. In HDM-AAs sputumwas induced 24 hours before (T0 and 24 hours (T24 after the challenge. Concentration of uPA and PAI-1 in induced sputumwere determined using immunoenzymatic assays. At T0 in HDM-AAs mean sputum uPA (151±96 pg/ml and PAI-1(4341±1262 pg/ml concentrations were higher than in HC (18.8±6.7 pg/ml; p=0.0002 and 596±180 pg/ml; p<0.0001; foruPA and PAI-1 respectively. After allergen challenge further increase in sputum uPA (187±144 pg/ml; p=0.03 and PAI-1(6252±2323 pg/ml; p<0.0001 concentrations were observed. Moreover, in Dp challenged, but not in saline challengedHDM-AAs the mean uPA/PAI-1 ratio decreased significantly at T24. No significant increase in the studied parameters werefound in sham challenged patients. In HDM-AAs allergen exposure leads to activation of the plasmin system in the airways.Greater increase of the PAI-1 concentration than uPA concentration after allergen challenge may promote airway remodelingand play an important role in the development of bronchial hyperreactivity.

Krzysztof Kowal,

2010-04-01

153

Concentrations of plasminogen activator inhibitor-1 (PAI-1 and urokinase plasminogen activator (uPA in induced sputum of asthma patients after allergen challenge.  

Directory of Open Access Journals (Sweden)

Full Text Available Urokinase plasminogen activator (uPA and its inhibitor (PAI-1 are involved in tiisue remodeling and repair processes associated with acute and chronic inflammation. The aim of the study was to evaluate the effect of allergen challenge on concentration of uPA and PAI-1 in induced sputum of house dust mite allergic asthmatics (HDM-AAs. Thirty HDM-AAs and ten healthy persons (HCswere recruited for the study. In 24 HDM-AAs bronchial challenge with Dermatophagoides pteronyssinus (Dp and in 6 HDM-AAs sham challenege with saline were performed. In HDM-AAs sputum was induced 24 hours before (T0 and 24 hours (T24 after the challenge. Concentration of uPA and PAI-1 in induced sputum were determined using immunoenzymatic assays. At T0 in HDM-AAs mean sputum uPA (151 Â? 96 pg/ml and PAI-1 (4341 Â? 1262 pg/ml concentrations were higher than in HC (18.8 Â? 6.7 pg/ml; p=0.0002 and 596 Â? 180 pg/ml; p<0.0001; for uPA and PAI-1 respectively. After allergen challenge further increase in sputum uPA (187 Â? 144 pg/ml; p=0.03 and PAI-1 (6252 Â? 2323 pg/ml; p<0.0001 concentrations were observed. Moreover, in Dp challenged, but not in saline challenged HDM-AAs the mean uPA/PAI-1 ratio decreased significantly at T24. No significant increase in the studied parameters were found in sham challenged patients. In HDM-AAs allergen exposure leads to activation of the plasmin system in the airways. Greater increase of the PAI-1 concentration than uPA concentration after allergen challenge may promote airway remodeling and play an important role in the development of bronchial hyperreactivity.

Marcin Moniuszko

2011-04-01

154

Ischaemia-reperfusion injury impairs tissue plasminogen activator release in man  

DEFF Research Database (Denmark)

AimsIschaemia-reperfusion (IR) injury causes endothelium-dependent vasomotor dysfunction that can be prevented by ischaemic preconditioning. The effects of IR injury and preconditioning on endothelium-dependent tissue plasminogen activator (t-PA) release, an important mediator of endogenous fibrinolysis, remain unknown.Methods and resultsIschaemia-reperfusion injury (limb occlusion at 200 mmHg for 20 min) was induced in 22 healthy subjects. In 12 subjects, IR injury was preceded by local or remote ischaemic preconditioning (three 5 min episodes of ipsilateral or contralateral limb occlusion, respectively) or sham in a randomized, cross-over trial. Forearm blood flow (FBF) and endothelial t-PA release were assessed using venous occlusion plethysmography and venous blood sampling during intra-arterial infusion of acetylcholine (5-20 µg/min) or substance P (2-8 pmol/min). Acetylcholine and substance P caused dose-dependent increases in FBF (P

Pedersen, Christian MØller; Barnes, Gareth

2011-01-01

155

Effect of recombinant tissue-type plasminogen activator on acute myocardial infarction  

International Nuclear Information System (INIS)

Radionuclide studies were performed in 18 patients with acute myocardial infarction receiving i.v. injection of recombinant tissue-type plasminogen activator (rt-PA) within 12 hr after an attack. Thallium-201 single photon emission computed tomography revealed that infarct size decreased by 42% in the rt-PA treated group, as compared with 25% in the control group. Left ventricular ejection fraction, as found on first-pass radionuclide angiography with Tc-99m PYP, was significantly higher in the rt-PA treated group than the control group (49% vs 38%). Radionuclide imagings were helpful in confirming myocardial salvage after rt-PA intravenous therapy. It was also considered necessary to perform rt-PA therapy as early as possible after an acute myocardial attack. (N.K.)

156

DNA rearrangement causes hepatocarcinogenesis in albumin-plasminogen activator transgenic mice.  

Science.gov (United States)

Hepatocyte-directed production of urokinase-type plasminogen activator (uPA) in transgenic mice is hepatotoxic. Infrequently, hepatocytes arise that do not express uPA, due to physical loss of transgene DNA, and these cells clonally repopulate the entire liver within 3 months of birth. Surprisingly, hepatic tumors appear in these mice beginning at 8 months of age despite the fact that uPA is not oncogenic or genotoxic. Analysis of the transgene locus reveals that tumors arise only from a particular subclass of transgene-deficient cells in which the entire transgene array, and possibly a significant amount of flanking DNA, is deleted. Considering that all transgene-deficient regenerative nodules undergo extensive replication but only a subset gives rise to tumors, we propose that loss of genomic DNA, not mitogenesis per se, is a primary carcinogenic determinant in this model of hepatocarcinogenesis. Images PMID:1454842

Sandgren, E P; Palmiter, R D; Heckel, J L; Brinster, R L; Degen, J L

1992-01-01

157

Structure of the human plasminogen activator inhibitor 1 gene: nonrandom distribution of introns  

International Nuclear Information System (INIS)

Plasminogen activator inhibitor 1 (PAI-1) is the primary inhibitor of tissue-type plasminogen activator and thus performs an essential role in the regulation of the fibrinolytic process. It is a member of a large family of serine protease inhibitors (serpins). The authors determined the structure of the PAI-1 gene in order to more completely investigate the relationship of PAI-1 to other serpins and, at the same time, to begin to delineate structure-function relations in PAI-1 itself. A human genomic cosmid DNA library was screened and found to contain two independent clones, each harboring the entire PAI-1 gene. Restriction site mapping, electron microscopic inspection of heteroduplexes, and nucleotide sequence analysis all demonstrate that the PAI-1 gene is approximately 12.2 kilobase pairs in length and consists of nine exons and eight introns. All intron-exon boundaries are in accord with the GT-AG rule, including a cryptic acceptor splice site found in intron 7. The intron-exon pattern of the PAI-1 gene is distinct from that of most other serpins except that intron 3 of PAI-1 occupies an identical position as intron E of ovalbumin. Comparison of the authors data with the proposed subdomain structure of serpins suggests that seven of the eight introns may occupy a nonrandom position in the gene. These introns either delineate boundaries of individual structural subdomains or are located in random coil regions of the protein. Transcription of the PAI-1 gene in cultn. Transcription of the PAI-1 gene in cultured vascular endothelial cells results in two distinct mRNA species. The data suggest that these two transcripts arise by alternative polyadenylation

158

RNA aptamers as conformational probes and regulatory agents for plasminogen activator inhibitor-1  

DEFF Research Database (Denmark)

The hallmark of serpins is the ability to undergo the so-called "stressed-to-relaxed" switch during which the surface-exposed reactive center loop (RCL) becomes incorporated as strand 4 in central beta-sheet A. RCL insertion drives not only the inhibitory reaction of serpins with their target serine proteases but also the conversion to the inactive latent state. RCL insertion is coupled to conformational changes in the flexible joint region flanking beta-sheet A. One interesting serpin is plasminogen activator inhibitor-1 (PAI-1), a fast and specific inhibitor of the serine proteases tissue-type and urokinase-type plasminogen activator. Via its flexible joints' region, native PAI-1 binds vitronectin and relaxed, protease-complexed PAI-1 certain endocytosis receptors. From a library of 35-nucleotides long 2'-fluoropyrimidine-containing RNA oligonucleotides, we have isolated two aptamers binding PAI-1 by the flexible joint region with low nanomolar K(D) values. One of the aptamers exhibited measurable binding to native PAI-1 only, while the other also bound relaxed PAI-1. While none of the aptamers inhibited the antiproteolytic effect of PAI-1, both aptamers inhibited vitronectin binding and the relaxed PAI-1-binding aptamer also endocytosis receptor binding. The aptamer binding exclusively to native PAI-1 increased the half-life for the latency transition to more than 6 h, manyfold more than vitronectin. Contact with Lys124 in the flexible joint region was critical for strong inhibition of the latency transition and the lack of binding to relaxed PAI-1. We conclude that aptamers yield important information about the serpin conformational switch and, because they can compete with high-affinity protein-protein interactions, may provide leads for pharmacological intervention.

Madsen, Jeppe B; Dupont, Daniel Miotto

2010-01-01

159

Purification and characterization of active and latent forms of recombinant plasminogen activator inhibitor 1 produced in Escherichia coli.  

Science.gov (United States)

Plasminogen activator inhibitor 1 (PAI-1), the principal physiological inhibitor of tissue plasminogen activator (tPA), is a protein of 379 amino acids and belongs to the SERPIN family of serine protease inhibitors. We have previously described methods to express [Sisk et al. (1990) Gene 96, 305-309] and purify [Reilly et al. (1990) J. Biol. Chem. 265, 9570-9574] a highly active form of the protein in substantial amounts, from Escherichia coli. Further analyses of this material showed the presence of small but significant amounts of latent rPAI-1. The present paper describes for the first time purification of latent and active forms of rPAI-1 from a single preparation, as well as the functional and structural characteristics of the two forms. Latent rPAI-1, which has properties similar to the latent forms described by other groups, was separated from active rPAI-1 by high-resolution ion-exchange chromatography or by affinity chromatography using immobilized anhydrotrypsin. It had low intrinsic activity (circular dichroism spectral analyses. However, they did exhibit differences in tryptophan fluorescence, suggesting tertiary structural differences between the two forms. PMID:1382596

Seetharam, R; Dwivedi, A M; Duke, J L; Hayman, A C; Walton, H L; Huckins, N R; Kamerkar, S M; Corman, J I; Woodeshick, R W; Wilk, R R

1992-10-20

160

Plasminogen activator inhibitor 1 is elevated in the children of men with premature myocardial infarction.  

Science.gov (United States)

To assess whether plasminogen activator inhibitor 1 (PAI-1) activity is elevated in the progeny of young coronary men, 193 young subjects were recruited and divided into two groups. Group A consisted of 104 children whose fathers had suffered a myocardial infarction before the age of 55 ("cases"). Eighty-nine young subjects matched for age, sex, body mass index (BMI) and smoking habits without familial history of coronary artery disease (CAD) served as controls (group B). Children with a family history of diabetes mellitus or hypertension were excluded from both groups. We measured PAI-1 activity, tissue-type plasminogen activator (t-PA) antigen, a2-antiplasmin, fibrinogen, lipids and apolipoproteins in both groups. PAI-1 activity levels were also determined in the men who suffered a premature myocardial infarction 4 months after their discharge. PAI-1 activity levels were higher in cases compared to controls (3.13 +/- 1.9 vs 2.17 +/- 1.9 U/ml, p = 0.0014). t-PA antigen and a2-antiplasmin did not differ significantly between the two groups, while fibrinogen, total cholesterol, low-density lipoprotein cholesterol, apolipoprotein B and lipoprotein(a) were significantly higher in group A. PAI-1 was positively correlated with triglycerides (r = 0.22, p = 0.024), apolipoprotein B (r = 0.21, p = 0.039) and fibrinogen (r = 0.22, p = 0.029) in cases and with BMI in both cases (r = 0.37, p = 0.0003) and controls (r = 0.23, p = 0.044). In stepwise multiple regression analysis, only apolipoprotein B (p = 0.008) and BMI (p = 0.0014) were significant determinants of PAI-1 activity in cases. There was also a positive correlation between PAI-1 activity levels of the affected fathers and their children (r = 0.30, p = 0.01). The present data support the hypothesis that elevated PAI-1 levels in the offspring of men with premature myocardial infarction impair their fibrinolytic capacity contributing to their familial predisposition to CAD. PMID:8883280

Rallidis, L S; Megalou, A A; Papageorgakis, N H; Trikas, A G; Chatzidimitriou, G I; Tsitouris, G K

1996-09-01

161

Biochemical properties of recombinant mutants of nonglycosylated single chain urokinase-type plasminogen activator.  

Science.gov (United States)

The role of glycosylation on the enzymatic properties of single chain urokinase-type plasminogen activator (scu-PA) was investigated by site-specific mutagenesis of the glycosylated Asn-302 residu to Gln. In addition, the role of the NH2-terminal polypeptide chain and of the Cys-148 to Cys-279 interchain disulphide bond on the activity of non-glycosylated scu-PA was investigated. Therefore, variants of recombinant scu-PA (rscu-PA) were produced by transfecting Chinese hamster ovary cells with cDNA encoding rscu-PA N302Q (rscu-PA with Asn-302 to Gln mutation), rscu-PA C279A,N302Q (rscu-PA with Cys-279 to Ala and Asn-302 to Gln mutations) or rscu-PA del(N2-F157)C279A,N302Q (rscu-PA C279A,N302Q with deletion of Asn-2 through Phe-157). These mutants were purified to homogeneity from conditioned cell culture medium and were obtained essentially as single chain molecules with specific activities on fibrin plates of (mean +/- S.E.; n = 6) 45,000 +/- 5000. IU/mg, 19,000 +/- 800 IU/mg and < or = 100 IU/mg for rscu-PA N302Q, rscu-PA C279A,N302Q and rscu-PA del(N2-F157)C279A,N302Q, respectively, as compared to 64,000 +/- 2600 IU/mg for wild-type rscu-PA obtained in the same expression system. Plasmin quantitatively converts rscu-PA N302Q and rscu-PA C279A,N302Q to amidolytically active two-chain derivatives with a specific activity of 56,000 IU/mg and 32,000 IU/mg, respectively, as compared to 75,000 IU/mg for wild-type rscu-PA. Plasminogen activation as a function of time was comparable for rscu-PA N302Q and wild-type rscu-PA, and somewhat slower for rscu-PA C279A,N302Q. In a human plasma milieu in vitro, consisting of a 125I-fibrin labeled plasma clot submerged in plasma, 50 percent clot lysis in 2 h required 2.2 micrograms/ml rscu-PA N302Q and 6.0 micrograms/ml rscu-PA C279A,N302Q, as compared to 3.2 micrograms/ml wild-type rscu-PA. In contrast, rscu-PA del(N2-F157)C279A,N302Q was not converted to an amidolytically active two chain derivative by plasmin, and did not induce significant plasminogen activation in purified systems or clot lysis in a human plasma milieu. Following bolus injections in hamsters, the initial half-lives (1.8-2.6 min) and the plasma clearances (0.6-1.5 ml min-1) were comparable for wild-type rscu-PA and for the three rscu-PA mutants. These results suggest that the fibrinolytic activity in a plasma milieu in vitro and the in vivo turnover of rscu-PA are not markedly affected by the absence of carbohydrate.(ABSTRACT TRUNCATED AT 400 WORDS) PMID:1390910

Li, X K; Lijnen, H R; Nelles, L; Hu, M H; Collen, D

1992-09-01

162

A Mechanism for Assembly of Complexes of Vitronectin and Plasminogen Activator Inhibitor-1 from Sedimmentation Velocity Analysis*  

OpenAIRE

Plasminogen activator inhibitor-1 (PAI-1) and vitronectin are cofactors involved in pathological conditions such as injury, inflammation, and cancer, during which local levels of PAI-1 are increased and the active serpin forms complexes with vitronectin. These complexes become deposited into surrounding tissue matrices, where they regulate cell adhesion and peri-cellular proteolysis. The mechanism for their co-localization has not been elucidated. We hypothesize that PAI-1-vitronectin complex...

Minor, Kenneth H.; Schar, Christine R.; Blouse, Grant E.; Shore, Joseph D.; Lawrence, Daniel A.; Schuck, Peter; Peterson, Cynthia B.

2005-01-01

163

Successful thrombolytic therapy with recombinant tissue-type plasminogen activator for massive pulmonary embolism -A case report-  

OpenAIRE

Massive pulmonary embolism is associated with significant perioperative morbidity and mortality. We report here on a case of a 69-year-old man who suffered a massive pulmonary embolism with pulseless electrical activity during knee arthroscopic surgery. After a diagnosis was made by performing transthoracic echocardiography, the patient was treated with recombinant tissue-type plasminogen activator. The patient was transferred to the intensive care unit after his hemodynamic status improved. ...

Kim, Min Soo; Kim, Kye-min; Woo, Seung-hoon; Lim, Yun Hee; Yon, Jun Heum; Jeon, Seung-gyu

2010-01-01

164

Binding and activation of host plasminogen on the surface of Francisella tularensis  

Directory of Open Access Journals (Sweden)

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.

Whitt Michael A

2010-03-01

165

Characterization of indium-111 labeled recombinant tissue plasminogen activator for the imaging of thrombi  

International Nuclear Information System (INIS)

The in vitro functional properties of recombinant tissue plasminogen activator (rt-PA), its biodistribution in mice, and its pharmacokinetics and clot localization properties in dogs have been investigated after labeling rt-PA with 111In. The rt-PA was coupled with the cyclic anhydride of DTPA using standard methodology. Amidolytic and fibrinolytic assays showed retention of protein activity when rt-PA was conjugated with an average of one DTPA group or less per molecule. Size exclusion HPLC showed each preparation to be radiochemically pure with 111In bound exlusively to the attached DTPA groups. Biodistribution in mice showed major accumulation of activity in the liver and kidneys. After administration of 0.5-1.0 mg of the labeled protein to dogs, blood activity decreased with a half time of approximately 5 min in agreement with previous reports of rapid blood clearance. Largely because of decreased blood levels, clot:blood ratios of labeled protein increased rapidly, in one study reaching 6.3 after 31 min, and satisfactory images of fibrin thrombi were obtained. The rt-PA may be labeled with 111In without destroying the ability of the protein to loacalize n clot and images of forming clot can be obtained with this agent within 1 h after administration. (orig.)

166

Characterization of indium-111 labeled recombinant tissue plasminogen activator for the imaging of thrombi  

Energy Technology Data Exchange (ETDEWEB)

The in vitro functional properties of recombinant tissue plasminogen activator (rt-PA), its biodistribution in mice, and its pharmacokinetics and clot localization properties in dogs have been investigated after labeling rt-PA with /sup 111/In. The rt-PA was coupled with the cyclic anhydride of DTPA using standard methodology. Amidolytic and fibrinolytic assays showed retention of protein activity when rt-PA was conjugated with an average of one DTPA group or less per molecule. Size exclusion HPLC showed each preparation to be radiochemically pure with /sup 111/In bound exlusively to the attached DTPA groups. Biodistribution in mice showed major accumulation of activity in the liver and kidneys. After administration of 0.5-1.0 mg of the labeled protein to dogs, blood activity decreased with a half time of approximately 5 min in agreement with previous reports of rapid blood clearance. Largely because of decreased blood levels, clot:blood ratios of labeled protein increased rapidly, in one study reaching 6.3 after 31 min, and satisfactory images of fibrin thrombi were obtained. The rt-PA may be labeled with /sup 111/In without destroying the ability of the protein to loacalize n clot and images of forming clot can be obtained with this agent within 1 h after administration.

Hnatowich, D.J.; Virzi, F.; Doherty, P.W.; Wilson, J.; Rosa, J.; Ansell, J.E.

1987-12-01

167

Tumor marker utility and prognostic relevance of cathepsin B, cathepsin L, urokinase-type plasminogen activator, plasminogen activator inhibitor type-1, CEA and CA 19-9 in colorectal cancer  

OpenAIRE

Abstract Background Cathepsin B and L (CATB, CATL), urokinase-type plasminogen activator (uPA) and its inhibitor PAI-1 play an important role in colorectal cancer invasion. The tumor marker utility and prognostic relevance of these proteases have not been evaluated in the same experimental setting and compared with that of CEA and CA-19-9. Methods Protease, CEA and CA 19-9 serum or plasma levels were determined in 56 patients with colorectal cancer, 25 patients ...

De Paoli Massimo; Hritz István; Molnár László D; István Gábor; Cardin Romilda; Farinati Fabio; Herszényi László; Plebani Mario; Tulassay Zsolt

2008-01-01

168

Targeted delivery of tissue plasminogen activator by binding to silica-coated magnetic nanoparticle  

Directory of Open Access Journals (Sweden)

Full Text Available Jyh-Ping Chen,1 Pei-Ching Yang,1 Yunn-Hwa Ma,2 Su-Ju Tu,3 Yu-Jen Lu1,41Department of Chemical and Materials Engineering, 2Department of Physiology and Pharmacology, 3Department of Medical Imaging and Radiological Sciences, Chang Gung University, Kwei-San, Taoyuan, Taiwan, Republic of China; 4Department of Neurosurgery, Chang Gung Memorial Hospital, Kwei-San, Taoyuan, Taiwan, Republic of ChinaBackground and methods: Silica-coated magnetic nanoparticle (SiO2-MNP prepared by the sol-gel method was studied as a nanocarrier for targeted delivery of tissue plasminogen activator (tPA. The nanocarrier consists of a superparamagnetic iron oxide core and an SiO2 shell and is characterized by transmission electron microscopy, Fourier transform infrared spectroscopy, X-ray diffraction, superconducting quantum interference device, and thermogravimetric analysis. An amine-terminated surface silanizing agent (3-aminopropyltrimethoxysilane was used to functionalize the SiO2 surface, which provides abundant —NH2 functional groups for conjugating with tPA.Results: The optimum drug loading is reached when 0.5 mg/mL tPA is conjugated with 5 mg SiO2-MNP where 94% tPA is attached to the carrier with 86% retention of amidolytic activity and full retention of fibrinolytic activity. In vitro biocompatibility determined by lactate dehydrogenase release and cell proliferation indicated that SiO2-MNP does not elicit cytotoxicity. Hematological analysis of blood samples withdrawn from mice after venous administration indicates that tPA-conjugated SiO2-MNP (SiO2-MNP-tPA did not alter blood component concentrations. After conjugating to SiO2-MNP, tPA showed enhanced storage stability in buffer and operation stability in whole blood up to 9.5 and 2.8-fold, respectively. Effective thrombolysis with SiO2-MNP-tPA under magnetic guidance is demonstrated in an ex vivo thrombolysis model where 34% and 40% reductions in blood clot lysis time were observed compared with runs without magnetic targeting and with free tPA, respectively, using the same drug dosage. Enhanced penetration of SiO2-MNP-tPA into blood clots under magnetic guidance was confirmed from microcomputed tomography analysis.Conclusion: Biocompatible SiO2-MNP developed in this study will be useful as a magnetic targeting drug carrier to improve clinical thrombolytic therapy.Keywords: magnetic nanoparticles, drug delivery, thrombolysis, tissue plasminogen activator, silica

Chen JP

2012-09-01

169

Differential effects of tissue plasminogen activator and streptokinase on infarct size and on rate of enzyme release: influence of early infarct related artery patency  

OpenAIRE

BACKGROUND: The recent international GUSTO trial of 41,021 patients with acute myocardial infarction demonstrated improved 90-min infarct related artery patency as well as reduced mortality in patients treated with an accelerated regimen of tissue plasminogen activator, compared to patients treated with streptokinase. A regimen combining tissue plasminogen activator and streptokinase yielded intermediate results. The present study investigated the effects of treatment on infarct size and enzy...

Baardman, T.; Hermens, W. T.; Molhoek, G. P.; Grollier, G.; Pfisterer, M. E.; Simoons, M. L.; Lenderink, T.

1996-01-01

170

Elevated levels of plasminogen activators in the pathogenesis of delayed radiation damage in rat cervical spinal cord in vivo  

International Nuclear Information System (INIS)

The pathophysiology of the cellular basis of radiation-induced demyelination and white-matter necrosis of the central nervous system (CNS) is poorly understood. Preliminary data suggest that tissue damage is partly mediated through changes in the proteolytic enzymes. In this study, we irradiated rat cervical spinal cords with single doses of 24 Gy of 18 MV photons or 20 MeV electrons and measured the levels of plasminogen activators at days 2, 7, 30, 60, 90, 120, 130 and 145 after irradiation, using appropriate controls at each time. Fibrin zymography revealed fibrinolytic bands representing molecular weights of 68,000 and 48,000 in controls and irradiated samples; these bands increased significantly at days 120, 130 and 145 after irradiation. Inhibition of these enzymatic bands with specific antibodies against tissue-type plasminogen activator (tPA) and amiloride, an inhibitor for urokinase plasminogen activator (uPA), confirmed that these bands were tPA and uPA. Enzymatic levels quantified by densitometry showed a twofold elevation in the levels of tPA and more than a tenfold increase in uPA after 120 days' irradiation. Activity of uPA was increased threefold by day 2 and increased steadily with time compared to nonirradiated control samples. Enzyme-linked immunosorbent assay (ELISA) also showed a threefold increase in the tPA content in the extracts of irradiated rat cervical spinal cords at days 120, 130 and 145. This study adds additional information to the propoy adds additional information to the proposed role of plasminogen activators in the pathogenic pathways of radiation damage in the CNS. 38 refs., 6 figs

171

Elevated levels of plasminogen activators in the pathogenesis of delayed radiation damage in rat cervical spinal cord in vivo  

Energy Technology Data Exchange (ETDEWEB)

The pathophysiology of the cellular basis of radiation-induced demyelination and white-matter necrosis of the central nervous system (CNS) is poorly understood. Preliminary data suggest that tissue damage is partly mediated through changes in the proteolytic enzymes. In this study, we irradiated rat cervical spinal cords with single doses of 24 Gy of 18 MV photons or 20 MeV electrons and measured the levels of plasminogen activators at days 2, 7, 30, 60, 90, 120, 130 and 145 after irradiation, using appropriate controls at each time. Fibrin zymography revealed fibrinolytic bands representing molecular weights of 68,000 and 48,000 in controls and irradiated samples; these bands increased significantly at days 120, 130 and 145 after irradiation. Inhibition of these enzymatic bands with specific antibodies against tissue-type plasminogen activator (tPA) and amiloride, an inhibitor for urokinase plasminogen activator (uPA), confirmed that these bands were tPA and uPA. Enzymatic levels quantified by densitometry showed a twofold elevation in the levels of tPA and more than a tenfold increase in uPA after 120 days` irradiation. Activity of uPA was increased threefold by day 2 and increased steadily with time compared to nonirradiated control samples. Enzyme-linked immunosorbent assay (ELISA) also showed a threefold increase in the tPA content in the extracts of irradiated rat cervical spinal cords at days 120, 130 and 145. This study adds additional information to the proposed role of plasminogen activators in the pathogenic pathways of radiation damage in the CNS. 38 refs., 6 figs.

Sawaya, R.; Rayford, A.; Kono, S.; Rao, J.S.; Ang, K.K.; Feng, Y.; Stephens, L.C. [Univ. of Texas, Houston, TX (United States)

1994-06-01

172

Tissue Plasminogen Activator Alters Intracellular Sequestration of Zinc through Interaction with the Transporter ZIP4  

Energy Technology Data Exchange (ETDEWEB)

Glutamatergic neurons contain free zinc packaged into neurotransmitter-loaded synaptic vesicles. Upon neuronal activation, the vesicular contents are released into the synaptic space, whereby the zinc modulates activity of postsynaptic neurons though interactions with receptors, transporters and exchangers. However, high extracellular concentrations of zinc trigger seizures and are neurotoxic if substantial amounts of zinc reenter the cells via ion channels and accumulate in the cytoplasm. Tissue plasminogen activator (tPA), a secreted serine protease, is also proepileptic and excitotoxic. However, tPA counters zinc toxicity by promoting zinc import back into the neurons in a sequestered form that is nontoxic. Here, we identify the zinc influx transporter, ZIP4, as the pathway through which tPA mediates the zinc uptake. We show that ZIP4 is upregulated after excitotoxin stimulation of the mouse, male and female, hippocampus. ZIP4 physically interacts with tPA, correlating with an increased intracellular zinc influx and lysosomal sequestration. Changes in prosurvival signals support the idea that this sequestration results in neuroprotection. These experiments identify a mechanism via which neurons use tPA to efficiently neutralize the toxic effects of excessive concentrations of free zinc.

Emmetsberger, Jaime; Mirrione, Martine M.; Zhou, Chun; Fernandez-Monreal, Monica; Siddiq, Mustafa M.; Ji, Kyungmin; Tsirka, Stella E. (SBU)

2010-09-17

173

Sustained thrombolysis with DNA-recombinant tissue type plasminogen activator in rabbits  

International Nuclear Information System (INIS)

Tissue type plasminogen activator (t-PA) is an effective thrombolytic agent in experimental animals. The duration of the thrombolytic effect of infused t-PA is unknown. The authors compared the duration of the thrombolytic effect of t-PA with streptokinase by measuring the lysis of 125I-fibrin-labeled thrombi in rabbit jugular veins at different times after a bolus injection of the fibrinolytic agents. The pharmacodynamics of both thrombolytic agents were determined in rabbits using a sensitive ex vivo fibrinolytic assay. Streptokinase and t-PA were given as a bolus dose of 15,000 U/kg. There was no detectable circulating fibrinolytic activity 30 minutes after the bolus dose of t-PA and 120 minutes after the bolus dose of streptokinase. The t-PA injection produced 34% thrombolysis at 30 minutes, 90% thrombolysis at 120 minutes, and 96% thrombolysis at 240 minutes. The streptokinase injection produced 17% thrombolysis at 30 minutes, 34% at 120 minutes, and 34% at 240 minutes. These observations indicate that the thrombolytic effect of t-PA is sustained beyond its time of clearance from the circulation whereas the thrombolytic effect of streptokinase closely parallels its activity in the circulation

174

Enhanced levels of urokinase plasminogen activator and its soluble receptor in common variable immunodeficiency  

DEFF Research Database (Denmark)

Common variable immunodeficiency (CVID) is a heterogeneous syndrome characterized by defective immunoglobulin production and high frequency of bacterial infections, autoimmunity and manifestations of chronic inflammation. The urokinase plasminogen activator (uPA), its cell bound and soluble receptor (uPAR, suPAR) have complex biological functions involving innate immune defense mechanisms and regulation of inflammation. Based on this dual role, we hypothesized that the uPA system could be affected in CVID, and examined expression of components of the uPA system in subgroups of CVID. All CVID-patients had increased plasma levels of suPAR with particularly high levels in those with splenomegaly and thrombocytopenia. Plasma uPA levels were also raised in these patients, and both suPAR and uPA levels correlated with the monocyte activation marker neopterin. Monocytes from CVID patients had increased expression of uPAR. We show an increased activation of the uPA system possibly contributing to the inflammatory phenotype seen in subgroups of CVID patients.

Fevang, BØrre; Eugen-Olsen, Jesper

2009-01-01

175

Urokinase-type plasminogen activator-deficient mice are predisposed to staphylococcal botryomycosis, pleuritis, and effacement of lymphoid follicles.  

OpenAIRE

Urokinase-type plasminogen activator (uPA) is thought to be an important mediator in the proteolytic degradation of extracellular matrix components observed in a wide variety of normal physiological and pathological conditions. However, the phenotype of a recently developed strain of urokinase-deficient (uPA-/-) mice appears to be normal when maintained under ideal nonstressful conditions. We report an outbreak of botryomycosis, an unusual staphylococcal infection, in a colony of uPA-deficien...

Shapiro, R. L.; Duquette, J. G.; Nunes, I.; Roses, D. F.; Harris, M. N.; Wilson, E. L.; Rifkin, D. B.

1997-01-01

176

Pretreatment Plasminogen Activator Inhibitor-1 (PAI-1) Level in the Failure of Streptokinase Therapy in Acute Myocardial Infaction  

OpenAIRE

Background: The risk for unsuccessful reperfusion after streptokinase therapy may be caused by the antifibrinolytic effect of platelet-derived type 1 plasminogen activator inhibitor (Pal-1) and antistreptokinase antibodies. This study aims to show the relation of pretreatment PAI-1 levels of patients with acute myocardial infarction treated with streptokinase and the outcome of fibrinolysis, emphasizing on reperfusion with and without considering pretreatment antistreptokinase antibodies. Met...

Shemirani, H.; Tajmirriahi, M.; Rezaei, A.; Oreisi, F.

2004-01-01

177

Thrombolysis with tissue plasminogen activator in acute myocardial infarction: no additional benefit from immediate percutaneous coronary angioplasty  

OpenAIRE

A randomised trial of 367 patients with acute myocardial infarction was performed to determine whether an invasive strategy combining thrombolysis with recombinant tissue-type plasminogen activator (rTPA), heparin, and acetylsalicylic acid, and immediate percutaneous transluminal coronary angioplasty (PTCA) would be superior to a noninvasive strategy with the same medical treatment but without immediate angiography and PTCA. Intravenous infusion of 100 mg rTPA was started within 5 h after ons...

Simoons, M. L.; Betriu, A.; Col, J. J.; Essen, R. Von; Lubsen, J.; Michel, P. L.; Rutsch, W. R.; Schmidt, W.; Thery, C.; Vahanian, A.; Willems, G. M.; Bono, D. P.; Dougherty, F. C.; Lambertz, H.; Meier, B.

1988-01-01

178

Challenging delivery of VLHL NS plasminogen activator inhibitor-1 by osmotic pumps in diabetic mouse: A case report  

OpenAIRE

ALZET® osmotic pumps are implantable devices used in animals for the continuous infusion of drugs or proteins at controlled rates from 1 day to 4 weeks. Pumps have been used successfully in a number of studies on the effects of controlled delivery of a wide range of experimental agents, independent of their properties. In the present study, use of these pumps was made in mice with diabetic nephropathy. Plasminogen activator inhibitor-1 (PAI-1) mediates diabetic nephropathy, which is characte...

Jankun, Jerzy

2012-01-01

179

Intrapleural Adenoviral Delivery of Human Plasminogen Activator Inhibitor–1 Exacerbates Tetracycline-Induced Pleural Injury in Rabbits  

OpenAIRE

Elevated concentrations of plasminogen activator inhibitor–1 (PAI-1) are associated with pleural injury, but its effects on pleural organization remain unclear. A method of adenovirus-mediated delivery of genes of interest (expressed under a cytomegalovirus promoter) to rabbit pleura was developed and used with lacZ and human (h) PAI-1. Histology, ?-galactosidase staining, Western blotting, enzymatic and immunohistochemical analyses of pleural fluids (PFs), lavages, and pleural mesothelial...

Karandashova, Sophia; Florova, Galina; Azghani, Ali O.; Komissarov, Andrey A.; Koenig, Kathy; Tucker, Torry A.; Allen, Timothy C.; Stewart, Kris; Tvinnereim, Amy; Idell, Steven

2013-01-01

180

Plasminogen Activator Pla of Yersinia pestis Utilizes Murine DEC-205 (CD205) as a Receptor to Promote Dissemination*  

OpenAIRE

Yersinia pestis, a Gram-negative bacterium that causes bubonic and pneumonic plague, is able to rapidly disseminate to other parts of its mammalian hosts. Y. pestis expresses plasminogen activator (PLA) on its surface, which has been suggested to play a role in bacterial dissemination. It has been speculated that Y. pestis hijacks antigen-presenting cells, such as macrophages (M?s) and dendritic cells, to be delivered to lymph nodes to initiate dissemination and infec...

Zhang, Shu-sheng; Park, Chae Gyu; Zhang, Pei; Bartra, Sara Schesser; Plano, Gregory V.; Klena, John D.; Skurnik, Mikael; Hinnebusch, B. Joseph; Chen, Tie

2008-01-01

181

Age-dependent neonatal intracerebral hemorrhage in plasminogen activator inhibitor 1 knockout mice.  

Science.gov (United States)

Intracerebral-intraventricular hemorrhages (ICH/IVH) in very preterm neonates are responsible for high mortality and subsequent disabilities. In humans, tissue plasminogen activator (t-PA) initiates fibrinolysis and activates endoluminal-endothelial receptors; dysfunction of the t-PA inhibitor (PAI-1) results in recurrent hemorrhages. We used PAI-1 knockout (PAI-1) mice to examine the role of t-PA in age-dependent intracranial hemorrhages as a possible model of preterm ICH/IVH. Intracortical injection of 2 ?L of phosphate-buffered saline produced a small traumatic injury and a high rate of hemorrhage in PAI-1 pups at postnatal day 3 (P3) or P5, whereas it had no effect in wild-type neonates. This resulted in white matter and cortical lesions, ventricle enlargement, hyperlocomotion, and altered cortical levels of serotonin and dopamine in the adult PAI mice. N-methyl-D-aspartate receptor blockers, plasmin- and matrix metalloproteinases inhibitors reduced hemorrhage and tissue lesions. In contrast to P3 to P5, no significant hemorrhages were induced in P10 PAI-1 pups and there were no behavioral or neurochemical alterations in adulthood. These data suggest that microvascular immaturity up to P5 in mice is a determinant factor required for t-PA-dependent vascular rupture. Neonatal PAI-1 mice could be a useful ICH/IVH model for studying the ontogenic window of vascular immaturity and vascular protection against later neurodisabilities. PMID:24709679

Leroux, Philippe; Omouendze, Priscilla L; Roy, Vincent; Dourmap, Nathalie; Gonzalez, Bruno J; Brasse-Lagnel, Carole; Carmeliet, Peter; Leroux-Nicollet, Isabelle; Marret, Stéphane

2014-05-01

182

The role of tissue-type plasminogen activator system in amphetamine-induced conditional place preference extinction and reinstatement.  

Science.gov (United States)

Extracellular serine proteases of the plasminogen activator family (tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA) may modulate synaptic adhesion and associate with learning behavior. Psychostimulants strongly induce their expression in the mesolimbic dopaminergic pathway, but cocaine preferentially induces uPA, whereas morphine and amphetamine preferentially induce tPA. tPA-expressing animals displayed enhanced conditional place preference (CPP) for amphetamine compared with uPA-overexpressing animals. Thus, modulation of the plasminogen system in the brain might be a potential target against drugs of abuse. In the present study, we aim to identify whether tPA is involved in the acquisition/learning phase or in the expression/retrieval phase of conditioned drug preference. For this purpose, animals were injected with lentiviruses expressing or silencing tPA in the NAc and place preference was assessed. We found that tPA expression is associated with acquisition of place preference and animals overexpressing tPA spend >87% of the time in the drug-associated compartment, compared with 60% for control animals. When ectopic expression of tPA has been inhibited by doxycycline during acquisition, animals do no more associate the environment with the drug. Suppression of endogenous tPA expression in animals treated with LV-siRNA fully suppresses place preference, and these animals appear to avoid the drug-associated box. tPA overexpression delays extinction, but priming with low doses of amphetamine reinstates place preference even after full extinction. Together, these data clearly indicate that tPA plays an important role in acquisition of amphetamine-induced CPP, but its role in CPP expression does not seem important. PMID:18256596

Bahi, Amine; Kusnecov, Alexander; Dreyer, Jean-Luc

2008-10-01

183

Streptococcal cell walls and synovial cell activation. Stimulation of synovial fibroblast plasminogen activator activity by monocytes treated with group A streptococcal cell wall sonicates and muramyl dipeptide  

OpenAIRE

Group A streptococcal peptidoglycan has previously been shown to be arthritogenic in rats and has been implicated as a structure present in a class of possible etiologic agents for rheumatoid arthritis. The present study reports that conditioned medium from human monocytes, after interaction with cell wall sonicates of four group A streptococcal strains, stimulates the plasminogen activator (PA) activity of nonrheumatoid synovial fibroblasts. Low concentrations of N- acetylmuramyl-L-alanyl-D ...

1982-01-01

184

A novel plasminogen activator from Agkistrodon blomhoffii Ussurensis venom (ABUSV-PA): Purification and characterization  

International Nuclear Information System (INIS)

A plasminogen activator with arginine ester hydrolysis activity (ABUSV-PA) has been identified and purified to homogeneity from Chinese Agkistrodon blomhoffii Ussurensis snake venom. ABUSV-PA, a monomeric protein with molecular mass of 27815.2 Da, was purified 180-fold with 0.02% recovery for protein and 3.6% recovery for esterase activity. ABUSV-PA reacts optimally with its substrate N ?-tosyl-L-arginine-methyl ester (TAME) at ?pH 7.5 and at 51 oC. Measurement from inductively coupled plasma-atomic emission spectroscopy (ICP-AES) reveals that ABUSV-PA is a Zn2+-containing protein with a stoichiometry of 1:1 [Zn2+]:[ABUSV-PA]. Analyses of esterase hydrolysis and UV absorption and CD spectra indicate that Zn2+ plays an important role in maintaining the structural integrity rather than the esterase activity of ABUSV-PA. Divalent metal ions, including Ca2+, Mg2+, Cu2+, Ni2+, Mn2+, and Co2+, increase the TAME hydrolysis activity of ABUSV-PA. A red-shift of the emission wavelengths of the synchronous fluorescence of ABUSV-PA, compared to those of free Tyr and Trp, indicates a conformation where the Tyr and Trp residues are in exposed hydrophilic environments. The presence of zinc increases the hydrophobicity of the conformational environments surrounding the Trp residues of ABUSV-PA and affects the secondary structure of ABUSV-PA, as proved by UV absocture of ABUSV-PA, as proved by UV absorption and CD spectroscopy

185

The use of intravenous recombinant tissue plasminogen activator in acute ischemic stroke.  

Science.gov (United States)

We sought to determine the frequency of use of intravenous (i.v.) recombinant tissue plasminogen activator (rt-PA) in patients presenting to our institution with acute ischemic stroke (AIS). This observational study involved keeping a log of all patients presenting to our institution with symptoms consistent with AIS who were potential candidates for emergency thrombolysis over a 3-year period. The log included brain computed tomography (CT) scan results, whether or not rt-PA was administered, and contraindications to thrombolysis. It also included each patient's time flow through the system, from symptom onset to decision time regarding (and administration of) thrombolytics. Over the 36-month period of the study, there were 142 patients who presented to the Emergency Department (ED) who initially were thought to be potential candidates for thrombolysis for AIS. Ninety-five (68.5%) of these 142 patients had a confirmed diagnosis of AIS. On further clarification of symptom onset, 77 (81%) of these 95 patients with AIS actually presented within 3 h, and 17 (22%) of these 77 patients met criteria for thrombolysis and had no contraindications. All 17 (100%) patients with AIS presenting within 3 h of onset and without contraindications received i.v. rt-PA in the ED. In conclusion, i.v. rt-PA can be administered for AIS within the 3-h window if a hospital is committed to providing this treatment. Thrombolysis remains a treatment for a minority of AIS patients. PMID:16183445

Kahn, Joseph H; Viereck, Jason; Kase, Carlos; Jeerakathil, Thomas; Romero, Rafael; Mehta, Supriya D; Kociol, Robb; Babikian, Viken

2005-10-01

186

TFRC and ACTB as the best reference genes to quantify Urokinase Plasminogen Activator in breast cancer  

Directory of Open Access Journals (Sweden)

Full Text Available Abstract Background Biomedical researchers have long looked for ways to diagnose and treat cancer patients at the early stages through biomarkers. Although conventional techniques are routinely applied in the detection of biomarkers, attitudes towards using Real-Time PCR techniques in detection of many biomarkers are increasing. Normalization of quantitative Real-Time PCR is necessary to validate non-biological alteration occurring during the steps of RNA quantification. Selection of variably expressed housekeeping genes (HKs will affect the validity of the data. The aim of the present study was to identify uniformly expressed housekeeping genes in order to use in the breast cancer gene expression studies. Urokinase Plasminogen Activator was used as a gene of interest. Findings The expression of six HKs (TFRC, GUSB, GAPDH, ACTB, HPRT1 and RPLP0 was investigated using geNorm and NormFinder softwares in forty breast tumor, four normal and eight adjacent tissues. RPLP0 and GAPDH revealed maximum M value, while TFRC demonstrated lowest M value. Conclusions In the present study the most and the least stable genes were TFRC and RPLP0 respectively. TFRC and ACTB were verified as the best combination of two genes for breast cancer quantification. The result of this study shows that in each gene expression analysis HKs selection should be done based on experiment conditions.

Abdoli Nasrin

2011-06-01

187

Factors predicting intracerebral hemorrhage of patients treated with intravenous recombinant tissue plasminogen activator  

International Nuclear Information System (INIS)

The use of recombinant tissue plasminogen activator (rt-PA) was approved in Japan in October 2005, and has had a marked effect on the treatment of patients presenting with acute ischemic stroke. Since the administration of rt-PA might cause intracerebral hemorrhage (ICH) and a poor prognosis, it is necessary to identify predictors of ICH after treatment with rt-PA. In this article, we examined 58 consecutive patients with acute ischemic stroke treated with intravenous rt-PA within 3 hours of symptom onset for 45 months, March 2006 to November 2009. In principle, we evaluated patients before and one day after rt-PA with MRI. We made a retrospective comparison of 21 patients with hemorrhagic change on CT and MRI T2* within 36 hours and 37 patients without hemorrhagic change. The rate of ICH with or without symptoms was increased with a higher National Institutes of Health Stroke Scale (NIHSS) and infarction range, defined by diffusion weighted imaging (DWI) Alberta Stroke Programme Early CT Score (ASPECTS). Major artery occlusion and reperfusion, including partial recanalization in MR angiography (MRA), were taken as factors in the hemorrhage group. In conclusion, DWI ASPECTS and NIHSS were useful predictors of ICH after rt-PA administration. (author)

188

Differential effects of low-dose tissue plasminogen activator and streptokinase on platelet aggregation.  

Science.gov (United States)

Despite increasing success with low-dose intra-arterial thrombolysis, early rethrombosis still occurs. Platelet aggregation is thought to play a major part in this process. We have therefore investigated the effects of recombinant tissue plasminogen activator (rt-PA) and streptokinase on platelet function at doses currently used for peripheral arterial thrombolysis. Platelet-rich plasma was stirred at 37 degrees C, with either streptokinase (100, 300 or 1000 units ml-1) or rt-PA (10 (T10), 30 (T30) and 100 (T100) mg l-1), with immediate addition of an agonist for platelet aggregation (thrombin, collagen, adenosine diphosphate (ADP) or adrenaline) at a predetermined threshold dose. Significant inhibition of collagen-induced and adrenaline-induced platelet aggregation was produced with rt-PA at all doses used (P less than 0.05). With adrenaline as the agonist, T100 produced disaggregation to a mean (s.d.) level of 26 per cent. Thrombin-stimulated platelet aggregation was significantly reduced by T100 (P less than 0.001) and T30 (P less than 0.01) only, disaggregation being dose-dependent and complete with T100. Using ADP as the agonist, T100 produced a significant reduction in maximum platelet aggregation (P less than 0.01), and disaggregation was achieved to a mean (s.d.) level of 48(13) per cent. Streptokinase did not produce any significant changes in any parameter of aggregation. PMID:2513082

Berridge, D C; Burgess-Wilson, M E; Westby, J C; Hopkinson, B R; Makin, G S

1989-10-01

189

Pericyte protection by edaravone after tissue plasminogen activator treatment in rat cerebral ischemia.  

Science.gov (United States)

Pericytes play a pivotal role in contraction, mediating inflammation and regulation of blood flow in the brain. In this study, changes of pericytes in the neurovascular unit (NVU) were examined in relation to the effects of exogenous tissue plasminogen activator (tPA) and a free radical scavenger, edaravone. Immunohistochemistry and Western blot analyses showed that the overlap between platelet-derived growth factor receptor ?-positive pericytes and N-acetylglucosamine oligomers (NAGO)-positive endothelial cells increased significantly at 4 days after 90 min of transient middle cerebral artery occlusion (tMCAO). The number of pericytes and the overlap with NAGO decreased with tPA but recovered with edaravone 4 days after tMCAO with proliferation. Thus, tPA treatment damaged pericytes, resulting in the detachment from astrocytes and a decrease in glial cell line-derived neurotrophic factor secretion. However, treatment with edaravone greatly improved tPA-induced damage to pericytes. The present study demonstrates that exogenous tPA strongly damages pericytes and destroys the integrity of the NVU, but edaravone treatment can greatly ameliorate such damage after acute cerebral ischemia in rats. PMID:24938625

Deguchi, Kentaro; Liu, Ning; Liu, Wentao; Omote, Yoshio; Kono, Syoichiro; Yunoki, Taijun; Deguchi, Shoko; Yamashita, Toru; Ikeda, Yoshio; Abe, Koji

2014-11-01

190

Angiotensinogen and Plasminogen Activator Inhibitor-1 Gene Polymorphism in Relation to Renovascular Disease  

International Nuclear Information System (INIS)

The present study was designed to evaluate angiotensinogen (AGT) M235T and plasminogen activator inhibitor-1 (PAI-1) (4G/5G) polymorphisims in relation to the occurrence of atherosclerotic renal artery stenosis (ARAS) and recurrent stenosis. In this study, 30 patients were enrolled after angiographic demonstration of ARAS; 100 healthy subjects for AGT polymorphism and 80 healthy subjects for PAI-1 polymorphism were considered the control group. The patients were followed for a mean 46.1 ± 9.2 months. The patients had significantly higher frequencies of the MT genotype and the T allele than control group (?2 = 18.2, p 2 = 11.5 p 2= 2.45, p = 0.29 and ?2 = 0.019, p = 0.89). There were no significant differences in the genotype and allele findings for the patients with and without restenosis (p > 0.05). The C-reactive protein (CRP) level was higher in the patients with restenosis than in the patients without restenosis (7.694 ± 0.39 mg/L and 1.56 ± 1.08 mg/L) (p = 0.001). Our results suggest that the M235T MT genotype and T allele might be associated with increased risk of atherosclerotic renal artery stenosis. The CRP level might be an independent predictor for recurrent stenosis

191

Binding of tissue plasminogen activator to human umbilical vein endothelial cells  

Energy Technology Data Exchange (ETDEWEB)

The binding of purified, recombinant tissue plasminogen activator (tPA) to human umbilical vein endothelial cells (HUVEC) was studied in vitro using immunofluorescence as well as radiolabeled tPA. Immunofluorescence was performed on HUVEC grown on round glass coverslips using rabbit anti-human tPA and fluorescein-conjugated anti-rabbit immunoglobulin. Positive fluorescence was observed only after incubation of HUVEC with tPA. HUVEC were grown to confluence in 24-well tissue culture plates, washed, and incubated with a constant amount of /sup 125/I-tPA and various concentrations of unlabeled tPA. The binding of tPA to HUVEC was found to be specific, saturable, and reversible. Scatchard analysis yielded as equilibrium constant (K/sub eq/) of 4.2 x 10/sup 6/ M/sup -1/ and 1.2 x 10/sup 7/ binding sites per cell. Binding was inhibited by positively charged amino acids and by D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone but not by carbohydrates including mannose, galactose, N-acetyl glucosamine and N-acetyl galactosamine. Neat human plasma abrogates but does not totally inhibit binding of tPA to HUVEC. Binding was neither enhanced nor inhibited by fibronectin. Although the affinity of binding of tPA to HUVEC is low, the endothelial cell may be involved in regulating plasma levels of tPA in vivo which may have therapeutic significance.

Beebe, D.P.

1987-05-01

192

Pneumatic Displacement of a Dense Submacular Hemorrhage with or without Tissue Plasminogen Activator  

Directory of Open Access Journals (Sweden)

Full Text Available Background: To evaluate the efficacy of treating a dense submacular hemorrhage withpneumatic displacement with or without tissue plasminogen activator (tPA.Methods: Twenty-four patients with a dense submacular hemorrhage were treated withintravitreal expansile gas, with or without an intravitreal injection of tPA, inorder to displace the submacular blood. The main outcome measurementsinclude preoperative and postoperative visual acuity, postoperative fluoresceinangiography (FAG results and additional postoperative treatments.Results: Total or subtotal subfoveal blood displacement was achieved in all 24 eyes.After a mean follow-up of 15.5 months (range 6-50 months, final visualacuity had improved two or more lines in 11 (45.8% of the 24 eyes, andmeasured 20/100 or better in 10 (4l.7% of the 11 eyes. Based on the FAGresults for 14 cases, nine eyes (64.3% received additional postoperativelaser treatment. Final visual acuity of 20/100 or better was achieved in four(40% of the 10 eyes, with a choroidal neovascular membrane (CNVMdetected on FAG, and dye leakage not detected in three (75% of the foureyes.Conclusions: Pneumatic displacement, with or without intravitreal injection of tPA, seemsuseful in displacing dense submacular hemorrhage and facilitating visualimprovement, although the visual result is often limited by the progression ofthe underlying macular disease. In patients with age-related macular degeneration,more treatable CNVM may be detected on postoperative FAG.

Po-Min Yang

2005-12-01

193

Successful treatment of mobile right atrial thrombus and acute pulmonary embolism with intravenous tissue plasminogen activator.  

Science.gov (United States)

An 89-year-old woman came with symptoms of progressively worsening dyspnoea at rest over the preceding week. She was normotensive, had elevated jugular venous pressure and clear lungs. ECG revealed atrial fibrillation with the rapid ventricular rate. Labs were significant for markedly elevated pro-brain natriuretic peptide of 43,000 pg/mL and troponin-T of 1 ng/mL. An urgent 2D echocardiogram was obtained, which revealed the severely dilated right atrium and a large linear mobile mass in the right atrium consistent with a thrombus. An emergent CT scan revealed multiple bilateral pulmonary emboli. She received intravenous tissue plasminogen activator. Repeat echocardiogram performed 6 h later showed no evidence of the right atrial thrombus. She was subsequently maintained on intravenous heparin and transitioned to Coumadin. Early recognition of this rare but potentially fatal complication is important as prompt treatment measures can help in preventing life-threatening complications of the right atrial thrombus. PMID:23892824

Bajaj, R; Ramanakumar, Ajay; Mamidala, Suresh; Kumar, Deepti

2013-01-01

194

Targeting of peptide conjugated magnetic nanoparticles to urokinase plasminogen activator receptor (uPAR) expressing cells  

DEFF Research Database (Denmark)

Ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles are currently being used as a magnetic resonance imaging (MRI) contrast agent in vivo, mainly by their passive accumulation in tissues of interest. However, a higher specificity can ideally be achieved when the nanoparticles are targeted towards cell specific receptors and this may also facilitate specific drug delivery by an enhanced target-mediated endocytosis. We report efficient peptide-mediated targeting of magnetic nanoparticles to cells expressing the urokinase plasminogen activator receptor (uPAR), a surface biomarker for poor patient prognosis shared by several cancers including breast, colorectal, and gastric cancers. Conjugation of a uPAR specific targeting peptide onto polyethylene glycol (PEG) coated USPIO nanoparticles by click chemistry resulted in a five times higher uptake in vitro in a uPAR positive cell line compared to nanoparticles carrying a non-binding control peptide. In accordance with specific receptor-mediated recognition, a low uptake was observed in the presence of an excess of ATF, a natural ligand for uPAR. The uPAR specific magnetic nanoparticles can potentially provide a useful supplement for tumor patient management when combined with MRI and drug delivery.

Hansen, Line; Larsen, Esben Kjær Unmack

2013-01-01

195

Relationship between expression of urokinase-type plasminogen activator and tumor angiogenesis in epithelial ovarian carcinoma  

Directory of Open Access Journals (Sweden)

Full Text Available Objective To explore the expression of urokinase-type plasminogen activator(uPA in epithelial ovarian carcinoma(EOC and the relationship of the expression to microvessel density.Methods SP immunohistochemical staining was performed to determine the expression of uPA in ovarian cancer(85 cases,borderline ovarian tumor(16 cases,benign ovarian tumor(39 cases and normal ovarian tissue(24 cases.Microvessels in ovarian cancer were marked by CD34,and microvessel density(MVD was determined by direct count.The relationship between uPA and MVD was analyzed.The 85 patients with epithelial ovarian cancer were followed up.Results The highest positive rate of uPA existed in the patients with EOC.There was a correlation between the expression of uPA and MVD and the differentiation of EOC,clinical stage,lymphatic metastasis,omental metastasis,and 5 years survival rate.A significant positive correlation was found between the expression of uPA in EOC and MVD(r=0.56,P=0.02.Conclusion uPA may promote the angiogenesis of EOC,and participate in the occurrence,development,invasion and metastasis of ovarian cancer.The detection of uPA and MVD may be used as an indicator of biological behaviors and prognosis of ovarian cancer.

Ping NAN

2011-06-01

196

Resolution of acute onset hemichorea-hemiballismus after treatment with intravenous tissue plasminogen activator.  

Science.gov (United States)

Hyperkinetic movement disorders are uncommon after acute ischemic stroke. Since these movement disorders are rarely the initial manifestation of acute cerebral ischemia, their presence may result in diagnostic uncertainty or it may inappropriately delay intravenous thrombolytic therapy for ischemic stroke. Hemichorea-hemiballism (HC-HB) is one of the more frequently encountered hyperkinetic movement disorders occurring in conjunction with stroke. Although HC-HB may result from a stroke mimic, the acute onset should prompt rapid evaluation and consideration for the presence of stroke along with its time-dependent therapies including recombinant tissue plasminogen activator (rtPA). In this article, we describe a case of a patient with acute cerebral ischemia presenting clinically with HC-HB, who was given intravenous rtPA therapy despite an initially negative, early diffusion-weighted magnetic resonance imaging (MRI). Follow-up brain MRI performed 24 hours after the initiation of thrombolytic therapy confirmed acute infarction in the contralateral striatum. The patient had near-complete resolution of her HC-HB on discharge and had no complications related to the administration of intravenous rtPA. PMID:24167646

McCollum, D; Silvers, S; Dawson, S B; Barrett, K M

2013-07-01

197

Aggregation and retention of human urokinase type plasminogen activator in the yeast endoplasmic reticulum  

Directory of Open Access Journals (Sweden)

Full Text Available Abstract Background Secretion of recombinant proteins in yeast can be affected by their improper folding in the endoplasmic reticulum and subsequent elimination of the misfolded molecules via the endoplasmic reticulum associated protein degradation pathway. Recombinant proteins can also be degraded by the vacuolar protease complex. Human urokinase type plasminogen activator (uPA is poorly secreted by yeast but the mechanisms interfering with its secretion are largely unknown. Results We show that in Hansenula polymorpha overexpression worsens uPA secretion and stimulates its intracellular aggregation. The absence of the Golgi modifications in accumulated uPA suggests that aggregation occurs within the endoplasmic reticulum. Deletion analysis has shown that the N-terminal domains were responsible for poor uPA secretion and propensity to aggregate. Mutation abolishing N-glycosylation decreased the efficiency of uPA secretion and increased its aggregation degree. Retention of uPA in the endoplasmic reticulum stimulates its aggregation. Conclusions The data obtained demonstrate that defect of uPA secretion in yeast is related to its retention in the endoplasmic reticulum. Accumulation of uPA within the endoplasmic reticulum disturbs its proper folding and leads to formation of high molecular weight aggregates.

Smirnov Vladimir N

2002-10-01

198

Role of plasminogen activator inhibitor type-1 in radiation-induced normal tissues injury  

International Nuclear Information System (INIS)

Radiotherapy is an essential tool for cancer treatment, but there is a balance between benefits and risks related to the use of ionizing radiation: the objective is to deliver a maximum dose to the tumour to destroy or to sterilize it while protecting surrounding normal tissues. Radio-induced damages to normal tissues are therefore a limiting factor when increasing the dose delivered to the tumour. One of the objectives of this research thesis is to bring to the fore a relationship between the initiation of lesions and the development of late damages, more particularly in the intestine, and to identify the involved molecular actors and their inter-connectivity. After a first part presenting ionizing radiation, describing biological effects of ionizing radiation and their use in radiotherapy, presenting the intestine and the endothelium and discussing the intestine radio-sensitivity, discussing the radio-induced intestine damages and radiotherapy-induced complications, and presenting the plasminogen activator inhibitor (PAI-1) and its behaviour in presence of ionizing radiation, two articles are reproduced. The first one addresses the effect of a pharmacological inhibition and of genetic deficiency in PAI-1 on the evolution of radio-induced intestine lesions. The second one discusses the fact that radio-induced PAI-1-related death of endothelial cells determines the severity of early radio-induced intestine lesions

199

Hemorrhagic transformation in focal cerebral ischemia: influence of time to artery reopening and tissue plasminogen activator.  

Science.gov (United States)

We used an adaptation of a well-established rat model of middle cerebral artery occlusion (MCAO) that is both minimally invasive and reproducible to determine the effects of time to reperfusion and administration of tissue plasminogen activator (t-PA) on the development of hemorrhagic transformation (HT) in a rat model of acute stroke. Animals were randomized to receive either t-PA 10 mg/kg (29 rats) or an equal volume of saline (29 rats) over 20 minutes, beginning 5 minutes before reperfusion. Time to artery reopening varied between 1 and 24 hours after MCAO in both groups. At 18-24 hours after ischemia, the animals were sacrificed and their brains were preserved for analysis of HT. Logistic regression was used to determine the influence of time on HT risk and calculate the time at which 50% of animals developed HT (HT50%). At 24 hours, HT was present in 17 of 29 animals in each group and was significantly influenced by the time of artery reopening: 3 (15%) of 20 animals reperfused less than 3 hours after onset of ischemia and 32 (84%) of 38 reperfused 3 or more hours after the onset of ischemia (pHT50% between groups. Time to artery reopening is an important determinant of HT risk in this model of cerebral ischemia. This model may have utility in developing strategies to reduce HT formation after thrombolytic therapy in patients with acute stroke. PMID:10030763

Fagan, S C; Garcia, J H

1999-02-01

200

Identification of genes in the ovine endometrium regulated by interferon tau independent of signal transducer and activator of transcription 1.  

Science.gov (United States)

Interferon tau (IFNtau), a type I IFN produced by the conceptus trophectoderm, increases many type I IFN-stimulated genes (ISGs) in the ovine uterine endometrial stroma and glandular epithelium (GE) using signal transducer and activator of transcription 1 (STAT1)-dependent pathways. Most ISGs are not induced or increased by IFNtau in the STAT1-negative endometrial luminal epithelium (LE). The objective was to identify genes regulated by IFNtau in a STAT1-independent manner using DNA microarray and human cell lines. The RNA from human 2fTGH and U3A (STAT1 null 2fTGH) cell lines, stimulated for 24 h with nothing or recombinant ovine IFNtau, was profiled using an Affymetrix human genome U95Av2 microarray. In 2fTGH cells, IFNtau increased the expression of 101 genes at least 2-fold, including IFN-inducible 56-kDa protein (IFI56), ISG12 or p27, and guanylate binding protein isoform I (GBP-2). In U3A cells, IFNtau increased expression of 66 genes at least 2-fold, including Wnt7a. Steady-state levels of IFI56, ISG12, GBP-2, and Wnt7a mRNAs increased in the ovine uterine endometrium between d 10 and 16 of pregnancy but not during the estrous cycle. GBP-2 and IFI56 mRNAs were expressed only in endometrial stroma, ISG12 in both LE and GE, and Wnt7a only in LE of the ovine uterus. Intrauterine infusion of ovine IFNtau increased expression of all four genes in the endometrium of cyclic ewes. Therefore, IFNtau does regulate genes independent of STAT1 in the endometrial LE and U3A cells and dependent on STAT1 in the endometrial stroma and 2fTGH cells. These IFNtau -stimulated genes may be important in establishment of uterine receptivity to the embryo and conceptus implantation given their stage-specificity in endometrium across diverse species. PMID:12960022

Kim, Seokwoon; Choi, Youngsok; Bazer, Fuller W; Spencer, Thomas E

2003-12-01

201

Enhancing the Function of CD34+ Cells by Targeting Plasminogen Activator Inhibitor-1  

Science.gov (United States)

Previously, we showed that transient inhibition of TGF- ?1 resulted in correction of key aspects of diabetes-induced CD34+ cell dysfunction. In this report, we examine the effect of transient inhibition of plasminogen activator inhibitor-1 (PAI-1), a major gene target of TGF-?1 activation. Using gene array studies, we examined CD34+ cells isolated from a cohort of longstanding diabetic individuals, free of microvascular complications despite suboptimal glycemic control, and found that the cells exhibited reduced transcripts of both TGF-?1 and PAI-1 compared to age, sex, and degree of glycemic control-matched diabetic individuals with microvascular complications. CD34+ cells from diabetic subjects with microvascular complications consistently exhibited higher PAI-1 mRNA than age-matched non-diabetic controls. TGF- ?1 phosphorodiamidate morpholino oligo (PMO) reduced PAI-1 mRNA in diabetic (p<0.01) and non-diabetic (p=0.05) CD34+ cells. To reduce PAI-1 in human CD34+ cells, we utilized PAI-1 siRNA, lentivirus expressing PAI-1 shRNA or PAI-1 PMO. We found that inhibition of PAI-1 promoted CD34+ cell proliferation and migration in vitro, likely through increased PI3(K) activity and increased cGMP production. Using a retinal ischemia reperfusion injury model in mice, we observed that recruitment of diabetic CD34+ cells to injured acellular retinal capillaries was greater after PAI-1-PMO treatment compared with control PMO-treated cells. Targeting PAI-1 offers a promising therapeutic strategy for restoring vascular reparative function in defective diabetic progenitors. PMID:24223881

Hazra, Sugata; Stepps, Valerie; Bhatwadekar, Ashay D.; Caballero, Sergio; Boulton, Michael E.; Higgins, Paul J.; Nikonova, Elena V.; Pepine, Carl J.; Thut, Catherine; Finney, Eva M.; Stone, David J.; Bartelmez, Stephen H.; Grant, Maria B.

2013-01-01

202

Biochemical mechanism of action of a diketopiperazine inactivator of plasminogen activator inhibitor-1.  

DEFF Research Database (Denmark)

XR5118 [(3 Z,6 Z )-6-benzylidine-3-(5-(2-dimethylaminoethyl-thio-))-2-(thienyl)methylene-2,5-dipiperazinedione hydrochloride] can inactivate the anti-proteolytic activity of the serpin plasminogen activator inhibitor-1 (PAI-1), a potential therapeutic target in cancer and cardiovascular diseases. Serpins inhibit their target proteases by the P(1) residue of their reactive centre loop (RCL) forming an ester bond with the active-site serine residue of the protease, followed by insertion of the RCL into the serpin's large central beta-sheet A. In the present study, we show that the RCL of XR5118-inactivated PAI-1 is inert to reaction with its target proteases and has a decreased susceptibility to non-target proteases, in spite of a generally increased proteolytic susceptibility of specific peptide bonds elsewhere in PAI-1. The properties of XR5118-inactivated PAI-1 were different from those of the so-called latent form of PAI-1. Alanine substitution of several individual residues decreased the susceptibility of PAI-1 to XR5118. The localization of these residues in the three-dimensional structure of PAI-1 suggested that the XR5118-induced inactivating conformational change requires mobility of alpha-helix F, situated above beta-sheet A, and is in agreement with the hypothesis that XR5118 binds laterally to beta-sheet A. These results improve our understanding of the unique conformational flexibility of serpins and the biochemical basis for using PAI-1 as a therapeutic target. Udgivelsesdato: 2003-Aug-1

Einholm, Anja P; Pedersen, Katrine E

2003-01-01

203

Optimizing production of recombinant tissue plasminogen activator in non-pathogenic Leishmania by two genetic constructs  

Directory of Open Access Journals (Sweden)

Full Text Available "nBackground: Recombinant tissue plasminogen activator (rt-PA is one of the most important thrombolytic agents used in patients with vascular occlusions such as acute ischemic stroke or myocardial infarction. A variety of recombinant protein expression systems have been developed for heterologous gene expression in prokaryotic and eukaryotic hosts. In recent years, Leishmania tarentolae (L. tarentolae, a non-pathogenic trypanosomatid protozoa, has come under consideration because of its safety and immunogenicity as a vaccine vector and special attributes in the expression of complex proteins. This study was done to improve rt-PA expression in this protozoon and create the opportunity for the replacement of rt-PA gene with other genes for the production of other complex proteins."n "n Methods: Two expression cassettes were used for the integration of two copies of t-PA cDNA, one copy in each cassette, into the parasite genome by electroporation. The transformed clones were selected by antibiotic resistancy. The expression of active secreted rt-PA was confirmed by Western blot analysis and Chromolize assay."n "n Results: Appearance of a 64 kD band in nitrocellulose membrane in the Western blot analysis confirmed the presence of full-length rt-PA in the culture media. Chromolize assay showed the expression levels of active recombinant t-PA in single and double transfected L. tarentolae clones- 375 IU/ml and 480 IU/ml of the culture media, respectively."n "n Conclusion: The produced rt-PA in the culture media containing the transfected cells was at least seven times higher than what has been reported in previous studies on L. tarentolae or on some other eukaryotic systems.

Hemayatkar M

2011-02-01

204

Oviduct-specific expression of tissue plasminogen activator in laying hens  

Scientific Electronic Library Online (English)

Full Text Available SciELO Brazil | Language: English Abstract in english Egg-laying hens are important candidate bioreactors for pharmaceutical protein production because of the amenability of their eggs for protein expression. In this study, we constructed an oviduct-specific vector containing tissue plasminogen activator (tPA) protein and green fluorescent protein (pL- [...] 2.8OVtPAGFP) and assessed its expression in vitro and in vivo. Oviduct epithelial and 3T3 cells were cultured and transfected with pL-2.8OVtPAGFP and pEGP-N1 (control vector), respectively. The pL-2.8OVtPAGFP vector was administered to laying hens via a wing vein and their eggs and tissues were examined for tPA expression. The oviduct-specific vector pL-2.8OVtPAGFP was expressed only in oviduct epithelial cells whereas pEGP-N1 was detected in oviduct epithelial and 3T3 cells. Western blotting detected a 89 kDa band corresponding to tPA in egg white and oviduct epithelial cells, thus confirming expression of the protein. The amount of tPAGFP in eggs ranged 9 to 41 ng/mL on the third day after vector injection. The tPA expressed in egg white and oviduct epithelial cells showed fibrinolytic activity, indicating that the protein was expressed in active form. GFP was observed only in oviducts, with no detection in heart, muscle, liver and intestine. This is the first study to report the expression of tPA in egg white and oviduct epithelial cells using an oviduct-specific vector.

Hubdar Ali, Kaleri; Liu, Xiang; Jueken, Aniwashi; Shiyong, Xu.

205

Antibody-mediated targeting of the urokinase-type plasminogen activator proteolytic function neutralizes fibrinolysis in vivo  

DEFF Research Database (Denmark)

Urokinase-type plasminogen activator (uPA) plays a central role in tissue remodeling processes. Most of our understanding of the role of uPA in vivo is derived from studies using gene-targeted uPA-deficient mice. To enable in vivo studies on the specific interference with uPA functionality in mouse models, we have now developed murine monoclonal antibodies (mAbs) directed against murine uPA by immunization of uPA-deficient mice with the recombinant protein. Guided by enzyme-linked immunosorbent assay, Western blotting, surface plasmon resonance, and enzyme kinetic analyses, we have selected two highly potent and inhibitory anti-uPA mAbs (mU1 and mU3). Both mAbs recognize epitopes located on the B-chain of uPA that encompasses the catalytic site. In enzyme activity assays in vitro, mU1 blocked uPA-catalyzed plasminogen activation as well as plasmin-mediated pro-uPA activation, whereas mU3 only was directed against the first of these reactions. We additionally provide evidence that mU1, but not mU3, successfully targets uPA-dependent processes in vivo. Hence, systemic administration of mU1 (i) rescued mice treated with a uPA-activable anthrax protoxin and (ii) impaired uPA-mediated hepatic fibrinolysis in tissue-type plasminogen activator (tPA)-deficient mice, resulting in a phenotype mimicking that of uPA;tPA double deficient mice. Importantly, this is the first report demonstrating specific antagonist-directed targeting of mouse uPA at the enzyme activity level in a normal physiological process in vivo.

Lund, Ida K; Jögi, Annika

2008-01-01

206

Soluble Urokinase-Type Plasminogen Activator Receptor Levels in Patients With Schizophrenia  

DEFF Research Database (Denmark)

BACKGROUND: The etiology of schizophrenia remains largely unknown but alterations in the immune system may be involved. In addition to the psychiatric symptoms, schizophrenia is also associated with up to 20 years reduction in life span. Soluble urokinase-type plasminogen activator receptor (suPAR) is a protein that can be measured in blood samples and reflects the levels of inflammatory activity. It has been associated with mortality and the development of type 2 diabetes and cardiovascular disease. METHODS: suPAR levels in patients with schizophrenia were compared to healthy controls from the Danish Blood Donor Study. SuPAR levels were dichotomized at >4.0 ng/ml, which is considered the threshold for low grade inflammation. A multiple logistic regression model was used and adjusted for age, sex, and current smoking. RESULTS: In total we included 1009 subjects, 105 cases with schizophrenia (10.4%) and 904 controls (89.6%). The mean suPAR values were 4.01 ng/ml (SD = 1.43) for the cases vs 1.91 ng/ml (SD = 1.35) for the controls (P 4.0 ng/ml yielded: schizophrenia, OR: 46.15 95% CI 22.69-93.87, P < .001; age, OR: 1.02 95% CI 0.99-1.02, P = .15; male sex, OR: 0.70 95% CI 0.35-1.36, P = .29; and current smoking, OR: 3.51 95% CI 1.78-6.94, P < .001. CONCLUSIONS: Patients with schizophrenia had significantly higher suPAR levels than healthy controls. Further studies are warranted to clarify if elevated suPAR levels are involved in the pathophysiology of schizophrenia and/or the increased mortality found in patients with schizophrenia.

Nielsen, Jimmi; RØge, Rasmus

2014-01-01

207

Plasminogen activator inhibitor 1, fibroblast apoptosis resistance, and aging-related susceptibility to lung fibrosis.  

Science.gov (United States)

Idiopathic pulmonary fibrosis (IPF) is a fatal lung disorder with unknown cause and no effective treatment. The incidence of and mortality from IPF increase with age, suggesting that advanced age is a major risk factor for IPF. The mechanism underlying the increased susceptibility of the elderly to IPF, however, is unknown. In this study, we show for the first time that the protein level of plasminogen activator inhibitor 1 (PAI-1), a protease inhibitor which plays an essential role in the control of fibrinolysis, was significantly increased with age in mouse lung homogenate and lung fibroblasts. Upon bleomycin challenge, old mice experienced augmented PAI-1 induction and lung fibrosis as compared to young mice. Most interestingly, we show that fewer (myo)fibroblasts underwent apoptosis and more (myo)fibroblasts with increased level of PAI-1 accumulated in the lung of old than in young mice after bleomycin challenge. In vitro studies further demonstrate that fibroblasts isolated from lungs of old mice were resistant to H2O2 and tumor necrosis factor alpha-induced apoptosis and had augmented fibrotic responses to TGF-?1, compared to fibroblasts isolated from young mice. Inhibition of PAI-1 activity with a PAI-1 inhibitor, on the other hand, eliminated the aging-related apoptosis resistance and TGF-?1 sensitivity in isolated fibroblasts. Moreover, we show that knocking down PAI-1 in human lung fibroblasts with PAI-1 siRNA significantly increased their sensitivity to apoptosis and inhibited their responses to TGF-?1. Together, the results suggest that increased PAI-1 expression may underlie the aging-related sensitivity to lung fibrosis in part by protecting fibroblasts from apoptosis. PMID:25451236

Huang, Wen-Tan; Akhter, Hasina; Jiang, Chunsun; MacEwen, Mark; Ding, Qiang; Antony, Veena; Thannickal, Victor John; Liu, Rui-Ming

2015-01-01

208

Soluble urokinase plasminogen activator receptor: an indicator of pneumonia severity in children.  

Science.gov (United States)

Enhanced level of soluble urokinase plasminogen activator receptor (suPAR) level has been associated with activation of the immune system. It may be a novel biomarker for pneumonia severity, yet data on this subject are limited. In the present study we seek to determine the suPAR level in hospitalized children with community-acquired pneumonia (CAP), its correlation with pneumonia severity, and to compare the suPAR level between pneumonia and healthy conditions. The study encompassed a total of 596 children: 447 with pneumonia and 119 healthy. suPAR was measured in 227 out of the 447 pneumonia patients and in all healthy subjects. We used clinical indicators (fever, time for defeverscence, heart and breath rate, saturation, and length of antibiotic treatment and of hospitalization) and laboratory indicators (CRP, procalcitonin, white blood cell count, and sodium) to assess the CAP severity. The finding were that the suPAR concentration in children with pneumonia was significantly higher (median 7.11 ng/mL) than in healthy individuals (4.68 ng/mL). We found a positive correlation between the suPAR and the following factors: fever, time for defeverscence, length of hospital stay, and elevated CRP and procalcitonin levels. There was a reverse correlation with sodium concentration and capillary blood saturation. Moreover, the suPAR level was significantly higher in children with a severe course of pneumonia compared with those having non-severe pneumonia (7.79 vs. 6.87 ng/mL; p?=?0.006). In conclusion, suPAR elevation is observed in pneumonia and may reflect its severity. PMID:25315615

Wrotek, A; Jackowska, T; Pawlik, K

2015-01-01

209

Rapid formation of an anion-sensitive active site in stoichiometric complexes of streptokinase and human [Glu1]plasminogen.  

OpenAIRE

We have examined the time-dependent appearance of amidolytic activity in equimolar complexes of streptokinase (SK) and human [Glu1]plasminogen (HPg) under various conditions. When stoichiometric levels of the two proteins are incubated and assayed in hypotonic buffers at 4 degrees C, amidolytic activity toward the chromogenic substrate D-Val-Leu-Lys-p-nitroanilide (S-2251), within the resulting complex, appears with an observed first-order rate constant of 1.03 +/- 0.06/min. On the other hand...

Chibber, B. A.; Radek, J. T.; Morris, J. P.; Castellino, F. J.

1986-01-01

210

The miR-143/-145 cluster regulates plasminogen activator inhibitor-1 in bladder cancer  

DEFF Research Database (Denmark)

Background:Upregulation of the proto-oncogene plasminogen activator inhibitor-1 (PAI-1) is a common hallmark of various solid tumours, but the mechanisms controlling its expression are not fully understood.Methods:We investigate microRNAs (miRNAs) regulating PAI-1 in a panel of normal bladder urothelial biopsies, superficial Ta bladder tumours and invasive T1-T4 tumours using expression microarrays and qRT-PCR. The prognostic implications of PAI-1 deregulation are established by tissue microarray staining of non-muscle-invasive bladder tumours. MicroRNA repression of PAI-1 is assayed by ectopic miRNA expression, argonaute immunoprecipitation and luciferase assays.Results:We found that the miR-143/-145 cluster is downregulated in all stages of bladder cancer and inversely correlated with PAI-1 expression. Mature miR-143 and miR-145 are coordinately expressed, and both directly target the PAI-1 3'UTR, leading to reduced PAI-1 mRNA and protein levels. Furthermore, we show that PAI-1 and miR-145 levels may serve as useful prognostic markers for non-muscle-invasive bladder tumours for which accurate progressive outcome is currently difficult to predict.Conclusion:This report provides the first evidence for direct miRNA regulation of PAI-1 in bladder cancer. We also demonstrate mRNA co-targeting by a cluster of non-family miRNAs, and suggest miR-145 and PAI-1 as clinically relevant biomarkers in bladder cancer.British Journal of Cancer advance online publication, 22 November 2011; doi:10.1038/bjc.2011.520 www.bjcancer.com

Villadsen, S B; Bramsen, Jesper Bertram

2012-01-01

211

Simultaneous administration of recombinant tissue plasminogen activator and edaravone in acute cerebral ischemic stroke patients.  

Science.gov (United States)

Among the 1052 patients admitted to our hospital because of cerebral infarction between January 1, 2007, and December 31, 2010, we report the treatment outcomes of 48 patients (4.6% of all patients) who received recombinant tissue plasminogen activator (rt-PA) therapy (simultaneously combined with edaravone) within 3 hours after the onset of infarction. Twenty (41.7%) patients started receiving edaravone before rt-PA administration, and 28 patients (58.3%) started receiving rt-PA and edaravone simultaneously. The patients had an average age of 73.5 years (range, 55-93 years; male:female, 32:16). Medical histories included hypertension, diabetes mellitus, dyslipidemia, arterial fibrillation, and a smoking history in 23 (47.8%), 7 (14.6%), 8 (16.7%), 29 (60.4%), and 8 (16.7%) of patients, respectively. Regarding the treatment outcome of the therapy, the National Institutes of Health Stroke Scale score, which was 15 points before rt-PA administration, showed a statistically significant improvement to 8 points after rt-PA administration (P < .001). The modified Rankin Scale scores at 90 days after treatment were as follows: 0 in 12 patients (25.0%), 1 in 11 patients (22.9%), 2 in 7 patients (14.6%), 3 in 5 patients (10.4%), 4 in 6 patients (12.5%), 5 in 5 patients (10.4%), and 6 in 2 patients (4.2%). The occluded blood vessel reopened completely in 30 patients (62.5%) and partially in 5 patients (10.4%). Asymptomatic hemorrhage over the entire brain developed in 2 patients (4.2%). Thus, rt-PA therapy in combination with edaravone improved the recanalization rate, reduced the incidence of intracranial hemorrhage, and improved functional prognosis. PMID:25307430

Takenaka, Katsunobu; Kato, Masayasu; Yamauti, Keita; Hayashi, Katsuhiko

2014-01-01

212

Localization of human malignant tumors with radioiodinated recombinant tissue plasminogen activator  

International Nuclear Information System (INIS)

Human recombinant tissue plasminogen activator (tPA), labeled with 131I(1.1 to 6.2 mCi) by the iodogen method, was administered intravenously to 15 patients with various soft-tissue malignant tumors after blocking of thyroidal radioiodine uptake. Gamma camera imaging was performed 4 and 24 hr after injection; three patients were also imaged 5 days following injection. We observed accumulation of radioactivity in primary and secondary lesions in 11 patients. In this preliminary study we did not detect any definite association between the magnitude of uptake and type of tumor. Tumors were usually visualized already after 4 hr but the uptake was more intense at 24 hr. The target-to-nontarget ratios at 24 hr, determined by computer analysis of stored images, varied from less than 1.2 to 2.1. This is the first demonstration of accumulation of radiolabeled tPA in malignant tissue. We do not know the mechanism of the uptake but because tPA is known to be avidly bound to fibrin, a component of the stroma of many malignant tumors, it is possible that [131I]tPA is bound to fibrin rather than taken up by the malignant cell; various possible cell uptake mechanisms are discussed. Due to the relatively early maximal uptake of this radiopharmaceutical it will be possible to substitute 123I for 131I, a possibility suggesting a potential clinical use of radioiodinated tPA for the detection of malignant tumors of various originrigin

213

Technetium-99m modified recombinant tissue plasminogen activator to detect pulmonary embolism: a pilot study  

Energy Technology Data Exchange (ETDEWEB)

Full text: Technetium 99m labelled modified recombinant tissue plasminogen activator ({sup 99m}Tc rt PA) has been shown to have a high sensitivity and specificity in the detection of venous thrombosis in the lower limbs in both symptomatic and asymptomatic patients. This study was performed to test the sensitivity of {sup 99m}Tc rt PA scinitigraphy in the detection of pulmonary embolism (PE). Ten patients with a high clinical suspicion of PE and a very high probability lung scan (i.e. greater than three segmental areas of ventilation perfusion mismatch) were injected with 500 MBq of {sup 99m}Tc rt PA with imaging of the legs and thorax taking place four hours post injection. Planar images of the legs were obtained, with both planar and SPECT images (using a double headed Picker Prism 2000) of the thorax being obtained. All images were viewed by nuclear medicine physicians. Of the ten patients with presumed PE, only two patients had abnormal SPECT Tc-99 rt-PA studies of the lungs. Only one of these patients had an abnormal planar image. In this group of patients, {sup 99m}Tc rt PA has a sensitivity of 20 per cent in the detection of PE. Possible causes for this low sensitivity include the small size of the thrombi in lungs and the significant scatter into the lung caused by hepatic {sup 99m}Tc rt-PA uptake. In the images of the legs, five patients had abnormal scans indicating the presence of DVT in 50 per cent of patients. This study has demonstrated that the sensitivity of {sup 99m}Tc rt PA for the detection of PE is too low to be clinically useful In addition, the expected high prevalence of peripheral venous thrombosis in patients with PE has been confirmed.

Paszkowski, A.L.; Parkes, P.L.; Quinn, R.J.; Butler, S.P. [St George Hospital, Kogarah, NSW, (Australia). Department of Nuclear Medicine

1997-09-01

214

Technetium-99m modified recombinant tissue plasminogen activator to detect pulmonary embolism: a pilot study  

International Nuclear Information System (INIS)

Full text: Technetium 99m labelled modified recombinant tissue plasminogen activator (99mTc rt PA) has been shown to have a high sensitivity and specificity in the detection of venous thrombosis in the lower limbs in both symptomatic and asymptomatic patients. This study was performed to test the sensitivity of 99mTc rt PA scinitigraphy in the detection of pulmonary embolism (PE). Ten patients with a high clinical suspicion of PE and a very high probability lung scan (i.e. greater than three segmental areas of ventilation perfusion mismatch) were injected with 500 MBq of 99mTc rt PA with imaging of the legs and thorax taking place four hours post injection. Planar images of the legs were obtained, with both planar and SPECT images (using a double headed Picker Prism 2000) of the thorax being obtained. All images were viewed by nuclear medicine physicians. Of the ten patients with presumed PE, only two patients had abnormal SPECT Tc-99 rt-PA studies of the lungs. Only one of these patients had an abnormal planar image. In this group of patients, 99mTc rt PA has a sensitivity of 20 per cent in the detection of PE. Possible causes for this low sensitivity include the small size of the thrombi in lungs and the significant scatter into the lung caused by hepatic 99mTc rt-PA uptake. In the images of the legs, five patients had abnormal scans indicating the presence of DVT in 50 per cent of patients. This study has demonper cent of patients. This study has demonstrated that the sensitivity of 99mTc rt PA for the detection of PE is too low to be clinically useful In addition, the expected high prevalence of peripheral venous thrombosis in patients with PE has been confirmed

215

Localization of human malignant tumors with radioiodinated recombinant tissue plasminogen activator  

Energy Technology Data Exchange (ETDEWEB)

Human recombinant tissue plasminogen activator (tPA), labeled with /sup 131/I(1.1 to 6.2 mCi) by the iodogen method, was administered intravenously to 15 patients with various soft-tissue malignant tumors after blocking of thyroidal radioiodine uptake. Gamma camera imaging was performed 4 and 24 hr after injection; three patients were also imaged 5 days following injection. We observed accumulation of radioactivity in primary and secondary lesions in 11 patients. In this preliminary study we did not detect any definite association between the magnitude of uptake and type of tumor. Tumors were usually visualized already after 4 hr but the uptake was more intense at 24 hr. The target-to-nontarget ratios at 24 hr, determined by computer analysis of stored images, varied from less than 1.2 to 2.1. This is the first demonstration of accumulation of radiolabeled tPA in malignant tissue. We do not know the mechanism of the uptake but because tPA is known to be avidly bound to fibrin, a component of the stroma of many malignant tumors, it is possible that (/sup 131/I)tPA is bound to fibrin rather than taken up by the malignant cell; various possible cell uptake mechanisms are discussed. Due to the relatively early maximal uptake of this radiopharmaceutical it will be possible to substitute 123I for 131I, a possibility suggesting a potential clinical use of radioiodinated tPA for the detection of malignant tumors of various origin.

Karonen, S.L.; Aronen, H.; Liewendahl, K.; Nikkinen, P.; Maentylae, M.Li.; Lindgren, J.

1988-07-01

216

Tissue Plasminogen Activator Potently Stimulates Pleural Effusion via an MCP-1 Dependent Mechanism.  

Science.gov (United States)

Pleural infection is common. Evacuation of infected pleural fluid is essential for successful treatment but is often difficult due to adhesions/loculations within the effusion and the viscosity of the fluid. Intrapleural delivery of tissue plasminogen activator (tPA, to break the adhesions) and deoxyribonuclease (DNase, to reduce fluid viscosity) has recently been shown to improve clinical outcomes in a large randomized study of pleural infection. Clinical studies of intrapleural fibrinolytic therapy consistently showed subsequent production of large effusions, the mechanism(s) of which are unknown. We aimed to determine the mechanism by which tPA induces exudative fluid formation. Intrapleural tPA, with or without DNase, significantly induced pleural fluid accumulation in CD1 mice (tPA alone: median (IQR) 53.5 (30-355) µL) compared to DNase or vehicle controls (both; 0.0 (0.0-0.0) µL) after 6 hrs. Fluid induction was reproduced after intrapleural delivery of streptokinase and urokinase, indicating a class effect. Pleural fluid monocyte chemotactic protein (MCP)-1 levels strongly correlated with effusion volume (r=0.7302, p=0.003) and were significantly higher than MCP-1 levels in corresponding sera. Mice treated with anti-MCP1 antibody 30µg (p<0.0001) or MCP-1 receptor antagonist (p=0.0049) demonstrated a significant decrease in tPA-induced pleural fluid formation. Our data implicate MCP-1 as the key molecule governing tPA-induced fluid accumulation. The role of MCP-1 in the development of other exudative effusions warrants examination. PMID:25474480

Lansley, Sally M; Cheah, Hui Min; Varano Della Vergiliana, Julius F; Chakera, Aron; Lee, Yc Gary

2014-12-01

217

Induction of urokinase-type plasminogen activator receptor by IL-1 beta.  

Science.gov (United States)

Extensive tissue remodeling occurs in survivors of acute lung injury, leading to nearly normal histology and physiology in the majority of individuals, whereas others suffer significant impairment due to the development of pulmonary fibrosis. Alveolar epithelial cells play a central role in the repair process. They are strategically located to directly participate in the solubilization of intraalveolar fibrin deposits, and have the capacity to promote fibrinolysis. We have previously reported that interleukin-1 beta (IL-1 beta), an important inflammatory mediator in acute lung injury, upregulates urokinase-type plasminogen activator expression by human A549 cells (1). In this work, we show that IL-1 beta increases cell-surface plasmin generation, mediated in part by increased expression of urokinase receptor (u-PAR). Northern blot analyses demonstrated that IL-1 beta rapidly induces accumulation of u-PAR messenger RNA (mRNA) in a dose-dependent fashion, and that this effect is blocked by actinomycin. The IL-1 beta-mediated increase in u-PAR mRNA is inhibited by: (1) the relatively specific protein kinase C (PKC) inhibitors 1-(5-isoquinoline sulfonyl)-2-methylpiperazine (H7) and calphostin C; and (2) prolonged pretreatment of cells with phorbol myristate acetate (PMA), suggesting that PKC is an important component of the signaling pathway. Okadaic acid, an inhibitor of serine/threonine phosphatases, markedly potentiates the effect of IL-1 beta on u-PAR mRNA levels. In contrast, dexamethasone, in concentrations as low as 10(-8) M, completely blocks the IL-1 beta-mediated increase in u-PAR mRNA. Half-life experiments show that dexamethasone has no effect on u-PAR mRNA stability. Aldosterone, at concentrations in which it binds primarily to the mineralocorticoid receptor, has no effect on u-PAR expression, suggesting that the glucocorticoid effect is due to a transrepressive mechanism. In summary, IL-1 beta increases cell-surface plasmin generation in A549 cells by coordinately upregulating urokinase and u-PAR expression. Transcriptional activation of the u-PAR gene involves PKC-dependent mechanisms, and glucocorticoid suppression is probably due to interactions between the glucocorticoid receptor and another transcriptional activating system such as activator protein-1 (AP-1) and/or nuclear factor-kB (NF-kB). PMID:9191470

Hasegawa, T; Sorensen, L; Dohi, M; Rao, N V; Hoidal, J R; Marshall, B C

1997-06-01

218

Discrimination of different forms of the murine urokinase plasminogen activator receptor on the cell surface using monoclonal antibodies  

DEFF Research Database (Denmark)

The urokinase plasminogen activator receptor (uPAR) is a versatile three-domain GPI-anchored protein, which binds urokinase plasminogen activator (uPA) and thereby focalises plasminogen activation on the cell surface. Generation of a proteolytic potential is essential in both normal physiological and pathological extracellular tissue remodelling processes. uPA can also cleave uPAR, resulting in liberation of the amino-terminal domain I, which encompasses binding sites for both uPA and the adhesion molecule, vitronectin. In order to localise the different uPAR forms on the plasma membrane of murine monocyte macrophage-like P388D.1 cells, we have now generated and characterised two high-affinity murine mAbs, mR3 and mR4, raised against murine uPAR. mR3 was found to recognise an epitope located in domain I of uPAR. Surface plasmon resonance analyses and cell binding studies revealed that this mAb was able to bind preformed complexes of murine pro-uPA and murine uPAR. In contrast, mR4 recognises domains II-III inuPAR and does not bind preformed pro-uPA-uPAR complexes in similar analyses. Immunofluorescence microscopy of P388D.1 cells revealed that mR3 stained the cells equally well in the presence or absence of saturation with the amino-terminal fragment of uPA, ATF. However, the signal intensity obtained using another uPAR domain I specific mAb, mR1, was significantly reduced upon ATF saturation. Furthermore, when adding ATF, mR4 selectively stained the cleaved receptor. Applying these newly generated mAbs, we additionally demonstrated that cleaved and intact uPAR was evenly distributed on the surface of these cells Udgivelsesdato: 2008/11/30

Rasch, M.G.; Pass, J.

2008-01-01

219

Involvement of tissue plasminogen activator "tPA" in ethanol-induced locomotor sensitization and conditioned-place preference.  

Science.gov (United States)

Ethanol is one of the most abused drugs in the western societies. It is well established that mesolimbic dopaminergic neurons mediate the rewarding properties of ethanol. In our previous studies we have shown that the serine protease tissue plasminogen activator (tPA) is involved in the rewarding properties of morphine and amphetamine. In the current study, we investigated the role of tPA in ethanol-induced behavioral sensitization and conditioned-place preference (CPP). Ethanol treatment dose-dependently induced tPA enzymatic activity in the nucleus accumbens (NAc). In addition, ethanol-induced increase in tPA activity was completely inhibited by pre-treatment with the dopamine D1 and D2 receptor antagonists SCH23390 and raclopride respectively. Furthermore, ethanol-induced locomotor stimulation, behavioral sensitization and conditioned-place preference were enhanced following tPA over-expression in the NAc using a lentiviral vector. In contrast, tPA knock down in the NAc with specific shRNA blocked the rewarding properties of ethanol. The defect of locomotor stimulation in shRNA-injected mice was reversed by microinjections of exogenous recombinant tPA into the nucleus accumbens. Collectively, these results indicate, for the first time, that activation of tPA is effective in enhancing the rewarding effects of ethanol. Targeting the tissue plasminogen activator system would provide new therapeutic approaches to the treatment of alcoholism. PMID:21945298

Bahi, Amine; Dreyer, Jean-Luc

2012-01-01

220

Synergistic and multidimensional regulation of plasminogen activator inhibitor type 1 expression by transforming growth factor type ? and epidermal growth factor  

Energy Technology Data Exchange (ETDEWEB)

The major physiological inhibitor of plasminogen activator, type I plasminogen activator inhibitor (PAI-1), controls blood clotting and tissue remodeling events that involve cell migration. Transforming growth factor type ? (TGF?) and epidermal growth factor (EGF) interact synergistically to increase PAI-1 mRNA and protein levels in human HepG2 and mink Mv1Lu cells. Other growth factors that activate tyrosine kinase receptors can substitute for EGF. EGF and TGF? regulate PAI-1 by synergistically activating transcription, which is further amplified by a decrease in the rate of mRNA degradation, the latter being regulated only by EGF. The combined effect of transcriptional activation and mRNA stabilization results in a rapid 2-order of magnitude increase in the level of PAI-1. TGF? also increases the sensitivity of the cells to EGF, thereby recruiting the cooperation of EGF at lower than normally effective concentrations. The contribution of EGF to the regulation of PAI-1 involves the MAPK pathway, and the synergistic interface with the TGF? pathway is downstream of MEK1/2 and involves phosphorylation of neither ERK1/2 nor Smad2/3. Synergism requires the presence of both Smad and AP-1 recognition sites in the promoter. This work demonstrates the existence of a multidimensional cellular mechanism by which EGF and TGF? are able to promote large and rapid changes in PAI-1 expression.

Song, Xiaoling; Thalacker, F.W.; Nilsen-Hamilton, Marit

2012-04-06

221

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

OpenAIRE

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-methylpent...

Lund, Leif R.; Green, Kirsty A.; Stoop, Allart A.; Ploug, Michael; Almholt, Kasper; Lilla, Jennifer; Nielsen, Boye S.; Christensen, Ib J.; Craik, Charles S.; Werb, Zena; Danø, Keld; Rømer, John

2006-01-01

222

Treatment of TBI with Collagen Scaffolds and Human Marrow Stromal Cells Increases the Expression of Tissue Plasminogen Activator  

OpenAIRE

This study examines the effects of combination therapy of collagen scaffolds and human marrow stromal cells (hMSCs) on the expression of tissue plasminogen activator (tPA) after traumatic brain injury (TBI) in rats. Adult male Wistar rats (n=48) were injured with controlled cortical impact and treated either with scaffolds suffused with hMSCs (3×106) or hMSCs (3×106) alone transplanted into the lesion cavity 1 week after TBI. A control group was treated with saline. Neurological function wa...

Mahmood, Asim; Qu, Changsheng; Ning, Riuzuo; Wu, Hongtao; Goussev, Anton; Xiong, Ye; Irtenkauf, Susan; Li, Yi; Chopp, Michael

2011-01-01

223

Outcomes of Intravenous Tissue Plasminogen Activator for Acute Ischemic Stroke in Patients Aged 90 Years or Older  

OpenAIRE

Although age is a major risk factor for stroke, physicians are often reluctant to use thrombolytic agents in those who are very old. No published study provides detailed information on the use of intravenous tissue plasminogen activator (tPA) in patients aged 90 years or older. We retrospectively reviewed the use of intravenous tPA for patients 90 years or older who were admitted with acute ischemic stroke to the hospital at 4 academic centers from March 1, 1999, to March 1, 2008. We reviewed...

Mateen, Farrah J.; Nasser, Majeed; Spencer, Byron R.; Freeman, William D.; Shuaib, Ashfaq; Demaerschalk, Bart M.; Wijdicks, Eelco F. M.

2009-01-01

224

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

DEFF Research Database (Denmark)

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 to a degree indistinguishable from that observed in Plg-deficient mice, and completely blocks the activity of pKal, but not uPA and tPA in wound extracts. These findings demonstrate that an additional plasminogen activator provides sufficient plasmin activity to sustain the healing process albeit at decreased speed in the absence of uPA, tPA and galardin-sensitive MMPs and suggest that pKal plays a role in plasmin generation.

Lund, Leif R; Green, Kirsty A

2006-01-01

225

Radiation-Induced Hypomethylation Triggers Urokinase Plasminogen Activator Transcription in Meningioma Cells  

Directory of Open Access Journals (Sweden)

Full Text Available Our previous studies have shown the role of radiation-induced urokinase plasminogen activator (uPA expression in the progression of meningioma. In the present study, we investigated whether modulation of DNA methylation profiles could regulate uPA expression. Initially, radiation treatment was found to induce hypomethylation in meningioma cells with a decrease in DNA (cytosine-5-methyltransferase 1 (DNMT1 and methyl-CpG binding domain protein (MBD expression. However, oxidative damage by H2O2 or pretreatment of irradiated cells with N-acetyl cysteine (NAC did not show any influence on these proteins, thereby indicating a radiation-specific change in the methylation patterns among meningioma cells. Further, we identified that hypomethylation is coupled to an increase in uPA expression in these cells. Azacytidine treatment induced a dose-dependent surge of uPA expression, whereas pre-treatment with sodium butyrate inhibited radiation-induced uPA expression, which complemented our prior results. Methylation-specific polymerase chain reaction on bisulfite-treated genomic DNA revealed a diminished methylation of uPA promoter in irradiated cells. Transfection with small hairpin RNA (shRNA-expressing plasmids targeting CpG islands of the uPA promoter showed a marked decline in uPA expression with subsequent decrease in invasion and proliferation of meningioma cells. Further, radiation treatment was found to recruit SP1 transcription factor, which was abrogated by shRNA treatment. Analysis on signaling events demonstrated the activation of MAP kinase kinase (MEK-extracellular signal-regulated kinase (ERK in radiation-treated cells, while U0126 (MEK/ERK inhibitor blocked hypomethylation, recruitment of SP1, and uPA expression. In agreement with our in vitro data, low DNMT1 levels and high uPA were found in intracranial tumors treated with radiation compared to untreated tumors. In conclusion, our data suggest that radiation-mediated hypomethylation triggers uPA expression in meningioma cells.

Kiran Kumar Velpula

2013-02-01

226

A technique to screen plant extracts for anti-inflammatory activity on ovine neutrophils  

OpenAIRE

Neutrophils play an essential role in host defense and inflammation. In recent years, several studies in men and dairy animals have shown that administration of natural compounds can influence the immune system. In the present study the extracts of six patented protected plants, named C1, C2, C3, C4, C5, C6, have been screened for their effect on ovine neutrophils. Three, clinically healthy, non lactating, non pregnant sheep were used. Freshly isolated neutrophils were incubated with extracts...

Stefanon, B.; Sgorlon, S.; Colitti, M.; Farinacci, M.

2010-01-01

227

Regulation of plasminogen activation by TGF-? in cultured human retinal endothelial cells  

OpenAIRE

BACKGROUND/AIMS—Regulation of plasmin mediated extracellular matrix degradation by vascular endothelial cells is important in the development of angiogenesis. The aim was to determine whether transforming growth factor ? (TGF-?) affected the regulation of components of the plasminogen system by human retinal endothelial cells, in order to define more clearly the role of TGF-? in retinal angiogenesis in the context of diabetes mellitus.?METHODS—Human retinal endothelial cells (HREC) ...

Wileman, S.; Booth, N.; Moore, N.; Redmill, B.; Forrester, J.; Knott, R.

2000-01-01

228

Thrombolysis by intravenous tissue plasminogen activator (t-PA). Current status and future direction  

International Nuclear Information System (INIS)

In Japan, the intravenous tissue plasminogen activator (t-PA) Alteplase (0.6 mg/kg) administration of the within 3 h of the onset of acute ischemic stroke was approved for therapeutic use in the year 2006. t-PA induces thrombolysis in patients with acute ischemic stroke, and this method has gradually gained recognition among physicians and the general population. However, the number of patients who were treated using Alteplase is low (4,000-5,000 patients/year), and this figure accounts for only 2-3% of the annual number of cases of ischemic stroke. There is little doubt that Alteplase treatment is a potentially effective modality for some patients with acute ischemic stroke. The post-marketing surveillance of 4,749 Japanese patients treated using Alteplase showed that 33% of the patients had modified Rankin scale (mRS) scores of 0-1, 17% of patients died and 4.5% presented with symptomatic intracerebral hemorrhage (ICH); these results were comparable to those from other countries. The expansion of the therapeutic time window has been a matter of concern. The investigators of the European Cooperative Acute Stroke Study (ECASS) have reported that there was significant improvement in the clinical outcomes of patients with acute ischemie stroke when Alteplase was administered 3-4.5 h after the onset of the symptoms. Mismatches in perfusion- and diffusion-weighted (DW) magnetic resonance imaging (MRI) images have been used for selecting patients 3 h after the onset of symecting patients 3 h after the onset of symptoms, and the findings from MRI, dwimages (DWI) and MR angiography are practical predictors of t-PA therapy within 3 h of onset. The Middle Cerebral Artery Embolism Local Fibrinolytic Intervention Trial (MELT) Japan study showed that local intra-arterial fibrinolysis is effective in patients with embolic MCA occlusion within 6 h of the onset of symptoms. Combining the initiation of intravenous t-PA administration with further intra-arterial fibrinolysis or mechanical thrombolectomy may improve the recanalization rate. Thrombolysis in combination with ultrasound-enhanced clot lysis is another attractive therapy. In Japan the neuroprotective agent edaravone (radical scavenger) is commonly used in combination with t-PA, and it is expected to decrease the hemorrhagic transformation after t-PA administration. Acute cerebral ischemic symptoms may occasionally precede thoracic aortic dissection. Thoracic aortic dissection after t-PA administration may prove to be fatal, and it is an important disorder that must be differentially diagnosed. (author)

229

Characterisation of urokinase plasminogen activator receptor variants in human airway and peripheral cells  

Directory of Open Access Journals (Sweden)

Full Text Available Abstract Background Expression of the urokinase plasminogen activator receptor (UPAR has been shown to have clinical relevance in various cancers. We have recently identified UPAR as an asthma susceptibility gene and there is evidence to suggest that uPAR may be upregulated in lung diseases such as COPD and asthma. uPAR is a key receptor involved in the formation of the serine protease plasmin by interacting with uPA and has been implicated in many physiological processes including proliferation and migration. The current aim was to determine key regulatory regions and splice variants of UPAR and quantify its expression in primary human tissues and cells (including lung, bronchial epithelium (HBEC, airway smooth muscle (HASM and peripheral cells. Results Using Rapid Amplification of cDNA Ends (RACE a conserved transcription start site (-42 to -77 relative to ATG was identified and multiple transcription factor binding sites predicted. Seven major splice variants were identified (>5% total expression, including multiple exon deletions and an alternative exon 7b (encoding a truncated, soluble, 229aa protein. Variants were differentially expressed, with a high proportion of E7b usage in lung tissue and structural cells (55–87% of transcripts, whereas classical exon 7 (encoding the GPI-linked protein was preferentially expressed in peripheral cells (~80% of transcripts, often with exon 6 or 5+6 deletions. Real-time PCR confirmed expression of uPAR mRNA in lung, as well as airway and peripheral cell types with ~50–100 fold greater expression in peripheral cells versus airway cells and confirmed RACE data. Protein analysis confirmed expression of multiple different forms of uPAR in the same cells as well as expression of soluble uPAR in cell supernatants. The pattern of expression did not directly reflect that seen at the mRNA level, indicating that post-translational mechanisms of regulation may also play an important role. Conclusion We have identified multiple uPAR isoforms in the lung and immune cells and shown that expression is cell specific. These data provide a novel mechanism for uPAR regulation, as different exon splicing may determine uPAR function e.g. alternative E7b results in a soluble isoform due to the loss of the GPI anchor and exon deletions may affect uPA (ligand and/or integrin binding and therefore influence downstream pathways. Expression of different isoforms within the lung should be taken into consideration in studies of uPAR in respiratory disease.

Sayers Ian

2009-07-01

230

Plasminogen activator inhibitor-1 removal using dextran sulphate columns. Evidence of PAI-1 homeostasis.  

LENUS (Irish Health Repository)

Patients with high plasma plasminogen activator inhibitor-1 (PAI-1) antigen levels are prone to develop thrombosis. Lowering PAI-1 levels may offer a therapeutic option and help to better understand PAI-1 metabolism. We examined the effect on plasma PAI-1 levels of LDL-apheresis using dextran sulphate (DS) columns in 12 patients (9 male, 3 female, 49 +\\/- 10 years) with heterozygous familial hypercholesterolaemia and coronary artery disease. One plasma volume equivalent (2.3-4.0 l) was treated during each procedure (at flow rates of 23 +\\/- 2 ml\\/min). Lipids and PAI-1 antigen levels were measured in plasma before and immediately after 19 aphereses (once in 7 patients, twice in 3 patients and three times in 2 patients) and also at 3 and 7 days post apheresis in five of these patients and in the column eluates from 8 of these patients. DS-apheresis reduced plasma cholesterol (50 +\\/- 8%), triglyceride (45 +\\/- 27%), apolipoprotein B (59 +\\/- 10%) and PAI-1 antigen levels from 10.2 +\\/- 5.2 to 6.0 +\\/- 3.1 ng\\/ml (P = 0.005). The PAI-I changes were independent of circadian variation. PAI-I bound to the DS-columns (3.51 +\\/- 1.03 ng\\/ml filtered plasma) and the percent of filtered PAI-1 that was bound correlated inversely (r = -0.81, P < 0.02) with basal PAI-1 levels indicating a high affinity saturable binding process. In four patients, plasma PAI-1 levels post-apheresis were higher than expected based on the amount of PAI-removed by the DS columns. The difference between the expected and actual PAI-1 level post apheresis, reflecting PAI-1 secretion or extracellular redistribution, correlated inversely with basal PAI-1 levels (r = -0.83, P = 0.01). PAI-1 levels returned to baseline pre-apheresis values 7 days post apheresis. PAI-1 antigen may be removed from plasma without adverse effect, resulting temporarily in its extracellular redistribution and restoration to baseline levels over one week. PAI-1 redistribution particularly when baseline pre-apheresis values were low may reflect a homeostatic mechanism to maintain sufficient PAI-1 levels. Procedures that could selectively remove PAI-1 from plasma may offer a treatment option for those with very high plasma PAI-1 levels and thrombosis.

Maher, Vincent M G

2009-08-01

231

Tissue plasminogen activator reduces the elevated intraocular pressure induced by prednisolone in sheep.  

Science.gov (United States)

We have previously shown that tissue plasminogen activator (tPA) injected in the vitreous of sheep, reduced or prevented the elevation of the intraocular pressure (IOP) normally produced by the instillation of 1% prednisolone. We now report the effect of tPA when injected into the anterior chamber (AC) in amounts of 0.01, 0.001 and 0.0001 ?g diluted in a volume of 50 ?L. Lyophilized tPA, obtained as Actilyse(®) 50 mg from Boehringer Ingelheim containing arginine was utilized. The Actilyse was diluted in balanced salt solution to obtain the desired amount of tPA in 50 ?L. An identical solution containing only arginine was prepared to inject into the contralateral eye as a control. Six sheep of the Corriedale breed were selected. At the beginning of the study all eyes received instillation of 1% prednisolone 3 times/day for 10 days to elevate their IOP from 10 mm Hg to about 23 mm Hg. Then, 0.0001 ?g was injected into one of the eyes and its effect was followed for up to 55:00 h while the instillation of prednisolone continued in both eyes. The same protocol was implemented for the 0.001 and 0.01 ?g amounts after extended washout and IOP was over 22 mm Hg. The injection of 0.0001 ?g into the AC had no effect on an IOP of 23.0 mm Hg at 6:00 and 30:00 h after injection. 0.001 ?g of tPA reduced IOP from 23.1 to 18.6 mm Hg at 6:00 h but IOP recovered to 22.3 mm Hg at 30:00 h. Injection of 0.01 ?g produced a marked and prolonged reduction of IOP. From a baseline of 23.0, IOP was reduced to 14.0, 14.7, 21.2, and 20.9 mm Hg at 5.0, 23.0, 27.0 and 45.5 h, respectively. The 0.423 ?g of arginine, which is associated with 0.01 ?g tPA, was injected alone and had no effect. Recombinant human tPA injected in the AC is effective in reversing steroid-induced IOP elevation in sheep. The reduction of IOP elevation may be the result of an effect on extra-cellular matrix turnover in the TM. These findings suggest that tPA may by useful as a therapeutic agent in steroid-induced glaucomas. PMID:25304217

Candia, Oscar A; Gerometta, Rosana M; Danias, John

2014-11-01

232

Plasminogen carbohydrate side chains in receptor binding and enzyme activation: A study of C6 glioma cells and primary cultures of rat hepatocytes  

Energy Technology Data Exchange (ETDEWEB)

The human (Glu1)-plasminogen carbohydrate isozymes, plasminogen type I (Pg 1) and plasminogen type II (Pg 2), were separated by chromatography and studied in cell binding experiments at 4{degrees}C with primary cultures of rat hepatocytes and rat C6 glioma cells. In both cell systems, Pg 1 and Pg 2 bound to an equivalent number of receptors, apparently representing the same population of surface molecules. The affinity for Pg 2 was slightly higher. With hepatocytes, the KD for Pg 1 was 3.2 +/- 0.2 microM, and the KD for Pg 2 was 1.9 +/- 0.1 microM, as determined from Scatchard transformations of the binding isotherms. The Bmax was approximately the same for both isozymes. With C6 cells, the KD for Pg 1 was 2.2 +/- 0.1 microM vs. 1.5 +/- 0.2 microM for Pg 2. Again, the Bmax was similar with both isozymes. 125I-Pg 1 and 125I-Pg 2 were displaced from specific binding sites by either nonradiolabeled isozyme. The KI for Pg 2 was slightly lower than the KI for Pg 1 with hepatocytes (0.9 vs. 1.3 microM) and with C6 cells (0.6 vs. 1.1 microM). No displacement was detected with miniplasminogen at concentrations up to 5.0 microM. Activation of Pg 1 and Pg 2 by recombinant two-chain tissue-plasminogen activator (rt-PA) was enhanced by hepatocyte cultures. The enhancing effect was greater with Pg 2. Hepatocyte cultures did not affect the activation of miniplasminogen by rt-PA or the activation of plasminogen by streptokinase. Unlike the hepatocytes, C6 cells did not enhance the activation of plasminogen by rt-PA or streptokinase; however, plasmin generated in the presence of C6 cells reacted less readily with alpha 2-antiplasmin.

Hall, S.W.; VandenBerg, S.R.; Gonias, S.L. (Univ. of Virginia Health Sciences Center, Charlottesville (USA))

1990-07-01

233

Successful thrombolysis of a thrombosed prosthetic mitral valve using a synthetic tissue plasminogen activator: a case report  

Directory of Open Access Journals (Sweden)

Full Text Available Abstract Introduction Prosthetic valve thrombosis is a rare but life-threatening condition that requires careful evaluation and prompt treatment. While surgical intervention remains the gold standard, thrombolytic therapy is now emerging as a potential substitute. Various thrombolytic treatments including streptokinase, urokinase and recombinant tissue plasminogen activators have been reported with variable success rates. However, the data on the use of tenecteplase (a synthetic tissue plasminogen activator is limited. Case presentation A 44-year-old Middle Eastern man with a previously implanted prosthetic mitral valve presented with exertional dyspnea and orthopnea. Investigations revealed a thrombosed prosthetic mitral valve. Successful thrombolysis was achieved using tenecteplase which lead to the complete restoration of valve function with no risk to the patient. Conclusion Prosthetic valve thrombosis is a rare but life threatening condition, the diagnosis of which requires a high index of suspicion. Tenecteplase can be used successfully in the management of such cases. It has proved to be useful with no extra risk to the patient.

Al-Fadhli Jamal

2010-08-01

234

Soluble urokinase plasminogen activator receptor levels are associated with severity of fibrosis in nonalcoholic fatty liver disease.  

Science.gov (United States)

The identification of individuals with severe liver fibrosis among patients with chronic liver disease is of major importance when evaluating prognosis, potential risk for complications, and when deciding treatment strategies. Although percutaneous liver biopsy is still considered a "gold standard" for staging of liver fibrosis, attempts to find reliable noninvasive markers of liver fibrosis are frequent. Inflammation is essential for the progression of fibrosis. The urokinase plasminogen activator and its receptor have been associated with hepatic inflammation and fibrosis in mice. High serum concentrations of soluble urokinase plasminogen activator receptor (suPAR) are suggested to be involved in inflammation, tissue remodeling, and cancer metastasis. Here, we evaluated serum suPAR as a noninvasive test to detect liver fibrosis in 82 well-characterized patients with nonalcoholic fatty liver disease (NAFLD), and in 38 untreated patients with chronic hepatitis C virus (HCV) infection at the time of their first liver biopsy. suPAR levels were increased in chronic liver disease compared with blood donors (P marker related to fibrosis and prognosis rather than reflecting inflammation. PMID:25445207

Sjöwall, Christopher; Martinsson, Klara; Cardell, Kristina; Ekstedt, Mattias; Kechagias, Stergios

2014-10-01

235

Urokinase-type plasminogen activator receptor (uPAR) as a promising new imaging target : potential clinical applications  

DEFF Research Database (Denmark)

Urokinase-type plasminogen activator receptor (uPAR) has been shown to be of special importance during cancer invasion and metastasis. However, currently, tissue samples are needed for measurement of uPAR expression limiting the potential as a clinical routine. Therefore, non-invasive methods are needed. In line with this, uPAR has recently been identified as a very promising imaging target candidate. uPAR consists of three domains attached to the cell membrane via a glycosylphosphatidylinositol (GPI) anchor and binds it natural ligand uPA with high affinity to localize plasminogen activation at the cell surface. Due to the importance of uPAR in cancer invasion and metastasis, a number of high-affinity ligands have been identified during the last decades. These ligands have recently been used as starting point for the development of a number of ligands for imaging of uPAR using various imaging modalities such as optical imaging, magnetic resonance imaging, single photon emission computer tomography (SPECT) and positron emission topography (PET). In this review, we will discuss recent advances in the development of uPAR-targeted imaging ligands according to imaging modality. In addition, we will discuss the potential future clinical application for uPAR imaging as a new imaging biomarker.

Persson, Morten; Kjaer, Andreas

2013-01-01

236

Reactive oxygen species regulate urokinase plasminogen activator expression and cell invasion via mitogen-activated protein kinase pathways after treatment with hepatocyte growth factor in stomach cancer cells  

OpenAIRE

Abstract Background Reactive oxygen species (ROS) are closely associated with the intracellular signal cascade, thus strongly implicating involvement in tumor progression. However, the mechanism by which ROS are generated and how ROS target downstream molecules to trigger tumor metastasis is unclear. In this study, we investigated the underlying signal pathways in ROS-induced urokinase plasminogen activator (uPA) expression in the human gastric cancer cells, NUGC-3 and MKN-28...

Kim Jae-Ryong; Kim Sang; Lee Kyung

2009-01-01

237

Prohemorrhagic and bleeding time activities of recombinant tissue plasminogen activator, heparin, aspirin, and a glycoprotein IIb/IIIa antagonist.  

Science.gov (United States)

Intracerebral hemorrhage (ICH) is the most serious side effect of antithrombotic agents, especially in cases of cerebrovascular disease. In the present study, we compared the exacerbation of ICH and prolongation of bleeding time (BT) in guinea pigs with recombinant tissue plasminogen activator (rt-PA), heparin, aspirin, and FK419, a novel nonpeptide platelet glycoprotein (GP) IIb/IIIa receptor antagonist. ICH was induced by injection of bacterial collagenase into the caudate nucleus; BT was measured with a Simplate R device. Neither heparin nor aspirin prolonged BT. In contrast, rt-PA at the highest dose used in the study did prolong BT, and FK419 caused a dose-dependent prolongation of BT. Moreover, rt-PA and heparin increased the degree of ICH in a dose-dependent manner, leading to death in more than half of the animals treated with higher doses of these drugs. These findings show that the prohemorrhagic mechanisms underlying the prolongation of BT differ from those in collagenase-induced ICH, and that the risk of an agent with antithrombotic effects potentiating hemorrhage in the collagenase-induced model of ICH more closely parallels that in stroke patients than does the effect of the agent on BT. The findings also suggest that antiplatelet agents, including FK419, may be safer than thrombolytic or anticoagulant agents for use in patients at risk for ICH, such as those with stroke or cerebral aneurysm. PMID:16305324

Mihara, Kayoko; Aoki, Toshiaki; Moriguchi, Akira; Maeda, Masashi; Furuichi, Yasuhisa; Matsuoka, Nobuya; Mutoh, Seitaro

2005-11-01

238

Pharmacokinetics of antigen and activity of recombinant tissue-type plasminogen activator after infusion in healthy volunteers.  

Science.gov (United States)

Pharmacokinetics of recombinant tissue plasminogen activator (rt-PA, large-scale process) were determined based on antigen and activity after infusion of 0.25 mg/kg in 8 healthy volunteers. Plasma antigen was measured using enzyme-linked immunosorbent assay (ELISA) with and without treatment of blood at collection with D-Phe-Pro-Arg-CH2Cl (P PACK); activity was quantified in acidified plasma both on fibrin plates and in a chromogenic assay. Highest rt-PA concentrations were measured in ELISA with P PACK-treated samples, yielding the following pharmacokinetic parameters (2-compartment model, mean +/- S.D.): Cmax = 973 +/- 133 ng/ml, CL = 687 ml/min, dominant half-life t1/2 alpha = 3.3 +/- 0.4 min, t1/2 beta = 26 +/- 12 min, V1 = 3.9 +/- 0.6 l and Vss = 7.2 +/- 1.0 l. The other assays yielded lower rt-PA concentrations, which affected clearance and volume parameters but not t1/2 beta and t1/2 beta. Linear regressions of the fibrin plate and chromogenic assay results vs. ELISA yielded excellent correlations (R greater than 0.96, n = 55-57) but slopes of 0.76 and 0.64, respectively. This indicates that about 25-35% of rt-PA antigen in thawed plasma samples are not detected in activity assays, due at least partially to in vitro binding of rt-PA by proteinase inhibitors. PMID:3132929

Seifried, E; Tanswell, P; Rijken, D C; Barrett-Bergshoeff, M M; Su, C A; Kluft, C

1988-03-01

239

Interrelations between blood-brain barrier permeability and matrix metalloproteinases are differently affected by tissue plasminogen activator and hyperoxia in a rat model of embolic stroke  

OpenAIRE

Abstract Background In ischemic stroke, blood-brain barrier (BBB) regulations, typically involving matrix metalloproteinases (MMPs) and inhibitors (TIMPs) as mediators, became interesting since tissue plasminogen activator (tPA)-related BBB breakdown with risk of secondary hemorrhage was considered to involve these mediators too. Despite high clinical relevance, detailed interactions are purely understood. After a pilot study addressing hyperoxia as potential neuroprotective ...

Michalski Dominik; Hobohm Carsten; Weise Christopher; Pelz Johann; Heindl Marita; Kamprad Manja; Kacza Johannes; Härtig Wolfgang

2012-01-01

240

PULMONARY LOCALIZATION AND EXPRESSION OF PLASMINOGEN ACTIVATOR INHIBITOR-1 (PAI-1) IN HEALTHY OR HYPERTENSIVE RATS EXPOSED TO PARTICULATE MATTER (PM)  

Science.gov (United States)

PULMONARY LOCALIZATION AND EXPRESSION OF PLASMINOGEN ACTIVATOR INHIBITOR-1 (PAI-1) IN HEALTHY OR HYPERTENSIVE RATS EXPOSED TO PARTICULATE MATTER (PM). GS Backus1, R Vincent2, UP Kodavanti2, 1Curriculum in Toxicology, UNC, Chapel Hill; 2NHEERL, ORD, US EPA, Research Triangle Park,...

241

Signal transduction in the platelet activation induced by IgG anti-streptokinase and anisoylated plasminogen-streptokinase activator complex.  

Science.gov (United States)

Streptokinase (SK) is one of the plasminogen activators currently used in therapeutics. SK antibodies may appear in the blood after thrombolytic therapy with SK or after-hemolytic streptococci infection. Such antibodies may both activate platelets and neutralize the ability of SK to convert plasminogen into plasmin. We previously demonstrated that platelet activation induced by the combination of IgG anti-SK and anisoylated plasminogen-SK activator complex (APSAC) is mediated by Fc gamma RIIa1 receptor. However, the mechanism by which IgG anti-SK and APSAC (or SK) transduce an activating signal across the platelet plasma membrane remains unknown. We have demonstrated in the present study that the platelet aggregation induced by the combination of IgG anti-SK and APSAC is accompanied by an increase in inositol phosphate, Ca2+ mobilization and thromboxane (Tx) A2 generation. Neomycin, erbstatin and GF 109203X, which inhibit phospholipase C (PLC), protein tyrosine kinase (PTK) and protein kinase C (PKC) activities, respectively, abolished platelet aggregation induced by IgG anti-SK plus APSAC, indicating the pivotal roles of the PLC, PTK and PKC pathways in this immunological activation. In addition, TxA2 generation is also important since aspirin, a cyclooxygenase inhibitor and SQ 29548, a TxA2 receptor antagonist, showed significant inhibition of the platelet response. The contribution of released ADP was confirmed using apyrase, which significantly inhibited IgG anti-SK plus APSAC-induced platelet aggregation. Finally, WEB 2086, a platelet-activating factor (PAF) receptor antagonist, was not effective, indicating that PAF is not involved in this process. APSAC- or SKinduced platelet activation may limit the therapeutic effectiveness of the drug and may contribute to the pathogenesis of early reocclusion. The study of the mechanism leading to APSAC-induced platelet activation could be relevant for a better understanding of the physiopathology of immune complex disorder diseases and thrombolytic treatment failure. PMID:16793641

1997-04-01

242

Expression of matrix metalloproteinases-1, -2, and -9; tissue inhibitors of matrix metalloproteinases-1 and -2; cathepsin B; urokinase plasminogen activator; and plasminogen activator inhibitor, type I in skull base chordoma.  

Science.gov (United States)

Little is known about proteinase expression in skull base chordoma, a rare bone tumor exhibiting local invasiveness. Using immunohistochemical techniques, we investigated the expression of matrix metalloproteinases (MMPs)-1, -2, and -9; tissue inhibitors of matrix metalloproteinases (TIMPs)-1 and -2; cathepsin B (CatB); urokinase plasminogen activator (uPA); and plasminogen activator inhibitor, type I (PAI1), in 45 patients with skull base chordoma (45 primary and 25 autologous recurrent lesions). We compared these data with clinicopathologic parameters and the expression of cell differentiation markers. MMP-1, MMP-2, TIMP-1, CatB, uPA, and PAI1 were frequently expressed, and there was a significant correlation in the expression of some proteinases. Immunoreactivity for MMP-1, MMP-2, CatB, and uPA was significantly higher in lesions exhibiting tumor infiltration of host bone than in those without such components. Expression of MMP-1, TIMP-1, CatB, and uPA was associated with that of low-molecular-weight cytokeratin (CAM5.2). There were no differences in proteinase expression in 25 pairs of primary and their recurrent lesions, and proteinase expression did not predict local recurrences. However, patients with higher expression of both MMP-1 and uPA showed worse prognosis compared with the others. In conclusion, expression of some proteinases correlated with CAM5.2 expression and seemed to play an important role in a synergistic manner in the invasion process in skull base chordoma. The authors believe that elevated expression of MMP-1 and uPA can be used to identify patients with a worse prognosis in skull base chordoma. PMID:17949787

Naka, Takahiko; Kuester, Doerthe; Boltze, Carsten; Schulz, Torss-Oliver; Samii, Amir; Herold, Christian; Ostertag, Helmut; Roessner, Albert

2008-02-01

243

Binding areas of urokinase-type plasminogen activator-plasminogen activator inhibitor-1 complex for endocytosis receptors of the low-density lipoprotein receptor family, determined by site-directed mutagenesis  

DEFF Research Database (Denmark)

Some endocytosis receptors related to the low-density lipoprotein receptor, including low-density lipoprotein receptor-related protein-1A, very-low-density lipoprotein receptor, and sorting protein-related receptor, bind protease-inhibitor complexes, including urokinase-type plasminogen activator (uPA), plasminogen activator inhibitor-1 (PAI-1), and the uPA-PAI-1 complex. The unique capacity of these receptors for high-affinity binding of many structurally unrelated ligands renders mapping of receptor-binding surfaces of serpin and serine protease ligands a special challenge. We have mapped the receptor-binding area of the uPA-PAI-1 complex by site-directed mutagenesis. Substitution of a cluster of basic residues near the 37-loop and 60-loop of uPA reduced the receptor-binding affinity of the uPA-PAI-1 complex approximately twofold. Deletion of the N-terminal growth factor domain of uPA reduced the affinity 2-4-fold, depending on the receptor, and deletion of both the growth factor domain and the kringle reduced the affinity sevenfold. The binding affinity of the uPA-PAI-1 complex to the receptors was greatly reduced by substitution of basic and hydrophobic residues in alpha-helix D and alpha-helix E of PAI-1. The localization of the implicated residues in the 3D structures of uPA and PAI-1 shows that they form a continuous receptor-binding area spanning the serpin as well as the A-chain and the serine protease domain of uPA. Our results suggest that the 10-100-fold higher affinity of the uPA-PAI-1 complex compared with the free components depends on the bonus effect of bringing the binding areas on uPA and PAI-1 together on the same binding entity.

Skeldal, Sune; Larsen, Jakob Vejby

2006-01-01

244

Immunoradiometric quantitation of tissue plasminogen activator-related antigen in human plasma: crypticity phenomenon and relationship to plasma fibrinolysis  

Energy Technology Data Exchange (ETDEWEB)

A two-site immunoradiometric assay for tissue plasminogen activator (tPA) antigen has been developed using immunoaffinity purified antibody. Various treatments enhanced the detection of tPA antigen in the plasma samples. Maximum detection was obtained by acidification of plasma to pH 4.8 to 6.5 or addition of 0.5 mol/L of L-lysine or L-arginine. Acidification or addition of lysine to plasma is also required for maximum immunoadsorption of plasma tPA antigen on anti-tPA-Ig-sepharose. These results indicate that plasma tPA antigen is partially cryptic to antibody in untreated plasma. The plasma tPA antigen isolated by immunoadsorption of either untreated plasma or acidified plasma on anti-tPA-Ig-sepharose consists mainly of a 100-kd plasminogen activator species as determined by fibrin-agar zymography. The 100-kd activity is possibly a tPA:inhibitor complex. A standardized sample preparation method was conveniently adopted by mixing 3 vol of plasma and 1 vol of 2 mol/L of L-lysine for the assay. Reconstitution and recovery studies showed that the method is specific and permits full detection of both free tPA and tPA:inhibitor complex. The validity of the assay is further supported by the finding that the spontaneous plasma fibrinolysis previously demonstrated to be dependent on plasma tPA antigen is correlated with tPA antigen content. Using the standardized assay, we found that tPA antigen concentrations in 16 blood bank plasmas are equivalent to 3.7 to 20 ng of 60 kd tPA/mL. In all the plasma tested, more than half of the antigen is undetected unless the plasma is treated as described above.

Wun, T.C.; Capuano, A.

1987-05-01

245

Immunoradiometric quantitation of tissue plasminogen activator-related antigen in human plasma: crypticity phenomenon and relationship to plasma fibrinolysis  

International Nuclear Information System (INIS)

A two-site immunoradiometric assay for tissue plasminogen activator (tPA) antigen has been developed using immunoaffinity purified antibody. Various treatments enhanced the detection of tPA antigen in the plasma samples. Maximum detection was obtained by acidification of plasma to pH 4.8 to 6.5 or addition of 0.5 mol/L of L-lysine or L-arginine. Acidification or addition of lysine to plasma is also required for maximum immunoadsorption of plasma tPA antigen on anti-tPA-Ig-sepharose. These results indicate that plasma tPA antigen is partially cryptic to antibody in untreated plasma. The plasma tPA antigen isolated by immunoadsorption of either untreated plasma or acidified plasma on anti-tPA-Ig-sepharose consists mainly of a 100-kd plasminogen activator species as determined by fibrin-agar zymography. The 100-kd activity is possibly a tPA:inhibitor complex. A standardized sample preparation method was conveniently adopted by mixing 3 vol of plasma and 1 vol of 2 mol/L of L-lysine for the assay. Reconstitution and recovery studies showed that the method is specific and permits full detection of both free tPA and tPA:inhibitor complex. The validity of the assay is further supported by the finding that the spontaneous plasma fibrinolysis previously demonstrated to be dependent on plasma tPA antigen is correlated with tPA antigen content. Using the standardized assay, we found that tPA antigen concentrations in 16 blood bank plasmas are equivalent to 3.7 to 20 ng of 60 kd s are equivalent to 3.7 to 20 ng of 60 kd tPA/mL. In all the plasma tested, more than half of the antigen is undetected unless the plasma is treated as described above

246

Degradation of fibrin and elastin by intact human alveolar macrophages in vitro. Characterization of a plasminogen activator and its role in matrix degradation.  

OpenAIRE

Fibrin deposition is prominent in the histopathology of a number of inflammatory lung diseases. Plasmin, activated locally in the lung, can degrade not only this fibrin but potentially structural proteins important to normal lung architecture. Because alveolar macrophages are prominent in inflammatory processes of the lung, we examined the plasminogen activator (PA) activity of human alveolar macrophages. Intact alveolar macrophages from each of 10 healthy subjects expressed PA activity. Ther...

Chapman, H. A.; Stone, O. L.; Vavrin, Z.

1984-01-01

247

Stroke in a child safely treated with intravenous tissue plasminogen activator and edaravone, a free radical scavenger.  

Science.gov (United States)

An 11-year-old female felt discomfort in her head, and left hemispheric syndrome occurred shortly thereafter. At presentation, her National Institutes of Health stroke scale (NIHSS) score was 13, and a magnetic resonance imaging scan revealed acute brain infarction in the left thalamus. She was immediately treated with the intravenous administration of tissue plasminogen activator (IV t-PA) followed by edaravone, a free radical scavenger. Two hours after IV t-PA, her symptoms dramatically resolved and her NIHSS score decreased to 5. No adverse events were observed. She was the youngest patient treated with IV t-PA in Japan, and would be the youngest treated in most developed countries. An optimal treatment for stroke in children has not been established, and this case highlights the urgent need to examine the safety and efficacy of IV t-PA and edaravone therapy for ischemic stroke in children. PMID:21421331

Baba, Haruhisa; Sugimori, Hiroshi; Nanishi, Etsuro; Nagata, Hazumu; Lee, Sooyoung; Kuwashiro, Takahiro; Hashizume, Makoto

2012-11-01

248

Effect of Vitreoscilla hemoglobin expression on growth and specific tissue plasminogen activator productivity in recombinant Chinese hamster ovary cells  

Energy Technology Data Exchange (ETDEWEB)

Previous studies suggest that secretion of cloned proteins synthesized by recombinant Chinese hamster ovary (CHO) cells can be adenosine triphosphate (ATP) limited. Other research indicates that the presence of cloned Vitreoscilla hemoglobin (VHb) enhances ATP production in oxygen-limited Escherichia coli. To evaluate the influence of VHb expression on recombinant CHO cell productivity, the vhb gene has been fused to the mouse mammary tumor virus (MMTV) promoter and cloned in a CHO cell line previously engineered to express human tissue plasminogen activator (tPA). Western blot analysis confirms dexamethasone-inducible VHb expression in all of the clones tested. Batch cultivation experiments with one VHb-expressing clone and the parental CHO-tPA cells show a reduced specific growth rate in the VHb-expressing cells. The VHb-expressing clone exhibits specific tPA production 40 to 100% greater than the parental CHO-tPA culture.

Pendse, G.J.; Bailey, J.E. (California Inst. of Tech., Pasadena, CA (United States). Dept. of Chemical Engineering)

1994-12-01

249

Cryopreserved recombinant tissue plasminogen activator for the restoration of occluded central venous access devices in pediatric oncology patients  

International Nuclear Information System (INIS)

Thrombolytic therapy with urokinase 5000 units has been the standard therapy for restoration of thrombosed central catheters. However, with the decreased availability of urokinase, alternatives needed to be sought. The aim of the study was to determine the efficacy, bioactivity, dwell time and cost of cryopreserved recombinant tissue plasminogen activator (rTPA) in the restoration of occluded central venous access devices. For children 10kg, a dose of 1 mg was used. The dwell time was 1-2 hours. Of the 40 courses of rTPA, 39 fully restored central venous line patency (97%). Successful courses were instilled for an average of 1 hour. Cryopreserved rTPA appears to be safe and effective in the dose used to restore the patency of occluded central venous access devices in pediatric oncology patients. (author)

250

Inhibition of urokinase plasminogen activator “uPA” activity alters ethanol consumption and conditioned place preference in mice  

Science.gov (United States)

Urokinase plasminogen activator, uPA, is a serine protease implicated in addiction to drugs of abuse. Using its specific inhibitor, B428, we and others have characterized the role of uPA in the rewarding properties of psychostimulants, including cocaine and amphetamine, but none have examined the role of uPA in ethanol use disorders. Therefore, in the current study, we extended our observations to the role of uPA in ethanol consumption and ethanol-induced conditioned place preference. The general aim of the present series of experiments was to investigate the effects of the administration of the B428 on voluntary alcohol intake and ethanol conditioned reward. A two-bottle choice, unlimited-access paradigm was used to compare ethanol intake between vehicle- and 3, 10, and 30 mg/kg B428-administered mice. For this purpose, the mice were presented with an ethanol solution (2.5%–20%) and water, at each concentration for 4 days, and their consumption was measured daily. Consumption of saccharin and quinine solutions was also measured. Systemic administration of B428 dose-dependently decreased ethanol intake and preference. Additionally, B428 mice did not differ from vehicle mice in their intake of graded solutions of tastants, suggesting that the uPA inhibition did not alter taste function. Also, ethanol metabolism was not affected following B428 injection. More importantly, 1.5 g/kg ethanol-induced conditioned place preference acquisition was blocked following B428 administration. Taken together, our results are the first to implicate uPA inhibition in the regulation of ethanol consumption and preference, and suggest that uPA may be considered as a possible therapeutic drug target for alcoholism and abstinence. PMID:25258509

Al Maamari, Elyazia; Al Ameri, Mouza; Al Mansouri, Shamma; Bahi, Amine

2014-01-01

251

Inhibition of urokinase plasminogen activator "uPA" activity alters ethanol consumption and conditioned place preference in mice.  

Science.gov (United States)

Urokinase plasminogen activator, uPA, is a serine protease implicated in addiction to drugs of abuse. Using its specific inhibitor, B428, we and others have characterized the role of uPA in the rewarding properties of psychostimulants, including cocaine and amphetamine, but none have examined the role of uPA in ethanol use disorders. Therefore, in the current study, we extended our observations to the role of uPA in ethanol consumption and ethanol-induced conditioned place preference. The general aim of the present series of experiments was to investigate the effects of the administration of the B428 on voluntary alcohol intake and ethanol conditioned reward. A two-bottle choice, unlimited-access paradigm was used to compare ethanol intake between vehicle- and 3, 10, and 30 mg/kg B428-administered mice. For this purpose, the mice were presented with an ethanol solution (2.5%-20%) and water, at each concentration for 4 days, and their consumption was measured daily. Consumption of saccharin and quinine solutions was also measured. Systemic administration of B428 dose-dependently decreased ethanol intake and preference. Additionally, B428 mice did not differ from vehicle mice in their intake of graded solutions of tastants, suggesting that the uPA inhibition did not alter taste function. Also, ethanol metabolism was not affected following B428 injection. More importantly, 1.5 g/kg ethanol-induced conditioned place preference acquisition was blocked following B428 administration. Taken together, our results are the first to implicate uPA inhibition in the regulation of ethanol consumption and preference, and suggest that uPA may be considered as a possible therapeutic drug target for alcoholism and abstinence. PMID:25258509

Al Maamari, Elyazia; Al Ameri, Mouza; Al Mansouri, Shamma; Bahi, Amine

2014-01-01

252

Plasminogen Activator Inhibitor-1 Regulates LPS-Induced TLR4/MD-2 Pathway Activation and Inflammation in Alveolar Macrophages.  

Science.gov (United States)

Toll-like receptor 4 (TLR4) and myeloid differentiation protein 2 (MD-2) are the main lipopolysaccharide (LPS) binding receptors that respond to inflammatory stimuli and mediate NF-kappa B (NF-?B) signaling pathway in macrophages. We have previously shown that plasminogen activator inhibitor-1 (PAI-1) deletion increased lung injury induced by intratracheal instillation of LPS through downregulation of TLR4 negative regulators. However, the mechanisms by which PAI-1 regulates lung inflammation are largely unknown. The aim of this study is to assess the relationship between PAI-1 and TLR4 signaling pathways in LPS-induced NR8383 cells inflammatory reaction. The results showed that the levels of PAI-1, TNF-?, and IL-1? protein were increased remarkably in NR8383 cell supernatants after LPS stimulation. PAI-1 gene knockdown reduced TNF-? and IL-1? levels in cell supernatants and inhibited the NF-?B p65 protein expression in NR8383 cells. The upregulated mRNA and protein expressions of TLR4, MD-2, and myeloid differentiation protein (MyD88) induced by LPS were attenuated after PAI-1 gene knockdown. Conversely, overexpression of PAI-1 in NR8383 cells not only resulted in additional mRNA and protein production of PAI-1, TLR4, MD-2, and MyD88, it also aggravated the inflammatory response induced by LPS. In conclusion, PAI-1 contributes to the regulation of LPS-induced inflammatory response in NR8383 cells, likely by affecting the TLR4-MD-2/NF-?B signaling transduction pathway. PMID:25342286

Ren, Weiying; Wang, Zhonghui; Hua, Feng; Zhu, Lei

2015-02-01

253

Pretreatment Plasminogen Activator Inhibitor-1 (PAI-1 Level in the Failure of Streptokinase Therapy in Acute Myocardial Infaction  

Directory of Open Access Journals (Sweden)

Full Text Available Background: The risk for unsuccessful reperfusion after streptokinase therapy may be caused by the antifibrinolytic effect of platelet-derived type 1 plasminogen activator inhibitor (Pal-1 and antistreptokinase antibodies. This study aims to show the relation of pretreatment PAI-1 levels of patients with acute myocardial infarction treated with streptokinase and the outcome of fibrinolysis, emphasizing on reperfusion with and without considering pretreatment antistreptokinase antibodies. Methods: Pretreatment PAI-1 and antistreptokinase antibodies levels of 61 patients with acute myocardial infarction, treated with streptokinase, were determined by an enzyme- linked immunosorbent assay kit method. Failure of thrombolysis with streptokinase was present when reperfusion was unsuccessful as assessed by noninvasive reperfusion criteria. Results: Mean pretreatment PAI-1 level of patients was 29.72 ± 4.74 ng/ml. Thrombolysis with streptokinase failed significantly with higher pretreatment PAI-1 levels (p < 0.05 in all patients and patients with negative pretreatment antistreptokinase antibodies. Conclusion: We showed that higher on reperfusion in patients with acute myocardial infarction, with and without considering pretreatment antistreptokinase pretreatment PAI-1 levels were associated with significant failure of streptokinase therapy with the emphasis antibodies (anti-SK. It seems that by estimating PAI-1>25ng/l and antistreptokinase antibodies higher than normal levels in patients before the start of streptokinase therapy, candidates for potentially unsuccessful streptokinase therapy (with failed reperfusion can be identified in advance and an alternative therapy such as primary angioplasty with better immediate results can be started. Keywords: Plasminogen Activator Inhibitor, Streptokinase, Myocardial Infarction.

H Shemirani

2004-08-01

254

Two distinct expression patterns of urokinase, urokinase receptor and plasminogen activator inhibitor-1 in colon cancer liver metastases  

DEFF Research Database (Denmark)

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 liver metastases, we found intense expression of uPAR, uPA-mRNA and PAI-1 in primarily stromal cells at the metastases periphery, and in an expression pattern similar to that found in the primary tumours. In the remaining 9 liver metastases, uPAR and uPA-mRNA were only seen associated with the presence of necrosis within the liver metastases. In addition, PAI-1-immunoreactivity was in all liver metastases seen in hepatocytes at the metastases periphery. Interestingly, the former 5 liver metastases positive for uPAR, uPA mRNA and PAI-1 at the metastasis periphery all had a predominantly desmoplastic reaction, 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 may have implication for the treatment of patients with metastatic disease.

Illemann, Martin; Bird, Nigel

2009-01-01

255

Tonic arterial chemoreceptor activity contributes to cardiac sympathetic activation in mild ovine heart failure.  

Science.gov (United States)

Heart failure (HF) is associated with a large increase in cardiac sympathetic nerve activity (CSNA), which has detrimental effects on the heart and promotes arrhythmias and sudden death. There is increasing evidence that arterial chemoreceptor activation plays an important role in stimulating renal sympathetic nerve activity (RSNA) and muscle sympathetic nerve activity in HF. Given that sympathetic nerve activity to individual organs is differentially controlled, we investigated whether tonic arterial chemoreceptor activation contributes to the increased CSNA in HF. We recorded CSNA and RSNA in conscious normal sheep and in sheep with mild HF induced by rapid ventricular pacing (ejection fraction bursts (100 heart beats)(-1), P burst incidence [93 ± 4 versus 92 ± 4 bursts (100 heart beats)(-1), n = 7], although due to the bradycardia the RSNA burst frequency was decreased (90 ± 8 versus 77 ± 7 bursts min(-1), P < 0.001). In normal sheep, chemoreceptor deactivation reduced heart rate without a significant effect on CSNA or RSNA. In summary, deactivation of peripheral chemoreceptors during HF reduced the elevated levels of CSNA, indicating that tonic arterial chemoreceptor activation plays a critical role in stimulating the elevated CSNA in HF. PMID:24928955

Xing, Daniel T; May, Clive N; Booth, Lindsea C; Ramchandra, Rohit

2014-08-01

256

1H NMR structural characterization of a recombinant kringle 2 domain from human tissue-type plasminogen activator  

International Nuclear Information System (INIS)

The kringle 2 domain of human tissue-type plasminogen activator (t-PA) has been characterized via 1H NMR spectroscopy at 300 and 620 MHz. The experiments were performed on the isolated domain obtained by expression of the 174-263 portion of t-PA in Escherichia coli. The spectrum of t-Pa kringle 2 is characteristic of a globular structure and shows overall similarity to that of the plasminogen (PGN) kringle 4. Spectral comparison with human and bovine PGN kringle 4 identified side-chain resonances from Leu46, which afford a fingerprint of kringle folding, and from most of the aromatic ring spin systems. Ligand-binding studies confirm that t-PA kringle 2 binds L-lysine with an association constant Ka ? 11.9 mM-1. The data indicate that homologous or conserved residues relative to those that compose the lysine-binding sites of PGN kringles 1 and 4 are involved in the binding of L-lysine to t-PA kringle 2. These include Tyr36 and, within the kringle inner loop, Trp62, His64, Trp72, and Tyr74. Several labile NH protons of t-PA kringle 2 exhibit retarded H-exchange kinetics, requiring more than a week in 2H2O for full deuteration in the presence of L-lysine at 37 degree C. This reveals that kringle 2 is endowed with a compact, dynamically stable conformation. Proton Overhauser experiments in 1H2O, centered on well-resolved NH resonub>O, centered on well-resolved NH resonances between 9.8 and 12 ppm, identify signals arising from the His48a imidazole NH3 proton and the three Trp indole NH1 protons. Overall, the data indicate a highly structured conformation for the recombinant t-PA kringle 2 that is closely related to that of the previously investigated PGN kringles 1, 4, and 5

257

Soluble urokinase plasminogen activator receptor is a marker of dysmetabolism in HIV-infected patients receiving highly active antiretroviral therapy  

DEFF Research Database (Denmark)

Circulating soluble urokinase plasminogen activator receptor (suPAR) reflects the immune and pro-inflammatory status of the HIV-infected patient. Highly active antiretroviral therapy (HAART) suppresses suPAR. Independent of the immune response to HAART, suPAR remains elevated in some HIV-infected patients, reflecting possibly a low-grade pro-inflammatory state. Low-grade inflammation has been implicated in insulin resistance and other features of dysmetabolism. Accordingly it is hypothesized that circulating suPAR is associated with the metabolic status of HIV-infected patients on HAART. Fasting plasma suPAR was determined in 36 normoglycaemic HIV-infected patients on HAART (n = 18 lipodystrophic, and n = 18 non-lipodystrophic) who had estimated insulin sensitivity (Rd) and non-oxidative glucose disposal (NOGM) by euglycaemic hyperinsulinaemic clamps, indirect calorimetry, and glucose tracer infusion. Five patients had circadian suPAR concentrations measured (24 hr, 20 min-intervals). suPAR and non-HDL-cholesterol were higher and Rd, NOGM, and limb fat were lower in lipodystrophic patients than in non-lipodystrophic patients (P < 0.05). suPAR correlated positively with non-HDL-cholesterol and inversely with Rd, NOGM and limb fat (P < 0.005, n = 36). suPAR also correlated positively with leukocyte count and TNF-alpha (P < 0.01, n = 36) but not with IL-6. In multiple regression analyses suPAR was a stronger predictor of dysmetabolism than TNF-alpha and IL-6. Circadian suPAR did not systematically fluctuate. In conclusion, suPAR may reflect the metabolic status of the HIV-infected patient on HAART, thus linking low-grade inflammation, immune constitution, lipid and glucose metabolism, and fat redistribution. Circadian suPAR concentration appeared stable, suggesting that sampling schedule does not affect measurement. Further studies addressing whether suPAR predicts lipodystrophy and dysmetabolism in HIV-infected patients are warranted. J. Med. Virol. 80:209-216, 2008. (c) 2007 Wiley-Liss, Inc.

Andersen, Ove; Eugen-Olsen, Jesper

2008-01-01

258

Transforming growth factor beta stimulates urokinase-type plasminogen activator and DNA synthesis, but not prostaglandin E2 production, in human synovial fibroblasts.  

OpenAIRE

Transforming growth factor beta (TGF-beta) is usually associated with matrix formation and tissue repair; in contrast, cellular expression of the serine proteinase, urokinase-type plasminogen activator (u-PA) is often correlated with tissue remodeling, as well as with cell migration and transformation. We report here that purified recombinant human TGF-beta (greater than or equal to 300 pg/ml) can stimulate rapidly (within 2 h) the u-PA activity of nonrheumatoid synovial fibroblast-like cells...

Hamilton, J. A.; Piccoli, D. S.; Leizer, T.; Butler, D. M.; Croatto, M.; Royston, A. K.

1991-01-01

259

Plasminogen carbohydrate side chains in receptor binding and enzyme activation: A study of C6 glioma cells and primary cultures of rat hepatocytes  

International Nuclear Information System (INIS)

The human [Glu1]-plasminogen carbohydrate isozymes, plasminogen type I (Pg 1) and plasminogen type II (Pg 2), were separated by chromatography and studied in cell binding experiments at 4 degrees C with primary cultures of rat hepatocytes and rat C6 glioma cells. In both cell systems, Pg 1 and Pg 2 bound to an equivalent number of receptors, apparently representing the same population of surface molecules. The affinity for Pg 2 was slightly higher. With hepatocytes, the KD for Pg 1 was 3.2 +/- 0.2 microM, and the KD for Pg 2 was 1.9 +/- 0.1 microM, as determined from Scatchard transformations of the binding isotherms. The Bmax was approximately the same for both isozymes. With C6 cells, the KD for Pg 1 was 2.2 +/- 0.1 microM vs. 1.5 +/- 0.2 microM for Pg 2. Again, the Bmax was similar with both isozymes. 125I-Pg 1 and 125I-Pg 2 were displaced from specific binding sites by either nonradiolabeled isozyme. The KI for Pg 2 was slightly lower than the KI for Pg 1 with hepatocytes (0.9 vs. 1.3 microM) and with C6 cells (0.6 vs. 1.1 microM). No displacement was detected with miniplasminogen at concentrations up to 5.0 microM. Activation of Pg 1 and Pg 2 by recombinant two-chain tissue-plasminogen activator (rt-PA) was enhanced by hepatocyte cultures. The enhancing effect was greater with Pg 2. Hepatocyte cultures did not affect the activation of miniplasminogen by rt-PA or the activation of plasminogen by streptokinase. Unlike the hepatocytes, C6 cells did not enhance the aepatocytes, C6 cells did not enhance the activation of plasminogen by rt-PA or streptokinase; however, plasmin generated in the presence of C6 cells reacted less readily with alpha 2-antiplasmin

260

Inhibition of urokinase plasminogen activator “uPA” activity alters ethanol consumption and conditioned place preference in mice  

Directory of Open Access Journals (Sweden)

Full Text Available Elyazia Al Maamari,* Mouza Al Ameri, Shamma Al Mansouri, Amine Bahi*Department of Anatomy, College of Medicine and Health Sciences, United Arab Emirates University, Al Ain, United Arab Emirates*These authors contributed equally to this workAbstract: Urokinase plasminogen activator, uPA, is a serine protease implicated in addiction to drugs of abuse. Using its specific inhibitor, B428, we and others have characterized the role of uPA in the rewarding properties of psychostimulants, including cocaine and amphetamine, but none have examined the role of uPA in ethanol use disorders. Therefore, in the current study, we extended our observations to the role of uPA in ethanol consumption and ethanol-induced conditioned place preference. The general aim of the present series of experiments was to investigate the effects of the administration of the B428 on voluntary alcohol intake and ethanol conditioned reward. A two-bottle choice, unlimited-access paradigm was used to compare ethanol intake between vehicle- and 3, 10, and 30 mg/kg B428-administered mice. For this purpose, the mice were presented with an ethanol solution (2.5%–20% and water, at each concentration for 4 days, and their consumption was measured daily. Consumption of saccharin and quinine solutions was also measured. Systemic administration of B428 dose-dependently decreased ethanol intake and preference. Additionally, B428 mice did not differ from vehicle mice in their intake of graded solutions of tastants, suggesting that the uPA inhibition did not alter taste function. Also, ethanol metabolism was not affected following B428 injection. More importantly, 1.5 g/kg ethanol-induced conditioned place preference acquisition was blocked following B428 administration. Taken together, our results are the first to implicate uPA inhibition in the regulation of ethanol consumption and preference, and suggest that uPA may be considered as a possible therapeutic drug target for alcoholism and abstinence.Keywords: B428, CPP, two-bottle choice

Al Maamari E

2014-09-01

261

Transformation phenotype of polyoma virus-transformed rat fibroblasts: plasminogen activator production is modulated by the growth state of the cells and regulated by the expression of an early viral gene function.  

OpenAIRE

The expression of two transformation parameters, namely, ability to grow in agar and plasminogen activator production, was studied in several rat fibroblasts transformed by either wild-type or thermo-sensitive (tsa and ts25) polyoma viruses. The production of plasminogen activator was found to be dependent upon the growth state of the infected cells during a period of several days after infection. The analysis of the transformed phenotype of 25 tsa transformants and of 19 ts25 transformants i...

Perbal, B.

1980-01-01

262

Increased soluble urokinase plasminogen activator receptor (suPAR) is associated with thrombosis and inhibition of plasmin generation in paroxysmal nocturnal hemoglobinuria (PNH) patients  

OpenAIRE

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired genetic disorder of the bone marrow that produces intravascular hemolysis, proclivity to venous thrombosis, and hematopoietic failure. Mutation in the PIG-A gene of a hematopoietic stem cell abrogates synthesis of glycosylphosphoinositol (GPI) anchors and expression of all GPI-anchored proteins on the surface of progeny erythrocytes, leukocytes, and platelets. Urokinase plasminogen activator receptor (uPAR), a GPI-linked protein express...

Sloand, Elaine M.; Pfannes, Loretta; Scheinberg, Phillip; More, Kenneth; Wu, Colin O.; Horne, Mcdonald; Young, Neal S.

2008-01-01

263

Evaluation of a weight-adjusted single-bolus plasminogen activator in patients with myocardial infarction: a double-blind, randomized angiographic trial of lanoteplase versus alteplase.  

OpenAIRE

BACKGROUND: Lanoteplase (nPA) is a rationally designed variant of tissue plasminogen activator with greater fibrinolytic potency and slower plasma clearance than alteplase. METHODS AND RESULTS: InTIME (Intravenous nPA for Treatment of Infarcting Myocardium Early), a multicenter, double-blind, randomized, double-placebo angiographic trial, evaluated the dose-response relationship and safety of single-bolus, weight-adjusted lanoteplase. Patients (n=602) presenting within 6 hours of acute myocar...

Den Heijer, P.; Vermeer, F.; Ambrosioni, E.; Sadowski, Z.; Lopez-sendon, J. L.; Von Essen, R.; Beaufils, P.; Thadani, U.; Adgey, J.; Pierard, Luc; Brinker, J.; Davies, R. F.; Smalling, R. W.; Wallentin, L.; Caspi, A.

1998-01-01

264

A systemic non-lytic state and local thrombolytic failure of anistreplase (anisoylated plasminogen streptokinase activator complex, APSAC) in acute myocardial infarction.  

OpenAIRE

The relation between coronary thrombolysis and coagulation variables after administration of anistreplase (anisoylated plasminogen streptokinase activator complex, APSAC) was studied in patients with an acute myocardial infarction. Fifty eight consecutive patients with acute myocardial infarction were given 30 U of anistreplase intravenously within 4 hours of the onset of symptoms. A fall in the plasma concentration fibrinogen to less than 1.0 g/l 90 minutes after administration of anistrepla...

Bru?gemann, J.; Meer, J.; Takens, B. H.; Hillege, H.; Lie, K. I.

1990-01-01

265

Gene therapy to promote thromboresistance: Local overexpression of tissue plasminogen activator to prevent arterial thrombosis in an in vivo rabbit model  

OpenAIRE

Tissue-type plasminogen activator (tPA) catalyzes the rate-limiting initial step in the fibrinolytic cascade. Systemic infusion of tPA has become the standard of care for acute myocardial infarction. However, even the relatively short-duration protocols currently employed have encountered significant hemorrhagic complications, as well as complications from rebound thrombosis. Gene therapy offers a method of local high-level tPA expression over a prolonged time period to avoid both systemic he...

Waugh, J. M.; Kattash, M.; Li, J.; Yuksel, E.; Kuo, M. D.; Lussier, M.; Weinfeld, A. B.; Saxena, R.; Rabinovsky, E. D.; Thung, S.; Woo, S. L. C.; Shenaq, S. M.

1999-01-01

266

Outcome after systemic thrombolysis is predicted by age and stroke severity: an open label experience with recombinant tissue plasminogen activator and tirofiban  

OpenAIRE

Stroke patients can recover upon intravenous thrombolysis but remain impaired in lacking recanalization. We sought to investigate the clinical effect of systemic thrombolysis with an intravenous bolus of 20 mg recombinant tissue plasminogen activator (rtPA) and an infusion of body-weight adjusted tirofiban for 48 hours in acute stroke. This prospective, open label study, included 192 patients (68±13 years, 50% males) treated between 1 January 2005 and 31 December 2007. The neurological defic...

Seitz, Rudiger J.; Judith Sukiennik; Mario Siebler

2012-01-01

267

Elevated plasma plasminogen activator inhibitor type-1 is an independent predictor of coronary microvascular dysfunction in hypertension  

International Nuclear Information System (INIS)

Elevated plasma plasminogen activator inhibitor-1 (PAI-1) is related to cardiovascular events, but its role in subclinical coronary microvascular dysfunction remains unknown. Thus, in the present study it was investigated whether elevated plasma PAI-1 activity is associated with coronary microvascular dysfunction in hypertensive patients. Thirty patients with untreated essential hypertension and 10 age-matched healthy controls were studied prospectively. Myocardial blood flow (MBF) was measured by using 15O-water positron emission tomography. Clinical variables associated with atherosclerosis (low-density lipoprotein-cholesterol, high-density lipoprotein (HDL)-cholesterol, triglyceride, homeostasis model assessment (HOMA-IR), and PAI-1 activity) were assessed to determine their involvement in coronary microvascular dysfunction. Adenosine triphosphate (ATP)-induced hyperemic MBF and coronary flow reserve (CFR) were significantly lower in hypertensive patients than in healthy controls (ATP-induced MBF: 2.77±0.82 vs 3.49±0.71 ml·g-1·min-1; p<0.02 and CFR: 2.95±1.06 vs 4.25±0.69; p<0.001). By univariate analysis, CFR was positively correlated with HDL-cholesterol (r=0.46, p<0.02), and inversely with HOMA-IR (r=-0.39, p<0.05) and PAI-1 activity (r=-0.61, p<0.001). By multivariate analysis, elevated PAI-1 activity remained a significant independent determinant of diminished CFR. Elevated plasma PAI-1 activity was independently associat activity was independently associated with coronary microvascular dysfunction, which suggests that plasma PAI-1 activity is an important clue linking hypofibrinolysis to the development of atherosclerosis. (author)

268

Localization and significance of urokinase plasminogen activator and its receptor in placental tissue from intrauterine, ectopic and molar pregnancies  

DEFF Research Database (Denmark)

Urokinase plasminogen activator receptor (uPAR) is a membrane-anchored protein with urokinase plasminogen activator (uPA) as the ligand. This complex induces proteolysis and remodelling of maternal decidua during placental implantation. The presence of uPAR on trophoblasts is supposed to promote adhesion, migration and invasion. In cancer tissue, high levels of uPAR are correlated with a poor prognosis. This immunohistochemical study shows the localization of uPA and uPAR in a prospective design with stereological sampling of fetal and maternal tissue from normal, ectopic and hydatidiform molar (HM) pregnancies. Cytokeratin and Ki67 were used as markers for trophoblasts and proliferating cells. Membrane-bound uPAR was observed on villous non-proliferating intermediate trophoblasts (IT) within cell columns in intrauterine and ectopic pregnancies. The corresponding proliferating IT with cytological atypia sprouting from the chorionic villi in HM was uPAR-negative. uPA but not uPAR was observed in anchoring distal IT at the attachment-point to the basal plate. In the placental bed, extravillous interstitial trophoblasts were uPA-positive but uPAR-negative. The trophoblast giant cells were both uPA- and uPAR-negative. In relation to the maternal vessels, a focal distribution for uPA and uPAR was present in the endovascular and perivascular trophoblasts. The intraluminal trophoblasts overlying endothelial cells were uPAR-positive only. In maternal tissue from intrauterine and molar pregnancies, uPAR was seen in the decidual cells in a zone facing the anchoring villi and the fibrinoid lesions with embedded trophoblasts. In contrast, the stromal cells of the fallopian tube without a decidual reaction facing the implanted gestation were uPAR-negative. Non-invaded decidual, myometrial and muscular tissue of the pregnant uterus and fallopian tube was extensively positive for uPA whereas 'pseudodecidual' cells from the intrauterine evacuate in patients with an ectopic pregnancy only showed a focal and scanty reaction for uPA. When trophoblast invasion of the decidua was present, the decidual cells were uPA-negative. A semi-quantitative assessment of the receptor was estimated in villous IT within cell columns in normal and molar pregnancies but, in conclusion, quantitative evaluation of uPAR cannot be used to predict development of post-molar persistent trophoblastic disease (PTD).

Floridon, C; Nielsen, O

1999-01-01

269

Radio-enzymatic-assay method of plasminogen  

International Nuclear Information System (INIS)

Plasmin obtained from activating plasminogen is used to hydrolyze 125I-fibrin into soluble 125I-polypeptide, thus developing a radioenzymatic-assay method of plasminogen. The activity of plasmin can be measured and the content of 125I-fibrin can be determined by the method. This method is simple, quick, specific and sensitive for evaluating thrombophilia in clinical study of thrombin disease

270

Hemothorax under thrombolytic therapy with recombinant tissue: plasminogen activator (rt-PA) in a 16-year-old girl.  

Science.gov (United States)

We present the case of a 16-year-old girl with an extended thrombosis of the femoral and iliac vein and the inferior vena cava during pleuropneumonia; predisposing risk factors for thrombophilia were: use of contraceptives, nicotine abuse and congenital deficiency of antithrombin III (not previously diagnosed). Thrombolytic therapy with recombinant tissue plasminogen activator (rt-PA; initial dose: 0.08 mg/kg/h) was started. 2 days later--after diagnosis of an extended hemothorax: 1500 ml blood were obtained after thoracocentesis, transfusion of packed red blood cells was necessary--rt-PA was stopped, with only heparin (400 U/kg/d) being administered. 36 h later--the thrombosis had not yet changed--the thrombolytic therapy with rt-PA was continued in a markedly reduced dose (0.015 mg/kg/d) with no further bleeding complications. 8 days later--after successful thrombolysis--t-PA was stopped, heparin was given for another 10 days, then cumarin was administered orally. PMID:10650854

Varnholt, V; Ringe, H; Nietsch, L; Gaedicke, G

1999-12-01

271

A flexible multidomain structure drives the function of the urokinase-type plasminogen activator receptor (uPAR)  

DEFF Research Database (Denmark)

The urokinase-type plasminogen activator receptor (uPAR) provides a rendezvous between proteolytic degradation of the extracellular matrix and integrin mediated adhesion to vitronectin. These processes are however tightly linked as the high-affinity binding of urokinase regulates the binding of uPAR to matrix-embedded vitronectin. Although crystal structures exist to define the corresponding static bi- and trimolecular receptor complexes it is evident that the dynamic property of uPAR plays a decisive role for its function. In the present study, we combine small angle X-ray scattering, hydrogen-deuterium exchange, and surface plasmon resonance to develop a structural model describing the allosteric regulation of uPAR. We show that the flexibility of its N-terminal domain provides the key for understanding this allosteric mechanism. Importantly, our model has direct implications for understanding uPAR-assisted cell adhesion and migration as well as for translational research including targeted intervention therapy and non-invasive tumor imaging in vivo.

Mertens, Haydyn D.T.; Kjærgaard, Magnus

2012-01-01

272

A flexible multidomain structure drives the function of the urokinase-type plasminogen activator receptor (uPAR).  

Science.gov (United States)

The urokinase-type plasminogen activator receptor (uPAR) provides a rendezvous between proteolytic degradation of the extracellular matrix and integrin-mediated adhesion to vitronectin. These processes are, however, tightly linked because the high affinity binding of urokinase regulates the binding of uPAR to matrix-embedded vitronectin. Although crystal structures exist to define the corresponding static bi- and trimolecular receptor complexes, it is evident that the dynamic property of uPAR plays a decisive role in its function. In the present study, we combine small angle x-ray scattering, hydrogen-deuterium exchange, and surface plasmon resonance to develop a structural model describing the allosteric regulation of uPAR. We show that the flexibility of its N-terminal domain provides the key for understanding this allosteric mechanism. Importantly, our model has direct implications for understanding uPAR-assisted cell adhesion and migration as well as for translational research, including targeted intervention therapy and non-invasive tumor imaging in vivo. PMID:22896701

Mertens, Haydyn D T; Kjaergaard, Magnus; Mysling, Simon; Gårdsvoll, Henrik; Jørgensen, Thomas J D; Svergun, Dmitri I; Ploug, Michael

2012-10-01

273

A Flexible Multidomain Structure Drives the Function of the Urokinase-type Plasminogen Activator Receptor (uPAR)*  

Science.gov (United States)

The urokinase-type plasminogen activator receptor (uPAR) provides a rendezvous between proteolytic degradation of the extracellular matrix and integrin-mediated adhesion to vitronectin. These processes are, however, tightly linked because the high affinity binding of urokinase regulates the binding of uPAR to matrix-embedded vitronectin. Although crystal structures exist to define the corresponding static bi- and trimolecular receptor complexes, it is evident that the dynamic property of uPAR plays a decisive role in its function. In the present study, we combine small angle x-ray scattering, hydrogen-deuterium exchange, and surface plasmon resonance to develop a structural model describing the allosteric regulation of uPAR. We show that the flexibility of its N-terminal domain provides the key for understanding this allosteric mechanism. Importantly, our model has direct implications for understanding uPAR-assisted cell adhesion and migration as well as for translational research, including targeted intervention therapy and non-invasive tumor imaging in vivo. PMID:22896701

Mertens, Haydyn D. T.; Kjaergaard, Magnus; Mysling, Simon; Gårdsvoll, Henrik; Jørgensen, Thomas J. D.; Svergun, Dmitri I.; Ploug, Michael

2012-01-01

274

Plasminogen activator inhibitor-1, factor V, factor II and methylenetetrahydrofolate reductase polymorphisms in women with recurrent miscarriage.  

Science.gov (United States)

The present study investigated the association between genetic polymorphisms of selected thrombophilic factors with recurrent miscarriage (RM). The genetic polymorphisms for plasminogen activator inhibitor-1 4G/5G (PAI-1), Factor V Leiden (FVL), Factor II G20210A (FII) and methylenetetrahydrofolate reductase MTHFR C677T were determined in 186 RM women and 129 healthy women. In RM women, the frequency of heterozygosity for PAI-1 5G/4G (31%) was significantly higher than in controls (5G/4G: 22%) whereas no difference was found in the case of homozygosity 4G/4G and 5G/5G. The frequencies of genotype G/A for FVL and FII were significantly higher in RM women (FVL, 10%; FII, 8%) than in controls (FVL, 3%; FII, 2%). No difference was found in the case of MTHFR C677T. The polymorphisms of FVL and FII should be screened in RM women, whereas PAI-1 seems to be weakly associated with RM. The role of MTHFR C677T polymorphisms without hyperhomocysteinemia appears negligible. PMID:24484533

Pietropolli, A; Giuliani, E; Bruno, V; Patrizi, L; Piccione, E; Ticconi, C

2014-04-01

275

Distribution patterns of Gd-DTPA-enhanced magnetic resonance imaging after intravenous tissue plasminogen activator therapy for acute myocardial infarction  

International Nuclear Information System (INIS)

In patients who received thrombolytic therapy for acute myocardial infarction (AMI), we observed 3 distinct patterns in gadolinium diethylenetriamine pentaacetic acid (Gd-DTPA)-enhanced magnetic resonance (MR) imaging. To clarify the significance of these distribution patterns of Gd-DTPA, 20 consecutive patients underwent Gd-DTPA-enhanced MR imaging 7-10 days after AMI. All of the patients received intravenous recombinant tissue plasminogen activator (IVTPA) within 6 h of onset. Echocardiograms were obtained prior to and serially over 10 days, and interpreted for regional wall motion. Coronary angiograms were obtained the day before discharge. None of the 6 patients with a closed infarct-related artery, and 9 of the 14 patients with an open artery, demonstrated subendocardial enhancement (p<0.05). All of these latter 9 patients demonstrated a significant improvement in wall motion between days 1 and 10 after AMI. In contrast, only 1 of the 7 patients with transmural enhancement and none of the 4 patients with non-homogeneous enhancement demonstrated improvement of wall motion on day 10 (p<0.05). We concluded that subendocardial enhancement was a fair prognostic sign for restoration of regional cardiac function in patients who received IVTPA during AMI. (author)

276

Dissolution of emboli in rats with experimental cerebral thromboembolism by recombinant human tissue plasminogen activator (TD-2061)  

International Nuclear Information System (INIS)

Tissue plasminogen activator (t-PA) is frequently administered clinically as thrombolytic therapy. We injected recombinant t-PA into rats with cerebral 125I-labeled blood clot emboli to evaluate the dissolutive effect of recombinant human single-chain t-PA (rt-PA; TD-2061) on such emboli and to examine the possibility of improving neurological damage in patients with cerebral thrombosis. When rt-PA was given intravenously at a dose of 350,000 IU/kg 2 minutes before embolization, radioactivity in the affected cerebral hemisphere decreased to 20% of that in the vehicle control 2 hours after embolization. A significant decrease in radioactivity in the cerebral hemisphere was also found on the administration of 700,000 IU/kg of rt-PA 30 or 60 minutes after embolization, but not when rt-PA was administered 2 minutes after embolization. Marked inhibition of abnormal behavior such as hemiplegia was seen on treatment with rt-PA 2 minutes before embolization, but not at all when rt-PA treatment was given 30 or 60 minutes after embolization. The findings suggest that rt-PA can dissolve blood clot emboli in cerebral vessels and that prompt thrombolytic therapy is important to minimize neurological dysfunction in cases of cerebral thromboembolism

277

Left ventricular apical thrombus after systemic thrombolysis with recombinant tissue plasminogen activator in a patient with acute ischemic stroke  

Directory of Open Access Journals (Sweden)

Full Text Available Abstract Background Thrombolysis with recombinant tissue plasminogen activator (rtPA is an established treatment in acute stroke. To prevent rethrombosis after rtPA therapy, secondary anticoagulation with heparin is commonly performed. However, the recommended time-point and extent of heparin treatment vary and are not well investigated. Case presentation We report a 61-year-old man who developed an acute global aphasia and right-sided hemiparesis. Cranial CT was normal and systemic thrombolytic therapy with tPA was started 120 minutes after symptom onset. Low-dose subcutaneous heparin treatment was initiated 24 hours later. Transthoracic echocardiography (TTE 12 hours after admission showed slightly reduced left ventricular ejection fraction (LVEF but was otherwise normal. 48 hours later the patient suddenly deteriorated with clinical signs of dyspnea and tachycardia. TTE revelead a large left ventricular apical thrombus as well as a reduction of LVEF to 20 %. Serial further TTE investigations demonstrated a complete resolution of the thrombus and normalisation of LVEF within two days. Conclusion Our case demonstrates an intracardiac thrombus formation following rtPA treatment of acute stroke, probably caused by secondary hypercoagulability. Rethrombosis or new thrombus formation might be an underestimated complication of rtPA therapy and potentially explain cases of secondary stroke progression.

Baumann Gert

2005-05-01

278

The Effect of Recombinant Tissue Plasminogen Activator (r-tPA on Quantitation of Neutralising Anti-Streptokinase Antibodies  

Directory of Open Access Journals (Sweden)

Full Text Available The measurement of anti-streptokinase antibodies can distinguish the patients who may benefit from streptokinase and those who should be treated with some other thrombolytic regimen. Neutralising titration test is a commonly used classical assay for measuring anti-streptokinase antibodies and in this assay the ability of anti-streptokinase antibodies in patients' sera in preventing the lytic effect of streptokinase is assessd. As we showed previously the presence of r-tPA in the serum may interefere with the neutralising titration test, we investigated this interference in vitro. The level of neutralising anti-streptokinase antibodies in a serum sample with known levels of the antibodies were measured in absence and presence of increasing amount of r-tPA including therapeutic values. Increasing amount of r-tPA in vitro induced a sudden reduction in the measurable titre of anti-streptokinase antibody levels in a serum with elevated levels of anti-streptokinase antibodies. This effect of r-tPA, which is through activation of plasminogen has not been reported previously. We suggest this assay is unsuitable for clinical diagnosis of anti-streptokinase antibodies.

Afshin Afshari

2004-10-01

279

Dissolution of emboli in rats with experimental cerebral thromboembolism by recombinant human tissue plasminogen activator (TD-2061)  

Energy Technology Data Exchange (ETDEWEB)

Tissue plasminogen activator (t-PA) is frequently administered clinically as thrombolytic therapy. We injected recombinant t-PA into rats with cerebral {sup 125}I-labeled blood clot emboli to evaluate the dissolutive effect of recombinant human single-chain t-PA (rt-PA; TD-2061) on such emboli and to examine the possibility of improving neurological damage in patients with cerebral thrombosis. When rt-PA was given intravenously at a dose of 350,000 IU/kg 2 minutes before embolization, radioactivity in the affected cerebral hemisphere decreased to 20% of that in the vehicle control 2 hours after embolization. A significant decrease in radioactivity in the cerebral hemisphere was also found on the administration of 700,000 IU/kg of rt-PA 30 or 60 minutes after embolization, but not when rt-PA was administered 2 minutes after embolization. Marked inhibition of abnormal behavior such as hemiplegia was seen on treatment with rt-PA 2 minutes before embolization, but not at all when rt-PA treatment was given 30 or 60 minutes after embolization. The findings suggest that rt-PA can dissolve blood clot emboli in cerebral vessels and that prompt thrombolytic therapy is important to minimize neurological dysfunction in cases of cerebral thromboembolism.

Hara, T.; Iwamoto, M.; Ogawa, H.; Yamamoto, A.; Tomikawa, M. (Research Institute, Daiichi Pharmaceutical Co., Ltd., Tokyo, (Japan))

1990-08-15

280

Distribution patterns of Gd-DTPA-enhanced magnetic resonance imaging after intravenous tissue plasminogen activator therapy for acute myocardial infarction  

Energy Technology Data Exchange (ETDEWEB)

In patients who received thrombolytic therapy for acute myocardial infarction (AMI), we observed 3 distinct patterns in gadolinium diethylenetriamine pentaacetic acid (Gd-DTPA)-enhanced magnetic resonance (MR) imaging. To clarify the significance of these distribution patterns of Gd-DTPA, 20 consecutive patients underwent Gd-DTPA-enhanced MR imaging 7-10 days after AMI. All of the patients received intravenous recombinant tissue plasminogen activator (IVTPA) within 6 h of onset. Echocardiograms were obtained prior to and serially over 10 days, and interpreted for regional wall motion. Coronary angiograms were obtained the day before discharge. None of the 6 patients with a closed infarct-related artery, and 9 of the 14 patients with an open artery, demonstrated subendocardial enhancement (p<0.05). All of these latter 9 patients demonstrated a significant improvement in wall motion between days 1 and 10 after AMI. In contrast, only 1 of the 7 patients with transmural enhancement and none of the 4 patients with non-homogeneous enhancement demonstrated improvement of wall motion on day 10 (p<0.05). We concluded that subendocardial enhancement was a fair prognostic sign for restoration of regional cardiac function in patients who received IVTPA during AMI. (author).

Fukuzawa, Shigeru; Watanabe, Hiroyuki; Shimada, Kazuhiro; Katagiri, Nakoto; Ozawa, Shun (Funabashi Municipal Medical Center, Chiba (Japan))

1994-03-01

281

The liberated domain I of urokinase plasminogen activator receptor--a new tumour marker in small cell lung cancer  

DEFF Research Database (Denmark)

The prognosis of small cell lung cancer (SCLC) remains poor with a 5-year survival rate of 4-6%. In non-small cell lung cancer (NSCLC), high levels of intact and cleaved forms of the receptor for urokinase plasminogen activator (uPAR) are significantly associated with short overall survival. Our aim was therefore to determine the prognostic value of the different uPAR forms in blood from SCLC patients. Serum samples from 92 treatment naive SCLC patients were analysed. Intact uPAR, uPAR(I-III), intact and cleaved uPAR, uPAR(I-III) + uPAR(II-III) and the liberated domain I, uPAR(I) were measured using time-resolved fluorescence immunoassays (TR-FIA 1-3). Assessment of association of the uPAR forms to overall survival (OS) was done using Cox regression analysis adjusted for clinical covariates [age, gender, stage, lactate dehydrogenase (LDH), WHO performance status (PS)]. Multivariate survival analysis demonstrated that high levels of uPAR(I) were significantly (p = 0.009) associated with short overall survival (OS). Patients with uPAR(I) levels above the second tertile had a hazard ratio (HR) of 1.9 (95% confidence interval (CI): 1.1-3.3), compared to patients with levels below the first tertile. High serum uPAR(I) levels are associated with short OS in SCLC patient, independent of LDH and PS.

Almasi, Charlotte E; Drivsholm, Lars

2013-01-01

282

Urokinase type plasminogen activator mediates Interleukin-17-induced peripheral blood mesenchymal stem cell motility and transendothelial migration.  

Science.gov (United States)

Mesenchymal stem cells (MSCs) have the potential to migrate toward damaged tissues increasing tissue regeneration. Interleukin-17 (IL-17) is a proinflammatory cytokine with pleiotropic effects associated with many inflammatory diseases. Although IL-17 can modulate MSC functions, its capacity to regulate MSC migration is not well elucidated so far. Here, we studied the role of IL-17 on peripheral blood (PB) derived MSC migration and transmigration across endothelial cells. IL-17 increased PB-MSC migration in a wound healing assay as well as cell mobilization from collagen gel. Concomitantly IL-17 induced the expression of urokinase type plasminogen activator (uPA) without affecting matrix metalloproteinase expression. The incremented uPA expression mediated the capacity of IL-17 to enhance PB-MSC migration in a ERK1,2 MAPK dependent way. Also, IL-17 induced PB-MSC migration alongside with changes in cell polarization and uPA localization in cell protrusions. Moreover, IL-17 increased PB-MSC adhesion to endothelial cells and transendothelial migration, as well as increased the capacity of PB-MSC adhesion to fibronectin, in an uPA-dependent fashion. Therefore, our data suggested that IL-17 may act as chemotropic factor for PB-MSCs by incrementing cell motility and uPA expression during inflammation development. PMID:25433194

Krsti?, Jelena; Obradovi?, Hristina; Jaukovi?, Aleksandra; Oki?-?or?evi?, Ivana; Trivanovi?, Drenka; Kukolj, Tamara; Mojsilovi?, Slavko; Ili?, Vesna; Santibañez, Juan F; Bugarski, Diana

2015-02-01

283

Massive Pulmonary Embolism: Treatment with Thrombus Fragmentation and Local Fibrinolysis with Recombinant Human-Tissue Plasminogen Activator  

International Nuclear Information System (INIS)

Purpose: To report the results of thrombus fragmentation in combination with local fibrinolysis using recombinant human-tissue plasminogen activator (rtPA) in patients with massive pulmonary embolism. Methods: Five patients with massive pulmonary embolism were treated with thrombus fragmentation followed by intrapulmonary injection of rtPA. Clot fragmentation was performed with a guidewire, angiographic catheter, and balloon catheter. Three patients had undergone recent surgery; one of them received a reduced dosage of rtPA. Results: All patients survived and showed clinical improvement with a resultant significant (p < 0.05) decrease in the pulmonary blood pressure (mean systolic pulmonary blood pressure before treatment, 49 mmHg; 4 hr after treatment, 28 mmHg). Angiographic follow-up in three patients revealed a decrease in thrombus material and an increase in pulmonary perfusion. Two patients developed retroperitoneal hematomas requiring transfusion. Conclusion: Clot fragmentation and local fibrinolysis with rtPA was an effective therapy for massive pulmonary embolism. Bleeding at the puncture site was a frequent complication

284

Effect of dipeptidyl peptidase-4 inhibitor, vildagliptin on plasminogen activator inhibitor-1 in patients with diabetes mellitus.  

Science.gov (United States)

Dipeptidyl peptidase-4 (DPP-4) inhibitors may affect the serum levels of plasminogen activator inhibitor-1 (PAI-1) associated with triglyceride (TG) metabolism, which is a prognostic factor for cardiovascular disease, in diabetic patients. We conducted an 8-week, prospective, randomized study in which we assigned type 2 diabetic patients who were inadequately controlled with antidiabetic therapy to the vildagliptin group (50 mg bid, n = 49) or the control group (n = 49). The primary efficacy parameter was the change in the serum level of PAI-1, and the secondary end point was the change in the serum levels of TG-rich lipoproteins. In the vildagliptin group, significant decrease of the serum PAI-1 level by 16.3% (p changes were observed in the control group. Multivariate regression analyses identified the absolute change from the baseline (?) of the PAI-1, but not that of the fasting blood glucose or hemoglobin A1c, as independent predictors of the ?TG, ? remnant-like particle cholesterol, and ? apolipoprotein B. In conclusion, treatment of type 2 diabetes with vildagliptin might prevent the progression of atherosclerotic cardiovascular disease in diabetic patients by decreasing the serum PAI-1 levels and improving TG metabolism. PMID:25637323

Tani, Shigemasa; Takahashi, Atsuhiko; Nagao, Ken; Hirayama, Atsushi

2015-02-15

285

Bacterial endotoxin enhances colorectal cancer cell adhesion and invasion through TLR-4 and NF-kappaB-dependent activation of the urokinase plasminogen activator system.  

LENUS (Irish Health Repository)

Perioperative exposure to lipopolysaccharide (LPS) is associated with accelerated metastatic colorectal tumour growth. LPS directly affects cells through Toll-like receptor 4 (TLR-4) and the transcription factor NF-kappaB. The urokinase plasminogen activator (u-PA) system is intimately implicated in tumour cell extracellular matrix (ECM) interactions fundamental to tumour progression. Thus we sought to determine if LPS directly induces accelerated tumour cell ECM adhesion and invasion through activation of the u-PA system and to elucidate the cellular pathways involved. Human colorectal tumour cell lines were stimulated with LPS. u-PA concentration, u-PA activity, active u-PA, surface urokinase plasminogen activator receptor (u-PAR) and TLR-4 expression were assessed by ELISA, colorimetric assay, western blot analysis and flow cytometry respectively. In vitro tumour cell vitronectin adhesion and ECM invasion were analysed by vitronectin adhesion assay and ECM invasion chambers. u-PA and u-PAR function was inhibited with anti u-PA antibodies or the selective u-PA inhibitors amiloride or WXC-340, TLR-4 by TLR-4-blocking antibodies and NF-kappaB by the selective NF-kappaB inhibitor SN-50. LPS upregulates u-PA and u-PAR in a dose-dependent manner, enhancing in vitro tumour cell vitronectin adhesion and ECM invasion by >40% (P<0.01). These effects were ameliorated by u-PA and u-PAR inhibition. LPS activates NF-kappaB through TLR-4. TLR-4 and NF-kappaB inhibition ameliorated LPS-enhanced u-PA and u-PAR expression, tumour cell vitronectin adhesion and ECM invasion. LPS promotes tumour cell ECM adhesion and invasion through activation of the u-PA system in a TLR-4- and NF-kappaB-dependent manner.

Killeen, S D

2009-05-19

286

A regulatory hydrophobic area in the flexible joint region of plasminogen activator inhibitor-1, defined with fluorescent activity-neutralizing ligands. Ligand-induced serpin polymerization.  

DEFF Research Database (Denmark)

We have characterized the neutralization of the inhibitory activity of the serpin plasminogen activator inhibitor-1 (PAI-1) by a number of structurally distinct organochemicals, including compounds with environment-sensitive spectroscopic properties. In contrast to latent and reactive center-cleaved PAI-1 and PAI-1 in complex with urokinase-type plasminogen activator (uPA), active PAI-1 strongly increased the fluorescence of the PAI-1-neutralizing compounds 1-anilinonaphthalene-8-sulfonic acid and 4,4'-dianilino-1,1'-bisnaphthyl-5,5'-disulfonic acid. The fluorescence increase could be competed by all tested nonfluorescent neutralizers, indicating that all neutralizers bind to a common hydrophobic area preferentially accessible in active PAI-1. Activity neutralization proceeded through two consecutive steps as follows: first step is conversion to forms displaying substrate behavior toward uPA, and second step is to forms inert to uPA. With some neutralizers, the second step was associated with PAI-1 polymerization. Vitronectin reduced the susceptibility to the neutralizers. Changes in sensitivity to activity neutralization by point mutations were compatible with the various neutralizers having overlapping, but not identical, binding sites in the region around alpha-helices D and E and beta-strand 1A, known to act as a flexible joint when beta-sheet A opens and the reactive center loop inserts as beta-strand 4A during reaction with target proteinases. The defined binding area may be a target for development of compounds for neutralizing PAI-1 in cancer and cardiovascular diseases. Udgivelsesdato: 2001-Apr-20

Egelund, R; Einholm, A P

2001-01-01

287

Activated human neutrophils rapidly release the chemotactically active D2D3 form of the urokinase-type plasminogen activator receptor (uPAR/CD87).  

Science.gov (United States)

The urokinase-type plasminogen activator receptor (uPAR/CD87) exists both in cell-bound and soluble forms. Neutrophils contain extensive intracellular pools of uPAR that are translocated to the plasma membrane upon activation. In the present study, we investigated the ability of human neutrophils to shed uPAR from cell surface following activation and addressed the possible involvement of the released receptor in the inflammatory response. We first observed that the spontaneous release of suPAR by resting neutrophils was strongly and rapidly (within minutes) enhanced by calcium ionophore ionomycin and to a lesser extent when cells were primed with TNF-alpha and then stimulated with fMLP or IL-8. We demonstrated that suPAR is produced by resting and activated neutrophils predominantly as a truncated form devoid of N-terminal D1 domain (D2D3 form) that lacks GPI anchor. Migration of formyl peptide receptor-like 1 (FPRL1)-transfected human embryonic kidney (HEK) 293 cells toward the supernatants harvested from activated neutrophils was significantly diminished when D2D3 form of suPAR was immunodepleted from the supernatants. We conclude that activated neutrophils release the chemotactically active D2D3 form of suPAR that acts as a ligand of FPRL1. Interestingly, we present evidence that GPI-specific phospholipase D (GPI-PLD) that has previously been shown to shed uPAR in cancer cells is not involved in suPAR release from human neutrophils. We suggest that production of the chemotactically active D2D3 form of suPAR by activated human neutrophils in vivo could contribute to the recruitment of monocytes and other formyl peptide receptors-expressing cells to the sites of acute inflammation where neutrophil accumulation and activation occur. PMID:18830568

Pliyev, Boris K

2009-01-01

288

Expression and large-scale production of the biochemically active human tissue-plasminogen activator in hairy roots of Oriental melon (Cucumis melo).  

Science.gov (United States)

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. PMID:22018738

Kim, Sung-Ryong; Sim, Joon-Soo; Ajjappala, Hemavathi; Kim, Yong-Hwan; Hahn, Bum-Soo

2012-01-01

289

Tumor marker utility and prognostic relevance of cathepsin B, cathepsin L, urokinase-type plasminogen activator, plasminogen activator inhibitor type-1, CEA and CA 19-9 in colorectal cancer  

Directory of Open Access Journals (Sweden)

Full Text Available Abstract Background Cathepsin B and L (CATB, CATL, urokinase-type plasminogen activator (uPA and its inhibitor PAI-1 play an important role in colorectal cancer invasion. The tumor marker utility and prognostic relevance of these proteases have not been evaluated in the same experimental setting and compared with that of CEA and CA-19-9. Methods Protease, CEA and CA 19-9 serum or plasma levels were determined in 56 patients with colorectal cancer, 25 patients with ulcerative colitis, 26 patients with colorectal adenomas and 35 tumor-free control patients. Protease, CEA, CA 19-9 levels have been determined by ELISA and electrochemiluminescence immunoassay, respectively; their sensitivity, specificity, diagnostic accuracy have been calculated and correlated with clinicopathological staging. Results The protease antigen levels were significantly higher in colorectal cancer compared with other groups. Sensitivity of PAI-1 (94%, CATB (82%, uPA (69%, CATL (41% were higher than those of CEA or CA 19-9 (30% and 18%, respectively. PAI-1, CATB and uPA demonstrated a better accuracy than CEA or CA 19-9. A combination of PAI-1 with CATB or uPA exhibited the highest sensitivity value (98%. High CATB, PAI-1, CEA and CA 19-9 levels correlated with advanced Dukes stages. CATB (P = 0.0004, CATL (P = 0.02, PAI-1 (P = 0.01 and CA 19-9 (P = 0.004 had a significant prognostic impact. PAI-1 (P = 0.001, CATB (P = 0.04 and CA 19-9 (P = 0.02 proved as independent prognostic variables. Conclusion At the time of clinical detection proteases are more sensitive indicators for colorectal cancer than the commonly used tumor markers. Determinations of CATB, CATL and PAI-1 have a major prognostic impact in patients with colorectal cancer.

De Paoli Massimo

2008-07-01

290

Urokinase-type plasminogen activator deficiency (uPA-KO) prevented carotid artery ligation-induced vascular remodeling in mice.  

Science.gov (United States)

It has been demonstrated that urokinase-type plasminogen activator (uPA) plays an important role in vascular remodeling. This study was designed to determine whether uPA deficiency (KO) affects carotid artery ligation-induced vessel remodeling and the interaction with angiotensin II (Ang II). Ligation of the left common carotid artery in 6-month-old wild-type (C57 black/6J) mice for 4 weeks induced a concentric remodeling with vessel wall thickening, characterized by cell proliferation in neointima, media, and adventitia, and with lumen narrowing without a significant enlargement of overall vessel dimension. Intima lesions were characterized by alpha-actin positive smooth muscle cell (SMC) proliferation in a matrix background. No detectable presence of MAC-3 positive macrophages existed in the vascular wall. The ligation-induced vascular neointimal formation and adventitial proliferation, but not lumen narrowing and media expansion, were completely prevented in age-matched uPA-KO mice. Chronic infusion of Ang II (1.44 mg/kg per day) via a subcutaneously implanted osmotic minipump did not significantly affect the gross morphology of the nonligated carotid artery from both wild-type and uPA-KO mice, but it enhanced ligation-induced vascular remodeling. However, in the presence of Ang II, uPA deficiency had no effects on ligation-induced mophermetric change, but it partially and significantly reduced cell proliferation. These data indicate that uPA may play a critical role in ligation-induced vessel remodeling. Ang II may activate other mechanisms independent of uPA to exacerbate ligation-induced vascular remodeling. PMID:17240317

Martin-McNulty, Baby; Zhang, Lening; da Cunha, Valdeci; Vincelette, Jon; Rutledge, John C; Vergona, Ronald; Sullivan, Mark E; Wang, Yi-Xin

2007-02-01

291

SERPINE2, an inhibitor of plasminogen activators, is highly expressed in the human endometrium during the secretory phase  

Directory of Open Access Journals (Sweden)

Full Text Available Abstract Background SERPINE2, also known as protease nexin-1, belongs to the serine protease inhibitor (SERPIN superfamily. It is one of the potent SERPINs that modulates the activity of plasminogen activators (PAs. PAs and their SERPIN inhibitors, such as SERPINB2 and SERPINE1, were expressed in the human endometrium and were implicated in implantation. However, expression data about SERPINE2 in the human endometrium is still unknown. Thus, we conducted an investigation to reveal the spatiotemporal and cellular expression of SERPINE2 in the human uterus during the menstrual cycle. Methods Seven patients who underwent a hysterectomy and samples of 120 archived patients' endometrial curettage or parts of the uterus that were formalin-fixed and embedded in paraffin. Western blotting was performed to evaluate the specificity and sensitivity of the antibody. Immunohistochemistry was conducted to localize the SERPINE2 expression site. Quantitative analysis was conducted to evaluate expression levels of SERPINE2 in various sub-phases of the menstrual cycle. Results The SERPINE2 protein was primarily detected in the uterine fluid during the mid- and late-secretory phases of the menstrual cycle. It was predominantly expressed in the luminal and glandular epithelium, less in the myometrium, and only dispersedly in certain stromal cells throughout the menstrual cycle. A quantitative analysis of expression levels of SERPINE2 in the glandular epithelium revealed that it was highly expressed in the endometrium during the secretory phase compared to the proliferative phase. Conclusions The SERPINE2 protein is highly expressed in the endometrium during the secretory phase, indicating that it may participate in tissue remodeling involved in implantation.

Hwu Yuh-Ming

2011-03-01

292

Hypoxia dysregulates the production of adiponectin and plasminogen activator inhibitor-1 independent of reactive oxygen species in adipocytes  

International Nuclear Information System (INIS)

Low plasma levels of adiponectin (hypoadiponectinemia) and elevated circulating concentrations of plasminogen activator inhibitor (PAI)-1 are causally associated with obesity-related insulin resistance and cardiovascular disease. However, the mechanism that mediates the aberrant production of these two adipokines in obesity remains poorly understood. In this study, we investigated the effects of hypoxia and reactive oxygen species (ROS) on production of adiponectin and PAI-1 in 3T3-L1 adipocytes. Quantitative PCR and immunoassays showed that ambient hypoxia markedly suppressed adiponectin mRNA expression and its protein secretion, and increased PAI-1 production in mature adipocytes. Dimethyloxallyl glycine, a stabilizer of hypoxia-inducible factor 1? (HIF-1?), mimicked the hypoxia-mediated modulations of these two adipokines. Hypoxia caused a modest elevation of ROS in adipocytes. However, ablation of intracellular ROS by antioxidants failed to alleviate hypoxia-induced aberrant production of adiponectin and PAI-1. On the other hand, the antioxidants could reverse hydrogen peroxide (H2O2)-induced dysregulation of adiponectin and PAI-1 production. H2O2 treatment decreased the expression levels of peroxisome proliferator-activated receptor gamma (PPAR?) and CCAAT/enhancer binding protein (C/EBP?), but had no effect on HIF-1?, whereas hypoxia stabilized HIF-1? and decreased expression of C/EBP?, but not PPAR?. Taken togf C/EBP?, but not PPAR?. Taken together, these data suggest that hypoxia and ROS decrease adiponectin production and augment PAI-1 expression in adipocytes via distinct signaling pathways. These effects may contribute to hypoadiponectinemia and elevated PAI-1 levels in obesity, type 2 diabetes, and cardiovascular diseases

293

Role of the amino-terminal region of streptokinase in the generation of a fully functional plasminogen activator complex probed with synthetic peptides.  

OpenAIRE

The mechanism whereby fragments of streptokinase (SK) derived from its N terminus (e.g., SK1-59 or SK1-63) enhance the low plasminogen (PG)-activating ability of other fragments, namely SK64-386, SK60-414, SK60-387, and SK60-333 (reported previously), has been investigated using a synthetic peptide approach. The addition of either natural SK1-59, or chemically synthesized SK16-59, at saturation (about 500-fold molar excess) generated amidolytic and PG activation capabilities in equimolar mixt...

Nihalani, D.; Kumar, R.; Rajagopal, K.; Sahni, G.

1998-01-01

294

Reduced cardiac expression of plasminogen activator inhibitor 1 and transforming growth factor ?1 in obese Zucker rats by perindopril  

Science.gov (United States)

Objective: To determine whether angiotensin converting enzyme inhibition by perindopril can reduce cardiac transforming growth factor ?1 (TGF?1) and plasminogen activator inhibitor 1 (PAI-1) and therefore control collagen accumulation in an animal model with the metabolic syndrome such as the obese Zucker rat (OZR). Animals: Male OZR (group 1, n ?=? 10); OZR treated with perindopril (group 2, n ?=? 10); and lean Zucker rats (group 3, n ?=? 10). Methods: During six months, group 2 received 3 mg/kg/day of perindopril orally and group 1 and group 3 were given a vehicle. Hearts were processed for pathology studies including immunohistochemical analysis with antibodies to PAI-1, TGF?1, collagen type I, and collagen type III. Results: Group 2 had lower blood pressure (126.7 (2) v 148.6 (2.7) mm Hg, p < 0.01) than untreated OZR and had decreased cardiac PAI-1 (3.6 (0.4) v 13.5 (1.7)% of positive area/field, p < 0.01), TGF?1 in myocytes (0.13 (0.1) v 9.14 (4.7)%/area, p < 0.01) and in interstitium (19.8 (6.8) v 178.9 (27.4) positive cells/area, p < 0.01), collagen I (3 (0.8) v 13.3 (1)%/area, p < 0.01), collagen III (5 (0.6) v 9.5 (0.9)%/area, p < 0.01), and collagen I to collagen III ratio (0.59 (0.13) v 1.40 (0.15) p < 0.01) compared with untreated OZR. Conclusion: These results suggest that perindopril reduces cardiac PAI-1 and TGF?1 and ameliorates cardiac fibrosis in a rat model with multiple cardiovascular risk factors. PMID:15604340

Toblli, J E; Cao, G; DeRosa, G; Forcada, P

2005-01-01

295

Hyperacute thrombolysis with recombinant tissue plasminogen activator of acute ischemic stroke: Feasibility and effectivity from an Indian perspective  

Directory of Open Access Journals (Sweden)

Full Text Available Given the constraints of resources, thrombolysis for acute ischemic stroke (AIS is under evaluation in developing countries like India, especially in areas such as western Utter Pradesh, where it is overly crowded and there is poor affordability. Aim: This study was done to evaluate recombinant tissue plasminogen activator r-tpa in acute ischemic stroke in hyper acute phase, in selected patients of western Utter Pradesh, in terms of feasibility and effectivity. Design: Open, non randomized study. Materials and Methods: Thirty two patients were classified using Trial of ORG 10172 in Acute Stroke treatment (TOAST criteria (large artery atherosclerotic = 8; cardio embolic = 6; small vessel occlusion = 14; other determined etiology = 2; undetermined etiology = 2. The mean time to reach the hospital was 2 h (1.15-3.0, the mean door to CT scan 20 min (10-40 and door to r-tpa injection was 30 min (24-68. The National Institute of Health Stroke Scale (NIHSS scores ranged from 11-22 (mean 15.5 +2.7. The dose of r-tpa administered was 0.9 mg/kg. Results: Twenty one patients (65.6% showed significant improvement on the NIHSS score, at 48 h (4 points. (Mean change = 10; range = 4-17. At one month, 25 (78% recorded improvement on the Barthel index (mean change = 45%. One developed frontal lobe hemorrhage and another developed recurrent stroke; one died of aspiration; and four showed no improvement. Modified Rankin score (m RS was administered at the end of three months to 28 patients (90%; however, the rest could not be directly observed. The average modified Rankin Score was 1.2 (0-2. Conclusions: Hyperacute thrombolysis was found feasible and effective in selected patients with AIS from western Utter Pradesh and who had poor affordability.

Sharma S

2008-01-01

296

Low vs standard dose of recombinant tissue plasminogen activator in treating East Asian patients with acute ischemic stroke  

Directory of Open Access Journals (Sweden)

Full Text Available Background : Intravenous recombinant tissue plasminogen activator (rtPA has been approved to treat eligible patients with acute ischemic stroke within 4.5 hours of onset. The rationale for using a lower dose in Asian patients came from concerns about intracerebral hemorrhage because of the racial differences in blood coagulation-fibrinolysis factors. Aim : The aim of this systemic review was to compare the data from previous studies to address the efficacy and safety of using low-dose vs standard-dose rtPA in treating patients with acute ischemic stroke. Material and Methods : Previous studies were searched and analyzed. The confidence interval was calculated at 95%. Baseline characteristics and outcomes of the patients were compared between two doses of rtPA (0.6 vs 0.9 mg/kg, using Z test for two independent proportions. Results : Patients who received standard-dose rtPA had significantly higher favorable outcome at 3 months (33.1 vs 47.2%, P<0.0001, without significant difference in the rates of symptomatic intracerebral hemorrhage (3.5 vs 4.3%, P = 0.42 and mortality (13.1 vs 11.7%, P = 0.56. However, patients in the low-dose group were older and had more severe stroke. Conclusions : Patients receiving standard-dose rtPA seem to have higher rates of favorable outcome. However, there were significant differences in baseline characteristics between the two groups. A further, well-designed, randomized study in the same population is still needed to clarify the suspected benefit of the standard dose for East Asian patients.

Dharmasaroja Pornpatr

2011-01-01

297

Transcatheter Thrombolysis with High-Dose Bolus Tissue Plasminogen Activator in Iatrogenic Arterial Occlusion after Femoral Arterial Catheterization  

International Nuclear Information System (INIS)

Purpose: To assess the efficacy of percutaneous local thrombolysis with high-dose bolus recombinant tissue plasminogen activator (rt-PA) in patients with acute limb ischemia due to arterial thrombosis after cardiac catheterization.Methods: We treated eight patients (7 men; mean age 56 years) with thrombotic occlusion of both the common femoral artery (CFA) and external iliac artery (EIA) in six patients and of the CFA only in two patients. Two 5 mg boluses of rt-PA were injected into the proximal clot through a 5 Fr end-hole catheter and subsequently two additional boluses of 5 mg rt-PA were given through a catheter with multiple side-holes. In case of a significant amount of residual thrombus, a continuous infusion of 2.5 mg/hr of rt-PA was started.Results: Successful lysis was achieved in all patients. The mean duration of lysis was 2 hr 41 min. The mean total amount of rt-PA delivered was 23.16 mg. In four patients unmasked flow-limited dissections confined to the CFA were managed by prolonged balloon dilatation, while in the remaining four patients with extension of the dissection to the external iliac artery one or two Easy Wallstents were implanted. There was prompt relief of lower limb ischemic symptoms and signs in all patients. Two groin hematomas were conservatively treated.Clinical and color Doppler flow imaging follow-up with a mean duration of 15 months, showed no reappearance of ischemic symptoms or development of restenosis in any of the patients. One pf restenosis in any of the patients. One patient died 6 months after thrombolysis.Conclusions: Transcatheter thrombolysis with high-dose bolus rt-PA is a safe and effective treatment inpatients with iatrogenic arterial occlusion after femoral catheterization. Underlying dissections should be treated by prolonged balloon dilatation but stent implantation is often required

298

Levels of microRNA-181b and plasminogen activator inhibitor-1 are associated with hypertensive disorders complicating pregnancy.  

Science.gov (United States)

The aim of the present study was to explore the association between the expression of microRNA (miRNA)-181b and plasminogen activator inhibitor-1 (PAI-1) in the placental tissue of pregnant females with a hypertensive disorder complicating pregnancy (HDCP). Placental tissue samples were obtained from 48 patients with HDCP and 40 females with a normal pregnancy. The levels of miRNA-181b and PAI-1 mRNA were determined by the reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The expression of PAI-1 protein was analyzed by western blotting. Vascular smooth muscle cells (VSMCs) were transfected with the pEGP-miRNA-181b plasmid using Lipofectamine(®) 2000. Transfection efficiency was confirmed by immunohistochemical analysis. The levels of miRNA-181b in the placental tissue of patients with HDCP were lower than those in the control group, whereas the levels of PAI-1 mRNA in the placental tissue of patients with HDCP were higher than those in the control group. The expression of the PAI-1 protein in the HDCP group was higher than that in the control group. Following transfection of VSMCs with plasmid pGCMV/EGFP/miRNA-181b, the levels of PAI-1 mRNA were reduced while the levels of miRNA-181 were upregulated. Furthermore, the expression levels of PAI-1 protein were lower than those in the control group. The levels of miRNA-181b and PAI-1 mRNA were strongly associated with HDCP. Thus, miRNA-181b may play an important role in the regulation of PAI-1. PAI-1 and miRNA-181b may be novel biomarkers to be used in HDCP therapy. PMID:25289053

Chen, Yan-Shan; Shen, Ling; Mai, Rui-Qin; Wang, Ying

2014-11-01

299

Coagulation factors, fibrinogen and plasminogen activator inhibitor-1, are differentially regulated by yellow fever virus infection of hepatocytes.  

Science.gov (United States)

Yellow fever virus (YFV) infection poses a great risk to un-vaccinated individuals living or traveling in the endemic regions of Africa and South America. It is estimated that approximately 30,000 people die each year of this disease. The liver is the main target of YFV, where as many as 80% of the hepatocytes may become involved in the infection. The overwhelming infection of the liver is associated with the observed hemorrhagic disease manifestations such as petechiae, ecchymoses, and hematemesis which are all thought to be linked with the observed coagulation abnormalities that include prolonged clotting times, reduction in clotting factors, fibrin-split products (D-dimers) and elevated prothrombin times. Many factors involved in the coagulation pathway are produced by hepatocytes, such as fibrinogen (FBG) and plasminogen activator inhibitor-1 (PAI-1). Both of these proteins have been indicated in another flavivirus related disease, dengue, as having roles related to the bleeding abnormalities observed and overall outcome of infection. In this study we wanted to determine if FBG and PAI-1 expression levels by human hepatocytes was disrupted or altered by infection with either wild-type Asibi or vaccine strain17-D YFVs. Our findings indicate that YFV infection does affect the transcriptional and translational expression of FBG and PAI-1 in human hepatocytes and that these results are further affected by IL-6 during early stages of infection. These results may lead to further understanding of the molecular mechanism associated with bleeding abnormalities observed during late stage YFV infection. PMID:23639427

Woodson, Sara E; Freiberg, Alexander N; Holbrook, Michael R

2013-08-01

300

Treatment of TBI with collagen scaffolds and human marrow stromal cells increases the expression of tissue plasminogen activator.  

Science.gov (United States)

This study examines the effects of combination therapy of collagen scaffolds and human marrow stromal cells (hMSCs) on the expression of tissue plasminogen activator (tPA) after traumatic brain injury (TBI) in rats. Adult male Wistar rats (n=48) were injured with controlled cortical impact and treated either with scaffolds suffused with hMSCs (3×10(6)) or hMSCs (3×10(6)) alone transplanted into the lesion cavity 1 week after TBI. A control group was treated with saline. Neurological function was assessed using the Morris Water Maze test (MWM) and modified Neurological Severity Scores (mNSS). The rats were sacrificed 14 days after TBI and brain samples were processed for immunohistochemical analysis and quantitative Western blot and quantitative real-time polymerase chain reaction (qRT-PCR) studies. Enhanced functional improvement was observed on both the mNSS and MWM tests in the scaffold+hMSC-treated group compared to the other two groups. Immunostaining with anti-human mitochondrial antibody (E5204) showed more hMSCs in the injury zone of the scaffold+hMSC group compared to the hMSC-alone group. Triple staining showed that more neurons were tPA-positive in the scaffold+hMSC group compared to the other two groups (p<0.05). Western blot analysis and qRT-PCR showed that scaffold+hMSC and hMSC-alone treatment enhanced the expression of tPA compared to controls (p<0.05), but tPA expression was significantly greater in the scaffold+hMSC group. The induction of tPA by hMSCs after TBI may be one of the mechanisms involved in promoting functional improvement after TBI. PMID:21355820

Mahmood, Asim; Qu, Changsheng; Ning, Riuzuo; Wu, Hongtao; Goussev, Anton; Xiong, Ye; Irtenkauf, Susan; Li, Yi; Chopp, Michael

2011-07-01

301

Binding of /sup 131/I-labeled tissue-type plasminogen activator on de-endothelialized lesions in rabbits  

Energy Technology Data Exchange (ETDEWEB)

Tissue-type plasminogen activator (t-PA) which has a high affinity for fibrin in the clot, was labeled with /sup 131/I by the iodogen method, and its binding to de-endothelialized lesions in the rabbit was measured to assess the detectability of thrombi. The de-endothelialized lesion was induced in the abdominal aorta with a Fogarty 4F balloon catheter. Two hours after the de-endothelialization, /sup 131/I-labeled t-PA (125 +- 46 ..mu..Ci) was injected intravenously. The initial half-life of the agent in blood (n=12) was 2.9 +- 0.4 min. The degree of binding of /sup 131/I-labeled t-PA to the de-endothelialized lesion was evaluated at 15 min (n=6) or at 30 min (n=6) after injection of the agent. In spite of the retention of the biochemical properties of /sup 131/I-labeled t-PA and the presence of fibrin deposition at the de-endothelialized lesion, the binding of t-PA to the lesion was not sufficiently strong. Lesion-to-control ratios (cpmgcpmg) were 1.65 +- 0.40 (at 15 min) and 1.39 +- 1.31 (at 30 min), and lesion-to-blood ratios were 1.39 +- 0.32 (at 15 min) and 1.36 +- 0.23 (at 30 min). These results suggest that radiolabeled t-PA may be inappropriate as a radiopharmaceutical for the scintigraphic detection of a pre-existing thrombotic lesion.

Uehara, A.; Isaka, Y.; Kimura, K.; Kozuka, T.; Etani, H.; Yoneda, S.; Kamada, T.; Nobuhara, M.

1987-10-01

302

Binding of 131I-labeled tissue-type plasminogen activator on de-endothelialized lesions in rabbits  

International Nuclear Information System (INIS)

Tissue-type plasminogen activator (t-PA) which has a high affinity for fibrin in the clot, was labeled with 131I by the iodogen method, and its binding to de-endothelialized lesions in the rabbit was measured to assess the detectability of thrombi. The de-endothelialized lesion was induced in the abdominal aorta with a Fogarty 4F balloon catheter. Two hours after the de-endothelialization, 131I-labeled t-PA (125 ± 46 ?Ci) was injected intravenously. The initial half-life of the agent in blood (n=12) was 2.9 ± 0.4 min. The degree of binding of 131I-labeled t-PA to the de-endothelialized lesion was evaluated at 15 min (n=6) or at 30 min (n=6) after injection of the agent. In spite of the retention of the biochemical properties of 131I-labeled t-PA and the presence of fibrin deposition at the de-endothelialized lesion, the binding of t-PA to the lesion was not sufficiently strong. Lesion-to-control ratios (cpm/g/cpm/g) were 1.65 ± 0.40 (at 15 min) and 1.39 ± 1.31 (at 30 min), and lesion-to-blood ratios were 1.39 ± 0.32 (at 15 min) and 1.36 ± 0.23 (at 30 min). These results suggest that radiolabeled t-PA may be inappropriate as a radiopharmaceutical for the scintigraphic detection of a pre-existing thrombotic lesion. (orig.)

303

Interactions of plasminogen activator inhibitor-1 with vitronectin involve an extensive binding surface and induce mutual conformational rearrangements  

DEFF Research Database (Denmark)

In order to explore early events during the association of plasminogen activator inhibitor-1 (PAI-1) with its cofactor vitronectin, we have applied a robust strategy that combines protein engineering, fluorescence spectroscopy, and rapid reaction kinetics. Fluorescence stopped-flow experiments designed to monitor the rapid association of PAI-1 with vitronectin indicate a fast, concentration-dependent, biphasic binding of PAI-1 to native vitronectin but only a monophasic association with the somatomedin B (SMB) domain, suggesting that multiple phases of the binding interaction occur only when full-length vitronectin is present. Nonetheless, in all cases, the initial fast interaction is followed by slower fluorescence changes attributed to a conformational change in PAI-1. Complementary experiments using an engineered, fluorescently silent PAI-1 with non-natural amino acids showed that concomitant structural changes occur as well in native vitronectin. Furthermore, we have measured the effect of vitronectin on the rate of insertion of the reactive center loop into beta-sheet A of PAI-1 during reaction with target proteases. With a variety of PAI-1 variants, we observe that both full-length vitronectin and the SMB domain have protease-specific effects on the rate of loop insertion but that the two exhibit clearly different effects. These results support a model for PAI-1 binding to vitronectin in which the interaction surface extends beyond the region of PAI-1 occupied by the SMB domain. In support of this model are recent results that define a PAI-1-binding site on vitronectin that lies outside the somatomedin B domain (Schar, C. R., Blouse, G. E., Minor, K. H., and Peterson, C. B. (2008) J. Biol. Chem. 283, 10297-10309) and the complementary site on PAI-1 (Schar, C. R., Jensen, J. K., Christensen, A., Blouse, G. E., Andreasen, P. A., and Peterson, C. B. (2008) J. Biol. Chem. 283, 28487-28496).

Blouse, Grant E; Dupont, Daniel Miotto

2009-01-01

304

Transcriptional Regulation of Urokinase-type Plasminogen Activator Receptor by Hypoxia-Inducible Factor 1 Is Crucial for Invasion of Pancreatic and Liver Cancer  

Directory of Open Access Journals (Sweden)

Full Text Available Angioinvasion is critical for metastasis with urokinase-type plasminogen activator receptor (uPAR and tumor hypoxia-activated hypoxia-inducible factor 1 (HIF-1 as key players. Transcriptional control of uPAR expression by HIF has never been reported. The aim of the present study, therefore, was to test whether tumor hypoxia-induced HIF expression may be linked to transcriptional activation of uPAR and dependent angioinvasion. We used human pancreatic cancer cells and a model of parental and derived HIF-1?-deficient mouse liver cancer cell lines and performed Northern blot analysis, nuclear runoff assays, electrophoretic mobility shift assay, polymerase chain reaction-generated deletion mutants, luciferase assays, Matrigel invasion assays, and in vivo angioinvasion assays in the chorioallantoic membrane of fertilized chicken eggs. Urokinase-type plasminogen activator receptor promoter analysis resulted in four putative HIF binding sites. Hypoxia strongly induced de novo transcription of uPAR mRNA. With sequential deletion mutants of the uPAR promoter, it was possible to identify one HIF binding site causing a nearly 200-fold increase in luciferase activity. Hypoxia enhanced the number of invading tumor cells in vitro and in vivo. In contrast, HIF-1?-deficient cells failed to upregulate uPAR expression, to activate luciferase activity, and to invade on hypoxia. Taken together, we show for the first time that uPAR is under transcriptional control of HIF and that this is important for hypoxia-induced metastasis.

Peter Büchler

2009-02-01

305

Fructose-induced steatosis in mice: role of plasminogen activator inhibitor-1, microsomal triglyceride transfer protein and NKT cells.  

Science.gov (United States)

Plasminogen activator inhibitor-1 (PAI-1) is an acute-phase protein known to be involved in alcoholic liver disease and hepatic fibrosis. In the present study, the hypothesis that PAI-1 is causally involved in the onset of fructose-induced hepatic steatosis was tested in a mouse model. Wild-type C57BL/6J and PAI-1?/? mice were fed with 30% fructose solution or water for 8 weeks. Markers of hepatic steatosis, expression of PAI-1, apolipoprotein B (ApoB), cluster of differentiation 1d (CD1d), markers of natural killer T (NKT) cells, protein levels of phospho-c-Met and tumor necrosis factor-? (TNF-?) were determined. Activity of the microsomal triglyceride transfer protein (MTTP) was measured in liver tissue. In comparison with water controls, chronic intake of 30% fructose solution caused a significant increase in hepatic triglycerides, PAI-1 expression and plasma alanine aminotransferase levels in wild-type mice. This effect of fructose feeding was markedly attenuated in PAI-1?/? mice. Despite no differences in portal endotoxin levels and hepatic TNF-? protein levels between fructose-fed groups, the protective effect of the loss of PAI-1 against the onset of fructose-induced steatosis was associated with a significant increase in phospho-c-Met, phospho Akt, expression of ApoB and activity of MTTP in livers of PAI-1?/? mice in comparison with fructose-fed wild types. Moreover, in PAI-1?/? mice, expressions of CD1d and markers of CD1d-reactive NKT cells were markedly higher than in wild-type mice; however, expression of markers of activation of CD1d-reactive NKT cells (eg, interleukin-15 and interferon-?) were only found to be increased in livers of fructose-fed PAI-1?/? mice. Taken together, these data suggest that PAI-1 has a causal role in mediating the early phase of fructose-induced liver damage in mice through signaling cascades downstream of Kupffer cells and TNF-?. PMID:21423135

Kanuri, Giridhar; Spruss, Astrid; Wagnerberger, Sabine; Bischoff, Stephan C; Bergheim, Ina

2011-06-01

306

Angiotensin II Increases Urokinase-Type Plasminogen Activator Expression and Induces Aneurysm in the Abdominal Aorta of Apolipoprotein E-Deficient Mice  

OpenAIRE

Urokinase-type plasminogen activator (uPA) is increased in human abdominal aortic aneurysm (AAA). Chronic infusion of angiotensin II (Ang II) results in AAA in apolipoprotein E-deficient mice. We tested the hypothesis that Ang II infusion results in an elevation of uPA expression contributing to aneurysm formation. Ang II or vehicle was infused by osmotic pumps into apoE-KO mice. All mice treated with Ang II developed a localized expansion of the suprarenal aorta (75% increase in outer diamet...

Wang, Yi-xin; Martin-mcnulty, Baby; Freay, Ana D.; Sukovich, Drew A.; Halks-miller, Meredith; Li, Wei-wei; Vergona, Ronald; Sullivan, Mark E.; Morser, John; Dole, William P.; Deng, Gary G.

2001-01-01

307

Tissue-type plasminogen activator–mediated shedding of astrocytic low-density lipoprotein receptor–related protein increases the permeability of the neurovascular unit  

OpenAIRE

The low-density lipoprotein receptor–related protein (LRP) is a member of the LDL receptor gene family that binds several ligands, including tissue-type plasminogen activator (tPA). tPA is found in blood, where its primary function is as a thrombolytic enzyme, and in the central nervous system where it mediates events associated with cell death. Cerebral ischemia induces changes in the neurovascular unit (NVU) that result in brain edema. We investigated whether the interaction between tPA a...

Polavarapu, Rohini; Gongora, Maria Carolina; Yi, Hong; Ranganthan, Sripriya; Lawrence, Daniel A.; Strickland, Dudley; Yepes, Manuel

2007-01-01

308

Caseinolytic activity of fruit extract from Opuntia ficus-indica on bovine, caprine, and ovine Sodium Caseinates  

OpenAIRE

The rates and extents of hydrolysis of RS- and â-caseins from bovine, caprine, and ovine sodium caseinates produced by an enzymatic extract of the fruit of Opuntia ficusindica, (L.) Miller were evaluated and compared with those produced by a commercial animal rennet. A mechanistic model based on a pseudo-first-order enzymatic reaction, in the presence of first-order deactivation of the enzyme, was postulated and successfully fitted to the experimental data. The animal rennet e...

Pintado, Ana I.; Macedo, Angela C.; Teixeira, Grimaneza; Pais, Salome?; Clemente, Alda; Malcata, F. Xavier

2001-01-01

309

Effects of cytokine-suppressive anti-inflammatory drugs on inflammatory activation in ex vivo human and ovine fetal membranes.  

Science.gov (United States)

Intrauterine infection and inflammation are responsible for the majority of early (PTBs). Anti-inflammatory agents, delivered intra-amniotically together with antibiotics, may be an effective strategy for preventing PTB. In this study, the effects of four cytokine-suppressive anti-inflammatory drugs (CSAIDs: N-acetyl cysteine (NAC), SB239063, TPCA-1 and NEMO binding domain inhibitor (NBDI)) were assessed on human and ovine gestational membrane inflammation. Full-thickness membranes were collected from healthy, term, human placentas delivered by Caesarean section (n=5). Using a Transwell model, they were stimulated ex vivo with ?-irradiation-killed Escherichia coli applied to the amniotic face. Membranes from near-term, ovine placentas were stimulated in utero with lipopolysaccharide, Ureaplasma parvum or saline control and subjected to explant culture. The effects of treatment with CSAIDs or vehicle (1% DMSO) on accumulation of PGE2 and cytokines (human interleukin 6 (IL6), IL10 and TNF?; ovine IL8 (oIL8)) were assessed in conditioned media at various time points (3-20 ?h). In human membranes, the IKK? inhibitor TPCA-1 (7 ??M) and p38 MAPK inhibitor SB239063 (20 ??M) administered to the amniotic compartment were the most effective in inhibiting accumulation of cytokines and PGE2 in the fetal compartment. NAC (10 ?mM) inhibited accumulation of PGE2 and IL10 only; NBDI (10 ??M) had no significant effect. In addition to the fetal compartment, SB239063 also exerted consistent and significant inhibitory effects in the maternal compartment. TPCA-1 and SB239063 suppressed oIL8 production, while all CSAIDs tested suppressed ovine PGE2 production. These results support the further investigation of intra-amniotically delivered CSAIDs for the prevention of inflammation-mediated PTB. PMID:24493151

Stinson, Lisa F; Ireland, Demelza J; Kemp, Matthew W; Payne, Matthew S; Stock, Sarah J; Newnham, John P; Keelan, Jeffrey A

2014-03-01

310

Urokinase plasminogen activator released by alveolar epithelial cells modulates alveolar epithelial repair in vitro.  

Science.gov (United States)

Intra-alveolar fibrin is formed following lung injury and inflammation and may contribute to the development of pulmonary fibrosis. Fibrin turnover is altered in patients with pulmonary fibrosis, resulting in intra-alveolar fibrin accumulation, mainly due to decreased fibrinolysis. Alveolar type II epithelial cells (AEC) repair the injured alveolar epithelium by migrating over the provisional fibrin matrix. We hypothesized that repairing alveolar epithelial cells modulate the underlying fibrin matrix by release of fibrinolytic activity, and that the degree of fibrinolysis modulates alveolar epithelial repair on fibrin. To test this hypothesis we studied alveolar epithelial wound repair in vitro using a modified epithelial wound repair model with human A549 alveolar epithelial cells cultured on a fibrin matrix. In presence of the inflammatory cytokine interleukin-1beta, wounds increase by 800% in 24 hours mainly due to detachment of the cells, whereas in serum-free medium wound areas decreases by 22.4 +/- 5.2% (p < 0.01). Increased levels of D-dimer, FDP and uPA in the cell supernatant of IL-1beta-stimulated A549 epithelial cells indicate activation of fibrinolysis by activation of the plasmin system. In presence of low concentrations of fibrinolysis inhibitors, including specific blocking anti-uPA antibodies, alveolar epithelial repair in vitro was improved, whereas in presence of high concentrations of fibrinolysis inhibitors, a decrease was observed mainly due to decreased spreading and migration of cells. These findings suggest the existence of a fibrinolytic optimum at which alveolar epithelial repair in vitro is most efficient. In conclusion, uPA released by AEC alters alveolar epithelial repair in vitro by modulating the underlying fibrin matrix. PMID:16411403

Van Leer, Coretta; Stutz, Monika; Haeberli, André; Geiser, Thomas

2005-12-01

311

Heparin-binding epidermal growth factor-like growth factor: p91 activation induction of plasminogen activator/inhibitor, and tubular morphogenesis in human microvascular endothelial cells.  

Science.gov (United States)

Epidermal growth factor (EGF) or transforming growth factor-alpha (TGF-alpha) stimulates cell migration, proliferation and the formation of tube-like structures of human microvascular endothelial cells in culture. Heparin-binding EGF-like growth factor(HB-EGF), which shows 35% homology with EGF/TGF-alpha, is a member of the EGF family, and it is ubiquitous in many tissues and organs. We examined whether or not HB-EGF induced angiogenic responses in human microvascular endothelial cells. HB-EGF inhibited the binding of (125) I-EGF to the EGF receptor and induced autophosphorylation of the receptor on endothelial cells. Exogenous HB-EGF induced the loss of more than 70% of the EGF receptor from the cell surface within 30 min, with similar kinetics to that of EGF. The level of c-fos mRNA markedly increased at 30 min in response to HB-EGF as well as EGF. A gel shift assay demonstrated the activation of the transcription factor p91 by HB-EGF and EGF. This factor directly interacts with EGF receptor and mediates the activation of c-fos gene promoter. HB-EGF enhanced the mRNA expression of tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1) mRNA. However, the enhancement of t-PA and PAI-1 by HB-EGF was less than that by EGF. Heparitinase/chlorate, which digests the heparan sulfate proteoglycan of the endothelial cell surface, restored both t-PA and PAI-1 mRNA levels in response to HB-EGF in a manner similar to that by EGF. HB-EGF at 10 ng/ml developed tube-like structures in type I collagen gel at similar levels to that of EGF at 10 ng/ml, suggesting that HB-EGF is also a potent angiogenic factor in the model system for angiogenesis. The tubulogenesis activity of HB-EGF is discussed in relation to the expression of the t-PA and PAI-1 genes. PMID:8609052

Ushiro, S; Ono, M; Izumi, H; Kohno, K; Taniguchi, N; Higashiyama, S; Kuwano, M

1996-01-01

312

Plasminogen activator inhibitor 1: Mechanisms of its synergistic regulation by growth factors  

Energy Technology Data Exchange (ETDEWEB)

My research is on the synergistic regulation of PAI-1 by EGF and TGF-?. The mechanism of synergistic regulation of PAI-1 by EGF and TGF-? are addressed. Methods are described for effective identification of RNA accessible sites for antisense oligodexoxynucleotides (ODNs) and siRNA. In this study effective AS-ODN sequences for both Lcn2 and Bcl2 were identified by in vitro tiled microarray studies. Our results suggest that hybridization of ODN arrays to a target mRNA under physiological conditions might be used as a rapid and reliable in vitro method to accurately identify targets on mRNA molecules for effective antisense and potential siRNA activity in vivo.

Song, Xiaoling

2011-12-01

313

Effect of shear stress on intrinsic CHO culture state and glycosylation of recombinant tissue-type plasminogen activator protein.  

Science.gov (United States)

Shear stress in suspension culture was investigated as a possible manipulative parameter for the control of glycosylation of the recombinant tissue-type plasminogen activator protein (r-tPA) produced by recombinant Chinese hamster ovary (CHO) cell culture, grown in protein-free media. Resulting fractions of partially glycosylated, Type II, and fully glycosylated, Type I, r-tPA protein were monitored as a direct function of the shear characteristics of the culture environment. The shear-induced response of CHO culture to levels of low shear stress, where exponential growth was not obtained, and to higher levels of shear stress, which resulted in extensive cell death, were examined through manipulation of the bioreactor stirring velocity. Both apparent and intrinsic cell growth, metabolite consumption, byproduct and r-tPA production, and r-tPA glycosylation, from a variable site-occupancy standpoint, were monitored throughout. Kinetic analyses revealed a shear-stress-induced alteration of cellular homeostasis resulting in a nonlinear dependency of metabolic yield coefficients and an intrinsic cell lysis kinetic constant on shear stress. Damaging levels of shear stress were used to investigate the shear dependence of cell death and lysis, as well as the effects on the intrinsic growth rate of the culture. Kinetic models were also developed on the basis of the intrinsic state of the culture and compared to traditional models. Total r-tPA production was maximized under moderate shear conditions, as was the viable CHO cell density of the culture. However, Type II r-tPA production and the fraction of Type II glycoform production ratio was maximized under damaging levels of shear stress. Analyses of biomass production yield coefficients coupled with a plug-flow reactor model of glycan addition in the endoplasmic reticulum (ER) were used to propose an overall mechanism of decreased r-tPA protein site-occupancy glycosylation with increasing shear stress. Decreased residence time of r-tPA in the ER as a result of increased protein synthesis related to shear protection mechanisms is proposed to limit contact of site Asn184 with the membrane-bound oligosaccharyltransferase enzyme in the ER. PMID:12892482

Senger, Ryan S; Karim, M Nazmul

2003-01-01

314

Collagenase and tissue plasminogen activator production in developing rat calvariae: normal progression despite fetal exposure to microgravity  

Science.gov (United States)

Exposure to zero gravity has been shown to cause a decrease in bone formation. This implicates osteoblasts as the gravity-sensing cell in bone. Osteoblasts also are known to produce neutral proteinases, including collagenase and tissue plasminogen activator (tPA), which are thought to be important in bone development and remodeling. The present study investigated the effects of zero gravity on development of calvariae and their expression of collagenase and tPA. After in utero exposure to zero gravity for 9 days on the NASA STS-70 space shuttle mission, the calvariae of rat pups were examined by immunohistochemistry for the presence and location of these two proteinases. The ages of the pups were from gestational day 20 (G20) to postnatal (PN) day 35. Both collagenase and tPA were found to be present at all ages examined, with the greatest amount of both proteinases present in the PN14 rats. At later ages, high amounts were maintained for tPA but collagenase decreased substantially between ages PN21 to PN35. The location of collagenase was found to be associated with bone-lining cells, osteoblasts, osteocytes, and in the matrix along cement lines. In contrast, tPA was associated with endothelial cells lining the blood vessels entering bone. The presence and developmental expression of these two proteinases appeared to be unaffected by the exposure to zero gravity. The calvarial thickness of the pups was also examined; again the exposure to zero gravity showed little to no effect on the growth of the calvariae. Notably, from G20 to PN14, calvarial thickness increased dramatically, reaching a plateau after this age. It was apparent that elevated collagenase expression correlated with rapid bone growth in the period from G20 to PN14. To conclude, collagenase and tPA are present during the development of rat calvariae. Despite being produced by the same cell in vitro, i.e., the osteoblast, they are located in distinctly different places in bone in vivo. Their presence, developmental expression, and quantity do not seem to be affected by a brief exposure to zero gravity in utero.

Davis, B. A.; Sipe, B.; Gershan, L. A.; Fiacco, G. J.; Lorenz, T. C.; Jeffrey, J. J.; Partridge, N. C.

1998-01-01

315

Significance of the plasminogen activator inhibitor of placental type (PAI-2) in pregnancy.  

Science.gov (United States)

Marked changes in the hemostasis system, especially increases in PAI-2, are observed during pregnancy and at delivery. This inhibitor is produced by placental trophoblasts and by macrophages. PAI-2 occurs in two forms, a LMW and a HMW form. LMW PAI-2 is intracellular, HMW PAI-2 is secreted. PAI-2 inhibits both u-PA and two-chain t-PA. PAI-2 seems to be involved in the processes of invasion and remodeling of fetal and uterine tissues. It may protect against premature placental separation and secure hemostasis at parturition. Excess levels of PAI-2 in amniotic fluid may protect membranes from premature rupture. An imbalance between fibrinolytic activators and inhibitors may also be related to intracranial hemorrhage in premature infants. During preeclampsia t-PA and PAI-1 levels are markedly increased in plasma, and in cases of intrauterine growth retardation, u-PA and PAI-2 levels are decreased. While elevated PAI-1 concentrations might be helpful markers of severity of preeclampsia, decreased PAI-2 levels seem to indicate decreased placental function and intrauterine growth retardation. PMID:9834009

Astedt, B; Lindoff, C; Lecander, I

1998-01-01

316

In vivo kinetics of 99mTc labeled recombinant tissue plasminogen activator in rabbits  

International Nuclear Information System (INIS)

Our previous studies demonstrated that 99mTc labeled recombinant tissue plasminiogen activator (rt-PA) retained high affinity with fibrin in vitro but showed unexpectedly low uptake in fresh thrombi in vivo. The present study was performed to determine the in vivo kinetics of radiolabeled t-PA in the rabbit. Sequential images and blood samples after the intravenous administration of 99mTc labeled rt-PA in thrombus-bearing rabbits were taken. The radioactivity and immunological level of t-PA and PAI-1 in the solution eluted to each fraction by gel permeation chromatography were measured by means of a well scintillation counter and enzyme-linked immunosorbent assay (ELISA). Most of the radioactivity was eluted in the fraction (Fr.7) of larger molecular weight than that (Fr.9) of intact t-PA. The level of intact rt-PA was increased with a regimen involving the preadministration of cold rt-PA which was followed by the administration of hot rt-PA. The level of PAI-1 in plasma showed an increased rebound 15 minutes after the intravenous injection. These results suggest two possible reasons why rt-PA retains high affinity with fibrin in vitro, once radiolabeled, but was ineffective in delineating fresh thrombi with a gamma camera: 1) some plasma components such as PAI-1 combine with circulating radiolabeled rt-PA and form a larger molecule immediately and/or 2) radiolabeled rt-PA is modulated as a consequence of the radiolabeling and forms a larger mole the radiolabeling and forms a larger molecule than intact rt-PA. (author)

317

In vivo kinetics of [sup 99m]Tc labeled recombinant tissue plasminogen activator in rabbits  

Energy Technology Data Exchange (ETDEWEB)

Our previous studies demonstrated that [sup 99m]Tc labeled recombinant tissue plasminiogen activator (rt-PA) retained high affinity with fibrin in vitro but showed unexpectedly low uptake in fresh thrombi in vivo. The present study was performed to determine the in vivo kinetics of radiolabeled t-PA in the rabbit. Sequential images and blood samples after the intravenous administration of [sup 99m]Tc labeled rt-PA in thrombus-bearing rabbits were taken. The radioactivity and immunological level of t-PA and PAI-1 in the solution eluted to each fraction by gel permeation chromatography were measured by means of a well scintillation counter and enzyme-linked immunosorbent assay (ELISA). Most of the radioactivity was eluted in the fraction (Fr.7) of larger molecular weight than that (Fr.9) of intact t-PA. The level of intact rt-PA was increased with a regimen involving the preadministration of cold rt-PA which was followed by the administration of hot rt-PA. The level of PAI-1 in plasma showed an increased rebound 15 minutes after the intravenous injection. These results suggest two possible reasons why rt-PA retains high affinity with fibrin in vitro, once radiolabeled, but was ineffective in delineating fresh thrombi with a gamma camera: (1) some plasma components such as PAI-1 combine with circulating radiolabeled rt-PA and form a larger molecule immediately and/or (2) radiolabeled rt-PA is modulated as a consequence of the radiolabeling and forms a larger molecule than intact rt-PA. (author).

Itoh, Kazuo; Ieko, Masahiro; Hiraguchi, Etsuro; Kitayama, Hide; Tsukamoto, Eriko (Hokkaido Univ., Sapporo (Japan). School of Medicine)

1994-08-01

318

Cholinergic signal activated renin angiotensin system associated with cardiovascular changes in the ovine fetus  

OpenAIRE

Aim: Cholinergic regulation is important in the control of cardiovascular and endocrine responses. The mechanisms behind cardiovascular responses induced by cholinergic activation are explored by studying hormonal systems, including renin-angiotensin and vasopressin (VP). Results: In chronically prepared fetal sheep, intravenous infusion of the cholinergic agonist carbachol increased fetal systolic, diastolic, and mean arterial pressure accompanied with bradycardia at near-term. Although int...

Geng, Chunsong; Mao, Caiping; Wu, Lei; Cheng, Yu; Liu, Rulu; Chen, Bingxin; Chen, Ling; Zhang, Lubo; Xu, Zhice

2010-01-01

319

Probiotic in rennet paste can affect lipase activity of rennet and lipolysis in ovine cheese  

OpenAIRE

Lambs were subjected to three different feeding regimes (mother suckling MS, artificial rearing AR, and artificial rearing with 7log10 cfu/ml Lactobacillus acidophilus supplementation to the milk substitute ARLb) and slaughtered at 20d and 40d of age for each feeding treatment. Lambs abomasa were processed to rennet paste and lipases activity was evaluated. Rennet paste was used for Pecorino cheese production. Free fatty acids (FFAs) and conjugated linoleic acids (CLAs) were detected in chees...

Marzia Albenzio; Rosaria Marino; Mariangela Caroprese; Antonella Santillo

2010-01-01

320

Inhibitory effect of berberine on the invasion of human lung cancer cells via decreased productions of urokinase-plasminogen activator and matrix metalloproteinase-2  

International Nuclear Information System (INIS)

Berberine, a compound isolated from medicinal herbs, has been reported with many pharmacological effects related to anti-cancer and anti-inflammation capabilities. In this study, we observed that berberine exerted a dose- and time-dependent inhibitory effect on the motility and invasion ability of a highly metastatic A549 cells under non-cytotoxic concentrations. In cancer cell migration and invasion process, matrix-degrading proteinases are required. A549 cell treated with berberine at various concentrations showed reduced ECM proteinases including matrix metalloproteinase-2 (MMP2) and urokinase-plasminogen activator (u-PA) by gelatin and casein zymography analysis. The inhibitory effect is likely to be at the transcriptional level, since the reduction in the transcripts levels was corresponding to the proteins. Moreover, berberine also exerted its action via regulating tissue inhibitor of metalloproteinase-2 (TIMP-2) and urokinase-plasminogen activator inhibitor (PAI). The upstream mediators of the effect involved c-jun, c-fos and NF-?B, as evidenced by reduced phosphorylation of the proteins. These findings suggest that berberine possesses an anti-metastatic effect in non-small lung cancer cell and may, therefore, be helpful in clinical treatment

321

Inhibition of plasminogen activator inhibitor-1 binding to endocytosis receptors of the low-density-lipoprotein receptor family by a peptide isolated from a phage display library  

DEFF Research Database (Denmark)

The functions of the serpin PAI-1 (plasminogen activator inhibitor-1) are based on molecular interactions with its target proteases uPA and tPA (urokinase-type and tissue-type plasminogen activator respectively), with vitronectin and with endocytosis receptors of the low-density-lipoprotein family. Understanding the significance of these interactions would be facilitated by the ability to block them individually. Using phage display, we have identified the disulfide-constrained peptide motif CFGWC with affinity for natural human PAI-1. The three-dimensional structure of a peptide containing this motif (DVPCFGWCQDA) was determined by liquid-state NMR spectroscopy. A binding site in the so-called flexible joint region of PAI-1 was suggested by molecular modelling and validated through binding studies with various competitors and site-directed mutagenesis of PAI-1. The peptide with an N-terminal biotin inhibited the binding of the uPA-PAI-1 complex to the endocytosis receptors low-density-lipoprotein-receptor-related protein 1A (LRP-1A) and very-low-density-lipoprotein receptor (VLDLR) in vitro and inhibited endocytosis of the uPA-PAI-1 complex in U937 cells. We conclude that the isolated peptide represents a novel approach to pharmacological interference with the functions of PAI-1 based on inhibition of one specific molecular interaction.

Jensen, Jan Kristian; Malmendal, Anders

2006-01-01

322

Inhibition of plasminogen activator inhibitor-1 binding to endocytosis receptors of the low density lipoprotein receptor family by a peptide isolated from a phage displayed library  

DEFF Research Database (Denmark)

The functions of the serpin plasminogen activator inhibitor-1 (PAI-1) are based on molecular interactions with its target proteases urokinase-type and tissue-type plasminogen activator (uPA and tPA), with vitronectin, and with endocytosis receptors of the low density lipoprotein family. Understanding the significance of these interactions would be facilitated by the ability to block them individually. Using phage display, we have identified the disulphide constrained peptide motif CFGWC with affinity for natural human PAI-1. The three-dimensional structure of a peptide containing this motif (DVPCFGWCQDA) was determined by NMR. A binding site in the so-called flexible joint region of PAI-1 was suggested by molecular modelling and validated through binding studies with various competitors and site-directed mutagenesis of PAI-1. The peptide with an N-terminal biotin inhibited the binding of the uPA-PAI-1 complex to the endocytosis receptors low density lipoprotein receptor-related protein (LRP-1A) and very low density lipoprotein receptor (VLDLR) in vitro and inhibited endocytosis of the uPA-PAI-1 complex in U937 cells. We conclude that the isolated peptide represents a novel approach to pharmacological interference with the functions of PAI-1 based on inhibition of one specific molecular interaction.

Jensen, Jan K.; Malmendal, Anders

2006-01-01

323

Angiotensin II induces secretion of plasminogen activator inhibitor 1 and a tissue metalloprotease inhibitor-related protein from rat brain astrocytes  

Energy Technology Data Exchange (ETDEWEB)

The present study investigates angiotensin (Ang) II effects on secretory protein synthesis in brain astrocytes cultured from neonatal and 21-day-old rats. Ang II-induced changes in the de novo synthesis of (35S)methionine-labeled secretory proteins were visualized using two-dimensional NaDodSO4/PAGE. Astrocytes from 21-day-old rat brain possess specific high-affinity receptors for Ang II. These cells express two Ang II-induced secretory proteins with Mr 55,000 (AISP-55K) and Mr 30,000 (AISP-30K), which were time- and dose-dependent (EC50, 1 nM). (Sar1, Ile8)Ang II (where Sar is sarcosine) inhibited Ang II-induced secretion of AISP-55K but not AISP-30K. N-terminal amino acid sequencing indicates that AISP-55K is identical to rat plasminogen activator inhibitor 1, whereas AISP-30K exhibits 72-81% identity to three closely related proteins: human tissue inhibitor of metalloproteases, a rat phorbol ester-induced protein, and the murine growth-responsive protein 16C8. Immunofluorescent staining with rat plasminogen activator inhibitor 1 antibody was induced in the majority of cells in culture after Ang II treatment of astrocytes from 21-day-old rat brains. Absence of this response to Ang II in astrocytes from neonatal rat brain provides evidence that this action of Ang II on astrocytes is developmentally regulated.

Olson, J.A. Jr.; Shiverick, K.T.; Ogilvie, S.; Buhi, W.C.; Raizada, M.K. (Univ. of Florida College of Medicine, Gainesville (USA))

1991-03-01

324

Angiotensin II induces secretion of plasminogen activator inhibitor 1 and a tissue metalloprotease inhibitor-related protein from rat brain astrocytes  

International Nuclear Information System (INIS)

The present study investigates angiotensin (Ang) II effects on secretory protein synthesis in brain astrocytes cultured from neonatal and 21-day-old rats. Ang II-induced changes in the de novo synthesis of [35S]methionine-labeled secretory proteins were visualized using two-dimensional NaDodSO4/PAGE. Astrocytes from 21-day-old rat brain possess specific high-affinity receptors for Ang II. These cells express two Ang II-induced secretory proteins with Mr 55,000 (AISP-55K) and Mr 30,000 (AISP-30K), which were time- and dose-dependent (EC50, 1 nM). [Sar1, Ile8]Ang II (where Sar is sarcosine) inhibited Ang II-induced secretion of AISP-55K but not AISP-30K. N-terminal amino acid sequencing indicates that AISP-55K is identical to rat plasminogen activator inhibitor 1, whereas AISP-30K exhibits 72-81% identity to three closely related proteins: human tissue inhibitor of metalloproteases, a rat phorbol ester-induced protein, and the murine growth-responsive protein 16C8. Immunofluorescent staining with rat plasminogen activator inhibitor 1 antibody was induced in the majority of cells in culture after Ang II treatment of astrocytes from 21-day-old rat brains. Absence of this response to Ang II in astrocytes from neonatal rat brain provides evidence that this action of Ang II on astrocytes is developmentally regulated

325

Pneumatic displacement with perfluoropropane gas and intravitreal tissue plasminogen activator for subretinal subfoveal hemorrhage after focal laser photocoagulation in central serous chorioretinopathy.  

Science.gov (United States)

Objective. To report the visual and anatomic outcomes of pneumatic displacement with perfluoropropane (C3F8) gas and intravitreal tissue plasminogen activator (IVTPA) for subretinal subfoveal hemorrhage after focal laser photocoagulation in central serous chorioretinopathy (CSCR). Method. Interventional, retrospective case report of one eye (one patient). Outcome measures included visual acuity (VA), central macular thickness (CMT), and size of the lesion at two weeks of followup. Fluorescein angiography (FA) and optical coherent tomography (OCT) were used to measure anatomic outcomes. Results. A 35-year-old man with history of chronic CSCR received focal laser photocoagulation in the right eye two days before presentation. At initial examination, VA was 20/200 (ETDRS chart), CMT was 398??, and a subretinal subfoveal hemorrhage was seen. Tissue plasminogen activator (tPA) at a dose of 25?µg/0.1?mL was injected intravitreally before intravitreal C3F8 injection, and prone positioning was indicated postoperatively. At 24 hours, the hemorrhage had been displaced inferiorly and VA improved to 20/100. Two weeks later, VA improved to 20/80, CMT decreased to 225??, and the hemorrhage decreased without foveal involvement. Conclusions. The technique seems safe and effective in treating visually significant subretinal subfoveal hemorrhage. PMID:25485161

Al Rubaie, Khalid; Espinoza, Juan V; Lasave, Andres F; Savino-Zari, Dario; Arevalo, Fernando A; Arevalo, J Fernando

2014-01-01

326

Angiotensin II induces secretion of plasminogen activator inhibitor 1 and a tissue metalloprotease inhibitor-related protein from rat brain astrocytes.  

Science.gov (United States)

The present study investigates angiotensin (Ang) II effects on secretory protein synthesis in brain astrocytes cultured from neonatal and 21-day-old rats. Ang II-induced changes in the de novo synthesis of [35S]methionine-labeled secretory proteins were visualized using two-dimensional NaDodSO4/PAGE. Astrocytes from 21-day-old rat brain possess specific high-affinity receptors for Ang II. These cells express two Ang II-induced secretory proteins with Mr 55,000 (AISP-55K) and Mr 30,000 (AISP-30K), which were time- and dose-dependent (EC50, 1 nM). [Sar1, Ile8]Ang II (where Sar is sarcosine) inhibited Ang II-induced secretion of AISP-55K but not AISP-30K. N-terminal amino acid sequencing indicates that AISP-55K is identical to rat plasminogen activator inhibitor 1, whereas AISP-30K exhibits 72-81% identity to three closely related proteins: human tissue inhibitor of metalloproteases, a rat phorbol ester-induced protein, and the murine growth-responsive protein 16C8. Immunofluorescent staining with rat plasminogen activator inhibitor 1 antibody was induced in the majority of cells in culture after Ang II treatment of astrocytes from 21-day-old rat brains. Absence of this response to Ang II in astrocytes from neonatal rat brain provides evidence that this action of Ang II on astrocytes is developmentally regulated. PMID:2000398

Olson, J A; Shiverick, K T; Ogilvie, S; Buhi, W C; Raizada, M K

1991-03-01

327

Plasminogen activator inhibitor type 1 gene is located at region q21.3-q22 of chromosome 7 and genetically linked with cystic fibrosis  

International Nuclear Information System (INIS)

The regional chromosomal location of the human gene for plasminogen activator inhibitor type 1 (PAI1) was determined by three independent methods of gene mapping. PAI1 was localized first to 7cen-q32 and then to 7q21.3-q22 by Southern blot hybridization analysis of a panel of human and mouse somatic cell hybrids with a PAI1 cDNA probe and in situ hybridization, respectively. The authors frequent HindIII restriction fragment length polymorphism (RFLP) of the PAI1 gene with an information content of 0.369. In family studies using this polymorphism, genetic linkage was found between PAI1 and the loci for erythropoietin (EPO), paraoxonase (PON), the met protooncogene (MET), and cystic fibrosis (CF), all previously assigned to the middle part of the long arm of chromosome 7. The linkage with EPO was closest with an estimated genetic distance of 3 centimorgans, whereas that to CF was 20 centimorgans. A three-point genetic linkage analysis and data from previous studies showed that the most likely order of these loci is EPO, PAI1, PON, (MET, CF), with PAI1 being located centromeric to CF. The PAI1 RFLP may prove to be valuable in ordering genetic markers in the CF-linkage group and may also be valuable in genetic analysis of plasminogen activation-related diseases, such as certain thromboembolic disorders and cancer

328

Protection of single-chain urokinase-type plasminogen activator (scu-PA) in aprotinin treated cardiac surgical patients undergoing cardiopulmonary bypass.  

Science.gov (United States)

Intraoperative high-dose aprotinin administration has been shown to reduce the intra-and postoperative blood loss in cardiac surgery. The haemostatic effect has been attributed to platelet preserving properties and to inhibition of contact activation reducing thrombotic and fibrinolytic activity during and after cardiopulmonary bypass (CPB). Here we report on the effects of aprotinin on urokinase-type plasminogen activator, especially on the protection of the zymogen single-chain urokinase-type plasminogen activator (scu-PA). scu-PA occurs cell associated as well as free in the circulation (concentration 50 pM, half-life 5 min), and is potentially activated by kallikrein and plasmin, both potent targets for aprotinin. The generated active two-chain u-PA (tcu-PA) is a powerful activator of fibrinolysis. Sixteen male patients undergoing myocardial revascularization were randomly assigned to aprotinin treatment (A) or control group (C). Plasma concentration of total u-PA antigen and of the specific forms scu-PA(zymogen) and tcu-PA(active enzyme) were measured at different stages intraoperatively and two hours postoperatively. After an initial drop due to haemodilution at the onset of CPB, the concentrations of circulating u-PA forms restored intraoperatively in A, but remained subnormal in C until the end of the observation period. The concentration of total u-PA antigen of shed mediastinal blood was both in A and C two-fold higher than in the circulation, but the antigen was preserved as the zymogen scu-PA in A and largely converted to an inactive, non activatable form in C. Intra- and postoperative blood losses were less than half the amount in A as compared to C.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7482410

Spannagl, M; Dooijewaard, G; Dietrich, W; Kluft, C

1995-05-01

329

Effect of malondialdehyde-acetaldehyde-protein adducts on the protein kinase C-dependent secretion of urokinase-type plasminogen activator in hepatic stellate cells.  

Science.gov (United States)

Previous studies from our laboratory have shown that malondialdehyde-acetaldehyde-protein adducts (MAA adducts) are formed in hepatocytes of ethanol-fed rats and directly influence the hepatic stellate cells (HSCs) to induce their secretion of chemokines and to up-regulate their expression of adhesion molecules. Since protein kinase C (PKC) is known to play a major role in many diverse intracellular signal transduction processes, we investigated whether MAA adducts influence the function of HSCs via a PKC-dependent pathway. HSCs in culture were exposed to MAA adducts, and PKC activity was determined. We observed a time- and concentration-dependent activation of PKC when cultures were exposed to BSA-MAA as compared with unmodified BSA. Using PKC isoform-specific inhibitors, we also showed that BSA-MAA induces the activation of a specific isoform of PKC, PKC-alpha, in HSCs. No activation of PKC was observed when HSCs were exposed to other aldehyde adducts such as BSA-acetaldehyde or BSA-malondialdehyde, indicating that the effects of MAA adducts on HSCs were somewhat specific. We further examined whether the observed increase in PKC activation induced by MAA adducts in HSCs, in turn, causes a functional effect. We observed that BSA-MAA induces the increased secretion of urokinase-type plasminogen activator, a key component of the plasmin-generating system, and that PKC activation is necessary for this enhanced urokinase-type plasminogen activator secretion. These results indicate that MAA adducts via a PKC-mediated pathway may regulate plasmin-mediated matrix degradation in the liver, thereby contributing to the progression of hepatic fibrosis. PMID:11853706

Kharbanda, Kusum K; Shubert, Kris A; Wyatt, Todd A; Sorrell, Michael F; Tuma, Dean J

2002-02-01

330

Effects of urokinase-type plasminogen activator in the acquisition, expression and reinstatement of cocaine-induced conditioned-place preference.  

Science.gov (United States)

Cocaine and many other psychostimulants strongly induce urokinase-type plasminogen activator (uPA) expression in the mesolimbic dopaminergic pathway, which plays a major role in drug-mediated behavioral plasticity [Bahi A, Boyer F, Gumy C, Kafri T, Dreyer JL. In vivo gene delivery of urokinase-type plasminogen activator with regulatable lentivirus induces behavioral changes in chronic cocaine administration. Eur J Neurosci 2004;20:3473-88; Bahi A, Boyer F, Kafri T, Dreyer JL. Silencing urokinase in the ventral tegmental area in vivo induces changes in cocaine-induced hyperlocomotion. J Neurochem 2006;98:1619-31; Bahi A, Dreyer JL. Overexpression of plasminogen activators in the nucleus accumbens enhances cocaine-, amphetamine- and morphine-induced reward and behavioral sensitization. Genes Brain Behav 2007]. In this study, the role of mesolimbic dopamine (DA) pathways in the development of cocaine reward was examined by conditioned-place preference in rats with bilateral intra-accumbens injections of uPA-expressing lentiviral vectors. We show that overexpression of uPA in the Nac significantly augments cocaine-induced place preference. Furthermore, while this did not affect the ability of preference to be extinguished, reinstatement with a low dose of cocaine produced significantly greater preference to the cocaine-associated context. Once CPP had been established, and the preference extinguished, reinstatement induced by a priming dose of cocaine was facilitated by uPA. Inhibition of this serine protease expression using doxycycline abolished the augmented acquisition produced by overexpression of uPA but not the expression of the cocaine-induced CPP. When uPA is inhibited during the acquisition phase, animals no longer demonstrate place preference for the environment previously paired with cocaine. B428, a specific uPA inhibitor does not affect drug reinstatement after extinction if uPA has been activated during acquisition, a clear indication that uPA is involved in the acquisition phase of conditioned-place preference. Our results suggest that that increased uPA expression with repeated drug exposure produces conditions for enhanced acquisition of cocaine-induced CPP, indicating that cocaine-induced CPP and reinstatement may be dependent on active extracellular uPA. PMID:18436315

Bahi, Amine; Kusnecov, Alexander W; Dreyer, Jean-Luc

2008-08-01

331

Inhibitory effect of a synthetic prostacyclin analogue, beraprost, on urokinase-type plasminogen activator expression in RC-K8 human lymphoma cells.  

Science.gov (United States)

Plasminogen activation by urokinase-type plasminogen activator (uPA) is implicated in tumor invasion and metastasis by the breakdown of extracellular matrix. We have recently demonstrated the inhibitory effect of cAMP on uPA gene transcription in RC-K8 human lymphoma cells (Biochim Biophys Acta 1268: 293-9, 1995). Prostacyclin produced by endothelial cells is shown to increase cellular cAMP levels by activating adenylate cyclase. We, therefore, examined the effect of a stable analogue of prostacyclin, Beraprost, on uPA production in RC-K8 cells. uPA activity gradually increased in the conditioned medium with time. Beraprost (0.1 nM-1.0 microM) inhibited uPA accumulation in a dose-dependent manner without affecting cell viability. Fibrinzymography demonstrated that high and low molecular forms of uPA were present in the conditioned medium and that after Beraprost-treatment all forms of uPA decreased and no PA/PA inhibitor complex was present. Northern blot analysis revealed that after exposure to Beraprost, uPA mRNA levels increased transiently and then rapidly decreased to below control levels. Treatment with Beraprost resulted in a rapid activation of cellular cyclic AMP-dependent protein kinase (PKA). Beraprost completely negated uPA gene expression induced by phorbol myristate acetate, an activator of protein kinase C (PKC). These results suggest that Beraprost inhibits uPA production by suppressing uPA gene expression through the PKA pathway and that PKA-mediated signals are dominant in uPA gene expression as compared to those medicated by PKC. This inhibition of uPA expression by a prostacyclin analogue may be an important fact to explain the mechanism of anti-metastatic effects of prostacyclin. PMID:8822588

Nomura, N; Niiya, K; Shinbo, M; Ozawa, T; Hayakawa, Y; Higashiyama, K; Fujimaki, M; Sakuragawa, N

1996-06-01

332

Statins suppress glucose-induced plasminogen activator inhibitor-1 expression by regulating RhoA and nuclear factor-?B activities in cardiac microvascular endothelial cells.  

Science.gov (United States)

The aim of this study was to investigate the possible proinflammatory signaling pathways involved in statin inhibition of glucose-induced plasminogen activator inhibitor-1 (PAI-1) expression in cardiac microvascular endothelial cells (CMECs). Primary rat CMECs were grown in the presence of 5.7 or 23 mmol/L glucose. PAI-1 mRNA and protein expression levels were measured by realtime polymerase chain reaction, Western blotting and enzyme-linked immunosorbent assay, respectively. A pull-down assay was performed to determine RhoA activity. I?B? protein expression was measured by Western blotting, nuclear factor (NF)-?B activation was detected by electrophoretic mobility shift assay and its transcription activity was determined by a dual luciferase reporter gene assay. PAI-1 mRNA and protein expression levels were both increased with high glucose concentrations, but they were significantly suppressed by simvastatin and atorvastatin treatment (P pyrophosphate (10 ?mol/L) but not farnesyl pyrophosphate (10 ?mol/L). Such effects were similar to those of a RhoA inhibitor, C3 exoenzyme (5 ?g/mL), inhibitors of RhoA kinase (ROCK), Y-27632 (10 ?mol/L) and hydroxyfasudil (10 ?mol/L) and an NF-?B inhibitor, BAY 11-7082 (5 ?mol/L). High glucose-induced RhoA and NF-?B activations in CMECs were both significantly inhibited by statins (P < 0.01). Simvastatin and atorvastatin equally suppress high glucose-induced PAI-1 expression. These effects of statins may occur partly by regulating the RhoA/ROCK-NF-?B pathway. The multifunctional roles of statins may be particularly beneficial for patients with metabolic syndrome. PMID:23479762

Ni, Xiao-Qing; Zhu, Jian-Hua; Yao, Ning-Hua; Qian, Juan; Yang, Xiang-Jun

2013-01-01

333

Interleukin-1? enhances the aggressive behavior of pancreatic cancer cells by regulating the ?6?1-integrin and urokinase plasminogen activator receptor expression  

Directory of Open Access Journals (Sweden)

Full Text Available Abstract Background In human pancreatic cancer progression, the ?6?1-integrin is expressed on cancer cell surface during invasion and metastasis formation. In this study, we investigated whether interleukin (IL-1? induces the alterations of integrin subunits and urokinase plasminogen activator/urokinase plasminogen activator receptor (uPA/uPAR expression in pancreatic cancer cells. We hypothesize that the alterations of integrin subunits and uPA/uPAR expression make an important role in signaling pathways responsible for biological behavior of pancreatic cancer cells. Results IL-1? upregulated the expression of ?6 and ?1 integrins without any alterations of ?5 and ?v integrins expression. IL-1? also induced enhancement in the expression of uPA/uPAR in pancreatic cancer cells. IL-1? enhanced the proliferation, adhesion, and migration in pancreatic cancer cells, and IL-1?-induced alterations of uPA/uPAR expression correlated with the increased the migration of pancreatic cancer cells. Upregulation of ?6 integrin subunit and uPA/uPAR correlated with the activation of Ras and downstream extracellular signal-regulated kinase (ERK pathways. IL-1?-induced activation of Ras and downstream ERK can be inhibited by using inhibitory antibodies against ?6 and ?1 integrin and uPAR, consistent with the inhibition of proliferation, adhesion and migration of pancreatic cancer cells. Immunohistochemical analysis demonstrated a significant association between strong expressions of ?6 integrin with uPAR in pancreatic cancer specimens. Furthermore, the strong expression of ?6 integrin and uPAR was found to be independent prognosticator in pancreatic cancer patients. Conclusion Based on these findings, we conclude that IL-1? can induce selective upregulation of ?6?1-integrin and uPA/uPAR in pancreatic cancer cells and these changes may modulate the aggressive functions of pancreatic cancer.

Takeyama Hiromitsu

2006-02-01

334

Staurosporine induces ganglion cell differentiation in part by stimulating urokinase-type plasminogen activator expression and activation in the developing chick retina  

International Nuclear Information System (INIS)

Highlights: ? Staurosporine mediates stimulation of RGC differentiation in vitro cultured retinal neuroblasts. ? Staurosporine mediates uPA activation during RGC differentiation in vitro. ? Inhibition of uPA blocks the staurosporine mediated RGC differentiation both in vitro and in ovo. ? Thus, uPA may play a role in the staurosporine-mediated stimulation of RGC differentiation. -- Abstract: Here, we investigated whether staurosporine-mediated urokinase-type plasminogen activator (uPA) activation is involved in retinal ganglion cell (RGC) differentiation. Retinal cells were isolated from developing chick retinas at embryonic day 6 (E6). Relatively few control cells grown in serum-free medium started to form processes by 12 h. In contrast, staurosporine-treated cells had processes within 3 h, and processes were evident at 8 h. Immunofluorescence staining showed that Tuj-1-positive cells with shorter neurites could be detected in control cultures at 18 h, whereas numerous Tuj-1 positive ganglion cells with longer neuritic extensions were seen in staurosporine-treated cultures. BrdU-positive proliferating cells were more numerous in control cultures than in staurosporine-treated cultures, and the BrdU staining was not detected in post-mitotic Tuj-1 positive ganglion cells. Western blotting of cell lysates showed that staurosporine induced high levels of the active form of uPA. The staurosporine-induced uPA signal was localized predominantly in the soma, neurites and axons of Tuj-1-positive ganglion cells. Amiloride, an inhibitor of uPA, markedly reduced staurosporine-induced Tuj-1 staining, neurite length, neurite number, and uPA staining versus controls. In developing retinas in ovo, amiloride administration remarkably reduced the staurosporine-induced uPA staining and RGC differentiation. Taken together, our in vitro and in vivo data collectively indicate that uPA plays a role in the staurosporine-mediated stimulation of RGC differentiation.

335

Posttranslational, reversible O-glycosylation is stimulated by high glucose and mediates plasminogen activator inhibitor-1 gene expression and Sp1 transcriptional activity in glomerular mesangial cells.  

Science.gov (United States)

Metabolic flux through the hexosamine biosynthetic pathway (HBP) is increased in the presence of high glucose (HG) and potentially stimulates the expression of genes associated with the development of diabetic nephropathy. A number of synthetic processes are coupled to the HBP, including enzymatic intracellular O-glycosylation (O-GlcNAcylation), the addition of single O-linked N-acetylglucosamine monosaccharides to serine or threonine residues. Despite much data linking flow through the HBP and gene expression, the exact contribution of O-GlcNAcylation to HG-stimulated gene expression remains unclear. In glomerular mesangial cells, HG-stimulated plasminogen activator inhibitor-1 (PAI-1) gene expression requires the HBP and the transcription factor, Sp1. In this study, the specific role of O-GlcNAcylation in HG-induced PAI-1 expression was tested by limiting this modification with a dominant-negative O-linked N-acetylglucosamine transferase, by overexpression of neutral beta-N-acetylglucosaminidase, and by knockdown of O-linked beta-N-acetylglucosamine transferase expression by RNA interference. Decreasing O-GlcNAcylation by these means inhibited the ability of HG to increase endogenous PAI-1 mRNA and protein levels, the activity of a PAI-1 promoter-luciferase reporter gene, and Sp1 transcriptional activation. Conversely, treatment with the beta-N-acetylglucosaminidase inhibitor, O-(2-acetamido-2-deoxy-d-glucopyranosylidene)amino-N-phenylcarbamate, in the presence of normal glucose increased Sp1 O-GlcNAcylation and PAI-1 mRNA and protein levels. These findings demonstrate for the first time that among the pathways served by the HBP, O-GlcNAcylation, is obligatory for HG-induced PAI-1 gene expression and Sp1 transcriptional activation in mesangial cells. PMID:16365142

Goldberg, Howard J; Whiteside, Catharine I; Hart, Gerald W; Fantus, I George

2006-01-01

336

Mycoplasma fermentans binds to and invades HeLa cells: involvement of plasminogen and urokinase.  

Science.gov (United States)

Adherence of Mycoplasma fermentans to HeLa cells followed saturation kinetics, required a divalent cation, and was enhanced by preincubation of the organism at 37 degrees C for 1 h in a low-osmolarity solution. Proteolytic digestion, choline phosphate, or anti-choline phosphate antibodies partially inhibited the adherence, supporting the notion that M. fermentans utilizes at least two surface components for adhesion, a protease-sensitive surface protein and a phosphocholine-containing glycolipid. Plasminogen binding to M. fermentans greatly increased the maximal adherence of the organism to HeLa cells. Anti-plasminogen antibodies and free plasminogen inhibited this increase. These observations suggest that in the presence of plasminogen the organism adheres to novel sites on the HeLa cell surface, which are apparently plasminogen receptors. Plasminogen-bound M. fermentans was detected exclusively on the cell surface of the infected HeLa cells. Nevertheless, plasminogen binding in the presence of the urokinase-type plasminogen activator (uPA) promoted the invasion of HeLa cells by M. fermentans. The latter finding indicates that the invasiveness of M. fermentans does not result from binding plasminogen but from activation of the bound plasminogen to plasmin. Cholesterol depletion and sequestration with beta-cyclodextrin and filipin, respectively, did not affect the capacity of M. fermentans to adhere, but invasion of HeLa cells by uPA-activated plasminogen-bound M. fermentans was impaired, suggesting that lipid rafts are implicated in M. fermentans entry. PMID:15321992

Yavlovich, Amichai; Katzenell, Avigail; Tarshis, Mark; Higazi, Abd A-R; Rottem, Shlomo

2004-09-01

337

Exploring soluble urokinase plasminogen activator receptor and its relationship with arterial stiffness in a bi-ethnic population: the SAfrEIC-study  

DEFF Research Database (Denmark)

INTRODUCTION: Elevated soluble urokinase-type plasminogen activator receptor (suPAR) indicates an inflammatory state caused by conditions such as HIV and cancer. Recently suPAR was identified as an indicator of cardiovascular disease (CVD). CVD is highly prevalent in black South Africans, but the potential role of suPAR is unknown. We investigated suPAR as a possible marker of arterial stiffness in Africans and Caucasians. METHODS: This study involved 207 Africans and 314 Caucasians (aged 20-70yrs). C-reactive protein (CRP) and suPAR were determined in fasting blood samples. We measured blood pressure, pulse wave velocity (PWV) and Windkessel arterial compliance (Cwk). RESULTS: Africans displayed higher suPAR, CRP, PWV and lower Cwk (p

Schutte, Aletta E; Myburgh, Anélda

2012-01-01

338

Plasminogen activator inhibitor-1 deficiency augments visceral mesothelial organization, intrapleural coagulation, and lung restriction in mice with carbon black/bleomycin-induced pleural injury.  

Science.gov (United States)

Local derangements of fibrin turnover and plasminogen activator inhibitor (PAI)-1 have been implicated in the pathogenesis of pleural injury. However, their role in the control of pleural organization has been unclear. We found that a C57Bl/6j mouse model of carbon black/bleomycin (CBB) injury demonstrates pleural organization resulting in pleural rind formation (14 d). In transgenic mice overexpressing human PAI-1, intrapleural fibrin deposition was increased, but visceral pleural thickness, lung volumes, and compliance were comparable to wild type. CBB injury in PAI-1(-/-) mice significantly increased visceral pleural thickness (P intrapleural fibrin deposition, PAI-1 deficiency promotes profibrogenic alterations of the mesothelium that exacerbate pleural organization and lung restriction. PMID:24024554

Tucker, Torry A; Jeffers, Ann; Alvarez, Alexia; Owens, Shuzi; Koenig, Kathleen; Quaid, Brandon; Komissarov, Andrey A; Florova, Galina; Kothari, Hema; Pendurthi, Usha; Mohan Rao, L Vijaya; Idell, Steven

2014-02-01

339

In vivo distribution of Tc-99m labeled recombinant tissue-type plasminogen activator in control and thrombus-bearing rats  

International Nuclear Information System (INIS)

In vivo distribution of Tc-99m labeled recombinant tissue-type plasminogen activator (Tc-99m-rt-PA) was studied in control rats and thrombus-bearing rats. To compare fibrin binding in vivo with that in vitro, Tc-99m-rt-PA binding to fibrin gel in vitro was also imaged. Rapid blood clearance and accumulation into the liver and kidneys were observed in both control and thrombus-bearing rats. Accumulation in the stomach, which indicates instability of labeled rt-PA in vivo, was very low until two hours after injection. Tc-99m-rt-PA accumulation in the clots was higher than that in skeletal and heart muscles, although it was lower than in blood, liver, and kidneys. Administration of aprotinin, an antifibrinolytic agent, significantly prolonged clot accumulation of Tc-99m-rt-PA at 30 minutes after injection. These results suggest that fibrinolysis is responsible for the low rt-PA concentration in the clots. A scintigram of a thrombus-bearing rat demonstrated increased radioactivity at the clot forming site. On the other hand, Tc-99m-labeled human albumin, which was used as a control, was not accumulated in the clot. Tc-99m-rt-PA binding to fibrin gel in vitro was clearly imaged. By comparison, in vivo fibrin binding of Tc-99m-rt-PA was much lower than in vitro. The reasons for low thrombus uptake in vivo may be: (1) biochemical inactivation of extrinsically administered rt-PA by t-PA inhibitor; (2) fibrinolysis by rt-PA activated plasminogen. Overcoming these limitations wlasminogen. Overcoming these limitations will enable Tc-99m-rt-PA to reach the stage of clinical trials. (author)

340

Nicotine stimulates urokinase-type plasminogen activator receptor expression and cell invasiveness through mitogen-activated protein kinase and reactive oxygen species signaling in ECV304 endothelial cells  

Energy Technology Data Exchange (ETDEWEB)

Urokinase-type plasminogen activator receptor (uPAR) expression is elevated during inflammation, tissue remodeling and in many human cancers. This study investigated the effect of nicotine, a major alkaloid in tobacco, on uPAR expression and cell invasiveness in ECV304 endothelial cells. Nicotine stimulated uPAR expression in a dose-dependent manner and activated extracellular signal-regulated kinases-1/2 (Erk-1/2), c-Jun amino-terminal kinase (JNK) and p38 mitogen activated protein kinase (MAPK). Specific inhibitors of MEK-1 (PD98059) and JNK (SP600125) inhibited the nicotine-induced uPAR expression, while the p38 MAPK inhibitor SB203580 did not. Expression vectors encoding dominant negative MEK-1 (pMCL-K97M) and JNK (TAM67) also prevented nicotine-induced uPAR promoter activity. The intracellular hydrogen peroxide (H{sub 2}O{sub 2}) content was increased by nicotine treatment. The antioxidant N-acetylcysteine prevented nicotine-activated production of reactive oxygen species (ROS) and uPAR expression. Furthermore, exogenous H{sub 2}O{sub 2} increased uPAR mRNA expression. Deleted and site-directed mutagenesis demonstrated the involvement of the binding sites of transcription factor nuclear factor-kappaB (NF-?B) and activator protein (AP)-1 in the nicotine-induced uPAR expression. Studies with expression vectors encoding mutated NF-?B signaling molecules and AP-1 decoy confirmed that NF-?B and AP-1 were essential for the nicotine-stimulated uPAR expression. MAPK (Erk-1/2 and JNK) and ROS functioned as upstream signaling molecules in the activation of AP-1 and NF-?B, respectively. In addition, ECV304 endothelial cells treated with nicotine displayed markedly enhanced invasiveness, which was partially abrogated by uPAR neutralizing antibodies. The data indicate that nicotine induces uPAR expression via the MAPK/AP-1 and ROS/NF-?B signaling pathways and, in turn, stimulates invasiveness in human ECV304 endothelial cells. -- Highlights: ? Endothelial cells treated with nicotine displayed enhanced invasiveness. ? Nicotine induces uPAR expression and, in turn, stimulates invasiveness. ? MAPK/AP-1 and ROS/NF-?B signals are involved in nicotine-induced uPAR.

Khoi, Pham Ngoc; Park, Jung Sun; Kim, Nam Ho; Jung, Young Do, E-mail: ydjung@chonnam.ac.kr

2012-03-01

341

Nicotine stimulates urokinase-type plasminogen activator receptor expression and cell invasiveness through mitogen-activated protein kinase and reactive oxygen species signaling in ECV304 endothelial cells  

International Nuclear Information System (INIS)

Urokinase-type plasminogen activator receptor (uPAR) expression is elevated during inflammation, tissue remodeling and in many human cancers. This study investigated the effect of nicotine, a major alkaloid in tobacco, on uPAR expression and cell invasiveness in ECV304 endothelial cells. Nicotine stimulated uPAR expression in a dose-dependent manner and activated extracellular signal-regulated kinases-1/2 (Erk-1/2), c-Jun amino-terminal kinase (JNK) and p38 mitogen activated protein kinase (MAPK). Specific inhibitors of MEK-1 (PD98059) and JNK (SP600125) inhibited the nicotine-induced uPAR expression, while the p38 MAPK inhibitor SB203580 did not. Expression vectors encoding dominant negative MEK-1 (pMCL-K97M) and JNK (TAM67) also prevented nicotine-induced uPAR promoter activity. The intracellular hydrogen peroxide (H2O2) content was increased by nicotine treatment. The antioxidant N-acetylcysteine prevented nicotine-activated production of reactive oxygen species (ROS) and uPAR expression. Furthermore, exogenous H2O2 increased uPAR mRNA expression. Deleted and site-directed mutagenesis demonstrated the involvement of the binding sites of transcription factor nuclear factor-kappaB (NF-?B) and activator protein (AP)-1 in the nicotine-induced uPAR expression. Studies with expression vectors encoding mutated NF-?B signaling molecules and AP-1 decoy confirmed that NF-?B and AP-1 were essential for the nicotine-stimulated uPAR expression. MAPK (Erk-1/2 and JNK) and ROS functioned as upstream signaling molecules in the activation of AP-1 and NF-?B, respectively. In addition, ECV304 endothelial cells treated with nicotine displayed markedly enhanced invasiveness, which was partially abrogated by uPAR neutralizing antibodies. The data indicate that nicotine induces uPAR expression via the MAPK/AP-1 and ROS/NF-?B signaling pathways and, in turn, stimulates invasiveness in human ECV304 endothelial cells. -- Highlights: ? Endothelial cells treated with nicotine displayed enhanced invasiveness. ? Nicotine induces uPAR expression and, in turn, stimulates invasiveness. ? MAPK/AP-1 and ROS/NF-?B signals are involved in nicotine-induced uPAR.

342

Structural mapping of the active site specificity determinants of human tissue-type plasminogen activator. Implications for the design of low molecular weight substrates and inhibitors.  

Science.gov (United States)

The recent structure determination of the catalytic domain of tissue-type plasminogen activator (tPA) suggested residue Arg174 could play a role in P3/P4 substrate specificity. Six synthetic chromogenic tPA substrates of the type R-Xaa-Gly-Arg-p-nitroanilide, in which R is an N-terminal protection group, were synthesized to test this property. Although changing the residue Xaa (in its L or D form) at position P3 from the hydrophobic Phe to an acidic residue, Asp or Glu, gave no improvement in catalytic efficiency, comparative analysis of the substrates indicated a preference for an acidic substituent occupying the S3 site when the S4 site contains a hydrophobic or basic moiety. The 2.9 A structure determination of the catalytic domain of human tPA in complex with the bis-benzamidine inhibitor 2, 7-bis-(4-amidinobenzylidene)-cycloheptan-1-one reveals a three-site interaction, salt bridge formation of the proximal amidino group of the inhibitor with Asp189 in the primary specificity pocket, extensive hydrophobic surface burial, and a weak electrostatic interaction between the distal amidino group of the inhibitor and two carbonyl oxygens of the protein. The latter position was previously occupied by the guanidino group of Arg174, which swings out to form the western edge of the S3 pocket. These data suggest that the side chain of Arg174 is flexible, and does not play a major role in the S4 specificity of tPA. On the other hand, this residue would modulate S3 specificity, and may be exploited to fine tune the specificity and selectivity of tPA substrates and inhibitors. PMID:9268299

Renatus, M; Bode, W; Huber, R; Stürzebecher, J; Prasa, D; Fischer, S; Kohnert, U; Stubbs, M T

1997-08-29

343

Factors affecting the plasmin-plasminogen system in milk obtained from three Greek dairy sheep breeds with major differences in milk production capacity.  

Science.gov (United States)

The purpose of this study was to evaluate the effect of breed, stage of lactation, and health status of the udder on the plasmin-plasminogen system in ovine milk. A total of 38 ewes were used from 3 breeds [Boutsiko (n = 12), Chios (n = 12), and a synthetic breed (50% Boutsiko, 25% Arta, and 25% Chios, n = 14)] with major differences in their genetic potential with respect to milk yield. Milk samples were collected every 2 wk throughout the lactation period and were analyzed for fat, protein, lactose, and somatic cell count (SCC). In addition, milk plasmin (PL), plasminogen (PG), and plasminogen activator (PA) activities were determined. The Chios breed had the greatest average daily milk yield, the synthetic breed had an intermediate milk yield, and ewes of the Boutsiko breed had the lowest milk yield. Milk samples obtained from the Boutsiko breed had similar PL and PA activities, compared with those obtained from the other 2 breeds. The ratio of PG:PL was less in milk samples from the Boutsiko breed compared with the other 2 breeds, indicative of an increased rate of conversion of PG to PL for this breed. There was no correlation between PL activity and daily milk yield in ewes from all 3 breeds. Activities of PL, PG, and PA were greater in ovine milk with elevated SCC (>300,000/mL) compared with activities in milk with low SCC (<300,000/mL). The ratio of PG:PL was less in the high-SCC group compared with the low-SCC group, which indicates an increased rate of conversion of PG to PL for the high-SCC group. There was a decrease in PG and PA activities as well as in the PG:PL ratio in late lactation milk (mo 5 to 6) when compared with early or mid lactation milk (mo 1 to 4). Thus, the PL-PG system is affected by breed, stage of lactation, and the health status of the udder. No relationship was found between PL activity and daily milk yield in the 3 Greek dairy sheep breeds. Plasmin is not a marker for gradual involution in the Greek sheep breeds studied. PMID:17582110

Theodorou, G; Kominakis, A; Rogdakis, E; Politis, I

2007-07-01

344

Fluorescence correlation spectroscopy and photon counting histogram on membrane proteins: functional dynamics of the glycosylphosphatidylinositol-anchored urokinase plasminogen activator receptor.  

Science.gov (United States)

The oligomerization of glycosylphosphatidylinositol-anchored proteins is thought to regulate their association with membrane microdomains, subcellular sorting, and activity. However, these mechanisms need to be comprehensively explored in living, unperturbed cells, without artificial clustering agents, and using fluorescent protein-tagged chimeras that are fully biologically active. We expressed in human embryo kidnay 293 (HEK293) cells a biologically active chimera of the urokinase plasminogen activator receptor (uPAR), the uPAR-mEGFP-GPI. We also produced HEK293/D2D3-mEGFP-GPI cells expressing the truncated form of the receptor, lacking biological activity. We studied the dynamics and oligomerization of the two proteins, combining fluorescence correlation spectroscopy (FCS) and photon counting histogram (PCH) analyses, and using subclones with homogenously low expression levels. Overall, the mobile fractions of the two proteins, constituted by monomers and dimers, had comparable diffusion coefficients. However, the diffusion coefficient decreased in monomer-enriched fractions only for the active receptor, suggesting that uPAR monomers might be preferentially engaged in multiprotein transmembrane signaling complexes. Our approach helps in limiting the alteration of the data due to out-of-focus effects and in minimizing the overestimation of the molecular brightness. In addition to a careful design of the cellular model, it gives reliable estimates of diffusion coefficients and oligomerization of GPI-anchored proteins, in steady-state conditions, at low expression levels, and in live, unperturbed cells. PMID:18601539

Malengo, Gabriele; Andolfo, Annapaola; Sidenius, Nicolai; Gratton, Enrico; Zamai, Moreno; Caiolfa, Valeria R

2008-01-01

345

Low doses of 3-aminobenzamide, a poly(ADP-ribose polymerase inhibitor, stimulate angiogenesis by regulating expression of urokinase type plasminogen activator and matrix metalloprotease 2  

Directory of Open Access Journals (Sweden)

Full Text Available Abstract Background Poly(ADP-Ribose polymerase (PARP activity has been demonstrated fundamental in many cellular processes, including DNA repair, cell proliferation and differentiation. In particular, PARP activity has been recently found to affect proliferation, migration, and tube formation of human umbilical vein endothelial cells. In recent times, PARP inhibitors have entered in clinical trials to potentiate cancer treatments by preventing DNA repair, but little is known about the effects performed by different drug concentrations on neoangiogenesis, an essential step in tumor growth. Methods Human umbilical vein endothelial cells were treated with 3 aminobenzamide (3ABA, a PARP inhibitor, and tested for several different cellular parameters. Results Here we present in vitro evidence that a low concentration of 3ABA (50 ?M, stimulates angiogenesis by decreasing fibrinolytic activity, carried out by urokinase-type plasminogen activator (uPA, and by enhancing matrix metalloprotease-2 (MMP-2 gelatinolytic activity, in fibroblast growth factor-2-stimulated endothelial cells. These unbalanced pathways modify in vitro angiogenic steps, inhibiting chemoinvasion and stimulating tubulogenic activity. Conclusions Our results suggest that the proangiogenic effect of low concentrations of 3ABA alerts on the efficacy of PARP inhibitors to potentiate anticancer therapy. Moreover, they indicate that endothelial chemoinvasion and tubulogenesis depend on distinct proteolytic pathways.

Caldini Riccardo

2011-05-01

346

Staurosporine induces ganglion cell differentiation in part by stimulating urokinase-type plasminogen activator expression and activation in the developing chick retina  

Energy Technology Data Exchange (ETDEWEB)

Highlights: Black-Right-Pointing-Pointer Staurosporine mediates stimulation of RGC differentiation in vitro cultured retinal neuroblasts. Black-Right-Pointing-Pointer Staurosporine mediates uPA activation during RGC differentiation in vitro. Black-Right-Pointing-Pointer Inhibition of uPA blocks the staurosporine mediated RGC differentiation both in vitro and in ovo. Black-Right-Pointing-Pointer Thus, uPA may play a role in the staurosporine-mediated stimulation of RGC differentiation. -- Abstract: Here, we investigated whether staurosporine-mediated urokinase-type plasminogen activator (uPA) activation is involved in retinal ganglion cell (RGC) differentiation. Retinal cells were isolated from developing chick retinas at embryonic day 6 (E6). Relatively few control cells grown in serum-free medium started to form processes by 12 h. In contrast, staurosporine-treated cells had processes within 3 h, and processes were evident at 8 h. Immunofluorescence staining showed that Tuj-1-positive cells with shorter neurites could be detected in control cultures at 18 h, whereas numerous Tuj-1 positive ganglion cells with longer neuritic extensions were seen in staurosporine-treated cultures. BrdU-positive proliferating cells were more numerous in control cultures than in staurosporine-treated cultures, and the BrdU staining was not detected in post-mitotic Tuj-1 positive ganglion cells. Western blotting of cell lysates showed that staurosporine induced high levels of the active form of uPA. The staurosporine-induced uPA signal was localized predominantly in the soma, neurites and axons of Tuj-1-positive ganglion cells. Amiloride, an inhibitor of uPA, markedly reduced staurosporine-induced Tuj-1 staining, neurite length, neurite number, and uPA staining versus controls. In developing retinas in ovo, amiloride administration remarkably reduced the staurosporine-induced uPA staining and RGC differentiation. Taken together, our in vitro and in vivo data collectively indicate that uPA plays a role in the staurosporine-mediated stimulation of RGC differentiation.

Kim, Yeoun-Hee [Department of Biology, College of Natural Sciences, Kyungpook National University, Daegu 702-701 (Korea, Republic of); Chang, Yongmin [Department of Molecular Medicine, Kyungpook National University College of Medicine, Kyungpook National University, 200 Dongduk-Ro Jung-Gu, Daegu 700-714 (Korea, Republic of); Jung, Jae-Chang, E-mail: jcjung@knu.ac.kr [Department of Biology, College of Natural Sciences, Kyungpook National University, Daegu 702-701 (Korea, Republic of)

2012-06-22

347

Macrophage inhibitory cytokine-1 (MIC-1) and subsequent urokinase-type plasminogen activator mediate cell death responses by ribotoxic anisomycin in HCT-116 colon cancer cells.  

Science.gov (United States)

Ribosome-inactivating stresses possess a potent regulatory activity against tumor cell progression. In this study, we demonstrated that macrophage inhibitory cytokine-1 (MIC-1) and its associated signals determined the colon cancer cell response to the chemical ribotoxic stress. The ribotoxic stress agent anisomycin-induced MIC-1 gene expression which was involved in the ribotoxin-induced apoptotic pathway. MIC-1 was also a critical inducer of apoptosis-related gene products such as activated urokine-type plasminogen activator (PLAU) and PLAU receptor (uPAR). When MIC-1 or PLAU action was repressed in the tumor cells, the chemical ribotoxic stress triggered a survival-related MAP kinase such as ERK. Mechanistically, gene expression of apoptosis-mediator MIC-1 was enhanced by activating transcription factor 3 (ATF-3) via the p38 MAP kinase signaling pathway. Moreover, both promoter activity and mRNA stability of MIC-1 gene were up-regulated by ribotoxic anisomycin via the p38 MAP kinase signaling pathway. In conclusion, ribotoxic anisomycin-induced MIC-1 expression via p38-ATF3 pathway and subsequent apoptosis while suppressing survival ERK signal in the colon cancer cells. The results of this study provide mechanistic insight into tumor cell decision for death or survival pathways in response to ribosome-disrupting stresses from chemotherapeutics. PMID:19540205

Yang, Hyun; Choi, Hye Jin; Park, Seong Hwan; Kim, Jong Sik; Moon, Yuseok

2009-11-01

348

Intracoronary thrombolysis with an acylated streptokinase-plasminogen activator (BRL 26921) in patients with acute myocardial infarction.  

Science.gov (United States)

The fibrinolytic efficacy and systemic effects on coagulation variables of intracoronary administration of an acylated streptokinase-plasminogen complex (BRL 26921) were assessed in 23 patients with an acute transmural myocardial infarction. The infarct vessel was totally occluded in 22 patients and subtotally stenosed in 1 patient. Reperfusion was achieved in a total of 17 patients (74%), in 2 patients with the use of a guide wire. Reperfusion time in those patients treated with BRL 26921 alone amounted to 42 +/- 37 minutes. Reocclusion occurred in two patients subsequently. Four patients died; in two of these, intracoronary thrombolysis was unsuccessful. Reptilase time increased from 13 +/- 3 to 49 +/- 31 seconds (p less than 0.001), fibrinogen levels decreased from 280 +/- 65 to 126 +/- 76 mg% (p less than 0.001). Factor V decreased from 96 +/- 11 to 53 +/- 26% (p less than 0.001), and factor VIII from 99 +/- 1 to 55 +/- 36% (p less than 0.001). Peripheral hyperplasminemia, defined as a reduction of fibrinogen (less than 100 mg%) with a reduction of factor V and VIII (less than 75%) simultaneously occurred in eight patients. Six (75%) of these 8 patients demonstrated reperfusion, whereas 9 (64%) of 14 patients without peripheral hyperplasminemia were also successfully reperfused. Bleeding complications occurred in two patients who demonstrated hyperplasminemia. Thus, effective intracoronary thrombolysis could be achieved with only minor effects on peripheral coagulation variables in the majority of patients. PMID:6429226

Kasper, W; Erbel, R; Meinertz, T; Drexler, M; Rückel, A; Pop, T; Prellwitz, W; Meyer, J

1984-08-01

349

Distribution of trichloroacetic acid precipitated radioactivity in rabbits after intravenous injection of 125I-labelled single-chain human recombinant urokinase-type plasminogen activator  

International Nuclear Information System (INIS)

Objective: To study tissue distribution profiles of trichloroacetic acid (TCA) precipitated radioactivity in rabbits following intravenous injection of 125I-labelled single-chain human recombinant urokinase-type plasminogen activator (125I-rh-sc-uPA). Methods: 125I-rh-su-uPA was prepared by Iodogen method and was purified by Saphacryl HR S-200. The radio-chemical purity was measured by reverse phase high performance liquid chromatography (RP-HPLC). Biological activity of labelled protein was measured by thrombolytic activity on fibrin plate. Results: The identification of 125I-rh-sc-uPA revealed that it was a 94.4% radio-chemically purified thrombolytic active protein. The AUC of radioactivity gradients in tissues and other specimens were as follows: kidney>urine>plasma>liver>spleen>adrenal gland>bone marrow>heart>urinary bladder>lungs>gonad>small intestinal wall>fat>lymph node>gall bladder>thymus gland>intestinal content>muscle>brain>feces in colon. Conclusion: The highest TCA precipitated radioactivity was found in plasma following I.V. of 125I-rh-u-PA, which might be favorable for the drug action. The main excretion route was urinary system

350

Ochratoxin A inhibits the production of tissue factor and plasminogen activator inhibitor-2 by human blood mononuclear cells: Another potential mechanism of immune-suppression  

International Nuclear Information System (INIS)

The mycotoxin ochratoxin A (OTA), an ubiquitous contaminant of food products endowed with a wide spectrum of toxicity, affects several functions of mononuclear leukocytes. Monocytes/macrophages play a major role in fibrin accumulation associated with immune-inflammatory processes through the production of tissue factor (TF) and plasminogen activator inhibitor 2 (PAI-2). We studied the effect of OTA on TF and PAI-2 production by human blood mononuclear cells (MNC). The cells were incubated for 3 or 18 h at 37 deg. C with non toxic OTA concentrations in the absence and in the presence of lipopolysaccharide (LPS) or other inflammatory agents. TF activity was measured by a one-stage clotting test. Antigen assays were performed by specific ELISAs in cell extracts or conditioned media and specific mRNAs were assessed by RT-PCR. OTA had no direct effect on TF and PAI-2 production by MNC. However, OTA caused a dose-dependent reduction in LPS-induced TF (activity, antigen and mRNA) and PAI-2 (antigen and mRNA) production with > 85% inhibition at 1 ?g/ml. Similar results were obtained when monocyte-enriched preparations were used instead of MNC. TF production was also impaired by OTA (1 ?g/ml) when MNC were stimulated with phorbol myristate acetate (98% inhibition), IL-1? (83%) or TNF-? (62%). The inhibition of TF and PAI-2 induction might represent a hitherto unrecognized mechanism whereby OTA exerts immunosuppressant activity

351

Modulation of ovine SBD-1 expression by 17beta-estradiol in ovine oviduct epithelial cells  

Directory of Open Access Journals (Sweden)

Full Text Available Abstract Background Mucosal epithelia, including those of the oviduct, secrete antimicrobial innate immune molecules (AIIMS. These have bactericidal/bacteriostatic functions against a variety of pathogens. Among the AIIMs, sheep ?-defensin-1 (SBD-1 is one of the most potent. Even though the SBD-1 is an important AIIM and it is regulated closely by estrogenic hormone, the regulation mechanism of 17?-estradiol has not been clearly established. We investigated the effects of E2 and agonist or inhibitor on ovine oviduct epithelial cells in regard to SBD-1 expression using reverse transcription quantitative PCR (RT-qPCR. In addition, three different pathways were inhibited separately or simultaneously to confirm the effect of different inhibitors in the regulation mechanism. Results 17beta-estradiol (E2 induced release of SBD-1 in ovine oviduct epithelial cells. SBD-1 expression was mediated through G-protein-coupled receptor 30 (GPR30 and Estrogen Receptors (ERs activation in ovine oviduct epithelial cell. Inhibition of gene expression of protein kinase A (PKA, protein kinase C (PKC, and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-?B led to a decreased SBD-1 expression. Conclusions Taken together, E2-induced up-regulation of SBD-1 expressions were GPR30-dependent during prophase and ERs-dependent during later-stage in ovine oviduct epithelial cells, and we assume that the effect was completed by the PKA, PKC, and NF-?B pathways simultaneous.

Wen Shiyong

2012-08-01

352

Increased soluble urokinase plasminogen activator receptor (suPAR) is associated with thrombosis and inhibition of plasmin generation in paroxysmal nocturnal hemoglobinuria (PNH) patients.  

Science.gov (United States)

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired genetic disorder of the bone marrow that produces intravascular hemolysis, proclivity to venous thrombosis, and hematopoietic failure. Mutation in the PIG-A gene of a hematopoietic stem cell abrogates synthesis of glycosylphosphoinositol (GPI) anchors and expression of all GPI-anchored proteins on the surface of progeny erythrocytes, leukocytes, and platelets. Urokinase plasminogen activator receptor (uPAR), a GPI-linked protein expressed on neutrophils, mediates endogenous thrombolysis through a urokinase-dependent mechanism. Here we show that membrane GPI-anchored uPAR is decreased or absent on granulocytes and platelets of patients with PNH, while soluble uPAR (suPAR) levels are increased in patients' plasma. Serum suPAR concentrations correlated with the number of GPI-negative neutrophils and were highest in patients who later develop thrombosis. In vitro, suPAR is released from PNH hematopoietic cells and from platelets upon activation, suggesting that these cells are the probable source of plasma suPAR in the absence of GPI anchor synthesis and trafficking of uPAR to the cell membrane. In vitro, the addition of recombinant suPAR results in a dose-dependent decrease in the activity of single-chain urokinase. We hypothesized that suPAR, prevents the interaction of urokinase with membrane-anchored uPAR on residual normal cells. PMID:18954937

Sloand, Elaine M; Pfannes, Loretta; Scheinberg, Phillip; More, Kenneth; Wu, Colin O; Horne, McDonald; Young, Neal S

2008-12-01

353

Dissecting the effect of RNA aptamer binding on the dynamics of plasminogen activator inhibitor 1 using hydrogen/deuterium exchange mass spectrometry  

DEFF Research Database (Denmark)

RNA aptamers, selected from large synthetic libraries, are attracting increasing interest as protein ligands, with potential uses as prototype pharmaceuticals, conformational probes, and reagents for specific quantification of protein levels in biological samples. Very little is known, however, about their effects on protein conformation and dynamics. We have employed hydrogen/deuterium exchange (HDX) mass spectrometry to study the effect of RNA aptamers on the structural flexibility of the serpin plasminogen activator inhibitor-1 (PAI-1). The aptamers have characteristic effects on the biochemical properties of PAI-1. In particular, they are potent inhibitors of the structural transition of PAI-1 from the active state to the inactive, so-called latent state. This transition is one of the largest conformational changes of a folded protein domain without covalent modification. Binding of the aptamers to PAI-1 is associated with substantial and widespread protection against deuterium uptake in PAI-1. The aptamers induce protection against exchange with the solvent both in the protein-aptamer interface as well as in other specific areas. Interestingly, the aptamers induce substantial protection against exchange in ?-helices B, C and I. This observation substantiates the relevance of structural instability in this region for transition to the latent state and argues for involvement of flexibility in regions not commonly associated with regulation of latency transition in serpins.

Trelle, Morten B; Dupont, Daniel Miotto

2014-01-01

354

Dissecting the Effect of RNA Aptamer Binding on the Dynamics of Plasminogen Activator Inhibitor 1 Using Hydrogen/Deuterium Exchange Mass Spectrometry  

DEFF Research Database (Denmark)

RNA aptamers, selected from large synthetic libraries, are attracting increasing interest as protein ligands, with potential uses as prototype pharmaceuticals, conformational probes, and reagents for specific quantification of protein levels in biological samples. Very little is known, however, about their effects on protein conformation and dynamics. We have employed hydrogen/deuterium exchange (HDX) mass spectrometry to study the effect of RNA aptamers on the structural flexibility of the serpin plasminogen activator inhibitor-1 (PAI-1). The aptamers have characteristic effects on the biochemical properties of PAI-1. In particular, they are potent inhibitors of the structural transition of PAI-1 from the active state to the inactive, so-called latent state. This transition is one of the largest conformational changes of a folded protein domain without covalent modification. Binding of the aptamers to PAI-1 is associated with substantial and widespread protection against deuterium uptake in PAI-1. The aptamers induce protection against exchange with the solvent both in the protein-aptamer interface as well as in other specific areas. Interestingly, the aptamers induce substantial protection against exchange in ?-helices B, C and I. This observation substantiates the relevance of structural instability in this region for transition to the latent state and argues for involvement of flexibility in regions not commonly associated with regulation of latency transition in serpins.

Trelle, Morten B; Dupont, Daniel Miotto

2014-01-01

355

Tissue-type plasminogen activator–mediated shedding of astrocytic low-density lipoprotein receptor–related protein increases the permeability of the neurovascular unit  

Science.gov (United States)

The low-density lipoprotein receptor–related protein (LRP) is a member of the LDL receptor gene family that binds several ligands, including tissue-type plasminogen activator (tPA). tPA is found in blood, where its primary function is as a thrombolytic enzyme, and in the central nervous system where it mediates events associated with cell death. Cerebral ischemia induces changes in the neurovascular unit (NVU) that result in brain edema. We investigated whether the interaction between tPA and LRP plays a role in the regulation of the permeability of the NVU during cerebral ischemia. We found that the ischemic insult induces shedding of LRP's ectodomain from perivascular astrocytes into the basement membrane. This event associates with the detachment of astrocytic end-feet processes and the formation of areas of perivascular edema. The shedding of LRP's ectodomain is significantly decreased in tPA deficient (tPA?/?) mice, is increased by incubation with tPA, and is inhibited by the receptor-associated protein (RAP). Furthermore, treatment with either RAP or anti-LRP IgG results in a faster recovery of motor activity and protection of the integrity of the NVU following middle cerebral artery occlusion (MCAO). Together, these results implicate tPA/LRP interactions as key regulators of the integrity of the NVU. PMID:17170123

Polavarapu, Rohini; Gongora, Maria Carolina; Yi, Hong; Ranganthan, Sripriya; Lawrence, Daniel A.; Strickland, Dudley

2007-01-01

356

EFFECTS ON HUMAN PLASMINOGEN CONFORMATION AND ACTIVATION RATE CAUSED BY INTERACTION WITH VEK-30, A PEPTIDE DERIVED FROM THE GROUP A STREPTOCOCCAL M-LIKE PROTEIN (PAM)*  

Science.gov (United States)

In vertebrates, fibrinolysis is primarily carried out by the serine protease plasmin (Pm), which is derived from activation of the zymogen precursor, plasminogen (Pg). One of the most distinctive features of Pg/Pm is the presence of five homologous kringle (K) domains. These structural elements possess conserved Lys-binding sites (LBS) that facilitate interactions with substrates, activators, inhibitors and receptors. In human Pg (hPg), K2 displays weak Lys affinity, however the LBS of this domain has been implicated in an atypical interaction with the N-terminal region of a bacterial surface protein known as PAM (Pg-binding group A streptococcal M-like protein). A direct correlation has been established between invasiveness of group A streptococci and their ability to bind Pg. It has been previously demonstrated that a 30-residue internal peptide (VEK-30) from the N-terminal region of PAM competitively inhibits binding of the full-length parent protein to Pg. We have attempted to determine the effects of this ligand-protein interaction on the regulation of Pg zymogen activation and conformation. Our results show minimal effects on the sedimentation velocity coefficients (S°20,w) of Pg when associated to VEK-30 and a direct relationship between the concentration of VEK-30 or PAM and the activation rate of Pg. These results are in contrast with the major conformational changes elicited by small-molecule activators of Pg, and point towards a novel mechanism of Pg activation that may underlie group A streptococcal (GAS) virulence. PMID:20152941

Figuera-Losada, Mariana; Ranson, Marie; Sanderson-Smith, Martina L.; Walker, Mark J.; Castellino, Francis J.; Prorok, Mary

2010-01-01

357

Circulating intact and cleaved forms of the urokinase-type plasminogen activator receptor : Biological variation, reference intervals and clinical useful cut-points  

DEFF Research Database (Denmark)

BACKGROUND: High levels of circulating forms of the urokinase-type plasminogen activator receptor (uPAR) are significantly associated to poor prognosis in cancer patients. Our aim was to determine biological variations and reference intervals of the uPAR forms in blood, and in addition, to test the clinical relevance of using these as cut-points in colorectal cancer (CRC) prognosis. METHODS: uPAR forms were measured in citrated and EDTA plasma samples using time-resolved fluorescence immunoassays. Diurnal, intra- and inter-individual variations were assessed in plasma samples from cohorts of healthy individuals. Reference intervals were determined in plasma from healthy individuals randomly selected from a Danish multi-center cross-sectional study. A cohort of CRC patients was selected from the same cross-sectional study. RESULTS: The reference intervals showed a slight increase with age and women had~20% higher levels. The intra- and inter-individual variations were ~10 % and ~20-30 %, respectively and themeasured levels of the uPAR forms were within the determined 95% reference intervals. No diurnal variation was found. Applying the normal upper limit of the reference intervals as cut-point for dichotomizing CRC patients revealed significantly decreased overall survival of patients with levels above this cut-point of any uPAR form. CONCLUSIONS: The reference intervals for the different uPAR forms are valid and the upper normal limits are clinically relevant cut-points for CRC prognosis.

SØrensen, Tine Thurison; Christensen, Ib J

2014-01-01

358

Plasminogen activator inhibitor-I-related regulation of procollagen I (?1 and ?2) by antitransforming growth factor-?1 treatment during radiation-impaired wound healing  

International Nuclear Information System (INIS)

Purpose: Plasminogen activator inhibitor (PAI)-1 mediates transforming growth factor-?1 (TGF-?1)-related signaling by stimulating collagen Type I synthesis in radiation-impaired wound healing. The regulation of ?(I)-procollagen is contradictory in fibroblasts of different fibrotic lesions. It is not known whether anti-TGF-?1 treatment specifically inhibits ?(I)-procollagen synthesis. We used an experimental wound healing study to address anti-TGF-?1-associated influence on ?(I)-procollagen synthesis. Methods and Materials: A free flap was transplanted into the preirradiated (40 Gy) or nonirradiated neck region of Wistar rats: Group 1 (n = 8) surgery alone; Group 2 (n = 14) irradiation and surgery; Group 3 (n = 8) irradiation and surgery and anti-TGF-?1 treatment. On the 14th postoperative day, skin samples were processed for fibroblast culture, in situ hybridization for TGF-?1, immunohistochemistry, and immunoblotting for PAI-1, ?1/?2(I)-procollagen. Results: Anti-TGF-?1 significantly reduced TGF-?1 mRNA (p 1 treatment in vivo significantly reduced ?1(I)-procollagen protein (p 2(I)-procollagen expression. Conclusion: These results emphasize anti-TGF-?11 treatment to reduce radiation-induced fibrosis by decreasing ?1(I)-procollagen synthesis in vivo. ?1(I)-procollagen and ?2(I)-procollagen might be differentially regulated by anti-TGF-?1 treatment. Increased TGF-? signaling in irradiated skin fibroblasts seemed to be reversible, as shown by a reduction in PAI-1 expression after anti-TGF-?1 treatment

359

Expression of matrix metalloproteinase (MMP)-1, MMP-2, MMP-9, cathepsin B, and urokinase plasminogen activator in non-skull base chordoma.  

Science.gov (United States)

We analyzed the expression of proteases and the clinicopathologic significance in non-skull base chordoma (NSBC). By using immunohistochemical techniques, we studied the expression of matrix metalloproteinase (MMP)-1, MMP-2, MMP-9, cathepsin B (CatB), and urokinase plasminogen activator (uPA) in 29 NSBCs and compared these data with clinicopathologic parameters and the expression of cell differentiation markers. Expression of MMP-1 (P = .092), MMP-2 (P = .041), and CatB (P = .058) was associated with nuclear pleomorphism, a previously described adverse prognostic indicator. Expression of cytokeratin 8 correlated with that of MMP-1 (P = .005), MMP-2 (P = .002), and uPA (P = .032). Patients with higher MMP-2 expression had a poorer prognosis than those with lower MMP-2 expression (P = .013). We believe that NSBCs with nuclear pleomorphism or stronger epithelial character have a higher invasive ability than those without. In addition, high MMP-2 expression was an indicator of an unfavorable clinical outcome in NSBC. PMID:15539385

Naka, Takahiko; Boltze, Carsten; Kuester, Doerthe; Schulz, Torss-Oliver; Samii, Amir; Herold, Christian; Ostertag, Helmut; Roessner, Albert

2004-12-01

360

Prevalence of coagulation factor XIII and plasminogen activator inhibitor-1 gene polymorphisms among Egyptian women suffering from unexplained primary recurrent miscarriage.  

Science.gov (United States)

Recurrent miscarriage (RM) is an obstetric challenge. Polymorphisms of factor XIII (FXIII) and plasminogen activator inhibitor-1 (PAI-1) may cause an imbalance between coagulation and fibrinolysis that can end in RM. The aim of the work was to determine the prevalence of FXIII Val34Leu and PAI-1 4G/5G gene polymorphisms in Egyptian women presenting with unexplained primary first trimester RM. Genotyping of 120 unexplained primary first trimester RM patients and 130 healthy controls by polymerase chain reaction (PCR) amplification of target genes followed by the allele-specific restriction enzyme digestion (RFLP technique). Among the cases, 67.5% of individuals had wild-type FXIII; 21.7% were heterozygous and 10.8% were homozygous for the FXIII Val34Leu polymorphism. Among controls, the proportions were 89.2%, 8.5% and 2.3% respectively. In addition, comparison between the two groups regarding Leu and 4G allele frequencies showed statistically significant differences (P values=0.0001 and 0.027 respectively). RM is more frequent in women with combined polymorphisms than in women with a single gene polymorphism (RR=3.91; OR=4.51; 95% CI=1.79-11.38; P=0.002). FXIII Val34Leu and PAI-1 4G/5G polymorphisms are prevalent in Egyptian women, with unexplained primary first trimester RM and combined polymorphisms statistically increasing the risk. PMID:24702949

Elmahgoub, Iman Rifaat; Afify, Reham Abdelaleem; Abdel Aal, Asmaa Ahmed; El-Sherbiny, Walid Sayed

2014-06-01

361

Gene Therapy to Promote Thromboresistance: Local Overexpression of Tissue Plasminogen Activator to Prevent Arterial Thrombosis in an in vivo Rabbit Model  

Science.gov (United States)

Tissue-type plasminogen activator (tPA) catalyzes the rate-limiting initial step in the fibrinolytic cascade. Systemic infusion of tPA has become the standard of care for acute myocardial infarction. However, even the relatively short-duration protocols currently employed have encountered significant hemorrhagic complications, as well as complications from rebound thrombosis. Gene therapy offers a method of local high-level tPA expression over a prolonged time period to avoid both systemic hemorrhage and local rebound thrombosis. To examine the impact of local tPA overexpression, an adenoviral vector expressing tPA was created. The construct was characterized functionally in vitro, and the function of the vector was confirmed in vivo by delivery to the rabbit common femoral artery. Systemic coagulation parameters were not perturbed at any of the doses examined. The impact of local overexpression of tPA on in vivo thrombus formation was examined subsequently in a stasis/injury model of arterial thrombosis. The construct effectively prevented arterial thrombosis in treated animals, whereas viral and nonviral controls typically developed occluding thrombi. This construct thus offers a viable technique for promoting a locally thromboresistant small-caliber artery.

Waugh, J. M.; Kattash, M.; Li, J.; Yuksel, E.; Kuo, M. D.; Lussier, M.; Weinfeld, A. B.; Saxena, R.; Rabinovsky, E. D.; Thung, S.; Woo, S. L. C.; Shenaq, S. M.

1999-02-01

362

Interrelations between blood-brain barrier permeability and matrix metalloproteinases are differently affected by tissue plasminogen activator and hyperoxia in a rat model of embolic stroke  

Directory of Open Access Journals (Sweden)

Full Text Available Abstract Background In ischemic stroke, blood-brain barrier (BBB regulations, typically involving matrix metalloproteinases (MMPs and inhibitors (TIMPs as mediators, became interesting since tissue plasminogen activator (tPA-related BBB breakdown with risk of secondary hemorrhage was considered to involve these mediators too. Despite high clinical relevance, detailed interactions are purely understood. After a pilot study addressing hyperoxia as potential neuroprotective co-treatment to tPA, we analyzed interrelations between BBB permeability (BBB-P, MMPs and TIMPs. Findings Rats underwent embolic middle cerebral artery occlusion (eMCAO and treatment with normobaric (NBO or hyperbaric oxygen (HBO, tPA, tPA+HBO, or no treatment. BBB-P was assessed by intravenously applied FITC-albumin at 4 or 24 hours. MMP-2/-9 and TIMP-1/-2 serum levels were determined at 5 or 25 hours. Time point-corrected partial correlations were used to explore interrelations of BBB-P in ischemic regions (extra-/intravasal FITC-albumin ratio and related serum markers. BBB-P correlated positively with MMP-2 and MMP-9 in controls, whereas hyperoxia led to an inverse association, most pronounced for HBO/MMP-9 (r = -0.606; P Conclusions HBO was found to reverse the positively directed interrelation of BBB-P and MMPs after eMCAO, but this effect failed to sustain in the expected amount when HBO and tPA were given simultaneously.

Michalski Dominik

2012-01-01

363

Prospective evaluation of {sup 99m}Tc-labelled recombinant tissue plasminogen activator in the detection of lower limb deep venous thrombosis in asymptomatic high risk patients  

Energy Technology Data Exchange (ETDEWEB)

Full text: The purpose of this study was to compare the accuracy of this new technique against the accepted ``gold standard`` of contrast venography in 36 asymptomatic high risk patients. A kit formulation has been devised in which {sup 99m}Tc is labelled to rt- PA where the plasminogen binding site has been permanently inhibited but the fibrin binding site remained active. Kit preparation prior to injection takes five minutes. Scintigraphic imaging is performed at four hours post-injection (10 min/scan for thighs and calves). The results of scintigraphic imaging were then compared to those of contrast venography. 36 consecutive asymptomatic high risk patients (17 total hip, 19 total knee replacements) underwent both a contrast venogram on the operated leg and scintigraphic scan 7-10 days following operation. For the purpose of this analysis, each venogram was divided into a proximal and a distal segment. Venograms were interpreted as being positive, negative or uninterpretable in each segment. Similar analysis of the scintigraphic scans was performed except that all segments were considered to be of diagnostic quality. Fifty seven segments were able to be analysed. Of the 13 thrombosed segments (1 proximal, 12 calf ), 12 had positive scans; in the 44 non-thrombosed segments, 40 had negative scans. In detecting lower limb DVT, scanning had a sensitivity of 92% and a specificity of 91%. Scintigraphic scanning with inhibited {sup 99m}Tc rt-PA permits accurate detection of thrombus in high risk patients.

Butler, S.P.; Rahman, T.; Boyd, S.J.; Parkes, S.L.; Quinn, R.J. [St George Hospital, Kogarah, NSW (Australia)

1998-03-01

364

A milk protein gene promoter directs the expression of human tissue plasminogen activator cDNA to the mammary gland in transgenic mice  

Energy Technology Data Exchange (ETDEWEB)

Whey acidic protein (WAP) is a major whey protein in mouse milk. Its gene is expressed in the lactating mammary gland and is inducible by steroid and peptide hormones. A series of transgenic mice containing a hybrid gene in which human tissue plasminogen activator (tPA) cDNA is under the control of the murine WAP gene promoter had previously been generated. In this study, 21 tissues from lactating and virgin transgenic female mice containing the WAP-tPA hybrid gene were screened for the distribution of murine WAP and human tPA transcripts. Like the endogenous WAP RNA, WAP-tPA RNA was expressed predominantly in mammary gland tissue and appeared to be inducible by lactation. Whereas WAP transcripts were not detected in 22 tissues of virgin mice, low levels of WAP-tPA RNA, which were not modulated during lactation, were found in tongue, kidney, and sublingual gland. These studies demonstrate that the WAP gene promoter can target the expression of a transgene to the mammary gland and that this expression is inducible during lactation.

Pittius, C.W.; Hennighausen, L.; Lee, E.; Westphal, H.; Nicols, E.; Vitale, J.; Gordon, K. (National Institutes of Health, Bethesda, MD (USA))

1988-08-01

365

Coagulation alterations due to local fibrinolytic therapy with recombinant tissue-type plasminogen activator (rt-PA) in patients with peripheral arterial occlusive disease  

International Nuclear Information System (INIS)

Purpose. To determine the systemic effects of local fibrinolytic therapy with low-dose recombinant tissue-type plasminogen activator (rt-PA). Methods. Ten patients received intrathrombal infusion of 20 mg rt-PA and heparin for local thrombolysis and had subsequent percutaneous transluminal angioplasty (PTA). Eight controls underwent PTA and received heparin alone. We measured t-PA, D-Dimer, and fibrinogen levels before, directly after, and 20, 40, and 60 min and 24 hr after therapy. Results. In the thrombolysis group the t-PA level peaked immediately after infusion and then declined within 1 hr. D-Dimer increased and remained elevated, whereas in the control group only t-PA levels increased, and only after 24 hr. Fibrinogen remained within the normal range in both groups. Eight of ten patients in the thrombolysis group and seven of eight with PTA had clinical improvement after the procedure. Conclusions. The increase in D-Dimer in the rt-PA group indicates a good local fibrinolytic effect. The fact that fibrinogen levels remained unchanged indicates that there is a lack of systemic fibrinogenolysis

366

TISSUE INHIBITOR OF METALLOPROTEINASE 1, MATRIX METALLOPROTEINASE 9, ALPHA-1 ANTITRYPSIN, METALLOTHIONEIN AND UROKINASE TYPE PLASMINOGEN ACTIVATOR RECEPTOR IN SKIN BIOPSIES FROM PATIENTS AFFECTED BY AUTOIMMUNE BLISTERING DISEASES  

Directory of Open Access Journals (Sweden)

Full Text Available Introduction: Proteinases and proteinase inhibitors have been described to play a role in autoimmune skin blistering diseases. We studied skin lesional biopsies from patients affected by several autoimmune skin blistering diseases for proteinases and proteinase inhibitors. Methods: We utilized immunohistochemistry to evaluate biopsies for alpha-1-antitrypsin, human matrix metalloproteinase 9 (MMP9, human tissue inhibitor of metalloproteinases 1 (TIMP-1, metallothionein and urokinase type plasminogen activator receptor (uPAR. We tested 30 patients affected by endemic pemphigus, 30 controls from the endemic area, and 15 normal controls. We also tested 30 biopsies from patients with bullous pemphigoid (BP, 20 with pemphigus vulgaris (PV, 8 with pemphigus foliaceus, and 14 with dermatitis herpetiformis (DH. Results: Contrary to findings in the current literature, most autoimmune skin blistering disease biopsies were negative for uPAR and MMP9. Only some chronic patients with El Bagre-EPF were positive to MMP9 in the dermis, in proximity to telocytes. TIMP-1 and metallothionein were positive in half of the biopsies from BP patients at the basement membrane of the skin, within several skin appendices, in areas of dermal blood vessel inflammation and within dermal mesenchymal-epithelial cell junctions.

Ana Maria Abreu Velez

2013-07-01

367

Plasminogen activator inhibitor type I may contribute to transient, non-specific changes in immunity in the subacute phase of murine tuberculosis.  

Science.gov (United States)

Tuberculosis, caused by Mycobacterium (M.) tuberculosis, is a devastating infectious disease causing many deaths worldwide. Non-specific host defense mechanisms such as the coagulation and fibrinolytic system may give insight in possible new therapeutic targets. Plasminogen activator inhibitor type-1 (PAI-1), an important regulator of inflammation and fibrinolysis, might be of interest as tuberculosis patients have elevated plasma levels of PAI-1. In this study we set out to investigate the role of PAI-1 during tuberculosis in vivo. Wildtype (WT) and PAI-1 deficient (PAI-1?/?) mice were intranasally infected with M. tuberculosis H37rv and sacrificed after 2, 5 and 29 weeks. Five weeks post-infection, bacterial loads in lungs of PAI-1?/? mice were significantly higher compared to WT mice, while no differences were seen 2 and 29 weeks post-infection. At two weeks post-infection increased influx of macrophages and lymphocytes was observed. PAI-1 deficiency was associated with a reduced cytokine response in the lungs; however, upon stimulation with tuberculin purified protein derivative (PPD), PAI-1?/? splenocytes released increased levels of IFN-? compared to WT. No clear differences were found between PAI-1?/? and WT mice at 29 weeks after infection. In conclusion, these data suggest that PAI-1 contributes to transient, non-specific changes in immunity during the early phase of murine tuberculosis. PMID:22484384

Kager, Liesbeth M; van der Windt, Gerritje J W; Wieland, Catharina W; Florquin, Sandrine; van 't Veer, Cornelis; van der Poll, Tom

2012-08-01

368

Prognostic value of plasma soluble urokinase plasminogen activator receptor (suPAR) in Danish patients with recurrent epithelial ovarian cancer (REOC).  

DEFF Research Database (Denmark)

The level of the soluble urokinase plasminogen activator receptor (suPAR) is elevated in tumour tissue from several types of cancer. This is the first study aiming to predict the prognosis for survival by the use of a pre-chemotherapeutic plasma suPAR value in 71 patients with recurrent epithelial ovarian cancer (REOC). For determination of suPAR, pre-chemotherapeutic blood samples from the patients with REOC were processed into plasma (EDTA) within one working day from venipuncture. The plasma suPAR level is not correlated with performance status (p=0.41), FIGO stage (p=0.09), treatment-free interval (TFI) of 12 months (p=0.26), site of recurrence (peritoneum, p=0.50 or pelvis, p=0.44), age (p=0.43), or serum CA125 (p=0.09). Univariate as well as multivariate analyses cannot demonstrate that high pre-chemotherapeutic levels of plasma suPAR (p=0.22, p=0.80) are associated with shorter survival of REOC patients. Multivariate analysis showed that only TFI of 12 months (p=0.001) and performance score status of 2 (p=0.02) were independent prognostic factors. Our study indicates that pre-chemotherapeutic measurement of plasma suPAR level in REOC patients may not be useful to identify a subgroup of patients with poor prognosis.

Begum, Farah Diba; HØgdall, Estrid V S

2006-01-01

369

Usefulness of Soluble Urokinase Plasminogen Activator Receptor to Predict Repeat Myocardial Infarction and Mortality in Patients With ST-Segment Elevation Myocardial Infarction Undergoing Primary Percutaneous Intervention  

DEFF Research Database (Denmark)

The plasma level of the inflammatory biomarker soluble urokinase plasminogen activator receptor (suPAR) is an independent predictor of cardiovascular disease and all-cause mortality in healthy subjects. The prognostic capability of suPAR, its temporal course, and its relation to plasma C-reactive protein (CRP) in patients with ST-segment elevation myocardial infarction undergoing primary percutaneous intervention (PCI) is unknown. Therefore, the plasma suPAR and CRP levels were measured in 296 consecutive patients with ST-segment elevation myocardial infarction admitted for primary PCI at baseline and every 6 to 8 hours thereafter until the cardiac biomarker levels had peaked. The end points were all-cause mortality and fatal or nonfatal recurrent myocardial infarction (MI). During a median follow-up period of 5.75 years, 69 deaths and 48 nonfatal and 14 fatal recurrent MIs occurred. All-cause mortality increased significantly from 8.1% to 41.5% across increasing quartiles of suPAR levels at the end of follow-up (log-rank p

Lyngbæk, Stig; Marott, Jacob L

2012-01-01

370

Identification of an ovine atadenovirus gene whose product activates the viral E2 promoter: possible involvement of E2F-1  

International Nuclear Information System (INIS)

Activation of the adenoviral E2 promoter is an early step in adenovirus gene expression. For members of the mast- and aviadenoviruses, this requires induction of the cellular transcription factor E2F by virally encoded gene products such as E1A, E4orf6/7 and orf22/GAM-1. The newly recognized genus atadenovirus, of which the ovine isolate OAdV is the prototype, lacks any sequence homology to those genes. To find a possible link between E2 promoter activation and OAdV gene expression, we utilized a screening method to search for genes within the OAdV genome that were capable of stimulating the viral E2 promoter. One such gene, E43, was identified within the proposed E4 region toward the right-hand end of the OAdV genome. The E43 gene product was also found to be capable of stimulating E2F-1-dependent gene expression. A closer inspection of the E2 promoter revealed the presence of a non-palindromic E2F binding site within the OAdV E2 promoter. Mutation of this site markedly reduced both E2F-1- and E43-dependent promoter activation. Moreover, a direct protein-protein interaction of the E43 gene product with E2F, but not with the retinoblastoma protein pRb, suggested a possible cooperation between these two proteins in activating the E2 promoter. The importance of the E43 gene product for virus replication is also underlined by the finding that an OAdV recombinant with a functionally inactivated E43 gene showed severely inhibited virus growthirus growth

371

Overexpression of Hsp27 in a human melanoma cell line: regulation of E-cadherin, MUC18/MCAM, and plasminogen activator (PA) system.  

Science.gov (United States)

Hsp27 is considered a potential marker for cell differentiation in diverse tissues. Several aspects linked to the differentiation process and to the transition from high to low metastatic potential were analyzed in melanoma cells transfected with Hsp27. E-cadherin plays a central role in cell differentiation, migration, and normal development. Loss of expression or function of E-cadherin has been documented in a variety of human malignancies. We observed by fluorescence-activated cell sorter (FACS) as well as immunofluorescence (IF) analysis a pronounced expression of E-cadherin in Hsp27-transfected A375 melanoma cells compared with control melanoma cells. The expression of the adhesion molecule MUC18/MCAM correlates directly with the metastatic potential of melanoma cells. In contrast to wild-type and neotransfected melanoma cells, in Hsp27-transfected cells the expression of MUC18/MCAM could not be detected by FACS and IF analysis. The plasminogen activator (PA) system plays a central role in mediating extracellular proteolysis and also in nonproteolytic events such as cell adhesion, migration, and transmembrane signaling. Hsp27 transfectants revealed elevated messenger ribonucleic acid expression of the urokinase-type PA (uPA) and its inhibitor, PA inhibitor type 1, which might indicate a neutralization effect of the proteolytic activity of uPA. Control cells failed to express both these molecules. The influence of Hsp27 expression on uPA activity and the involvement of E-cadherin could be demonstrated by use of anti-E-cadherin-blocking antibody. Our data provide evidence for an inhibitory-regulatory role of Hsp27 in tumor progression as found in our system. PMID:14984058

Aldrian, Silke; Kindas-Mügge, Ingela; Trautinger, Franz; Fröhlich, Ilse; Gsur, Andrea; Herbacek, Irene; Berger, Walter; Micksche, Michael

2003-01-01

372

Spatio-temporal course of macrophage-like cell accumulation after experimental embolic stroke depending on treatment with tissue plasminogen activator and its combination with hyperbaric oxygenation  

Directory of Open Access Journals (Sweden)

Full Text Available Inflammation following ischaemic stroke attracts high priority in current research, particularly using human-like models and long-term observation periods considering translational aspects. The present study aimed on the spatio-temporal course of macrophage-like cell accumulation after experimental thromboembolic stroke and addressed microglial and astroglial reactions in the ischaemic border zone. Further, effects of tissue plasminogen activator (tPA as currently best treatment for stroke and the potentially neuroprotective co-administration of hyperbaric oxygen (HBO were investigated. Rats underwent middle cerebral artery occlusion and were assigned to control, tPA or tPA+HBO. Twenty-four hours, 7, 14 and 28 days were determined as observation time points. The accumulation of macrophage-like cells was semiquantitatively assessed by CD68 staining in the ischaemic area and ischaemic border zone, and linked to the clinical course. CD11b, ionized calcium binding adaptor molecule 1 (Iba, glial fibrillary acidic protein (GFAP and Neuronal Nuclei (NeuN were applied to reveal delayed glial and neuronal alterations. In all groups, the accumulation of macrophage-like cells increased distinctly from 24 hours to 7 days post ischaemia. tPA+HBO tended to decrease macrophage-like cell accumulation at day 14 and 28. Overall, a trend towards an association of increased accumulation and pronounced reduction of the neurological deficit was found. Concerning delayed inflammatory reactions, an activation of microglia and astrocytes with co-occurring neuronal loss was observed on day 28. Thereby, astrogliosis was found circularly in contrast to microglial activation directly in the ischaemic area. This study supports previous data on long-lasting inflammatory processes following experimental stroke, and additionally provides region-specific details on glial reactions. The tendency towards a decreasing macrophage-like cell accumulation after tPA+HBO needs to be discussed critically since neuroprotective properties were recently ascribed to long-term inflammatory processes.

D. Michalski

2012-05-01

373

N-Acetyl-seryl-aspartyl-lysyl-proline inhibits TGF-beta-mediated plasminogen activator inhibitor-1 expression via inhibition of Smad pathway in human mesangial cells.  

Science.gov (United States)

Recent large clinical trials indicate that angiotensin-converting enzyme inhibitors (ACE-I) attenuate the detrimental outcome of progressive renal disease. The hemoregulatory tetrapeptide N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP, AcSDKP) is hydrolyzed by ACE, and plasma Ac-SDKP level is increased by fivefold after treatment with ACE-I. Ac-SDKP was found to ameliorate cardiac and renal fibrosis in hypertensive animal models. However, the molecular mechanisms by which Ac-SDKP mediates anti-fibrotic effects remain unclear. This study is an examination of the interaction between Ac-SDKP and transforming growth factor-beta (TGF-beta), one of the key cytokines in the progression of renal disease, in human mesangial cells. Ac-SDKP inhibited TGF-beta1-induced plasminogen activator inhibitor-1 (PAI-1) and alpha2 (I) collagen mRNA. Ac-SDKP suppressed not only TGF-beta1-induced Smad2 phosphorylation at Ser-465/467 in a dose-dependent manner, but also the nuclear accumulation of receptor-regulated Smads (R-Smad), Smad2 and Smad3. As expected, Ac-SDKP inhibited TGF-beta-responsive Smad-dependent luciferase reporters, 3TP-luc and 4xSBE-luc. Immunofluorescence analysis revealed that the inhibitory Smad, Smad7, was exported to the cytoplasm from the nucleus by the treatment with Ac-SDKP. These findings provide novel evidence that Ac-SDKP inhibits TGF-beta signal transduction through the suppression of R-Smad activation via nuclear export of Smad7, highlighting an alternative mechanism involved in the reno-protective efficacy of ACE-I. PMID:12660320

Kanasaki, Keizo; Koya, Daisuke; Sugimoto, Toshiro; Isono, Motohide; Kashiwagi, Atsunori; Haneda, Masakazu

2003-04-01

374

An Anti-Urokinase Plasminogen Activator Receptor Antibody (ATN-658 Blocks Prostate Cancer Invasion, Migration, Growth, and Experimental Skeletal Metastasis In Vitro and In Vivo  

Directory of Open Access Journals (Sweden)

Full Text Available Urokinase plasminogen activator receptor (uPAR is a multidomain protein that plays important roles in the growth, invasion, and metastasis of a number of cancers. In the present study, we examined the effects of administration of a monoclonal anti-uPAR antibody (ATN-658 on prostate cancer progression in vitro and in vivo. We examined the effect of treatment of ATN-658 on human prostate cancer cell invasion, migration, proliferation, and regulation of intracellular signaling pathways. For in vivo studies, PC-3 cells (1 x 106 were inoculated into the right flank of male Balb C nu/nu mice through subcutaneous or through intratibial route (2 x 105 of male Fox Chase severe combined immunodeficient mice to monitor the effect on tumor growth and skeletal metastasis. Treatment with ATN-658 resulted in a significant dose-dependent decrease in PC-3 cell invasion and migration without affecting cell doubling time. Western blot analysis showed that ATN-658 treatment decreased the phosphorylation of serine/threonine protein kinase B (AKT, mitogen-activated protein kinase (MAPK, and focal adhesion kinase (FAK without affecting AKT, MAPK, and FAK total protein expression. In in vivo studies, ATN-658 caused a significant decrease in tumor volume and a marked reduction in skeletal lesions as determined by Faxitron x-ray and micro-computed tomography. Immunohistochemical analysis of subcutaneous and tibial tumors showed a marked decrease in the levels of expression of pAKT, pMAPK, and pFAK, consistent with the in vitro observations. Results from these studies provide compelling evidence for the continued development of ATN-658 as a potential therapeutic agent for the treatment of prostate and other cancers expressing uPAR.

Shafaat A. Rabbani

2010-10-01

375

Hydrogen/deuterium exchange mass spectrometry reveals specific changes in the local flexibility of plasminogen activator inhibitor 1 upon binding to the somatomedin B domain of vitronectin  

DEFF Research Database (Denmark)

The native fold of plasminogen activator inhibitor 1 (PAI-1) represents an active metastable conformation that spontaneously converts to an inactive latent form. Binding of the somatomedin B domain (SMB) of the endogenous cofactor vitronectin to PAI-1 delays the transition to the latent state and increases the thermal stability of the protein dramatically. We have used hydrogen/deuterium exchange mass spectrometry to assess the inherent structural flexibility of PAI-1 and to monitor the changes induced by SMB binding. Our data show that the PAI-1 core consisting of ?-sheet B is rather protected against exchange with the solvent, while the remainder of the molecule is more dynamic. SMB binding causes a pronounced and widespread stabilization of PAI-1 that is not confined to the binding interface with SMB. We further explored the local structural flexibility in a mutationally stabilized PAI-1 variant (14-1B) as well as the effect of stabilizing antibody Mab-1 on wild-type PAI-1. The three modes of stabilizing PAI-1 (SMB, Mab-1, and the mutations in 14-1B) all cause a delayed latency transition, and this effect was accompanied by unique signatures on the flexibility of PAI-1. Reduced flexibility in the region around helices B, C, and I was seen in all three cases, which suggests an involvement of this region in mediating structural flexibility necessary for the latency transition. These data therefore add considerable depth to our current understanding of the local structural flexibility in PAI-1 and provide novel indications of regions that may affect the functional stability of PAI-1.

Trelle, Morten Beck; Hirschberg, Daniel

2012-01-01