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Sample records for ovine plasminogen activator

  1. Insulin and insulin-like growth factor I exert different effects on plasminogen activator production or cell growth in the ovine thyroid cell line OVNIS.

    Science.gov (United States)

    Degryse, B; Maisonobe, F; Hovsépian, S; Fayet, G

    1991-11-01

    Insulin and Insulin-like Growth Factor I (IGF-I) are evaluated for their capacity to affect cell proliferation and plasminogen activator (PA) activity production in an ovine thyroid cell line OVNIS. Insulin at physiological and supraphysiological doses induces cell proliferation and increases PA activity. IGF-I, which is also clearly mitogenic for these cells, surprisingly does not modulate PA activity. The results indicate that the growth promoting effect is mediated through the insulin and IGF-I receptors whereas PA activity is solely regulated via the insulin receptors. PMID:1802921

  2. Insulin and insulin-like growth factor I exert different effects on plasminogen activator production or cell growth in the ovine thyroid cell line OVNIS.

    Science.gov (United States)

    Degryse B; Maisonobe F; Hovsépian S; Fayet G

    1991-11-01

    Insulin and Insulin-like Growth Factor I (IGF-I) are evaluated for their capacity to affect cell proliferation and plasminogen activator (PA) activity production in an ovine thyroid cell line OVNIS. Insulin at physiological and supraphysiological doses induces cell proliferation and increases PA activity. IGF-I, which is also clearly mitogenic for these cells, surprisingly does not modulate PA activity. The results indicate that the growth promoting effect is mediated through the insulin and IGF-I receptors whereas PA activity is solely regulated via the insulin receptors.

  3. Plasminogen activation and cancer

    DEFF Research Database (Denmark)

    Danø, Keld; Behrendt, N.; Hoyer-Hansen, G.; Johnsen, Morten; Lund, L. R.; Ploug, M.; Nielsen, John Rømer

    2005-01-01

    Breakdown of the extracellular matrix is crucial for cancer invasion and metastasis. It is accomplished by the concerted action of several proteases, including the serine protease plasmin and a number of matrix metalloproteases.The activity of each of these proteases is regulated by an array of activators, inhibitors and cellular receptors.Thus, the generation of plasmin involves the pro-enzyme plasminogen, the urokinase type plasminogen activator uPA and its pro-enzyme pro-uPA, the uPA inhibito...

  4. PLASMINOGEN ACTIVATOR OF YERSINIA PESTIS

    Directory of Open Access Journals (Sweden)

    V. V. Evseeva

    2015-04-01

    Full Text Available Plague has been the cause of three pandemics and has led to the death of millions of people. Plague is a typical zoonosis caused by Yersinia pestis that circulates in populations of wild rodents inhabiting natural plague foci on all continents except for Australia. Transmission of plague is provided by flea bites. Circulation of Y. pestis in natural plague foci is supported by a numerous of pathogenicity factors. This review explores one of them, plasminogen activator Pla. This protein is one of representatives of omptins, a family of enterobacterial outer membrane proteases that are responsible for colonization of specific organs or even infection generalization as a result of successful overcoming of the host innate immunity. The review reflects the history of its discovery and studying of its genetic control, biosynthesis, isolation and purification, physicochemical properties. Highly purified preparations of plasminogen activator are deficient in enzymatic activities but renaturation in the presence of Y. pestis lipooligosaccharide restores enzymatic properties of Pla. This pathogenicity factor is absent in representatives of the most ancient phylogenetic group of the plague pathogen, bv. caucasica, while the ancestor of other groups of Y. pestis subsp. microtus obtained in result of horizontal transfer Pla isoform with characteristics similar to properties of omptins from the less virulent enterobacteria. After that in the course of microevolution the “classic” isoform of Pla with increased protease activity was selected that is typical of all highly virulent for humans strains of Y. pestis subsp. pestis. The “classic” isoform of Pla Y. pestis is functionally similar to mammalian plasminogen activators transforming plasminogen into plasmin with the help of limited proteolysis. Pla protease activating plasminogen and also degrading the main plasmin inhibitor — ?2-antiplasmin and, respectively, determining Y. pestis ability to lyse fibrin clots preventing bacteria dissemination after bites of infected fleas or subcutaneous challenge is believed to be the main Y. pestis factor responsible for generalization of infectious process. Pla-mediated ability of Y. pestis for selective binding with extracellular matrix and basal membranes may promote further hydrolysis of these structures by the host’s plasmin and overcoming tissue barriers by the pathogen. Y. pestis plasminogen activator also hydrolyses C3 complement component, human antimicrobial peptide — cathelicidin LL-37 and such cytokines as tumor necrosis factor ?, interferon ?, interleukin 8 and protein 1 of monocyte chemotaxis. The main endogenic TFPI tissue factor pathway inhibitor also highly susceptible to proteolytic action of Pla, and efficiency of TFPI inactivation is much higher than efficacy of plasminogen activation. The review also debates the possibility of using Pla as a molecular target for prophylaxis and treatment of plague. 

  5. PLASMINOGEN ACTIVATOR OF YERSINIA PESTIS

    Directory of Open Access Journals (Sweden)

    V. V. Evseeva

    2015-04-01

    Full Text Available Plague has been the cause of three pandemics and has led to the death of millions of people. Plague is a typical zoonosis caused by Yersinia pestis that circulates in populations of wild rodents inhabiting natural plague foci on all continents except for Australia. Transmission of plague is provided by flea bites. Circulation of Y. pestis in natural plague foci is supported by a numerous of pathogenicity factors. This review explores one of them, plasminogen activator Pla. This protein is one of representatives of omptins, a family of enterobacterial outer membrane proteases that are responsible for colonization of specific organs or even infection generalization as a result of successful overcoming of the host innate immunity. The review reflects the history of its discovery and studying of its genetic control, biosynthesis, isolation and purification, physicochemical properties. Highly purified preparations of plasminogen activator are deficient in enzymatic activities but renaturation in the presence of Y. pestis lipooligosaccharide restores enzymatic properties of Pla. This pathogenicity factor is absent in representatives of the most ancient phylogenetic group of the plague pathogen, bv. caucasica, while the ancestor of other groups of Y. pestis subsp. microtus obtained in result of horizontal transfer Pla isoform with characteristics similar to properties of omptins from the less virulent enterobacteria. After that in the course of microevolution the “classic” isoform of Pla with increased protease activity was selected that is typical of all highly virulent for humans strains of Y. pestis subsp. pestis. The “classic” isoform of Pla Y. pestis is functionally similar to mammalian plasminogen activators transforming plasminogen into plasmin with the help of limited proteolysis. Pla protease activating plasminogen and also degrading the main plasmin inhibitor — α2-antiplasmin and, respectively, determining Y. pestis ability to lyse fibrin clots preventing bacteria dissemination after bites of infected fleas or subcutaneous challenge is believed to be the main Y. pestis factor responsible for generalization of infectious process. Pla-mediated ability of Y. pestis for selective binding with extracellular matrix and basal membranes may promote further hydrolysis of these structures by the host’s plasmin and overcoming tissue barriers by the pathogen. Y. pestis plasminogen activator also hydrolyses C3 complement component, human antimicrobial peptide — cathelicidin LL-37 and such cytokines as tumor necrosis factor α, interferon γ, interleukin 8 and protein 1 of monocyte chemotaxis. The main endogenic TFPI tissue factor pathway inhibitor also highly susceptible to proteolytic action of Pla, and efficiency of TFPI inactivation is much higher than efficacy of plasminogen activation. The review also debates the possibility of using Pla as a molecular target for prophylaxis and treatment of plague. 

  6. Heat inactivation of native plasmin, plasminogen and plasminogen activators in bovine milk: a revisited study

    OpenAIRE

    Denis, Thierry; Humbert, Gérard; Gaillard, Jean-Luc

    2001-01-01

    Thermal inactivation, at temperatures between 60 °C and 140 °C, of native plasmin, plasminogen and plasminogen activators were studied in bovine milk using improved enzymatic assays. While measured heat inactivation kinetic of plasmin and plasminogen were in line with previously reported values, plasminogen activators were, surprisingly, found to be as heat sensitive as plasmin and plasminogen in a milk system containing proteins with free SH groups. Activation energies (Ea) for the heat dena...

  7. Plasminogen activation and cancer

    DEFF Research Database (Denmark)

    Danø, Keld; Behrendt, N.; Hoyer-Hansen, G.; Johnsen, Morten; Lund, L. R.; Ploug, M.; Nielsen, John Rømer

    2005-01-01

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

  8. Liposomal nanocarriers for plasminogen activators.

    Science.gov (United States)

    Koudelka, Stepan; Mikulik, Robert; Mašek, Josef; Raška, Milan; Turánek Knotigová, Pavlína; Miller, Andrew D; Turánek, Jaroslav

    2016-04-10

    Several plasminogen activators (PAs) have been found effective in treating different thromboembolic diseases. However, administration of conventional thrombolytic therapy is limited by a low efficacy of present formulations of PAs. Conventional treatments using these therapeutic proteins are associated with several limitations including rapid inactivation and clearance, short half-life, bleeding complications or non-specific tissue targeting. Liposome-based formulations of PAs such as streptokinase, tissue-plasminogen activator and urokinase have been developed to improve the therapeutic efficacy of these proteins. Resulting liposomal formulations were found to preserve the original activity of PAs, promote their selective delivery and improve thrombus targeting. Therapeutic potential of these liposome-based PAs has been demonstrated successfully in various pre-clinical models in vivo. Reductions in unwanted side effects (e.g., hemorrhage or immunogenicity) as well as enhancements of efficacy and safety were achieved in comparison to currently existing treatment options based on conventional formulations of PAs. This review summarizes present achievements in: (i) preparation of liposome-based formulations of various PAs, (ii) development of PEGylated and targeted liposomal PAs, (iii) physico-chemical characterization of these developed systems, and (iv) testing of their thrombolytic efficacy. We also look to the future and the imminent arrival of theranostic liposomal formulations to move this field forward. PMID:26876783

  9. Does plasminogen activator inhibitor-1 drive lymphangiogenesis?

    DEFF Research Database (Denmark)

    Bruyère, Françoise; Melen-Lamalle, Laurence; Blacher, Silvia; Detry, Benoît; Masset, Anne; Lecomte, Julie; Lambert, Vincent; Maillard, Catherine; Høyer-Hansen, Gunilla; Lund, Leif R; Foidart, Jean-Michel; Noël, Agnès

    2010-01-01

    The purpose of this study is to explore the function of plasminogen activator inhibitor-1 (PAI-1) during pathological lymphangiogenesis. PAI-1, the main physiological inhibitor of plasminogen activators is involved in pathological angiogenesis at least by controlling extracellular proteolysis and by regulating endothelial cell survival and migration. Protease system's role in lymphangiogenesis is unknown yet. Thus, based on its important pro-angiogenic effect, we hypothesized that PAI-1 may regu...

  10. Thrombolytic effect of a plasminogen-plasminogen activator chimera in a photochemically induced thrombosis (PIT) model.

    OpenAIRE

    Matsuno, H.; Uematsu, T.; Nakashima, M

    1993-01-01

    The thrombolytic effects of the plasminogen/plasminogen activator chimera (SUN9216), comprising the fibrin-binding kringle 1 domain of plasminogen and two kringle and the serine protease domain of the wild-type tissue plasminogen activator (t-PA) including a modification of the mannose glycosylation on the kringle 1 of t-PA (PK1 delta FE1X), was compared with tht of t-PA by use of a photochemically induced thrombus (PIT) in the rat femoral artery. When SUN9216 was administered either as an i....

  11. Polymerization of plasminogen activator inhibitor-1.

    OpenAIRE

    Zhou, A.; Faint, R.; Charlton, P.; Dafforn, T.R; Carrell, R. W.; Lomas, D.A.

    2001-01-01

    The activity of the serine proteinase inhibitor (serpin) plasminogen activator inhibitor-1 (PAI-1) is controlled by the intramolecular incorporation of the reactive loop into beta-sheet A with the generation of an inactive latent species. Other members of the serpin superfamily can be pathologically inactivated by intermolecular linkage between the reactive loop of one molecule and beta-sheet A of a second to form chains of polymers associated with diverse diseases. It has long been believed ...

  12. Design of a novel chimeric tissue plasminogen activator with favorable Vampire bat plasminogen activator properties.

    Science.gov (United States)

    Kazemali, MohammadReza; Majidzadeh-A, Keivan; Sardari, Soroush; Saadatirad, Amir Hossein; Khalaj, Vahid; Zarei, Najmeh; Barkhordari, Farzaneh; Adeli, Ahmad; Mahboudi, Fereidoun

    2014-12-01

    Fibrinolytic agents are widely used in treatment of the thromboembolic disorders. The new generations like recombinant tissue plasminogen activator (t-PA, alteplase) are not showing promising results in clinical practice in spite of displaying specific binding to fibrin in vitro. Vampire bat plasminogen activator (b-PA) is a plasminogen activator with higher fibrin affinity and specificity in comparison to t-PA resulting in reduced probability of hemorrhage. b-PA is also resistant to plasminogen activator inhibitor-1 (PAI-1) showing higher half-life compared to other variants of t-PA. However, its non-human origin was a driving force to design a human t-PA with favorable properties of b-PA. In the present study, we designed a chimeric t-PA with desirable b-PA properties and this new molecule was called as CT-b. The construct was prepared through kringle 2 domain removal and replacement of t-PA finger domain with b-PA one. In addition, the KHRR sequence at the initial part of protease domain was replaced by four alanine residues. The novel construct was integrated in Pichia pastoris genome by electroporation. Catalytic activity was investigated in the presence and absence of fibrin. The purified protein was analyzed by western blot. Fibrin binding and PAI resistance assays were also conducted. The activity of the recombinant protein in the presence of fibrin was 1560 times more than its activity in the absence of fibrin, showing its higher specificity to fibrin. The fibrin binding of CT-b was 1.2 fold more than t-PA. In addition, it was inhibited by PAI enzyme 44% less than t-PA. Although the presented data demonstrate a promising in vitro activity, more in vivo studies are needed to confirm the therapeutic advantage of this novel plasminogen activator. PMID:25442953

  13. Does plasminogen activator inhibitor-1 drive lymphangiogenesis?

    DEFF Research Database (Denmark)

    Bruyère, Françoise; Melen-Lamalle, Laurence

    2010-01-01

    The purpose of this study is to explore the function of plasminogen activator inhibitor-1 (PAI-1) during pathological lymphangiogenesis. PAI-1, the main physiological inhibitor of plasminogen activators is involved in pathological angiogenesis at least by controlling extracellular proteolysis and by regulating endothelial cell survival and migration. Protease system's role in lymphangiogenesis is unknown yet. Thus, based on its important pro-angiogenic effect, we hypothesized that PAI-1 may regulate lymphangiogenesis associated at least with metastatic dissemination of cancer cells. To address this issue, we studied the impact of PAI-1 deficiency in various murine models of tumoral lymphangiogenesis. Wild-type PAI-1 proficient mice were used as controls. We provide for the first time evidence that PAI-1 is dispensable for tumoral lymphangiogenesis associated with breast cancers either induced by mammary carcinoma cell injection or spontaneously appearing in transgenic mice expressing the polyomavirus middle Tantigen (PymT) under the control of a mouse mammary tumor virus long-terminal repeat promoter (MMTV-LTR). We also investigated inflammation-related lymphatic vessel recruitment by using two inflammatory models. PAI-1 deficiency did neither affect the development of lymphangioma nor burn-induced corneal lymphangiogenesis. These novel data suggest that vascular remodelling associated with lymphangiogenesis and angiogenesis involve different molecular determinants. PAI-1 does not appear as a potential therapeutic target to counteract pathological lymphangiogenesis.

  14. Inactivation of plasminogen activator inhibitor by oxidants

    Energy Technology Data Exchange (ETDEWEB)

    Lawrence, D.A.; Loskutoff, D.J.

    1986-10-21

    The rapidly acting plasminogen activator inhibitor (PAI) purified from cultured bovine endothelial cells (BAEs) was inactivated during iodination with chloramine T and other oxidizing iodination systems. Inactivation was observed in the absence of iodine, suggesting that the loss of activity resulted from the oxidizing conditions employed. In an attempt to further study the nature of this inactivation, the PAI was treated with chloramine T under conditions that specifically oxidize methionine and cystein residues. Both PAI inhibitory activity and the ability of the PAI to form complexes with tissue-type PA were decreased in a dose-dependent manner by such treatment. PAI activity was measured with the lysis of /sup 125/I-labelled fibrin. The reductase is a DTT-dependent enzyme that specifically converts methionine sulfoxide to methionine. Little activity was restored by either the reductase or DTT alone. These results indicate that the oxidation of at least one critical methionine residue is responsible for the loss of PAI activity upon iodination. In this respect, the BAE PAI resembles ..cap alpha../sub 1/-protease inhibitor, a well-characterized elastase inhibitor that also is inactivated by oxidants. Both inhibitors are members of the serine protease inhibitor superfamily (Serpins), and both have a methionine residue in their reactive center.

  15. Inactivation of plasminogen activator inhibitor by oxidants

    International Nuclear Information System (INIS)

    The rapidly acting plasminogen activator inhibitor (PAI) purified from cultured bovine endothelial cells (BAEs) was inactivated during iodination with chloramine T and other oxidizing iodination systems. Inactivation was observed in the absence of iodine, suggesting that the loss of activity resulted from the oxidizing conditions employed. In an attempt to further study the nature of this inactivation, the PAI was treated with chloramine T under conditions that specifically oxidize methionine and cystein residues. Both PAI inhibitory activity and the ability of the PAI to form complexes with tissue-type PA were decreased in a dose-dependent manner by such treatment. PAI activity was measured with the lysis of 125I-labelled fibrin. The reductase is a DTT-dependent enzyme that specifically converts methionine sulfoxide to methionine. Little activity was restored by either the reductase or DTT alone. These results indicate that the oxidation of at least one critical methionine residue is responsible for the loss of PAI activity upon iodination. In this respect, the BAE PAI resembles ?1-protease inhibitor, a well-characterized elastase inhibitor that also is inactivated by oxidants. Both inhibitors are members of the serine protease inhibitor superfamily (Serpins), and both have a methionine residue in their reactive center

  16. Human monocytes can produce tissue-type plasminogen activator

    OpenAIRE

    1989-01-01

    Evidence has previously been presented that monocytes and macrophages produce urokinase-type plasminogen activator. We have shown for the first time that human monocytes, when stimulated appropriately in vitro, can produce tissue type-plasminogen activator (t-PA) of 70 kD. Detection of t-PA mRNA was consistent with the biochemical and immunological characterization of t-PA produced by human monocytes.

  17. Influence of Plasminogen Activator Inhibitor Type 1 on Choroidal Neovascularization

    OpenAIRE

    Lambert, Vincent; Munaut, Carine; Frankenne, F.; Bajou, Khalid; Gerard, R; Carmeliet, P; Defresne, Marie-Paule; Foidart, Jean-Michel; Rakic, Jean-Marie; Noël, Agnès

    2001-01-01

    High levels of the plasminogen activators, but also their inhibitor, plasminogen activator inhibitor 1 (PAI-1), have been documented in neovascularization of severe ocular pathologies such as diabetic retinopathy or age-related macular degeneration (AMD). AMD is the primary cause of irreversible photoreceptors loss, and current therapies are limited. PAI-1 has recently been shown to be essential for tumoral angiogenesis. We report here that deficient PAI-1 expression in mice prevented the dev...

  18. The extracellular matrix proteins laminin and fibronectin contain binding domains for human plasminogen and tissue plasminogen activator

    DEFF Research Database (Denmark)

    Moser, T L; Enghild, J J; Pizzo, S V; Stack, M S

    1993-01-01

    This study describes the binding of plasminogen and tissue-type plasminogen activator (t-PA) to the extracellular matrix proteins fibronectin and laminin. Plasminogen bound specifically and saturably to both fibronectin and laminin immobilized on microtiter wells, with Kd(app) values of 115 and 18 nM, respectively. Limited proteolysis by endoproteinase V8 coupled with ligand blotting analysis showed that both plasminogen and t-PA preferentially bind to a 55-kDa fibronectin fragment and a 38-kDa ...

  19. Tissue plasminogen activator in central nervous system physiology and pathology

    OpenAIRE

    Melchor, Jerry P.; Strickland, Sidney

    2005-01-01

    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.

  20. The Glycosylation of Plasminogen Activator Inhibitor-1

    DEFF Research Database (Denmark)

    Skottrup, Peter Durand; Pedersen, Katrine Egelund; Christensen, Anni; Thøgersen, Ida; Andreasen, Peter; Enghild, Jan Johannes

    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...

  1. The Glycosylation of Plasminogen Activator Inhibitor-1

    DEFF Research Database (Denmark)

    Skottrup, Peter; Pedersen, Katrine Egelund; Christensen, Anni; Thøgersen, Ida; Andreasen, Peter; Enghild, Jan J.

    2002-01-01

    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...

  2. A role for tissue plasminogen activator in thrombotic thrombocytopenic purpura.

    Science.gov (United States)

    Hoirisch-Clapauch, Silvia; Nardi, Antonio Egidio

    2014-12-01

    Thrombotic thrombocytopenic purpura (TTP) is a life-threatening disease characterized by generalized microvascular occlusion. TTP has been related to severe deficiency of ADAMTS13, an enzyme that cleaves von Willebrand factor multimers into less adhesive molecules. However, ADAMTS13 deficiency correlates poorly with severity of thrombocytopenia or microangiopathic hemolysis, with the frequency of neurologic complications or the response to plasma exchange. Also, some patients with severe hereditary ADAMTS13 deficiency consistently relapse every few weeks, whereas others remain asymptomatic into their forties. Taken together, these findings suggest that an additional element is missing in the pathophysiology of TTP. We postulate that both low ADAMTS13 activity and low tissue-plasminogen activator activity are required to trigger TTP attacks. Tissue-plasminogen activator end product, plasmin, extensively degrades von Willebrand factor, breaking-down the bonds between platelets and the blood vessel wall, so that low tissue-plasminogen activator activity prevents a mechanism similar to that of ADAMTS13. The hypothesis that low tissue-plasminogen activator activity plays an important role in TTP pathogenesis is further substantiated by TTP comorbidity. Problems prevalent in patients with TTP attacks or with long-term TTP remission, including increased body mass index, major depression, cognitive abnormalities, hypertension, and premature death, are somehow associated with low tissue-plasminogen activator activity. PMID:25459148

  3. Photonic Activation of Plasminogen induced by low dose UVB

    DEFF Research Database (Denmark)

    Correia, Manuel Guiherme L.P. Marins; Snabe, Torben; Thiagarajan, Viruthachalam; Petersen, Steffen; Sara R. R. Campos, Sara R. R.; Baptista, António M; Neves Petersen, Teresa

    2015-01-01

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

  4. Photonic Activation of Plasminogen induced by low dose UVB

    DEFF Research Database (Denmark)

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

    2015-01-01

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

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

    OpenAIRE

    Saif-Ali Riyadh; Ismail Ikram S; Al-Hamodi Zaid; Ahmed Khaled A; Muniandy Sekaran

    2011-01-01

    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 acti...

  6. 4G/5G Plasminogen Activator Inhibitor-1 Polymorphisms and Haplotypes Are Associated with Pneumonia

    OpenAIRE

    Yende, Sachin; Angus, Derek C.; Ding, Jingzhong; NEWMAN, ANNE B.; John A. Kellum; Li, Rongling; Ferrell, Robert. E.; Zmuda, Joseph; Kritchevsky, Stephen B.; Harris, Tamara B; Garcia, Melissa; Yaffe, Kristine; Wunderink, Richard G.

    2007-01-01

    Rationale: Plasminogen activator inhibitor (PAI)-1 inhibits urokinase and tissue plasminogen activator, required for host response to infection. Whether variation within the PAI-1 gene is associated with increased susceptibility to infection is unknown.

  7. Mechanisms regulating plasminogen activators in transformed retinal ganglion cells

    OpenAIRE

    Rock, Nathan; Chintala, Shravan K

    2008-01-01

    Irreversible loss of retinal ganglion cells (RGCs) is a major clinical issue in glaucoma, but the mechanisms that lead to RGC death are currently unclear. We have previously reported that elevated levels of tissue plasminogen activator (tPA) and urokinase plasminogen activator (uPA) cause the death of RGCs in vivo and transformed retinal ganglion cells (RGC-5) in vitro. Yet, it is unclear how secreted proteases such as tPA and uPA directly cause RGCs' death. In this study, by employing RGC-5 ...

  8. Aspirin inhibits vascular plasminogen activator activity in vivo. Studies utilizing a new assay to quantify plasminogen activator activity.

    OpenAIRE

    Levin, R.I.; Harpel, P. C.; Weil, D.; Chang, T S; Rifkin, D. B.

    1984-01-01

    Vascular or tissue-type plasminogen activator (TPA) is a key enzyme in physiologic fibrinolysis. To study the role of prostaglandins in modulating the synthesis and release of TPA in vivo, we prospectively studied the effect of aspirin (650 mg/d X 2) on TPA activity in 13 human subjects before and after 10 min of forearm venous occlusion. TPA activity was quantified by a newly developed enzyme-linked immunosorbent assay that both measures and differentiates between TPA and urokinase (UK)-like...

  9. Plasminogen activator inhibitor type 1 regulates microglial motility and phagocytic activity

    OpenAIRE

    Jeon Hyejin; Kim Jong-Heon; Kim Jae-Hong; Lee Won-Ha; Lee Myung-Shik; Suk Kyoungho

    2012-01-01

    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 chromatog...

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

    DEFF Research Database (Denmark)

    Stæhr, Mette; Buhl, Kristian Bergholt

    2015-01-01

    In nephrotic syndrome, aberrant glomerular filtration of plasminogen and conversion to active plasmin in pre-urine is thought to activate proteolytically ENaC and contribute to sodium retention and edema. The ENaC blocker amiloride is an off-target inhibitor of urokinase-type plasminogen activator (uPA) in vitro. It was hypothesized that uPA is abnormally filtered to pre-urine and is inhibited in urine by amiloride in nephrotic syndrome. This was tested by determination of Na+-balance, uPA protein and activity and amiloride concentration in urine from rats with puromycin aminonucleoside (PAN) induced nephrotic syndrome. Urine samples from 6 adult and 18 pediatric patients with nephrotic syndrome were analyzed for uPA activity and protein. PAN-treatment induced significant proteinuria in rats which coincided with increased urine uPA protein and activity, increased urine protease activity and total plasminogen/plasmin concentration and Na+ retention. Amiloride (2mg/kg/24h) concentration in urine was in the range 10-20 µmol/L and reduced significantly urine uPA activity, plasminogen activation, protease activity and sodium retention in PAN rats, while proteinuria was not altered. In paired urine samples, uPA protein was significantly elevated in urine from children with active nephrotic syndrome compared to remission phase. In 6 adult nephrotic patients, urine uPA protein and activity correlated positively with 24h urine protein excretion. In conclusion, nephrotic syndrome is associated with aberrant filtration of uPA across the injured glomerular barrier. Amiloride inhibits urine uPA activity which attenuates plasminogen activation and urine protease activity in vivo. Urine uPA is a relevant target for amiloride in vivo.

  11. Treatment of subretinal hemorrhages with tissue plasminogen activator.

    Science.gov (United States)

    Borrillo, J L; Regillo, C D

    2001-06-01

    Submacular hemorrhages pose a danger to visual acuity. Intervention may help prevent or mitigate severe visual loss. Pneumatic displacement and vitrectomy with direct evacuation are two methods of treating submacular hemorrhages. Tissue plasminogen activator may be an important adjunct to these techniques. PMID:11389348

  12. The plasminogen activator system modulates sympathetic nerve function

    OpenAIRE

    Schaefer, Ulrich; Machida, Takuji; Vorlova, Sandra; Strickland, Sidney; Levi, Roberto

    2006-01-01

    Sympathetic neurons synthesize and release tissue plasminogen activator (t-PA). We investigated whether t-PA modulates sympathetic activity. t-PA inhibition markedly reduced contraction of the guinea pig vas deferens to electrical field stimulation (EFS) and norepinephrine (NE) exocytosis from cardiac synaptosomes. Recombinant t-PA (rt-PA) induced exocytotic and carrier-mediated NE release from cardiac synaptosomes and cultured neuroblastoma cells; this was a plasmin-independent effect but wa...

  13. Proteases induce secretion of collagenase and plasminogen activator by fibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    Werb, Z.; Aggeler, J.

    1978-04-01

    We have observed that treatment of rabbit synovial fibroblasts with proteolytic enzymes can induce secretion of collagenase (EC 3.4.24.7) and plasminogen activator (EC 3.4.21.-). Cells treated for 2 to 24 hr with plasmin, trypsin, chymotrypsin, pancreatic elastase, papain, bromelain, thermolysin, or ..cap alpha..-protease but not with thrombin or neuraminidase secreted detectable amounts of collagenase within 16 to 48 hr. Treatment of fibroblasts with trypsin also induced secretion of plasminogen activator. Proteases initiated secretion of collagenase (up to 20 units per 10/sup 6/ cells per 24 hr) only when treatment produced decreased cell adhesion. Collagenase production did not depend on continued presence of proteolytic activity or on subsequent cell adhesion, spreading, or proliferation. Routine subculturing with crude trypsin also induced collagenase secretion by cells. Secretion of collagenase was prevented and normal spreading was obtained if the trypsinized cells were placed into medium containing fetal calf serum. Soybean trypsin inhibitor, ..cap alpha../sub 1/-antitrypsin, bovine serum albumin, collagen, and fibronectin did not inhibit collagenase production. Although proteases that induced collagenase secretion also removed surface glycoprotein, the kinetics of induction of cell protease secretion were different from those for removal of fibronectin. Physiological inducers of secretion of collagenase and plasminogen activator by cells have not been identified. These results suggest that extracellular proteases in conjunction with plasma proteins may govern protease secretion by cells.

  14. Recombinant tissue plasminogen activator following paediatric cataract surgery

    OpenAIRE

    Mehta, J.; Adams, G.

    2000-01-01

    BACKGROUND—The use of recombinant tissue plasminogen activator (r-TPA) has been advocated in the treatment of postsurgical fibrinous membrane formation following cataract surgery in adults. Its use in paediatric cases is not well documented.?METHOD—A retrospective review of paediatric cataract extractions performed at Moorfields Eye Hospital between 1 January 1997 and 4 April 1999 was carried out.?RESULTS—Cataract extractions were performed in 37 patients, 22 in males 15 in females. Four (9.2...

  15. Bicyclic Peptide Inhibitor of Urokinase-Type Plasminogen Activator

    DEFF Research Database (Denmark)

    Roodbeen, Renée; Paaske, Berit; Jiang, Longguang; Jensen, Jan Kristian; Christensen, Anni; Nielsen, Jakob T; Huang, Mingdong; Mulder, Frans A.A.; Nielsen, Niels Christian; Andreasen, Peter; Jensen, Knud Jørgen

    2013-01-01

    The development of protease inhibitors for pharmacological intervention has taken a new turn with the use of peptide-based inhibitors. Here, we report the rational design of bicyclic peptide inhibitors of the serine protease urokinase-type plasminogen activator (uPA), based on the established monocyclic peptide, upain-2. It was successfully converted to a bicyclic peptide, without loss of inhibitory properties. The aim was to produce a peptide cyclised by an amide bond with an additional stabili...

  16. Low activity of plasminogen activator: a common feature of non- iatrogenic comorbidities of schizophrenia.

    Science.gov (United States)

    Hoirisch-Clapauch, Silvia; Nardi, Antonio E

    2015-01-01

    Understanding the pathogenesis of non-iatrogenic comorbidities of schizophrenia may provide insights into the pathogenesis of schizophrenia itself. First-episode, drug-naïve schizophrenia patients are at high risk of thromboembolic events, diseases related to substance abuse, sexual dysfunction, reproductive disorders, inflammatory and autoimmune diseases, as well as complications of hyperinsulinemia or hyperhomocysteinemia. This review focuses on the role of reduced plasminogen activator activity in non-iatrogenic comorbidity of schizophrenia. By preventing thrombus dissolution, low tissue plasminogen activator activity increases the risk of thrombotic events. Components of the plasminogen activator system also play a key role in reproduction. Both illicit drugs and tobacco increase plasminogen activator levels in the central nervous system, which seems to relieve symptoms of the mental disorder. Chronic alcoholism, sexual dysfunction, inflammatory and autoimmune disorders, and complications of hyperinsulinemia or hyperhomocysteinemia are somehow related to low plasminogen activator activity. Plasminogen activator mediates several neurochemical processes that seem to prevent or reverse gray-matter atrophy seen in first-episode schizophrenia patients. Such processes include cleavage of brain-derived neurotrophic factor precursor to an anti-apoptotic neurotrophin and activation of N-methyl-D-aspartate receptor. Controlled, randomized studies are needed to determine if measures aimed at correcting plasminogen activator activity can improve the quality of life, reduce morbidity and mortality rates, and particularly improve the course of schizophrenia. PMID:25714971

  17. Crystal Structure of the Michaelis Complex between Tissue-type Plasminogen Activator and Plasminogen Activators Inhibitor-1.

    Science.gov (United States)

    Gong, Lihu; Liu, Min; Zeng, Tu; Shi, Xiaoli; Yuan, Cai; Andreasen, Peter A; Huang, Mingdong

    2015-10-23

    Thrombosis is a leading cause of death worldwide. Recombinant tissue-type plasminogen activator (tPA) is the Food and Drug Administration-approved thrombolytic drug. tPA is rapidly inactivated by endogenous plasminogen activator inhibitor-1 (PAI-1). Engineering on tPA to reduce its inhibition by PAI-1 without compromising its thrombolytic effect is a continuous effort. Precise details, with atomic resolution, of the molecular interactions between tPA and PAI-1 remain unknown despite previous extensive studies. Here, we report the crystal structure of the tPA·PAI-1 Michaelis complex, which shows significant differences from the structure of its urokinase-type plasminogen activator analogue, the uPA·PAI-1 Michaelis complex. The PAI-1 reactive center loop adopts a unique kinked conformation. The structure provides detailed interactions between tPA 37- and 60-loops with PAI-1. On the tPA side, the S2 and S1? pockets open up to accommodate PAI-1. This study provides structural basis to understand the specificity of PAI-1 and to design newer generation of thrombolytic agents with reduced PAI-1 inactivation. PMID:26324706

  18. 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)

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

    DEFF Research Database (Denmark)

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

    2014-01-01

    In conditions with albuminuria, plasminogen is aberrantly filtered across the glomerular barrier and activated along the tubular system to plasmin. In the collecting duct, plasmin activates epithelial sodium channels (ENaC) proteolytically. Hyperactivity of ENaC could link microalbuminuria/proteinuria to resistant hypertension. Amiloride, an ENaC inhibitor, inhibits urokinase-type plasminogen activator. We hypothesized that amiloride (1) reduces blood pressure (BP); (2) attenuates plasminogen-to...

  20. Secretion of platelet-activating factor by periovulatory ovine follicles

    International Nuclear Information System (INIS)

    Secretion of platelet-activating factor (PAF) in vitro by ovine follicles and ovarian interstitium obtained at various times before, during and after the endogenous preovulatory surge of luteinizing hormone (LH) and ovulation was quantified by radioimmunoassay. Release of PAF by the preovulatory follicle increased within 2 h after initiation of the surge of LH. Capacity for secretion of PAF was greatest at the time of ovulation, then declined thereafter. Production of PAF by ovarian interstitium throughout the periovulatory period was relatively low and did not change with time. It appears that PAF could act as an intrafollicular mediator in the mechanisms of ovulation and(or) luteinization

  1. Plasminogen activator in the rodent brain

    International Nuclear Information System (INIS)

    The cellular origin(s), the biochemical properties and the developmental pattern of the protease activator (PA) were investigated in the rodent brain. PA activity was localized in frozen brain sections by a novel autoradiographic technique. PA levels and electrophoretic mobility were determined in homogenates prepared from major regions of the developing and the mature brain, and both the localization and the specific activity of the enzyme were examined in X-irradiated brain regions. PA activity was shown to be correlated with cell bodies in neuronal-enriched regions and also with endothelial, meningeal and ependymal layers. PA levels increased in a transient manner and at different rates and time periods in the various brain regions that were analyzed. PA in neuronal, but not in epithelial cell layers was affected by X-irradiation and one of the brain PA species had a similar molecular weight to that of neuroblastoma cells. The authors' findings suggest that in the brain PA is produced by neurons and by epithelial cells, and that it may have additional functions to that of thrombolysis both in the developing and the mature brain. (Auth.)

  2. Stimulation of radiation-impaired plasminogen activator release by phorbol ester in aortic endothelial cells

    International Nuclear Information System (INIS)

    Ionizing radiation has been reported to affect the fibrinolytic activity of exposed tissue. With cultured bovine aortic endothelial cells, radiation suppresses the release of plasminogen activator to the conditioned media, with a concomitant increase in intracellular plasminogen activator. Thus study was undertaken to determine whether radiation-impaired plasminogen activator release can be modified by phorbol ester. We exposed cultured bovine aortic endothelial cells to a sterilizing dose of 10 Gy of gamma-rays and found the treatment led to cell injury, as evidenced by an increased release of prelabeled chromium, and to a reduction of plasminogen activator in the conditioned media with elevated intracellular plasminogen activator in irradiated cells. Phorbol ester enhanced plasminogen activator activity in both sham-irradiated and irradiated endothelial cells. It was interesting to note that the increased plasminogen activator in phorbol ester-stimulated sham-irradiated cells was largely retained inside the cell, while it was released to the conditioned media in irradiated cells. Apparently, altered plasminogen activator activity of radiation-sterilized endothelial cells can be modified by exogenous stimuli

  3. Comparative molecular analysis of ovine and bovine Streptococcus uberis isolates.

    Science.gov (United States)

    Gilchrist, T L; Smith, D G E; Fitzpatrick, J L; Zadoks, R N; Fontaine, M C

    2013-02-01

    Streptococcus uberis causes clinical and subclinical mastitis in cattle and sheep, but it is unknown whether the composition of Strep. uberis populations differs between host species. To address this, we characterized a collection of bovine and ovine Strep. uberis isolates with shared geographical and temporal origins by means of an expanded multilocus sequence typing scheme. Among 14 ovine and 35 bovine isolates, 35 allelic profiles were detected. Each allelic profile was associated with a single host species and all but one were new to the multilocus sequence typing database. The median number of new alleles per isolate was higher for ovine isolates than for bovine isolates. None of the ovine isolates belonged to the global clonal complexes 5 or 143, which are commonly associated with bovine mastitis and which have a wide geographical distribution. Ovine isolates also differed from bovine isolates in carriage of plasminogen activator genes, with significantly higher prevalence of pauB in ovine isolates. Isolates that were negative for yqiL, one of the targets of multilocus sequence typing, were found among ovine and bovine isolates and were not associated with a specific sequence type or global clonal complex. One bovine isolate carried a gapC allele that was probably acquired through lateral gene transfer, most likely from Streptococcus salivarius. We conclude that ovine isolates are distinct from bovine isolates of Strep. uberis, and that recombination between isolates from different host species or bacterial species could contribute to changes in virulence gene profiles with relevance for vaccine development. PMID:23200465

  4. Measurement of human tissue-type plasminogen activator by a two-site immunoradiometric assay

    Energy Technology Data Exchange (ETDEWEB)

    Rijken, D.C. (Univ. of Leuven, Belgium); Juhan-Vague, I.; De Cock, F.; Collen, D.

    1983-02-01

    A two-site immunoradiometric assay for human extrinsic (tissue-type) plasminogen activator was developed by using rabbit antibodies raised against plasminogen activator purified from human melanoma cell culture fluid. Samples of 100 ..mu..l containing 1 to 100 ng/ml plasminogen activator were incubated in the wells of polyvinyl chloride microtiter plates coated with antibody. The amount of bound extrinsic plasminogen activator was quantitated by the subsequent binding of /sup 125/I-labeled affinospecific antibody. The mean level of plasma samples taken at rest was 6.6 +/- 2.9 ng/ml (n = 54). This level increased approximately threefold by exhaustive physical exercise, venous occlusion, or infusion of DDAVP. Extrinsic plasminogen activator in plasma is composed of a fibrin-adsorbable and active component (1.9 +/- 1.1 ng/ml, n = 54, in resting conditions) and an inactive component that does not bind to a fibrin clot (probably extrinsic plasminogen activator-proteinase inhibitor complexes). The fibrin-adsorbable fraction increased approximately fivefold to eightfold after physical exercise, venous occlusion, or DDAVP injections. Potential applications of the immunoradiometric assay are illustrated by the measurement of extrinsic plasminogen activator in different tissue extracts, body fluids, and cell culture fluids and in oocyte translation products after injection with mRNA for plasminogen activator.

  5. Measurement of human tissue-type plasminogen activator by a two-site immunoradiometric assay

    International Nuclear Information System (INIS)

    A two-site immunoradiometric assay for human extrinsic (tissue-type) plasminogen activator was developed by using rabbit antibodies raised against plasminogen activator purified from human melanoma cell culture fluid. Samples of 100 ?l containing 1 to 100 ng/ml plasminogen activator were incubated in the wells of polyvinyl chloride microtiter plates coated with antibody. The amount of bound extrinsic plasminogen activator was quantitated by the subsequent binding of 125I-labeled affinospecific antibody. The mean level of plasma samples taken at rest was 6.6 +/- 2.9 ng/ml (n = 54). This level increased approximately threefold by exhaustive physical exercise, venous occlusion, or infusion of DDAVP. Extrinsic plasminogen activator in plasma is composed of a fibrin-adsorbable and active component (1.9 +/- 1.1 ng/ml, n = 54, in resting conditions) and an inactive component that does not bind to a fibrin clot (probably extrinsic plasminogen activator-proteinase inhibitor complexes). The fibrin-adsorbable fraction increased approximately fivefold to eightfold after physical exercise, venous occlusion, or DDAVP injections. Potential applications of the immunoradiometric assay are illustrated by the measurement of extrinsic plasminogen activator in different tissue extracts, body fluids, and cell culture fluids and in oocyte translation products after injection with mRNA for plasminogen activator

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

    Energy Technology Data Exchange (ETDEWEB)

    Shaw, George J [Department of Emergency Medicine, University of Cincinnati College of Medicine, Cincinnati, OH 45267-0769 (United States); Dhamija, Ashima [Department of Emergency Medicine, University of Cincinnati College of Medicine, Cincinnati, OH 45267-0769 (United States); Bavani, Nazli [Department of Emergency Medicine, University of Cincinnati College of Medicine, Cincinnati, OH 45267-0769 (United States); Wagner, Kenneth R [Department of Neurology, University of Cincinnati College of Medicine, Cincinnati, OH 45267-0769 (United States); Holland, Christy K [Department of Biomedical Engineering, University of Cincinnati College of Medicine, Cincinnati, OH 45267-0769 (United States)

    2007-06-07

    Stroke is a devastating disease and a leading cause of death and disability. Currently, the only FDA approved therapy for acute ischemic stroke is the intravenous administration of the thrombolytic medication, recombinant tissue plasminogen activator (tPA). However, this treatment has many contraindications and can have dangerous side effects such as intra-cerebral hemorrhage. These treatment limitations have led to much interest in potential adjunctive therapies, such as therapeutic hypothermia (T {<=} 35 deg. C) and ultrasound enhanced thrombolysis. Such interest may lead to combining these therapies with tPA to treat stroke, however little is known about the effects of temperature on the thrombolytic efficacy of tPA. In this work, we measure the temperature dependence of the fractional clot mass loss {delta}m(T) resulting from tPA exposure in an in vitro human clot model. We find that the temperature dependence is well described by an Arrhenius temperature dependence with an effective activation energy E{sub eff} of 42.0 {+-} 0.9 kJ mole{sup -1}. E{sub eff} approximates the activation energy of the plasminogen-to-plasmin reaction of 48.9 kJ mole{sup -1}. A model to explain this temperature dependence is proposed. These results will be useful in predicting the effects of temperature in future lytic therapies.

  7. 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

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

    Science.gov (United States)

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

    2007-06-01

    Stroke is a devastating disease and a leading cause of death and disability. Currently, the only FDA approved therapy for acute ischemic stroke is the intravenous administration of the thrombolytic medication, recombinant tissue plasminogen activator (tPA). However, this treatment has many contraindications and can have dangerous side effects such as intra-cerebral hemorrhage. These treatment limitations have led to much interest in potential adjunctive therapies, such as therapeutic hypothermia (T 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.

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

    DEFF Research Database (Denmark)

    Knoop, Ann; Andreasen, Peter A; Andersen, J A; Hansen, S; Lænkholm, A-V; Simonsen, A C W; Andersen, J; Overgaard, Jens; Rose, Caspar

    1998-01-01

    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 ...

  10. Biochemical Importance of Glycosylation of Plasminogen Activator Inhibitor-1

    DEFF Research Database (Denmark)

    Gils, Ann; Pedersen, Katrine Egelund; Skottrup, Peter Durand; Christensen, Anni; Naessens, Dominik; Deinum, Johanna; Enghild, Jan Johannes; Declerck, Paul; Andreasen, Peter

    2003-01-01

    The serpin plasminogen activator inhibitor-1 (PAI-1) is a potential target for anti-thrombotic and anti-cancer therapy. PAI-1 has 3 potential sites for N-linked glycosylation. We demonstrate here that PAI-1 expressed recombinantly or naturally by human cell lines display a heterogeneous glycosyla......The serpin plasminogen activator inhibitor-1 (PAI-1) is a potential target for anti-thrombotic and anti-cancer therapy. PAI-1 has 3 potential sites for N-linked glycosylation. We demonstrate here that PAI-1 expressed recombinantly or naturally by human cell lines display a heterogeneous...... glycosylation pattern of the sites at N209 and N265, while that at N329 is not utilised. The IC(50)-values for inactivation of PAI-1 by 4 monoclonal antibodies differed strongly between glycosylated PAI-1 and non-glycosylated PAI-1 expressed in E. coli. For 3 antibodies, an overlap of the epitopes with the...... glycosylation sites could be excluded as explanation for the differential reactivity. The latency transition of non-glycosylated, but not of glycosylated PAI-1, was strongly accelerated by a non-ionic detergent. The different biochemical properties of glycosylated and non-glycosylated PAI-1 depended...

  11. Schistosomes Enhance Plasminogen Activation: The Role of Tegumental Enolase

    Science.gov (United States)

    Figueiredo, Barbara C.; Da'dara, Akram A.; Oliveira, Sergio C.; Skelly, Patrick J.

    2015-01-01

    Schistosoma mansoni is a blood fluke parasite that causes schistosomiasis, a debilitating disease of global public health importance. These relatively large parasites are able to survive prolonged periods in the human vasculature without inducing stable blood clots around them. We show here that the intravascular life stages (schistosomula and adult males and females) can all promote significant plasminogen (PLMG) activation in the presence of tissue plasminogen activator (tPA). This results in the generation of the potent fibrinolytic agent plasmin which could degrade blood clots forming around the worms in vivo. We demonstrate that S. mansoni enolase (SmEno) is a host-interactive tegumental enzyme that, in recombinant form, can bind PLMG and promote its activation. Like classical members of the enolase protein family, SmEno can catalyze the interconversion of 2-phospho-D-glycerate (2-PGA) and phosphoenolpyruvate (PEP). The enzyme has maximal activity at pH 7.5, requires Mg2+ for optimal activity and can be inhibited by NaF but not mefloquin. Suppressing expression of the SmEno gene significantly diminishes enolase mRNA levels, protein levels and surface enzyme activity but, surprisingly, does not affect the ability of the worms to promote PLMG activation. Thus, while SmEno can enhance PLMG activation, our analysis suggests that it is not the only contributor to the parasite’s ability to perform this function. We show that the worms possess several other PLMG-binding proteins in addition to SmEno and these may have a greater importance in schistosome-driven PLMG activation. PMID:26658895

  12. Levels of plasminogen activator inhibitor type 1 and urokinase plasminogen activator receptor in non-small cell lung cancer as measured by quantitative ELISA and semiquantitative immunohistochemistry

    DEFF Research Database (Denmark)

    Pappot, Helle; Skov, Birgit Guldhammer; Pyke, Charles; Grøndahl-Hansen, Jan

    1997-01-01

    The components of the plasminogen activation system have been reported to have prognostic impact in several cancer types, e.g. breast-, colon-, gastric- and lung cancer. Most of these studies have used quantification by enzyme-linked immunosorbent assay (ELISA) on tumour tissue extracts. However, results in non-small cell lung cancer (NSCLC) studies obtained by quantitative ELISA and semiquantitative immunohistochemistry differ. If the prognostic value of the components of the plasminogen activa...

  13. Bovine embryos produce a urokinase-type plasminogen activator.

    Science.gov (United States)

    Berg, D A; Menino, A R

    1992-01-01

    The type of plasminogen activator (PA) secreted by bovine embryos was identified. Day 12-14 embryos were collected from estrus-synchronized, superovulated, and naturally mated crossbred beef cows. Embryos were left intact (E) or microdissected into component embryonic discs (ED) and trophoblastic vesicles (TV). Intact embryos, ED, and TV were pre-cultured for 2 days in Minimum Essential Medium Alpha (MEM alpha) with 10% heat-inactivated fetal calf serum, washed in serum-free MEM alpha, and cultured individually for 5 days in 50 microliters microdrops of MEM alpha with 15 mg/ml bovine serum albumin. At 24 hr intervals, E, ED, and TV were observed for tissue morphology and transferred to fresh microdrops, and medium was recovered and frozen at -20 degrees C. At the end of culture, blastocoelic fluid (BF) and embryonic tissues were recovered and frozen at -20 degrees C. Plasminogen activator concentrations in medium, tissues, and BF were determined by using a caseinolytic assay. Antibodies to urokinase-type PA (anti-uPA) and tissue-type PA (anti-tPA), and the urokinase inhibitor, amiloride (AMR), were used to identify the type of PA produced by bovine embryonic tissues. Intact embryos and TV released more PA (P less than 0.05) than ED, and tissues exhibiting expanded blastocoels released less PA (P less than 0.05) than tissues with collapsed blastocoels. Blastocoelic fluid from TV exhibited more PA (P less than 0.05) activity than from ED. Treatment with anti-uPA decreased PA activity (P less than 0.05) in pooled medium and tissues from E compared to treatment with nonspecific immunoglobulins and anti-tPA.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1562322

  14. Thrombin induction of plasminogen activator-inhibitor in cultured human endothelial cells.

    OpenAIRE

    Gelehrter, T. D.; Sznycer-Laszuk, R

    1986-01-01

    We have examined the effect of thrombin on the activity of plasminogen activator (PA) and plasminogen activator-inhibitor (PA-I) in medium conditioned by primary cultures of human umbilical vein endothelial cells. PA activity was measured by fibrinolytic and esterolytic assays, and total tissue-type PA (tPA) antigen by radioimmunoassay. Net PA-I activity was assayed by titration of human urokinase esterolytic activity. Incubation of confluent endothelial cell cultures with thrombin for 24 h c...

  15. Bicyclic Peptide Inhibitor of Urokinase-Type Plasminogen Activator

    DEFF Research Database (Denmark)

    Roodbeen, Renée; Paaske, Berit; Jiang, Longguang; Jensen, Jan Kristian; Christensen, Anni; Nielsen, Jakob T; Huang, Mingdong; Mulder, Frans A.A.; Nielsen, Niels Christian; Andreasen, Peter; Jensen, Knud Jørgen

    2013-01-01

    The development of protease inhibitors for pharmacological intervention has taken a new turn with the use of peptidebased inhibitors. Here, we report the rational design of bicyclic peptide inhibitors of the serine protease urokinase-type plasminogen activator (uPA), based on the established...... monocyclic peptide, upain-2. It was successfully converted to a bicyclic peptide, without loss of inhibitory properties. The aim was to produce a peptide cyclised by an amide bond with an additional stabilising across-the-ring covalent bond. We expected this bicyclic peptide to exhibit a lower entropic...... burden upon binding. Two bicyclic peptides were synthesised with affinities similar to that of upain-2, and their binding energetics were evaluated by isothermal titration calorimetry. Indeed, compared to upain-2, the bicyclic peptides showed reduced loss of entropy upon binding to uPA. We also...

  16. Physicochemical characteristics of magnetic microspheres containing tissue plasminogen activator

    Energy Technology Data Exchange (ETDEWEB)

    Xie Yumei [Neurocritical Care and Acute Stroke Program, Department of Neurology and Neurosurgery, University of Chicago Pritzker School of Medicine, Chicago, IL 60637 (United States); Kaminski, Michael D. [Chemical Engineering Division, Argonne National Laboratory, Argonne, IL 60439 (United States); Torno, Michael D. [Neurocritical Care and Acute Stroke Program, Department of Neurology and Neurosurgery, University of Chicago Pritzker School of Medicine, Chicago, IL 60637 (United States); Finck, Martha R. [Chemical Engineering Division, Argonne National Laboratory, Argonne, IL 60439 (United States); Liu Xianqiao [Neurocritical Care and Acute Stroke Program, Department of Neurology and Neurosurgery, University of Chicago Pritzker School of Medicine, Chicago, IL 60637 (United States); Rosengart, Axel J. [Neurocritical Care and Acute Stroke Program, Department of Neurology and Neurosurgery, University of Chicago Pritzker School of Medicine, Chicago, IL 60637 (United States)]. E-mail: arosenga@neurology.bsd.uchicago.edu

    2007-04-15

    As a first step toward improving the treatment of stroke, we are developing a magnetic carrier system to target tissue plasminogen activator (tPA) to a thrombosis. We report the characterization of biodegradable microspheres containing tPA and magnetic iron oxide. The resultant microspheres were superparamagnetic with a magnetization of 6.9-8.7emu/g. We encapsulated 5% tPA by mass which eluted from the microspheres to produce a solution concentration of 5.3-19.6{mu}g/mL in tPA, which exceeds the theoretical thrombolysis concentration. Although smaller microspheres will be necessary for in vivo experiments, we have shown that sufficient tPA can be encapsulated and released in a magnetic matrix.

  17. Structural and functional peculiarities of plasminogen activator inhibitor PAI-1

    Directory of Open Access Journals (Sweden)

    Kondratuk A. S.

    2010-07-01

    Full Text Available PAI-1, an important component of the hemostasis system, is a specific inhibitor of both urokinase type and tissue type plasminogen activators. PAI-1 belongs to the serpin family. The interaction between somatomedin-like domain of vitronectin and PAI-1 leads to stabilization of the latter. PAI-1 latency transition is related to the conformational changes in the reactive central loop. The inhibitory mechanism of PAI-1 is in accordance with the classic scheme of serpin action. PAI-1 blocks the adhesion mediated by UPA and integrins, so this inhibitor plays an important role in adhesion process and angiogenesis. An altered PAI-1level is associated with the development of cardiovascular diseases, kidney fibrosis, diabetis, cancerogenesis.

  18. Physicochemical characteristics of magnetic microspheres containing tissue plasminogen activator

    Science.gov (United States)

    Xie, Yumei; Kaminski, Michael D.; Torno, Michael D.; Finck, Martha R.; Liu, Xianqiao; Rosengart, Axel J.

    2007-04-01

    As a first step toward improving the treatment of stroke, we are developing a magnetic carrier system to target tissue plasminogen activator (tPA) to a thrombosis. We report the characterization of biodegradable microspheres containing tPA and magnetic iron oxide. The resultant microspheres were superparamagnetic with a magnetization of 6.9-8.7 emu/g. We encapsulated 5% tPA by mass which eluted from the microspheres to produce a solution concentration of 5.3- 19.6 ?g/mL in tPA, which exceeds the theoretical thrombolysis concentration. Although smaller microspheres will be necessary for in vivo experiments, we have shown that sufficient tPA can be encapsulated and released in a magnetic matrix.

  19. Keeping the blood flowing—plasminogen activator genes and feeding behavior in vampire bats

    Science.gov (United States)

    Tellgren-Roth, Åsa; Dittmar, Katharina; Massey, Steven E.; Kemi, Cecilia; Tellgren-Roth, Christian; Savolainen, Peter; Lyons, Leslie A.; Liberles, David A.

    2009-01-01

    The blood feeding vampire bats emerged from New World leaf-nosed bats that fed on fruit and insects. Plasminogen activator, a serine protease that regulates blood coagulation, is known to be expressed in the saliva of Desmodus rotundus (common vampire bat) and is thought to be a key enzyme for the emergence of blood feeding in vampire bats. To better understand the evolution of this biological function, we studied the plasminogen activator (PA) genes from all vampire bat species in light of their feeding transition to bird and subsequently mammalian blood. We include the rare species Diphylla ecaudata and Diaemus youngi, where plasminogen activator had not previously been studied and demonstrate that PA gene duplication observed in Desmodus is not essential to the vampire phenotype, but relates to the emergence of predominant mammalian blood feeding in this species. Plasminogen activator has evolved through gene duplication, domain loss, and sequence evolution leading to change in fibrin-specificity and susceptibility to plasminogen activator inhibitor-1. Before undertaking this study, only the four plasminogen activator isoforms from Desmodus were known. The evolution of vampire bat plasminogen activators can now be linked phylogenetically to the transition in feeding behavior among vampire bat species from bird to mammalian blood.

  20. Urokinase plasminogen activator and plasminogen activator inhibitor type-1 in nonsmall-cell lung cancer: relation to prognosis and angiogenesis

    DEFF Research Database (Denmark)

    Offersen, Birgitte Vrou; Pfeiffer, Per

    2007-01-01

    BACKGROUND: Urokinase plasminogen activator (uPA) and plasminogen activator inhibitor type-1 (PAI-1) have previously been suggested as prognostic markers in nonsmall-cell lung carcinomas (NSCLC). We investigate whether uPA and PAI-1 are prognostic markers in NSCLC and whether they are related to angiogenesis. MATERIALS AND METHODS: Frozen tumour tissue from surgical specimens from 118 previously untreated patients diagnosed with NSCLC in the period 1984-1991 were investigated. All patients were treated with surgery, and no chemo- or radiotherapy was given. UPA and PAI-1 levels were assessed using a sandwich ELISA method. RESULTS: Both uPA and PAI-1 were independent of classical histopathological parameters as well as of microvessel density and vascular pattern. Using death within the first 5 years as endpoint, neither of the factors were prognostic markers in univariate analysis, however, significantly higher levels of uPA and PAI-1 were seen in tumours with an angiogenic vascular pattern. In multivariate analysis, high disease stage (P<0.0001), adenocarcinoma (P=0.007), old age (P=0.02), and presence of an angiogenic pattern (P=0.05) were identified as independent markers of death within 5 years. CONCLUSIONS: The present study investigated the prognostic role of the protein levels of uPA and PAI-1 in 118 tumour specimens from patients diagnosed with NSCLC. Neither of the factors were identified as prognostic markers when evaluated with survival as endpoint. However, in tumours previously identified as non-angiogenic we found significantly lower contents of both uPA and PAI-1 as compared to angiogenic tumours, thus we hypothesize that uPA and PAI-1 stimulate angiogenesis in NSCLC. Udgivelsesdato: 2007-Apr

  1. Simvastatin suppresses dexamethasone-induced secretion of plasminogen activator inhibitor-1 in human bone marrow adipocytes

    OpenAIRE

    Baba Hideo; Fukushima Tatsuya; Goto Hisataka; Hozumi Akira; Osaki Makoto; Sakamoto Kazutaka; Shindo Hiroyuki

    2011-01-01

    Abstract Background Osteonecrosis of the femoral head is a common complication of high-dose glucocorticoid treatment. Intravascular thrombosis is thought to be associated with the ischemic state of the femoral head. Plasminogen activator inhibitor-1 (PAI-1) is an adipokine, which are physiologically active substances secreted from visceral and subcutaneous adipocytes. PAI-1 suppresses fibrinolysis by binding tissue-type plasminogen activator. Several reports have described the relationship be...

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

    DEFF Research Database (Denmark)

    Sehestedt, T; Lyngbæk, S

    2011-01-01

    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.

  3. Intracameral tissue plasminogen activator to prevent severe fibrinous effusion after congenital cataract surgery

    OpenAIRE

    Siatiri, H; Beheshtnezhad, A H; Asghari, H.; Siatirit, N; S. Moghimi; Piri, N

    2005-01-01

    Background/aims: To evaluate the efficacy of intracameral recombinant tissue plasminogen activator (r-TPA) in prevention of fibrinous effusion after lensectomy, anterior vitrectomy, and posterior chamber intraocular lens (PCIOL) implantation in patients with congenital cataract.

  4. Roles for chloride ion and fibrinogen in the activation of [Glu1]plasminogen in human plasma.

    OpenAIRE

    Gaffney, P J; Urano, T.; de Serrano, V S; Mahmoud-Alexandroni, M; Metzger, A R; Castellino, F J

    1988-01-01

    Using two-dimensional immunoelectrophoresis and an antibody to alpha 2-antiplasmin, we assessed the plasmin generated in serum under different conditions as the plasmin-alpha 2-antiplasmin complex. Activation in serum of human [Glu1]plasminogen ([Glu1]Pg) by recombinant tissue plasminogen activator was inhibited by the normal serum levels of Cl- and was enhanced by physiological levels of fibrinogen in the presence or absence of Cl-. These results agree with the recognized ability of Cl- to i...

  5. Urokinase plasminogen activator receptor on invasive cancer cells: A prognostic factor in distal gastric adenocarcinoma

    DEFF Research Database (Denmark)

    Alpizar, Warner Enrique Alpizar; Christensen, Ib Jarle; Santoni-Rugiu, Eric; Skarstein, Arne; Ovrebo, Kjell; Laerum, Ole Didrik; Illemann, Martin; Lærum, Ole D

    2012-01-01

    Gastric cancer is the second cancer causing death worldwide. The five-year survival for this malignancy is below 25% and few parameters have shown an impact on the prognosis of the disease. The receptor for urokinase plasminogen activator (uPAR) is involved in extracellular matrix degradation by mediating cell surface associated plasminogen activation, and its presence on gastric cancer cells is linked to micrometastasis and poor prognosis. Using immunohistochemistry, the prognostic significance...

  6. Human prostate carcinoma cells express enzymatic activity that converts human plasminogen to the angiogenesis inhibitor, angiostatin

    DEFF Research Database (Denmark)

    Gately, S; Twardowski, P; Stack, M S; Patrick, M; Boggio, L; Cundiff, D L; Schnaper, H W; Madison, L; Volpert, O; Bouck, N; Enghild, J; Kwaan, H C; Soff, G A

    1996-01-01

    Angiostatin is an inhibitor of angiogenesis and metastatic growth that is found in tumor-bearing animals and can be generated in vitro by the proteolytic cleavage of plasminogen. The mechanism by which angiostatin is produced in vivo has not been defined. We now demonstrate that human prostate carcinoma cell lines (PC-3, DU-145, and LN-CaP) express enzymatic activity that can generate bioactive angiostatin from purified human plasminogen or plasmin. Affinity purified PC-3-derived angiostatin inh...

  7. Tissue-type plasminogen activator in somatostatin cells of rat pancreas and hypothalamus

    DEFF Research Database (Denmark)

    Kristensen, P; Larsson, L I; Danø, K; Nielsen, Jens Høiriis

    1987-01-01

    Plasminogen activators (PAs) proteolytically convert plasminogen to plasmin, which, in turn, can degrade most proteins. This system has been implicated in a variety of biological processes. Using immunocytochemical methods, we here describe the localization of tissue-type PA (t-PA) in rat somatostatin cells. In the pancreatic islets, a low number of strongly t-PA-immunoreactive cells was found. By sequential staining, we found these cells to constitute a subpopulation of the somatostatin cells. ...

  8. Pneumatic displacement without tissue plasminogen activator in premacular subhyaloid hemorrhage

    Directory of Open Access Journals (Sweden)

    Rumita S. Kadarisman

    2007-06-01

    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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2010-08-13

    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.

  10. Plasminogen activator inhibitor-1 in the evolution of stroke

    Directory of Open Access Journals (Sweden)

    Jovanovi? Zagorka B.

    2004-01-01

    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.

  11. Complexes between tissue-type plasminogen activator and proteinase inhibitors in human plasma, identified with an immunoradiometric assay

    International Nuclear Information System (INIS)

    Extrinsic (tissue-type) plasminogen activator antigen in human plasma, as measured by a two-site immunoradiometric assay, is composed of a fibrin-adsorbable and a nonadsorbable fraction. Gel filtration on Ultrogel AcA 44 in 1.6M KSCN of the fibrin-adsorbable fraction showed a peak with M/sub r/ approx. =70,000, which contained plasminogen activator activity and was assumed to represent free extrinsic plasminogen activator. The nonadsorbable fraction showed a broad peak with M/sub r/ approx. =140,000 without plasminogen activator activity. Overnight incubation at 370C of postexercise plasma revealed a shift of the M/sub r/ approx. =70,000 peak to the M/sub r/ approx. =140,000 position, suggesting that the M/sub r/ approx. =140,000 peak consists of extrinsic plasminogen activator-protease inhibitor complex(es). ?2-Antiplasmin is the main inhibitor of extrinsic plasminogen activator in plasma and is probably responsible for the generation of the M/sub r/ approx. =140,000 component. A possible involvement of other plasma proteinase inhibitors was explored by incubation of 125I-labeled extrinsic plasminogen activator in ?2-antiplasmin-depleted plasma. A complex was formed with a t1/2 of about 1 hr, which was identified by immunoprecipitation as extrinsic plasminogen activator-?2-antiplasmin complex. Additional evidence for the presence of extrinsic plasminogen activator complexes with ?2-antiplasmin and ?1-antitrypsin in plasma was obtained from two-site immunoradiometric assays. It was concluded that plasma contains both free extrinsic plasminogen activator and plasminogen activator complexes with ?2-antiplasmin and ?1-antitrypsin. These complexes are also present in plasma collected on the active site inhibitor, D-Phe-Pro-Arg-CH2Cl, at rest and after exercise and are therefore assumed to circulate in vivo

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

    Directory of Open Access Journals (Sweden)

    Sebastian Zukowski

    2008-06-01

    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.

  13. Secretion of tissue-type plasminogen activator and plasminogen activator inhibitor by Rickettsia conorii- and Rickettsia rickettsii-infected cultured endothelial cells.

    OpenAIRE

    Drancourt, M.; Alessi, M.C.; Levy, P. Y.; Juhan-Vague, I; Raoult, D

    1990-01-01

    Hemostasis abnormalities have been described in patients with Mediterranean spotted fever and Rocky Mountain spotted fever. Evidence of the activation of the fibrinolytic system has been obtained in both diseases. After experimental Rocky Mountain spotted fever, an elevated level of fibrinogen was found in parallel with the activation of the fibrinolytic system and transient elevation of the tissue-type plasminogen activator. Later protein is mainly synthesized by endothelial cells. The abili...

  14. Hyperthermia stimulates plasminogen activator inhibitor type 1 expression in human umbilical vein endothelial cells in vitro.

    OpenAIRE

    Wojta, J.; Holzer, M.; Hufnagl, P.; Christ, G.; Hoover, R. L.; Binder, B R

    1991-01-01

    The effect of exposure to hyperthermia on the fibrinolytic potential of human umbilical vein endothelial cells (HUVEC) in culture was studied. HUVEC responded to exposure to 42 degrees C with a time-dependent increase in plasminogen activator inhibitor type 1 (PAI-1) activity and antigen accompanied by a four- to fivefold increase in PAI-1 specific m-RNA and a decrease in tissue-type plasminogen activator (t-PA) antigen. The effect of 8 hours exposure to hyperthermia on PAI-1 activity and ant...

  15. Receptor-mediated endocytosis of plasminogen activators and activator/inhibitor complexes

    DEFF Research Database (Denmark)

    Andreasen, P A; Sottrup-Jensen, L

    1994-01-01

    Recent findings have elucidated the mechanism for clearance from the extracellular space of the two types of plasminogen activators, urokinase-type plasminogen activator (u-PA) and tissue-type plasminogen activator (t-PA), and their type-1 inhibitor (PAI-1). Activator/PAI-1 complexes and uncomplexed t-PA bind to the multi-ligand receptors alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein (alpha 2MR) and epithelial glycoprotein 330 (gp330). These receptors mediate endocytosis and degradation of u-PA/PAI-1 complex bound to the glycosyl phosphatidyl inositol-anchored urokinase receptor (u-PAR) on cell surfaces, and participate, in cooperation with other receptors, in hepatic clearance of activator/PAI-1 complexes and uncomplexed t-PA from blood plasma. The alpha 2MR- and gp330-mediated endocytosis of a ligand (u-PA/PAI-1 complex) initially bound to another receptor (u-PAR) is a novel kind of interaction between membrane receptors. Binding to alpha 2MR and gp330 is a novel kind of molecular recognition of serine proteinases and serpins.

  16. Targeting of tumor cells by cell surface urokinase plasminogen activator-dependent anthrax toxin.

    Science.gov (United States)

    Liu, S; Bugge, T H; Leppla, S H

    2001-05-25

    Urokinase plasminogen activator receptor (uPAR) binds pro-urokinase plasminogen activator (pro-uPA) and thereby localizes it near plasminogen, causing the generation of active uPA and plasmin on the cell surface. uPAR and uPA are overexpressed in a variety of human tumors and tumor cell lines, and expression of uPAR and uPA is highly correlated to tumor invasion and metastasis. To exploit these characteristics in the design of tumor cell-selective cytotoxins, we constructed mutated anthrax toxin-protective antigen (PrAg) proteins in which the furin cleavage site is replaced by sequences cleaved specifically by uPA. These uPA-targeted PrAg proteins were activated selectively on the surface of uPAR-expressing tumor cells in the presence of pro-uPA and plasminogen. The activated PrAg proteins caused internalization of a recombinant cytotoxin, FP59, consisting of anthrax toxin lethal factor residues 1-254 fused to the ADP-ribosylation domain of Pseudomonas exotoxin A, thereby killing the uPAR-expressing tumor cells. The activation and cytotoxicity of these uPA-targeted PrAg proteins were strictly dependent on the integrity of the tumor cell surface-associated plasminogen activation system. We also constructed a mutated PrAg protein that selectively killed tissue plasminogen activator-expressing cells. These mutated PrAg proteins may be useful as new therapeutic agents for cancer treatment. PMID:11278833

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

    DEFF Research Database (Denmark)

    Oxlund, Christina S; Buhl, KB

    2014-01-01

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

  18. Staphylokinase Control of Staphylococcus aureus Biofilm Formation and Detachment Through Host Plasminogen Activation.

    Science.gov (United States)

    Kwiecinski, Jakub; Peetermans, Marijke; Liesenborghs, Laurens; Na, Manli; Björnsdottir, Halla; Zhu, Xuefeng; Jacobsson, Gunnar; Johansson, Bengt R; Geoghegan, Joan A; Foster, Timothy J; Josefsson, Elisabet; Bylund, Johan; Verhamme, Peter; Jin, Tao

    2016-01-01

    Staphylococcus aureus biofilms, a leading cause of persistent infections, are highly resistant to immune defenses and antimicrobial therapies. In the present study, we investigated the contribution of fibrin and staphylokinase (Sak) to biofilm formation. In both clinical S. aureus isolates and laboratory strains, high Sak-producing strains formed less biofilm than strains that lacked Sak, suggesting that Sak prevents biofilm formation. In addition, Sak induced detachment of mature biofilms. This effect depended on plasminogen activation by Sak. Host-derived fibrin, the main substrate cleaved by Sak-activated plasminogen, was a major component of biofilm matrix, and dissolution of this fibrin scaffold greatly increased susceptibility of biofilms to antibiotics and neutrophil phagocytosis. Sak also attenuated biofilm-associated catheter infections in mouse models. In conclusion, our results reveal a novel role for Sak-induced plasminogen activation that prevents S. aureus biofilm formation and induces detachment of existing biofilms through proteolytic cleavage of biofilm matrix components. PMID:26136471

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

    Directory of Open Access Journals (Sweden)

    Kavian Ghandehari

    2013-04-01

    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.

  20. Urokinase-type plasminogen activator does not affect in vitro bovine embryo development and quality.

    Science.gov (United States)

    Krania, Fotini; Dovolou, Eleni; Rekkas, Constantinos A; Heras, Sonia; Pappas, Ioannis; Soom, Ann Van; Amiridis, Georgios S

    2015-06-01

    The effects of modification of the in vitro embryo culture media (IVC) with the addition of urokinase-type plasminogen activator (u-PA) on the yield and/or quality of bovine embryos were examined in two experiments. In Experiment 1, denuded embryos were cultured in semi-defined synthetic oviductal fluid (SOF) for seven days, while in Experiment 2 embryos were co-cultured with cumulus cell monolayer in a serum-containing SOF medium. Plasminogen activator activity (PAA) and plasminogen activator inhibition (PAI) were determined in all spent IVC media. At the activity used (5 IU/ml), u-PA had no effect either on in vitro embryo production rates or on embryo quality as revealed by gene expression analysis of 10 important mRNA transcripts related to apoptosis, oxidation, implantation and metabolism. PAA and PAI analysis indicated the need for wellbalanced plasminogen activators and inhibitors as a culture environment for embryo development. However, more research is needed to unveil the mechanism by which u-PA is involved in in vitro embryo production systems. PMID:26051263

  1. Plasminogen activator inhibitor type 1 regulates microglial motility and phagocytic activity

    Directory of Open Access Journals (Sweden)

    Jeon Hyejin

    2012-06-01

    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.

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

    Directory of Open Access Journals (Sweden)

    Merih Ozgen

    2012-11-01

    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.

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

    DEFF Research Database (Denmark)

    Pedersen, Katrine E; Einholm, Anja P; Christensen, Anni; Schack, Lotte; Wind, Troels; Kenney, John M; Andreasen, Peter A

    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...

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

    Scientific Electronic Library Online (English)

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

    2012-11-01

    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. 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.

  5. Presence of urokinase-type plasminogen activator (u-PA) in tissue extracts of antrochoanal polyp.

    Science.gov (United States)

    Yamashiro, Y; Nakamura, M; Huang, G W; Kosugi, T

    1992-09-01

    Using a biochemical technique, the authors characterized and identified a plasminogen activator (PA) derived from tissue extracts of antrochoanal polyp (AP) and paranasal mucous membrane (PMM) with chronic sinusitis. The results of fibrin zymography indicated that the tissue extracts of AP revealed two lytic zones and that those of PMM revealed a single lytic zone on fibrin-agarose plates. One of the AP zones exhibited the same relative mobility as the PMM zone (molecular weight: 65 kd), while the other AP zone had a smaller molecular weight (about 54 kd). Goat immunoglobulin G (IgG) fraction of antihuman uterine tissue-type plasminogen activator (t-PA) inhibited the 65-kd lytic zones of AP and PMM. Antihuman low-molecular-weight urokinase inhibited only the 54-kd lytic zone of AP, and nonspecific goat IgG failed to inhibit any of the lytic zones. On the other hand, 10(-2) mol trans 4-(aminomethyl)cyclohexane-carboxylic acid (t-AMCHA) inhibited all of the lytic zones. No lytic zones could be observed on plasminogen-free fibrin-agarose plates. These findings confirmed that the tissue extracts of PMM contained t-PA, and that those of AP contained both t-PA and urokinase-type plasminogen activator (u-PA). In addition, it appeared that u-PA in inflammatory tissue was related to proliferative changes of the mucous membrane. PMID:1518351

  6. Analysis of five streptokinase formulations using the euglobulin lysis test and the plasminogen activation assay

    Scientific Electronic Library Online (English)

    L.T., Couto; J.L., Donato; G. de, Nucci.

    2004-12-01

    Full Text Available Streptokinase, a 47-kDa protein isolated and secreted by most group A, C and G ß-hemolytic streptococci, interacts with and activates human protein plasminogen to form an active complex capable of converting other plasminogen molecules to plasmin. Our objective was to compare five streptokinase form [...] ulations commercially available in Brazil in terms of their activity in the in vitro tests of euglobulin clot formation and of the hydrolysis of the plasmin-specific substrate S-2251™. Euglobulin lysis time was determined using a 96-well microtiter plate. Initially, human thrombin (10 IU/ml) and streptokinase were placed in individual wells, clot formation was initiated by the addition of plasma euglobulin, and turbidity was measured at 340 nm every 30 s. In the second assay, plasminogen activation was measured using the plasmin-specific substrate S-2251™. Streptase™ was used as the reference formulation because it presented the strongest fibrinolytic activity in the euglobulin lysis test. The Unitinase™ and Solustrep™ formulations were the weakest, showing about 50% activity compared to the reference formulation. All streptokinases tested activated plasminogen but significant differences were observed. In terms of total S-2251™ activity per vial, Streptase™ (75.7 ± 5.0 units) and Streptonase™ (94.7 ± 4.6 units) had the highest activity, while Unitinase™ (31.0 ± 2.4 units) and Strek™ (32.9 ± 3.3 units) had the weakest activity. Solustrep™ (53.3 ± 2.7 units) presented intermediate activity. The variations among the different formulations for both euglobulin lysis test and chromogenic substrate hydrolysis correlated with the SDS-PAGE densitometric results for the amount of 47-kDa protein. These data show that the commercially available clinical streptokinase formulations vary significantly in their in vitro activity. Whether these differences have clinical implications needs to be investigated.

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

    DEFF Research Database (Denmark)

    Bøtkjær, Kenneth Alrø; Fogh, Sarah; Bekes, Erin C; Chen, Zhuo; Blouse, Grant E; Jensen, Janni M; Mortensen, Kim; Huang, Mingdong; Deryugina, Elena; Quigley, James P; Declerck, Paul J; Andreasen, Peter A

    2011-01-01

    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 a...

  8. In vitro stimulation of plasminogen activator release from vein walls by adrenaline.

    OpenAIRE

    Kjaeldgaard, A; Kjaeldgaard, M

    1986-01-01

    The effect of adrenaline on plasminogen activator release was studied in vitro in human vein biopsy specimens, in which the fibrinolytic activity was determined according to the fibrin slide technique. The tissue slides were covered with a thin fibrin film containing 10(-9) and 10(-7) M adrenaline and exposed for 30 to 60 minutes. In both concentrations highly significant (p less than 0.001) enhancement of fibrinolytic activity was shown, and the enhancement of fibrinolysis was most pronounce...

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

    Directory of Open Access Journals (Sweden)

    Gruzdeva O

    2013-08-01

    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

  10. 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

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

    DEFF Research Database (Denmark)

    Nielsen, Jimmi; Røge, Rasmus; Pristed, Sofie Gry; Viuff, Anne Grethe; Ullum, Henrik; Thørner, Lise Wegner; Werge, Thomas; Vang, Torkel

    2015-01-01

    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 dis...

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

    OpenAIRE

    Lobov, Sergei

    2004-01-01

    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...

  13. 2-Amidino Analogs of Glycine-Amiloride Conjugates: Inhibitors of Urokinase-type Plasminogen Activator

    OpenAIRE

    Massey, Archna P.; Harley, William R.; Pasupuleti, Nagarekha; GORIN, FREDRIC A.; Nantz, Michael H

    2012-01-01

    The relative non-toxicity of the diuretic amiloride, coupled with its selective inhibition of the protease urokinase plasminogen activator (uPA), makes this compound class attractive for structure-activity studies. Herein we substituted the C(2)-acylguanidine of C(5)-glycyl-amiloride with amidine and amidoxime groups. The data show the importance of maintaining C(5)-hydrophobicity. The C(5)-benzylglycine analogs containing either C(2)-acylguanidine or amidine inhibited uPA with an IC50 rangin...

  14. Candesartan reduces the hemorrhage associated with delayed tissue plasminogen activator treatment in rat embolic stroke

    OpenAIRE

    Ishrat, Tauheed; Pillai, Bindu; Ergul, Adviye; Hafez, Sherif; Susan C. Fagan

    2013-01-01

    We have previously reported that angiotensin receptor blockade reduces reperfusion hemorrhage in a suture occlusion model of stroke, despite increasing matrix metalloproteinase (MMP-9) activity. We hypothesized that candesartan will also decrease hemorrhage associated with delayed (6h) tissue plasminogen activator (tPA) administration after embolic stroke, widening the therapeutic time window of tPA. Adult male Wistar rats were subjected to embolic middle cerebral artery occ...

  15. Complexes between tissue-type plasminogen activator and proteinase inhibitors in human plasma, identified with an immunoradiometric assay

    Energy Technology Data Exchange (ETDEWEB)

    Rijken, D.C. (Univ. of Leuven, Belgium); Juhan-Vague, I.; Collen, D.

    1983-02-01

    Extrinsic (tissue-type) plasminogen activator antigen in human plasma, as measured by a two-site immunoradiometric assay, is composed of a fibrin-adsorbable and a nonadsorbable fraction. Gel filtration on Ultrogel AcA 44 in 1.6M KSCN of the fibrin-adsorbable fraction showed a peak with M/sub r/ approx. =70,000, which contained plasminogen activator activity and was assumed to represent free extrinsic plasminogen activator. The nonadsorbable fraction showed a broad peak with M/sub r/ approx. =140,000 without plasminogen activator activity. Overnight incubation at 37/sup 0/C of postexercise plasma revealed a shift of the M/sub r/ approx. =70,000 peak to the M/sub r/ approx. =140,000 position, suggesting that the M/sub r/ approx. =140,000 peak consists of extrinsic plasminogen activator-protease inhibitor complex(es). ..cap alpha../sub 2/-Antiplasmin is the main inhibitor of extrinsic plasminogen activator in plasma and is probably responsible for the generation of the M/sub r/ approx. =140,000 component. A possible involvement of other plasma proteinase inhibitors was explored by incubation of /sup 125/I-labeled extrinsic plasminogen activator in ..cap alpha../sub 2/-antiplasmin-depleted plasma. A complex was formed with a t1/2 of about 1 hr, which was identified by immunoprecipitation as extrinsic plasminogen activator-..cap alpha../sub 2/-antiplasmin complex. Additional evidence for the presence of extrinsic plasminogen activator complexes with ..cap alpha../sub 2/-antiplasmin and ..cap alpha../sub 1/-antitrypsin in plasma was obtained from two-site immunoradiometric assays. It was concluded that plasma contains both free extrinsic plasminogen activator and plasminogen activator complexes with ..cap alpha../sub 2/-antiplasmin and ..cap alpha../sub 1/-antitrypsin. These complexes are also present in plasma collected on the active site inhibitor, D-Phe-Pro-Arg-CH/sub 2/Cl, at rest and after exercise and are therefore assumed to circulate in vivo. (JMT)

  16. Simultaneous Combined Intravenous Recombinant Tissue Plasminogen Activator and Endovascular Therapy for Hyperacute Middle Cerebral Artery M1 occlusion

    OpenAIRE

    Toyota, S.; Sugiura, S; Iwaisako, K.

    2011-01-01

    We investigated the efficacy and safety of combined intravenous (IV) recombinant tissue plasminogen activator (rtPA) and simultaneous endovascular therapy (ET) for hyperacute middle cerebral artery (MCA) M1 occlusion.

  17. 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)

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

    2011-06-01

    Full Text Available 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.

  18. Urokinase plasminogen activator receptor is expressed in invasive cells in gastric carcinomas from high- and low-risk countries

    DEFF Research Database (Denmark)

    Alpízar-Alpízar, Warner; Nielsen, Boye Schnack; Sierra, Rafaela; Illemann, Martin; Ramírez, Jose A; Arias, Adriana; Durán, Sundry; Skarstein, Arne; Ovrebo, Kjell; Lund, Leif R; Laerum, Ole D

    2010-01-01

    Gastric cancer is the second cancer causing death worldwide. Both incidence and mortality rates vary according to geographical regions. The receptor for urokinase plasminogen activator (uPAR) is involved in extracellular matrix degradation by mediating cell surface associated plasminogen activation, and its presence on gastric cancer cells is linked to micro-metastasis and poor prognosis. Immunohistochemical analyses of a set of 44 gastric cancer lesions from Costa Rica showed expression of uPAR...

  19. Tissue-type plasminogen activator-binding RNA aptamers inhibiting low-density lipoprotein receptor family-mediated internalisation

    DEFF Research Database (Denmark)

    Bjerregaard, N; Bøtkjær, K A; Helsen, N; Andreasen, P A; Dupont, Daniel Miotto

    2015-01-01

    Recombinant tissue-type plasminogen activator (tPA, trade name Alteplase), currently the only drug approved by the US Food and Drug Administration and the European Medicines Agency for the treatment of cerebral ischaemic stroke, has been implicated in a number of adverse effects reportedly mediated by interactions with the low-density lipoprotein (LDL) family receptors, including neuronal cell death and an increased risk of cerebral haemorrhage. The tissue-type plasminogen activator is the princ...

  20. Urokinase plasminogen activator receptor on invasive cancer cells: A prognostic factor in distal gastric adenocarcinoma

    DEFF Research Database (Denmark)

    Alpizar, Warner Enrique Alpizar; Christensen, Ib Jarle; Santoni-Rugiu, Eric; Skarstein, Arne; Ovrebo, Kjell; Laerum, Ole Didrik; Illemann, Martin; Lærum, Ole D

    2012-01-01

    PAR was expressed by neoplastic cells, macrophages, myofibroblasts and neutrophils in both intestinal and diffuse subtypes. No association was demonstrated between the expression of uPAR on cancer cells and histological subtype (p = 0.64) or TNM stage (p = 0.75). Univariate analysis revealed a significant......Gastric cancer is the second cancer causing death worldwide. The five-year survival for this malignancy is below 25% and few parameters have shown an impact on the prognosis of the disease. The receptor for urokinase plasminogen activator (uPAR) is involved in extracellular matrix degradation by...... mediating cell surface associated plasminogen activation, and its presence on gastric cancer cells is linked to micrometastasis and poor prognosis. Using immunohistochemistry, the prognostic significance of uPAR was evaluated in tissue samples from a retrospective series of 95 gastric cancer patients. u...

  1. Abrogation of Plasminogen Activator Inhibitor-1-Vitronectin Interaction Ameliorates Acute Kidney Injury in Murine Endotoxemia

    OpenAIRE

    Kamlesh K. Gupta; Donahue, Deborah L.; Sandoval-Cooper, Mayra J.; Castellino, Francis J.; Ploplis, Victoria A

    2015-01-01

    Sepsis-induced acute kidney injury (AKI) contributes to the high mortality and morbidity in patients. Although the pathogenesis of AKI during sepsis is poorly understood, it is well accepted that plasminogen activator inhibitor-1 (PAI-1) and vitronectin (Vn) are involved in AKI. However, the functional cooperation between PAI-1 and Vn in septic AKI has not been completely elucidated. To address this issue, mice were utilized lacking either PAI-1 (PAI-1?/?) or expressing a PAI-1-mutant (PAI-1R...

  2. The myofibroblast is the predominant plasminogen activator inhibitor-1-expressing cell type in human breast carcinomas

    DEFF Research Database (Denmark)

    Offersen, Birgitte Vrou; Nielsen, Boye Schnack; Høyer-Hansen, Gunilla; Rank, Fritz; Hamilton-Dutoit, Stephen; Overgaard, Jens; Andreasen, Peter A

    2003-01-01

    The tumor level of plasminogen activator inhibitor-1 (PAI-1) is an informative biochemical marker of a poor prognosis in several cancer types. However, the tumor biological functions of PAI-1 and the identity of PAI-1-expressing cells are controversial. With the aim of immunohistochemically localizing PAI-1 in formalin-fixed, paraffin-embedded invasive ductal breast carcinoma samples, we raised new polyclonal antibodies against PAI-1 from different expression systems. The antibodies were affinit...

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

    OpenAIRE

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

    2003-01-01

    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...

  4. Plasminogen activators and their inhibitors in synovial fluids from normal, osteoarthritis, and rheumatoid arthritis knees.

    OpenAIRE

    Belcher, C.; Fawthrop, F.; Bunning, R; Doherty, M.

    1996-01-01

    OBJECTIVES: To establish baseline concentrations of plasminogen activators and their inhibitors in normal knee synovial fluids, and to compare them with well characterised osteoarthritis (OA) and rheumatoid arthritis (RA) knee fluids. METHODS: A total of 26 normal subjects, 71 patients with OA, and 17 patients with RA underwent knee aspiration. Patients with OA were subclassified according to presence of nodal generalised OA (NGOA) and synovial fluid calcium pyrophosphate crystals. Clinical a...

  5. Prognostic value of urokinase plasminogen activator in primary breast carcinoma: comparison of two immunoassay methods.

    OpenAIRE

    Bouchet, C; Spyratos, F.; Hacène, K.; Durcos, L.; Bécette, V.; Oglobine, J.

    1998-01-01

    Urokinase-type plasminogen activator (uPA) is a potentially important prognostic factor in breast cancer for identifying patients at high risk of recurrence. This retrospective study assessed two enzyme-linked immunosorbent assay (ELISA) methods measuring uPA antigen levels in 499 primary breast cancer cytosols. Both uPA methods were applied to cytosols used routinely for oestrogen (ER) and progesterone (PgR) receptor assays. uPA was determined using a classical ELISA method (Imubind; America...

  6. Stereotactic fibrinolysis of spontaneous intracerebral hematoma using infusion of recombinant tissue plasminogen activator

    OpenAIRE

    Nasser José Augusto; Falavigna Asdrubal; Bezerra Márcio; Martinez Victor; Freitas Gabriel; Alaminos Armando; Bonatelli Antônio; Ferraz Fernando

    2002-01-01

    PURPOSE: The authors present a prospective study on 10 patients with stereotactic infusion of tissue plasminogen activator (rtPA) intraparenchimal hemorrhage. METHODS: Between 1999 and 2000, 10 patients with deep seated hematomas in the basal ganglia were selected for stereotactic infusion of rtPA and spontaneous clot drainage. RESULTS: All cases had about 80% reduction of the hematoma volume in the CT scan at the third day. The intracranial pressure was normalized by the third day too. There...

  7. Urokinase-type plasminogen activator receptor (uPAR) as a promising new imaging target

    DEFF Research Database (Denmark)

    Persson, Morten; Kjaer, Andreas

    2013-01-01

    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...

  8. Hormonal regulation of plasminogen activator production in porcine kidney cells (LLC-PK1)

    International Nuclear Information System (INIS)

    An attempt is made to study the molecular events regulating and accompanying the hormonally modulated plasminogen activator (PA) gene expression. The model system of interest is a porcine kidney cell line, LLC-PK1, responding to calcitonin in the PA production. The plasminogen activator secreted by calcitonin treated pig kidney cells has been purified, characterized, and compared with human urinary urokinase. The purified enzyme resembles the 53 k MW components of human urokinase. PA induction in LLC-PK1 cells is sensitive to inhibition by actinomycin D, suggesting the enhanced transcription of PA-mRNA. This hypothesis was tested by measuring PA-mRNA sequences in Xenopus oocyte system which showed a 15-20 fold enhanced PA synthesis when supplied with poly (A)+ RNA from induced cells, above that obtained from uninduced cell RNA. A pleiotropic response to calcitonin in LLC-PK1 cells has been examined using two-dimensional gel electrophoresis of 35S methionine labeled polypeptides. A set of twelve intracellular polypeptides, ten induced and two repressed, has been identified in calcitonin stimulated cells. One of the induced polypeptides has been identified as plasminogen activator by two dimensional tryptic peptide mapping. Other calcitonin induced polypeptides have been identified as cytokeratins by their solubility properties and cross-reactivity with antiserum against human keratin. The results indicate that the experimental system presented here is a useful and valid one for the study of molecular events regulating and accompanying the hormonally modulated PA gene expression

  9. Penta-L-lysine Potentiates Fibrin-Independent Activity of Human Tissue Plasminogen Activator.

    Science.gov (United States)

    Rehan, Mohammad; Sagar, Amin; Sharma, Vandna; Mishra, Sanskruti; Ashish; Sahni, Girish

    2015-10-22

    The therapeutic action of tissue plasminogen activator (t-PA) is a two-step process: (1) binding to lysine-rich fibrin (Km event) and (2) converting local plasminogen into plasmin (Kcat event). Overcoming limitations of other structural biophysics methods, we wanted to employ small-angle X-ray scattering (SAXS) to visualize what shape changes occur/accompany t-PA activation, but the prime hurdle was the polydisperse nature of the fibrin, which occluded scattering information from t-PA. Earlier, larger polylysine peptides have been used to potentiate activation of t-PA, so while screening short polylysine peptides as alternatives to fibrin or larger peptides, we found that penta-polylysine (P5) specifically activates t-PA in a dose-dependent manner, averaging to almost 3-fold more than in the absence of any peptide. SAXS data analysis confirmed that P5 does not induce association of t-PA molecules, and a narrower peak profile of the Kratky plot indicated that P5 binding quenches inherent motion in t-PA. Shape reconstruction of t-PA ? P5 revealed that P5 closes the "gap" between the two gross domains of t-PA, viz. fused F/E, K1 and K2 domains, and the P domain. Docking experiments suggested that, while other polylysine peptides preferentially interacted with the surfaces of kringle domains, P5 "slipped into" the gap/groove between K2 and P domains, thereby mediating a substantial increase in the number of long-range interactions between the K2 domain and exosites in the P domain. We report here dissection of shape events involved in between Km/Kcat steps of t-PA activation, which can pave the way toward the search for small molecule function regulator(s) of t-PA. PMID:26447340

  10. Host Plasminogen Activator Inhibitor-1 Promotes Human Skin Carcinoma Progression in a Stage-Dependent Manner

    Directory of Open Access Journals (Sweden)

    Catherine Maillard

    2005-01-01

    Full Text Available Angiogenesis and tumor expansion are associated with extracellular matrix remodeling and involve various proteases such as the plasminogen (Pig/plasminogen activator (PA system. Recently, several experimental data have implicated the plasminogen activator inhibitor-1 (PAI-1 in tumor angiogenesis in murine systems. However, little is known about PAI-1 functions in human skin carcinoma progression. By generating immunodeficient mice (in Rag-1-/- or nude background deleted for PAI-1 gene (PAI-1-/- , we have evaluated the impact of host PAI-1 deficiency on the tumorigenicity of two malignant human skin keratinocyte cell lines HaCaT II-4 and HaCaT A5-RT3 forming low-grade and high-grade carcinomas, respectively. When using the surface transplantation model, angiogenesis and tumor invasion of these two cell lines are strongly reduced in PAI-1-deficient mice as compared to the wild-type control animals. After subcutaneous injection in PAI-1-/- mice, the tumor incidence is reduced for HaCaT II-4 cells, but not for those formed by HaCaT A5-RT3 cells. These data indicate that PAI-1 produced by host cells is an important contributor to earlier stages of human skin carcinoma progression. It exerts its tumor-promoting effect in a tumor stage-dependent manner, but PAI-1 deficiency is not sufficient to prevent neoplastic growth of aggressive tumors of the human skin.

  11. Variable Resistance to Plasminogen Activator Initiated Fibrinolysis for Intermediate-Risk Pulmonary Embolism

    Science.gov (United States)

    Stubblefield, William B.; Alves, Nathan J.; Rondina, Matthew T.; Kline, Jeffrey A.

    2016-01-01

    Background We examine the clinical significance and biomarkers of tissue plasminogen activator (tPA)-catalyzed clot lysis time (CLT) in patients with intermediate-risk pulmonary embolism (PE). Methods Platelet-poor, citrated plasma was obtained from patients with PE. Healthy age- and sex-matched patients served as disease-negative controls. Fibrinogen, ?2-antiplasmin, plasminogen, thrombin activatable fibrinolysis inhibitor (TAFI), plasminogen activator Inhibitor 1 (PAI-1), thrombin time and D-dimer were quantified. Clotting was induced using CaCl2, tissue factor, and phospholipid. Lysis was induced using 60 ng/mL tPA. Time to 50% clot lysis (CLT) was assessed by both thromboelastography (TEG) and turbidimetry (A405). Results Compared with disease-negative controls, patients with PE exhibited significantly longer mean CLT on TEG (+2,580 seconds, 95% CI 1,380 to 3,720 sec). Patients with PE and a short CLT who were treated with tenecteplase had increased risk of bleeding, whereas those with long CLT had significantly worse exercise tolerance and psychometric testing for quality of life at 3 months. A multivariate stepwise removal regression model selected PAI-1 and TAFI as predictive biomarkers of CLT. Conclusion The CLT from TEG predicted increased risk of bleeding and clinical failure with tenecteplase treatment for intermediate-risk PE. Plasmatic PAI-1 and TAFI were independent predictors of CLT. PMID:26866684

  12. Plasminogen interaction with Trypanosoma cruzi

    Scientific Electronic Library Online (English)

    Laura, Almeida; Gilmer, Vanegas; Marina, Calcagno; Juan Luis, Concepción; Luisana, Avilan.

    2004-02-01

    Full Text Available The ability of Trypanosoma cruzi to interact with plasminogen, the zimogenic form of the blood serin protease plasmin, was examined. Immunohistochemistry studies revealed that both forms, epimastigotes and metacyclic trypomastigotes, were able to fix plasminogen in a lysine dependant manner. This in [...] teraction was corroborated by plasminogen activation studies. Both forms of the parasite enhanced the plasminogen activation by tissue-type plasminogen activator.The maximal enhancements obtained were 15-fold and 3.4-fold with epimastigotes and metacyclic trypomastigotes, respectively, as compared to plasminogen activation in absence of cells. Ligand-blotting analysis of proteins extracted with Triton X-114 from a microsomal fraction of epimastigotes revealed at least five soluble proteins and one hydrophobic protein able to bind plasminogen.

  13. Prognostic significance of urokinase plasminogen activator and plasminogen activator inhibitor-1 mRNA expression in lymph node- and hormone receptor-positive breast cancer

    International Nuclear Information System (INIS)

    One of the most thoroughly studied systems in relation to its prognostic relevance in patients with breast cancer, is the plasminogen activation system that comprises of, among others, the urokinase Plasminogen Activator (uPA) and its main inhibitor, the Plasminogen Activator Inhibitor-1 (PAI-1). In this study, we investigated the prognostic value of uPA and PAI-1 at the mRNA level in lymph node- and hormone receptor-positive breast cancer. The study included a retrospective series of 87 patients with hormone-receptor positive and axillary lymph node-positive breast cancer. All patients received radiotherapy, adjuvant anthracycline-based chemotherapy and five years of tamoxifen treatment. The median patient age was 54 and the median follow-up time was 79 months. Distant relapse occurred in 30 patients and 22 patients died from breast cancer during follow-up. We investigated the prognostic value of uPA and PAI-1 at the mRNA level as measured by real-time quantitative RT-PCR. uPA and PAI-1 gene expression was not found to be correlated with any of the established clinical and pathological factors. Metastasis-free Survival (MFS) and Breast Cancer specific Survival (BCS) were significantly shorter in patients expressing high levels of PAI-1 mRNA (p < 0.0001; p < 0.0001; respectively). In Cox multivariate analysis, the level of PAI-1 mRNA appeared to be the strongest prognostic factor for MFS (Hazard Ratio (HR) = 10.12; p = 0.0002) and for BCS (HR = 13.17; p = 0.0003). Furthermore, uPA gene expression was not significantly associated neither with MFS (p = 0.41) nor with BCS (p = 0.19). In a Cox-multivariate regression analysis, uPA expression did not demonstrate significant independent prognostic value. These findings indicate that high PAI-1 mRNA expression represents a strong and independent unfavorable prognostic factor for the development of metastases and for breast cancer specific survival in a population of hormone receptor- and lymph node-positive breast cancer patients

  14. 2-Amidino analogs of glycine-amiloride conjugates: inhibitors of urokinase-type plasminogen activator.

    Science.gov (United States)

    Massey, Archna P; Harley, William R; Pasupuleti, NagaRekha; Gorin, Fredric A; Nantz, Michael H

    2012-04-01

    The relative non-toxicity of the diuretic amiloride, coupled with its selective inhibition of the protease urokinase plasminogen activator (uPA), makes this compound class attractive for structure-activity studies. Herein we substituted the C(2)-acylguanidine of C(5)-glycyl-amiloride with amidine and amidoxime groups. The data show the importance of maintaining C(5)-hydrophobicity. The C(5)-benzylglycine analogs containing either C(2)-acylguanidine or amidine inhibited uPA with an IC(50) ranging from 3 to 7 ?M and were cytotoxic to human U87 malignant glioma cells. PMID:22366654

  15. Tissue-type plasminogen activator promotes murine myofibroblast activation through LDL receptor–related protein 1–mediated integrin signaling

    OpenAIRE

    Hu, Kebin; Wu, Chuanyue; Wendy M. Mars; Liu, Youhua

    2007-01-01

    The activation of interstitial fibroblasts to become ?-SMA–positive myofibroblasts is an essential step in the evolution of chronic kidney fibrosis, as myofibroblasts are responsible for the production and deposition of the ECM components that are a hallmark of the disease. Here we describe a signaling pathway that leads to this activation. Tissue-type plasminogen activator (tPA) promoted TGF-?1–mediated ?-SMA and type I collagen expression in rat kidney interstitial fibroblasts. This fibroge...

  16. Characterization of a recombinant chimeric plasminogen activator with enhanced fibrin binding.

    Science.gov (United States)

    Jiao, J; Yu, M; Ru, B

    2001-04-01

    A recombinant chimeric plasminogen activator (GHRP-scu-PA-32K), consisting of the tetrapeptide Gly-His-Arg-Pro fused to the N-terminus of the low-molecular single-chain urokinase-type plasminogen activator (Leu144-Leu411), was produced by expression in CHO cells. The stable expression cell line was selected for large-scale expression. The product was purified by antibody-Sepharose affinity chromatography with a recovery of 67%. The apparent molecular weight of purified GHRP-scu-PA-32K was 33 kDa according to SDS-PAGE. Its specific activity was 150000 IU/mg protein according to fibrin plate determination. The conversion of single-chain to two-chain molecules mediated by plasmin was comparable for GHRP-scu-PA-32K (K(m)=4.9 microM, k(2)=0.35 s(-1)) and scu-PA-32K. The activation of plasminogen by GHRP-scu-PA-32K (K(m)=1.02 microM, k(2)=0.0028 s(-1)) was also similar to that of scu-PA-32K. The fibrin binding of GHRP-scu-PA-32K was 2.5 times higher than that of scu-PA-32K at a fibrin concentration of 3.2 mg/ml. In contrast to scu-PA-32K in vitro 125I-fibrin-labeled plasma clot lysis, GHRP-scu-PA had a higher thrombolytic potency, whereas it depleted less fibrinogen in plasma. These results show that GHRP-scu-PA-32K as expected is a potential thrombolytic agent. PMID:11295444

  17. Immunohistochemical localization of urokinase-type plasminogen activator, urokinase-type plasminogen activator receptor and ?2-antiplasmin in human corneal perforation: a case report

    Directory of Open Access Journals (Sweden)

    Sugioka Koji

    2012-11-01

    Full Text Available Abstract Background Corneal ulceration leading to perforation is associated with infectious and non-infectious destructive conditions in the cornea. The fibrinolytic (plasminogen/plasmin system is considered to contribute to tissue remodeling in the wound healing process and it is believed to play an important role in proteolysis and fibrosis. To determine the localization of urokinase-type plasminogen activator (u-PA, u-PA receptor (u-PAR and ?2-antiplasmin (?2AP in the tissue of a corneal perforation, we investigated immunohistochemical expressions of u-PA, u-PAR, ?2AP, CD68, and ?-smooth muscle actin (?-SMA in a patient with corneal perforation that developed from an ulcer of no clear cause. Case presentation The patient was a 77-year-old woman who presented with a perforated corneal ulcer in her right eye. The cause of her corneal ulcer was unknown. Double immunohistochemistry was performed for the combinations of u-PA with u-PAR, CD68 or ?-SMA and ?2AP with CD68 or ?-SMA to detect the localization of u-PA and ?2AP. u-PA and u-PAR co-localization was seen in the corneal ulceration area. u-PA was mainly observed in CD68-positive cells and in some ?-SMA positive cells. On the other hand, ?2AP was not expressed in CD68-positive cells, but was expressed in ?-SMA positive cells. Conclusion We identified expression of the u-PA/u-PAR complex and ?2AP in a patient with a corneal ulcer. These two molecules are believed to play a crucial role in inflammatory cell recruitment, ECM synthesis and degradation during corneal wound healing.

  18. Exposure of mcf-7 breast cancer cells to electromagnetic fields up-regulates the plasminogen activator system.

    Science.gov (United States)

    Girgert, Rainer; Emons, Günter; Hanf, Volker; Gründker, Carsten

    2009-04-01

    Effects of electromagnetic fields (EMFs) on the incidence of breast cancer (BC) have been proposed by a number of epidemiological studies. The molecular mechanism of the impact of EMFs on cells is not yet clear, although changes in gene expression have been reported in various cellular systems. In this investigation, the interference of low-frequency EMFs with the plasminogen activator system was examined in BC cells.MCF-7 BC cells from 2 different sources were exposed to highly homogeneous 50-Hz EMFs. Changes in gene expression were analyzed by reverse transcriptase-polymerase chain reaction.In MCF-7 cells exposed to 1.2 microT EMF expression of the urokinase plasminogen activator gene and of plasminogen-activator inhibitor-1 was markedly increased. The expression of the receptor for urokinase plasminogen activator was only marginally increased in 1 of the 2 tested cell lines and expression of the tissue plasminogen activator was at least slightly down-regulated in BC cells exposed to EMFs.EMFs may be able to increase the metastatic potential of breast tumors. The use of our newly established exposure system for EMFs may allow us to study the signaling processes involved in the induction of a metastatic phenotype of breast cancer cells. PMID:19407555

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

    Directory of Open Access Journals (Sweden)

    Shaltiel Shmuel

    2003-07-01

    Full Text Available Abstract Background The plasminogen activator inhibitor-1 (PAI-1 spontaneously converts from an inhibitory into a latent form. Specificity of PAI-1 is mainly determined by its reactive site (Arg346-Met347, which interacts with serine residue of tissue-type plasminogen activator (tPA with concomitant formation of SDS-stable complex. Other sites may also play roles in determining the specificity of PAI-1 toward serine proteases. Results To understand more about the role of distal hinge for PAI-1 specificities towards serine proteases and for its conformational transition, wild type PAI-1 and its mutants were expressed in baculovirus system. WtPAI-1 was found to be about 12 fold more active than the fibrosarcoma PAI-1. Single site mutants within the Asp355-Arg356-Pro357 segment of PAI-1 yield guanidine activatable inhibitors (a that can still form SDS stable complexes with tPA and urokinase plasminogen activator (uPA, and (b that have inhibition rate constants towards plasminogen activators which resemble those of the fibrosarcoma inhibitor. More importantly, latency conversion rate of these mutants was found to be ~3–4 fold faster than that of wtPAI-1. We also tested if Glu351 is important for serine protease specificity. The functional stability of wtPAI-1, Glu351Ala, Glu351Arg was about 18 ± 5, 90 ± 8 and 14 ± 3 minutes, respectively, which correlated well with both their corresponding specific activities (84 ± 15 U/ug, 112 ± 18 U/ug and 68 ± 9 U/ug, respectively and amount of SDS-stable complex formed with tPA after denatured by Guanidine-HCl and dialyzed against 50 mM sodium acetate at 4°C. The second-order rate constants of inhibition for uPA, plasmin and thrombin by Glu351Ala and Glu351Arg were increased about 2–10 folds compared to wtPAI-1, but there was no change for tPA. Conclusion The Asp355-Pro357 segment and Glu351 in distal hinge are involved in maintaining the inhibitory conformation of PAI-1. Glu351 is a specificity determinant of PAI-1 toward uPA, plasmin and thrombin, but not for tPA.

  20. Hormonal regulation of plasminogen activator production in porcine kidney cells (LLC-PK/sub 1/)

    Energy Technology Data Exchange (ETDEWEB)

    Sudol, M.

    1983-01-01

    An attempt is made to study the molecular events regulating and accompanying the hormonally modulated plasminogen activator (PA) gene expression. The model system of interest is a porcine kidney cell line, LLC-PK/sub 1/, responding to calcitonin in the PA production. The plasminogen activator secreted by calcitonin treated pig kidney cells has been purified, characterized, and compared with human urinary urokinase. The purified enzyme resembles the 53 k MW components of human urokinase. PA induction in LLC-PK/sub 1/ cells is sensitive to inhibition by actinomycin D, suggesting the enhanced transcription of PA-mRNA. This hypothesis was tested by measuring PA-mRNA sequences in Xenopus oocyte system which showed a 15-20 fold enhanced PA synthesis when supplied with poly (A)/sup +/ RNA from induced cells, above that obtained from uninduced cell RNA. A pleiotropic response to calcitonin in LLC-PK/sub 1/ cells has been examined using two-dimensional gel electrophoresis of /sup 35/S methionine labeled polypeptides. A set of twelve intracellular polypeptides, ten induced and two repressed, has been identified in calcitonin stimulated cells. One of the induced polypeptides has been identified as plasminogen activator by two dimensional tryptic peptide mapping. Other calcitonin induced polypeptides have been identified as cytokeratins by their solubility properties and cross-reactivity with antiserum against human keratin. The results indicate that the experimental system presented here is a useful and valid one for the study of molecular events regulating and accompanying the hormonally modulated PA gene expression.

  1. Serum-stable RNA aptamers to urokinase-type plasminogen activator blocking receptor binding

    DEFF Research Database (Denmark)

    Dupont, Daniel Miotto; Madsen, Jeppe Buur; Hartmann, Roland Karl; Tavitian, Bertrand; Ducongé, Frédéric; Kjems, Jørgen; Andreasen, Peter André

    2010-01-01

    The serine proteinase urokinase-type plasminogen activator (uPA) is widely recognized as a potential target for anticancer therapy. Its association with cell surfaces through the uPA receptor (uPAR) is central to its function and plays an important role in cancer invasion and metastasis. In the current study, we used systematic evolution of ligands by exponential enrichment (SELEX) to select serum-stable 2'-fluoro-pyrimidine-modified RNA aptamers specifically targeting human uPA and blocking the...

  2. Inhibitory mechanism of peptides and antibodies targeting murine urokinase-type plasminogen activator

    DEFF Research Database (Denmark)

    Liu, Zhuo

    2012-01-01

    Urokinase-type plasminogen activator (uPA) is a serine protease that plays an important role in many pathophysiological processes. It is also a marker for a poor prognosis in several cancer types. In order to investigate the efficiency of uPA inhibitors which might be candidates for therapeutic drugs, a detailed mechanistic understanding must be obtained. One peptide and two antibodies were studied in this thesis. First, an engineered cyclic peptide, mupain-1-16 with an unnatural amino acid in t...

  3. 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)

    Egelund, R; Einholm, A P; Pedersen, K E; Nielsen, R W; Christensen, A; Deinum, J; Andreasen, Peter A

    2001-01-01

    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 a...

  4. 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 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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1990-07-15

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

  6. Roles of tissue plasminogen activator and its inhibitor in proliferative diabetic retinopathy

    Directory of Open Access Journals (Sweden)

    Shu-Ling Wu

    2014-10-01

    Full Text Available AIM:To investigate the role of tissue plasminogen activator (t-PA and plasminogen activator inhibitor (PAI in proliferative diabetic retinopathy (PDR and to discuss the correlations among t-PA, PAI and vascular endothelial growth factor (VEGF expressions.METHODS: A total of 36 vitreous samples were collected from 36 patients with PDR (PDR group, and 17 vitreous samples from 17 patients with idiopathic macular hole were used as control. The concentrations of t-PA, PAI and VEGF in samples were determined by ELISA method. The correlations among t-PA, PAI and VEGF expressions were discussed.RESULTS: The concentrations of t-PA, PAI and VEGF in the PDR group were significantly higher than those in the control group (P<0.001. The t-PA and PAI expressions were highly correlated with the VEGF expression (P<0.001.CONCLUSION:In addition to VEGF, a variety of bioactive substances, such as t-PA and PAI, are involved in the pathogenesis involved in the angiogenesis of PDR. VEGF can activate t-PA expression, resulting in collagen tissue degradation and angiogenesis. VEGF may also activate the mechanism for endogenous anti-neovascularization.

  7. Expression of urokinase plasminogen activator, its receptor and type-1 inhibitor in malignant and benign prostate tissue

    DEFF Research Database (Denmark)

    Usher, Pernille Autzen; Thomsen, Ole Frøkjær; Iversen, Peter; Johnsen, Morten; Brünner, Nils; Høyer-Hansen, Gunilla; Andreasen, Peter; Danø, Keld; Nielsen, Boye Schnack

    2005-01-01

    The plasminogen activation (PA) cascade participates in degradation of extracellular matrix during cancer invasion. We have studied the expression of urokinase-type plasminogen activator (uPA) mRNA, uPA receptor (uPAR) mRNA and immunoreactivity, and type-1 plasminogen activator inhibitor (PAI-1) m......RNAs was predominantly seen in cells identified as macrophages, which in most of the carcinomas (approximately 90%) were located in the interstitial tissue between the tumor cell islands, while in most of the benign hyperplasias they were located in the lumen of the glands and were in only a few cases...... vascular structures and in a few macrophages located in the interstitial tissue of both malignant and benign lesions. A similar expression pattern was found for PAI-1 immunoreactivity. In 8 of the 16 carcinomas, all 3 components were present, and in several areas colocalization was observed in stromal...

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

    DEFF Research Database (Denmark)

    Pedersen, Katrine E; Einholm, Anja P

    2003-01-01

    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

  9. 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)

    Jensen, Jan Kristian; Malmendal, Anders; Schiøtt, Birgit; Skeldal, Sune; Pedersen, Katrine E; Celik, Leyla; Nielsen, Niels Chr.; Andreasen, Peter A; Wind, Troels

    2006-01-01

    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...

  10. A recombinant chimeric plasminogen activator with high affinity for fibrin has increased thrombolytic potency in vitro and in vivo.

    OpenAIRE

    Runge, M. S.; QUERTERMOUS, T.; Zavodny, P J; Love, T W; Bode, C.; Freitag, M.; Shaw, S. Y.; Huang, P. L.; Chou, C C; Mullins, D.

    1991-01-01

    A recombinant plasminogen activator with high fibrin affinity and specificity was expressed by transfecting hybridoma cells with a plasmid that combines sequence coding for low molecular mass (32 kDa) single-chain urokinase-type plasminogen activator [scuPA(32kDa)] and anti-fibrin monoclonal antibody 59D8. The expression of the recombinant molecule [r-scuPA(32kDa)-59D8] was optimized by replacing the 3' untranslated region (initially that of high molecular mass scuPA) in the plasmid with the ...

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

    DEFF Research Database (Denmark)

    Illemann, Martin; Bird, Nigel; Majeed, Ali; Laerum, Ole D; Lund, Leif R; Danø, Keld; Nielsen, Boye Schnack

    2009-01-01

    Metastatic growth and invasion by colon cancer cells in the liver requires the ability of the cancer cells to interact with the new tissue environment. Plasmin(ogen) is activated on cell surfaces by urokinase-type PA (uPA), and is regulated by uPAR and plasminogen activator inhibitor-1 (PAI-1). To compare the expression patterns of uPA, uPAR and PAI-1 in colon cancer with that in their liver metastases, we analysed matched samples from 14 patients. In all 14 primary colon cancers, we found upreg...

  12. 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)

    Witzel I

    2014-11-01

    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

  13. 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)

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

    DEFF Research Database (Denmark)

    Jensen, P H; Cressey, L I

    1994-01-01

    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.

  15. Angiostatic activity of human plasminogen fragments is highly dependent on glycosylation.

    Science.gov (United States)

    Santos, Ivan Carlos; Silbiger, Vivian Nogueira; Higuchi, Débora Ayame; Gomes, Maria Aparecida; Barcelos, Lucíola Silva; Teixeira, Mauro Martins; Lopes, Mirian Teresa Paz; Cardoso, Valbert Nascimento; Lima, Mercia Paula; Araujo, Ronaldo Carvalho; Pesquero, João Bosco; Pesquero, Jorge Luiz

    2010-02-01

    To assess the importance of carbohydrate moieties to the anti-angiogenic activity of plasminogen fragments, we cloned the fragment corresponding to amino acids Val(79) to Thr(346) (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. (99m)Technecium-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. PMID:19961492

  16. Tissue Plasminogen Activator Coating on Implant Surfaces Reduces Staphylococcus aureus Biofilm Formation.

    Science.gov (United States)

    Kwiecinski, Jakub; Na, Manli; Jarneborn, Anders; Jacobsson, Gunnar; Peetermans, Marijke; Verhamme, Peter; Jin, Tao

    2015-01-01

    Staphylococcus aureus biofilm infections of indwelling medical devices are a major medical challenge because of their high prevalence and antibiotic resistance. As fibrin plays an important role in S. aureus biofilm formation, we hypothesize that coating of the implant surface with fibrinolytic agents can be used as a new method of antibiofilm prophylaxis. The effect of tissue plasminogen activator (tPA) coating on S. aureus biofilm formation was tested with in vitro microplate biofilm assays and an in vivo mouse model of biofilm infection. tPA coating efficiently inhibited biofilm formation by various S. aureus strains. The effect was dependent on plasminogen activation by tPA, leading to subsequent local fibrin cleavage. A tPA coating on implant surfaces prevented both early adhesion and later biomass accumulation. Furthermore, tPA coating increased the susceptibility of biofilm infections to antibiotics. In vivo, significantly fewer bacteria were detected on the surfaces of implants coated with tPA than on control implants from mice treated with cloxacillin. Fibrinolytic coatings (e.g., with tPA) reduce S. aureus biofilm formation both in vitro and in vivo, suggesting a novel way to prevent bacterial biofilm infections of indwelling medical devices. PMID:26519394

  17. Urokinase plasminogen activator receptor on invasive cancer cells: A prognostic factor in distal gastric adenocarcinoma

    DEFF Research Database (Denmark)

    Alpizar, Warner Enrique Alpizar; Christensen, Ib Jarle

    2012-01-01

    Gastric cancer is the second cancer causing death worldwide. The five-year survival for this malignancy is below 25% and few parameters have shown an impact on the prognosis of the disease. The receptor for urokinase plasminogen activator (uPAR) is involved in extracellular matrix degradation by mediating cell surface associated plasminogen activation, and its presence on gastric cancer cells is linked to micrometastasis and poor prognosis. Using immunohistochemistry, the prognostic significance of uPAR was evaluated in tissue samples from a retrospective series of 95 gastric cancer patients. uPAR was expressed by neoplastic cells, macrophages, myofibroblasts and neutrophils in both intestinal and diffuse subtypes. No association was demonstrated between the expression of uPAR on cancer cells and histological subtype (p = 0.64) or TNM stage (p = 0.75). Univariate analysis revealed a significant association between the expression of uPAR on tumor cells in the peripheral invasion zone and overall survival of gastric cancer patients (HR = 2.16; 95% CI: 1.13-4.14; p = 0.02). Multivariate analysis showed that uPAR immunoreactivity in cancer cells at the invasive front is an independent prognostic factor for overall survival in gastric cancer (HR = 2.39; 95% CI: 1.22-4.69; p = 0.011). In consequence, scoring of uPAR-positive cancer cells may be a direct measure for the invasive potential of gastric adenocarcinomas.

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

    Directory of Open Access Journals (Sweden)

    Marzia Albenzio

    2010-01-01

    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.

  19. Proteolytic cleavage of urokinase-type plasminogen activator by stromelysin-1 (MMP-3).

    Science.gov (United States)

    Ugwu, F; Van Hoef, B; Bini, A; Collen, D; Lijnen, H R

    1998-05-19

    Matrix metalloproteinase-3 (MMP-3, or stromelysin-1) specifically hydrolyzes the Glu143-Leu144 peptide bond in 45-kDa single-chain urokinase-type plasminogen activator (scu-PA) and in its two-chain (tcu-PA) derivative, yielding a 17-kDa NH2-terminal domain comprising the u-PA receptor (u-PAR) binding site and a 32-kDa COOH-terminal moiety containing the serine proteinase domain of u-PA. The conversion is completely abolished in the presence of the MMP inhibitors EDTA or 1,10-phenanthroline. Biospecific interaction analysis indicates that binding of MMP-3 occurs through the 32-kDa fragment. The 32-kDa fragment derived from scu-PA (scu-PA-32k) has a specific activity of 32k) with a specific activity of 79 000 IU/mg. tcu-PA and tcu-PA-32k moieties derived from scu-PA-32k by plasmin or from tcu-PA by MMP-3 have comparable amidolytic activities toward the chromogenic substrate S-2444 (kcat/Km of 110 and 160 mM-1 s-1, respectively) and similar plasminogen activating activities in a coupled chromogenic substrate assay. Specific binding of the 17-kDa NH2-terminal domain to THP-1 monocytoid cells is completely abolished by competition with scu-PA but is not affected by scu-PA-32k (residual binding of 88 +/- 9% (mean +/- SEM; n = 3) with 25-fold molar excess). Thus, MMP-3 removes a functional NH2-terminal u-PAR-binding domain from u-PA without affecting its enzymatic properties. PMID:9585535

  20. Acceleration of tissue plasminogen activator-mediated thrombolysis by magnetically powered nanomotors.

    Science.gov (United States)

    Cheng, Rui; Huang, Weijie; Huang, Lijie; Yang, Bo; Mao, Leidong; Jin, Kunlin; ZhuGe, Qichuan; Zhao, Yiping

    2014-08-26

    Dose control and effectiveness promotion of tissue plasminogen activator (t-PA) for thrombolysis are vitally important to alleviate serious side effects such as hemorrhage in stroke treatments. In order to increase the effectiveness and reduce the risk of stroke treatment, we use rotating magnetic nanomotors to enhance the mass transport of t-PA molecules at the blood clot interface for local ischemic stroke therapy. The in vitro experiments demonstrate that, when combined with magnetically activated nanomotors, the thrombolysis speed of low-concentration t-PA (50 ?g mL(-1)) can be enhanced up to 2-fold, to the maximum lysis speed at high t-PA concentration. Based on the convection enhanced transport theory due to rotating magnetic nanomotors, a theoretical model is proposed and predicts the experimental results reasonably well. The validity and efficiency of this enhanced treatment has been demonstrated in a rat embolic model. PMID:25006696

  1. Procollagen-III in serum, plasminogen activation and fibronectin in bronchoalveolar lavage fluid during and following irradiation of human lung

    International Nuclear Information System (INIS)

    In the search for predictors of late radiation-induced lung injury we studied procollagen type III peptide concentration (P-III-P) in serum as well as fibronectin and plasminogen activation in bronchoalveolar lavage (BAL) fluid during and following irradiation of human lung. The patients received either high-dose hemithorax irradiation for pleural mesothelioma (11 patients) or high-dose irradiation with individually shaped fields for non-small cell lung cancer (12 patients). The severity of radiation fibrosis was assessed clinically from CT scans 6 months and 12 months after treatment. Four scores were used: severe, moderate, mild, or normal. Radiological lung injury varied from 'severe' (9 patients) to near absence of injury-'normal' (6 patients). Serum levels of P-III-P, when measured weekly during the 5-week period of radiotherapy or at several time-points after treatment, did not show consistent changes, nor did the levels correlate with the score for radiation fibrosis as assessed by CT scanning. Changes in fibronectin levels or in markers of plasminogen activation in BAL fluid did not correlate with the development of late lung injury. The levels of BAL fluid plasmin and plasminogen activator as assessed zymographically, but not the free net enzyme values, showed a tendency to be elevated in patients with severe radiation-induced lung injury, suggesting a possible role for inhibitors of the plasminogen activation cascade in the process of radiation-induced lung injury

  2. Successful thrombolysis using recombinant tissue plasminogen activator in cases of severe pulmonary embolism with mobile thrombi in the right atrium

    OpenAIRE

    Şatiroğlu, Ömer; Durakoğlugil, Murtaza Emre; Uğurlu, Yavuz; Şahin, İsmail; Doğan, Sitki; Ergül, Elif; KARADAĞ, Zakir; Bostan, Mehmet

    2014-01-01

    Hereby, we report two cases of acute pulmonary embolism with concomitant right-sided thrombus, which were successfully treated using recombinant tissue plasminogen activator (rtPA). These patients had life-threatening acute right ventricular failure, which dramatically improved within hours following thrombolysis. These cases emphasize the clinical utility of rtPA for the treatment of life-threatening pulmonary embolism.

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

    DEFF Research Database (Denmark)

    Almasi, Charlotte E; Drivsholm, Lars; Pappot, Helle; Høyer-Hansen, Gunilla; Christensen, Ib J

    2013-01-01

    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...

  4. Effect of retinoic acid on plasminogen activator expression in human melanoma cells.

    Science.gov (United States)

    Alexander, C L; Edward, M; MacKie, R M

    1999-08-01

    The plasminogen activation system comprises various proteases that contribute to the invasive potential and metastatic spread of the tumour cell. Two such proteases are tissue-type (tPA) and urokinase-type (uPA) plasminogen activators. Both these enzymes convert plasminogen into the active zymogen plasmin, which has a broad substrate specificity and is capable of degrading a wide range of extracellular matrix molecules. In this study, we examined the effect of retinoic acid (RA) on uPA and tPA secretion in the highly metastatic C8161 and the poorly metastatic Hs294T human melanoma cell lines using a specific enzyme-linked immunosorbent assay (ELISA) detection system, and correlated this production with RA receptor (RAR) expression. Over a range of dilutions, we were able to show that the highly metastatic C8161 cells secreted 0.95 ng of uPA/cell compared with 4.41 fg/cell for the Hs294T cells, whereas the Hs294T cells secreted 24.5 fg of tPA/cell compared with 4.35 fg/cell for the C8161 cells. On exposure of the cells to RA (10(-10)-10(-5) M) for 4 days, uPA secretion was increased 3.4-fold in the C8161 cell line and 1.6-fold in the Hs294T cell line using 10(-8) M RA. In addition, tPA expression was increased in both cell lines by 3.7-fold in the C8161 cells and 3.8-fold in the Hs294T cells with 10(-6) M RA treatment. Increases in PA expression by RA have been reported to involve RAR alpha and RAR beta expression. We were able to detect RAR beta and gamma expression in both cell lines, with and without RA treatment, but were unable to detect expression of RAR alpha. This suggests that another mechanism must exist to regulate the RA modulation of tPA and uPA secretion in these cell lines that does not require RAR alpha expression. PMID:10504054

  5. Plasminogen activator inhibitor-1 suppresses endogenous fibrinolysis in a canine model of pulmonary embolism

    International Nuclear Information System (INIS)

    Plasminogen activator inhibitor-1 (PAI-1), the specific, fast-acting inhibitor of tissue-type plasminogen activator (t-PA), binds to fibrin and has been found in high concentrations within arterial thrombi. These findings suggest that the localization of PAI-1 to a thrombus protects that same thrombus from fibrinolysis. In this study, clot-bound PAI-1 was assessed for its ability to suppress clot lysis in vivo. Autologous, canine whole blood clots were formed in the presence of increasing amounts of activated PAI-1 (0-30 micrograms/ml). Approximately 6-8% of the PAI-1 bound to the clots under the experimental conditions. Control and PAI-1-enriched clots containing iodine-125-labeled fibrin (ogen) were homogenized, washed to remove nonbound elements, and delivered to the lungs of anesthetized dogs where the homogenates subsequently underwent lysis by the endogeneous fibrinolytic system. 125I-labeled fibrin degradation products appeared in the blood of control animals within 10 minutes and were maximal by 90 minutes. PAI-1 reduced fibrin degradation product release in a dose-responsive manner at all times between 30 minutes and 5 hours (greater than or equal to 76% inhibition at 30 minutes, PAI-1 greater than or equal to 6 micrograms/ml). PAI-1 also suppressed D-dimer release from clots containing small amounts of human fibrin (ogen). t-PA administration attenuated the effects of PAI-1, whereas latent PAI-1 (20 micrograms/ml) had no effect on clot lysis. Blood levels of PA and PAI activity remained unaltered during these experiments. The results indicate that PAI-1 markedly inhibits endogenous fibrinolysis in vivo and, moreover, suggest that the localization of PAI-1 to a forming thrombus is an important physiological mechanism for subsequent thrombus stabilization

  6. Tissue-type plasminogen activator induces synaptic vesicle endocytosis in cerebral cortical neurons.

    Science.gov (United States)

    Yepes, M; Wu, F; Torre, E; Cuellar-Giraldo, D; Jia, D; Cheng, L

    2016-04-01

    The release of the serine proteinase tissue-type plasminogen activator (tPA) from the presynaptic terminal of cerebral cortical neurons plays a central role in the development of synaptic plasticity, adaptation to metabolic stress and neuronal survival. Our earlier studies indicate that by inducing the recruitment of the cytoskeletal protein βII-spectrin and voltage-gated calcium channels to the active zone, tPA promotes Ca(2+)-dependent translocation of synaptic vesicles (SVs) to the synaptic release site where they release their load of neurotransmitters into the synaptic cleft. Here we used a combination of in vivo and in vitro experiments to investigate whether this effect leads to depletion of SVs in the presynaptic terminal. Our data indicate that tPA promotes SV endocytosis via a mechanism that does not require the conversion of plasminogen into plasmin. Instead, we show that tPA induces calcineurin-mediated dynamin I dephosphorylation, which is followed by dynamin I-induced recruitment of the actin-binding protein profilin II to the presynaptic membrane, and profilin II-induced F-actin formation. We report that this tPA-induced sequence of events leads to the association of newly formed SVs with F-actin clusters in the endocytic zone. In summary, the data presented here indicate that following the exocytotic release of neurotransmitters tPA activates the mechanism whereby SVs are retrieved from the presynaptic membrane and endocytosed to replenish the pool of vesicles available for a new cycle of exocytosis. Together, these results indicate that in murine cerebral cortical neurons tPA plays a central role coupling SVs exocytosis and endocytosis. PMID:26820595

  7. Selection and characterization of camelid nanobodies towards urokinase-type plasminogen activator

    DEFF Research Database (Denmark)

    Kaczmarek, Jakub; Skottrup, Peter Durand

    2015-01-01

    Urokinase-type plasminogen activator (uPA) is a trypsin-like serine protease that plays a vital role in extracellular conversion of inactive plasminogen into catalytically active plasmin. Activated plasmin facilitates the release of several proteolytic enzymes, which control processes like pericellular proteolysis and remodeling of ECM. uPA and the receptor uPAR, are overexpressed in a number of malignant tumours and uPA/uPAR play major roles in adhesion, migration, invasion and metastasis of cancer cells. Elevated levels of uPA have been reported as a risk biomarker for disease relapse, increased cancer malignancy and poor survival prognosis. For these reasons uPA is considered an important target for anticancer drug therapy. In this study we isolated two camel single domain antibodies (nanobodies) from a naïve library by phage display. The nanobody sequences were sequence-optimized for Escherichia coli expression, cloned into the pET22-B(+) vector system, expressed in BL-21 cells and purified from the periplasmic fraction by IMAC. ELISA tests demonstrated that the purified nanobodies were specific for uPA when tested towards other trypsin-like serine proteases. The apparent affinities of the nanobodies were determined by competitive ELISA to 80 nM and 522 nM, respectively. The best binder did not inhibit uPA (nAb-C3), however the lowest affinity binder (nAb-C8) was able to inhibit the uPA-mediated cleavage of the substrate S-2444. The results validate the naïve library as a resource for retrieval of relevant lead molecules and the novel uPA-nanobodies can be useful pharmacological tools to study uPA structure–function relationships.

  8. Plasminogen activation system in oral cancer: Relevance in prognosis and therapy (Review).

    Science.gov (United States)

    Wyganowska-?wi?tkowska, Marzena; Jankun, Jerzy

    2015-07-01

    Research on carcinogenesis and progress in cancer treatment have reduced mortality of cancer patients. Mortality rates decreased by 1.5% per year from 2001 through 2010 for most types of cancer in men and women. However, oral cancer is still a significant global health problem since incidence and mortality rates are increasing. Oral cavity cancer is ranked the 8th in men and the 14th in women based on data collected between 2006 and 2010 by the National Institute of Health. Furthermore, an increasing incidence of head and neck neoplasms, particularly the tongue cancer among young adults has been reported recently. It is most likely due to increasing human papillomavirus (HPV) infection or the early start of tobacco and alcohol consumption. Treatment of oral cancer patients is mainly surgical and often leads to esthetic and functional deformities, with severe impact on the quality of life. Thus, novel form of treatments and selection of patients with high and low risk of mortality is of high priority for clinical studies. The expression of proteolytic enzymes in tumor and stromal tissues has been shown to have prognostic significance in many human cancers and inhibiting proteolysis can reduce tumor growth in many in vivo and in vitro models. Plasmin, with its activators and inhibitors are of great importance in many human malignances and collectively are called plasminogen activation system (PAS). In this comprehensive review we examine expression, possible prognostic markers and importance for therapy of the PAS members in oral cancer. Literature review suggests that overexpression of urokinase and its receptor are markers of poor outcome, thus, their inhibition can be explored in oral cancer therapy. Role of plasminogen activator inhibitor (PAI-1) is complex and depends on its concentration. Overexpression of PAI-1 favors angiogenesis, metastasis and poor prognosis, although when applied in very high concentrations it inhibits angiogenesis and tumor growth, the phenomenon is described as the PAI-1 paradox. PMID:26004216

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

    DEFF Research Database (Denmark)

    Bager, René; Kristensen, Thomas Kielsgaard

    2012-01-01

    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.

  10. 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)

    Krzysztof Kowal,

    2010-04-01

    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.

  11. 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)

    Marcin Moniuszko

    2011-04-01

    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.

  12. Hemorrhagic Transformation after Tissue Plasminogen Activator Reperfusion Therapy for Ischemic Stroke: Mechanisms, Models, and Biomarkers.

    Science.gov (United States)

    Wang, Wei; Li, Mingchang; Chen, Qianxue; Wang, Jian

    2015-12-01

    Intracerebral hemorrhagic transformation (HT) is well recognized as a common cause of hemorrhage in patients with ischemic stroke. HT after acute ischemic stroke contributes to early mortality and adversely affects functional recovery. The risk of HT is especially high when patients receive thrombolytic reperfusion therapy with tissue plasminogen activator, the only available treatment for ischemic stroke. Although many important publications address preclinical models of ischemic stroke, there are no current recommendations regarding the conduct of research aimed at understanding the mechanisms and prediction of HT. In this review, we discuss the underlying mechanisms for HT after ischemic stroke, provide an overview of the models commonly used for the study of HT, and discuss biomarkers that might be used for the early detection of this challenging clinical problem. PMID:25367883

  13. 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.)

  14. Subconjunctival and topical application of recombinant tissue plasminogen activator in rabbits

    Directory of Open Access Journals (Sweden)

    José Ricardo de Abreu Reggi

    2015-02-01

    Full Text Available Purpose: To quantify fibrin degradation products after topical and subconjunctival administration of recombinant tissue plasminogen activator in rabbits. Methods: Fibrin formation was induced in the anterior chamber in 25 rabbits. Subsequently, five rabbits received an injection of r-TPA (positive control in the anterior chamber, another 10 received a subconjunctival injection of r-TPA, and the remaining 10 received instillations of topical r-TPA. Afterwards, samples of aqueous humor were collected and semi-quantitative analysis of fibrin degradation products (FDP was performed. Results: No statistical differences were noted between the treatment and control groups at any time point. Fibrin degradation products semi-quantification showed statistical improvement in the control group and the subconjunctival group. Conclusion: Fibrin degradation products were observed in the anterior chamber after subconjunctival administration of r-TPA. However, it was probably not sufficient to cause fibrin degradation. Topical r-TPA did not effectively absorb anterior chamber fibrin.

  15. Ischaemia-reperfusion injury impairs tissue plasminogen activator release in man

    DEFF Research Database (Denmark)

    Pedersen, Christian MØller; Barnes, Gareth

    2011-01-01

    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

  16. Basilar Artery Thrombosis in a Child Treated With Intravenous Tissue Plasminogen Activator and Endovascular Mechanical Thrombectomy

    DEFF Research Database (Denmark)

    Fink, Jakob; Sonnenborg, Laura

    2013-01-01

    Basilar artery occlusion in children is rare. It has a high mortality and morbidity if recanalization is not achieved before extensive brainstem infarction has occurred. An 11-year-old boy presented with a clinical and radiological "top-of-the-basilar" syndrome. Intravenous tissue plasminogen activator was administered, and the patient was immediately referred to the regional stroke center. Subsequent mechanical thrombectomy using a Solitaire stent (Solitaire FR stent; ev3, Irvine, CA, USA) resulted in clot removal and recanalization of the basilar artery 4 hours after stroke onset. The patient made a full clinical recovery. To the authors' knowledge this is the first report on basilar artery occlusion in a child treated with "bridging" therapy, the combination of intravenous thrombolysis and endovascular thrombectomy. If the diagnosis can be made within the time window for intravenous thrombolysis (4.5 hours), the present case suggests that bridging therapy in pediatric basilar artery occlusion can be safe and effective.

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

    Energy Technology Data Exchange (ETDEWEB)

    Loskutoff, D.J.; Linders, M.; Keijer, J.; Veerman, H.; van Heerikhuizen, H.; Pannekoek, H.

    1987-06-30

    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 cultured vascular endothelial cells results in two distinct mRNA species. The data suggest that these two transcripts arise by alternative polyadenylation.

  18. 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 cultured vascular endothelial cells results in two distinct mRNA species. The data suggest that these two transcripts arise by alternative polyadenylation

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

    DEFF Research Database (Denmark)

    Madsen, Jeppe B; Dupont, Daniel Miotto

    2010-01-01

    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.

  20. Plasminogen activator inhibitor 1 is an intracellular inhibitor of furin proprotein convertase.

    Science.gov (United States)

    Bernot, Denis; Stalin, Jimmy; Stocker, Pierre; Bonardo, Bernadette; Scroyen, Ilse; Alessi, Marie-Christine; Peiretti, Franck

    2011-04-15

    Proprotein convertases (PCs) are a family of serine proteases that are involved in the post-translational processing and activation of a wide range of regulatory proteins. The upstream role of PCs in the control of many physiological and pathological processes generates a growing interest in understanding their regulation. Here, we demonstrate that the serine protease inhibitor plasminogen activator inhibitor 1 (PAI-1) forms an SDS-stable complex with the PC furin, which leads to the inhibition of the intra-Golgi activity of furin. It is known that elevated PAI-1 plasma levels are correlated with the occurrence of the metabolic syndrome and type 2 diabetes, and we show that PAI-1 reduces the furin-dependent maturation and activity of the insulin receptor and ADAM17: two proteins involved in the onset of these metabolic disorders. In addition to demonstrating that PAI-1 is an intracellular inhibitor of furin, this study also provides arguments in favor of an active role for PAI-1 in the development of metabolic disorders. PMID:21406565

  1. 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 absorption and CD spectroscopy

  2. Improvement of Psychotic Symptoms and the Role of Tissue Plasminogen Activator.

    Science.gov (United States)

    Hoirisch-Clapauch, Silvia; Nardi, Antonio E

    2015-01-01

    Tissue plasminogen activator (tPA) mediates a number of processes that are pivotal for synaptogenesis and remodeling of synapses, including proteolysis of the brain extracellular matrix, degradation of adhesion molecules, activation of neurotrophins, and activation of the N-methyl-d-aspartate receptor. Abnormalities in these processes have been consistently described in psychotic disorders. In this paper, we review the physiological roles of tPA, focusing on conditions characterized by low tPA activity, which are prevalent in schizophrenia. We then describe how tPA activity is influenced by lifestyle interventions and nutritional supplements that may ameliorate psychotic symptoms. Next, we analyze the role of tPA in the mechanism of action of hormones and medications effective in mitigating psychotic symptoms, such as pregnenolone, estrogen, oxytocin, dopamine D3 receptor antagonists, retinoic acid, valproic acid, cannabidiol, sodium nitroprusside, N-acetyl cysteine, and warfarin. We also review evidence that tPA participates in the mechanism by which electroconvulsive therapy and cigarette smoking may reduce psychotic symptoms. PMID:26593907

  3. Improvement of Psychotic Symptoms and the Role of Tissue Plasminogen Activator

    Directory of Open Access Journals (Sweden)

    Silvia Hoirisch-Clapauch

    2015-11-01

    Full Text Available Tissue plasminogen activator (tPA mediates a number of processes that are pivotal for synaptogenesis and remodeling of synapses, including proteolysis of the brain extracellular matrix, degradation of adhesion molecules, activation of neurotrophins, and activation of the N-methyl-d-aspartate receptor. Abnormalities in these processes have been consistently described in psychotic disorders. In this paper, we review the physiological roles of tPA, focusing on conditions characterized by low tPA activity, which are prevalent in schizophrenia. We then describe how tPA activity is influenced by lifestyle interventions and nutritional supplements that may ameliorate psychotic symptoms. Next, we analyze the role of tPA in the mechanism of action of hormones and medications effective in mitigating psychotic symptoms, such as pregnenolone, estrogen, oxytocin, dopamine D3 receptor antagonists, retinoic acid, valproic acid, cannabidiol, sodium nitroprusside, N-acetyl cysteine, and warfarin. We also review evidence that tPA participates in the mechanism by which electroconvulsive therapy and cigarette smoking may reduce psychotic symptoms.

  4. Endogenous Nitric Oxide Contributes to Bradykinin-Stimulated Glucose Uptake but Attenuates Vascular Tissue-Type Plasminogen Activator Release

    OpenAIRE

    Pretorius, Mias; Brown, Nancy J.

    2010-01-01

    Bradykinin causes vasodilation, stimulates tissue-type plasminogen activator (t-PA) release and, in rodents, increases muscle glucose uptake. Although bradykinin causes vasodilation partly by activating nitric-oxide synthase (NOS), the role of nitric oxide in regulating bradykinin-stimulated t-PA release is uncertain. This study examined the effect of high-dose NOS inhibition on bradykinin-stimulated t-PA release and glucose uptake in humans. We studied 24 healthy (12 women and 12 men), overw...

  5. Abrogation of Plasminogen Activator Inhibitor-1-Vitronectin Interaction Ameliorates Acute Kidney Injury in Murine Endotoxemia

    Science.gov (United States)

    Gupta, Kamlesh K.; Donahue, Deborah L.; Sandoval-Cooper, Mayra J.; Castellino, Francis J.; Ploplis, Victoria A.

    2015-01-01

    Sepsis-induced acute kidney injury (AKI) contributes to the high mortality and morbidity in patients. Although the pathogenesis of AKI during sepsis is poorly understood, it is well accepted that plasminogen activator inhibitor-1 (PAI-1) and vitronectin (Vn) are involved in AKI. However, the functional cooperation between PAI-1 and Vn in septic AKI has not been completely elucidated. To address this issue, mice were utilized lacking either PAI-1 (PAI-1?/?) or expressing a PAI-1-mutant (PAI-1R101A/Q123K) in which the interaction between PAI-1 and Vn is abrogated, while other functions of PAI-1 are retained. It was found that both PAI-1?/? and PAI-1R101A/Q123K mice are associated with decreased renal dysfunction, apoptosis, inflammation, and ERK activation as compared to wild-type (WT) mice after LPS challenge. Also, PAI-1?/? mice showed attenuated fibrin deposition in the kidneys. Furthermore, a lack of PAI-1 or PAI-1-Vn interaction was found to be associated with an increase in activated Protein C (aPC) in plasma. These results demonstrate that PAI-1, through its interaction with Vn, exerts multiple deleterious mechanisms to induce AKI. Therefore, targeting of the PAI-1-Vn interaction in kidney represents an appealing therapeutic strategy for the treatment of septic AKI by not only altering the fibrinolytic capacity but also regulating PC activity. PMID:25799354

  6. Presence of urokinase plasminogen activator, its inhibitor and receptor in small cell lung cancer and non-small cell lung cancer

    DEFF Research Database (Denmark)

    Pappot, H.; Pfeiffer, P.; Grøndahl Hansen, J.; Skov, B.G.

    1997-01-01

    Spreading of cancer cells is dependent on the combined action of several proteolytic enzymes, such as serine proteases, comprising the urokinase pathway of plasminogen activation. Previous studies of lung cancer indicate that expression, localization and prognostic impact of the components of the...... plasminogen activation system differ in the different non-small cell lung cancer (NSCLC) types, whereas the expression of the components in small cell lung cancer (SCLC) has only sparingly been investigated. In the present study we investigate the presence of the components of the plasminogen activation...... the clinical parameters. This is the first report of a study using a quantitative method to compare levels of the components of the plasminogen activation system in tissue extracts from the two major lung cancer groups. The study shows that uPA, PAI-1 and uPAR are present in SCLC-tissue, suggesting...

  7. Promotion of Wound Healing by an Agonist of Adenosine A2A Receptor Is Dependent on Tissue Plasminogen Activator.

    Science.gov (United States)

    Montesinos, M Carmen; Desai-Merchant, Avani; Cronstein, Bruce N

    2015-12-01

    Impaired wound healing, as it occurs in diabetes mellitus or long-term corticoid treatment, is commonly associated with disability, diminished quality of life, and high economic costs. Selective agonists of the A2A receptor subtype of adenosine, an endogenous regulator of inflammation, promote tissue repair in animal models, both healthy and with impaired healing. Plasmin-mediated proteolysis of fibrin and other matrix proteins is essential for cell migration at sites of injury. Since adenosine A2A receptor activation increases plasminogen activator release from macrophages and mast cells, we studied the effect of a selective agonist, CGS-21680, on full-thickness excisional wound closure in wild-type, urokinase plasminogen activator (uPA)-deficient, and tissue plasminogen activator (tPA)-deficient mice. Wound closure was impaired in tPA- and uPA-deficient mice as compared with wild-type mice, and topical application of CGS-21680 significantly increased the rate at which wounds closed in wild-type mice and uPA-deficient mice, but not in tPA-deficient mice. Immunostaining of tissue sections showed that tPA was present in endothelial cells and histiocytes by day 3 post-wound and also by day 6. In contrast, uPA was more prominent in these cell types only by day 6 post-wound. Our results confirm that plasminogen activation contributes to wound repair and are consistent with the hypothesis that adenosine A2A receptor activation promotes wound closure by a mechanism that depends upon tPA, but not uPA. Moreover, our results suggest that topical adenosine A2A receptor agonists may be useful in promotion of wound closure in patients with impaired wound healing. PMID:25991438

  8. Tissue-type plasminogen activator-binding RNA aptamers inhibiting low-density lipoprotein receptor family-mediated internalisation

    DEFF Research Database (Denmark)

    Bjerregaard, N; BØtkjær, K A

    2015-01-01

    Recombinant tissue-type plasminogen activator (tPA, trade name Alteplase), currently the only drug approved by the US Food and Drug Administration and the European Medicines Agency for the treatment of cerebral ischaemic stroke, has been implicated in a number of adverse effects reportedly mediated by interactions with the low-density lipoprotein (LDL) family receptors, including neuronal cell death and an increased risk of cerebral haemorrhage. The tissue-type plasminogen activator is the principal initiator of thrombolysis in human physiology, an effect that is mediated directly via localised activation of the plasmin zymogen plasminogen at the surface of fibrin clots in the vascular lumen. Here, we sought to identify a ligand to tPA capable of inhibiting the relevant LDL family receptors without interfering with the fibrinolytic activity of tPA. Systematic evolution of ligands by exponential enrichment (SELEX) was employed to isolate tPA-binding RNA aptamers, which were characterised in biochemical assays of tPA association to low density lipoprotein receptor-related protein-1 (LRP-1, an LDL receptor family member); tPA-mediated in vitro and ex vivo clot lysis; and tPA-mediated plasminogen activation in the absence and presence of a stimulating soluble fibrin fragment. Two aptamers, K18 and K32, had minimal effects on clot lysis, but were able to efficiently inhibit tPA-LRP-1 association and LDL receptor family-mediated endocytosis in human vascular endothelial cells and astrocytes. These observations suggest that coadministration alongside tPA may be a viable strategy to improve the safety of thrombolytic treatment of cerebral ischaemic stroke by restricting tPA activity to the vascular lumen.

  9. Bioconjugation of recombinant tissue plasminogen activator to magnetic nanocarriers for targeted thrombolysis

    Directory of Open Access Journals (Sweden)

    Yang HW

    2012-10-01

    Full Text Available Hung-Wei Yang,1,* Mu-Yi Hua,1,* Kun-Ju Lin,2,* Shiaw-Pyng Wey,3 Rung-Ywan Tsai,4 Siao-Yun Wu,5 Yi-Ching Lu,5 Hao-Li Liu,6 Tony Wu,7 Yunn-Hwa Ma5 1Chang Gung Molecular Medicine Research Center, Department of Chemical and Materials Engineering, 2Molecular Imaging Center, Department of Nuclear Medicine, Chang Gung Memorial Hospital, Kuei-Shan, Tao-Yuan, Taiwan, Republic of China; 3Department of Medical Imaging and Radiological Sciences, 4Electronics and Optoelectronics Research Laboratories, Industrial Technology Research Institute, Hsin-chu, Taiwan, Republic of China; 5Department of Physiology and Pharmacology and Healthy Aging Research Center, 6Department of Electrical Engineering, Chang Gung University, Kuei-Shan, Tao-Yuan, Taiwan, Republic of China; 7Department of Neurology, Chang Gung University College of Medicine and Memorial Hospital, Tao-Yuan, Taiwan, Republic of China*These authors contributed equally to this workAbstract: Low-toxicity magnetic nanocarriers (MNCs composed of a shell of poly [aniline-co-N-(1-one-butyric acid aniline] over a Fe3O4 magnetic nanoparticle core were developed to carry recombinant tissue plasminogen activator (rtPA in MNC-rtPA for targeted thrombolysis. With an average diameter of 14.8 nm, the MNCs exerted superparamagnetic properties. Up to 276 µg of active rtPA was immobilized per mg of MNCs, and the stability of the immobilized rtPA was greatly improved during storage at 4°C and 25°C. In vitro thrombolysis testing with a tubing system demonstrated that magnet-guided MNC-rtPA showed significantly improved thrombolysis compared with free rtPA and reduced the clot lysis time from 39.2 ± 3.2 minutes to 10.8 ± 4.2 minutes. In addition, magnet-guided MNC-rtPA at 20% of the regular rtPA dose restored blood flow within 15–25 minutes of treatment in a rat embolism model without triggering hematological toxicity. In conclusion, this improved system is based on magnetic targeting accelerated thrombolysis and is potentially amenable to therapeutic applications in thromboembolic diseases.Keywords: thrombolysis, recombinant tissue plasminogen activator, magnetic nanocarriers, magnetic targeting, targeting therapy

  10. Increased expression of urokinase plasminogen activator and its cognate receptor in human seminomas

    International Nuclear Information System (INIS)

    The urokinase plasminogen activating system (uPAS) is implicated in neoplastic progression and high tissue levels of uPAS components correlate with a poor prognosis in different human cancers. Despite that, relative few studies are available on the expression and function of the uPAS components in human seminomas. In the present study we characterized the expression of the urokinase plasminogen activator (uPA), its cognate receptor (uPAR) and the uPA inhibitors PAI-1 and PAI-2 in normal human testis and seminomas. The expression of the above genes was evaluated by means of quantitative RT-PCR, western blot, zymographic analysis and immunohistochemistry. Quantitative RT-PCR analysis of 14 seminomas demonstrated that uPA and uPAR mRNAs were, with respect to control tissues, increased in tumor tissues by 3.80 ± 0.74 (p < 0.01) and 6.25 ± 1.18 (p < 0.01) fold, respectively. On the other hand, PAI-1 mRNA level was unchanged (1.02 ± 0.24 fold), while that of PAI-2 was significantly reduced to 0.34 ± 0.18 (p < 0.01) fold. Western blot experiments performed with protein extracts of three seminomas and normal tissues from the same patients showed that uPA protein levels were low or undetectable in normal tissues and induced in tumor tissues. On the same samples, zymographic analysis demonstrated increased uPA activity in tumor tissue extracts. Western blot experiments showed that also the uPAR protein was increased in tumor tissues by 1.83 ± 0.15 fold (p < 0.01). The increased expression of uPA and uPAR was further confirmed by immunohistochemical staining performed in 10 seminomas and autologous uninvolved peritumoral tissues. Finally, variation in the mRNA level of PAI-1 significantly correlated with tumor size. We demonstrated the increased expression of uPA and uPAR in human seminomas with respect to normal testis tissues, which may be relevant in testicular cancer progression

  11. Simvastatin suppresses dexamethasone-induced secretion of plasminogen activator inhibitor-1 in human bone marrow adipocytes

    Directory of Open Access Journals (Sweden)

    Baba Hideo

    2011-04-01

    Full Text Available Abstract Background Osteonecrosis of the femoral head is a common complication of high-dose glucocorticoid treatment. Intravascular thrombosis is thought to be associated with the ischemic state of the femoral head. Plasminogen activator inhibitor-1 (PAI-1 is an adipokine, which are physiologically active substances secreted from visceral and subcutaneous adipocytes. PAI-1 suppresses fibrinolysis by binding tissue-type plasminogen activator. Several reports have described the relationship between PAI-1 and steroid-induced osteonecrosis of the femoral head, and the preventive effects of lipid-lowering agents (statins against steroid-induced osteonecrosis of the femoral head. We previously reported that adipokines and dexamethasone induced PAI-1 secretion from bone marrow adipocytes. The purpose of the present study is to examine the effects of simvastatin on PAI-1 secretion from human bone marrow adipocytes in vitro. Methods Primary bone marrow adipocytes were extracted from collagenase-treated bone marrow fluid obtained from the femoral necks of 40 patients (6 men, 34 women; age range, 52-81 years undergoing hip joint replacement surgery. After suspended culture with or without dexamethasone or simvastatin, PAI-1 mRNA expression was assessed by real-time RT-PCR. Total PAI-1 protein secretion in culture medium was assessed by enzyme-linked immunosorbent assay. Results PAI-1 mRNA expression was up-regulated by 388% (P = 0.002 with dexamethasone, and down-regulated by 45% (P = 0.002 with simvastatin, as compared to control levels. Dexamethasone increased total PAI-1 secretion by 166% (P = 0.001 and simvastatin decreased total PAI-1 secretion by 64% (P = 0.002. No significant changes were observed in adiponectin mRNA expression and secretion by dexamethasone and simvastatin, while pre-treatment with simvastatin reversed dexamethasone induced PAI-1 secretion by 89%, as compared to control levels. Conclusion The present study confirmed the suppressive effects of simvastatin on PAI-1 expression and secretion from bone marrow adipocytes. Furthermore, pre-treatment with simvastatin reversed dexamethasone induced PAI-1 secretion. Simvastatin may thus exhibit preventive effects against steroid-induced osteonecrosis of the femoral head by suppressing PAI-1 secretion.

  12. PGE2 reduces MMP-14 and increases plasminogen activator inhibitor-1 in cardiac fibroblasts.

    Science.gov (United States)

    Kassem, Kamal M; Clevenger, Margarette H; Szandzik, David L; Peterson, Edward; Harding, Pamela

    2014-10-01

    Prostaglandin E2 (PGE2) is elevated during cardiac injury and we have previously shown that mice lacking the PGE2 EP4 receptor display dilated cardiomyopathy (DCM) with increased expression of the membrane type matrix metalloproteinase, MMP-14. We thus hypothesized that PGE2 regulates expression of MMP-14 and also affects fibroblast migration. Primary cultures of neonatal rat ventricular fibroblasts (NVFs) were used to test the effects of PGE2. Gene and protein expression was assessed by real time RT-PCR and Western blot, MMP activity was determined by zymography and migration of NVF was assessed by motility in a transwell system. PGE2 reduced expression of MMP-14 and these effects were antagonized by an EP4 antagonist. An EP4 agonist mimicked the effect of PGE2. PGE2 also increased mRNA and protein levels of plasminogen activator inhibitor-1 (PAI-1), an inhibitor of MMP activation. However, PGE2-stimulation of PAI-1 was mediated by the EP1/EP3 receptor and not EP4. Migration of NVF was assessed by motility in a transwell system. Treatment of NVFs with PGE2 reduced the number of cells migrating toward 10% FCS. Treatment with the EP2 agonist also reduced migration but did not affect MMP-14 expression or PAI-1. Our results suggest that PGE2 utilizes different receptors and mechanisms to ultimately decrease MMP expression and NVF migration. PMID:25263346

  13. Tissue-type plasminogen activator triggers the synaptic vesicle cycle in cerebral cortical neurons.

    Science.gov (United States)

    Wu, Fang; Torre, Enrique; Cuellar-Giraldo, David; Cheng, Lihong; Yi, Hong; Bichler, Edyta K; García, Paul S; Yepes, Manuel

    2015-12-01

    The active zone (AZ) is a thickening of the presynaptic membrane where exocytosis takes place. Chemical synapses contain neurotransmitter-loaded synaptic vesicles (SVs) that at rest are tethered away from the synaptic release site, but after the presynaptic inflow of Ca(+2) elicited by an action potential translocate to the AZ to release their neurotransmitter load. We report that tissue-type plasminogen activator (tPA) is stored outside the AZ of cerebral cortical neurons, either intermixed with small clear-core vesicles or in direct contact with the presynaptic membrane. We found that cerebral ischemia-induced release of neuronal tPA, or treatment with recombinant tPA, recruits the cytoskeletal protein ?II-spectrin to the AZ and promotes the binding of SVs to ?II-spectrin, enlarging the population of SVs in proximity to the synaptic release site. This effect does not require the generation of plasmin and is followed by the recruitment of voltage gated calcium channels (VGCC) to the presynaptic terminal that leads to Ca(+2)-dependent synapsin I phosphorylation, freeing SVs to translocate to the AZ to deliver their neurotransmitter load. Our studies indicate that tPA activates the SV cycle and induces the structural and functional changes in the synapse that are required for successful neurotransmission. PMID:26126868

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2010-09-17

    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.

  15. Urokinase plasminogen activator receptor is expressed in invasive cells in gastric carcinomas from high- and low-risk countries

    DEFF Research Database (Denmark)

    Alpízar-Alpízar, Warner; Nielsen, Boye Schnack; Sierra, Rafaela; Illemann, Martin; Ramírez, Jose A; Arias, Adriana; Durán, Sundry; Skarstein, Arne; Ovrebo, Kjell; Lund, Leif R; Laerum, Ole D

    2010-01-01

    expression pattern of uPAR in gastric cancers from a high-risk country (Costa Rica) with a low-risk country (Norway). We found uPAR on gastric cancer cells in 24 of 44 cases (54%) from Costa Rica and in 13 of 23 cases (56%) from Norway. uPAR was seen in macrophages and neutrophils in all cases. We also......Gastric cancer is the second cancer causing death worldwide. Both incidence and mortality rates vary according to geographical regions. The receptor for urokinase plasminogen activator (uPAR) is involved in extracellular matrix degradation by mediating cell surface associated plasminogen activation......, and its presence on gastric cancer cells is linked to micro-metastasis and poor prognosis. Immunohistochemical analyses of a set of 44 gastric cancer lesions from Costa Rica showed expression of uPAR in cancer cells in both intestinal subtype (14 of 27) and diffuse subtype (10 of 17). We compared the...

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

    Science.gov (United States)

    Hansen, Line; Unmack Larsen, Esben Kjær; Nielsen, Erik Holm; Iversen, Frank; Liu, Zhuo; Thomsen, Karen; Pedersen, Michael; Skrydstrup, Troels; Nielsen, Niels Chr.; Ploug, Michael; Kjems, Jørgen

    2013-08-01

    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.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. Electronic supplementary information (ESI) available. See DOI: 10.1039/c3nr32922d

  17. Soluble urokinase plasminogen activator receptor levels are elevated and associated with complications in patients with type 1 diabetes

    DEFF Research Database (Denmark)

    Theilade, S; Lyngbaek, S; Hansen, T W; Eugen-Olsen, J; Fenger, M; Rossing, P; Jeppesen, J L

    2015-01-01

    OBJECTIVES: Soluble urokinase plasminogen activator receptor (suPAR) is a marker of inflammation and endothelial dysfunction. We investigated the associations between suPAR and diabetes, including diabetes duration and complications, in patients with type 1 diabetes. DESIGN, SETTING AND SUBJECTS: From 2009 to 2011, 667 patients with type 1 diabetes and 51 nondiabetic control subjects were included in a cross-sectional study at Steno Diabetes Center, Gentofte, Denmark. suPAR levels were measured ...

  18. Intraocular Lens Opacification following Intracameral Injection of Recombinant Tissue Plasminogen Activator to Treat Inflammatory Membranes after Cataract Surgery

    OpenAIRE

    Fung, Simon S. M.; Sykakis, Evripidis; Islam, Niaz M.; Zambarakji, Hadi J.; Khoramnia, Ramin; Auffarth, Gerd U; Parmar, Dipak N.

    2015-01-01

    Purpose. To report 7 cases of intraocular lens (IOL) opacification following treatment of postoperative anterior chamber fibrin with recombinant tissue plasminogen activator (rtPA) after cataract surgery. Methods. Retrospective case series of 7 eyes in 7 patients who developed IOL opacification after receiving rtPA for anterior chamber inflammatory membrane formation resulting from phacoemulsification cataract surgery. Three explanted IOLs were investigated with light microscopy, histochemica...

  19. Quantitative PET of human urokinase-type plasminogen activator receptor with 64Cu-DOTA-AE105

    DEFF Research Database (Denmark)

    Persson, Morten; Madsen, Jacob; Østergaard, Søren; Jensen, Mette Munk; Jørgensen, Jesper Tranekjær; Juhl, Karina; Lehmann, Charlotte; Ploug, Michael; Kjaer, Andreas

    2012-01-01

    Expression levels of the urokinase-type plasminogen activator receptor (uPAR) represent an established biomarker for poor prognosis in a variety of human cancers. The objective of the present study was to explore whether noninvasive PET can be used to perform a quantitative assessment of expressi...... levels of uPAR across different human cancer xenograft models in mice and to illustrate the clinical potential of uPAR PET in future settings for individualized therapy....

  20. Prognostic significance of circulating intact and cleaved forms of urokinase plasminogen activator receptor in inoperable chemotherapy treated cholangiocarcinoma patients

    DEFF Research Database (Denmark)

    Grunnet, Mie; Christensen, I J; Lassen, Ulrik; Jensen, Lone Hummelshøj; Lydolph, M; Lund, I K; Thurison, T; Høyer-Hansen, G; Mau-Sørensen, Morten

    2014-01-01

    BACKGROUND: High levels of intact and cleaved forms of the urokinase-type plasminogen activator receptor (uPAR) in both tissue and blood are associated with poor survival in several cancer diseases. The prognostic significance of uPAR in cholangiocarcinoma is unknown. The aims of this study were to determine if pre-treatment serum levels of uPAR forms and a decrease in levels during chemotherapy are predictive of survival in patients with inoperable cholangiocarcinoma. DESIGN AND METHODS: Patien...

  1. Quantitative PET of human urokinase-type plasminogen activator receptor with 64Cu-DOTA-AE105

    DEFF Research Database (Denmark)

    Persson, Morten; Madsen, Jacob; Østergaard, Søren; Jensen, Mette Munk; Jørgensen, Jesper Tranekjær; Juhl, Karina; Lehmann, Charlotte; Ploug, Michael; Kjaer, Andreas

    2012-01-01

    Expression levels of the urokinase-type plasminogen activator receptor (uPAR) represent an established biomarker for poor prognosis in a variety of human cancers. The objective of the present study was to explore whether noninvasive PET can be used to perform a quantitative assessment of expression levels of uPAR across different human cancer xenograft models in mice and to illustrate the clinical potential of uPAR PET in future settings for individualized therapy.

  2. Localization of urokinase-type plasminogen activator in stromal cells in adenocarcinomas of the colon in humans.

    OpenAIRE

    Grøndahl-Hansen, J; Ralfkiaer, E; Kirkeby, L. T.; Kristensen, P.; Lund, L. R.; Danø, K.

    1991-01-01

    Human colon adenocarcinomas and adjacent normal colon tissues were stained immunohistochemically with three different monoclonal antibodies and one preparation of polyclonal antibodies against each of the two plasminogen activators, uPA (urokinase type) and tPA (tissue type). The staining patterns seen with the respective sets of antibodies were identical. In all of 10 cases, staining for uPA in the normal colon tissue was confined to scattered fibroblastlike cells in the lamina propria. Othe...

  3. Serum levels of soluble urokinase plasminogen activator receptor is associated with parasitemia in children with acute Plasmodium falciparum malaria infection

    DEFF Research Database (Denmark)

    Perch, M; Kofoed, Pe; Fischer, Torge; Có, F; Rombo, L; Aaby, P; Eugen-Olsen, J

    2004-01-01

    Serum levels of soluble urokinase plasminogen activator receptor (suPAR) are significantly elevated and of prognostic value in patients suffering from serious infectious diseases such as HIV and tuberculosis. Our objective was to investigate suPAR levels during symptomatic malaria infection and 7 days after treatment. Children younger than 6 years who presented with fever or other symptoms compatible with malaria were enrolled. Blood films and samples were collected on day 0 and day 7. Twenty-fi...

  4. Cloning and Expression of a Human Tissue Plasminogen Activator Variant:K2S in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Hamid Mir Mohammad Sadeghi

    2007-01-01

    Full Text Available The DNA sequence of Kringle-2 and serine protease domains of the human tissue plasminogen activator (reteplase, K2S was PCR amplified. This product was then cloned into the expression vector pET15b plasmid. The presence of the insert was confirmed by restriction digestion, PCR and determination of the nucleotide sequence. By using isopropyl ?-D thiogalactopyranoside (IPTG, reteplase was induced in E. coli BL21 cells and analyzed using polyacrylamide gel electrophoresis (PAGE.

  5. Heparanase procoagulant activity, factor Xa, and plasminogen activator inhibitor 1 are increased in shift work female nurses.

    Science.gov (United States)

    Nadir, Yona; Saharov, Gleb; Hoffman, Ron; Keren-Politansky, Anat; Tzoran, Inna; Brenner, Benjamin; Shochat, Tamar

    2015-07-01

    Epidemiologic studies indicate on an increased risk of cardiovascular disease and cancer in shift workers, although the underlying mechanism is obscure. Heparanase directly enhances tissue factor (TF) activity leading to increased factor Xa production and subsequent activation of the coagulation system. In the present study, a comparison of coagulation markers among healthy shift working (SW) vs. healthy daytime working (DW) female nurses was performed. Thirty SW and 30 DW female nurses were enrolled. For each of the 60 participants, blood was drawn between 7:00 and 8:00 a.m. and at least 8 h after the last work shift. Plasma was studied for coagulation marker that included TF/heparanase procoagulant activity, TF activity, heparanase procoagulant activity, heparanase level, factor Xa level, plasminogen activator inhibitor 1 (PAI-1), plasminogen, ?2-antiplasmin, fibrinogen, global protein C, von Willebrand factor, and D-dimer by chromogenic assays and enzyme-linked immunosorbent assays (ELISAs). Sleep quality was assessed by self-report according to the Pittsburgh Sleep Quality Index. The heparanase procoagulant activity increased by 2-fold and the TF/heparanase procoagulant activity increased by 1.5-fold in SW nurses compared to DW nurses (P?levels and PAI-1 levels were significantly higher among SW nurses compared to the DW group (22 vs. 18 ng/ml, P?level, and PAI-1 level were significantly higher in SW nurses compared to the DW group. These alterations of blood coagulation activation may potentially contribute to cardiovascular and cancer morbidity. PMID:25743687

  6. Biological and binding activities of ovine and porcine prolactins in porcine mammary tissue

    International Nuclear Information System (INIS)

    The concentration of prolactin receptors may play a critical role in regulating growth and development of the mammary gland during gestation and tumor development; however, the discrepancy between specific binding of ovine prolactin (oPRL) and porcine prolactin (pPRL) in porcine mammary tissue was disturbing. It was possible that 125I-oPRL may be an unsuitable ligand for the procine prolactin receptor. The validate the use of oPRL in binding assays, the biological and binding activities of oPRL and pPRL were compared. A lactogenic bioassay of pPRL was developed using porcine mammary explants cultured in Medium 199 containing insulin, cortisol, and pPRL. The potencies of oPRL and pPRL were compared using this bioassay. Oxidation of glucose and incorporation of glucose into lipids were similarly enhanced by physiological concentrations of both oPRL and pPRL. However, specific binding of 125I-oPRL was 20%, while less than 1% of 125I-pPRL was bound. 125I-oPRL bound to high affinity sites

  7. Combination Low-Dose Tissue-Type Plasminogen Activator Plus Annexin A2 for Improving Thrombolytic Stroke Therapy

    Science.gov (United States)

    Jiang, Yinghua; Fan, Xiang; Yu, Zhanyang; Liao, Zhengbu; Wang, Xiao-Shu; van Leyen, Klaus; Sun, Xiaochuan; Lo, Eng H.; Wang, Xiaoying

    2015-01-01

    Risk of hemorrhagic transformation, incomplete reperfusion, neurotoxicity, and a short treatment time window comprises major challenges for tissue plasminogen activator (tPA) thrombolytic stroke therapy. Improving tPA therapy has become one of the highest priorities in the stroke field. This mini review article focuses on our recent efforts aimed at evaluating a novel combination approach of low-dose tPA plus recombinant annexin A2 (rA2, a tPA, and plasminogen co-receptor), which might enhance tPA thrombolytic efficacy, while reducing its associated complications related to intracerebral hemorrhagic transformation. Results of our experimental studies using a focal embolic stroke model in rats support the feasibility of the combination approach and suggest the potential for successful clinical translation. PMID:26528130

  8. Bicyclic Peptide Inhibitor of Urokinase-Type Plasminogen Activator : Mode of Action

    DEFF Research Database (Denmark)

    Roodbeen, Renée; Paaske, Berit

    2013-01-01

    The development of protease inhibitors for pharmacological intervention has taken a new turn with the use of peptidebased inhibitors. Here, we report the rational design of bicyclic peptide inhibitors of the serine protease urokinase-type plasminogen activator (uPA), based on the established monocyclic peptide, upain-2. It was successfully converted to a bicyclic peptide, without loss of inhibitory properties. The aim was to produce a peptide cyclised by an amide bond with an additional stabilising across-the-ring covalent bond. We expected this bicyclic peptide to exhibit a lower entropic burden upon binding. Two bicyclic peptides were synthesised with affinities similar to that of upain-2, and their binding energetics were evaluated by isothermal titration calorimetry. Indeed, compared to upain-2, the bicyclic peptides showed reduced loss of entropy upon binding to uPA. We also investigated the solution structures of the bicyclic peptide by NMR spectroscopy to map possible conformations. An X-ray structure of the bicyclicpeptide– uPA complex confirmed an interaction similar to that for the previous upain-1/upain-2–uPA complexes. These physical studies of the peptide–protease interactions will aid future designs of bicyclic peptide protease inhibitors

  9. Annexin A2 in primary afferents contributes to neuropathic pain associated with tissue type plasminogen activator.

    Science.gov (United States)

    Yamanaka, H; Kobayashi, K; Okubo, M; Noguchi, K

    2016-02-01

    Annexin A2 (ANX2) is a calcium (Ca(2+))-binding protein that binds to acidic phospholipids and is known to play a crucial role in many cellular regulatory processes. In particular, ANX2 has been described as a crucial receptor for thrombolysis by the tissue-type plasminogen activator (tPA) and plasmin system. In the nervous system, tPA is involved in processes of neuronal plasticity such as hippocampal long-term potentiation (LTP) and in the dorsal horn pain in several pain models. We investigated detailed changes in expression of ANX2 after nerve injury and evaluated the interaction with tPA using the rat spared nerve injury (SNI) model. SNI-induced the expression of ANX2 in L4/5 dorsal root ganglia (DRG) neurons. In the spinal cord, constitutive ANX2-immunoreactivity was expressed in laminae I-II. Peripheral nerve injury increased the ANX2 immunoreactive terminals mainly in laminae I-V of the dorsal horn on the side ipsilateral to the nerve injury. Double-labeling analysis revealed the co-localization of ANX2 with tPA in the axons of primary afferents in the dorsal horn. Experimental inhibition of ANX2 and tPA interaction by intrathecal administration of homocysteine significantly prevented and reversed SNI-induced mechanical allodynia. Thus, alterations of ANX2 may be involved in tPA-dependent plasticity after peripheral nerve injury and have an important role in neuropathic pain. PMID:26642807

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

    Science.gov (United States)

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

    1989-10-01

    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

  11. 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

  12. Evaluation of Cerebral Perfusion in Patients Undergoing Intravenous Recombinant Tissue Plasminogen Activator Thrombolysis

    Science.gov (United States)

    HIRANO, Teruyuki

    2015-01-01

    Currently, the indication for thrombolytic therapy using intravenous recombinant tissue plasminogen activator (rt-PA) is restricted strictly to patients with acute ischemic stroke within 4.5 h of onset. The effect of rt-PA declines over time; therefore, we need to minimize the time delay while generating imaging information. The use of cerebral blood flow imaging is not recommended within this time window. Conversely, the balance of efficacy and the risk of bleeding complications differ among patients > 4.5 h after onset. Several ongoing studies are using mismatch concepts to extend the therapeutic time window for rt-PA. Long-awaited reliable software, such as RAPID and PMA, are now available to analyze computed tomography/magnetic resonance perfusion data. Patients with wake-up stroke (WUS) are another group that can be used to expand rt-PA candidates. Diffusion fluid- attenuated inversion recovery mismatch is a promising imaging surrogate to select good candidates with WUS. These trials will cause a therapeutic paradigm shift from time-based to tissue-based strategies in the near future. PMID:26369875

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

    DEFF Research Database (Denmark)

    Hansen, Line; Unmack Larsen, Esben Kjær

    2013-01-01

    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.

  14. Soluble Urokinase Plasminogen Activator Receptor as a Marker for Use of Antidepressants

    DEFF Research Database (Denmark)

    Haastrup, Eva; Grau, Katrine

    2014-01-01

    OBJECTIVES: Inflammation is involved in the pathogenesis of depression. A few cross-sectional population-based studies have found that depression is associated with increased levels of inflammatory markers. Soluble urokinase plasminogen activation receptor (suPAR) is known to be a stable marker for inflammation. We investigated the bidirectional association between suPAR levels and use of antidepressants. METHODS: suPAR level was measured in 9305 blood donors and analysed in relation to 5-years follow-up data on purchase of antidepressants and hospital diagnoses of depression from a nationwide Danish register. RESULTS: For men and women without prior use of antidepressants we found a significantly higher risk for incident use of antidepressants with higher suPAR values. For men, the risk of first use of antidepressants increased by 72% from the 1st to the 4th quartile (HR = 1.72, 95% CI: 1.11-2.69). For women, it increased by 108% from the 1st to the 4th quartile (HR = 2.08, 95% CI: 1.45-2.98). Previous use of antidepressants was also significantly associated with higher suPAR levels (p = 0.002). CONCLUSIONS: High suPAR levels are associated with an increased risk for both previous and future use of antidepressants in healthy men and women. High suPAR are also associated with increased risk for a hospital diagnosis of depression.

  15. Soluble urokinase plasminogen activator receptor as a marker for use of antidepressants

    DEFF Research Database (Denmark)

    Haastrup, Eva; Grau, Katrine

    2014-01-01

    OBJECTIVES: Inflammation is involved in the pathogenesis of depression. A few cross-sectional population-based studies have found that depression is associated with increased levels of inflammatory markers. Soluble urokinase plasminogen activation receptor (suPAR) is known to be a stable marker for inflammation. We investigated the bidirectional association between suPAR levels and use of antidepressants. METHODS: suPAR level was measured in 9305 blood donors and analysed in relation to 5-years follow-up data on purchase of antidepressants and hospital diagnoses of depression from a nationwide Danish register. RESULTS: For men and women without prior use of antidepressants we found a significantly higher risk for incident use of antidepressants with higher suPAR values. For men, the risk of first use of antidepressants increased by 72% from the 1st to the 4th quartile (HR?=?1.72, 95% CI: 1.11-2.69). For women, it increased by 108% from the 1st to the 4th quartile (HR?=?2.08, 95% CI: 1.45-2.98). Previous useof antidepressants was also significantly associated with higher suPAR levels (p?=?0.002). CONCLUSIONS: High suPAR levels are associated with an increased risk for both previous and future use of antidepressants in healthy men and women. High suPAR are also associated with increased risk for a hospital diagnosis of depression.

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

    Directory of Open Access Journals (Sweden)

    Abdoli Nasrin

    2011-06-01

    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.

  17. DNA repair and induction of plasminogen activator in human fetal cells treated with ultraviolet light

    International Nuclear Information System (INIS)

    Human fetal fibroblasts have been tested for development associated changes in DNA repair by utilizing nucleoid sedimentation as an assay for excision repair. Among skin fibroblasts the rate of excision repair was significantly higher in non-fetal cells than in fibroblasts derived from an 8 week fetus. Skin fibroblasts derived at 12 week gestation were more repair proficient than at 8 weeks. However, they exhibited a lower rate of repair than non-fetal cells. Enhancement of protease plasminogen activator (PA) was higher after u.v. irradiation in skin fibroblasts derived at 8 weeks than at 12 weeks gestation and was absent in non-fetal skin fibroblasts. Excision repair and PA inducibility depended on the tissue of origin in addition to gestational stage, as shown for skin and lung fibroblasts from the same 12 week fetus. The sedimentation velocity of nucleoids, prepared from unirradiated fibroblasts, in neutral sucrose gradients with or without ethidium bromide, indicated the presence of DNA strand breaks in fetal cells. It is proposed that reduced DNA repair in fetal cells may result from alterations in DNA supercoiling, and that persistent DNA strand breaks enhance transcription of PA gene(s). (author)

  18. Medicolegal Considerations with Intravenous Tissue Plasminogen Activator in Stroke: A Systematic Review

    Science.gov (United States)

    Bhatt, Archit; Safdar, Adnan; Majid, Arshad; Kassab, Mounzer

    2013-01-01

    Background. Intravenous tPA (tissue plasminogen activator) therapy remains underutilized in patients with Acute Ischemic Stroke (AIS). Anecdotal data indicates that physicians are increasingly liable for administering and for failure to administer tPA. Methods. An extensive search of Medline, Embase, Westlaw, LexisNexis Legal, and Google Scholar databases was performed. Case studies that involved malpractice litigation in ischemic stroke and thrombolytic therapy were analyzed systematically. Results. We identified 789 ischemic stroke litigation cases, of which 46 cases were related to intravenous tPA and stroke litigation. Case descriptions of 40 cases were available. Data for verdicts were available for 38 patients. The most frequent plaintiff claim was related to failure to administer intravenous tPA (38, 95%). Only 2 (5.0%) claim involved complications of treatment with tPA. Hospitals were defendants in majority of the 36 cases. Physicians were involved in 33 cases. While ED physicians were involved in 25 (60.52%) cases, neurologists were involved in 8 (20.0%) cases. There were 26 (65%) defendant-favored and 12 (30%) plaintiff-favored verdicts. Conclusion. Physicians and hospitals are at an increased risk of litigation in patients with AIS when in IV-tPA is being considered for treatment. While majority of the cases litigated were cases where tPA was not administered, only about 1 in 20 cases was litigated when complications occurred. PMID:24083048

  19. Is vascular imaging valuable prior to administration of intravenous tissue plasminogen activator?

    Science.gov (United States)

    Eichel, Roni; Cohen, Jose E; Gomori, John M; Ben-Hur, Tamir; Keigler, Galina; Leker, Ronen R

    2014-10-01

    Our goals were to explore whether performing computerized tomography angiography (CTA) prior to administration of tissue plasminogen activator (tPA) delays treatment and impacts outcome in patients with proximal middle cerebral artery occlusions (pMCAO). Patients with pMCAO with a National Institutes of Health Stroke scale (NIHSS) score >10 were identified from a prospective Stroke Registry. Patients underwent multi-parametric imaging studies whenever possible. Patients who underwent CTA were compared to those who only had non-contrast CT scan. Disability was measured with the modified Rankin Scale. Logistic regression was used to determine outcome modifiers. We included 73 patients (median age 73 years, 52% men) with moderate-severe stroke (median admission NIHSS 14). Of those, 44 underwent CTA and 29 did not. There were no differences between the groups in risk factor profile or baseline characteristics including stroke severity and door to needle, door to imaging or imaging to treatment times. At 90 days post-stroke there were no statistically significant differences in outcomes between the groups. On multivariate analysis, performing CTA had no impact on the chance of obtaining favorable outcome. In conclusion, CTA does not have a major impact on outcome in patients with pMCAO treated with tPA. Therefore, performing CTA should be considered on an individual basis prior to administration of tPA. PMID:24874695

  20. Evaluation of Cerebral Perfusion in Patients Undergoing Intravenous Recombinant Tissue Plasminogen Activator Thrombolysis.

    Science.gov (United States)

    Hirano, Teruyuki

    2015-10-15

    Currently, the indication for thrombolytic therapy using intravenous recombinant tissue plasminogen activator (rt-PA) is restricted strictly to patients with acute ischemic stroke within 4.5 h of onset. The effect of rt-PA declines over time; therefore, we need to minimize the time delay while generating imaging information. The use of cerebral blood flow imaging is not recommended within this time window. Conversely, the balance of efficacy and the risk of bleeding complications differ among patients > 4.5 h after onset. Several ongoing studies are using mismatch concepts to extend the therapeutic time window for rt-PA. Long-awaited reliable software, such as RAPID and PMA, are now available to analyze computed tomography/magnetic resonance perfusion data. Patients with wake-up stroke (WUS) are another group that can be used to expand rt-PA candidates. Diffusion fluid-attenuated inversion recovery mismatch is a promising imaging surrogate to select good candidates with WUS. These trials will cause a therapeutic paradigm shift from time-based to tissue-based strategies in the near future. PMID:26369875

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

    Science.gov (United States)

    Hansen, Line; Larsen, Esben Kjær Unmack; Nielsen, Erik Holm; Iversen, Frank; Liu, Zhuo; Thomsen, Karen; Pedersen, Michael; Skrydstrup, Troels; Nielsen, Niels Chr; Ploug, Michael; Kjems, Jørgen

    2013-09-01

    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. PMID:23835641

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

    Directory of Open Access Journals (Sweden)

    Po-Min Yang

    2005-12-01

    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.

  3. Dynamic structural and functional relationships in recombinant plasminogen activator inhibitor-1 (rPAI-1).

    Science.gov (United States)

    Vaughan, D E; Declerck, P J; Reilly, T M; Park, K; Collen, D; Fasman, G D

    1993-10-01

    The conformational characteristics of active, latent, and denatured recombinant plasminogen activator inhibitor-1 (rPAI-1) were compared using UV spectroscopy, spectrofluorimetry and circular dichroism (CD) techniques. The UV absorbance wavelength maxima in all preparations approximated 280 nm, while the extinction coefficients of active and latent rPAI-1 differed by up to 60%. When incubated at 37 degrees C, the A280 of latent rPAI-1 was quite stable while the A280 of active rPAI-1 spontaneously increased, eventually approximating that of latent rPAI-1. Alkali difference spectroscopy yielded markedly divergent titration patterns for active and latent rPAI-1, suggesting that the tyrosine residues present in active and latent rPAI-1 differ in terms of solvent exposure. At an excitation wavelength of 280 nm, active rPAI-1 exhibited the greatest relative fluorescence quantum yield. The relative fluorescence of latent and denatured rPAI-1 were less than that of active PAI-1, and the emission maxima of both species were slightly red-shifted in comparison to that of active rPAI-1, suggesting that at least one of the four tryptophan residues present in rPAI-1 is less exposed to the aqueous environment in the active form of the molecule. In contrast, the derived secondary structures based on CD of active and latent rPAI-1 were nearly identical, with both moieties exhibiting approx. 40% alpha-helix and 15% beta-sheet. Taken together, these spectroscopic data provide evidence supporting the hypothesis that active and latent PAI-1 differ in terms of their tertiary conformation and aromatic residue exposure, while their secondary structures appear generally comparable. Furthermore, denaturant-induced reactivation of latent rPAI-1 produces a partially active rPAI-1 with spectroscopic properties similar to that of latent rPAI-1, suggesting that denatured rPAI-1 more closely resembles the latent rPAI-1 conformation after refolding. The spontaneous spectroscopic changes observed in rPAI-1 may reflect conformational transitions that are critical to the regulation of endogenous PAI-1 activity. PMID:8399383

  4. Oviduct-specific expression of tissue plasminogen activator in laying hens

    Scientific Electronic Library Online (English)

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

    Full Text Available 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.

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

    DEFF Research Database (Denmark)

    Einholm, Anja P; Pedersen, Katrine E

    2003-01-01

    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

  6. Uncharged isocoumarin-based inhibitors of urokinase-type plasminogen activator

    Directory of Open Access Journals (Sweden)

    Deck Lorraine M

    2006-02-01

    Full Text Available Abstract Background Urokinase-type plasminogen activator (uPA plays a major role in extracellular proteolytic events associated with tumor cell growth, migration and angiogenesis. Consequently, uPA is an attractive target for the development of small molecule active site inhibitors. Most of the recent drug development programs aimed at nonpeptidic inhibitors targeted at uPA have focused on arginino mimetics containing amidine or guanidine functional groups attached to aromatic or heterocyclic scaffolds. There is a general problem of limited bioavailability of these charged inhibitors. In the present study, uPA inhibitors were designed on an isocoumarin scaffold containing uncharged substituents. Results 4-Chloro-3-alkoxyisocoumarins were synthesized in which the 3-alkoxy group contained a terminal bromine; these were compared with similar inhibitors that contained a charged terminal functional group. Additional variations included functional groups attached to the seven position of the isocoumarin scaffold. N- [3-(3-Bromopropoxy-4-chloro-1-oxo-1H-isochromen-7-yl]benzamide was identified as an uncharged lead inhibitor of uPA, Ki = 0.034 μM. Molecular modeling of human uPA with these uncharged inhibitors suggests that the bromine occupies the same position as positively charged arginino mimetic groups. Conclusion This study demonstrates that potent uncharged inhibitors of uPA can be developed based upon the isocoumarin scaffold. A tethered bromine in the three position and an aromatic group in the seven position are important contributors to binding. Although the aim was to develop compounds that act as mechanism-based inactivators, these inhibitors are competitive reversible inhibitors.

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

    DEFF Research Database (Denmark)

    Nielsen, Jimmi; RØge, Rasmus

    2015-01-01

    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.

  8. Prevention of obesity and insulin resistance in mice lacking plasminogen activator inhibitor 1.

    Science.gov (United States)

    Ma, Li-Jun; Mao, Su-Li; Taylor, Kevin L; Kanjanabuch, Talerngsak; Guan, YouFei; Zhang, YaHua; Brown, Nancy J; Swift, Larry L; McGuinness, Owen P; Wasserman, David H; Vaughan, Douglas E; Fogo, Agnes B

    2004-02-01

    Increased plasminogen activator inhibitor 1 (PAI-1) has been linked to not only thrombosis and fibrosis but also to obesity and insulin resistance. Increased PAI-1 levels have been presumed to be consequent to obesity. We investigated the interrelationships of PAI-1, obesity, and insulin resistance in a high-fat/high-carbohydrate (HF) diet-induced obesity model in wild-type (WT) and PAI-1-deficient mice (PAI-1(-/-)). Obesity and insulin resistance developing in WT mice on an HF diet were completely prevented in mice lacking PAI-1. PAI-1(-/-) mice on an HF diet had increased resting metabolic rates and total energy expenditure compared with WT mice, along with a marked increase in uncoupling protein 3 mRNA expression in skeletal muscle, likely mechanisms contributing to the prevention of obesity. In addition, insulin sensitivity was enhanced significantly in PAI-1(-/-) mice on an HF diet, as shown by euglycemic-hyperinsulinemic clamp studies. Peroxisome proliferator-activated receptor (PPAR)-gamma and adiponectin mRNA, key control molecules in lipid metabolism and insulin sensitivity, were maintained in response to an HF diet in white adipose tissue in PAI-1(-/-) mice, contrasting with downregulation in WT mice. This maintenance of PPAR-gamma and adiponectin may also contribute to the observed maintenance of body weight and insulin sensitivity in PAI-1(-/-) mice. Treatment in WT mice on an HF diet with the angiotensin type 1 receptor antagonist to downregulate PAI-1 indeed inhibited PAI-1 increases and ameliorated diet-induced obesity, hyperglycemia, and hyperinsulinemia. PAI-1 deficiency also enhanced basal and insulin-stimulated glucose uptake in adipose cells in vitro. Our data suggest that PAI-1 may not merely increase in response to obesity and insulin resistance, but may have a direct causal role in obesity and insulin resistance. Inhibition of PAI-1 might provide a novel anti-obesity and anti-insulin resistance treatment. PMID:14747283

  9. Interaction of mutants of tissue-type plasminogen activator with liver cells: effect of domain deletions.

    Science.gov (United States)

    Kuiper, J; Van't Hof, A; Otter, M; Biessen, E A; Rijken, D C; van Berkel, T J

    1996-02-01

    The fibrin-specific thrombolyticum tissue-type plasminogen activator (t-PA) has proven to be a potent drug in several clinical trials, but its clinical application is complicated by the rapid clearance of t-PA from the circulation. The rapid plasma clearance of t-PA results from the uptake of t-PA in the liver. t-PA consists of several domains which may be involved in the interaction with the liver. Three domain-deletion mutants, which were produced by the use of a cassette gene system, were studied in vivo and in vitro for their capacity to bind to the various types of rat liver cells. The three mutants lacked, in comparison to control t-PA, the epidermal growth factor (G) domain, the finger (F) domain or the G domain plus the first kringle (K1). The plasma clearance of the three mutants was slower than that of control t-PA. The slower plasma clearance resulted from a decreased liver uptake: 50 and 80% for t-PA mutants and control t-PA respectively. It was found that the K1 domain was of major importance for the uptake of t-PA by liver endothelial cells in vivo and in vitro. The high-affinity binding of t-PA (and t-PA mutants) to parenchymal liver cells depended largely on the presence of the G domain. Other domain(s), like the F, K2 or protease domain, may be responsible for low-affinity, t-PA-specific binding to rat parenchymal liver cells. PMID:8611154

  10. Preclinical evaluation of a urokinase plasminogen activator receptor-targeted nanoprobe in rhesus monkeys

    Science.gov (United States)

    Chen, Yushu; Gong, Li; Gao, Ning; Liao, Jichun; Sun, Jiayu; Wang, Yuqing; Wang, Lei; Zhu, Pengjin; Fan, Qing; Wang, Yongqiang Andrew; Zeng, Wen; Mao, Hui; Yang, Lily; Gao, Fabao

    2015-01-01

    Purpose To translate a recombinant peptide containing the amino-terminal fragment (ATF) of urokinase plasminogen activator receptor-targeted magnetic iron oxide (IO) nanoparticles (uPAR-targeted human ATF-IONPs) into clinical applications, we conducted a pilot study to evaluate the toxicity and pharmacokinetics of this nanoparticle in normal rhesus monkeys. Methods We assessed the changes in the following: magnetic resonance imaging (MRI) signals from pretreatment stage to 14 days posttreatment, serum iron concentrations from 5 minutes posttreatment to 12 weeks posttreatment, routine blood examination and serum chemistry analysis results from pretreatment stage to 12 weeks after administration, and results of staining of the liver with Perls’ Prussian Blue and hematoxylin–eosin at 24 hours and 3 months posttreatment in two rhesus monkeys following an intravenous administration of the targeted nanoparticles either with a polyethylene glycol (ATF-PEG-IONP) or without a PEG (ATF-IONP) coating. Results The levels of alkaline phosphatase, alanine transaminase, and direct bilirubin in the two monkeys increased immediately after the administration of the IONPs but returned to normal within 20 days and stayed within the normal reference range 3 months after the injection. The creatinine levels of the two monkeys stayed within the normal range during the study. In addition, red blood cells, white blood cells, hemoglobin level, and platelets remained normal during the 3 months of the study. Conclusion All of the results suggest that a transient injury in terms of normal organ functions, but no microscopic necrotic lesions, was observed at a systemic delivery dose of 5 mg/kg of iron equivalent concentration in the acute phase, and that no chronic toxicity was found 3 months after the injection. Therefore, we conclude that uPAR-targeted IONPs have the potential to be used as receptor-targeted MRI contrasts as well as theranostic agents for the detection and treatment of human cancers in future studies. PMID:26604745

  11. Plasma plasminogen activator inhibitor-1 predicts myocardial infarction in HIV-1-infected individuals

    DEFF Research Database (Denmark)

    Knudsen, Andreas; Katzenstein, Terese L

    2014-01-01

    OBJECTIVES:: Biomarkers of endothelial dysfunction, inflammation and coagulation are associated with atherosclerosis and cardiovascular disease, but their association and possible predictive value remain controversial among HIV-1-infected individuals. We sought to investigate the association of seven biomarkers with first-time myocardial infarction (MI) in an HIV-1-infected population. DESIGN:: A matched case-control study of 54 cases and 54 controls. METHODS:: We compared 54 HIV-1-infected patients with verified first-time MI and 54 HIV-1-infected controls matched for age, duration of antiretroviral therapy, sex, smoking and no known cardiovascular disease. Levels of high-sensitivity C-reactive protein, soluble endothelial selectin, soluble vascular cell adhesion molecule, soluble intercellular adhesion molecule, matrix metalloprotease 9, myeloperoxidase, and plasminogen activator inhibitor 1 (PAI-1) were measured using a Luminex assay in plasma samples from routine visits both 12 and 2 months prior to thecase patient's MI. RESULTS:: The two groups had similar HIV characteristics and traditional cardiovascular risk factors. In univariate analysis, PAI-1 levels were associated with MI, whereas none of the other markers showed any association.In multivariate analyses adjusting for the D:A:D risk score, HIV viral load and high-sensitivity C-reactive protein, PAI-1 levels in the highest quartile were associated with a six to seven-fold increased risk of MI in both samples. CONCLUSION:: High levels of PAI-1 were associated with risk of first-time MI in HIV-1-infected individuals independently of cardiovascular risk factors, HIV parameters and antiretroviral therapy. Therefore PAI-1 may be used for risk stratification and prediction of CHD, but further studies are needed.

  12. The hepatic clearance of recombinant tissue-type plasminogen activator decreases after an inflammatory stimulus

    Directory of Open Access Journals (Sweden)

    M.R. Nagaoka

    2000-01-01

    Full Text Available We have shown that tissue-type plasminogen activator (tPA and plasma kallikrein share a common pathway for liver clearance and that the hepatic clearance rate of plasma kallikrein increases during the acute-phase (AP response. We now report the clearance of tPA from the circulation and by the isolated, exsanguinated and in situ perfused rat liver during the AP response (48-h ex-turpentine treatment. For the sake of comparison, the hepatic clearance of a tissue kallikrein and thrombin was also studied. We verified that, in vivo, the clearance of 125I-tPA from the circulation of turpentine-treated rats (2.2 ± 0.2 ml/min, N = 7 decreases significantly (P = 0.016 when compared to normal rats (3.2 ± 0.3 ml/min, N = 6. The AP response does not modify the tissue distribution of administered 125I-tPA and the liver accounts for most of the 125I-tPA (>80% cleared from the circulation. The clearance rate of tPA by the isolated and perfused liver of turpentine-treated rats (15.5 ± 1.3 µg/min, N = 4 was slower (P = 0.003 than the clearance rate by the liver of normal rats (22.5 ± 0.7 µg/min, N = 10. After the inflammatory stimulus and additional Kupffer cell ablation (GdCl3 treatment, tPA was cleared by the perfused liver at 16.2 ± 2.4 µg/min (N = 5, suggesting that Kupffer cells have a minor influence on the hepatic tPA clearance during the AP response. In contrast, hepatic clearance rates of thrombin and pancreatic kallikrein were not altered during the AP response. These results contribute to explaining why the thrombolytic efficacy of tPA does not correlate with the dose administered.

  13. DNA repair and induction of plasminogen activator in human fetal cells treated with ultraviolet light

    International Nuclear Information System (INIS)

    We have tested human fetal fibroblasts for development associated changes in DNA repair by utilizing nucleoid sedimentation as an assay for excision repair. Among skin fibroblasts the rate of excision repair was significantly higher in non-fetal cells than in fibroblasts derived from an 8 week fetus; this was evident by a delay in both the relaxation and the restoration of DNA supercoiling in nucleoids after irradiation. Skin fibroblasts derived at 12 week gestation were more repair proficient than those derived at 8 week gestation. However, they exhibited a somewhat lower rate of repair than non-fetal cells. The same fetal and non-fetal cells were also tested for induction of the protease plasminogen activator (PA) after u.v. irradiation. Enhancement of PA was higher in skin fibroblasts derived at 8 week than in those derived at 12 week gestation and was absent in non-fetal skin fibroblasts. These results are consistent with our previous findings that in human cells u.v. light-induced PA synthesis is correlated with reduced DNA repair capacity. Excision repair and PA inducibility were found to depend on tissue of origin in addition to gestational stage, as shown for skin and lung fibroblasts from the same 12 week fetus. Lung compared to skin fibroblasts exhibited lower repair rates and produced higher levels of PA after irradiation. The sedimentation velocity of nucleoids, prepared from unirradiated fibroblasts, in neutral sucrose gradients with or without ethidium bromide, indicated the presence of DNA strand breaks in fetal cells. It is proposed that reduced DNA repair in fetal cells may result from alterations in DNA supercoiling, and that persistent DNA strand breaks enhance transcription of PA gene(s)

  14. Recombinant human erythropoietin reduces plasminogen activator inhibitor and ameliorates pro-inflammatory responses following trauma

    Directory of Open Access Journals (Sweden)

    M Mojtahedzadeh

    2011-05-01

    Full Text Available "n  "n Background and the purpose of the study: Besides its hematopoietic effects, erythropoietin (EPO by mobilization of iron and modulation of some inflammatory cytokines has antioxidant and anti-inflammatory properties. The purpose of this study was to evaluate these effects of erythropoietin and its impact on organ function in traumatized patients. "n Methods: Twenty-six ICU-admitted traumatized patients within 24 hrs after trauma were randomly assigned to the EPO (received EPO, 300 units/Kg/day and Control (not received EPO groups. The inflammatory biomarkers including Tumor Necrosis Factor alpha (TNF-α, Interleukin 1 (IL-1, Plasminogen Activator Inhibitor 1 (PAI-1 and Nitrotyrosine were recorded at the admission, 3, 6 and 9 days thereafter. Acute Physiology and Chronic Health Evaluation (APACHE II and Sequential Organ Failure Assessment (SOFA scores were also recorded. "n Results: Among 12 patients (EPO group TNF-α level at the day of 9 (P=0.046, and within EPO group at the days of 3 (P=0.026 ameliorate, 6 (P=0.016, and 9 (P=0.052 were significantly lowered. Level of IL-1 and PAI-1 decreased significantly at days of 3, 6 and 9 post intervention. Also there were significant differences between two groups in the SOFA score during three measured time intervals (the first, third and seventh days. "n Conclusion: From the results of this study it seems that injection of erythrocyte stimulating agent is well tolerated and inhibits the inflammatory response and oxidative stress following trauma.

  15. Ischaemia–reperfusion injury impairs tissue plasminogen activator release in man

    Science.gov (United States)

    Pedersen, Christian M.; Barnes, Gareth; Schmidt, Michael R.; Bøtker, Hans Erik; Kharbanda, Rajesh K.; Newby, David E.; Cruden, Nicholas L.

    2012-01-01

    Aims Ischaemia–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 results Ischaemia–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<0.05 for all). Substance P caused a dose-dependent increase in t-PA release (P<0.05 for all). Acetylcholine and substanceP-mediated vasodilatation and substanceP-mediated t-PA release were impaired following IR injury (P<0.05 for all). Neither local nor remote ischaemic preconditioning protected against the impairment of substance P-mediated vasodilatation or t-PA release. Conclusion Ischaemia–reperfusion injury induced substanceP-mediated, endothelium-dependent vasomotor and fibrinolytic dysfunction in man that could not be prevented by ischaemic preconditioning. Clinical Trial Registration Information: Reference number: NCT00789243, URL: http://clinicaltrials.gov/ct2/show/NCT00789243?term=NCT00789243&rank=1 PMID:21990263

  16. Segregation analysis of plasminogen activator inhibitor-1 and fibrinogen levels in the NHLBI family heart study.

    Science.gov (United States)

    Pankow, J S; Folsom, A R; Province, M A; Rao, D C; Williams, R R; Eckfeldt, J; Sellers, T A

    1998-10-01

    Elevated plasminogen activator inhibitor-1 (PAI-1) and fibrinogen concentrations are risk factors for coronary heart disease. We investigated environmental, familial, and genetic influences on PAI-1 antigen and fibrinogen concentrations in 2029 adults from 512 randomly ascertained families in 4 US communities. We used maximum-likelihood segregation analysis to fit several genetic and nongenetic modes of inheritance to the data to determine whether mendelian inheritance of a major gene could best explain the familial distributions of these 2 hemostatic factors. Age- and gender-adjusted familial correlations for PAI-1 antigen level averaged 0.16 in first-degree relatives (95% CI=0.11 to 0.21); the spouse correlation was positive but not statistically significant (r=0.10, 95% CI=-0.02 to 0.23). Complex segregation analysis indicated a major gene associated with higher PAI-1 concentrations in 65% of individuals from these families. Demographic, anthropometric, lifestyle, and metabolic characteristics together explained 37% to 47% of the variation in PAI-1 antigen levels, and the inferred major gene explained an additional 17% of the variance. Positive and statistically significant age- and gender-adjusted familial correlations in first-degree relatives indicated a possible heritable component influencing plasma fibrinogen concentration (r=0. 17, 95% CI=0.13 to 0.22); however, segregation analysis did not provide statistical evidence of a major gene controlling fibrinogen level. These family data suggest that there are modest familial and genetic effects on the concentration of PAI-1. PMID:9763527

  17. Tissue and urokinase plasminogen activators instigate the degeneration of retinal ganglion cells in a mouse model of glaucoma.

    Science.gov (United States)

    Chintala, Shravan K

    2016-02-01

    Elevated intraocular pressure (IOP) promotes the degeneration of retinal ganglion cells (RGCs) during the progression of Primary Open-Angle Glaucoma (POAG). However, the molecular mechanisms underpinning IOP-mediated degeneration of RGCs remain unclear. Therefore, by employing a mouse model of POAG, this study examined whether elevated IOP promotes the degeneration of RGCs by up-regulating tissue plasminogen activator (tPA) and urokinase plasminogen activator (uPA) in the retina. IOP was elevated in mouse eyes by injecting fluorescent-microbeads into the anterior chamber. Once a week, for eight weeks, IOP in mouse eyes was measured by using Tono-Pen XL. At various time periods after injecting microbeads, proteolytic activity of tPA and uPA in retinal protein extracts was determined by fibrinogen/plasminogen zymography assays. Localization of tPA and uPA, and their receptor LRP-1 (low-density receptor-related protein-1) in the retina was determined by immunohistochemistry. RGCs' degeneration was assessed by immunostaining with antibodies against Brn3a. Injection of microbeads into the anterior chamber led to a progressive elevation in IOP, increased the proteolytic activity of tPA and uPA in the retina, activated plasminogen into plasmin, and promoted a significant degeneration of RGCs. Elevated IOP up-regulated tPA and LRP-1 in RGCs, and uPA in astrocytes. At four weeks after injecting microbeads, RAP (receptor associated protein; 0.5 and 1.0 ?M) or tPA-Stop (1.0 and 4.0 ?M) was injected into the vitreous humor. Treatment of IOP-elevated eyes with RAP led to a significant decrease in proteolytic activity of both tPA and uPA, and a significant decrease in IOP-mediated degeneration of RGCs. Also, treatment of IOP-elevated eyes with tPA-Stop decreased the proteolytic activity of both tPA and uPA, and, in turn, significantly attenuated IOP-mediated degeneration of RGCs. Results presented in this study provide evidence that elevated IOP promotes the degeneration of RGCs by up-regulating the levels of proteolytically active tPA and uPA. PMID:26474495

  18. Serum soluble urokinase-type plasminogen activator receptor levels in male patients with acute exacerbation of schizophrenia.

    Science.gov (United States)

    Genc, Abdullah; Kalelioglu, Tevfik; Karamustafalioglu, Nesrin; Tasdemir, Akif; Genc, Esra Sena; Akkus, Mustafa; Emul, Murat

    2016-02-28

    Inflammatory abnormalities have been shown in the pathogenesis of schizophrenia. Soluble urokinase-type plasminogen activator receptor (suPAR) is a protein that is measurable in the circulating blood and reflects the inflammation in the body. We aimed to investigate serum suPAR levels in patients with schizophrenia who were in acute state and to compare with healthy controls. Forty five patients and 43 healthy controls were included in the study. We found no significant difference in suPAR levels between patients and controls, suggesting that suPAR as an inflammatory marker does not have a role in the inflammatory process of acute schizophrenia. PMID:26774189

  19. Acceleration of the thrombin inactivation of single chain urokinase-type plasminogen activator (pro-urokinase) by thrombomodulin.

    OpenAIRE

    de Munk, G A; Groeneveld, E; Rijken, D.C.

    1991-01-01

    The in vitro effects of thrombomodulin on the inactivation of single chain urokinase-type plasminogen activator (scu-PA) by thrombin were investigated by incubating scu-PA with varying concentrations of human thrombin, in both the absence and presence of soluble rabbit thrombomodulin. 50% inactivation of scu-PA occurred in 45 min at 160 ng/ml thrombin in the absence of thrombomodulin and at 4.6 ng/ml thrombin in the presence of thrombomodulin. No difference was found in either the absence or ...

  20. 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)

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

    2012-04-06

    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.

  1. Plasminogen Acquisition and Activation at the Surface of Leptospira Species Lead to Fibronectin Degradation ?

    OpenAIRE

    Vieira, Monica L; Vasconcellos, Silvio A.; Gonçales, Amane P; de Morais, Zenaide M; Nascimento, Ana L.T.O

    2009-01-01

    Pathogenic Leptospira species are the etiological agents of leptospirosis, a widespread disease of human and veterinary concern. In this study, we report that Leptospira species are capable of binding plasminogen (PLG) in vitro. The binding to the leptospiral surface was demonstrated by indirect immunofluorescence confocal microscopy with living bacteria. The PLG binding to the bacteria seems to occur via lysine residues because the ligation is inhibited by addition of the lysine analog 6-ami...

  2. Endotoxin-induced intravascular coagulation in rabbits: effect of tissue plasminogen activator vs urokinase of PAI generation, fibrin deposits and mortality

    OpenAIRE

    Paloma, M.J. (María José); Paramo, J A; Rocha, E.

    1995-01-01

    We have evaluated the effect of plasminogen activators (t-PA and urokinase) on an experimental model of disseminated intravascular coagulation (DIC) in rabbits by injection of 20 micrograms/kg/h of E. coli lipopolysaccharide during 6 h t-PA (0.2 mg/kg and 0.7 mg/kg), urokinase (3000 U/kg/h) and saline (control) were given simultaneously with endotoxin. Results indicated that urokinase and low dose of t-PA significantly reduced the increase of plasminogen activator inhibitor (PAI) activity obs...

  3. Specific binding of urinary-type plasminogen activator (u-PA) to vitronectin and its role in mediating u-PA-dependent adhesion of U937 cells

    DEFF Research Database (Denmark)

    Moser, T L; Enghild, J J; Pizzo, S V; Stack, M S

    1995-01-01

    The present paper described interactions of urinary-type plasminogen activator (u-PA) with isolated protein components of the extracellular matrix (ECM) using kinetic and ligand-blotting analyses, as well as adhesion studies with u-PA-saturated U937 monocytic cells. Kinetic analyses showed that fibronectin and laminin were moderately effective at decreasing activation of plasminogen by u-PA (3-4-fold decrease in kcat/Km), while activation was stimulated slightly by collagen types I and IV (2-4-f...

  4. Plasminogen in periodontitis and wound repair

    OpenAIRE

    Sulniute, Rima

    2013-01-01

    The plasminogen activator (PA) system plays a critical role in many physiological and pathological processes, such as fibrinolysis, extracellular matrix (ECM) degradation, wound healing, inflammation, and cancer. The key component of the PA system is plasmin, a broad-spectrum serine protease that is derived from its inactive form, plasminogen. The first aim of this thesis research was to determine the role of plasminogen in periodontitis, an inflammatory oral disease. The second aim was to ex...

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

    Directory of Open Access Journals (Sweden)

    Sayers Ian

    2009-07-01

    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.

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

    LENUS (Irish Health Repository)

    Maher, Vincent M G

    2009-08-01

    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.

  7. Preclinical evaluation of a urokinase plasminogen activator receptor-targeted nanoprobe in rhesus monkeys

    Directory of Open Access Journals (Sweden)

    Chen Y

    2015-10-01

    Full Text Available Yushu Chen,1 Li Gong,2 Ning Gao,3 Jichun Liao,1 Jiayu Sun,1 Yuqing Wang,1 Lei Wang,1 Pengjin Zhu,1 Qing Fan,1 Yongqiang Andrew Wang,4 Wen Zeng,2 Hui Mao,3 Lily Yang,5 Fabao Gao11Molecular Imaging Center, Department of Radiology, West China Hospital, Sichuan University, Chengdu, 2Sichuan Primed Bio-Tech Group Co, Ltd, Chengdu, People’s Republic of China; 3Department of Radiology and Imaging Sciences, Emory University School of Medicine, Atlanta, GA, 4Ocean NanoTech, LLC, San Diego, CA, 5Department of Surgery, Emory University School of Medicine, Atlanta, GA, USAPurpose: To translate a recombinant peptide containing the amino-terminal fragment (ATF of urokinase plasminogen activator receptor-targeted magnetic iron oxide (IO nanoparticles (uPAR-targeted human ATF-IONPs into clinical applications, we conducted a pilot study to evaluate the toxicity and pharmacokinetics of this nanoparticle in normal rhesus monkeys.Methods: We assessed the changes in the following: magnetic resonance imaging (MRI signals from pretreatment stage to 14 days posttreatment, serum iron concentrations from 5 minutes posttreatment to 12 weeks posttreatment, routine blood examination and serum chemistry analysis results from pretreatment stage to 12 weeks after administration, and results of staining of the liver with Perls’ Prussian Blue and hematoxylin–eosin at 24 hours and 3 months posttreatment in two rhesus monkeys following an intravenous administration of the targeted nanoparticles either with a polyethylene glycol (ATF-PEG-IONP or without a PEG (ATF-IONP coating.Results: The levels of alkaline phosphatase, alanine transaminase, and direct bilirubin in the two monkeys increased immediately after the administration of the IONPs but returned to normal within 20 days and stayed within the normal reference range 3 months after the injection. The creatinine levels of the two monkeys stayed within the normal range during the study. In addition, red blood cells, white blood cells, hemoglobin level, and platelets remained normal during the 3 months of the study.Conclusion: All of the results suggest that a transient injury in terms of normal organ functions, but no microscopic necrotic lesions, was observed at a systemic delivery dose of 5 mg/kg of iron equivalent concentration in the acute phase, and that no chronic toxicity was found 3 months after the injection. Therefore, we conclude that uPAR-targeted IONPs have the potential to be used as receptor-targeted MRI contrasts as well as theranostic agents for the detection and treatment of human cancers in future studies. Keywords: uPAR-IONP, nonhuman primates, transient harm, self-healing

  8. 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 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)

  9. Intravenous Tissue Plasminogen Activator Improves the Outcome in Very Elderly Korean Patients with Acute Ischemic Stroke

    Science.gov (United States)

    Choi, Jay Chol; Lee, Ji Sung; Park, Tai Hwan; Park, Sang-Soon; Cho, Yong-Jin; Park, Jong-Moo; Kang, Kyusik; Lee, Kyung Bok; Lee, Soo-Joo; Ko, Youngchai; Kim, Jae Guk; Lee, Jun; Cho, Ki-Hyun; Kim, Joon-Tae; Yu, Kyung-Ho; Lee, Byung-Chul; Oh, Mi-Sun; Cha, Jae-Kwan; Kim, Dae-Hyun; Nah, Hyun-Wook; Kim, Dong-Eog; Ryu, Wi-Sun; Kim, Beom Joon; Bae, Hee-Joon; Kim, Wook-Joo; Shin, Dong-Ick; Yeo, Min-Ju; Sohn, Sung Il; Hong, Jeong-Ho; Lee, Juneyoung; Hong, Keun-Sik

    2015-01-01

    Background and Purpose In a recent pooled analysis of randomized clinical trials (RCTs), intravenous tissue plasminogen activator (TPA) improves the outcome in patients aged ?80 years. However, it is uncertain whether the findings are applicable to clinical practice in Asian populations. Methods From a multicenter stroke registry database of Korea, we identified patients with acute ischemic stroke who were aged ? 80 years. Using multivariable analysis and propensity score (PS)-matched analyses, we assessed the effectiveness and safety of intravenous TPA within 4.5 hours. Results Among 2,334 patients who met the eligible criteria, 236 were treated with intravenous TPA (mean age, 83±5; median NIHSS, 13 [IQR, 8-17]). At discharge, the TPA group compared to the no-TPA group had a favorable shift on the modified Rankin Scale (mRS) score (multivariable analysis, OR [95% CI], 1.51 [1.17-1.96], P=0.002; PS-matched analysis, 1.54 [1.17-2.04], P=0.002) and was more likely to achieve mRS 0-1 outcome (multivariable analysis, 2.00 [1.32-3.03], P=0.001; PS-matched analysis, 1.59 [1.04-2.42], P=0.032). TPA treatment was associated with an increased risk of symptomatic intracranial hemorrhage (multivariable analysis, 5.45 [2.80-10.59], P<0.001; PS-matched analysis, 4.52 [2.24-9.13], P<0.001), but did not increase the in-hospital mortality (multivariable analysis, 0.86 [0.50-1.48], P=0.58; PS-matched analysis, 0.88 [0.52-1.47], P=0.61). Conclusions In the setting of clinical practice, intravenous TPA within 4.5 hours improved the functional outcome despite an increased risk of symptomatic intracranial hemorrhage in very elderly Korean patients. The findings, consistent with those from pooled analysis of RCTs, strongly support the use of TPA for this population. PMID:26437998

  10. Assessment of plasminogen synthesis in vitro by mouse tumor cells using a competition radioimmunoassay for mouse plasminogen

    International Nuclear Information System (INIS)

    A sensitive, specific competition radioimmunoassay for mouse plasmin(ogen) has been developed in order to determine whether mouse tumor cells can synthesize plasminogen in vitro. The rabbit anti-BALB/c mouse plasminogen antibodies used in the assay react with the plasminogen present in serum from BALB/c, C3H, AKR and C57BL/6 mice, and also recognized mouse plasmin. The competition radiommunoassay can detect as little as 50 ng of mouse plasminogen. No competition was observed with preparations of fetal calf, human and rabbit plasminogens. A variety of virus-transformed and mouse tumor cell lines were all found to contain less than 100 ng mouse plasminogen/mg of cell extract protein. Thus, if the plasminogen activator/plasmin system is important in the growth or movement of this group of tumor cells, the cells will be dependent upon the circulatory system of the host for their plasminogen supply. (Auth.)

  11. Platelet-activating factor induces ovine fetal pulmonary venous smooth muscle cell proliferation: role of epidermal growth factor receptor transactivation.

    Science.gov (United States)

    Zhou, Weilin; Ibe, Basil O; Raj, J Usha

    2007-06-01

    We have previously reported that platelet-activating factor (PAF) is present in very high levels in the ovine fetal lung and circulation and that PAF serves as an important physiological vasoconstrictor of the pulmonary circulation in utero. However, it is not known whether PAF stimulates pulmonary vascular smooth muscle cell (SMC) proliferation. In this study, we used ovine fetal pulmonary venous SMCs as our model system to study the effects and mechanisms of action of PAF on SMC proliferation. We found that PAF induced SMC proliferation in a dose-dependent manner. PAF also stimulated activation of both ERK and p38 but not c-Jun NH(2) terminal kinase (JNK) mitogen-activated protein (MAP) kinase pathways. PAF (10 nM) induced phosphorylation of epidermal growth factor receptor (EGFR). Specific inhibition of EGFR by AG-1478 and by the expression of a dominant-negative EGFR mutant in SMCs attenuated PAF-stimulated cell proliferation. Inhibition of heparin-binding EGF-like growth factor (HB-EGF) release by CRM-197 and inhibition of matrix metalloproteinases (MMP) by GM-6001 abolished PAF-induced MAP kinase activation and cell proliferation. Increased alkaline phosphatase (AP) activity after PAF treatment in AP-HB-EGF fusion construct-transfected SMCs indicated that PAF induced the release of HB-EGF within 1 min. Gelatin zymography data showed that PAF stimulated MMP-2 activity and MMP-9 activity within 1 min. These results suggest that PAF promotes pulmonary vascular SMC proliferation via transactivation of EGFR through MMP activation and HB-EGF, resulting in p38 and ERK activation and that EGFR transactivation is essential for the mitogenic effect of PAF in pulmonary venous SMC. PMID:17322418

  12. Lysosomal degradation of receptor-bound urokinase-type plasminogen activator is enhanced by its inhibitors in human trophoblastic choriocarcinoma cells

    DEFF Research Database (Denmark)

    Jensen, Poul Henning; Christensen, Erik Ilsø; Ebbesen, P.; Gliemann, Jørgen; Andreasen, Peter A.

    1990-01-01

    We have studied the effect of plasminogen activator inhibitors PAI-1 and PAI-2 on the binding of urokinase-type plasminogen activator (u-PA) to its receptor in the human choriocarcinoma cell line JAR. With 125I-labeled ligands in whole-cell binding assays, both uncomplexed u-PA and u-PA-inhibitor...... for the removal of u-PA after its complex formation with a specific inhibitor. The data suggest a novel mechanism by which receptor-mediated endocytosis is initiated by the binding of a secondary ligand. Full text...

  13. Mammalian protein secretion without signal peptide removal. Biosynthesis of plasminogen activator inhibitor-2 in U-937 cells

    International Nuclear Information System (INIS)

    Plasminogen activator inhibitor-2 (PAI-2) is a serine protease inhibitor that regulates plasmin generation by inhibiting urokinase and tissue plasminogen activator. The primary structure of PAI-2 suggests that it may be secreted without cleavage of a single peptide. To confirm this hypothesis we have studied the glycosylation and secretion of PAI-2 in human monocytic U-937 cells by metabolic labeling, immunoprecipitation, glycosidase digestion, and protein sequencing. PAI-2 is variably glycosylated on asparagine residues to yield intracellular intermediates with zero, one, two, or three high mannose-type oligosaccharide units. Secretion of the N-glycosylated species began by 1 h of chase and the secreted molecules contained both complex-type N-linked and O-linked oligosaccharides. Enzymatically deglycosylated PAI-2 had an electrophoretic mobility identical to that of the nonglycosylated precursor and also to that of PAI-2 synthesized in vitro in a rabbit reticulocyte lysate from synthetic mRNA derived from full length PAI-2 cDNA. The amino-terminal protein sequence of secreted PAI-2 began with the initiator methionine residue. These results indicate that PAI-2 is glycosylated and secreted efficiently without the cleavage of a signal peptide. PAI-2 shares this property with its nearest homologue in the serine protease inhibitor family, chicken ovalbumin, and appears to be the first well characterized example of this phenomenon among natural mammalian proteins

  14. Tissue-type plasminogen activator suppresses activated stellate cells through low-density lipoprotein receptor-related protein 1.

    Science.gov (United States)

    Kang, Liang-I; Isse, Kumiko; Koral, Kelly; Bowen, William C; Muratoglu, Selen; Strickland, Dudley K; Michalopoulos, George K; Mars, Wendy M

    2015-10-01

    Hepatic stellate cell (HSC) activation and trans-differentiation into myofibroblast (MFB)-like cells is key for fibrogenesis after liver injury and a potential therapeutic target. Recent studies demonstrated that low-density lipoprotein receptor-related protein 1 (LRP1)-dependent signaling by tissue-type plasminogen activator (t-PA) is a pro-fibrotic regulator of the MFB phenotype in kidney. This study investigated whether LRP1 signaling by t-PA is also relevant to HSC activation following injury. Primary and immortalized rat HSCs were treated with t-PA and assayed by western blot, MTT, and TUNEL. In vitro results were then verified using an in vivo, acute carbon tetrachloride (CCl4) injury model that examined the phenotype and recovery kinetics of MFBs from wild-type animals vs mice with a global (t-PA) or HSC-targeted (LRP1) deletion. In vitro, in contrast to kidney MFBs, exogenous, proteolytically inactive t-PA suppressed, rather than induced, activation markers in HSCs following phosphorylation of LRP1. This process was mediated by LRP1 as inhibition of t-PA binding to LRP1 blocked the effects of t-PA. In vivo, following acute injury, phosphorylation of LRP1 on activated HSCs occurred immediately prior to their disappearance. Mice lacking t-PA or LRP1 retained higher densities of activated HSCs for a longer time period compared with control mice after injury cessation. Hence, t-PA, an FDA-approved drug, contributes to the suppression of activated HSCs following injury repair via signaling through LRP1. This renders t-PA a potential target for exploitation in treating patients with fibrosis. PMID:26237273

  15. The Plasminogen Activator/Plasmin System Is Essential for Development of the Joint Inflammatory Phase of Collagen Type II-Induced Arthritis

    OpenAIRE

    Li, Jinan; Ny, Annelii; Leonardsson, Göran; Nandakumar, Kutty Selva; Holmdahl, Rikard; Ny, Tor

    2005-01-01

    The plasminogen activator (PA) system has been proposed to have important roles in rheumatoid arthritis. Here we have used the autoimmune collagen type II (CII)-induced arthritis (CIA) model and mice deficient for urokinase-type PA (uPA) or plasminogen to investigate the role of the PA system for development of arthritis. Our data revealed that uPA-deficient mice have a lower severity and incidence of CIA than wild-type mice. Furthermore, although >80% of wild-type control mice developed CIA,...

  16. Plasma soluble urokinase-type plasminogen activator receptor level is independently associated with coronary microvascular function in patients with non-obstructive coronary artery disease

    DEFF Research Database (Denmark)

    Mekonnen, Girum; Corban, Michel T; Hung, Olivia Y; Eshtehardi, Parham; Eapen, Danny J; Al-Kassem, Hatem; Rasoul-Arzrumly, Emad; Gogas, Bill D; McDaniel, Michael C; Pielak, Tomasz; Thorball, Christian W; Sperling, Laurence; Quyyumi, Arshed A; Samady, Habib

    2015-01-01

    BACKGROUND: Soluble urokinase-type plasminogen activator receptor (suPAR) is a novel biomarker released from leukocytes and endothelial cells that has been associated with atherosclerotic cardiovascular disease. We hypothesized that plasma suPAR level is an independent predictor of coronary micro...

  17. Soluble Urokinase Plasminogen Activator Receptor Level Is an Independent Predictor of the Presence and Severity of Coronary Artery Disease and of Future Adverse Events

    DEFF Research Database (Denmark)

    Eapen, Danny J; Manocha, Pankaj; Ghasemzedah, Nima; Patel, Riyaz S; Al Kassem, Hatem; Hammadah, Muhammad; Veledar, Emir; Le, Ngoc-Anh; Pielak, Tomasz; Thorball, Christian W; Velegraki, Aristea; Kremastinos, Dimitrios T; Lerakis, Stamatios; Sperling, Laurence; Quyyumi, Arshed A

    2014-01-01

    INTRODUCTION: Soluble urokinase plasminogen activator receptor (suPAR) is an emerging inflammatory and immune biomarker. Whether suPAR level predicts the presence and the severity of coronary artery disease (CAD), and of incident death and myocardial infarction (MI) in subjects with suspected CAD...

  18. Activation of Urokinase Plasminogen Activator and Its Receptor Axis Is Essential for Macrophage Infiltration in a Prostate Cancer Mouse Model

    Directory of Open Access Journals (Sweden)

    Jian Zhang

    2011-01-01

    Full Text Available Macrophages within the tumor microenvironment promote angiogenesis, extracellular matrix breakdown, and tumor cell migration, invasion, and metastasis. Activation of the urokinase plasminogen activator (uPA and its receptor (uPAR axis promotes prostate cancer tumorigenicity, invasion, metastasis, and survival within the tumor microenvironment. The link between macrophage infiltration and the uPA/uPAR axis in prostate cancer development has not been established, although it has been reported that uPA plays a critical role in monocyte and macrophage chemotaxis. In this study, murine prostate cancer RM-1 cells were subcutaneously inoculated into wild-type (WT, uPA?/?, and uPAR?/? mice. Tumor volume was significantly diminished in both uPA?/? and uPAR?/? mice compared with WT controls. Greater inhibition of tumor volume was also observed in uPA?/? mice compared with uPAR?/? mice, suggesting the important contribution of stromal-derived uPA to sustain the tumor growth. Immunohistochemical staining revealed that tumors in uPA?/? and uPAR?/? mice displayed significantly lower proliferative indices, higher apoptotic indices, and less neovascularity compared with the tumors in WT mice. Tumors in uPA?/? and uPAR?/? mice displayed significantly less macrophage infiltration as demonstrated by F4/80 staining and MAC3+ cell numbers by flow cytometry compared with the tumors from WT mice. These findings suggest that the uPA/uPAR axis acts in both autocrine and paracrine manners in the tumor microenvironment, and activation of uPA/uPAR axis is essential for macrophage infiltration into prostate tumors.

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

    DEFF Research Database (Denmark)

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

    2011-01-01

    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 investi...

  20. 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 tPA/mL. In all the plasma tested, more than half of the antigen is undetected unless the plasma is treated as described above

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

    Science.gov (United States)

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

    2015-06-01

    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 < 0.001). Patients with HCV had higher suPAR concentrations than patients with NAFLD (P < 0.002). suPAR levels were associated with the severity of fibrosis, particularly in NAFLD, but did not correlate with inflammation. Regarding the performance in predicting severity of fibrosis, suPAR was essentially as good as other commonly used noninvasive fibrosis scoring systems. The results in HCV confirm previous observations. However, this is the first study to investigate suPAR as a biomarker in NAFLD, and the results indicate that suPAR may constitute a severity marker related to fibrosis and prognosis rather than reflecting inflammation. PMID:25445207

  2. Overexpression of SERBP1 (Plasminogen activator inhibitor 1 RNA binding protein) in human breast cancer is correlated with favourable prognosis

    International Nuclear Information System (INIS)

    Plasminogen activator inhibitor 1 (PAI-1) overexpression is an important prognostic and predictive biomarker in human breast cancer. SERBP1, a protein that is supposed to regulate the stability of PAI-1 mRNA, may play a role in gynaecological cancers as well, since upregulation of SERBP1 was described in ovarian cancer recently. This is the first study to present a systematic characterisation of SERBP1 expression in human breast cancer and normal breast tissue at both the mRNA and the protein level. Using semiquantitative realtime PCR we analysed SERBP1 expression in different normal human tissues (n = 25), and in matched pairs of normal (n = 7) and cancerous breast tissues (n = 7). SERBP1 protein expression was analysed in two independent cohorts on tissue microarrays (TMAs), an initial evaluation set, consisting of 193 breast carcinomas and 48 normal breast tissues, and a second large validation set, consisting of 605 breast carcinomas. In addition, a collection of benign (n = 2) and malignant (n = 6) mammary cell lines as well as breast carcinoma lysates (n = 16) were investigated for SERBP1 expression by Western blot analysis. Furthermore, applying non-radioisotopic in situ hybridisation a subset of normal (n = 10) and cancerous (n = 10) breast tissue specimens from the initial TMA were analysed for SERBP1 mRNA expression. SERBP1 is not differentially expressed in breast carcinoma compared to normal breast tissue, both at the RNA and protein level. However, recurrence-free survival analysis showed a significant correlation (P = 0.008) between abundant SERBP1 expression in breast carcinoma and favourable prognosis. Interestingly, overall survival analysis also displayed a tendency (P = 0.09) towards favourable prognosis when SERBP1 was overexpressed in breast cancer. The RNA-binding protein SERBP1 is abundantly expressed in human breast cancer and may represent a novel breast tumour marker with prognostic significance. Its potential involvement in the plasminogen activator protease cascade warrants further investigation

  3. Urokinase plasminogen activator receptor is expressed in invasive cells in gastric carcinomas from high- and low-risk countries

    DEFF Research Database (Denmark)

    Alpízar-Alpízar, Warner; Nielsen, Boye S.

    2010-01-01

    Gastric cancer is the second cancer causing death worldwide. Both incidence and mortality rates vary according to geographical regions. The receptor for urokinase plasminogen activator (uPAR) is involved in extracellular matrix degradation by mediating cell surface associated plasminogen activation, and its presence on gastric cancer cells is linked to micro-metastasis and poor prognosis. Immunohistochemical analyses of a set of 44 gastric cancer lesions from Costa Rica showed expression of uPAR in cancer cells in both intestinal subtype (14 of 27) and diffuse subtype (10 of 17). We compared the expression pattern of uPAR in gastric cancers from a high-risk country (Costa Rica) with a low-risk country (Norway). We found uPAR on gastric cancer cells in 24 of 44 cases (54%) from Costa Rica and in 13 of 23 cases (56%) from Norway. uPAR was seen in macrophages and neutrophils in all cases. We also examined the nonneoplastic mucosa and found that uPAR was more frequently seen in epithelial cells located at the luminal edge of the crypts in cases with Helicobacter pylori infection than in similar epithelial cells in noninfected mucosa (p = 0.033; chi(2) = 4.54). In conclusion, the expression of uPAR in cancer cells in more than half of the gastric cancer cases suggests that their uPAR-positivity do not contribute to explain the different mortality rates between the 2 countries, however, the actual prevalence of uPAR-positive cancer cells in the gastric cancers may still provide prognostic information.

  4. Cloning and expression of a cDNA coding for a human monocyte-derived plasminogen activator inhibitor

    International Nuclear Information System (INIS)

    Human monocyte-derived plasminogen activator inhibitor (mPAI-2) was purified to homogeneity from the U937 cell line and partially sequenced. Oligonucleotide probes derived from this sequence were used to screen a cDNA library prepared from U937 cells. One positive clone was sequenced and contained most of the coding sequence as well as a long incomplete 3' untranslated region (1112 base pairs). This cDNA sequence was shown to encode mPAI-2 by hybrid-select translation. A cDNA clone encoding the remainder of the mPAI-2 mRNA was obtained by primer extension of U937 poly(A)+ RNA using a probe complementary to the mPAI-2 coding region. The coding sequence for mPAI-2 was placed under the control of the λ P/sub L/ promoter, and the protein expressed in Escherichia coli formed a complex with urokinase that could be detected immunologically. By nucleotide sequence analysis, mPAI-2 cDNA encodes a protein containing 415 amino acids with a predicted unglycosylated M/sub r/ of 46,543. The predicted amino acid sequence of mPAI-2 is very similar to placental PAI-2 and shows extensive homology with members of the serine protease inhibitor (serpin) superfamily. mPAI-2 was found to be more homologous to ovalbumin (37%) than the endothelial plasminogen activator inhibitor, PAI-1 (26%). The 3' untranslated region of the mPAI-2 cDNA contains a putative regulatory sequence that has been associated with the inflammatory mediators

  5. Mannose 6-Phosphate/Insulin-like Growth Factor 2 Receptor Limits Cell Invasion by Controlling ?V?3 Integrin Expression and Proteolytic Processing of Urokinase-type Plasminogen Activator Receptor

    OpenAIRE

    Schiller, Herbert B; Szekeres, Andreas; Binder, Bernd R.; Stockinger, Hannes; Leksa, Vladimir

    2009-01-01

    The multifunctional mannose 6-phosphate/insulin-like growth factor 2 receptor (M6P/IGF2R) is considered a tumor suppressor. We report here that RNA interference with M6P/IGF2R expression in urokinase-type plasminogen activator (uPA)/urokinase-type plasminogen activator receptor (uPAR) expressing human cancer and endothelial cells resulted in increased pericellular plasminogen activation, cell adhesion, and higher invasive potential through matrigel. M6P/IGF2R silencing led also to the cell su...

  6. Receptor-mediated endocytosis of plasminogen activators and activator/inhibitor complexes

    DEFF Research Database (Denmark)

    Andreasen, P A; Sottrup-Jensen, L; Kjøller, L; Nykjaer, A; Moestrup, S K; Petersen, C M; Gliemann, J

    1994-01-01

    uncomplexed t-PA bind to the multi-ligand receptors alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein (alpha 2MR) and epithelial glycoprotein 330 (gp330). These receptors mediate endocytosis and degradation of u-PA/PAI-1 complex bound to the glycosyl phosphatidyl inositol......-anchored urokinase receptor (u-PAR) on cell surfaces, and participate, in cooperation with other receptors, in hepatic clearance of activator/PAI-1 complexes and uncomplexed t-PA from blood plasma. The alpha 2MR- and gp330-mediated endocytosis of a ligand (u-PA/PAI-1 complex) initially bound to another receptor (u...

  7. Plasma concentrations of soluble urokinase-type plasminogen activator receptor are increased in patients with malaria and are associated with a poor clinical or a fatal outcome

    DEFF Research Database (Denmark)

    Ostrowski, Sisse R; Ullum, Henrik; Goka, Bamenla Q; Høyer-Hansen, Gunilla; Obeng-Adjei, George; Pedersen, Bente K; Akanmori, Bartholomew D; Kurtzhals, Jørgen

    2005-01-01

    BACKGROUND: Blood concentrations of soluble urokinase-type plasminogen activator receptor (suPAR) are increased in conditions with immune activation, and high concentrations of suPAR often predict a poor clinical outcome. This study explored the hypothesis that high plasma concentrations of suPAR are associated with disease severity in malaria. METHODS: At admission to the hospital, plasma concentrations of suPAR were measured by ELISA in samples from 645 African children with clinical symptoms ...

  8. Constriction of Carotid Arteries by Urokinase-Type Plasminogen Activator Requires Catalytic Activity and is Independent of NH2-Terminal Domains

    OpenAIRE

    Massey, Philip G.; Tanaka, Shinji; Buckler, Joshua M.; Jiang, Bo; McCourtie, Anton; Qian, Kun; Tom, Clifford; Stempien-Otero, April; Wen, Shan; Luttrell, Ian; Chitaley, Kanchan; Dichek, David A.

    2009-01-01

    Urokinase-type plasminogen activator (uPA) is expressed at increased levels in stenotic, atherosclerotic human arteries. However, the biological roles of uPA in the artery wall are poorly understood. Previous studies associate uPA with both acute vasoconstriction and chronic vascular remodeling and attribute uPA-mediated vasoconstriction to the kringle—not the catalytic—domain of uPA. We used an in vivo uPA overexpression model to test the hypothesis that uPA-induced vasoconstriction is a rev...

  9. Role of protein kinase C in tumor necrosis factor induction of endothelial cell urokinase-type plasminogen activator.

    Science.gov (United States)

    Niedbala, M J; Stein-Picarella, M

    1993-05-15

    Tumor necrosis factor (TNF) can promote endothelial cell transcription, synthesis, and secretion of urokinase plasminogen activator (uPA) augmenting extracellular matrix remodeling and influencing cellular differentiation. In this report, the role of the protein kinase C (PKC) pathway in mediating TNF induction of uPA in human umbilical vein endothelial cells is described. The PKC inhibitors (H-7, staurosporine, and calphostin C), but not HA-1004, inhibited TNF-induced uPA expression, synthesis, and secretion in a dose-dependent manner. Analysis of cell-free conditioned medium obtained from PKC inhibitor-treated cultures by micro-enzyme-linked immunosorbent assay methodologies using uPA- and plasminogen activator inhibitor type 1 (PAI-1)-specific monoclonal antibodies indicate that the decrease in uPA activity observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis zymography was a direct result of decreased extracellular uPA antigen and not a consequence of increased PAI-1 antigen. The effect of PKC inhibitors was specific for TNF-mediated increased uPA expression because cytokine induction of PAI-1 was not influenced by these agents. Northern blot analyses also showed that PKC inhibitor treatment of endothelial cells resulted in a decreased steady-state level of uPA mRNA with no measurable change in PAI-1 mRNA in cultures incubated with TNF. Downregulation of cellular PKC by 18 hours of phorbol myristate acetate (PMA) pretreatment of endothelial cell cultures abolished TNF-mediated extracellular uPA induction. This effect was specific for PMA because 4-alpha PMA pretreatment of cells, which does not stimulate PKC, was ineffective in altering TNF induction of endothelial cell uPA. Induction of PKC directly with PMA, mezerein, and (-)-octylindolactam V increased endothelial cell levels of extracellular uPA in a time- and dose-dependent manner. In addition, this increase in endothelial cell extracellular uPA activity mediated by PKC agonists could be inhibited with PKC inhibitors. Endothelial cells treated with TNF acquire the ability to invade extracellular matrix and reorganize into tube-like structures when grown on Matrigel-coated culture dishes, a behavior blocked by H-7, but not by HA 1004. In summary, these data implicate a role for the PKC pathway in the TNF-mediated induction of uPA expression, subsequent matrix remodeling, and the formation of tube-like structures, a process important in neovascularization, wound healing, and leukocyte extravasation. PMID:7683925

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

    Science.gov (United States)

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

    2016-02-01

    The plasminogen activator (PA) system consists of plasminogen activator inhibitor type 1 (PAI-1), urokinase-type plasminogen activator and its receptor (uPA and uPAR). PAI-1 inhibits the activation of uPA (which converts plasminogen to plasmin), and is involved in cancer invasion and metastasis, by remodeling the extracellular matrix (ECM) through regulating plasmin. Cancer stem cells (CSCs) are a small subset of cells within tumors, and are thought to be involved in tumor recurrence and metastasis. Considering these facts, we investigated the relationship between PAI-1 and cervical CSCs. We used ALDH1 as a marker of cervical CSCs. First, we demonstrated that culturing ALDH1-high cells and ALDH-low cells on collagen IV-coted plates increased their expression of active PAI-1 (ELISA), and these increases were suggested to be at mRNA expression levels (RT-qPCR). Secondly, we demonstrated PAI-1 was indeed involved in the ECM maintenance. With gelatin zymography assays, we found that ALDH1-high cells and ALDH-low cells expressed pro-matrix metalloproteinase-2 (pro-MMP-2) irrespective of their coatings. With gelatinase/collagenase assay kit, we confirmed that collagenase activity was increased when ALDH1-low cells were exposed to TM5275, a small molecule inhibitor of PAI-1. Putting the data together, we hypothesized that cancer cells adhered to basal membrane secrete abundant PAI-1, on the other hand, cancer cells (especially CSCs rather than non-CSCs) distant from basal membrane secrete less PAI-1, which makes the ECM surrounding CSCs more susceptible to degradation. Our study could be an explanation of conflicting reports, where some researchers found negative impacts of PAI-1 expression on clinical outcomes and others not, by considering the concept of CSCs. PMID:26676222

  11. Anti-tissue plasminogen activator (tPA) as an effective therapy of neonatal hypoxia-ischemia with and without inflammation.

    Science.gov (United States)

    Yang, Dianer; Kuan, Chia-Yi

    2015-04-01

    Hypoxic-ischemic brain injury is an important cause of neurodevelopmental deficits in neonates. Intrauterine infection and the ensuing fetal inflammatory responses augment hypoxic-ischemic brain injury and attenuate the efficacy of therapeutic hypothermia. Here, we review evidences from preclinical studies suggesting that the induction of brain parenchymal tissue-type plasminogen activator (tPA) plays an important pathogenic role in these conditions. Moreover, administration of a stable-mutant form of plasminogen activator inhibitor-1 called CPAI confers potent protection against hypoxic-ischemic injury with and without inflammation via different mechanisms. Besides intracerebroventricular injection, CPAI can also be administered into the brain using a noninvasive intranasal delivery strategy, adding to its applicability in clinical use. In sum, the therapeutic potential of CPAI in neonatal care merits further investigation with large-animal models of hypoxia-ischemia and cerebral palsy. PMID:25475942

  12. Induction of oxidative stress and inhibition of plasminogen activator inhibitor-1 production in endothelial cells following exposure to organic extracts of diesel exhaust particles and urban fine particles

    Energy Technology Data Exchange (ETDEWEB)

    Furuyama, Akiko; Koike, Eiko [National Institute for Environmental Studies, Inhalation toxicology Team, Tsukuba (Japan); Hirano, Seishiro [National Institute for Environmental Studies, Environmental Health Sciences Division, Tsukuba (Japan); Kobayashi, Takahiro [National Institute for Environmental Studies, Inhalation toxicology Team, Tsukuba (Japan); National Institute for Environmental Studies, Environmental Health Sciences Division, Tsukuba (Japan)

    2006-03-15

    Endothelial cells play important roles in anticoagulant and fibrinolytic systems. Recent studies suggest that increases in ambient particulate matter (PM) levels have been associated with an increase in mortality rate from cardiovascular diseases. We examined the production of heme oxygenase-1 (HO-1) and factors related to the fibrinolytic function by rat heart microvessel endothelial cells exposed to organic extracts of diesel exhaust particles (OE-DEP) and urban fine particles (OE-UFP) to investigate the direct effects of these soluble organic fractions in these PM on the fibrinolytic function of endothelial cells. The cell monolayer exposed to 10 {mu}g/ml OE-DEP produced a larger amount of HO-1 than cells exposed to 10 {mu}g/ml OE-UFP. OE-DEP and OE-UFP exposure reduced plasminogen activator inhibitor-1 (PAI-1) production by the cells but did not affect the production of thrombomodulin, tissue-type plasminogen activator, or urokinase-type plasminogen activator. Increased PAI-1 synthesis in response to treatment with 1.0 ng/ml tumor necrosis factor-{alpha} or 0.5 ng/ml transforming growth factor-{beta}1 was reduced by OE-DEP exposure. Suppression of PAI-1 production by OE-DEP exposure was mediated through oxidative stress and was independent of HO-1 activity. These results suggest that exposure to the soluble organic fraction of PM and DEP induced oxidative stress and reduced the PAI-1 production of endothelial cells. (orig.)

  13. Soluble urokinase plasminogen activator receptor levels are elevated and associated with complications in patients with type 1 diabetes

    DEFF Research Database (Denmark)

    Theilade, S; Lyngbaek, S

    2015-01-01

    OBJECTIVES: Soluble urokinase plasminogen activator receptor (suPAR) is a marker of inflammation and endothelial dysfunction. We investigated the associations between suPAR and diabetes, including diabetes duration and complications, in patients with type 1 diabetes. DESIGN, SETTING AND SUBJECTS: From 2009 to 2011, 667 patients with type 1 diabetes and 51 nondiabetic control subjects were included in a cross-sectional study at Steno Diabetes Center, Gentofte, Denmark. suPAR levels were measured with an enzyme-linked immunosorbent assay. MAIN OUTCOME MEASURES: The investigated diabetic complications were cardiovascular disease (CVD: previous myocardial infarction, revascularisation, peripheral arterial disease and stroke), autonomic dysfunction (heart rate variability during deep breathing <11 beats min(-1) ), albuminuria [urinary albumin excretion rate (UAER) ?30 mg/24 h] or a high degree of arterial stiffness (pulse wave velocity ?10 m s(-1) ). Analyses were adjusted for gender, age, systolic blood pressure, estimated glomerular filtration rate, UAER, glycated haemoglobin (HbA1c ), total cholesterol, body mass index, C-reactive protein, antihypertensive treatment and smoking. RESULTS: Soluble urokinase plasminogen activator receptor levels were lower in control subjects versus all patients, in control subjects versus normoalbuminuric patients (UAER <30 mg/24 h), in normoalbuminuric patients with short (<10 years) versus long diabetes duration and were increased with degree of albuminuria (adjusted P < 0.001 for all). Furthermore, suPAR levels were higher in patients with versus without CVD (n = 144; 21.3%), autonomic dysfunction (n = 369; 59.2%), albuminuria (n = 357; 53.1%) and a high degree of arterial stiffness (n = 298; 47.2%) (adjusted P ? 0.024). The adjusted odds ratio (95% confidence interval) values per 1 ln unit increase in suPAR were as follows: 2.5 (1.1-5.7) for CVD: 2.7 (1.2-6.2) for autonomic dysfunction; 3.8 (1.3-10.9) for albuminuria and 2.5 (1.1-6.1) for a high degree of arterial stiffness (P? 0.039). CONCLUSION: The suPAR level is higher in patients with type 1 diabetes and is associated with diabetes duration and complications independent of other risk factors. suPAR is a potential novel risk marker for the management of diabetes.

  14. Combined treatment with recombinant tissue plasminogen activator and dexamethasone phosphate-containing liposomes improves neurological outcome and restricts lesion progression after embolic stroke in rats

    OpenAIRE

    2012-01-01

    Variable efficacies have been reported for glucocorticoid drugs as anti-inflammatory treatment after stroke. We applied an alternative drug delivery strategy, by injection of dexamethasone phosphate-containing liposomes in combination with recombinant tissue plasminogen activator (rtPA), in an experimental stroke model, and tested the hypothesis that this approach improves behavioral recovery and reduces lesion growth. Rats were subjected to right middle cerebral artery occlusion with a blood...

  15. Mapping the topographic epitope landscape on the urokinase plasminogen activator receptor (uPAR) by surface plasmon resonance and X-ray crystallography

    DEFF Research Database (Denmark)

    Zhao, Baoyu; Gandhi, Sonu; Yuan, Cai; Luo, Zhipu; Li, Rui; GÃ¥rdsvoll, Henrik; de Lorenzi, Valentina; Sidenius, Nicolai; Huang, Mingdong; Ploug, Michael

    2015-01-01

    The urokinase-type plasminogen activator receptor (uPAR or CD87) is a glycolipid-anchored membrane protein often expressed in the microenvironment of invasive solid cancers and high levels are generally associated with poor patient prognosis (Kriegbaum et al., 2011 [1]). uPAR is organized as a dy...... these mAbs by X-ray crystallography alone and in complex with uPAR [deposited in the PDB database as 4QTH and 4QTI, respectively]....

  16. Protein S blocks the extrinsic apoptotic cascade in tissue plasminogen activator/N-methyl D-aspartate-treated neurons via Tyro3-Akt-FKHRL1 signaling pathway

    OpenAIRE

    Freeman Robert S; Griffin John H; Fernández José A; Zhong Zhihui; Barrett Theresa M; Guo Huang; Zlokovic Berislav V

    2011-01-01

    Abstract Background Thrombolytic therapy with tissue plasminogen activator (tPA) benefits patients with acute ischemic stroke. However, tPA increases the risk for intracerebral bleeding and enhances post-ischemic neuronal injury if administered 3-4 hours after stroke. Therefore, combination therapies with tPA and neuroprotective agents have been considered to increase tPA's therapeutic window and reduce toxicity. The anticoagulant factor protein S (PS) protects neurons from hypoxic/ischemic i...

  17. Cloning and Expression of a Human Tissue Plasminogen Activator Variant:K2S in Escherichia coli

    OpenAIRE

    Hamid Mir Mohammad Sadeghi; Kianoush Dormiani; Yahya Khazaie; Mohammad Rabbani; Fatemeh Moazen

    2007-01-01

    The DNA sequence of Kringle-2 and serine protease domains of the human tissue plasminogen activator (reteplase, K2S) was PCR amplified. This product was then cloned into the expression vector pET15b plasmid. The presence of the insert was confirmed by restriction digestion, PCR and determination of the nucleotide sequence. By using isopropyl ?-D thiogalactopyranoside (IPTG), reteplase was induced in E. coli BL21 cells and analyzed using polyacrylamide gel electrophoresis (PAGE).

  18. Distinctive binding modes and inhibitory mechanisms of two peptidic inhibitors of urokinase-type plasminogen activator with isomeric P1 residues

    DEFF Research Database (Denmark)

    Jiang, Longguang; Zhao, Baoyu; Xu, Peng; Sørensen, Hans Peter; Jensen, Jan Kristian; Christensen, Anni; Hosseini, Masood; Nielsen, Niels Christian; Jensen, Knud Jørgen; Andreasen, Peter; Huang, Mingdong

    2015-01-01

    Abstract Two isomeric piperidine derivatives (meta and para isomers) were used as arginine mimics in the P1 position of a cyclic peptidic inhibitor (CPAYSRYLDC) of urokinase-type plasminogen activator. The two resulting cyclic peptides showed vastly different affinities (?70 fold) to the target enzyme. X-ray crystal structure analysis showed that the two P1 residues were inserted into the S1 specificity pocket in indistinguishable manners. However, the rest of the peptides bound in entirely diff...

  19. Three-dimensional interplay among the ligand-binding domains of the urokinase-plasminogen-activator-receptor-associated protein, Endo180

    OpenAIRE

    Rivera-Calzada, Angel; Robertson, David; MacFadyen, John R.; Boskovic, Jasminka; Isacke, Clare M.; Llorca, Oscar

    2003-01-01

    Endo180, also known as the urokinase plasminogen activator receptor (uPAR)-associated protein (uPARAP), is one of the four members of the mannose receptor family, and is implicated in extracellular-matrix remodelling through its interactions with collagens, sugars and uPAR. The extracellular portion of Endo180 contains an amino-terminal cysteine-rich domain, a single fibronectin type II domain and eight C-type lectin-like domains. We have purified a soluble ...

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

    OpenAIRE

    Brügemann, J.; van der Meer, J.; Takens, B H; Hillege, H; Lie, K.I.

    1990-01-01

    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...

  1. Stereotactic fibrinolysis of spontaneous intracerebral hematoma using infusion of recombinant tissue plasminogen activator Fibrinólise com infusão de rtPA e drenagem estereotáxica de hematoma intracerebral espontâneo profundo

    OpenAIRE

    José Augusto Nasser; Asdrubal Falavigna; Márcio Bezerra; Victor Martinez; Gabriel Freitas; Armando Alaminos; Antônio Bonatelli; Fernando Ferraz

    2002-01-01

    PURPOSE: The authors present a prospective study on 10 patients with stereotactic infusion of tissue plasminogen activator (rtPA) intraparenchimal hemorrhage. METHODS: Between 1999 and 2000, 10 patients with deep seated hematomas in the basal ganglia were selected for stereotactic infusion of rtPA and spontaneous clot drainage. RESULTS: All cases had about 80% reduction of the hematoma volume in the CT scan at the third day. The intracranial pressure was normalized by the third day too. There...

  2. Tissue-Plasminogen Activator Attenuates Alzheimer's Disease-Related Pathology Development in APPswe/PS1 Mice.

    Science.gov (United States)

    ElAli, Ayman; Bordeleau, Maude; Thériault, Peter; Filali, Mohammed; Lampron, Antoine; Rivest, Serge

    2016-04-01

    Alzheimer's disease (AD) is the leading cause of dementia among elderly population. AD is characterized by the accumulation of beta-amyloid (A?) peptides, which aggregate over time to form amyloid plaques in the brain. Reducing soluble A? levels and consequently amyloid plaques constitute an attractive therapeutic avenue to, at least, stabilize AD pathogenesis. The brain possesses several mechanisms involved in controlling cerebral A? levels, among which are the tissue-plasminogen activator (t-PA)/plasmin system and microglia. However, these mechanisms are impaired and ineffective in AD. Here we show that the systemic chronic administration of recombinant t-PA (Activase rt-PA) attenuates AD-related pathology in APPswe/PS1 transgenic mice by reducing cerebral A? levels and improving the cognitive function of treated mice. Interestingly, these effects do not appear to be mediated by rt-PA-induced plasmin and matrix metalloproteinases 2/9 activation. We observed that rt-PA essentially mediated a slight transient increase in the frequency of patrolling monocytes in the circulation and stimulated microglia in the brain to adopt a neuroprotective phenotype, both of which contribute to A? elimination. Our study unraveled a new role of rt-PA in maintaining the phagocytic capacity of microglia without exacerbating the inflammatory response and therefore might constitute an interesting approach to stimulate the key populations of cells involved in A? clearance from the brain. PMID:26349911

  3. Glioma-derived plasminogen activator inhibitor-1 (PAI-1) regulates the recruitment of LRP1 positive mast cells.

    Science.gov (United States)

    Roy, Ananya; Coum, Antoine; Marinescu, Voichita D; Põlajeva, Jelena; Smits, Anja; Nelander, Sven; Uhrbom, Lene; Westermark, Bengt; Forsberg-Nilsson, Karin; Pontén, Fredrik; Tchougounova, Elena

    2015-09-15

    Glioblastoma (GBM) is a high-grade glioma with a complex microenvironment, including various inflammatory cells and mast cells (MCs) as one of them. Previously we had identified glioma grade-dependent MC recruitment. In the present study we investigated the role of plasminogen activator inhibitor 1 (PAI-1) in MC recruitment.PAI-1, a primary regulator in the fibrinolytic cascade is capable of forming a complex with fibrinolytic system proteins together with low-density lipoprotein receptor-related protein 1 (LRP1). We found that neutralizing PAI-1 attenuated infiltration of MCs. To address the potential implication of LRP1 in this process, we used a LRP1 antagonist, receptor-associated protein (RAP), and demonstrated the attenuation of MC migration. Moreover, a positive correlation between the number of MCs and the level of PAI-1 in a large cohort of human glioma samples was observed. Our study demonstrated the expression of LRP1 in human MC line LAD2 and in MCs in human high-grade glioma. The activation of potential PAI-1/LRP1 axis with purified PAI-1 promoted increased phosphorylation of STAT3 and subsequently exocytosis in MCs.These findings indicate the influence of the PAI-1/LRP1 axis on the recruitment of MCs in glioma. The connection between high-grade glioma and MC infiltration could contribute to patient tailored therapy and improve patient stratification in future therapeutic trials. PMID:26164207

  4. Treatment with recombinant tissue plasminogen activator (r-TPA) induces neutrophil degranulation in vitro via defined pathways.

    Science.gov (United States)

    Carbone, Federico; Vuilleumier, Nicolas; Bertolotto, Maria; Burger, Fabienne; Galan, Katia; Roversi, Gloria; Tamborino, Carmine; Casetta, Ilaria; Seraceni, Silva; Trentini, Alessandro; Dallegri, Franco; da Silva, Analina Raquel; Pende, Aldo; Artom, Nathan; Mach, François; Coen, Matteo; Fainardi, Enrico; Montecucco, Fabrizio

    2015-01-01

    Thrombolysis is recommended for reperfusion following acute ischemic stroke (AIS), but its effects on stroke-associated injury remain to be clarified. Here, we investigated the effects of recombinant tissue plasminogen activator (r-tPA) on neutrophil pathophysiology in vitro and in a case-control study with AIS patients submitted (n=60) or not (n=30) to thrombolysis. Patients underwent radiological and clinical examination as well as blood sampling at admission and after 1, 7 and 90days. In vitro, 30-min incubation with 0.1-1 mg/ml r-tPA induced neutrophil degranulation in different substrate cultures. Pre-incubation with kinase inhibitors and Western blot documented that degranulation was associated with activation of PI3K/Akt and ERK1/2 pathways in Teflon dishes and PI3K/Akt in polystyrene. In thrombolysed patients, a peak of neutrophil degranulation products (matrix metalloproteinase [MMP]-9, MMP-8, neutrophil elastase and myeloperoxidase), was shown during the first hours from drug administration. This was accompanied by serum augmentation of protective tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2. An increased rate of haemorrhagic transformations on day 1 after AIS was shown in thrombolysed patients as compared to non-thrombolysed controls. In conclusion, r-tPA treatment was associated with in vitro neutrophil degranulation, indicating these cells as potential determinants in early haemorrhagic complications after thrombolysis in AIS patients. PMID:25530154

  5. Risk factors associated with serum levels of the inflammatory biomarker soluble urokinase plasminogen activator receptor in a general population

    DEFF Research Database (Denmark)

    Haupt, Thomas H; Kallemose, Thomas

    2014-01-01

    The soluble urokinase plasminogen activator receptor (suPAR) is a biomarker of mortality risk in various patient populations. However, little is known about the implications of lifestyle for suPAR levels in the general population. Lifestyle, demographic, and cardiovascular disease (CVD) risk factor data were collected from 5,538 participants in the Danish population-based Inter99 study. Their suPAR levels were measured using a sandwich enzyme-linked immunosorbent assay. In the final adjusted model, smoking and morbid obesity were strongly associated with higher suPAR levels (P < 0.001). An unhealthy diet and alcohol abstinence in men were also associated with higher suPAR levels. Physical activity in leisure time had a modest impact on suPAR levels in univariate analysis, but not in the final adjusted model. In conclusion, smoking and morbid obesity were strongly associated with higher serum suPAR levels in this general population. Diet and alcohol consumption also seemed to impact suPAR levels. Lifestyle changes are likely to affect suPAR since ex-smokers had suPAR levels comparable to those of never-smokers.

  6. Risk Factors Associated with Serum Levels of the Inflammatory Biomarker Soluble Urokinase Plasminogen Activator Receptor in a General Population

    DEFF Research Database (Denmark)

    Haupt, Thomas H; Kallemose, Thomas

    2014-01-01

    The soluble urokinase plasminogen activator receptor (suPAR) is a biomarker of mortality risk in various patient populations. However, little is known about the implications of lifestyle for suPAR levels in the general population. Lifestyle, demographic, and cardiovascular disease (CVD) risk factor data were collected from 5,538 participants in the Danish population-based Inter99 study. Their suPAR levels were measured using a sandwich enzyme-linked immunosorbent assay. In the final adjusted model, smoking and morbid obesity were strongly associated with higher suPAR levels (P < 0.001). An unhealthy diet and alcohol abstinence in men were also associated with higher suPAR levels. Physical activity in leisure time had a modest impact on suPAR levels in univariate analysis, but not in the final adjusted model. In conclusion, smoking and morbid obesity were strongly associated with higher serum suPAR levels in this general population. Diet and alcohol consumption also seemed to impact suPAR levels. Lifestyle changes are likely to affect suPAR since ex-smokers had suPAR levels comparable to those of never-smokers.

  7. 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

  8. 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 associated with coronary microvascular dysfunction, which suggests that plasma PAI-1 activity is an important clue linking hypofibrinolysis to the development of atherosclerosis. (author)

  9. Meta-Analysis of the Association between Plasminogen Activator Inhibitor-1 4G/5G Polymorphism and Recurrent Pregnancy Loss

    Science.gov (United States)

    Li, Xuejiao; Liu, Yukun; Zhang, Rui; Tan, Jianping; Chen, Libin; Liu, Yinglin

    2015-01-01

    Background The association between plasminogen activator inhibitor-1 (PAI-1) 4G/5G polymorphism and recurrent pregnancy loss (RPL) risk is still contradictory. We thus performed a meta-analysis. Material/Methods Relevant studies were searched for in PubMed, Web of Science, Embase, and Cochrane Library. An odds ratio (OR) with a 95% confidence interval (CI) was used to assess the association between PAI-1 4G/5G polymorphism and RPL risk. Results A total of 22 studies with 4306 cases and 3076 controls were included in this meta-analysis. We found that PAI-1 4G/5G polymorphism was significantly associated with an increased RPL risk (OR=1.89; 95% CI 1.34–2.67; P=0.0003). In the subgroup analysis by race, PAI-1 4G/5G polymorphism was significantly associated with an increased RPL risk in Caucasians (OR=2.23; 95% CI 1.44–3.46; P=0.0003). However, no significant association was observed in Asians (OR=1.47; 95% CI 0.84–2.59; P=0.18). Conclusions In conclusion, this meta-analysis suggests that PAI-1 4G/5G polymorphism might be associated with RPL development in Caucasians. PMID:25862335

  10. 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)

  11. Specific identification of Lachesis muta muta snake venom using antibodies against the plasminogen activator enzyme, LV-PA.

    Science.gov (United States)

    Felicori, Liza F; Chávez-Olórtegui, Carlos; Sánchez, Eladio F

    2005-05-01

    Sandwich-type enzyme linked immunosorbent assays (ELISA) were developed to detect Lachesis muta muta (bushmaster) snake venom using antibodies against the plasminogen activator enzyme (LV-PA). Antibodies to LV-PA were obtained by immunization of one rabbit with the purified enzyme. The IgG fraction was purified from rabbit blood in a single step on a column of Sepharose-L. m. muta venom and used to coat the microtiter plates. The specificity of the assay was demonstrated by its capacity to correctly discriminate between the circulating antigens in mice that were experimentally inoculated with L. m. muta venom from those in mice inoculated with venoms from Bothrops atrox, B. brazili, B. castelnaudi, Bothriopsis taeniata, B. bilineata, Crotalus durissus ruruima and the antigenic Bothrops (AgB) and Crotalus (AgC) pools venoms used to produce Bothropic and Crotalic antivenoms at Fundacao Ezequiel Dias (FUNED). Measurable absorbance signals were obtained with 1.5 ng of venom per assay. The ELISA was used to follow the kinetic distribution of antigens in experimentally envenomed mice. PMID:15804530

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1990-08-15

    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.

  13. Treatment of hepatic venoocclusive disease with recombinant human tissue plasminogen activator or orthotopic liver transplantation after allogeneic bone marrow transplantation.

    Science.gov (United States)

    Hágglund, H; Ringdén, O; Ericzon, B G; Duraj, F; Ljungman, P; Lönnqvist, B; Winiarski, J; Tydén, G

    1996-10-27

    Ten allogeneic bone marrow transplant (BMT) recipients with hepatic venoocclusive disease (VOD) were treated with recombinant human tissue plasminogen activator (rt-PA). Two of them subsequently underwent orthotopic liver transplantation (OLT). One additional patient with VOD underwent OLT without prior rt-PA treatment. Treatment with rt-PA was started a median of 14 (1--35) days after BMT. The dose of rt-PA given to adults was 10-50 mg i.v. and that given to children was 3-10 mg i.v. Treatment was given for 2-4 days. In three patients, the dose was administered over a longer period or it was repeated. Four patients responded to rt-PA therapy and six did not. Eight patients suffered from hemorrhages, one intracranial and three gastrointestinal. Four patients required blood transfusions. Four had minor subcutaneous hemorrhages and/or epistaxis. One patient died of intracranial hemorrhage and five from hepatic and/or multiorgan failure. Two patients treated with rt-PA, 10 mg/day for 4 days, are alive; one is alive and well 3 months after BMT, the other has relapsed after 7 months. The three patients undergoing OLT died of chronic hepatic failure, cerebral edema, and pneumonia. Our experience suggests that rt-PA should not be administered in high doses and that the treatment should not be given over a longer period, because of the risk of severe hemorrhages. PMID:8900305

  14. 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)

    Baumann Gert

    2005-05-01

    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.

  15. 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

  16. Dengue Virus Nonstructural Protein 1-Induced Antibodies Cross-React with Human Plasminogen and Enhance Its Activation.

    Science.gov (United States)

    Chuang, Yung-Chun; Lin, Jessica; Lin, Yee-Shin; Wang, Shuying; Yeh, Trai-Ming

    2016-02-01

    Dengue virus (DENV) infection is the most common mosquito-borne viral disease, and it can cause life-threatening dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Abnormal activation of the coagulation and fibrinolysis system is one of the hallmarks of DHF/DSS. However, the mechanism underlying hemorrhage in DHF/DSS remains elusive. In previous studies, plasminogen (Plg) cross-reactive Abs, which can recognize DENV nonstructural protein (NS) 1, have been found in dengue patients. However, it is unclear whether these Abs are indeed induced by DENV NS1. Thus, we immunized mice with recombinant NS1 from both bacteria and drosophila to determine whether NS1 can induce Plg cross-reactive Abs. The results from the NS1-immunized mouse sera indicated that NS1 immunization induced Abs that could cross-react with Plg. To study the effects of these NS1-induced Plg cross-reactive Abs on fibrinolysis, we isolated several Plg cross-reactive anti-NS1 mAbs from these mice and found that some of them could enhance Plg activation. In addition, epitope mapping with a phage-displayed random peptide library revealed that one of these mAbs (2A5) could recognize NS1 C-terminal residues 305-311, which share sequence homology with Plg residues 590-597. A synthetic peptide of NS1 residues 305-311 could inhibit the binding of both 2A5 and its Fab to Plg and its enhanced activation. Thus, our results suggest that DENV NS1 can induce Plg cross-reactive Abs through molecular mimicry, which can enhance Plg activation and may contribute to the pathogenesis of DHF/DSS. PMID:26712948

  17. 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)

    Egelund, R; Einholm, A P

    2001-01-01

    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

  18. 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)

    Killeen, S D

    2009-05-19

    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.

  19. Respiratory burst function of ovine neutrophils

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    Fraser John F

    2009-05-01

    Full Text Available Abstract Background Respiratory burst function resulting in the release of reactive oxygen species such as superoxide anion (O2- from neutrophils is one of the key mechanisms of the innate immune system, and maladaptive control of this mechanism is thought to play a pivotal role in the development of pathologies such as acute lung injury and sepsis. Ovine models of these pathologies are limited by the poor understanding of ovine neutrophil respiratory burst function. Results Aspects of ovine neutrophil respiratory burst function to be characterised were: i the maximum rate of O2- generated (Vmax; ii the time taken to reach Vmax; iii the total amount of O2- generated during the reaction; and iv the duration of the reaction. As well as for unstimulated neutrophils, these aspects were also characterised after incubation with a priming agonist (platelet activating factor [PAF], tumour necrosis factor alpha [TNF-?] and lipopolysaccharides [LPS] activating agonists (N-formylmethionyl-leucyl-phenylalanine [fMLP] and phorbol 12-myristate 13-acetate [PMA] or a combination of a priming and an activating agonist. In the absence of priming or activating agonists, ovine neutrophils displayed a low level of respiratory burst function which was not enhanced by either PAF, TNF-?, LPS or fMLP, but was significantly enhanced by PMA. The PMA-induced respiratory burst function was further enhanced by pre-incubation with PAF, but not with TNF-? or LPS. By varying the length of pre-incubation with PAF it was demonstrated that this effect decreased as the duration of pre-incubation with PAF increased, and that PAF was enhancing PMA's effects rather than PMA enhancing PAF's effects. Conclusion This study successfully adapted a commonly used method of measuring human neutrophil respiratory burst function to characterise different aspects of ovine neutrophil respiratory burst function. This improved understanding of ovine neutrophils will facilitate the validitation of ovine biomedical models of human pathologies in which neutrophils have been implicated.

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

    DEFF Research Database (Denmark)

    List, K; Jensen, Ole Nørregaard; Bugge, T H; Lund, L. R.; Ploug, M; Danø, K; Behrendt, N

    2000-01-01

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

  1. 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

    International Nuclear Information System (INIS)

    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. 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. 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. 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

  2. Plasminogen activator inhibitor-1 deficiency ameliorates insulin resistance and hyperlipidemia but not bone loss in obese female mice.

    Science.gov (United States)

    Tamura, Yukinori; Kawao, Naoyuki; Yano, Masato; Okada, Kiyotaka; Matsuo, Osamu; Kaji, Hiroshi

    2014-05-01

    We previously demonstrated that plasminogen activator inhibitor-1 (PAI-1), an inhibitor of fibrinolysis, is involved in type 1 diabetic bone loss in female mice. PAI-1 is well known as an adipogenic factor induced by obesity. We therefore examined the effects of PAI-1 deficiency on bone and glucose and lipid metabolism in high-fat and high-sucrose diet (HF/HSD)-induced obese female mice. Female wild-type (WT) and PAI-1-deficient mice were fed with HF/HSD or normal diet for 20 weeks from 10 weeks of age. HF/HSD increased the levels of plasma PAI-1 in WT mice. PAI-1 deficiency suppressed the levels of blood glucose, plasma insulin, and total cholesterol elevated by obesity. Moreover, PAI-1 deficiency improved glucose intolerance and insulin resistance induced by obesity. Bone mineral density (BMD) at trabecular bone as well as the levels of osterix, alkaline phosphatase, and receptor activator of nuclear factor ?B ligand mRNA in tibia were decreased by HF/HSD in WT mice, and those changes by HF/HSD were not affected by PAI-1 deficiency. HF/HSD increased the levels of plasma TNF-? in both WT and PAI-1-deficient mice, and the levels of plasma TNF-? were negatively correlated with trabecular BMD in tibia of female mice. In conclusion, we revealed that PAI-1 deficiency does not affect the trabecular bone loss induced by obesity despite the amelioration of insulin resistance and hyperlipidemia in female mice. Our data suggest that the changes of BMD and bone metabolism by obesity might be independent of PAI-1 as well as glucose and lipid metabolism. PMID:24605827

  3. 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 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

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

    Directory of Open Access Journals (Sweden)

    Hwu Yuh-Ming

    2011-03-01

    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.

  5. Effect of varying days of tissue plasminogen activator therapy on the prevention of postsurgical adhesions in a rabbit model.

    Science.gov (United States)

    Dunn, R C; Mohler, M

    1993-03-01

    The depression of focal fibrinolytic activity in mesothelial or serosal tissues which results from a surgical ischemic insult can lead to the formation of permanent fibrinous adhesions. To assess the time course for the prevention of adhesion formation by recombinant tissue plasminogen activator (rt-PA), infusions of rt-PA were administered to rabbits every 12 hr for a total of 1, 2, 4, or 8 days. Animals in each rt-PA treatment group received a total cumulative dose of 16 mg. Control animals received saline with the same corresponding dosing regimens as the treated groups. Prior to dosing, all animals had a devascularizing injury to the uterine horns performed bilaterally by bipolar cauterization of the uterine mesenteric vascular arcade below the horns. A catheter was then placed from a subcutaneous port to the intraperitoneal space. rt-PA, or saline, was instilled intraperitoneally via the subcutaneous port. Adhesions were scored at the second laparotomy by assessing the percentage of the involvement of the uterine horns in adhesions. Mean score for adhesion formation in each placebo group was 35 to 40%. Mean score (+/- standard error of the mean) for rt-PA after 1 day of therapy was 13.1% +/- 3.89; after 2 days of therapy, 6.3% +/- 1.57; after 4 days of therapy, 3.8% +/- 1.57; and after 8 days of therapy, 6.9% +/- 2.3 (P < 0.01 compared to the control grouped mean for each day of treatment).(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8474240

  6. Independent prognostic value of angiogenesis and the level of plasminogen activator inhibitor type 1 in breast cancer patients

    DEFF Research Database (Denmark)

    Hansen, S; Overgaard, J

    2003-01-01

    Tumour angiogenesis and the levels of plasminogen activator inhibitor type 1 (PAI-1) are both informative prognostic markers in breast cancer. In cell cultures and in animal model systems, PAI-1 has a proangiogenic effect. To evaluate the interrelationship of angiogenesis and the PAI-1 level in breast cancer, we have evaluated the prognostic value of those factors in a total of 228 patients with primary, unilateral, invasive breast cancer, evaluated at a median follow-up time of 12 years. Microvessels were immunohistochemically stained by antibodies against CD34 and quantitated by the Chalkley counting technique. The levels of PAI-1 and its target proteinase uPA in tumour extracts were analysed by ELISA. The Chalkley count was not correlated with the levels of uPA or PAI-1. High values of uPA, PAI-1, and Chalkley count were all significantly correlated with a shorter recurrence-free survival and overall survival. In the multivariate analysis, the uPA level did not show independent prognostic impact for any ofthe analysed end points. In contrast, the risk of recurrence was independently and significantly predicted by both the PAI-1 level and the Chalkley count, with a hazard ratio (95% CI) of 1.6 (1.01-2.69) and 1.4 (1.02-1.81), respectively. For overall survival, the Chalkley count, but not PAI-1, was of significant independent prognostic value. The risk of death was 1.7 (1.30-2.15) for Chalkley counts in the upper tertile compared to the lower one. We conclude that the PAI-1 level and the Chalkley count are independent prognostic markers for recurrence-free survival in patients with primary breast cancer, suggesting that the prognostic impact of PAI-1 is not only based on its involvement in angiogenesis.

  7. Prognostic value analysis of urokinase-type plasminogen activator receptor in oral squamous cell carcinoma: an immunohistochemical study

    International Nuclear Information System (INIS)

    Oral squamous cell carcinoma (OSCC) represents the most common oral malignancy. Despite recent advances in therapy, up to 50% of the cases have relapse and/or metastasis. There is therefore a strong need for the identification of new biological markers able to predict the clinical behaviour of these lesions in order to improve quality of life and overall survival. Among tumour progression biomarkers, already known for their involvement in other neoplasia, a crucial role is ascribed to the urokinase-type plasminogen activator receptor (uPAR), which plays a multiple role in extracellular proteolysis, cell migration and tissue remodelling not only as a receptor for the zymogen pro-uPA but also as a component for cell adhesion and as a chemoattractant. The purpose of this study was to gain information on the expression of uPAR in OSCC and to verify whether this molecule can have a role as a prognostic/predictive marker for this neoplasia. In a retrospective study, a cohort of 189 OSCC patients was investigated for uPAR expression and its cellular localization by immunohistochemistry. As standard controls, 8 normal oral mucosal tissues free of malignancy, obtained from patients with no evidence or history of oral cavity tumours, were similarly investigated. After grouping for uPAR expression, OSCCs were statistically analyzed for the variables age, gender, histological grading (G), tumour size, recurrence, TNM staging and overall survival rate. In our immunohistochemical study, 74 cases (39.1%) of OSCC showed a mostly cytoplasmic positivity for uPAR, whereas 115 were negative. uPAR expression correlated with tumour differentiation grade and prognosis: percentage of positive cases was the greatest in G3 (70.4%) and patients positives for uPAR expression had an expectation of life lower than those for uPAR negatives. The results obtained in this study suggest a role of uPAR as a potential biomarker useful to identify higher risk subgroups of OSCC patients

  8. Preliminary Experience with Air Transfer of Patients for Rescue Endovascular Therapy after Failure of Intravenous Tissue Plasminogen Activator

    Science.gov (United States)

    TSUJIMOTO, Masanori; YOSHIMURA, Shinichi; ENOMOTO, Yukiko; YAMADA, Noriaki; MATSUMARU, Naoki; KUMADA, Keisuke; TOYODA, Izumi; OGURA, Shinji; IWAMA, Toru

    2015-01-01

    The present report describes our experience with air transfer of patients with acute ischemic stroke in whom intravenous tissue plasminogen activator (IV t-PA) failed for rescue endovascular therapy (EVT). Twenty-three consecutive patients in whom IV t-PA failed were transferred to our hospital for rescue EVT between February 2011 and April 2013. The amount of time required for transfer, distance, clinical outcomes, and complications were compared between patients transferred by ground (TG group; n = 17) and by air (TA group; n = 6). Computed tomography imaging on arrival revealed hemorrhagic transformation in 1 (5.9%) patient in the TG group, whereas none of the patients in the TA group developed any type of complication. The remaining 22 patients received rescue EVT. The elapsed time from the request call to arrival at our hospital did not significantly differ between the TG and TA groups (45.8 ± 4.9 min vs. 41.6 ± 2.3 min). However, the distance from the primary hospital to our institution was significantly longer for the TA group than for the TG group (38.8 ± 10.4 km vs. 13.5 ± 1.2 km, p = 0.001). The frequency of favorable outcomes (modified Rankin Scale 0–1 at 90 days after onset) in the TG and TA groups were 25.0% and 50.0%, respectively (p = 0.267). Air transfer for patients after IV t-PA failure allowed for more rapid delivery of patients over longer distances than ground transfer. PMID:25739430

  9. Association of plasminogen activator inhibitor-1 and angiotensin converting enzyme polymorphisms with recurrent pregnancy loss in Iranian women

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    Fatemeh Shakarami

    2015-10-01

    Full Text Available Background: Recurrent pregnancy loss (RPL defined by two or more failed pregnancies before 20 weeks of gestation. Several factors play a role in RPL including thrombophilic conditions which can be influenced by gene polymorphisms. Plasminogen activator inhibitor-1 (PAI-1 and angiotensin converting enzyme (ACE genes are closely related to fibrinolytic process, embryonic development and pregnancy success. Objective: The aim of this study was to investigate the relationship between RPL and common polymorphisms in ACE and PAI-1 genes. Materials and Methods: In this case control study, 100 women with recurrent abortions (at least two were selected as cases and 100 healthy women with two or more normal term deliveries without a history of abortion as controls. Total genomic DNA was isolated from blood leukocytes. The status of the PAI-1 4G/5G and ACE (D/I polymorphism was determined by PCR-RFLP. Results: Homozygosity for PAI-1 4G polymorphism was seen in 17 cases (17%, and 5 controls (5% (p=0.006 so patients with homozygote 4G mutation were significantly more prone to RPL in contrast to control group (OR: 4.63, % 95 CI: 1.55-13.84. In addition, 7 patients (7 %, and no one from the control group, were homozygote (I/I for ACE polymorphism (p=0.034, suggesting no significant associations between ACE D allele or DD genotype and RPL. Conclusion: Considering these results, because 4G/4G polymorphism for PAI-1 gene could be a thrombophilic variant leading to abortion, analysis of this mutation and other susceptibility factors are recommended in patients with RPL.

  10. Prognostic significance of circulating intact and cleaved forms of urokinase plasminogen activator receptor in inoperable chemotherapy treated cholangiocarcinoma patients

    DEFF Research Database (Denmark)

    Grunnet, M; Christensen, I J

    2014-01-01

    BACKGROUND: High levels of intact and cleaved forms of the urokinase-type plasminogen activator receptor (uPAR) in both tissue and blood are associated with poor survival in several cancer diseases. The prognostic significance of uPAR in cholangiocarcinoma is unknown. The aims of this study were to determine if pre-treatment serum levels of uPAR forms and a decrease in levels during chemotherapy are predictive of survival in patients with inoperable cholangiocarcinoma. DESIGN AND METHODS: Patients with inoperable cholangiocarcinoma were consecutively included in the training set (n=108). A test set included patients from a different hospital using similar treatment guidelines (n=60). Serum levels of the different uPAR forms were determined using time-resolved fluorescence immunoassays (TR-FIA). The Cox proportional hazards model was used for the uni- and multivariate survival analyses. RESULTS: Baseline level of uPAR(I-III)+uPAR(II-III) was an independent predictor of survival (HR=2.08, 95% CI:1.46-2.97, p<0.0001). Applying the linear predictor from the training set to the test set, it was validated that uPAR(I-III)+uPAR(II-III) predicted overall survival (p=0.049). A high level of uPAR(I-III)+uPAR(II-III) after 2cycles of chemotherapy was associated with poor survival (HR=1.79, 95% CI:1.08-2.97, p=0.023, n=57). This predictor, however, was not significant in the test set (p=0.21, 26 events in 27 patients). CONCLUSION: The baseline level of uPAR(I-III)+uPAR(II-III) is a predictor of survival in inoperable cholangiocarcinoma patients.

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

    Directory of Open Access Journals (Sweden)

    Dharmasaroja Pornpatr

    2011-01-01

    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.

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

    Directory of Open Access Journals (Sweden)

    Sharma S

    2008-01-01

    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.

  13. Plasminogen activator inhibitor-1 synthesis in the human hepatoma cell line Hep G2. Metformin inhibits the stimulating effect of insulin.

    OpenAIRE

    Anfosso, F; Chomiki, N; Alessi, M. C.; Vague, P; Juhan-Vague, I

    1993-01-01

    High plasma plasminogen activator inhibitor-1 (PAI-1) activity is associated with insulin resistance and is correlated with hyperinsulinemia. The cellular origin of plasma PAI-1 in insulin resistance is not known. The hepatoma cell line Hep G2 has been shown to synthesize PAI-1 in response to insulin. The aim of this study was to analyze the insulin-mediated response of PAI-1 and lipid synthesis in Hep G2 cells after producing an insulin-resistant state by decreasing insulin receptor numbers....

  14. Increase of cellular fibrinolysis in human lung cancer cell line by radiation. Relationship between urokinase-type plasminogen activator (uPA) and metastasis and invasion

    International Nuclear Information System (INIS)

    It is well known that urokinase-type plasminogen activator (uPA) activates fibrinolysis of tumor cells and accelerates their metastasis and invasion. Human adenosquamous cell line, AOI cells, were stimulated to produce and accumulate of uPA by radiation. In AOI cells, there was relationship between uPA production and accumulation and the radiation doses. It was suggested that radiation had the possibility to accelerate the metastasis and invasion by increasing the production and accumulation of uPA from cancer cells. (author)

  15. Interferon-tau activates multiple signal transducer and activator of transcription proteins and has complex effects on interferon-responsive gene transcription in ovine endometrial epithelial cells.

    Science.gov (United States)

    Stewart, M D; Stewart, D M; Johnson, G A; Vyhlidal, C A; Burghardt, R C; Safe, S H; Yu-Lee, L Y; Bazer, F W; Spencer, T E

    2001-01-01

    Interferon-tau (IFNtau), a type I IFN produced by sheep conceptus trophectoderm, is the signal for maternal recognition of pregnancy. Although it is clear that IFNtau suppresses transcription of the estrogen receptor alpha and oxytocin receptor genes and induces expression of various IFN-stimulated genes within the endometrial epithelium, little is known of the signal transduction pathway activated by the hormone. This study determined the effects of IFNtau on signal transducer and activator of transcription (STAT) activation, expression, DNA binding, and transcriptional activation using an ovine endometrial epithelial cell line. IFNtau induced persistent tyrosine phosphorylation and nuclear translocation of STAT1 and -2 (10 min to 48 h), but transient phosphorylation and nuclear translocation of STAT3, -5a/b, and -6 (10 to gene factor-3 and STAT1 homodimers formed and bound an IFN-stimulated response element (ISRE) and gamma-activated sequence (GAS) element, respectively. IFNtau increased transcription of GAS-driven promoters at 3 h, but suppressed their activity at 24 h. In contrast, the activity of an ISRE-driven promoter was increased at 3 and 24 h. These results indicate that IFNtau activates multiple STATs and has differential effects on ISRE- and GAS-driven gene transcription. PMID:11145571

  16. Enhanced poly(ADP-ribose) polymerase-1 activation contributes to recombinant tissue plasminogen activator-induced aggravation of ischemic brain injury in vivo.

    Science.gov (United States)

    Crome, Olaf; Doeppner, Thorsten R; Schwarting, Sönke; Müller, Barbara; Bähr, Mathias; Weise, Jens

    2007-06-01

    Recombinant tissue plasminogen activator (rt-PA) treatment improves functional outcome after acute ischemic stroke, inducing reperfusion by its thrombolytic activity. Conversely, there is evidence that rt-PA can mediate neuronal damage after ischemic brain injury in vivo. In addition to other mechanisms, enhancement of N-methyl-D-aspartate (NMDA) receptor signalling has been proposed to underlie rt-PA-mediated neurotoxicity. However, the role of poly(ADP-ribose) polymerase-1 (PARP-1) activation, which mediates postischemic excitotoxic cell death, in rt-PA-mediated aggravation of ischemic brain injury has not been established and was therefore addressed in this study. After permanent focal cerebral ischemia, intravenous rt-PA application significantly increased early postischemic PARP-1 activation within ischemic hemispheres and infarct volumes compared with control mice without affecting cerebral blood flow. Rt-PA induced increase in PARP-1 activation, and infarct volumes could be blocked by the PARP inhibitor 3-aminobenzamide. Moreover, the rt-PA-induced increase in PARP-1 activation was also prevented by the NMDA antagonist MK-801. In summary, we demonstrate that rt-PA treatment enhances postischemic PARP-1 activation, which contributes to rt-PA induced aggravation of ischemic brain injury in vivo. Furthermore, we provide evidence that NMDA receptor activation is required for rt-PA-mediated effects on postischemic PARP-1 activation. PMID:17455321

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

    OpenAIRE

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

    2001-01-01

    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 ex...

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

    DEFF Research Database (Denmark)

    Fevang, Børre; Eugen-Olsen, Jesper; Yndestad, Arne; Brosstad, Frank; Beiske, Klaus; Aukrust, Pål; Frøland, Stig S

    2009-01-01

    -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...

  19. Urokinase-mediated fibrinolysis in the synovial fluid of rheumatoid arthritis patients may be affected by the inactivation of single chain urokinase type plasminogen activator by thrombin

    OpenAIRE

    Braat, E; Jie, A; Ronday, H; Beekman, B; Rijken, D.

    2000-01-01

    BACKGROUND—Excessive fibrin deposition within the inflamed joints of rheumatoid arthritis (RA) patients suggests that local fibrinolysis is inefficient, which seems to be in contrast with the observed increased levels of urokinase type plasminogen activator (u-PA). Thrombin-mediated inactivation of single chain u-PA (scu-PA) into an inactive form called thrombin-cleaved two chain u-PA (tcu-PA/T) may provide a possible explanation for this contradiction.?AIM—To assess the occurrence of tcu-PA/...

  20. New transgenic evidence for a system of sympathetic axons able to express tissue plasminogen activator (t-PA) within arterial/arteriolar walls

    OpenAIRE

    Hao, Zhifang; Guo, Caiying; Jiang, Xi; Krueger, Susan; Pietri, Thomas; Dufour, Sylvie; Cone, Robert E.; O'Rourke, James

    2006-01-01

    Sympathetic axons embedded in a few arterioles and vasa vasora were recently shown to store tissue plasminogen activator (t-PA) in vesicles. But the extension of such t-PA axons to arteries and arterioles throughout the organism has not been verified. Confirmation of this anatomy would identify a second significant source of vessel wall t-PA. To visualize fine embedded axons independent of endothelium, we created a transgenic mouse whose expressions of the t-PA promoter and enhanced green flu...

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

    Energy Technology Data Exchange (ETDEWEB)

    Song, Xiaoling

    2011-12-01

    My research is on the synergistic regulation of PAI-1 by EGF and TGF-β. The mechanism of synergistic regulation of PAI-1 by EGF and TGF-β 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.

  2. Urokinase plasminogen activator receptor affects bone homeostasis by regulating osteoblast and osteoclast function

    DEFF Research Database (Denmark)

    Furlan, Federico; Galbiati, Clara

    2007-01-01

    The uPAR and its ligand uPA are expressed by both osteoblasts and osteoclasts. Their function in bone remodeling is unknown. We report that uPAR-lacking mice display increased BMD, increased osteogenic potential of osteoblasts, decreased osteoclasts formation, and altered cytoskeletal reorganization in mature osteoclasts. INTRODUCTION: Urokinase receptor (uPAR) is actively involved in the regulation of important cell functions, such as proliferation, adhesion, and migration. It was previously shown that the major players in bone remodeling, osteoblasts and osteoclasts, express uPAR and produce urokinase (uPA). The purpose of this study was to investigate the role of uPAR in bone remodeling. MATERIALS AND METHODS: In vivo studies were performed in uPAR knockout (KO) and wildtype (WT) mice on a C57Bl6/SV129 (75:25) background. Bone mass was analyzed by pQCT. Excised tibias were subjected to mechanical tests. UPAR KO calvaria osteoblasts were characterized by proliferation assays, RT-PCR for important proteins secreted during differentiation, and immunoblot for activator protein 1 (AP-1) family members. In vitro osteoclast formation was tested with uPAR KO bone marrow monocytes in the presence of macrophage-colony stimulating factor (M-CSF) and RANKL. Phalloidin staining in osteoclasts served to study actin ring and podosome formation. RESULTS: pQCT revealed increased bone mass in uPAR-null mice. Mechanical tests showed reduced load-sustaining capability in uPAR KO tibias. uPAR KO osteoblasts showed a proliferative advantage with no difference in apoptosis, higher matrix mineralization, and earlier appearance of alkaline phosphatase (ALP). Surface RANKL expression at different stages of differentiation was not altered. AP-1 components, such as JunB and Fra-1, were upregulated in uPAR KO osteoblasts, along with other osteoblasts markers. On the resorptive side, the number of osteoclasts formed in vitro from uPAR KO monocytes was decreased. Podosome imaging in uPAR KO osteoclasts revealed a defect in actin ring formation. CONCLUSIONS: The defective proliferation and differentiation of bone cells, coincident with both aberrant expression of transcription factors and cytoskeletal organization, are typical uPAR-dependent molecular phenotypes, and we have now shown their function in osteoblasts and osteoclasts function in vivo. Udgivelsesdato: 2007-Sep

  3. Plasminogen activator inhibitor-1 4G/5G polymorphism and retinopathy risk in type 2 diabetes: a meta-analysis

    Directory of Open Access Journals (Sweden)

    Zhang Tengyue

    2013-01-01

    Full Text Available Abstract Background Mounting evidence has suggested that plasminogen activator inhibitor-1 (PAI-1 is a candidate for increased risk of diabetic retinopathy. Studies have reported that insertion/deletion polymorphism in the PAI-1 gene may influence the risk of this disease. To comprehensively address this issue, we performed a meta-analysis to evaluate the association of PAI-1 4G/5G polymorphism with diabetic retinopathy in type 2 diabetes. Methods Data were retrieved in a systematic manner and analyzed using Review Manager and STATA Statistical Software. Crude odds ratios (ORs with 95% confidence intervals (CIs were used to assess the strength of associations. Results Nine studies with 1, 217 cases and 1, 459 controls were included. Allelic and genotypic comparisons between cases and controls were evaluated. Overall analysis suggests a marginal association of the 4G/5G polymorphism with diabetic retinopathy (for 4G versus 5G: OR 1.13, 95%CI 1.01 to 1.26; for 4G/4G versus 5G/5G: OR 1.30, 95%CI 1.04 to 1.64; for 4G/4G versus 5G/5G + 4G/5G: OR 1.26, 95%CI 1.05 to 1.52. In subgroup analysis by ethnicity, we found an association among the Caucasian population (for 4G versus 5G: OR 1.14, 95% CI 1.00 to 1.30; for 4G/4G versus 5G/5G: OR 1.33, 95%CI 1.02 to 1.74; for 4G/4G versus 5G/5G + 4G/5G: OR 1.41, 95%CI 1.13 to 1.77. When stratified by the average duration of diabetes, patients with diabetes histories longer than 10 years have an elevated susceptibility to diabetic retinopathy than those with shorter histories (for 4G/4G versus 5G/5G: OR 1.47, 95%CI 1.08 to 2.00. We also detected a higher risk in hospital-based studies (for 4G/4G versus 5G/5G+4G/5G: OR 1.27, 95%CI 1.02 to 1.57. Conclusions The present meta-analysis suggested that 4G/5G polymorphism in the PAI-1 gene potentially increased the risk of diabetic retinopathy in type 2 diabetes and showed a discrepancy in different ethnicities. A higher susceptibility in patients with longer duration of diabetes (more than 10 years indicated a gene-environment interaction in determining the risk of diabetic retinopathy.

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

    OpenAIRE

    Marzia Albenzio; Rosaria Marino; Mariangela Caroprese; Antonella Santillo

    2010-01-01

    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...

  5. Formyl-methyl-methylenetetrahydrofolate synthetase-(combined). An ovine protein with multiple catalytic activities.

    Science.gov (United States)

    Paukert, J L; Straus, L D; Rabinowitz, J C

    1976-08-25

    Formyltetrahydrofolate synthetase, methenyltetrahydrofolate cyclohydrolase, and methylenetetrahydrofolate dehydrogenase in sheep liver co-purify 200-fold to yield a homogeneous preparation containing a single protein species observed on discontinuous polyacrylamide gel electrophoresis in the presence of dodecyl sulfate. The synthetase and dehydrogenase activities migrate with the single protein band observed on polyacrylamide gel electrophoresis in two discontinuous buffer systems near pH 8. The protein sediments with a single symmetrical boundary with sedimentation coefficient (S20,w) = 8.30 S and elutes from molecular exclusion chromatography columns at a position corresponding to a diffusion coefficient (D20,w) = 3.99 X 10(-7) cm2 sec-1. Dodecyl sulfate-polyacrylamide gel electrophoresis of the protein intramolecularly cross-linked with dimethylsuberimidate shows one protein species of Mr = 218,000 in addition to the species of Mr = 108,500 characteristic of the unmodified protein. NH2-terminal analysis of the protein using 5-dimethylaminonaphthalene-1-sulfonyl (dansyl) chloride in the presence of dodecyl sulfate results in the recovery of a single dansyl amino acid, alanine. The three activities are thus shown to reside in a protein composed of two apparently identical subunits, and the trivial name formyl-methenyl-methylenetetrahydrofolate synthetase(combined) is proposed for this enzyme to suggest the multiple catalytic activities associated with the single protein species. PMID:956178

  6. 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

  7. 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

  8. Characteristics of ovine cytotoxic lymphocytes

    International Nuclear Information System (INIS)

    Experiments were conducted to examine characteristics of the effector cells responsible for cell-mediated cytotoxicity in the sheep. Conditions for the production and assay of ovine T cell growth factor (TCGF) activity were evaluated. Peripheral blood leukocytes (PBL) were stimulated with concanavalin A (Con A) in the presence of 2% autologous serum or serum-free media. A 28 h proliferation assay with 2.5 x 104 h Con A blasts per well was optimal for detection of TCGF. Peak TCGF activity occurred with a 30-37kD molecular weight fraction. Ovine PBL were used for in vitro generation of genetically-restricted cytotoxic T lymphocytes (CTL). Peripheral blood leukocytes from sheep that had been previously inoculated with live vaccinia virus were stimulated by being cultured in vitro on glutaraldehyde-fixed vaccinia-infected autologous skin fibroblasts. Cytotoxic T lymphocyte activity was assessed in a 6 h 51Cr-release assay on autologous and allogeneic fibroblasts targets. Killing was restricted to virus-infected autologous targets. In vitro generation of both anti-vaccinia and anti-TNP CTL activity could be enhanced by the addition of TCGF containing media from ConA-stimulated PBL

  9. High expression of urokinase plasminogen activator receptor (UPA-R) in acute myeloid leukemia (AML) is associated with worse prognosis.

    Science.gov (United States)

    Graf, Michaela; Reif, Susanne; Hecht, Karin; Pelka-Fleischer, Renate; Pfister, Karin; Schmetzer, Helga

    2005-05-01

    Urokinase-type plasminogen activator receptor (UPA-R; CD87) is a membrane protein responsible for plasmin expression on cells facilitating cellular extravasations and tissue invasions. We studied the expression of the UPA-R on bone marrow (BM) cells of 93 patients with acute myeloid leukemia at first diagnosis and 8 healthy probands as controls by FACS analysis using phycoerythrin (PE)-conjugated antibodies. A case was defined as UPA-R-positive (UPA-R+) if >20% of the gated cells expressed UPA-R. Whereas none of the 8 healthy BM samples was positive for the UPA-R, 32 (34%) of the 93 AML samples were UPA-R+. Expression of UPA-R was heterogeneous in different FAB types, however, with the highest expression rates in monocytic subtypes (FAB M4/M5): 18%/19%/30% of UPA-R+ cases were found in M1/M2 or M3, and 58%/80% of cases with M4 or M5 were UPA-R+. Proportions of UPA-R+ cells varied between 1% and 98% of the mononuclear cell fractions, with the highest proportions in M4/M5 subtypes (on average 27%/40% UPA-R+ cells) and the lowest expression in AML M2 (11% UPA-R+ cells). The density of expressed UPA-R, estimated as mean channel fluorescence activity, was highest in cases with AML M1 (mFI: 124) followed by M4 and M5 (mFI: 78/77) and lowest in AML M2 (mFI: 43). In sAML, higher proportions of UPA-R+ cases (8 of 18; 44%) compared to pAML (24 of 75; 32%) were found as well as higher proportions of UPA-R+ cells (27% vs. 19%). Separating our patients' cohort in cytogenetic risk groups, we could not detect significant differences in the UPA-R expression profiles. For evaluations of the clinical course of AML, only patients treated by the AML-CG protocol (n = 65) were included. In the group of patients who did not respond to AML-CG therapy, significantly higher proportions of UPA-R+ cells (31% vs. 14%, P = 0.0015, t-test) were found. By evaluating a cut-off value for the percentage of positive cells that allows the most significant separation and differentiation between cases with shorter or longer relapse-free survival times, we could show that patients with >26.5% UPA-R-positive cells were characterized by a significantly higher risk for relapse compared to cases with <26.5% positive cells (P = 0.05). In summary, our data show a high expression of the UPA-R in AML, especially in (myelo)monocytoid subtypes. Cases with higher proportions of UPA-R+ cells were characterized by a significant lower remission rate after AML-CG therapy and a higher risk for relapse. Although prospective trials are still lacking, UPA-R is a prognostically relevant factor independent from the karyotype. UPA-R positivity may identify subtypes of AML associated with a more aggressive clinical course. Thus due to lower remission probabilities in UPA-R+ cases, a more intensive induction therapy regimen could be considered. PMID:15849776

  10. Construction and characterization of a recombinant chimeric plasminogen activator consisting of a fibrin peptide and a low molecular mass single-chain urokinase.

    Science.gov (United States)

    Jiao, J; Yu, M; Ru, B

    2001-01-01

    A recombinant chimeric plasminogen activator (f beta/scuPA-32k), with a fibrin beta-chain peptide (comprising Gly15 through Arg 42) linked to the N-terminal of a low molecular mass (32 kDa) single-chain urokinase (scuPA-32k, comprising Leu144 through Leu 411) via a 50 amino acid linker sequence, was produced by expression the corresponding chimeric cDNA in Escherichia coli cells. After refolding in vitro, the chimeric protein was purified to homogeneity by zinc chelate-Sepharose chromatography, Sephacryl S200 chromatography and benzamidine-Sepharose chromatography in sequence. The apparent molecular mass was 36 kDa shown by SDS-PAGE analysis. The special activity was 87,000 IU/mg detected by fibrin plate determination. F beta/scuPA-32k could directly activate plasminogen following Michaelis-Menten kinetics with K(m) = 0.52 microM and k(2) = 0.0024 s(-1). Mediated by plasmin, the single-chain molecule could be converted to the active two-chain molecule. The chimeric protein had 3.3 times higher fibrin affinity than scuPA-32k in the fibrin concentration of 3.2 mg/mL, while the chimeric protein inhibited the fibrin clotting and platelet aggregation. F beta/scuPA-32k showed a higher thrombolytic potency in vitro plasma clot lysis than scuPA-32k and depleted less fibrinogen in plasma. These results showed that the chimeric protein had not only higher fibrinolytic activity but also anti-thrombus activity. Further evaluation of the thrombolytic potential in appropriate animal models is required. PMID:11879733

  11. Biochemical, thrombolytic and pharmacokinetic properties of rt-PA P47G, K49N, a substitution variant of human tissue-type plasminogen activator.

    Science.gov (United States)

    Nelles, L; Li, X K; Vanlinthout, I; De Cock, F; Lijnen, H R; Collen, D

    1992-04-01

    rt-PA P47G, K49N, a substitution variant of recombinant human tissue-type plasminogen activator (rt-PA), in which proline at position 47 and lysine at position 49 were replaced by glycine and asparagine respectively, was previously described by Ahern et al. (J Biol Chem 1990; 265:5540-5) to have an extended in vivo half-life with unaltered in vitro fibrinolytic properties. Because this variant might possess an increased in vivo thrombolytic potency, we have constructed its cDNA, expressed it in Chinese hamster ovary cells and determined its biochemical, thrombolytic and pharmacokinetic properties relative to those of home-made rt-PA and of alteplase (Actilyse). The specific fibrinolytic activities on fibrin plates were 160,000 +/- 17,000, 210,000 +/- 88,000 and 460,000 +/- 72,000 IU/mg (mean +/- SEM) for rt-PA P47G, K49N, rt-PA and alteplase, respectively, while the catalytic efficiencies for plasminogen activation (k2/Km) in the absence of fibrin were comparable (1.1 to 1.7 x 10(-3) microM-1s-1). Fibrin enhanced the rate of plasminogen activation by rt-PA P47G, K49N 100-fold and by both wild-type molecules 390-fold. Binding of the variant rt-PA to fibrin was significantly reduced, but its affinity for lysine-Sepharose was unaltered. In an in vitro clot lysis system, consisting of a radiolabeled human plasma clot submersed in plasma, 50% clot lysis in 2 h required 0.67 +/- 0.14 micrograms/ml rt-PA P47G, K49N, 0.36 +/- 0.01 micrograms/ml rt-PA and 0.17 +/- 0.01 micrograms/ml alteplase, respectively (mean +/- SEM; n = 3 or 4). At these doses residual fibrinogen levels at 2 h were in excess of 80%.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1631793

  12. Targeting tumor cell invasion and dissemination in vivo by an aptamer that inhibits urokinase-type plasminogen activator through a novel multifunctional mechanism

    DEFF Research Database (Denmark)

    Botkjaer, Kenneth A; Deryugina, Elena I

    2012-01-01

    Data accumulated over the latest two decades have established that the serine protease urokinase-type plasminogen activator (uPA) is a potential therapeutic target in cancer. When designing inhibitors of the proteolytic activity of serine proteases, obtaining sufficient specificity is problematic, because the topology of the proteases' active sites are highly similar. In an effort to generate highly specific uPA inhibitors with new inhibitory modalities, we isolated uPA-binding RNA aptamers by screening a library of 35 nucleotides long 2'-fluoro-pyrimidine RNA molecules using a version of human pro-uPA lacking the epidermal growth factor-like and kringle domains as bait. One pro-uPA-binding aptamer sequence, referred to as upanap-126, proved to be highly specific for human uPA. Upanap-126 delayed the proteolytic conversion of human pro-uPA to active uPA, but did not inhibit plasminogen activation catalyzed by two-chain uPA. The aptamer also inhibited the binding of pro-uPA to uPAR and the binding of vitronectin to the preformed pro-uPA/uPAR complex, both in cell-free systems and on cell surfaces. Furthermore, upanap-126 inhibited human tumor cell invasion in vitro in the Matrigel assay and in vivo in the chick embryo assay of cell escape from microtumors. Finally, upanap-126 significantly reduced the levels of tumor cell intravasation and dissemination in the chick embryo model of spontaneous metastasis. Together, our findings show that usage of upanap-126 represents a novel multifunctional mechanistic modality for inhibition of uPA-dependent processes involved in tumor cell spread.

  13. Targeting Tumor Cell Invasion and Dissemination In Vivo by an Aptamer That Inhibits Urokinase-type Plasminogen Activator through a Novel Multifunctional Mechanism

    DEFF Research Database (Denmark)

    Botkjaer, Kenneth A; Deryugina, Elena I

    2012-01-01

    Data accumulated over the latest two decades have established that the serine protease urokinase-type plasminogen activator (uPA) is a potential therapeutic target in cancer. When designing inhibitors of the proteolytic activity of serine proteases, obtaining sufficient specificity is problematic, because the topology of the proteases' active sites are highly similar. In an effort to generate highly specific uPA inhibitors with new inhibitory modalities, we isolated uPA-binding RNA aptamers by screening a library of 35 nucleotides long 2'-fluoro-pyrimidine RNA molecules using a version of human pro-uPA lacking the epidermal growth factor-like and kringle domains as bait. One pro-uPA-binding aptamer sequence, referred to as upanap-126, proved to be highly specific for human uPA. Upanap-126 delayed the proteolytic conversion of human pro-uPA to active uPA, but did not inhibit plasminogen activation catalyzed by two-chain uPA. The aptamer also inhibited the binding of pro-uPA to uPAR and the binding of vitronectin to the preformed pro-uPA/uPAR complex, both in cell-free systems and on cell surfaces. Furthermore, upanap-126 inhibited human tumor cell invasion in vitro in the Matrigel assay and in vivo in the chick embryo assay of cell escape from microtumors. Finally, upanap-126 significantly reduced the levels of tumor cell intravasation and dissemination in the chick embryo model of spontaneous metastasis. Together, our findings show that usage of upanap-126 represents a novel multifunctional mechanistic modality for inhibition of uPA-dependent processes involved in tumor cell spread.

  14. In vitro and in vivo antiangiogenic activity of a novel deca-peptide derived from human tissue-type plasminogen activator kringle 2

    Energy Technology Data Exchange (ETDEWEB)

    Su, Li; Xu, Xun; Zhao, Hui; Gu, Qing [Department of Ophthalmology, Shanghai First People' s Hospital, Affiliate of Shanghai Jiaotong University, No. 100 Haining Road, Shanghai 200080 (China); Zou, Haidong, E-mail: zouhaidong@hotmail.com [Department of Ophthalmology, Shanghai First People' s Hospital, Affiliate of Shanghai Jiaotong University, No. 100 Haining Road, Shanghai 200080 (China)

    2010-06-11

    A synthetic deca-peptide corresponding to the amino acid sequence Arg{sup 54}-Trp{sup 63} of human tissue-type plasminogen activator (t-PA) kringle 2 domain, named TKII-10, is produced and tested for its ability to inhibit endothelial cell proliferation, migration, tube formation in vitro, and angiogenesis in vivo. At the same time, another peptide TKII-10S composed of the same 10 amino acids as TKII-10, but in a different sequence, is also produced and tested. The results show that TKII-10 potently inhibits VEGF-stimulated endothelial cell migration and tube formation in a dose-dependent, as well as sequence-dependent, manner in vitro while it is inactive in inhibiting endothelial cell proliferation. Furthermore, TKII-10 potently inhibits angiogenesis in chick chorioallantoic membrane and mouse cornea. The middle four amino acids DGDA in their sequence play an important role in TKII-10 angiogenesis inhibition{sub .} These results suggest that TKII-10 is a novel angiogenesis inhibitor that may serve as a prototype for antiangiogenic drug development.

  15. In vitro and in vivo antiangiogenic activity of a novel deca-peptide derived from human tissue-type plasminogen activator kringle 2

    International Nuclear Information System (INIS)

    A synthetic deca-peptide corresponding to the amino acid sequence Arg54-Trp63 of human tissue-type plasminogen activator (t-PA) kringle 2 domain, named TKII-10, is produced and tested for its ability to inhibit endothelial cell proliferation, migration, tube formation in vitro, and angiogenesis in vivo. At the same time, another peptide TKII-10S composed of the same 10 amino acids as TKII-10, but in a different sequence, is also produced and tested. The results show that TKII-10 potently inhibits VEGF-stimulated endothelial cell migration and tube formation in a dose-dependent, as well as sequence-dependent, manner in vitro while it is inactive in inhibiting endothelial cell proliferation. Furthermore, TKII-10 potently inhibits angiogenesis in chick chorioallantoic membrane and mouse cornea. The middle four amino acids DGDA in their sequence play an important role in TKII-10 angiogenesis inhibition. These results suggest that TKII-10 is a novel angiogenesis inhibitor that may serve as a prototype for antiangiogenic drug development.

  16. Schistosoma bovis: plasminogen binding in adults and the identification of plasminogen-binding proteins from the worm tegument.

    Science.gov (United States)

    Ramajo-Hernández, Alicia; Pérez-Sánchez, Ricardo; Ramajo-Martín, Vicente; Oleaga, Ana

    2007-01-01

    Schistosoma bovis is a ruminant haematic parasite that lives for years in the mesenteric vessels of the host. The aim of this work was to investigate the ability of adult S. bovis worms to interact with plasminogen, a central component in the host fibrinolytic system. Confocal microscopy analysis revealed that plasminogen bound to the tegument surface of the male-but not female-S. bovis worms and that this binding was strongly dependent on lysine residues. It was also observed that a protein extract of the worm tegument (TG) had the capacity to generate plasmin and to enhance the plasmin generation by the tissue-type plasminogen activator. Proteomic analysis of the TG extract identified 10 plasminogen-binding proteins, among which the major ones were enolase, glyceraldehyde-3-phosphate dehydrogenase and actin. This study represents the first report about the binding of plasminogen to Schistosoma sp. proteins. PMID:16962583

  17. A comparison of ovine and equine antivenoms.

    Science.gov (United States)

    Sjostrom, L; al-Abdulla, I H; Rawat, S; Smith, D C; Landon, J

    1994-04-01

    Commercial antivenoms produced in horses were compared with monospecific antivenoms raised in sheep against Crotalus durissus terrificus, Crotalus atrox, Crotalus adamanteus, Micrurus fulvius fulvius, Naja naja, Naja kaouthia, Echis ocellatus, Vipera lebetina deserti, Vipera berus berus and Vipera ammodytes ammodytes venom. Antibodies raised by immunizing sheep with C. d. terrificus venom were more effective than their equine counterparts in preventing lethal toxicity in mice (ED50), in inhibiting the venom's pharmacological effects (haemolysis, platelet aggregation and coagulation), and in neutralizing phospholipase A2 activity. Comparison of one ovine and three equine F(ab)2 products raised against V. a. ammodytes venom showed that all were at least 95% pure; that all protected mice; and that all contained antibody populations directed against most components of V. a. ammodytes and V. b. berus venoms. The ovine antivenoms generally contained a higher concentration of specific antibodies than the equine products. Finally, the ovine antivenoms raised against E. ocellatus, V. lebetina deserti, V. b. berus, M. f. fulvius and N. naja venoms provided better in vivo protection to mice than the equine antivenoms, but the equine antivenoms to N. kaouthia and C. atrox were more protective than the ovine product. PMID:8052997

  18. Expression and characterization of clustered charge-to-alanine mutants of low M(r) single-chain urokinase-type plasminogen activator.

    Science.gov (United States)

    Ueshima, S; Holvoet, P; Lijnen, H R; Nelles, L; Seghers, V; Collen, D

    1994-01-01

    In an effort to modify the fibrinolytic and/or pharmacokinetic properties of recombinant low M(r) single-chain urokinase-type plasminogen activator (rscu-PA-32k), mutants were prepared by site-directed mutagenesis of clusters of charged amino acids with the highest solvent accessibility. The following mutants of rscu-PA-32k were prepared: LUK-2 (Lys 212, Glu 213 and Asp 214 to Ala), LUK-3 (Lys 243 and Asp 244 to Ala), LUK-4 (Arg 262, Lys 264, Glu 265 and Arg 267 to Ala), LUK-5 (Lys 300, Glu 301 and Asp 305 to Ala) and LUK-6 (Arg 400, Lys 404, Glu 405 and Glu 406 to Ala). The rscu-PA-32k moieties were expressed in High Five Trichoplasiani cells, and purified to homogeneity from the conditioned cell culture medium, with recoveries of 0.8 to 3.7 mg/l. The specific fibrinolytic activities (220,000 to 300,000 IU/mg), the rates of plasminogen activation by the single-chain moieties and the rates of conversion to two-chain moieties by plasmin were comparable for mutant and wild-type rscu-PA-32k moieties, with the exception of LUK-5 which was virtually inactive. Equi-effective lysis (50% in 2 h) of 60 microliters 125I-fibrin labeled plasma clots submerged in 0.5 ml normal human plasma was obtained with 0.7 to 0.8 microgram/ml of wild-type or mutant rscu-PA-32k, except with LUK-5 (no significant lysis with 16 micrograms/ml). Following bolus injection in hamsters, all rscu-PA-32k moieties had a comparably rapid plasma clearance (1.3 to 2.7 ml/min), as a result of a short initial half-life (1.4 to 2.5 min).(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8165632

  19. A suppressive effect of prostaglandin E2 on the expression of SERPINE1/plasminogen activator inhibitor-1 in human articular chondrocytes: An in vitro pilot study

    Directory of Open Access Journals (Sweden)

    Kayo Masuko

    2009-04-01

    Full Text Available Kayo Masuko1, Minako Murata2, Naoya Suematsu1, Kazuki Okamoto1, Kazuo Yudoh2, Hiroyuki Shimizu3, Moroe Beppu3, Hiroshi Nakamura4, Tomohiro Kato11Department of Biochemistry; 2Department of Frontier Medicine, Institute of Medical Science; 3Department of Orthopedic Surgery, St. Marianna University School of Medicine, Kawasaki-shi, Kanagawa, Japan; 4Department of Joint Disease and Rheumatism, Nippon Medical School, Bunkyo-ku, Tokyo, JapanAbstract: Prostaglandin E2 (PGE2 is expressed in articular joints with inflammatory arthropathy and may exert catabolic effects leading to cartilage degradation. As we observed in a preliminary experiment that PGE2 suppressed the expression of SERPINE1/plasminogen activator inhibitor (PAI-1 mRNA in chondrocytes, we focused on the effect of PGE2 on PAI-1 in a panel of cultured chondrocytes obtained from osteoarthritic patients. Specifically, articular cartilage specimens were obtained from patients with osteoarthritis who underwent joint surgery. Isolated chondrocytes were cultured in vitro as a monolayer and stimulated with PGE2. Stimulated cells and culture supernatants were analyzed using Western blotting and enzyme-linked immunosorbent assay. The results confirmed that the in vitro PGE2 stimulation suppressed the expression of PAI-1 in the tested chondrocyte samples. The inhibitory effect was partly abrogated by an antagonist of EP4 receptor of PGE2, but not by an EP2 antagonist. Although PGE2 induced activations of mitogen-activated protein kinases (MAPK, blocking of the MAPK did not abrogate the suppressive effect of PGE2, implying a distinct signaling pathway. In summary, prostaglandin is suggested to modulate the plasminogen system in chondrocytes. Further elucidation of the interaction might open a new avenue to understand the degradative process of cartilage.Keywords: chondrocyte, prostaglandin, PGE2, PAI-1

  20. Optimization of Crystals of an Inhibitory Antibody of Urokinase Plasminogen Activator Receptor (uPAR) with Hydrogen Peroxide and Low Protein Concentration

    Energy Technology Data Exchange (ETDEWEB)

    Li, Yongdong; Shi, Xiaoli; Parry, Graham; Chen, Liqing; Callahan, Jennifer A.; Mazar, Andrew P.; Huang, Mingdong (UAH); (Attenuon LLC); (Chinese Aca. Sci.)

    2010-07-19

    Optimization of protein crystal formation is often a necessary step leading to diffraction-quality crystals to enable collection of a full X-ray data set. Typical protein crystal optimization involves screening different components, e.g., pH, precipitants, and additives of the precipitant solution. Here we present an example using an inhibitory antibody of urokinase plasminogen activator receptor (uPAR) where such procedures did not yield diffracting crystals. In contrast, it was the treatment of the protein with hydrogen peroxide incubation and the protein concentration reduction that were found to be key factors in obtaining diffracting crystals. Final crystals diffracted to 1.75 {angstrom}, and belong to orthorhombic P212121 space group with unit cell parameters a = 37.162 {angstrom}, b = 84.474 {angstrom}, c = 134.030 {angstrom}, and contain one molecule of Fab fragment of anti-uro kinase receptor antibody in the asymmetric unit.

  1. Distinctive binding modes and inhibitory mechanisms of two peptidic inhibitors of urokinase-type plasminogen activator with isomeric P1 residues

    DEFF Research Database (Denmark)

    Jiang, Longguang; Zhao, Baoyu

    2015-01-01

    Abstract Two isomeric piperidine derivatives (meta and para isomers) were used as arginine mimics in the P1 position of a cyclic peptidic inhibitor (CPAYSRYLDC) of urokinase-type plasminogen activator. The two resulting cyclic peptides showed vastly different affinities (?70 fold) to the target enzyme. X-ray crystal structure analysis showed that the two P1 residues were inserted into the S1 specificity pocket in indistinguishable manners. However, the rest of the peptides bound in entirely different ways on the surface of the enzyme, and the two peptides have different conformations, despite the highly similar sequence. These results demonstrate how the subtle difference in P1 residue can dictate the exosite interactions and the potencies of peptidic inhibitors, and highlight the importance of the P1 residue for protease inhibition. This study provides important information for the development of peptidic agents for pharmacological intervention.

  2. Imaging of urokinase-type plasminogen activator receptor expression using a 64Cu-labeled linear peptide antagonist by microPET

    DEFF Research Database (Denmark)

    Li, Z.B.; Niu, G.; Wang, H.; He, L.; Yang, L.; Ploug, M.; Chen, X.

    2008-01-01

    PURPOSE: Malignant tumors are capable of degrading the surrounding extracellular matrix, resulting in local invasion or metastasis. Urokinase-type plasminogen activator (uPA) and its cell surface receptor (uPAR) are central molecules in one of the major protease systems involved in extracellular...... translation of this class of radiopharmaceuticals for uPAR-positive cancer detection and patient stratification for uPA/uPAR system-based cancer therapy Udgivelsesdato: 2008/8/1...... matrix degradation. Noninvasive imaging of this receptor in vivo with radiolabeled peptides that specifically target uPAR may therefore be useful to decipher the potential invasiveness of malignant lesions. EXPERIMENTAL DESIGN: In this study, we developed a (64)Cu-labeled uPAR-binding peptide for...

  3. Preoperative plasma plasminogen activator inhibitor type-1 and serum C-reactive protein levels in patients with colorectal cancer. The RANX05 Colorectal Cancer Study Group

    DEFF Research Database (Denmark)

    Nielsen, Hans Jørgen; Christensen, Ib Jarle; Sørensen, Steen; Moesgaard, F; Brünner, N

    2000-01-01

    statistical analysis including Dukes classification, gender, age, tumor location, perioperative blood transfusion, PAI-1 and CRP, plasma PAI-1 was a dependent prognostic variable, while serum CRP (P <.0001; HR: 1.4; 95% CI: 1.3-1.5) was found to be a Dukes independent prognostic variable. Similar analyses......BACKGROUND: Preoperative plasma plasminogen activator inhibitor-1 (PAI-1) is a prognostic variable in patients with colorectal cancer. It has been suggested, however, that plasma PAI-1 is a nonspecific prognostic parameter similar to the acute-phase reactant C-reactive protein (CRP). In the present...... study we analyzed the association between plasma PAI-1 and serum CRP in patients scheduled for elective resection of colorectal cancer. In addition, the prognostic value of PAI-1 and CRP was studied in this patient cohort. METHODS: PAI-1 and CRP were analyzed in citrated plasma and serum, respectively...

  4. Metformin therapy is associated with a decrease in plasma plasminogen activator inhibitor-1, lipoprotein(a), and immunoreactive insulin levels in patients with the polycystic ovary syndrome.

    Science.gov (United States)

    Velazquez, E M; Mendoza, S G; Wang, P; Glueck, C J

    1997-04-01

    Sixteen nondiabetic women with polycystic ovary syndrome (PCOS) aged 18 to 33 years were studied before and after 8 weeks on metformin (1.5 g/d) therapy to assess whether reducing hyperinsulinemia would reduce the levels of the major inhibitor of fibrinolysis, antigenic plasminogen activator inhibitor type 1 (PAI-1). Compared with six normal control women, PCOS women had a higher body mass index (BMI), waist to hip ratio, fasting insulin (Izero), insulin area under the curve during oral glucose tolerance testing (IA), glucose area under the curve during oral glucose tolerance testing (GA), IA/GA ratio, PAI-1, luteinizing hormone (LH) and ratio of LH to follicle-stimulating hormone (FSH), and free testosterone, and lower high-density lipoprotein (HDL) cholesterol (all P PCOS patients, reverses the hyperinsulinemia-driven endocrinopathy, decreases PAI-1, and decreases Lp(a), and should thus reduce the increased risk of atherothrombosis in PCOS. PMID:9109854

  5. Correlation between plasminogen activator inhibitor-1 (PAI-1) promoter 4G/5G polymorphism and metabolic/proinflammatory factors in polycystic ovary syndrome.

    Science.gov (United States)

    Sales, M F; Sóter, M O; Candido, A L; Fernandes, A P; Oliveira, F R; Ferreira, A C S; Sousa, M O; Ferreira, C N; Gomes, K B

    2013-10-01

    Polycystic Ovary Syndrome (PCOS) is the most common cause of subfertility associated to metabolic disorders. The aim of this study was to correlate metabolic and proinflammatory factors in women with PCOS. The frequency of Plasminogen Activator Inhibitor-1 (PAI-1) promoter 4?G/5?G polymorphism was also compared to healthy controls. We evaluated 79 PCOS and 79 healthy women. PAI-1 levels are positively correlated with proinflammatory factors in PCOS group. 4?G allele in PAI-1 gene was more frequent in PCOS and the 4G/4?G genotype was associated with increased PAI-1 levels. A correlation between insulin resistance and proinflammatory and overweight was also observed. C-reactive protein, serum levels of vascular cell adhesion molecule-1 (sVCAM-1), Lipid Accumulation Product (LAP) and vitamin D are good tools to evaluated factors associated to cardiovascular risk in women with PCOS. PMID:23898913

  6. Ovine TRIM5 Can Restrict Visna/Maedi Virus

    OpenAIRE

    P. Jáuregui; Crespo, H.; Glaria, I.; A. Contreras; De Andrés, D.; Amorena, B.; Towers, G. J.; Reina, R.

    2012-01-01

    The restrictive properties of tripartite motif-containing 5 alpha (TRIM5?) from small ruminant species have not been explored. Here, we identify highly similar TRIM5? sequences in sheep and goats. Cells transduced with ovine TRIM5? effectively restricted the lentivirus visna/maedi virus DNA synthesis. Proteasome inhibition in cells transduced with ovine TRIM5? restored restricted viral DNA synthesis, suggesting a conserved mechanism of restriction. Identification of TRIM5? active molecular sp...

  7. Transconjunctival sutureless vitrectomy with tissue plasminogen activator, gas and intravitreal bevacizumab in the management of predominantly hemorrhagic age-related macular degeneration

    Directory of Open Access Journals (Sweden)

    Luis Arias

    2010-02-01

    Full Text Available Luis Arias1,2, Jordi Monés11Institut de la Màcula i de la Retina, Centro Médico Teknon, Barcelona; 2Hospital Universitari de Bellvitge, L’Hospitalet de Llobregat, BarcelonaPurpose: To determine the efficacy and safety of treating predominantly hemorrhagic age-related macular degeneration (AMD with transconjunctival sutureless vitrectomy (TSV, tissue plasminogen activator (tPA, sulphur hexafluoride (SF6, and intravitreal bevacizumab.Methods: Retrospective study, consecutive case series. Patients with acute hemorrhagic AMD treated with 25- or 23-gauge TSV, subretinal or intravitreal tPA, fluid-air-SF6 exchange and intravitreal injection of bevacizumab. All operations were performed within the first 5 days after the start of symptoms, which consisted of visual acuity (VA loss and central scotoma.Results: Fifteen eyes from 15 patients were included. The patients’ mean age was 79.6 years, and the mean follow-up was 11.8 months. Five patients (33% were receiving oral anticoagulant treatment. At baseline, the mean VA (logMAR values was 1.5 (20/640 Snellen equivalent. At the last follow-up visit, the mean VA was 1.1 (20/250 (P < 0.0001; paired t-test. The submacular hemorrhage was successfully displaced in all the cases. Complications consisted of three cases of vitreous hemorrhage and a tear or the retinal pigment epithelium. Twelve cases (80% did not require further treatment during the follow-up period.Conclusion: A surgical approach with 25- or 23-gauge TSV, tPA, SF6 and intravitreal bevacizumab is an efficacious and safe procedure in patients with hemorrhagic AMD. Early treatment is advisable for obtaining the optimal outcome.Keywords: Hemorrhagic age-related macular degeneration, tissue plasminogen activator, intravitreal bevacizumab; transconjunctival sutureless vitrectomy

  8. Preparation of ultrasound microbubbles crosslinked to albumin nanoparticles packaged with tissue-type plasminogen activator gene plasmid and method of in vivo transfection

    Directory of Open Access Journals (Sweden)

    Jun J

    2011-03-01

    Full Text Available Ji Jun, Ji Shang-Yi, He Xia, Ling Wen-PingDepartment of Pathology, ShenZhen Sun Yat-Sen Cardiovascular Hospital, ShenZhen, GuangDong Province, People's Republic of ChinaAims: To observe the effect of constructed ultrasound microbubble crosslinked to albium nanoparticles packaged with tissue-type plasminogen activator (tPA gene plasmid on the in vivo transfection.Methods: The rabbits were chosen for all experiments. A highly expressive gene plasmid for tPA was constructed and packaged into a prepared nanoparticle with bovine serum albumin (BSA. This albium nanoparticle packaged with tPA gene plasmid was crosslinked to an ultrasound microbubble prepared with BSA and sucrose to form a nano-targeting vector system for tPA gene transfection. The transfection and effective expression of tPA in heart, liver, leg skeletal muscle and the cervical rib were detected with polyclonal antibodies to tPA using immunohistochemical method; the tPA level and D-dimer content of blood were also tested.Results: The expression of tPA could be seen in the tissues mentioned above, with the increase in blood tPA level and D-dimer content from 0.20 ± 0.05 µg/L and 81.76 ± 9.84 µg/L before the operation, to the higher levels of 0.44 ± 0.05 µg/L and 669.28 ± 97.74 µg/L after transfection.Conclusion: The nano-targeting vector system for tPA gene was contructed successfully. This provides a new theory and experimental method for the nano-targeted transgene.Keywords: tissue-type plasminogen activator, albium nanoparticle, ultrasound microbubble, targeting transfection 

  9. Cyclic AMP-mediated cytoskeletal effects in adrenal cells are modified by serum, insulin, insulin-like growth factor-I, and an antibody against urokinase plasminogen activator.

    Science.gov (United States)

    Hornsby, P J; Maghsoudlou, S S; Cheng, V; Cheng, C Y

    1989-12-01

    In adrenocortical cells in culture, increased intracellular cyclic AMP resulting from exposure to agents such as ACTH and cholera toxin causes a change in cell morphology termed 'retraction' or 'rounding'. The breakdown of actin-containing stress fibers in rounding suggested a role for microfilaments in steroidogenesis. Previously, we showed that cultured bovine adrenal cells under standard conditions (medium with 10% fetal bovine serum) do not round in response to intracellular cyclic AMP. Here, we show that these cells do round in defined, serum-free medium. Rounding was maximal within 1 h of addition of 1 nM cholera toxin and after 10 h most cells remained rounded. Cycloheximide at 100 micrograms/ml did not inhibit the response to cholera toxin. The rounding response was abolished when 10% fetal bovine serum, horse serum, or ether-extracted fetal bovine serum was included in the medium. The inhibitory effect of serum was not mimicked by growth factors with the exception that insulin and insulin-like growth factor-I (IGF-I), while not preventing rounding, accelerated the return of cells to a flattened morphology. A monoclonal antibody against urokinase plasminogen activator completely prevented rounding whereas a monoclonal antibody against tissue plasminogen activator had only a slight effect. Fluorescence visualization of F-actin with N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-phallacidin showed that rounding in cultured bovine adrenocortical cells resembles that defined earlier for human and rat adrenocortical cells and includes depolymerization of actin microfilaments. These cytoskeletal changes in adrenal cells are unlikely to play a role in steroidogenesis; however, they may be involved in tissue remodeling occurring as part of the indirect mitogenic effects of ACTH. PMID:2558936

  10. Mapping the topographic epitope landscape on the urokinase plasminogen activator receptor (uPAR) by surface plasmon resonance and X-ray crystallography.

    Science.gov (United States)

    Zhao, Baoyu; Gandhi, Sonu; Yuan, Cai; Luo, Zhipu; Li, Rui; Gårdsvoll, Henrik; de Lorenzi, Valentina; Sidenius, Nicolai; Huang, Mingdong; Ploug, Michael

    2015-12-01

    The urokinase-type plasminogen activator receptor (uPAR or CD87) is a glycolipid-anchored membrane protein often expressed in the microenvironment of invasive solid cancers and high levels are generally associated with poor patient prognosis (Kriegbaum et al., 2011 [1]). uPAR is organized as a dynamic modular protein structure composed of three homologous Ly6/uPAR domains (LU).This internally flexible protein structure of uPAR enables an allosteric regulation of the interactions with its two principal ligands: the serine protease urokinase-type plasminogen activator (uPA) and the provisional matrix protein vitronectin (Vn) (Mertens et al., 2012; Gårdsvoll et al., 2011; Madsen et al., 2007 [2-4]). The data presented here relates to the non-covalent trapping of one of these biologically relevant uPAR-conformations by a novel class of monoclonal antibodies (Zhao et al., 2015 [5]) and to the general mapping of the topographic epitope landscape on uPAR. The methods required to achieve these data include: (1) recombinant expression and purification of a uPAR-hybrid protein trapped in the desired conformation [patent; WO 2013/020898 A12013]; (2) developing monoclonal antibodies with unique specificities using this protein as antigen; (3) mapping the functional epitope on uPAR for these mAbs by surface plasmon resonance with a complete library of purified single-site uPAR mutants (Zhao et al., 2015; Gårdsvoll et al., 2006 [5,6]); and finally (4) solving the three-dimensional structures for one of these mAbs by X-ray crystallography alone and in complex with uPAR [deposited in the PDB database as 4QTH and 4QTI, respectively]. PMID:26504891

  11. 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-κB) and activator protein (AP)-1 in the nicotine-induced uPAR expression. Studies with expression vectors encoding mutated NF-κB signaling molecules and AP-1 decoy confirmed that NF-κB 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-κB, 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-κB 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-κB signals are involved in nicotine-induced uPAR.

  12. 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)

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

    2012-03-01

    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.

  13. 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)

    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

    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.

  14. 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

  15. Tissue plasminogen activator followed by antioxidant-loaded nanoparticle delivery promotes activation/mobilization of progenitor cells in infarcted rat brain.

    Science.gov (United States)

    Petro, Marianne; Jaffer, Hayder; Yang, Jun; Kabu, Shushi; Morris, Viola B; Labhasetwar, Vinod

    2016-03-01

    Inherent neuronal and circulating progenitor cells play important roles in facilitating neuronal and functional recovery post stroke. However, this endogenous repair process is rather limited, primarily due to unfavorable conditions in the infarcted brain involving reactive oxygen species (ROS)-mediated oxidative stress and inflammation following ischemia/reperfusion injury. We hypothesized that during reperfusion, effective delivery of antioxidants to ischemic brain would create an environment without such oxidative stress and inflammation, thus promoting activation and mobilization of progenitor cells in the infarcted brain. We administered recombinant human tissue-type plasminogen activator (tPA) via carotid artery at 3 h post stroke in a thromboembolic rat model, followed by sequential administration of the antioxidants catalase (CAT) and superoxide dismutase (SOD), encapsulated in biodegradable nanoparticles (nano-CAT/SOD). Brains were harvested at 48 h post stroke for immunohistochemical analysis. Ipsilateral brain slices from animals that had received tPA + nano-CAT/SOD showed a widespread distribution of glial fibrillary acidic protein-positive cells (with morphology resembling radial glia-like neural precursor cells) and nestin-positive cells (indicating the presence of immature neurons); such cells were considerably fewer in untreated animals or those treated with tPA alone. Brain sections from animals receiving tPA + nano-CAT/SOD also showed much greater numbers of SOX2- and nestin-positive progenitor cells migrating from subventricular zone of the lateral ventricle and entering the rostral migratory stream than in t-PA alone treated group or untreated control. Further, animals treated with tPA + nano-CAT/SOD showed far fewer caspase-positive cells and fewer neutrophils than did other groups, as well as an inhibition of hippocampal swelling. These results suggest that the antioxidants mitigated the inflammatory response, protected neuronal cells from undergoing apoptosis, and inhibited edema formation by protecting the blood-brain barrier from ROS-mediated reperfusion injury. A longer-term study would enable us to determine if our approach would assist progenitor cells to undergo neurogenesis and to facilitate neurological and functional recovery following stroke and reperfusion injury. PMID:26735970

  16. Three are better than one: plasminogen receptors as cancer theranostic targets.

    OpenAIRE

    Ceruti, Patrizia; Principe, Moitza; Capello, Michela; Cappello, Paola; Novelli, Francesco

    2013-01-01

    Activation of plasminogen on the cell surface initiates a cascade of protease activity with important implications for several physiological and pathological events. In particular, components of the plasminogen system participate in tumor growth, invasion and metastasis. Plasminogen receptors are in fact expressed on the cell surface of most tumors, and their expression frequently correlates with cancer diagnosis, survival and prognosis. Notably, they can trigger multiple specific immune resp...

  17. 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)

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

    2008-12-01

    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

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

    Directory of Open Access Journals (Sweden)

    Al Maamari E

    2014-09-01

    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

  19. Variation in the ovine PRKAG3 gene.

    Science.gov (United States)

    Yang, Guo; Zhou, Huitong; Wang, Ruoyu; Hickford, Jon

    2015-08-10

    The 5'AMP-activated protein kinase (AMPK) is a heterotrimeric enzyme that controls cellular energy homeostasis in response to environmental or nutritional stress. The PRKAG3 gene (PRKAG3) encodes the ?3 subunit of the AMPK. Variation in this gene has been found to be associated with meat quality traits in pigs. In this study, we used polymerase chain reaction-single stranded conformational polymorphism (PCR-SSCP) to investigate variation in exon 3 and exons 4-6 of ovine PRKAG3. In 160 New Zealand Suffolk sheep, two variant sequences (named a and b) were identified in the exon 3 region of the gene and three variant sequences (named A, B and C) were identified in the exon 4-6 region of the gene, respectively. A total of three nucleotide substitutions were revealed and these were located in intron 4, exon 4 and intron 5, respectively. The nucleotide substitution identified in the exon 4 (g.2656 C>T) could nominally lead to an amino acid substitution of tryptophan to arginine at position 230 (R230W) in ovine PRKAG3. In comparison with the PRKAG3 amino acid sequences in other species, this R230W substitution appeared to occur only in sheep. This is the first report of genetic variation in ovine PRKAG3, and the variation found in this study could be functionally important for AMPK activity, which in turn may affect meat quality traits in sheep. PMID:25967386

  20. Cytotoxicity of the Urokinase-Plasminogen Activator Inhibitor Carbamimidothioic Acid (4-Boronophenyl Methyl Ester Hydrobromide (BC-11 on Triple-Negative MDA-MB231 Breast Cancer Cells

    Directory of Open Access Journals (Sweden)

    Alessandra Longo

    2015-05-01

    Full Text Available BC-11 is an easily synthesized simple thiouronium-substituted phenylboronic acid, which has been shown to be cytotoxic on triple negative MDA-MB231 breast cancer cells by inducing a perturbation of cell cycle when administered at a concentration equal to its ED50 at 72 h (117 ?M. Exposure of cells to BC-11, either pre-absorbed with a soluble preparation of the N-terminal fragment of urokinase-plasminogen activator (uPa, or in co-treatment with two different EGFR inhibitors, indicated that: (i BC-11 acts via binding to the N-terminus of the enzyme where uPa- and EGF receptor-recognizing sites are present, thereby abrogating the growth-sustaining effect resulting from receptor binding; and (ii the co-presence of the EGFR inhibitor PD153035 potentiates BC-11’s cytotoxicity. Exposure of cells to a higher concentration of BC-11 corresponding to its ED75 at 72 h (250 ?M caused additional impairment of mitochondrial activity, the production of reactive oxygen species and promotion of apoptosis. Therefore, BC-11 treatment appears to show potential for the development of this class of compounds in the prevention and/or therapy of “aggressive” breast carcinoma.

  1. 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)

    Trelle, Morten B; Dupont, Daniel Miotto

    2014-01-01

    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.

  2. 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)

    Trelle, Morten B; Dupont, Daniel Miotto

    2014-01-01

    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.

  3. Interaction of ovine somatomedin and multiplication stimulating activity/rat insulin-like growth factor II with cultured skeletal muscle satellite cells

    International Nuclear Information System (INIS)

    The interactions of 125I-multiplication stimulating activity (MSA) and 125I-ovine somatomedin with receptors on skeletal muscle satellite cells are described. Specific binding of 125I-MSA/rIGF-II was inhibited by MSA/rIGF-II and oSm but not by insulin. Binding of 125I-oSm was inhibited by MSA/rIGF-II, oSm and insulin. In addition, 24-h pre-incubation of satellite cells with insulin increased the amount of 125I-MSA/rIGF-II bound, but insulin concentrations below 550 ?g/l had no effect on the subsequent binding of 125I-oSm. Preincubation of cultures with oSm or MSA/rIGF-II decreased the subsequent binding of 125I-oSm and 125I-MSA/rIGF-II. These preliminary experiments suggest that oSm is similar to IGF-I in its binding characteristics and that primary cultures of skeletal muscle satellite cells possess type I and type II IGF receptors. (author)

  4. Binding of human plasminogen to Borrelia burgdorferi.

    OpenAIRE

    Hu, L T; Perides, G; Noring, R; Klempner, M S

    1995-01-01

    We studied the binding of plasminogen to Borrelia burgdorferi, a spirochete which causes Lyme disease and produces no endogenous proteases which digest extracellular matrix proteins. Using 125I-labeled plasminogen, we demonstrated that B. burgdorferi bound human plasminogen and that this binding was inhibitable with unlabeled plasminogen. 125I-labeled plasminogen binding by B. burgdorferi was also inhibited by the lysine analog epsilon-aminocaproic acid. There was no significant difference in...

  5. Group B Streptococcus Hijacks the Host Plasminogen System to Promote Brain Endothelial Cell Invasion

    OpenAIRE

    Magalhães, V.; Andrade, E.; Alves, J.; Ribeiro, A.; Kim, K; M Lima; Trieu-Cuot, P.; Ferreira, P.

    2013-01-01

    Group B Streptococcus (GBS) is the leading cause of meningitis in neonates. We have previously shown that plasminogen, once recruited to the GBS cell surface and converted into plasmin by host-derived activators, leads to an enhancement of bacterial virulence. Here, we investigated whether plasmin(ogen) bound at the GBS surface contributes to blood-brain barrier penetration and invasion of the central nervous system. For that purpose, GBS strain NEM316 preincubated with or without plasminogen...

  6. Comparison of bezafibrate versus lovastatin for lowering plasma insulin, fibrinogen, and plasminogen activator inhibitor-1 concentrations in hyperlipemic heart transplant patients.

    Science.gov (United States)

    Zambrana, J L; Velasco, F; Castro, P; Concha, M; Vallés, F; Montilla, P; Jimenéz-Perepérez, J A; López-Miranda, J; Pérez-Jiménez, F

    1997-10-01

    Accelerated coronary artery disease is the most serious obstacle to long-term survival in heart transplant recipients. Hyperlipemia, hyperinsulinism, and changes in endothelial cell hemostatic function have been implicated in cardiac allograft vascular disease. Both lovastatin and bezafibrate are safe, effective, and well tolerated therapies for hyperlipidemia. Our study compares the effect of these lipid-lowering drugs in 21 patients with post-heart transplantation hyperlipidemia on different risk factors related to insulin resistance syndrome. Patients were given the same diet for 3 months, then randomized to lovastatin or bezafibrate for a period of 8 weeks, and crossed over to an additional 8 weeks of either bezafibrate or lovastatin. Baseline parameters were also compared with those of a control group of healthy subjects and after both periods of pharmacologic treatment. Transplant patients had higher insulin (35 +/- 3 vs 24 +/- 3 microIU/L), fibrinogen (298 +/- 15 vs 261 +/- 14 mg/dl), and plasminogen activator inhibitor-1 (PAI-1) (17 +/- 2 vs 11.7 +/- 2 arbitrary units/ml) plasma levels than controls. Significant decreases in insulin (-37 +/- 3%), fibrinogen (-12 +/- 4%), and PAI-1 plasma levels (-18 +/- 12%) were only observed after bezafibrate treatment. In conclusion, bezafibrate decreases plasma insulin, fibrinogen, and PAI-1 in hyperlipidemic heart transplant recipients. PMID:9381994

  7. 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)

    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

    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.

  8. 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)

    Michalski Dominik

    2012-01-01

    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.

  9. 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)

    SØrensen, Tine Thurison; Christensen, Ib J

    2015-01-01

    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.

  10. 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-?1 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

  11. A milk protein gene promoter directs the expression of human tissue plasminogen activator cDNA to the mammary gland in transgenic mice

    International Nuclear Information System (INIS)

    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

  12. 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)

    Ana Maria Abreu Velez

    2013-07-01

    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.

  13. Evidence for a pre-latent form of the serpin plasminogen activator inhibitor-1 with a detached beta-strand 1C

    DEFF Research Database (Denmark)

    Dupont, Daniel M; Blouse, Grant E

    2006-01-01

    Latency transition of plasminogen activator inhibitor-1 (PAI-1) occurs spontaneously in the absence of proteases and results in stabilization of the molecule through insertion of its reactive center loop (RCL) as a strand in beta-sheet A and detachment of beta-strand 1C (s1C) at the C-terminal hinge of the RCL. This is one of the largest structural rearrangements known for a folded protein domain without a concomitant change in covalent structure. Yet, the sequence of conformational changes during latency transition remains largely unknown. We have now mapped the epitope for the monoclonal antibody H4B3 to the cleft revealed upon s1C detachment and shown that H4B3 inactivates recombinant PAI-1 in a time-dependent manner. With fluorescence spectroscopy, we show that insertion of the RCL is accelerated in the presence of H4B3, demonstrating that the loss of activity is the result of latency transition. Considering that the epitope for H4B3 appears to be occluded by s1C in active PAI-1, this finding suggests theexistence of a pre-latent conformation on the path from active to latent PAI-1 characterized by at least partial detachment of s1C. Functional characterization of mutated PAI-1 variants suggests that a salt-bridge between Arg273 and Asp224 may stabilize the pre-latent conformation. The binding of H4B3 and of a peptide targeting the cleft revealed upon s1C detachment was hindered by the glycans attached to Asn267. Conclusively, we have provided evidence for the existence of an equilibrium between active PAI-1 and a pre-latent form, characterized by reversible detachment of s1C and formation of a glycan-shielded cleft in the molecule.

  14. Hypoxia inducible factor-1 mediates upregulation of urokinase-type plasminogen activator receptor gene transcription during hypoxia in cervical cancer cells.

    Science.gov (United States)

    Nishi, Hirotaka; Sasaki, Toru; Nagamitsu, Yuzo; Terauchi, Fumitoshi; Nagai, Takeshi; Nagao, Toshitaka; Isaka, Keiichi

    2016-02-01

    Hypoxia occurs during development of cervical cancer and is considered to correlate with its invasion. Hypoxia mediates tumor cells to have more invasive property in a variety of cancers. Urokinase plasminogen activator receptor (uPAR) which mediates invasion is considered to be induced by hypoxia. We sought to determine the regulators of uPAR expression during hypoxia in cervical cancer. We showed that cervical cancer cell lines, CaSki and CA, were more invasive under hypoxic condition (1% O2) than under normoxic condition (20% O2) by invasion assays. Using western blot analysis, hypoxia enhanced the endogenous hypoxia-inducible factor (HIF)-1? and uPAR protein expression. uPAR mRNA level was also upregulated by hypoxia using real-time RT-PCR. Overexpression of HIF-1? which is induced by hypoxia activated the transcriptional activity of the uPAR promoter by luciferase assays. HIF-1 protein bound the putative HIF-1 response element on the uPAR promoter using electrophoretic mobility shift analysis, and additional luciferase assays show that this is essential for uPAR transactivation by HIF-1. HIF-1 overexpression enhanced the endogenous uPAR expression and introduction of siRNA for HIF-1? diminishes uPAR expression during hypoxia. These results indicate the upregulation of uPAR by hypoxia in cervical cancer cells is mediated through HIF-1. In cervical cancer tissues, we also demonstrated that uPAR protein expression was detected in cervical cancer but not in normal cervix or cervical intraepithelial neoplasia (CIN) by immunohistopathological staining. Our results provide evidence that regulation of uPAR expression by HIF-1 represents a mechanism for cervical cancer invasion during hypoxia. PMID:26718775

  15. Preparation of excipient-free recombinant human tissue-type plasminogen activator by lyophilization from ammonium bicarbonate solution: an investigation of the two-stage sublimation phenomenon.

    Science.gov (United States)

    Overcashier, D E; Brooks, D A; Costantino, H R; Hsu, C C

    1997-04-01

    Dry, excipient-free recombinant human tissue-type plasminogen activator (tPA) powder was prepared by lyophilization from ammonium bicarbonate solution. Ammonium bicarbonate sublimes into ammonia, water, and carbon dioxide upon lyophilization, without causing measurable harm to the protein. There were approximately 4 mol of residual ammonium ion per mole of lyophilized tPA. Under certain lyophilization conditions, a large pressure increase in the lyophilizer chamber occurred, presenting a pressure control problem. Microscopy and sublimation rate measurements on the frozen matrix revealed that ice sublimation occurred first, followed by the sublimation of ammonium bicarbonate. Analysis of the sectioned frozen matrix indicated that the bicarbonate salt was evenly distributed throughout the vial, suggesting that the delay of ammonium bicarbonate sublimation was not due to hindrance by ice. In the two-stage process, ice sublimation proceeded according to zero-order kinetics, whereas ammonium bicarbonate sublimation followed a grain-burning (2/ 3-order) model and was governed by a higher activation enthalpy. In most cases, the sublimation rate of ammonium bicarbonate in the presence of tPA was lower than that in the absence of the protein. Sublimation activation enthalpy for ammonium bicarbonate in the presence of tPA was 26.1 +/- 3.8 kcal/mol, which was approximately 10 kcal/mol greater than that for the tPA-free system. Consistent with a prediction from our kinetic modeling, a 6-h extension of primary drying enabled us to conduct lyophilization while maintaining pressure control. PMID:9109048

  16. Effect of Temperature Downshift on the Transcriptomic Responses of Chinese Hamster Ovary Cells Using Recombinant Human Tissue Plasminogen Activator Production Culture

    Science.gov (United States)

    Bedoya-López, Andrea; Estrada, Karel; Sanchez-Flores, Alejandro; Ramírez, Octavio T.; Altamirano, Claudia; Segovia, Lorenzo; Miranda-Ríos, Juan; Trujillo-Roldán, Mauricio A.; Valdez-Cruz, Norma A.

    2016-01-01

    Recombinant proteins are widely used as biopharmaceuticals, but their production by mammalian cell culture is expensive. Hence, improvement of bioprocess productivity is greatly needed. A temperature downshift (TDS) from 37°C to 28–34°C is an effective strategy to expand the productive life period of cells and increase their productivity (qp). Here, TDS in Chinese hamster ovary (CHO) cell cultures, initially grown at 37°C and switched to 30°C during the exponential growth phase, resulted in a 1.6-fold increase in the qp of recombinant human tissue plasminogen activator (rh-tPA). The transcriptomic response using next-generation sequencing (NGS) was assessed to characterize the cellular behavior associated with TDS. A total of 416 (q > 0.8) and 3,472 (q > 0.9) differentially expressed transcripts, with more than a 1.6-fold change at 24 and 48 h post TDS, respectively, were observed in cultures with TDS compared to those at constant 37°C. In agreement with the extended cell survival resulting from TDS, transcripts related to cell growth arrest that controlled cell proliferation without the activation of the DNA damage response, were differentially expressed. Most upregulated genes were related to energy metabolism in mitochondria, mitochondrial biogenesis, central metabolism, and avoidance of apoptotic cell death. The gene coding for rh-tPA was not differentially expressed, but fluctuations were detected in the transcripts encoding proteins involved in the secretory machinery, particularly in glycosylation. Through NGS the dynamic processes caused by TDS were assessed in this biological system. PMID:26991106

  17. Irradiation-Induced Regulation of Plasminogen Activator Inhibitor Type-1 and Vascular Endothelial Growth Factor in Six Human Squamous Cell Carcinoma Lines of the Head and Neck

    International Nuclear Information System (INIS)

    Purpose: It has been shown that plasminogen activator inhibitor type-1 (PAI-1) and vascular endothelial growth factor (VEGF) are involved in neo-angiogenesis. The aim of this study was to investigate the irradiation-induced regulation of PAI-1 and VEGF in squamous cell carcinomas of the head and neck (SCCHN) cell lines of varying radiation sensitivity. Methods and Materials: Six cell lines derived from SCCHN were investigated in vitro. The colorimetric AlamarBlue assay was used to detect metabolic activity of cell lines during irradiation as a surrogate marker for radiation sensitivity. PAI-1 and VEGF secretion levels were measured by enzyme-linked immunosorbent assay 24, 48, and 72 h after irradiation with 0, 2, 6, and 10 Gy. The direct radioprotective effect of exogenous PAI-1 was measured using the clonogenic assay. For regulation studies, transforming growth factor-?1 (TGF-?1), hypoxia-inducible factor-1? (HIF-1?), hypoxia-inducible factor-2? (HIF-2?), or both HIF-1? and HIF-2? were downregulated using siRNA. Results: Although baseline levels varied greatly, irradiation led to a comparable dose-dependent increase in PAI-1 and VEGF secretion in all six cell lines. Addition of exogenous stable PAI-1 to the low PAI-1-expressing cell lines, XF354 and FaDu, did not lead to a radioprotective effect. Downregulation of TGF-?1 significantly decreased VEGF secretion in radiation-sensitive XF354 cells, and downregulation of HIF-1? and HIF-2? reduced PAI-1 and VEGF secretion in radiation-resistant SAS cells. Conclusions: Irradiation dose-dependently increased PAI-1 and VEGF secretion in all SCCHN cell lines tested regardless of their basal levels and radiation sensitivity. In addition, TGF-?1 and HIF-1? could be partly responsible for VEGF and PAI-1 upregulation after irradiation.

  18. 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)

    Shafaat A. Rabbani

    2010-10-01

    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.

  19. 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)

    Trelle, Morten Beck; Hirschberg, Daniel

    2012-01-01

    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.

  20. Protein S blocks the extrinsic apoptotic cascade in tissue plasminogen activator/N-methyl D-aspartate-treated neurons via Tyro3-Akt-FKHRL1 signaling pathway

    Directory of Open Access Journals (Sweden)

    Freeman Robert S

    2011-02-01

    Full Text Available Abstract Background Thrombolytic therapy with tissue plasminogen activator (tPA benefits patients with acute ischemic stroke. However, tPA increases the risk for intracerebral bleeding and enhances post-ischemic neuronal injury if administered 3-4 hours after stroke. Therefore, combination therapies with tPA and neuroprotective agents have been considered to increase tPA's therapeutic window and reduce toxicity. The anticoagulant factor protein S (PS protects neurons from hypoxic/ischemic injury. PS also inhibits N-methyl-D-aspartate (NMDA excitotoxicity by phosphorylating Bad and Mdm2 which blocks the downstream steps in the intrinsic apoptotic cascade. To test whether PS can protect neurons from tPA toxicity we studied its effects on tPA/NMDA combined injury which in contrast to NMDA alone kills neurons by activating the extrinsic apoptotic pathway. Neither Bad nor Mdm2 which are PS's targets and control the intrinsic apoptotic pathway can influence the extrinsic cascade. Thus, based on published data one cannot predict whether PS can protect neurons from tPA/NMDA injury by blocking the extrinsic pathway. Neurons express all three TAM (Tyro3, Axl, Mer receptors that can potentially interact with PS. Therefore, we studied whether PS can activate TAM receptors during a tPA/NMDA insult. Results We show that PS protects neurons from tPA/NMDA-induced apoptosis by suppressing Fas-ligand (FasL production and FasL-dependent caspase-8 activation within the extrinsic apoptotic pathway. By transducing neurons with adenoviral vectors expressing the kinase-deficient Akt mutant AktK179A and a triple FKHRL1 Akt phosphorylation site mutant (FKHRL1-TM, we show that Akt activation and Akt-mediated phosphorylation of FKHRL1, a member of the Forkhead family of transcription factors, are critical for FasL down-regulation and caspase-8 inhibition. Using cultured neurons from Tyro3, Axl and Mer mutants, we show that Tyro3, but not Axl and Mer, mediates phosphorylation of FHKRL1 that is required for PS-mediated neuronal protection after tPA/NMDA-induced injury. Conclusions PS blocks the extrinsic apoptotic cascade through a novel mechanism mediated by Tyro3-dependent FKHRL1 phosphorylation which inhibits FasL-dependent caspase-8 activation and can control tPA-induced neurotoxicity associated with pathologic activation of NMDA receptors. The present findings should encourage future studies in animal stroke models to determine whether PS can increase the therapeutic window of tPA by reducing its post-ischemic neuronal toxicity.

  1. Elevated urinary levels of urokinase-type plasminogen activator receptor (uPAR) in pancreatic ductal adenocarcinoma identify a clinically high-risk group

    International Nuclear Information System (INIS)

    The urokinase plasminogen activator receptor is highly expressed and its gene is amplified in about 50% of pancreatic ductal adenocarcinomas; this last feature is associated with worse prognosis. It is unknown whether the level of its soluble form (suPAR) in urine may be a diagnostic-prognostic marker in these patients. The urinary level of suPAR was measured in 146 patients, 94 pancreatic ductal adenocarcinoma and 52 chronic pancreatitis. Urine from 104 healthy subjects with similar age and gender distribution served as controls. suPAR levels were normalized with creatinine levels (suPAR/creatinine, ng/mg) to remove urine dilution effect. Urinary suPAR/creatinine values of pancreatic ductal adenocarcinoma patients were significantly higher (median 9.8; 25th-75th percentiles 5.3-20.7) than those of either healthy donors (median 0; 0-0.5) or chronic pancreatitis patients (median 2.7; 0.9-4.7). The distribution of values among cancer patients was widespread and asymmetric, 53% subjects having values beyond the 95th percentile of healthy donors. The values of suPAR/creatinine did not correlate with tumour stage, Ca19-9 or CEA levels. Higher values correlated with poor prognosis among non-resected patients at univariate analysis; multivariate Cox regression identified high urinary suPAR/creatinine as an independent predictor of poor survival among all cancer patients (odds ratio 2.10, p = 0.0023), together with tumour stage (stage III odds ratio 2.65, p = 0.0017; stage IV odds ratio 4.61, p < 0.0001) and female gender (odds ratio 1.85, p = 0.01). A high urinary suPAR/creatinine ratio represents a useful marker for the identification of a subset of patients with poorer outcome

  2. Association of the 4?g/5?g polymorphism of plasminogen activator inhibitor-1 gene with sudden sensorineural hearing loss. A case control study

    Directory of Open Access Journals (Sweden)

    Cho Seong

    2012-06-01

    Full Text Available Abstract Background The 5 G/5 G genotype of PAI-1 polymorphism is linked to decreased plasminogen activator inhibitor-1 (PAI-1 levels and it has been suggested that lower PAI-1 levels may provide protective effects on inflammation, local microcirculatory disturbance, and fibrotic changes, which are likely associated with development of sudden sensorineural hearing loss (SSNHL. Methods The association of the 4?G/5?G PAI-1 polymorphism with the development and clinical outcome of SSNHL is evaluated via a case control study. 103 patients with SSNHL and 113 age and sex-matched controls were enrolled at University of Ferrara, Italy and hearing loss outcome was measured at least 3?months after the onset of hearing loss. DNA was isolated from peripheral blood using the QIAamp kit and the 4?G/5?G polymorphism in the ?675 promoter region was genotyped with an allele-specific PCR. Genotype distribution was tested in patients and compared to controls by chi-square and odd-ratio analysis. The codominant and recessive models were used for the multiple logistic regression analyses of the PAI-1 gene allele. Results In this population, 5?G/5?G genotype had a two-time lower frequency in SSNHL patients compared to healthy controls (15.5% vs 30.1% and was associated with decreased odds compared to 4?G/5?G genotype (OR 0.37, 95% CI 0.19-0.75, p?=?0.005. In addition, the patients with 5?G/5?G genotype showed a trend of more than 2 times higher ratio of hearing recovery (> 20?dB after systemic corticosteroid treatment compared to 4?G/5?G genotype (OR 2.3, 95% CI 0.32 - 16.83, p?=?0.39, suggesting a better clinical outcome. Conclusions The 5?G/5?G genotype of PAI-1 may be associated with a reduced risk of SSNHL in the Italian population.

  3. Results of phase III clinical trial of 99mTc-labelled recombinant tissue plasminogen activator in the detection of deep venous thrombosis

    International Nuclear Information System (INIS)

    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 79 patients suspected of DVT. A kit formulation has been devised in which 99mTc is labelled to rt- PA where the plasminogen binding site has been permanently inhibited but the fibrin binding site remained active. Kit preparation 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. Mean thrombus age was 5.4 days. 58% patients were receiving intravenous heparin. Mean time interval between contrast venography and scanning was 20 hours. For the purpose of analysis, the leg was divided into proximal and distal segments for both the scintigraphic study and the contrast venography. Of the 14 thrombosed proximal segments, 13 had positive scans; in the 53 non-thrombosed proximal segments, 49 had negative scans. Thus in proximal vein thrombosis, scanning had a sensitivity of 93% and a specificity of 92%. Of the 36 thrombosed calf vein segments, 31 had positive scans; in the 30 non-thrombosed calf segments, 28 had negative scans. Thus in calf vein thrombosis, scanning has a sensitivity of 86% and a specificity of 93%. Scintigraphic scanning with this new radiopharmaceutical permits accurate detection of thrombus in both proximal and calf veins. The technique detects both fresh and aged thrombi and is unaffected by heparin administration. Further work in different patient groups will need to be performed to define its clinical usefulness

  4. Plasma levels of soluble urokinase-type plasminogen activator receptor (suPAR and early mortality risk among patients enrolling for antiretroviral treatment in South Africa

    Directory of Open Access Journals (Sweden)

    Bangani Nonzwakazi

    2007-05-01

    Full Text Available Abstract Background Serum concentrations of soluble urokinase-type plasminogen activator receptor (suPAR have a strong independent association with HIV-1-related mortality. The practical utility of plasma suPAR in assessing short-term all-cause mortality risk was evaluated in patients with advanced immunodeficiency enrolling in an antiretroviral treatment (ART programme in South Africa. Methods An enzyme-linked immunosorbent assay (ELISA was used to measure plasma concentrations of suPAR in patients at the time of enrolment to the ART programme. The association between plasma suPAR concentrations, baseline patient characteristics and cohort outcomes after 4 months of ART were determined. Results Patients (n = 293, 70% female had a median age of 33 years and were followed up for a median of 5 months from enrolment. The median CD4 cell count was 47 cells/?l (IQR = 22–72 and 38% of patients had WHO stage 4 disease. 218 (74% patients remained alive after 4 months of ART; 39 (13% died and 36 (12% were lost to the programme for other reasons. Patients who died had significantly higher plasma suPAR concentrations compared to those who either survived (P 10 suPAR concentrations were significantly associated with lower CD4 cell counts, WHO clinical stage 4 disease and male sex. In multivariate analysis to identify factors associated with death, log10 suPAR concentration was the most strongly associated variable (P Conclusion Plasma suPAR concentration was the strongest independent predictor of short-term mortality risk among patients with advanced immunodeficiency enrolling in this ART programme. However, lack of a discriminatory threshold did not permit this marker to be used to triage patients according to short-term mortality risk.

  5. Soluble urokinase-type plasminogen activator receptor (suPAR) as an independent factor predicting worse prognosis and extra-bone marrow involvement in multiple myeloma patients.

    Science.gov (United States)

    Rigolin, Gian Matteo; Tieghi, Alessia; Ciccone, Maria; Bragotti, Letizia Zenone; Cavazzini, Francesco; Della Porta, Matteo; Castagnari, Barbara; Carroccia, Rosanna; Guerra, Giovanni; Cuneo, Antonio; Castoldi, Gianluigi

    2003-03-01

    The urokinase-type plasminogen activator (uPA) system, which consists of a proteinase (uPA), a receptor (uPAR or CD87) and inhibitors, is involved in proteolysis, cell migration, tissue remodelling, angiogenesis and cell adhesion. Recent findings suggest that malignant plasma cells express uPA and uPAR. The expression of these factors could represent a process by which myeloma plasma cells interact with the bone marrow (BM) environment and influence important biological events such as bone matrix degradation, plasma cell invasion and homing and, possibly, clinical evolution. We evaluated uPAR (CD87) and its soluble form (suPAR) in 49 multiple myeloma (MM) patients and correlated their expression and levels with clinico-biological characteristics of the disease. Flow cytometric analysis demonstrated that CD87 was expressed in all MM patients. High CD87 expression was associated with higher intensity of expression of CD56 (P = 0.038), CD38 (P = 0.058) and CD138 (P = 0.054) and CD45bright positivity (P = 0.014). suPAR levels correlated positively with soluble serum CD138 (P = 0.001), creatinine (P = 0.001), beta2-microglobulin (P < 0.001), disease stage (P = 0.017) and extra-BM involvement (P = 0.002). In the 46 evaluable patients, multivariate analysis showed that high levels of suPAR (P = 0.0214) and disease stage (P = 0.0064) were predictive of extra-BM involvement. In multivariate Cox analysis, 13q deletion (P = 0.0278), high soluble serum CD138 (P = 0.0201) and high suPAR (P = 0.0229) were the only parameters that independently affected survival. We conclude that CD87 is expressed on myeloma plasma cells and that suPAR, which predicts extra-BM involvement and poor prognosis, possibly represents a molecule with a relevant role in the biology of MM. PMID:12648064

  6. Does plasminogen activator inhibitor-1 (PAI-1) control trophoblast invasion? : A study of fetal and maternal tissue in intrauterine, tubal and molar pregnancies

    DEFF Research Database (Denmark)

    Floridon, C; Nielsen, O

    2000-01-01

    Urokinase plasminogen activator, its receptor and the inhibitor PAI-1 are believed to control proteolysis and remodelling of maternal tissue during trophoblast invasion. This system appears to be strictly regulated in normal intrauterine pregnancies whereas tubal and molar pregnancies seem to be characterized by an uncontrolled excessive placental invasion. This study evaluates subcellular PAI-1 by immunohistochemistry in the villous placenta, in the basal plate and placental bed, and in the decidual compartments of normal, tubal and molar pregnancies. PAI-1 was present in villous syncytiotrophoblasts and co-localized focally with fibrin-type fibrinoid on the surface of the chorionic villi. Basal plate and placental bed extravillous interstitial trophoblasts, as well as vascular trophoblasts, were also PAI-1 positive. In the decidua parietalis, PAI-1 was observed in the cytoplasm of the non-invaded decidual cells. In the decidua basalis comprising the basal plate, PAI-1 was seen to be membrane-associated or confined to the extracellular matrix (ECM) facing the invasive front of anchoring villi. The ECM of decidua capsularis and chorion laeve displayed the most pronounced PAI-1 expression towards the maternal interface. In contrast, the majority of placental bed decidual cells adjacent to the interstitial and vascular trophoblasts were PAI-1 negative. Only a few stromal cells distant from the implantation site were PAI-1 positive in the tubal pregnancies and decidualization was not present. Likewise, excessive decidual necrosis and fibrinoid deposition devoid of PAI-1 was a common finding in complete molar pregnancies. These results suggest that PAI-1 defines specific extravillous invasive trophoblasts within the maternal decidua. Moreover, maternal cellular lack of PAI-1 in tubal pregnancies and excessive decidual necrosis in molar pregnancies indicate an uncontrolled placental invasion. The present data indicate that trophoblast invasion is primarily regulated by signals from decidual cells.

  7. Stereotactic fibrinolysis of spontaneous intracerebral hematoma using infusion of recombinant tissue plasminogen activator / Fibrinólise com infusão de rtPA e drenagem estereotáxica de hematoma intracerebral espontâneo profundo

    Scientific Electronic Library Online (English)

    José Augusto, Nasser; Asdrubal, Falavigna; Márcio, Bezerra; Victor, Martinez; Gabriel, Freitas; Armando, Alaminos; Antônio, Bonatelli; Fernando, Ferraz.

    2002-06-01

    Full Text Available OBJETIVO: Estudo prospectivo em 10 pacientes com infusão de trombolítico (rtPA) dentro do hematoma cerebral profundo supratentorial e drenagem estereotáxica. MÉTODO: Entre 1999 e 2000 10 pacientes com hematomas de profundidade foram selecionados para infusão de rtPA e drenagem do coágulo espontânea. [...] RESULTADO: Todos os casos obtiveram 80% de redução do volume do hematoma medidos por TC no terceiro dia. A pressão intracraniana estava normalizada no terceiro dia. Não houve complicações locais ou sistêmicas relacionadas com o uso deste trombolítico. Os resultados comparados foram mostrados pela Escala de Prognóstico de Glasgow com 6 pacientes em GrauV, 3 pacientes em Grau IV e 1 paciente em Grau III após três meses. CONCLUSÃO: Tratamento precoce e drenagem com técnica neurocirúrgica minimamente invasiva pode fazer estes pacientes terem uma recuperação da consciência mais rápida e assim serem reabilitados mais precocemente evitando complicações secundárias. Abstract in english PURPOSE: The authors present a prospective study on 10 patients with stereotactic infusion of tissue plasminogen activator (rtPA) intraparenchimal hemorrhage. METHODS: Between 1999 and 2000, 10 patients with deep seated hematomas in the basal ganglia were selected for stereotactic infusion of rtPA a [...] nd spontaneous clot drainage. RESULTS: All cases had about 80% reduction of the hematoma volume in the CT scan at the third day. The intracranial pressure was normalized by the third day too. There were no local or systemic complications with the use of this trombolitic. The results were shown by the Glasgow Outcome Scale with six patients in V, three in IV and one in III after 3 months. CONCLUSION: Early treatment and drainage with minimally invasive neurosurgery , can make these patients with deep-seated hematomas recover the consciousness and they can be rehabilitated earlier avoiding secondary complications.

  8. Urokinase-type plasminogen activator receptor (uPAR) expression is associated with T-stage and survival in urothelial carcinoma of the bladder

    DEFF Research Database (Denmark)

    Dohn, Line Hammer; Illemann, Martin

    2015-01-01

    OBJECTIVES: To evaluate the expression-and localization pattern of the urokinase-type plasminogen activator receptor (uPAR), focusing on its clinical implications in patients with urothelial neoplasia of the bladder treated with radical cystectomy. uPAR is a central molecule in tissue remodeling during cancer invasion and metastasis and is an established prognostic marker in cancer. The expression and localization of uPAR and its prognostic significance is only limitedly investigated in urothelial bladder neoplasia. MATERIALS AND METHODS: The expression-and localization pattern of uPAR was investigated in formalin-fixed paraffin-embedded tumor tissue from 149 patients treated with radical cystectomy between 1988 and 2005. uPAR expression was determined by immunohistochemistry and scored as either negative or positive. Separate values were obtained for cancer cells, macrophages, and myofibroblasts at the invasive front and tumor core, respectively. Statistical analyses were performed to evaluate the association of uPAR localization and score with clinicopathologic covariates and survival. RESULTS: uPAR positivity was seen in 122/137 (89%) and 118/149 (74%) of the neoplasias at the invasive front and tumor core, respectively. uPAR was primarily expressed by myofibroblasts and macrophages in the surrounding stroma as well as some cancer cells. A significant association between uPAR positivity and T-stage as well as grade was found for all 3 cell types in tumor core (P?0.04 for all comparisons). In univariate analysis, the uPAR positive group had a shorter survival than the uPAR negative group (hazard ratio = 2.39; 95% CI: 1.15-5.01; P = 0.020). CONCLUSIONS: The expression of uPAR is a possible prognostic marker that could be useful in identification of patients with aggressive, highly invasive tumors that could benefit from additional chemotherapy or more intensive follow-up after cystectomy.

  9. Soluble Urokinase Plasminogen Activator Receptor Level Is an Independent Predictor of the Presence and Severity of Coronary Artery Disease and of Future Adverse Events

    DEFF Research Database (Denmark)

    Eapen, Danny J; Manocha, Pankaj

    2014-01-01

    INTRODUCTION: Soluble urokinase plasminogen activator receptor (suPAR) is an emerging inflammatory and immune biomarker. Whether suPAR level predicts the presence and the severity of coronary artery disease (CAD), and of incident death and myocardial infarction (MI) in subjects with suspected CAD, is unknown. METHODS AND RESULTS: We measured plasma suPAR levels in 3367 subjects (67% with CAD) recruited in the Emory Cardiovascular Biobank and followed them for adverse cardiovascular (CV) outcomes of death and MI over a mean 2.1±1.1 years. Presence of angiographic CAD (?50% stenosis in ?1 coronary artery) and its severity were quantitated using the Gensini score. Cox's proportional hazard survival and discrimination analyses were performed with models adjusted for established CV risk factors and C-reactive protein levels. Elevated suPAR levels were independently associated with the presence of CAD (P<0.0001) and its severity (P<0.0001). A plasma suPAR level ?3.5 ng/mL (cutoff by Youden's index) predicted future risk of MI (hazard ratio [HR]=3.2; P<0.0001), cardiac death (HR=2.62; P<0.0001), and the combined endpoint of death and MI (HR=1.9; P<0.0001), even after adjustment of covariates. The C-statistic for a model based on traditional risk factors was improved from 0.72 to 0.74 (P=0.008) with the addition of suPAR. CONCLUSION: Elevated levels of plasma suPAR are associated with the presence and severity of CAD and are independent predictors of death and MI in patients with suspected or known CAD.

  10. Reduced uptake of plasminogen activators during formation of whole blood thrombi by a bovine polypeptide of uterine origin: in vitro study using Chandler's loop method

    International Nuclear Information System (INIS)

    The authors have previously isolated and purified by high performance liquid chromatography low molecular weight polypeptide of uterine origin (LMW-UDF) which promotes metastasis in tumor-bearing mice. In order to determine what effects this polypeptide could have on the coagulation systems they generated whole blood thrombi in vitro in the presence (1 ?g/ml - 100 ?g/ml) or absence of test protein. Using trace amount of radiolabeled plasminogen activators, 125I-Urokinase and 125I-pro-Urokinase (single chain urokinase) inhibition of uptake to the thrombi was observed vs control groups. Also, specific radioactivity of the thrombi pre-incubated with LMW-UDF was statistically significantly lower than control thrombi (p 125I-fibrinogen revealed no net increase or decrease of the uptake into the thrombi when LMW-UDF was used. Gross morphological differences were noted in the group with 1 ?g/ml LMW-UDF, where weight was significantly greater (243.56 mg +/- SD 50.44) and size was significantly larger (3.5 cm +/- SD 0.6) when compared to the control groups (61.45 mg +/- SD 10.26 and 0.8 cm +/- SD 0.3), respectively. It appears that LMW-UDF has an influence on the physical formation of whole blood thrombi and suggests that this effect is mediated possibly by the uptake of Urokinase and pro-Urokinase. The actual mechanism of the action is unknown but may be caused by altered fibrin cross-linking or platelet aggregation

  11. Prevention of delayed ischaemic deficits after aneurysmal subarachnoid haemorrhage by intrathecal bolus injection of tissue plasminogen activator (rTPA). A prospective study.

    Science.gov (United States)

    Seifert, V; Stolke, D; Zimmermann, M; Feldges, A

    1994-01-01

    Among a series of 224 patients with aneurysmal subarachnoid haemorrhage (SAH) admitted over a period of three years, 52 patients were prospectively treated with intrathecal tissue plasminogen activator (rTPA). All of these patients were admitted and operated on within 72 h after SAH. SAH was confirmed by CT scan and the volume of blood accumulated in the basal cisterns was graded according to Fisher's scale. All patients had a SAH according to Fisher's grade III, as a prerequisite for inclusion into the study. In 21 patients additional intraventricular bleeding was detectable on CT scan. The diagnosis of a single intracerebral aneurysm as the bleeding source was established by pan-angiography, which also excluded additional cerebro-vascular malformations. The control group consisted of 68 patients, which were also treated within 72 h after SAH. Age and sex distribution as well as the clinical patterns were comparable to the rTPA group. In all patients the aneurysm was clipped using standard microsurgical techniques. After the aneurysm had been excluded from the parent vessel, 10 mg of rTPA, dissolved in 10 ml of its solution fluid, were slowly instilled into the basal cisterns in the treatment group. In patients with additional severe intraventricular bleeding, 5-10 mg of rTPA were injected into the ventricles via an intraventricular catheter at the end of the operation. Apart from the intrathecal application of the thrombolytic substance, the surgical protocol was identical in the patients of the control group. During the postoperative period, the patients in both groups were examined neurologically and by transcranial Doppler on a daily basis.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7847131

  12. Results of phase III clinical trial of {sup 99m}Tc-labelled recombinant tissue plasminogen activator in the detection of deep venous thrombosis

    Energy Technology Data Exchange (ETDEWEB)

    Butler, S.P.; Boyd, S.J.; Parkes, S.L.; Quinn, R.J. [St George Hospital, Kogarah, NSW (Australia)

    1998-03-01

    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 79 patients suspected of DVT. A kit formulation has been devised in which {sup 99}mTc is labelled to rt- PA where the plasminogen binding site has been permanently inhibited but the fibrin binding site remained active. Kit preparation 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. Mean thrombus age was 5.4 days. 58% patients were receiving intravenous heparin. Mean time interval between contrast venography and scanning was 20 hours. For the purpose of analysis, the leg was divided into proximal and distal segments for both the scintigraphic study and the contrast venography. Of the 14 thrombosed proximal segments, 13 had positive scans; in the 53 non-thrombosed proximal segments, 49 had negative scans. Thus in proximal vein thrombosis, scanning had a sensitivity of 93% and a specificity of 92%. Of the 36 thrombosed calf vein segments, 31 had positive scans; in the 30 non-thrombosed calf segments, 28 had negative scans. Thus in calf vein thrombosis, scanning has a sensitivity of 86% and a specificity of 93%. Scintigraphic scanning with this new radiopharmaceutical permits accurate detection of thrombus in both proximal and calf veins. The technique detects both fresh and aged thrombi and is unaffected by heparin administration. Further work in different patient groups will need to be performed to define its clinical usefulness.

  13. Analgesic drug delivery via recombinant tissue plasminogen activator and microRNA-183-triggered opening of the blood-nerve barrier.

    Science.gov (United States)

    Yang, Shaobing; Krug, Susanne M; Heitmann, Johanna; Hu, Liu; Reinhold, Ann Kristin; Sauer, Solange; Bosten, Judith; Sommer, Claudia; Fromm, Michael; Brack, Alexander; Rittner, Heike L

    2016-03-01

    The peripheral nerve contains three barriers which include the blood-nerve barrier consisting of endoneurial vessels and the perineurium as well as autotypic junctions in Schwann cells. The perineurium prevents diffusion of perineurally injected drugs that can be used for selective regional pain control. It is composed of a basal membrane and layers of perineurial cells sealed by tight junction proteins like claudin-1. Claudin-1 expression and barrier function are regulated via low-density lipoprotein receptor-related protein (LRP-1). Perisciatic application of recombinant tissue plasminogen activator (rtPA) or the catalytically inactive rtPAi - both agonists of LRP-1 - reduced claudin-1 mRNA and protein expression in the rat nerve. This facilitated an increase of nociceptive thresholds after local application of hydrophilic opioids or the voltage gated sodium channel blocker (NaV1.7) ProToxin-II without apparent nerve toxicity. RtPA-induced barrier opening was mediated by LRP-1 and intracellularly by Erk phosphorylation. In silico, microRNA (miR)-rno-29b-2-5p and rno-miR-183-5p were identified as potential regulators of claudin-1 transcription in the rat. RtPA application increased miR-183-5p in the sciatic nerve. MiR-183-5p mimics functionally opened the perineurium and downregulated claudin-1 expression in vivo. In vitro, hsa-miR-183-3p mimics reduced claudin-1 expression in human HT-29/B6 cells. Overall, rtPA regulates perineurial barrier tightness via LRP-1, Erk phosphorylation and miR-183-5p/3p. This mechanism might serve as a new principle to facilitate drug delivery to peripheral nerves in humans. PMID:26735170

  14. Rescue localized intra-arterial thrombolysis for hyperacute MCA ischemic stroke patients after early non-responsive intravenous tissue plasminogen activator therapy

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Dong Joon; Kim, Dong Ik [Yonsei University College of Medicine, Department of Diagnostic Radiology, Seoul (Korea); Kim, Seo Hyun [Yonsei University Wonju College of Medicine, Department of Neurology, Wonju (Korea); Lee, Kyung Yeol [Yong Dong Severance Hospital, Department of Neurology, Seoul (Korea); Heo, Ji Hoe; Han, Sang Won [Yonsei University College of Medicine, Department of Neurology, Seoul (Korea)

    2005-08-01

    The outcome of patients who show no early response to intravenous (i.v.) tissue plasminogen activator (tPA) therapy is poor. The objective of this study was to evaluate the feasibility of rescue localized intra-arterial thrombolysis (LIT) therapy for acute ischemic stroke patients after an early non-responsive i.v. tPA therapy. Patients with proximal MCA occlusions who were treated by LIT (n=10) after failure of early response [no improvement or improvement of National Institute of Health Stroke Scale (NIHSS) scores of {<=}3] to i.v. tPA therapy (0.9 mg/kg - 10% bolus and 90% i.v. infusion over 60 min) were selected. The recanalization rates, incidence of post-thrombolysis hemorrhage and clinical outcomes [baseline and discharge NIHSS scores, mortality, 3 months Barthel index (BI) and modified Rankin score (mRS)] were evaluated. Rescue LIT therapy was performed on ten MCA occlusion patients (male:female=3:7, mean age 71 years). The mean time between the initiation of i.v. tPA therapy and the initiation of intra-arterial urokinase (i.a. UK) was 117{+-}25.0 min [time to i.v. tPA 137{+-}32 min; time to digital subtraction angiography (DSA) 221{+-}42 min; time to i.a. UK 260{+-}46 min]. The baseline NIHSS scores showed significant improvement at discharge (median from 18 to 6). Symptomatic hemorrhage and, consequent, mortality were noted in 2/10 (20%) patients. Three months good outcome was noted in 4/10 (40%, mRS 0-2) and 3/10 (30%, BI {>=}95). In conclusion, rescue LIT therapy can be considered as a treatment option for patients not showing early response to full dose i.v. tPA therapy. Larger scale studies for further validation of this protocol may be necessary. (orig.)

  15. Rescue localized intra-arterial thrombolysis for hyperacute MCA ischemic stroke patients after early non-responsive intravenous tissue plasminogen activator therapy

    International Nuclear Information System (INIS)

    The outcome of patients who show no early response to intravenous (i.v.) tissue plasminogen activator (tPA) therapy is poor. The objective of this study was to evaluate the feasibility of rescue localized intra-arterial thrombolysis (LIT) therapy for acute ischemic stroke patients after an early non-responsive i.v. tPA therapy. Patients with proximal MCA occlusions who were treated by LIT (n=10) after failure of early response [no improvement or improvement of National Institute of Health Stroke Scale (NIHSS) scores of ?3] to i.v. tPA therapy (0.9 mg/kg - 10% bolus and 90% i.v. infusion over 60 min) were selected. The recanalization rates, incidence of post-thrombolysis hemorrhage and clinical outcomes [baseline and discharge NIHSS scores, mortality, 3 months Barthel index (BI) and modified Rankin score (mRS)] were evaluated. Rescue LIT therapy was performed on ten MCA occlusion patients (male:female=3:7, mean age 71 years). The mean time between the initiation of i.v. tPA therapy and the initiation of intra-arterial urokinase (i.a. UK) was 117±25.0 min [time to i.v. tPA 137±32 min; time to digital subtraction angiography (DSA) 221±42 min; time to i.a. UK 260±46 min]. The baseline NIHSS scores showed significant improvement at discharge (median from 18 to 6). Symptomatic hemorrhage and, consequent, mortality were noted in 2/10 (20%) patients. Three months good outcome was noted in 4/10 (40%, mRS 0-2) and 3/10 (30%, BI ?95). In conclusion, rescue LIT therapy can be considered as a treatment option for patients not showing early response to full dose i.v. tPA therapy. Larger scale studies for further validation of this protocol may be necessary. (orig.)

  16. Stereotactic fibrinolysis of spontaneous intracerebral hematoma using infusion of recombinant tissue plasminogen activator Fibrinólise com infusão de rtPA e drenagem estereotáxica de hematoma intracerebral espontâneo profundo

    Directory of Open Access Journals (Sweden)

    José Augusto Nasser

    2002-06-01

    Full Text Available PURPOSE: The authors present a prospective study on 10 patients with stereotactic infusion of tissue plasminogen activator (rtPA intraparenchimal hemorrhage. METHODS: Between 1999 and 2000, 10 patients with deep seated hematomas in the basal ganglia were selected for stereotactic infusion of rtPA and spontaneous clot drainage. RESULTS: All cases had about 80% reduction of the hematoma volume in the CT scan at the third day. The intracranial pressure was normalized by the third day too. There were no local or systemic complications with the use of this trombolitic. The results were shown by the Glasgow Outcome Scale with six patients in V, three in IV and one in III after 3 months. CONCLUSION: Early treatment and drainage with minimally invasive neurosurgery , can make these patients with deep-seated hematomas recover the consciousness and they can be rehabilitated earlier avoiding secondary complications.OBJETIVO: Estudo prospectivo em 10 pacientes com infusão de trombolítico (rtPA dentro do hematoma cerebral profundo supratentorial e drenagem estereotáxica. MÉTODO: Entre 1999 e 2000 10 pacientes com hematomas de profundidade foram selecionados para infusão de rtPA e drenagem do coágulo espontânea. RESULTADO: Todos os casos obtiveram 80% de redução do volume do hematoma medidos por TC no terceiro dia. A pressão intracraniana estava normalizada no terceiro dia. Não houve complicações locais ou sistêmicas relacionadas com o uso deste trombolítico. Os resultados comparados foram mostrados pela Escala de Prognóstico de Glasgow com 6 pacientes em GrauV, 3 pacientes em Grau IV e 1 paciente em Grau III após três meses. CONCLUSÃO: Tratamento precoce e drenagem com técnica neurocirúrgica minimamente invasiva pode fazer estes pacientes terem uma recuperação da consciência mais rápida e assim serem reabilitados mais precocemente evitando complicações secundárias.

  17. Subconjunctival and topical application of recombinant tissue plasminogen activator in rabbits / Uso tópico e subconjuntival de ativador de plasminogênio tecidual recombinante em coelhos

    Scientific Electronic Library Online (English)

    José Ricardo de Abreu, Reggi; Richard Yudi, Hida; Milton Massato, Hida; Maria Cristina, Nishiwaki-Dantas; Hisashi, Suzuki.

    2015-02-01

    Full Text Available Objetivo: Quantificar produtos de degradação de fibrina (PDF) após uso tópico e subconjunctival de ativador de plasminogênio tecidual recombinante (r-TPA) em coelhos. Métodos: Formação de fibrina foi induzida na câmara anterior em 25 coelhos. Cinco coelhos foram submetidos a injeção intracameral de [...] r-TPA (controle positivo). Dez coelhos foram submetidos a injeção subconjuntival de r-TPA e dez coelhos foram submetidos a instilação tópica de r-TPA. Amostras de humor aquoso foram coletados e uma análise quantitativa dos produtos de degradação de fibrina foi realizada. Resultados: Não foi observado diferença estatisticamente significativa na degradação de fibrina em nenhum dos momentos estudados quando comparados com o controle. Porém foi observado diferença estatisticamente significante na quantificação do produtos de degradação de fibrina no grupo controle e no grupo subconjuntival. Conclusão: Produtos de degradação de fibrina foi observado nas amostras do grupo subconjunctival, porém, provavelmente não foi suficiente para degradar a fibrin presente. r-TPA tópico não foi efetivo em absorver fibrina na câmara anterior. Abstract in english Purpose: To quantify fibrin degradation products after topical and subconjunctival administration of recombinant tissue plasminogen activator in rabbits. Methods: Fibrin formation was induced in the anterior chamber in 25 rabbits. Subsequently, five rabbits received an injection of r-TPA (positive [...] control) in the anterior chamber, another 10 received a subconjunctival injection of r-TPA, and the remaining 10 received instillations of topical r-TPA. Afterwards, samples of aqueous humor were collected and semi-quantitative analysis of fibrin degradation products (FDP) was performed. Results: No statistical differences were noted between the treatment and control groups at any time point. Fibrin degradation products semi-quantification showed statistical improvement in the control group and the subconjunctival group. Conclusion: Fibrin degradation products were observed in the anterior chamber after subconjunctival administration of r-TPA. However, it was probably not sufficient to cause fibrin degradation. Topical r-TPA did not effectively absorb anterior chamber fibrin.

  18. Elevated urinary levels of urokinase-type plasminogen activator receptor (uPAR in pancreatic ductal adenocarcinoma identify a clinically high-risk group

    Directory of Open Access Journals (Sweden)

    Brocco Giorgio

    2011-10-01

    Full Text Available Abstract Background The urokinase plasminogen activator receptor is highly expressed and its gene is amplified in about 50% of pancreatic ductal adenocarcinomas; this last feature is associated with worse prognosis. It is unknown whether the level of its soluble form (suPAR in urine may be a diagnostic-prognostic marker in these patients. Methods The urinary level of suPAR was measured in 146 patients, 94 pancreatic ductal adenocarcinoma and 52 chronic pancreatitis. Urine from 104 healthy subjects with similar age and gender distribution served as controls. suPAR levels were normalized with creatinine levels (suPAR/creatinine, ng/mg to remove urine dilution effect. Results Urinary suPAR/creatinine values of pancreatic ductal adenocarcinoma patients were significantly higher (median 9.8; 25th-75th percentiles 5.3-20.7 than those of either healthy donors (median 0; 0-0.5 or chronic pancreatitis patients (median 2.7; 0.9-4.7. The distribution of values among cancer patients was widespread and asymmetric, 53% subjects having values beyond the 95th percentile of healthy donors. The values of suPAR/creatinine did not correlate with tumour stage, Ca19-9 or CEA levels. Higher values correlated with poor prognosis among non-resected patients at univariate analysis; multivariate Cox regression identified high urinary suPAR/creatinine as an independent predictor of poor survival among all cancer patients (odds ratio 2.10, p = 0.0023, together with tumour stage (stage III odds ratio 2.65, p = 0.0017; stage IV odds ratio 4.61, p Conclusions A high urinary suPAR/creatinine ratio represents a useful marker for the identification of a subset of patients with poorer outcome.

  19. Cyclo19,31[D-Cys19]-uPA19-31 is a potent competitive antagonist of the interaction of urokinase-type plasminogen activator with its receptor (CD87)

    OpenAIRE

    Magdolen, Viktor; Bürgle, Markus; Arroyo de Prada, Nuria; Schmiedeberg, Niko; Riemer, Christoph; Schroeck, Florian; Kellermann, Josef; Degitz, Klaus; Wilhelm, Olaf G.; SCHMITT, MANFRED; Kessler, Horst

    2001-01-01

    Urokinase-type plasminogen activator (uPA) represents a central molecule in pericellular proteolysis and is implicated in a variety of physiological and pathophysiological processes such as tissue remodelling, wound healing, tumor invasion, and metastasis. uPA binds with high affinity to a specific cell surface receptor, uPAR (CD87), via a well defined sequence within the N-terminal region of uPA (uPA(19-31)). This interaction directs the proteolytic activity of uPA to the cell surface which ...

  20. The mechanism of cancer-mediated conversion of plasminogen to the angiogenesis inhibitor angiostatin

    DEFF Research Database (Denmark)

    Gately, S; Twardowski, P; Stack, M S; Cundiff, D L; Grella, D; Castellino, F J; Enghild, J; Kwaan, H C; Lee, F; Kramer, R A; Volpert, O; Bouck, N; Soff, G A

    1997-01-01

    FSDs (N-acetyl-L-cysteine, D-penicillamine, captopril, L-cysteine, or reduced glutathione] generate angiostatin from plasminogen. An essential role of plasmin catalytic activity for angiostatin generation was identified by using recombinant mutant plasminogens as substrates. The wild-type recombinant...

  1. 9 CFR 113.301 - Ovine Ecthyma Vaccine.

    Science.gov (United States)

    2010-01-01

    ... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Ovine Ecthyma Vaccine. 113.301 Section... Virus Vaccines § 113.301 Ovine Ecthyma Vaccine. Ovine Ecthyma Vaccine shall be prepared from tissue... inoculation with virulent ovine ecthyma virus. Ovine Ecthyma Vaccine is exempt from the...

  2. Involvement of nitric oxide synthase in matrix metalloproteinase-9- and/or urokinase plasminogen activator receptor-mediated glioma cell migration

    International Nuclear Information System (INIS)

    Src tyrosine kinase activates inducible nitric oxide synthase (iNOS) and, in turn, nitric oxide production as a means to transduce cell migration. Src tyrosine kinase plays a key proximal role to control ?9?1 signaling. Our recent studies have clearly demonstrated the role of ?9?1 integrin in matrix metalloproteinase-9 (MMP-9) and/or urokinase plasminogen activator receptor (uPAR)-mediated glioma cell migration. In the present study, we evaluated the involvement of ?9?1 integrin-iNOS pathway in MMP-9- and/or uPAR-mediated glioma cell migration. MMP-9 and uPAR shRNAs and overexpressing plasmids were used to downregulate and upregulate these molecules, respectively in U251 glioma cells and 5310 glioma xenograft cells. The effect of treatments on migration and invasion potential of these glioma cells were assessed by spheroid migration, wound healing, and Matrigel invasion assays. In order to attain the other objectives we also performed immunocytochemical, immunohistochemical, RT-PCR, Western blot and fluorescence-activated cell sorting (FACS) analysis. Immunohistochemical analysis revealed the prominent association of iNOS with glioblastoma multiforme (GBM). Immunofluorescence analysis showed prominent expression of iNOS in glioma cells. MMP-9 and/or uPAR knockdown by respective shRNAs reduced iNOS expression in these glioma cells. RT-PCR analysis revealed elevated iNOS mRNA expression in either MMP-9 or uPAR overexpressed glioma cells. The migration potential of MMP-9- and/or uPAR-overexpressed U251 glioma cells was significantly inhibited after treatment with L-NAME, an inhibitor of iNOS. Similarly, a significant inhibition of the invasion potential of the control or MMP-9/uPAR-overexpressed glioma cells was noticed after L-NAME treatment. A prominent reduction of iNOS expression was observed in the tumor regions of nude mice brains, which were injected with 5310 glioma cells, after MMP-9 and/or uPAR knockdown. Protein expressions of cSrc, phosphoSrc and p130Cas were reduced with simultaneous knockdown of both MMP-9 and uPAR. Taken together, our results from the present and earlier studies clearly demonstrate that ?9?1 integrin-mediated cell migration utilizes the iNOS pathway, and inhibition of the migratory potential of glioma cells by simultaneous knockdown of MMP-9 and uPAR could be attributed to the reduced ?9?1 integrin and iNOS levels

  3. Atividade de plasmina e plasminogênio no leite longa vida com alta e baixa contagem de células somáticas durante o armazenamento / Activity of plasmin and plasminogen in ultra high temperature milk with high and low somatic cell counts during storage

    Scientific Electronic Library Online (English)

    Carlos Humberto, Corassin; Roice Eliana, Rosim; Carlos Augusto Fernandes de, Oliveira.

    2010-12-01

    Full Text Available O objetivo deste estudo foi avaliar o efeito da contagem de células somáticas (CCS) do leite na atividade de plasmina e plasminogênio durante o período de armazenamento do leite longa vida integral. Os leites crus foram categorizados em grupos de CCS de baixa (342.000-487.000 células mL-1) e alta co [...] ntagem (603.000-808.000 células mL-1). Dois lotes de leite longa vida em cada categoria de CCS foram analisados para determinação de plasmina e plasminogênio após 10, 30, 60, 90 e 120 dias de armazenamento em temperatura ambiente. Para a fabricação do leite longa vida, o leite cru foi submetido à pasteurização rápida seguida da esterilização industrial do leite por injeção de vapor pelo método direto e embalagem asséptica do produto. A CCS não apresentou efeitos sobre as características físico-químicas do leite cru, e nem sobre a atividade de plasmina e plasminogênio nos leites cru e longa vida, armazenados por 120 dias. Entretanto, independentemente da CCS, a atividade de plasmina e plasminogênio aumentou no leite longa vida ao longo do armazenamento, indicando a possibilidade de aumento da proteólise no produto durante sua vida de prateleira. Abstract in english This study aimed to evaluate the effect of somatic cell counts (SCC) in milk on plasmin and plasminogen activities of ultra high temperature (UHT) milk during storage. Raw milks were categorized in SCC groups of low (342,000-487,000 cells mL-1) and high cells (603,000-808,000 cells mL-1). Two replic [...] ates of UHT milks within each SCC category were analyzed for plasmin and plasminogen activities after 10, 30, 60, 90 and 120 days of storage at room temperature. For manufacture of UHT milk, raw milk was pasteurized and sterilized by direct vapor injection process, followed by aseptic packaging. SCC had no effect on physical-chemical characteristics of raw milk, and on plasmin or plasminogen activities in raw and UHT milks during 120 days of storage. However, independently of the SCC in raw milk, the activity of plasmin and plasminogen increased in UHT milk during storage, hence indicating a possible increase in proteolysis in the product during its shelf-life.

  4. Release of tissue-type plasminogen activator is induced in rats by leukotrienes C4 and D4, but not by prostaglandins E1, E2 and I2.

    OpenAIRE

    Tranquille, N.; Emeis, J.J.

    1988-01-01

    1. Acute release of plasminogen activator (PA) was studied in rat isolated hindleg system perfused with Tyrode solution. 2. Leukotriene C4 (LTC4) and LTD4 dose-dependently induced the release of PA, which plateaued at 160 nmol l-1 and 200 nmol l-1, respectively. The amount of PA released was about 1 iu ml-1. The effects of LTC4 and LTD4 were not additive. 3. The PA released was identified as tissue-type PA (t-PA) by quenching experiments using anti-human t-PA IgG, by fibrin autography, and by...

  5. Type 1 plasminogen activator inhibitor (PAI-1) in clear cell renal cell carcinoma (CCRCC) and its impact on angiogenesis, progression and patient survival after radical nephrectomy

    OpenAIRE

    Seidal Tomas; Wentzel-Larsen Tore; Zubac Dragomir P; Bostad Leif

    2010-01-01

    Abstract Background To examine the expression of type 1 plasminogen inhibitor (PAI-1) in clear cell renal cell carcinoma (CCRCC), and its possible association with microvessel density (MVD), the expression of thrombospondin-1 (TSP-1), nuclear grade, tumour stage, continuously coded tumour size (CCTS) and to assess the value of PAI as a prognostic marker in 162 patients with CCRCC treated with radical nephrectomy. Methods A total of 172 consecutive patients with CCRCC treated with radical neph...

  6. Interaction of Leptospira Elongation Factor Tu with Plasminogen and Complement Factor H: A Metabolic Leptospiral Protein with Moonlighting Activities

    OpenAIRE

    Wolff, Danielly G.; Castiblanco-Valencia, Mónica M.; Abe, Cecília M.; Monaris, Denize; de Morais, Zenaide M; Souza, Gisele O.; Vasconcellos, Sílvio A.; Isaac, Lourdes; Abreu, Patrícia A. E.; Barbosa, Angela S.

    2013-01-01

    The elongation factor Tu (EF-Tu), an abundant bacterial protein involved in protein synthesis, has been shown to display moonlighting activities. Known to perform more than one function at different times or in different places, it is found in several subcellular locations in a single organism, and may serve as a virulence factor in a range of important human pathogens. Here we demonstrate that Leptospira EF-Tu is surface-exposed and performs additional roles as a cell-surface receptor for ho...

  7. Increased Soluble Urokinase-Type Plasminogen Activator Receptor (suPAR) Levels in Plasma of Suicide Attempters.

    Science.gov (United States)

    Ventorp, Filip; Gustafsson, Anna; Träskman-Bendz, Lil; Westrin, Åsa; Ljunggren, Lennart

    2015-01-01

    The soluble form of the urokinase receptor, suPAR, has been suggested as a novel biomarker of low-grade inflammation. Activation of the immune system has been proposed to contribute to the development of depression and suicidal behavior. In order to identify depressed and suicidal individuals who could benefit from an anti-inflammatory treatment, a reliable biomarker of low-grade inflammation is vital. This study evaluates plasma suPAR levels as a biomarker of low-grade inflammation in patients with major depressive disorder and in patients who recently attempted suicide. The plasma suPAR and an established biomarker, C reactive protein (CRP) of suicide attempters (n = 54), depressed patients (n = 19) and healthy controls (n = 19) was analyzed with enzyme-linked immunosorbent assays. The biomarker attributes of sensitivity and sensibility were evaluated using ROC curve analysis. Both the depressed patients and suicide attempters had increased plasma suPAR. The levels of suPAR discriminated better between controls and suicide attempters than did CRP. In the future, plasma suPAR might be a superior prognosticator regarding outcome of treatment applying conventional antidepressants in conjunction with anti-inflammatory drugs. PMID:26451727

  8. Preoperative plasma plasminogen activator inhibitor type-1 and serum C-reactive protein levels in patients with colorectal cancer. The RANX05 Colorectal Cancer Study Group

    DEFF Research Database (Denmark)

    Nielsen, Hans JØrgen; Christensen, Ib Jarle

    2000-01-01

    BACKGROUND: Preoperative plasma plasminogen activator inhibitor-1 (PAI-1) is a prognostic variable in patients with colorectal cancer. It has been suggested, however, that plasma PAI-1 is a nonspecific prognostic parameter similar to the acute-phase reactant C-reactive protein (CRP). In the present study we analyzed the association between plasma PAI-1 and serum CRP in patients scheduled for elective resection of colorectal cancer. In addition, the prognostic value of PAI-1 and CRP was studied in this patient cohort. METHODS: PAI-1 and CRP were analyzed in citrated plasma and serum, respectively, obtained preoperatively from 594 patients. Patients who required preoperative blood transfusion received SAGM blood, in which soluble PAI-1 is not present. None of the patients received pre- or postoperative adjuvant chemotherapy, and all were followed in the outpatient clinic for at least 5 years or until death. The association of PAI-1 and CRP, respectively, with survival was tested using the median value of PAI-1 and the upper normal limit for CRP. Analyses were performed by inclusion of all patients, and in the subgroup of patients, who underwent curative resection. RESULTS: The median follow-up period was 6.8 (5.4-7.9) years. The median value of plasma PAI-1 was 35.8 ng/ml, and values greater than 94 nmol/L identified patients with increased CRP levels. Comparison of the molecules showed that PAI-1 was weakly correlated with CRP (r = .26; P <.0001). Both molecules showed a Dukes independent distribution. In univariate survival analyses high levels of PAI-1 were found associated with poor prognosis and low levels with good prognosis (P = .02, HR: 1.3). Similarly, high levels of CRP were found associated with poor prognosis and low levels with good prognosis (P <.0001, HR: 1.9). In a multivariate statistical analysis including Dukes classification, gender, age, tumor location, perioperative blood transfusion, PAI-1 and CRP, plasma PAI-1 was a dependent prognostic variable, while serum CRP (P <.0001; HR: 1.4; 95% CI: 1.3-1.5) was found to be a Dukes independent prognostic variable. Similar analyses, excluding patients with Dukes' D disease showed serum CRP to be an independent prognostic variable (P <.0001; HR: 1.3: 95% CI: 1.2-1.5). CONCLUSIONS: This study did not show a strong correlation between plasma PAI-1 and serum CRP in patients with colorectal cancer. Serum CRP was found to be a Dukes independent prognostic variable in this patient cohort, and was found to identify a subgroup of curatively resected patients at risk for short survival.

  9. Soluble urokinase plasminogen activator receptor is in contrast to high-sensitive C-reactive-protein associated with coronary artery calcifications in healthy middle-aged subjects

    DEFF Research Database (Denmark)

    SØrensen, Mette Hjortdal; Gerke, Oke

    2014-01-01

    OBJECTIVE: The main objective of this study was to investigate the association between two markers of low-grade inflammation; soluble urokinase plasminogen activator receptor (suPAR) and high-sensitive C-reactive protein (hs-CRP); and coronary artery calcification (CAC) score detected by cardiac computed tomography (CT) scan. DESIGN: A cross sectional study of 1126 randomly sampled middle-aged men and women. METHODS: CAC score was measured by a non-contrast cardiac CT scan and total 10-year cardiovascular mortality risk was estimated using the Systematic Coronary Risk Evaluation (SCORE). Plasma samples were analysed for suPAR and hs-CRP. The association of suPAR and hs-CRP to CAC was evaluated by logistic regression analyses adjusting for categorised SCORE. The additive effect of suPAR to SCORE was evaluated by comparing area under curve (AUC) and net reclassification improvement (NRI). RESULTS: The odds of being in a higher CAC category, i.e. having more severe CAC, increased 16% (odds ratio (OR) 1.16, p = 0.02) when plasma suPAR concentration increased 1 ng/ml, and this was more pronounced in women (OR 1.30, p = 0.01) than in men (OR 1.15, p = 0.05). In comparison, hs-CRP was not associated with CAC category (OR 1.00, p = 0.90). When adding suPAR to categorised SCORE, AUC increased from 0.66 to 0.70 (p = 0.04) in women and from 0.65 to 0.68 (p = 0.03) in men. NRI was significant in men (NRI 19.3%, 95% CI 6.1-32.6, p = 0.004) as well as in women (NRI 20.8%, 95%CI 1.0-40.7, p = 0.04), without significant gender difference. CONCLUSIONS: suPAR, but not hs-CRP, appeared to be associated with CAC score independently of SCORE. The association was strongest in women.

  10. Extracellular matrix biomarker, fibulin-1, is closely related to NT-proBNP and soluble urokinase plasminogen activator receptor in patients with aortic valve stenosis (the SEAS study)

    DEFF Research Database (Denmark)

    Kruger, Ruan; Rasmussen, Lars M

    2014-01-01

    BACKGROUND: Fibulin-1, a circulating extracellular matrix glycoprotein, has been associated with arterial disease and elevated N-terminal prohormone B-type natriuretic peptide (NT-proBNP) in diabetes. Soluble urokinase plasminogen activator receptor (suPAR), a marker of inflammation, has been associated with subclinical atherosclerosis. Therefore, we aimed to explore the interplay between these biomarkers and mild to moderate aortic valve stenosis (AS). METHODS: In 374 patients with mild to moderate AS, we investigated the relationship of fibulin-1 with NT-proBNP, levels of suPAR and the degree of AS at baseline and after one and four years of treatment with Simvastatin 40 mg and Ezetimibe 10 mg or placebo. RESULTS: During treatment, fibulin-1 became more closely associated with NT-proBNP (?year0?=?0.10, p?=?0.08, ?year1?=?0.16, p?=?0.005, ?year4?=?0.22, p<0.001) and suPAR (?year0?=?0.05, p?=?0.34, ?year1?=?0.16, p?=?0.006, ?year4?=?0.13, p?=?0.03) at the expense of the association to aortic valve area index (AVAI) (?year0?=?-0.14, p?=?0.005, ?year1?=?-0.08, p?=?0.11, ?year4?=?-0.06, p?=?0.22) independently of age, gender, creatinine, and serum aspartate aminotransferase (Adj.Ryear02?=?0.19, Adj.Ryear12?=?0.22, Adj.Ryear42?=?0.27). Fibulin-1 was unrelated to aortic regurgitation, left ventricular mass, and ejection fraction. In patients with baseline AVAI<0.58 cm2/m2 (median value), fibulin-1 was more closely associated to NT-proBNP (?year0?=?0.25, ?year1?=?0.21, ?year4?=?0.22, all p<0.01), and suPAR (?year0?=?0.09, p?=?0.26, ?year1?=?0.23, ?year4?=?0.21, both p<0.01) independently of age, gender, AST and treatment allocation. CONCLUSIONS: Increased levels of fibulin-1 were independently associated with higher levels of suPAR and NT-proBNP especially in patients with lower AVAI, suggesting that fibulin-1 may be an early marker of AS as well as cardiac fibrosis secondarily to elevated left ventricular hemodynamic load.

  11. Extracellular Matrix Biomarker, Fibulin-1, Is Closely Related to NT-proBNP and Soluble Urokinase Plasminogen Activator Receptor in Patients with Aortic Valve Stenosis (The SEAS Study)

    DEFF Research Database (Denmark)

    Kruger, Ruan; Rasmussen, Lars M

    2014-01-01

    BACKGROUND: Fibulin-1, a circulating extracellular matrix glycoprotein, has been associated with arterial disease and elevated N-terminal prohormone B-type natriuretic peptide (NT-proBNP) in diabetes. Soluble urokinase plasminogen activator receptor (suPAR), a marker of inflammation, has been associated with subclinical atherosclerosis. Therefore, we aimed to explore the interplay between these biomarkers and mild to moderate aortic valve stenosis (AS). METHODS: In 374 patients with mild to moderate AS, we investigated the relationship of fibulin-1 with NT-proBNP, levels of suPAR and the degree of AS at baseline and after one and four years of treatment with Simvastatin 40 mg and Ezetimibe 10 mg or placebo. RESULTS: During treatment, fibulin-1 became more closely associated with NT-proBNP (?year0?=?0.10, p?=?0.08, ?year1?=?0.16, p?=?0.005, ?year4?=?0.22, p<0.001) and suPAR (?year0?=?0.05, p?=?0.34, ?year1?=?0.16, p?=?0.006, ?year4?=?0.13, p?=?0.03) at the expense of the association to aortic valve area index (AVAI) (?year0?=?-0.14, p?=?0.005, ?year1?=?-0.08, p?=?0.11, ?year4?=?-0.06, p?=?0.22) independently of age, gender, creatinine, and serum aspartate aminotransferase (Adj.Ryear02?=?0.19, Adj.Ryear12?=?0.22, Adj.Ryear42?=?0.27). Fibulin-1 was unrelated to aortic regurgitation, left ventricular mass, and ejection fraction. In patients with baseline AVAI<0.58 cm2/m2 (median value), fibulin-1 was more closely associated to NT-proBNP (?year0?=?0.25, ?year1?=?0.21, ?year4?=?0.22, all p<0.01), and suPAR (?year0?=?0.09, p?=?0.26, ?year1?=?0.23, ?year4?=?0.21, both p<0.01) independently of age, gender, AST and treatment allocation. CONCLUSIONS: Increased levels of fibulin-1 were independently associated with higher levels of suPAR and NT-proBNP especially in patients with lower AVAI, suggesting that fibulin-1 may be an early marker of AS as well as cardiac fibrosis secondarily to elevated left ventricular hemodynamic load.

  12. Ultrasound-targeted transfection of tissue-type plasminogen activator gene carried by albumin nanoparticles to dog myocardium to prevent thrombosis after heart mechanical valve replacement

    Directory of Open Access Journals (Sweden)

    Ji J

    2012-06-01

    Full Text Available Ji Jun, Ji Shang-Yi, Yang Jian-An, He Xia, Yang Xiao-Han, Ling Wen-Ping, Chen Xiao-LingDepartment of Pathology and Cardiovascular Surgery, Shenzhen Sun Yat-Sen Cardiovascular Hospital, Shenzhen, Guangdong, People's Republic of ChinaBackground: There are more than 300,000 prosthetic heart valve replacements each year worldwide. These patients are faced with a higher risk of thromboembolic events after heart valve surgery and long-term or even life-long anticoagulative and antiplatelet therapies are necessary. Some severe complications such as hemorrhaging or rebound thrombosis can occur when the therapy ceases. Tissue-type plasminogen activator (t-PA is a thrombolytic agent. One of the best strategies is gene therapy, which offers a local high expression of t-PA over a prolonged time period to avoid both systemic hemorrhaging and local rebound thrombosis. There are some issues with t-PA that need to be addressed: currently, there is no up-to-date report on how the t-PA gene targets the heart in vivo and the gene vector for t-PA needs to be determined.Aims: To fabricate an albumin nano-t-PA gene ultrasound-targeted agent and investigate its targeting effect on prevention of thrombosis after heart mechanic valve replacement under therapeutic ultrasound.Methods: A dog model of mechanical tricuspid valve replacement was constructed. A highly expressive t-PA gene plasmid was constructed and packaged by nanoparticles prepared with bovine serum albumin. This nanopackaged t-PA gene plasmid was further cross-linked to ultrasonic microbubbles prepared with sucrose and bovine serum albumin to form the ultrasonic-targeted agent for t-PA gene transfection. The agent was given intravenously followed by a therapeutic ultrasound treatment (1 MHz, 1.5 w/cm2, 10 minutes of the heart soon after valve replacement had been performed. The expression of t-PA in myocardium was detected with multiclonal antibodies to t-PA by the indirect immunohistochemical method. Venous blood t-PA and D-dimer contents were tested before and 1, 2, 4, and 8 weeks after the operation.Results: The high expression of t-PA could be seen in myocardium with increases in blood t-PA and D-dimer contents and thrombosis was prevented 8 weeks after operation.Conclusion: We successfully fabricated an albumin nano-t-PA gene ultrasound-targeted agent that could prevent dog thrombosis after mechanical heart valve replacement. Our study provides an experimental basis for prevention of human thrombosis-related diseases.Keywords: albumin nanoparticles, ultrasonic microbubbles, valve replacement

  13. Synthesis of 99Tcm-HYNIC-AE105 and its application for imaging urokinase plasminogen activator receptors in human pancreatic cancer xenografts by gamma imaging

    International Nuclear Information System (INIS)

    Objective: To synthesize 99Tcm-labeled urokinase plasminogen activator receptor (uPAR)-binding peptide D-Cha-F-s-r-Y-L-W-S (AE105) (99Tcm-AE105), and evaluate its value as an uPAR imaging agent in pancreatic cancer xenograft models. Methods: 99Tcm-AE105 and 99Tcm-D-cha-F-s-r-Y-L-E-S (AE105mut) were synthesized by using Tricine as the coligand and SnCl2 as the reductant. Gamma imaging was performed on 2 groups of pancreatic cancer (bxpc-3 cells) xenograft mice models (30 mice in each group, by completely randomized design) post injection of 18.5 MBq (0.2 ml) 99Tcm-AE105 and 99Tcm-AE105mut, respectively. The tumor uptake (%ID/g) of the 2 groups was calculated and compared by two-sample t test. The uPAR expression was measured by immunohistochemistry. Correlation between uPAR expression and tumor uptake was analyzed by Pearson correlation analysis. Results: The labeling efficiency of 99Tcm-AE105 was (94.64±0.72)%. The radiochemical purity was (97.72± 1.73)% after purification and the stability was acceptable. The tumor could be visualized on gamma imaging at 2 h post injection, and better visualized at 4 h and 6 h in the 99Tcm-AE105 group. However, the tumor could not be visualized on gamma imaging in the 99Tcm-AE105mut group. The tumor uptake of 99Tcm-AE105 was higher than that of 99Tcm-AE105mut (4 h:(3.12±0.27) vs (1.65±0.53) %ID/g, 6 h: (2.98±0.15) vs (1.41±0.38) %ID/g; t=4.496, 6.787, both P<0.01). Significant correlation between tumor uptake of 99Tcm-AE105 and uPAR expression was found in 4-6 h (r =0.791, P<0.01). There was no correlation between 99Tcm-AE105mut uptake and uPAR expression in 4-6 h (r=-0.415, P>0.05). Conclusion: 99Tcm-AE105 could be synthesized with high radiochemical purity. The specific tumor uptake of 99Tcm-AE105, which correlates with uPAR expression in pancreatic cancer, makes it a potential clinical gamma imaging tracer for cancer detection and therapy monitoring. (authors)

  14. EMMPRIN/CD147 up-regulates urokinase-type plasminogen activator: implications in oral tumor progression

    International Nuclear Information System (INIS)

    An elevated level of EMMPRIN in cancer tissues have been correlated with tumor invasion in numerous cancers including oral cavity and larynx. Although EMMPRIN's effect has been generally attributed to its MMP inducing activity, we have previously demonstrated in breast cancer model that EMMPRIN can also enhance invasion by upregulating uPA. In this study, the role of EMMPRIN in regulating uPA and invasion was investigated in oral squamous cell carcinoma (OSCC) progression. Precancerous and invasive oral tumoral tissues were used as well as the corresponding cell lines, DOK and SCC-9 respectively. The paracrine regulation of uPA by EMMPRIN was investigated by treating culture cells with EMMPRIN-enriched membrane vesicles. UPA expression was analyzed by qPCR and immunostaining and the consequence on the invasion capacity was studied using modified Boyden chamber assay, in the presence or absence of EMMPRIN blocking antibody, the uPA inhibitor amiloride or the MMP inhibitor marimastat. OSCC tumors were shown to express more EMMPRIN and uPA compared to dysplastic lesions. The corresponding cell models, SCC-9 and DOK cells, displayed similar expression pattern. In both cell types EMMPRIN upregulated the expression of uPA as well as that of MMP-2 and MMP-9. EMMPRIN treatment led to a significant increase in cell invasion both in the invasive SCC-9 and in the less invasive dysplastic DOK cells, in an MMP and uPA dependent manner. Our results suggest that the upregulation of uPA contributes to EMMPRIN's effect in promoting oral tumor invasion

  15. Platelet Activation in Ovines Undergoing Sham Surgery or Implant of the Second Generation PediaFlow™ Pediatric Ventricular Assist Device

    OpenAIRE

    Johnson, Carl A.; Wearden, Peter D.; Kocyildirim, Ergin; Maul, Timothy M.; Woolley, Joshua R.; Ye, Sang-Ho; Strickler, Elise M.; Harvey S. Borovetz; Wagner, William R.

    2011-01-01

    The PediaFlow™ pediatric ventricular assist device (VAD) is a magnetically levitated turbodynamic pump under development for circulatory support of small children with a targeted flow rate range of 0.3 - 1.5 L/min. As the design of this device is refined, ensuring high levels of blood biocompatibility is essential. In this study we characterized platelet activation during the implantation and operation of a second generation prototype of the PediaFlow VAD (PF2) and also performed a series of ...

  16. Late results of catheter-directed recombinant tissue plasminogen activator fibrinolytic therapy of iliofemoral deep venous thrombosis / Resultados de longo prazo do tratamento fibrinolítico da trombose venosa iliacofemoral por infusão seletiva do ativador de plasminogênio tissular recombinante em baixas doses

    Scientific Electronic Library Online (English)

    Ivan Benaduce, Casella; Calógero, Presti; Ricardo, Aun; Joseph Elias, Benabou; Pedro, Puech-Leão.

    2007-02-01

    Full Text Available OBJETIVOS: Avaliar a eficácia da infusão seletiva por cateter do ativador de plasminogênio tecidual recombinante em baixas doses no tratamento da trombose venosa iliacofemoral e na prevenção da síndrome pós-trombótica. MÉTODO: Dezoito pacientes (de 260 avaliados) portadores de trombose venosa profun [...] da iliacofemoral sem evidência prévia de insuficiência venosa foram selecionados para terapia fibrinolítica e submetidos a infusão seletiva por cateter do ativador de plasminogênio tecidual recombinante na dose de 1mg/dl nos segmentos venosos trombóticos. RESULTADOS: Quatorze pacientes apresentaram fibrinólise efetiva; observamos correlação entre o grau de melhora clínica observado e a redução percentual do volume trombótico (P Abstract in english PURPOSE: To evaluate the efficacy of catheter-directed low-dose recombinant tissue-type plasminogen activator infusion in the treatment of iliofemoral deep venous thrombosis and prevention of post-thrombotic syndrome. METHOD: Eighteen patients (out of 260 evaluated) with acute iliofemoral deep venou [...] s thrombosis and no previous evidence of venous insufficiency were prospectively selected for thrombolytic therapy. Catheter-directed low-dose recombinant tissue-type plasminogen activator (1 mg/h) was infused into the thrombotic segments. RESULTS: Effective fibrinolysis was achieved in 14 of 18 cases, with correlation between effective fibrinolysis and major/complete resolution of acute signs and symptoms (P

  17. Angiotensin I-converting-enzyme (ACE)-inhibitory activity of tryptic peptides of ovine $\\beta$-lactoglobulin and of milk yoghurts obtained by using different starters

    OpenAIRE

    Chobert, Jean-Marc; El-Zahar, Khaled; Sitohy, Mahmoud; Dalgalarrondo, Michèle; Métro, François; Choiset, Yvan; Haertlé, Thomas

    2005-01-01

    Activité inhibitrice de l'enzyme de conversion de l'angiotensine I (ECA) de peptides trypsiques de la$\\beta$-lactoglobuline du lait de brebis et de yoghourts obtenus à partir de différents levains. Une activité inhibitrice de l'enzyme de conversion de l'angiotensine I (ECA) a été recherchée dans les peptides trypsiques de la $\\beta$-lactoglobuline du lait de brebis et dans des fractions issues de yoghourts fabriqués à partir de différents ferments lactiques. La $\\beta$-lactoglobuline ovine (u...

  18. Flavonol-enriched fraction from Vaccinium macrocarpon fruit inhibits matrix metalloproteinase-2, matrix metalloproteinase-9 and urokinase-type plasminogen activator expression in human prostate cancer cells in vitro

    Directory of Open Access Journals (Sweden)

    James MacPhee

    2014-11-01

    Full Text Available Background: Prostate cancer, amongst other cancer types has a genetic and environmental component, which can contribute to prostate cancer development and progression. Vaccinum macrocarpon (American cranberry is a botanical that contains several phytochemicals which have been suggested to play a role in preventing cardiovascular disease, cancer, and urinary tract infections as well as in the maintenance of oral health. Context and purpose of this study: This investigation evaluated the effects of a flavonolenriched fraction (FL from the American cranberry (Vaccinium macrocarpon containing quercetin and myricetin glycosides on matrix metalloproteinase (MMP and urokinase-type plasminogen activator (uPA activities and their associated regulatory proteins in DU145 human prostate cancer cells in vitro. Results: A flavonol-enriched fraction (FL was prepared from Vaccinium macrocarpon berries and the effect of this fraction on prostate cancer cell behaviour was assessed using biochemical and molecular approaches including cytotoxicity assays and Western blot analysis to determine protein expression. Cranberry FL decreased cellular viability of DU145 cells at a concentration of 25 ug/ml by 20% after 6 hours of treatment. Further investigations determined that associated with this cytotoxicity, cranberry FL decreases matrix metalloproteinase (MMP ( specifically MMP-2 and MMP-9 activity and urokinase plasminogen activator (uPA activity through effects on specific temporal MMP regulators and uPA regulators and by affecting either the phosphorylation status and/or expression of specific MAP kinase, PI-3 kinase, NF-kB and AP-1 pathway associated proteins. Conclusion: This study demonstrates, for the first time, the ability of Vaccinium macrocarpon flavonols to modulate cellular pathways associated with migration, invasion, and proliferation, suggesting that cranberry (Vaccinium macrocarpon is a viable candidate for further research as a natural product that may protect against certain cancers.

  19. Novel in vitro systems for prediction of veterinary drug residues in ovine milk and dairy products.

    Science.gov (United States)

    González-Lobato, L; Real, R; Herrero, D; de la Fuente, A; Prieto, J G; Marqués, M M; Alvarez, A I; Merino, G

    2014-01-01

    A new in vitro tool was developed for the identification of veterinary substrates of the main drug transporter in the mammary gland. These drugs have a much higher chance of being concentrated into ovine milk and thus should be detectable in dairy products. Complementarily, a cell model for the identification of compounds that can inhibit the secretion of drugs into ovine milk, and thus reduce milk residues, was also generated. The ATP-binding cassette transporter G2 (ABCG2) is responsible for the concentration of its substrates into milk. The need to predict potential drug residues in ruminant milk has prompted the development of in vitro cell models over-expressing ABCG2 for these species to detect veterinary drugs that interact with this transporter. Using these models, several substrates for bovine and caprine ABCG2 have been found, and differences in activity between species have been reported. However, despite being of great toxicological relevance, no suitable in vitro model to predict substrates of ovine ABCG2 was available. New MDCKII and MEF3.8 cell models over-expressing ovine ABCG2 were generated for the identification of substrates and inhibitors of ovine ABCG2. Five widely used veterinary antibiotics (marbofloxacin, orbifloxacin, sarafloxacin, danofloxacin and difloxacin) were discovered as new substrates of ovine ABCG2. These results were confirmed for the bovine transporter and its Y581S variant using previously generated cell models. In addition, the avermectin doramectin was described as a new inhibitor of ruminant ABCG2. This new rapid assay to identify veterinary drugs that can be concentrated into ovine milk will potentially improve detection and monitoring of veterinary drug residues in ovine milk and dairy products. PMID:24679113

  20. Cloning and characterization of a plasminogen-binding surface-associated enolase from Schistosoma bovis.

    Science.gov (United States)

    de la Torre-Escudero, Eduardo; Manzano-Román, Raúl; Pérez-Sánchez, Ricardo; Siles-Lucas, Mar; Oleaga, Ana

    2010-10-11

    Schistosoma bovis is a ruminant parasite able to survive prolonged periods in the vasculature of its host without either being cleared by the host defensive systems or inducing thrombotic or coagulation disturbances. This suggests that the parasite modulates both the immune and haemostatic host responses. Previous studies have shown that host plasminogen binds to the surface of S. bovis adult worms, and that a tegument extract from S. bovis fixes and activates host plasminogen, generating plasmin, which in turn could both inhibit blood clotting and dissolve clots. Enolase has been identified among the tegumental proteins that bind plasminogen. The aim of the present study is to determine the physiological role of the enolase found in the tegument of S. bovis adult worms as regards plasminogen-binding and activation, and to confirm its surface exposure on the parasite. The study included the cloning and sequencing of S. bovis enolase cDNA, collection of the corresponding recombinant protein and evaluation of its plasminogen-binding and activation activity, and an exploration of the expression and localization of native enolase in adult worms and lung schistosomulae. Here we show that S. bovis male adult worms express enolase on their tegumental surface and that this protein binds host plasminogen and increases its activation in the presence of host tissue plasminogen activator (t-PA). This suggests that the surface-associated enolase found here is a physiological receptor of plasminogen that plays a role in the activation of the host fibrinolytic system, most probably to avoid blood clot formation on the worm's surface. PMID:20609522

  1. The sGC activator inhibits the proliferation and migration, promotes the apoptosis of human pulmonary arterial smooth muscle cells via the up regulation of plasminogen activator inhibitor-2

    International Nuclear Information System (INIS)

    Background: Different types of pulmonary hypertension (PH) share the same process of pulmonary vascular remodeling, the molecular mechanism of which is not entirely clarified by far. The abnormal biological behaviors of pulmonary arterial smooth muscle cells (PASMCs) play an important role in this process. Objectives: We investigated the regulation of plasminogen activator inhibitor-2 (PAI-2) by the sGC activator, and explored the effect of PAI-2 on PASMCs proliferation, apoptosis and migration. Methods: After the transfection with PAI-2 overexpression vector and specific siRNAs or treatment with BAY 41-2272 (an activator of sGC), the mRNA and protein levels of PAI-2 in cultured human PASMCs were detected, and the proliferation, apoptosis and migration of PASMCs were investigated. Results: BAY 41-2272 up regulated the endogenous PAI-2 in PASMCs, on the mRNA and protein level. In PAI-2 overexpression group, the proliferation and migration of PASMCs were inhibited significantly, and the apoptosis of PASMCs was increased. In contrast, PAI-2 knockdown with siRNA increased PASMCs proliferation and migration, inhibited the apoptosis. Conclusions: PAI-2 overexpression inhibits the proliferation and migration and promotes the apoptosis of human PASMCs. Therefore, sGC activator might alleviate or reverse vascular remodeling in PH through the up-regulation of PAI-2. - Highlights: • sGC activator BAY41-2272 up regulated PAI-2 in PASMCs, on the mRNA and protein level. • PAI-2 overexpression inhibits the proliferation and migration of human PASMCs. • PAI-2 overexpression promotes the apoptosis of human PASMCs. • sGC activator might alleviate the vascular remodeling in pulmonary hypertension

  2. Hypoxia Represses ER-? Expression and Inhibits Estrogen-Induced Regulation of Ca2+-Activated K+ Channel Activity and Myogenic Tone in Ovine Uterine Arteries: Causal Role of DNA Methylation.

    Science.gov (United States)

    Chen, Man; Xiao, Daliao; Hu, Xiang-Qun; Dasgupta, Chiranjib; Yang, Shumei; Zhang, Lubo

    2015-07-01

    Previous in vivo study demonstrated that chronic hypoxia during gestation was associated with estrogen receptor-? (ER-?) gene repression in ovine uterine arteries. Yet, it remains undetermined whether hypoxia had a direct effect and if DNA methylation played a causal role in hypoxia-mediated ER-? gene repression. Thus, this study tested the hypothesis that prolonged hypoxia has a direct effect and increases promoter methylation resulting in ER-? gene repression and inhibition of estrogen-mediated adaptation of uterine vascular tone. Uterine arteries isolated from nonpregnant and pregnant sheep were treated ex vivo with 21.0% O2 and 10.5% O2 for 48 hours. Hypoxia significantly increased ER-? promoter methylation at both specificity protein-1 and upstream stimulatory factor binding sites, decreased specificity protein-1 and upstream stimulatory factor binding to the promoter, and suppressed ER-? expression in uterine arteries of pregnant animals. Of importance, the effects of hypoxia were blocked by a methylation inhibitor 5-aza-2'-deoxycytidine. In addition, hypoxia abrogated steroid hormone-mediated increase in ER-? expression and inhibited the hormone-induced increase in large-conductance Ca(2+)-activated K(+) channel activity and decrease in myogenic tone in uterine arteries of nonpregnant animals, which were reversed by 5-aza-2'-deoxycytidine. The results provide novel evidence of a direct effect of hypoxia on heightened promoter methylation that plays a causal role in ER-? gene repression and ablation of steroid hormone-mediated adaptation of uterine arterial large conductance Ca(2+)-activated K(+) channel activity and myogenic tone in pregnancy. PMID:25987666

  3. Topography of the high-affinity lysine binding site of plasminogen as defined with a specific antibody probe

    International Nuclear Information System (INIS)

    An antibody population that reacted with the high-affinity lysine binding site of human plasminogen was elicited by immunizing rabbits with an elastase degradation product containing kringles 1-3 (EDP I). This antibody was immunopurified by affinity chromatography on plasminogen-Sepharose and elution with 0.2 M 6-aminohexanoic acid. The eluted antibodies bound [125I]EDP I, [125I]Glu-plasminogen, and [125I]Lys-plasminogen in radioimmunoassays, and binding of each ligand was at least 99% inhibited by 0.2 M 6-aminohexanoic acid. The concentrations for 50% inhibition of [125I]EDP I binding by tranexamic acid, 6-aminohexanoic acid, and lysine were 2.6, 46, and l730 ?M, respectively. Similar values were obtained with plasminogen and suggested that an unoccupied high-affinity lysine binding site was required for antibody recognition. The antiserum reacted exclusively with plasminogen derivatives containing the EDP I region and did not react with those lacking an EDP I region, or with tissue plasminogen activator or prothrombin, which also contains kringles. By immunoblotting analyses, a chymotryptic degradation product of M/sub r/ 20,000 was derived from EDP I that retained reactivity with the antibody. ?2-Antiplasmin inhibited the binding of radiolabeled EDP I, Glu-plasminogen, or Lys-plasminogen by the antiserum, suggesting that the recognized site is involved in the noncovalent interaction of the inhibitor with plasminogen. The binding of [125I]EDP I to fibrin was also inhibited by the antiserum. The observations provide independent evidence for the role of the high-affinity lysine binding site in the functional interactions of plasminogen with its primary substrate and inhibitor

  4. Identification of peptides in functional Scamorza ovine milk cheese.

    Science.gov (United States)

    Albenzio, M; Santillo, A; Marino, R; Della Malva, A; Caroprese, M; Sevi, A

    2015-12-01

    Ovine bulk milk was used to produce Scamorza cheese with probiotics: either a mix of Bifidobacterium longum and Bifidobacterium lactis or Lactobacillus acidophilus as the probiotic strains. Peptides obtained from reverse phase-HPLC water-soluble extract of Scamorza cheeses were analyzed using a quadrupole time-of-flight liquid chromatography-mass spectrometry system. Identified fragments were derived from casein hydrolysis or probiotic bacterial enzymes; some of the fragments showed encrypted peptide sequences that shared structural homology with previously described bioactive peptides in ovine milk and dairy products. Bifidobacterium longum and B. lactis showed greater proteolytic potential both in terms of level of pH 4.6 water-soluble nitrogen extract and ability to generate peptides with potential biofunctionality. Fragments deriving from microbial enzymes may be regarded as tracing fragments useful for monitoring probiotic activity in functional Scamorza cheese. PMID:26409967

  5. Plasmin degradation of the alpha chain of fibrinogen/fibrin: improved activation constant and activity determination in assays for tissue plasminogen activator / Degradación por la plasmina de la cadena alfa del fibrinógeno/fibrina: mejoría de la constante de activación y determinación de la actividad en ensayos para el activador del plasminógeno tisular

    Scientific Electronic Library Online (English)

    Tatiana M., Garcés P; Alfonso, Quijano P.; Luis Fernando, Arbeláez R..

    2013-07-01

    Full Text Available Objetivos. El propósito de la presente investigación fue incrementar la eficacia de la formación del complejo terciario (fibrina-plasminógeno-activador tisular del plasminógeno) en el proceso de degradación de la estructura tridimensional del monómero de fibrina soluble. Materiales y métodos. El fib [...] rinógeno fue purificado de plasma humano, por seis precipitaciones repetidas, con diferentes concentraciones de etanol frío. El fibrinógeno fue convertido a desAAfibrinógeno por degradación con batroxobina. El plasminógeno humano fue purificado por cromatografías de afinidad e intercambio iónico y activado a plasmina con uroquinasa. El desAAfibrinogeno digerido fue preparado por digestión controlada con plasmina. Resultados. Este estudio demuestra que la cadena ? del desAAfibrinógeno, dificulta la formación del complejo terciario, por impedimentos estéricos, por lo cual la cadena ? se sometió a hidrólisis controlada con plasmina, facilitando así la determinación in vitro de la actividad del activador tisular del plasminógeno. Finalmente, la liberación del fibrinopéptido A por hidrólisis del fibrinógeno con batroxobina, fue confirmada, optimizada y evaluada por varios métodos. Conclusiones. El uso de desAAfibrinogeno digerido con plasmina da una constante de activación más estable en la formación del complejo terciario que el desAAfibrinógeno no digerido (fibrina-plasminogeno- activador tisular del plasminógeno). Abstract in english Objectives. The aim of this investigation was to increase the efficiency of ternary complex formation (fibrin-plasminogen-tissue-plasminogen activator) in the degradation process of the three-dimensional soluble fibrin monomer. Materials and methods. Fibrinogen was purified from human plasma by repe [...] ating precipitation six times, using different concentrations of cold ethanol. Fibrinogen was converted to DesAAfibrinogen by degradation with bathroxobin. Human plasminogen was purified by affinity and ion-exchange chromatography, and activated to plasmin by incubation with urokinase. Digested DesAAfibrinogen was prepared by controlled digestion with plasmin. Results. This study demonstrates that the ?-chains of DesAAfibrinogen sterically hinder the formation of the ternary complex and are first degraded by plasmin. The degradation of fibrin(ogen) facilitates the in vitro determination of tissue plasminogen activator activity. Finally, release of fibrinopeptide A from bathroxobin-cleaved fibrinogen was confirmed, optimized and evaluated by various methods. Conclusions. Use of digested desAAfibrinogen with plasmin yielded a more stable activation constant of the ternary complex than that of undigested DesAAfibrinogen.

  6. Ovine Mastitis Due to Histophilus ovis

    OpenAIRE

    Beauregard, M.; Higgins, R.

    1983-01-01

    The clinical, pathological and bacteriological findings related to ovine mastitis caused by Histophilus ovis are described. A high proportion of the ewes belonging to a flock were infected, but the source of the contamination could not be determined.

  7. NKp46 defines ovine cells that have characteristics corresponding to NK cells

    Directory of Open Access Journals (Sweden)

    Connelley Timothy

    2011-02-01

    Full Text Available Abstract Natural killer (NK cells are well recognized as playing a key role in innate immune defence through cytokine production and cytotoxic activity; additionally recent studies have identified several novel NK cell functions. The ability to study NK cells in the sheep has been restricted due to a lack of specific reagents. We report the generation of a monoclonal antibody specific for ovine NKp46, a receptor which in a number of mammals is expressed exclusively in NK cells. Ovine NKp46+ cells represent a population that is distinct from CD4+ and ??+ T-cells, B-cells and cells of the monocytic lineage. The NKp46+ cells are heterogenous with respect to expression of CD2 and CD8 and most, but not all, express CD16 - characteristics consistent with NK cell populations in other species. We demonstrate that in addition to populations in peripheral blood and secondary lymphoid organs, ovine NKp46+ populations are also situated at the mucosal surfaces of the lung, gastro-intestinal tract and non-gravid uterus. Furthermore, we show that purified ovine NKp46+ populations cultured in IL-2 and IL-15 have cytotoxic activity that could be enhanced by ligation of NKp46 in re-directed lysis assays. Therefore we conclude that ovine NKp46+ cells represent a population that by phenotype, tissue distribution and function correspond to NK cells and that NKp46 is an activating receptor in sheep as in other species.

  8. High-Resolution structure of the stable plasminogen activator inhibitor type-1 variant 14-1B in its proteinase-cleaved form: A new tool for detailed interaction studies and modeling

    Energy Technology Data Exchange (ETDEWEB)

    Jensen, J.; Gettins, P. (UIC)

    2008-10-22

    Wild-type plasminogen activator inhibitor type-1 (PAI-1) rapidly converts to the inactive latent state under conditions of physiological pH and temperature. For in vivo studies of active PAI-1 in cell culture and in vivo model systems, the 14-1B PAI-1 mutant (N150H-K154T-Q319L-M354I), with its stabilized active conformation, has thus become the PAI-1 of choice. As a consequence of the increased stability, the only two forms likely to be encountered are the active or the cleaved form, the latter either free or complexed with target proteinase. We hereby report the first structure of the stable 14-1B PAI-1 variant in its reactive center cleaved form, to a resolution of 2.0 {angstrom}. The >99% complete structure represents the highest resolved structure of free cleaved PAI-1. This high-resolution structure should be of great use for drug target development and for modeling protein-protein interactions such as those of PAI-1 with vitronectin.

  9. Pseudomonas aeruginosa toxin ExoU induces a PAF-dependent impairment of alveolar fibrin turnover secondary to enhanced activation of coagulation and increased expression of plasminogen activator inhibitor-1 in the course of mice pneumosepsis

    Directory of Open Access Journals (Sweden)

    Suassuna José HR

    2011-08-01

    Full Text Available Abstract Background ExoU, a Pseudomonas aeruginosa cytotoxin with phospholipase A2 activity, was shown to induce vascular hyperpermeability and thrombus formation in a murine model of pneumosepsis. In this study, we investigated the toxin ability to induce alterations in pulmonary fibrinolysis and the contribution of the platelet activating factor (PAF in the ExoU-induced overexpression of plasminogen activator inhibitor-1 (PAI-1. Methods Mice were intratracheally instilled with the ExoU producing PA103 P. aeruginosa or its mutant with deletion of the exoU gene. After 24 h, animal bronchoalveolar lavage fluids (BALF were analyzed and lung sections were submitted to fibrin and PAI-1 immunohistochemical localization. Supernatants from A549 airway epithelial cells and THP-1 macrophage cultures infected with both bacterial strains were also analyzed at 24 h post-infection. Results In PA103-infected mice, but not in control animals or in mice infected with the bacterial mutant, extensive fibrin deposition was detected in lung parenchyma and microvasculature whereas mice BALF exhibited elevated tissue factor-dependent procoagulant activity and PAI-1 concentration. ExoU-triggered PAI-1 overexpression was confirmed by immunohistochemistry. In in vitro assays, PA103-infected A549 cells exhibited overexpression of PAI-1 mRNA. Increased concentration of PAI-1 protein was detected in both A549 and THP-1 culture supernatants. Mice treatment with a PAF antagonist prior to PA103 infection reduced significantly PAI-1 concentrations in mice BALF. Similarly, A549 cell treatment with an antibody against PAF receptor significantly reduced PAI-1 mRNA expression and PAI-1 concentrations in cell supernatants, respectively. Conclusion ExoU was shown to induce disturbed fibrin turnover, secondary to enhanced procoagulant and antifibrinolytic activity during P. aeruginosa pneumosepsis, by a PAF-dependent mechanism. Besides its possible pathophysiological relevance, in vitro detection of exoU gene in bacterial clinical isolates warrants investigation as a predictor of outcome of patients with P. aeruginosa pneumonia/sepsis and as a marker to guide treatment strategies.

  10. Human plasminogen binding protein tetranectin

    DEFF Research Database (Denmark)

    Kastrup, J S; Rasmussen, H; Nielsen, B B; Larsen, I K; Holtet, T L; Graversen, Jonas Heilskov; Etzerodt, M; Thøgersen, H C

    The recombinant human plasminogen binding protein tetranectin (TN) and the C-type lectin CRD of this protein (TN3) have been crystallized. TN3 crystallizes in the tetragonal space group P4(2)2(1)2 with cell dimensions a = b = 64.0, c = 75.7 A and with one molecule per asymmetric unit. The crystals...... at least 2.5 A. A full data set has been collected to 3.0 A. The asymmetric unit contains one monomer of TN. Molecular replacement solutions for TN3 and TN have been obtained using the structure of the C-type lectin CRD of rat mannose-binding protein as search model. The rhombohedral space group...

  11. Type 1 plasminogen activator inhibitor (PAI-1 in clear cell renal cell carcinoma (CCRCC and its impact on angiogenesis, progression and patient survival after radical nephrectomy

    Directory of Open Access Journals (Sweden)

    Seidal Tomas

    2010-12-01

    Full Text Available Abstract Background To examine the expression of type 1 plasminogen inhibitor (PAI-1 in clear cell renal cell carcinoma (CCRCC, and its possible association with microvessel density (MVD, the expression of thrombospondin-1 (TSP-1, nuclear grade, tumour stage, continuously coded tumour size (CCTS and to assess the value of PAI as a prognostic marker in 162 patients with CCRCC treated with radical nephrectomy. Methods A total of 172 consecutive patients with CCRCC treated with radical nephrectomy were enrolled in the study. The expression of PAI-1, TSP-1 and factor VIII were analysed on formalin-fixed, paraffin-embedded tissues without knowledge of the clinical outcome. Ten cases, where PAI-1 immunohistochemistry was not possible due to technical problems and lack of material, were excluded. Sixty-nine patients (43% died of RCC, while 47 patients (29% died of other diseases. Median follow-up was 13.8 years for the surviving 46 patients (28%. Results Nine percent of the tumours showed PAI-1 positivity. High expression of PAI-1 was significantly inversely correlated with TSP-1 (p = 0.046 and directly with advanced stage (p = 0.008, high NG (3+4 (p = 0.002, tumour size (p = 0.011, microvessel density (p = 0.049 and disease progression (p = 0.002. In univariate analysis PAI-1 was a significant prognosticator of cancer-specific survival (CSS (p Conclusions PAI-1 was found to be an independently significant prognosticator of CSS and a promoter of tumour angiogenesis, aggressiveness and progression in CCRCC.

  12. A complete DNA sequence map of the ovine Major Histocompatibility Complex

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    Gao Jianfeng

    2010-08-01

    Full Text Available Abstract Background The ovine Major Histocompatibility Complex (MHC harbors clusters of genes involved in overall resistance/susceptibility of an animal to infectious pathogens. However, only a limited number of ovine MHC genes have been identified and no adequate sequence information is available, as compared to those of swine and bovine. We previously constructed a BAC clone-based physical map that covers entire class I, class II and class III region of ovine MHC. Here we describe the assembling of a complete DNA sequence map for the ovine MHC by shotgun sequencing of 26 overlapping BAC clones. Results DNA shotgun sequencing generated approximately 8-fold genome equivalent data that were successfully assembled into a finished sequence map of the ovine MHC. The sequence map spans approximately 2,434,000 nucleotides in length, covering almost all of the MHC loci currently known in the sheep and cattle. Gene annotation resulted in the identification of 177 protein-coding genes/ORFs, among which 145 were not previously reported in the sheep, and 10 were ovine species specific, absent in cattle or other mammals. A comparative sequence analyses among human, sheep and cattle revealed a high conservation in the MHC structure and loci order except for the class II, which were divided into IIa and IIb subregions in the sheep and cattle, separated by a large piece of non-MHC autosome of approximately 18.5 Mb. In addition, a total of 18 non-protein-coding microRNAs were predicted in the ovine MHC region for the first time. Conclusion An ovine MHC DNA sequence map was successfully assembled by shotgun sequencing of 26 overlapping BAC clone. This makes the sheep the second ruminant species for which the complete MHC sequence information is available for evolution and functional studies, following that of the bovine. The results of the comparative analysis support a hypothesis that an inversion of the ancestral chromosome containing the MHC has shaped the MHC structures of ruminants, as we currently observed in the sheep and cattle. Identification of relative large numbers of microRNAs in the ovine MHC region helps to provide evidence that microRNAs are actively involved in the regulation of MHC gene expression and function.

  13. 78 FR 68327 - Importation of Ovine Meat From Uruguay

    Science.gov (United States)

    2013-11-14

    ...will be monitoring the fresh meat export program, including...ruminant species, including cattle and sheep, and, therefore...the following phrases: ``beef and ovine meat,'' ``bovines and sheep,'' and ``bovine parts and ovine parts...importation of fresh ovine meat from Uruguay into the...

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

    Directory of Open Access Journals (Sweden)

    Miroslava Didiasova

    2014-11-01

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

  15. A serial fluoroscopy-guided thrombolytic therapy of a mechanical tricuspid prosthetic valve thrombosis with low-dose and ultra-slow infusion of tissue-type plasminogen activator.

    Science.gov (United States)

    Kalç?k, Macit; Gürsoy, Ozan Mustafa; Astarc?o?lu, Mehmet Ali; Özkan, Mehmet

    2014-07-01

    Prosthetic valve thrombosis is a life-threatening complication that is seen most commonly in patients with left-sided prosthetic valves. However, mechanical tricuspid valves carry the highest risk of thrombosis of any cardiac valve. Thrombolysis has been performed successfully in right-sided prosthetic valve thrombosis and has been recommended as the first-line treatment in these patients. Although two-dimensional and real-time three-dimensional transesophageal echocardiography are the gold standard imaging modalities for the diagnosis of prosthetic valve thrombosis, right-sided prosthetic valves may not be evaluated precisely. This is a serious problem during the follow-up of patients who receive thrombolytic therapy for tricuspid valve thrombosis. Fluoroscopy is an alternative noninvasive imaging method that provides valuable information about leaflet motion and may be used for such cases with restricted leaflets. Herein, we report a case of tricuspid valve thrombosis who was managed with low-dose (25 mg) and ultra-slow (25 hours) infusion of tissue-type plasminogen activator under the guidance of serial fluoroscopy. PMID:25080958

  16. Cancer cells overexpress mRNA of urokinase-type plasminogen activator, its receptor and inhibitors in human non-small-cell lung cancer tissue: analysis by Northern blotting and in situ hybridization.

    Science.gov (United States)

    Morita, S; Sato, A; Hayakawa, H; Ihara, H; Urano, T; Takada, Y; Takada, A

    1998-10-29

    The transcriptional localizations of urokinase-type plasminogen activator (uPA), its receptor (uPAR) and its inhibitors (PAI-1 and PAI-2), which are possibly involved in cancer metastasis, have not been determined in human lung cancer. To identify their regulation in primary non-small-cell lung cancer, we assayed mRNA levels by Northern blot analysis in 25 cases and determined the localizations of mRNA by in situ hybridization in 10 cases. The amounts of uPA and PAI-2 mRNA were significantly higher in cancerous relative to normal lung tissues. However, no significant difference was observed in uPAR and PAI-1 mRNA levels. All transcripts were present in cancer cells and were predominantly located in tumor edges in several cases. In addition, PAI-1 transcripts were more abundant in poorly and moderately differentiated carcinomas relative to well-differentiated carcinomas and PAI-2 transcripts were more abundant in squamous cell carcinomas than in adenocarcinomas. Thus, PAIs may be involved in modulation of malignant potency. Our results indicate that human non-small-cell lung cancer cells can autonomously express the mRNAs of uPA, uPAR and PAIs, which are possibly involved in metastasis. PMID:9766559

  17. Recanalization rate and clinical outcome of intravenous tissue plasminogen activator at 0.6 mg/kg and intra-arterial urokinase in acute ischemic stroke with large vessel occlusion

    International Nuclear Information System (INIS)

    We evaluated the recanalization rate and clinical outcome of intravenous tissue plasminogen activator (t-PA) and intra-arterial urokinase (PTA) in acute ischemic stroke with large vessel occlusion. The recanalization of the occlusion site and the ischemic change were evaluated with pre-and post-treated MRI and MR angiography (MRA). Total recanalization rates after the intravenous (IV) t-PA and the PTA therapy were 35.6 and 21.9%, respectively. These rates were 50.0 and 16.7% in the distal middle cerebral artery (MCA), 52.6 and 25.0% in the proximal MCA, 6.3 and 5.3% in the internal carotid artery (ICA), 25.0 and 26.3% in the basilar artery (BA), respectively. The rates of the symptomatic intracerebral hemorrhage after IV t-PA and PTA were 0 and 5.2%, respectively. The proportion of modified Rankin Scale (mRS) of 0 to 1 at 3 months after treatments were 17.4% in IV t-PA and 12.5% in PTA. Our results indicated better recanalization rate and outcome of MCA with t-PA than that of PTA. However, the recanalization rate of ICA and BA were very poor in both t-PA and PTA as yet. It is necessary to investigate newly strategies and/or modality for ICA and BA occlusion. (author)

  18. Experiencia de trombolisis sistematizada en infarto cerebral agudo en un hospital público de Chile / Thrombolysis for acute ischemic stroke with recombinant tissue plasminogen activator in a Chilean public hospital

    Scientific Electronic Library Online (English)

    Tatiana, Figueroa-Reyes; David, Sáez M; Eloy, Mansilla L; Rodrigo, Sánchez V; Jorge, Nogales-Gaete; Iris, Delgado B.

    2011-09-01

    Full Text Available [...] Abstract in english Background: The only accepted treatment for acute ischemic stroke is thrombolysis with recombinant tissue plasminogen activator (t-PA). It was implemented in Chile in 1996, although its use was mainly restricted in Chile to private clinics. Recently, at year 2009, we have implemented this treatment [...] in a public hospital. Aim: To describe the results of treatment of acute ischemic stroke with t-PA in a public hospital in Chile. Material and Methods: Prospective analysis of all eligible patients with acute ischemic stroke that were admitted within 4 hours of its onset and had no contraindications for thrombolysis. Results: In an eight months period, a total of 19 intravenous thrombolyses were performed in 12 males and seven females aged 28 to 79 years old. The mean lapse between onset of symptoms and onset of thrombolysis was 190 ± 57 min. Results were favorable, according to Rankin and National Institute of Health Stroke scales. Ninety days after treatment, 63% of patients had minimal or absent disability, 26% had moderate disability and only one (5%) had severe disability. One patient had a clinically not significant intracranial hemorrhage and one patient died six days after thrombolysis. Conclusions: These results indicate that thrombolysis can be successfully implemented in Chilean public hospitals. The limitations for its use in this setting are mostly administrative.

  19. Specificity of binding of the low density lipoprotein receptor-related protein to different conformational states of the clade E serpins plasminogen activator inhibitor-1 and proteinase nexin-1

    DEFF Research Database (Denmark)

    Jensen, Jan Kristian; Dolmer, Klavs

    2009-01-01

    The low density lipoprotein receptor-related protein (LRP) is the principal clearance receptor for serpins and serpin-proteinase complexes. The ligand binding regions of LRP consist of clusters of cysteine-rich approximately 40-residue complement-like repeats (CR), with cluster II being the principal ligand-binding region. To better understand the specificity of binding at different sites within the cluster and the ability of LRP to discriminate in vivo between uncomplexed and proteinase-complexed serpins, we have systematically examined the affinities of plasminogen activator inhibitor-1 (PAI-1) and proteinase nexin-1 (PN-1) in their native, cleaved, and proteinase-complexed states to (CR)(2) and (CR)(3) fragments of LRP cluster II. A consistent blue shift of the CR domain tryptophan fluorescence suggested a common mode of serpin binding, involving lysines on the serpin engaging the acidic region around the calcium binding site of the CR domain. High affinity binding of non-proteinase-complexed PAI-1 and PN-1 occurred to all fragments containing three CR domains (3-59 nm) and most that contain only two CR domains, although binding energies to different (CR)(3) fragments differed by up to 18% for PAI-1 and 9% for PN-1. No detectable difference in affinity was seen between native and cleaved serpin. However, the presence of proteinase in complex with the serpin enhanced affinity modestly and presumably nonspecifically. This may be sufficient to give preferential binding of such complexes in vivo at the relevant physiological concentrations.

  20. Experiencia de trombolisis sistematizada en infarto cerebral agudo en un hospital público de Chile Thrombolysis for acute ischemic stroke with recombinant tissue plasminogen activator in a Chilean public hospital

    Directory of Open Access Journals (Sweden)

    Tatiana Figueroa-Reyes

    2011-09-01

    Full Text Available Background: The only accepted treatment for acute ischemic stroke is thrombolysis with recombinant tissue plasminogen activator (t-PA. It was implemented in Chile in 1996, although its use was mainly restricted in Chile to private clinics. Recently, at year 2009, we have implemented this treatment in a public hospital. Aim: To describe the results of treatment of acute ischemic stroke with t-PA in a public hospital in Chile. Material and Methods: Prospective analysis of all eligible patients with acute ischemic stroke that were admitted within 4 hours of its onset and had no contraindications for thrombolysis. Results: In an eight months period, a total of 19 intravenous thrombolyses were performed in 12 males and seven females aged 28 to 79 years old. The mean lapse between onset of symptoms and onset of thrombolysis was 190 ± 57 min. Results were favorable, according to Rankin and National Institute of Health Stroke scales. Ninety days after treatment, 63% of patients had minimal or absent disability, 26% had moderate disability and only one (5% had severe disability. One patient had a clinically not significant intracranial hemorrhage and one patient died six days after thrombolysis. Conclusions: These results indicate that thrombolysis can be successfully implemented in Chilean public hospitals. The limitations for its use in this setting are mostly administrative.

  1. Thrombolytic and pharmacokinetic properties of human tissue-type plasminogen activator variants, obtained by deletion and/or duplication of structural/functional domains, in a hamster pulmonary embolism model.

    Science.gov (United States)

    Collen, D; Lijnen, H R; Vanlinthout, I; Kieckens, L; Nelles, L; Stassen, J M

    1991-02-12

    A pulmonary embolism model in hamsters was used for the quantitative evaluation of the thrombolytic and pharmacokinetic properties of variants of tissue-type plasminogen activator (t-PA). A 25 microliters 125I-fibrin labeled human plasma clot was made in vitro and injected into the jugular vein of heparinized hamsters. The extent of thrombolysis within 90 min was determined as the difference between the radioactivity injected in the jugular vein and that recovered in the heart and lungs. Recombinant t-PA (home-made rt-PA or Activase) infused intravenously over 60 min caused dose-dependent progressive thrombolysis. The results of thrombolytic potency (clot lysis in percent versus dose administered in mg/kg) and of specific thrombolytic activity (clot lysis in percent versus steady state plasma level in microgram/ml) were fitted with an exponentially transformed sigmoidal function y = 100 c/(1 + e-a(ax-eh] and the maximal percent lysis (c), the dose or plasma level at which maximal rate of lysis is achieved (b) and the maximal rate of lysis (z = 1/4 ac.eb) were determined. With rt-PA, these parameters were c = 72 +/- 6% (mean +/- SEM), b = 0.19 +/- 0.08 mg/kg, z = 68 +/- 25% lysis per mg/kg, with corresponding values of 87 +/- 5%, 0.07 +/- 0.03 mg/kg and 150 +/- 38% lysis per mg/kg for Activase (p = NS). Deletion of the finger and growth factor domains in rt-PA (rt-PA-delta FE) was not associated with marked alteration of the thrombolytic potency (c = 90 +/- 30%, b = 0.34 +/- 0.35 mg/kg, and z = 54 +/- 14% per mg/kg), but was associated with a significant reduction of the specific thrombolytic activity.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1905070

  2. Tumor necrosis factor alpha up-regulates in an autocrine manner the synthesis of plasminogen activator inhibitor type-1 during induction of monocytic differentiation of human HL-60 leukemia cells.

    Science.gov (United States)

    Lopez, S; Peiretti, F; Bonardo, B; Juhan-Vague, I; Nalbone, G

    2000-02-01

    Tumor necrosis factor-alpha (TNFalpha) critically regulates several cellular functions during monocyte/macrophage differentiation. We therefore investigated during the phorbol ester (phorbol 12-myristate 13-acetate (PMA))-induced monocyte/macrophage differentiation of the human HL-60 leukemia cells, if TNFalpha contributed to plasminogen activator inhibitor type-1 (PAI-1) synthesis that is initiated by a protein kinase Cbeta-extracellular signal-regulated kinase 2-dependent pathway (Lopez, S., Peiretti, F., Morange, P., Laouar, A., Fossat, C., Bonardo, B., Huberman, E., Juhan-Vague, I., and Nalbone, G. (1999) Thromb. Haemostasis 81, 415-422). Following PMA treatment, the level of TNFalpha mRNA strongly increased and appeared earlier than PAI-1 mRNA. An anti-TNFalpha antibody significantly inhibited the PMA-induced PAI-1 mRNA and protein levels. The recombinant human TNFalpha, which is inactive on native HL-60 cells in terms of PAI-1 synthesis, optimally potentiates it once HL-60 cells are committed into the differentiation process. The use of 1) the HL-525 cell line, a clone issued from HL-60 cells rendered resistant to PMA-induced differentiation, and 2) the transforming growth factorbeta-1/vitamin D3 differentiative mixture confirmed the relationships between the induction of differentiation and the potency of TNFalpha to up-regulate PAI-1 synthesis. In conclusion, we showed that during the induction of monocyte/macrophage differentiation, TNFalpha and PAI-1 gene expressions are activated and that synthesized TNFalpha up-regulates and prolongs, in an autocrine manner, the synthesis of PAI-1. PMID:10652289

  3. Mannheimia Species Associated with Ovine Mastitis▿

    OpenAIRE

    Omaleki, Lida; Barber, Stuart R.; Allen, Joanne L.; Browning, Glenn F

    2010-01-01

    Mannheimia glucosida, M. haemolytica, and M. ruminalis were isolated from cases of acute mastitis in ewes. M. glucosida was found to be a common cause of clinical mastitis in sheep. Selected phenotypic tests in addition to genotyping were needed to definitively identify Mannheimia species causing ovine mastitis.

  4. The Soluble Form of LR11 Protein Is a Regulator of Hypoxia-induced, Urokinase-type Plasminogen Activator Receptor (uPAR)-mediated Adhesion of Immature Hematological Cells*

    Science.gov (United States)

    Nishii, Keigo; Nakaseko, Chiaki; Jiang, Meizi; Shimizu, Naomi; Takeuchi, Masahiro; Schneider, Wolfgang J.; Bujo, Hideaki

    2013-01-01

    A key property of hematopoietic stem and progenitor cells (HSPCs) regarding differentiation from the self-renewing quiescent to the proliferating stage is their adhesion to the bone marrow (BM) niche. An important molecule involved in proliferation and pool size of HSPCs in the BM is the hypoxia-induced urokinase-type plasminogen activator receptor (uPAR). Here, we show that the soluble form (sLR11) of LR11 (also called SorLA or SORL1) modulates the uPAR-mediated attachment of HSPCs under hypoxic conditions. Immunohistochemical and mRNA expression analyses revealed that hypoxia increased LR11 expression in hematological c-Kit+ Lin? cells. In U937 cells, hypoxia induced a transient rise in LR11 transcription, production of cellular protein, and release of sLR11. Attachment to stromal cells of c-Kit+ Lin? cells of lr11?/? mice was reduced by hypoxia much more than of lr11+/+ animals. sLR11 induced the adhesion of U937 and c-Kit+ Lin? cells to stromal cells. Cell attachment was increased by sLR11 and reduced in the presence of anti-uPAR antibodies. Furthermore, the fraction of uPAR co-immunoprecipitated with LR11 in membrane extracts of U937 cells was increased by hypoxia. CoCl2, a chemical inducer of HIF-1?, enhanced the levels of LR11 and sLR11 in U937 cells. The decrease in hypoxia-induced attachment of HIF-1?-knockdown cells was largely prevented by exogenously added sLR11. Finally, hypoxia induced HIF-1? binding to a consensus binding site in the LR11 promoter. Thus, we conclude that sLR11 regulates the hypoxia-enhanced adhesion of HSPCs via an uPAR-mediated pathway that stabilizes the hematological pool size by controlling cell attachment to the BM niche. PMID:23486467

  5. The soluble form of LR11 protein is a regulator of hypoxia-induced, urokinase-type plasminogen activator receptor (uPAR)-mediated adhesion of immature hematological cells.

    Science.gov (United States)

    Nishii, Keigo; Nakaseko, Chiaki; Jiang, Meizi; Shimizu, Naomi; Takeuchi, Masahiro; Schneider, Wolfgang J; Bujo, Hideaki

    2013-04-26

    A key property of hematopoietic stem and progenitor cells (HSPCs) regarding differentiation from the self-renewing quiescent to the proliferating stage is their adhesion to the bone marrow (BM) niche. An important molecule involved in proliferation and pool size of HSPCs in the BM is the hypoxia-induced urokinase-type plasminogen activator receptor (uPAR). Here, we show that the soluble form (sLR11) of LR11 (also called SorLA or SORL1) modulates the uPAR-mediated attachment of HSPCs under hypoxic conditions. Immunohistochemical and mRNA expression analyses revealed that hypoxia increased LR11 expression in hematological c-Kit(+) Lin(-) cells. In U937 cells, hypoxia induced a transient rise in LR11 transcription, production of cellular protein, and release of sLR11. Attachment to stromal cells of c-Kit(+) Lin(-) cells of lr11(-/-) mice was reduced by hypoxia much more than of lr11(+/+) animals. sLR11 induced the adhesion of U937 and c-Kit(+) Lin(-) cells to stromal cells. Cell attachment was increased by sLR11 and reduced in the presence of anti-uPAR antibodies. Furthermore, the fraction of uPAR co-immunoprecipitated with LR11 in membrane extracts of U937 cells was increased by hypoxia. CoCl2, a chemical inducer of HIF-1?, enhanced the levels of LR11 and sLR11 in U937 cells. The decrease in hypoxia-induced attachment of HIF-1?-knockdown cells was largely prevented by exogenously added sLR11. Finally, hypoxia induced HIF-1? binding to a consensus binding site in the LR11 promoter. Thus, we conclude that sLR11 regulates the hypoxia-enhanced adhesion of HSPCs via an uPAR-mediated pathway that stabilizes the hematological pool size by controlling cell attachment to the BM niche. PMID:23486467

  6. Selective inhibition of GluN2D-containing N-methyl-D-aspartate receptors prevents tissue plasminogen activator-promoted neurotoxicity both in vitro and in vivo

    Directory of Open Access Journals (Sweden)

    Maubert Eric

    2011-10-01

    Full Text Available Abstract Background Tissue plasminogen activator (tPA exerts multiple functions in the central nervous system, depending on the partner with which it interacts. In particular, tPA acts as a positive neuromodulator of N-methyl-D-aspartate glutamatergic receptors (NMDAR. At the molecular level, it has been proposed that the pro-neurotoxicity mediated by tPA might occur through extrasynaptic NMDAR containing the GluN2D subunit. Thus, selective antagonists targeting tPA/GluN2D-containing NMDAR signaling would be of interest to prevent noxious effects of tPA. Results Here, we compared three putative antagonists of GluN2D-containing NMDAR and we showed that the new compound UBP145 ((2R*,3S*-1-(9-bromophenan-threne-3-carbonylpiperazine-2,3-dicarboxylic acid is far more selective for GluN2D subunits than memantine and PPDA (phenanthrene derivative (2S*, 3R*-1-(phenanthrene-2-carbonylpiperazine-2,3-dicarboxylic acid. Indeed, in vitro, in contrast to the two other compounds, UBP145 prevented NMDA toxicity only in neurons expressing GluN2D (ie, in cortical but not hippocampal neurons. Furthermore, in cultured cortical neurons, UBP145 fully prevented the pro-excitotoxic effect of tPA. In vivo, we showed that UBP145 potently prevented the noxious action of exogenous tPA on excitotoxic damages. Moreover, in a thrombotic stroke model in mice, administration of UBP145 prevented the deleterious effect of late thrombolysis by tPA. Conclusions In conclusion, tPA exerts noxious effects on neurons by acting on GluN2D-containing NMDAR and pharmacological antagonists of GluN2D-containing NMDAR could be used to prevent the ability of tPA to promote neurotoxicity.

  7. Conformational changes of ovine ?-1-proteinase inhibitor: The influence of heparin binding

    Science.gov (United States)

    Gupta, Vivek Kumar; Gowda, Lalitha R.

    2008-11-01

    ?-1-Proteinase inhibitor (?-1-PI), the archetypal serpin causes rapid, irreversible stoichiometric inhibition of redundant circulating serine proteases and is associated with emphysema, inflammatory response and maintenance of protease-inhibitor equilibrium in vascular and peri-vascular spaces. A homogenous preparation of heparin octasaccharide binds to ovine and human ?-1-PI and enhances their protease inhibitory activity phenomenally. Size-exclusion chromatography and dynamic light scattering experiments reveal that ovine ?-1-PI undergoes a decrease in the Stokes' radius upon heparin binding. A strong binding; characterizes this ?-1-PI-heparin interaction as revealed by the binding constant ( K?) 1.98 ± 0.2 × 10 -6 M and 2.1 ± 0.2 × 10 -6 M determined by fluorescence spectroscopy and equilibrium dialysis, respectively. The stoichiometry of heparin binding to ovine ?-1-PI was 1.1 ± 0.2:1. The Stern-Volmer constants ( Ksv) for heparin activated ovine and human ?-1-PI were found to be 5.13 × 10 -6 M and 5.67 × 10 -6 M, respectively, significantly higher than the native inhibitors. FTIR and CD spectroscopy project the systematic structural reorientations that ?-1-PI undergoes upon heparin binding characterized by a decrease in ?-helical content and a concomitant increase in ?-turn and random coil elements. It is likely that these conformational changes result in the movement of the ?-1-PI reactive site loop into an extended structure that is better poised to combat the cognate protease and accelerate the inhibition.

  8. Sensory and microbiological evaluation of traditional ovine ricotta cheese in modified atmosphere packaging

    Directory of Open Access Journals (Sweden)

    Isabella Mancuso

    2014-06-01

    Full Text Available Ovine ricotta cheese is a traditional Sicilian dairy product characterised by high humidity and a short shelf life (2-4 days when refrigerated. The increasing demand for fresh food has prompted manufacturers to develop special packaging techniques, such as modified atmosphere packaging (MAP, that can extend the shelf life and maintain the organoleptic characteristics of ovine ricotta cheese. The aim of the present study was to evaluate the shelf life of fresh MAP-packed ovine ricotta cheese by monitoring the microbiological, chemical, physical and organoleptic characteristics of the product. Samples of a single batch were packed in MAP or vacuum packed and stored at 4°C for 24 and 7 days, respectively. Water activity, pH, physicochemical parameters and microbiological characteristics were examined. A sensory panel rated the product’s main organoleptic characteristics (colour, odour, flavour and texture. Results showed that MAP controlled the development of any unwanted microflora, but did not affect the development of intrinsic lactic floras or chemical parameters. Sensory analysis revealed that overall the MAP-packed ricotta remained acceptable for up to 15 days of storage. The vacuum-packed ricotta cheese, however, showed a progressive deterioration in organoleptic characteristics from day 5 onward and therefore had a shorter shelf life. In conclusion, the ability of MAP to extend the shelf life of a traditional regional product (such as fresh ovine ricotta cheese guarantees consumers a quality product and provides opportunities for manufacturers to expand their markets beyond national boundaries.

  9. [The role of the sympathetic cholinergic pathway in the neurogenic control of the release of tissue-type plasminogen activator into the blood].

    Science.gov (United States)

    Bashkov, G B; Sergeev, I Iu; Medvedeva, N A; Makarov, V A

    1992-05-01

    In anesthetized cats, electric stimulation of the sympathetic chain induced vasoconstriction and release of t-PA from the vascular wall into the blood. Efferent stimulation of the sympathetic chain against the background of alpha-adrenoceptor blockade increased the blood flow and the t-PA activity. Atropin suppressed both the vascular relaxation and the t-PA release into the blood. A neurogenous mechanism controlling t-PA release from the vascular wall caused by activation of sympathetic cholinergic pathway and associated with excitation of vascular M-cholinoreceptors by acetylcholine, has been suggested. PMID:1334882

  10. Lytic efficacy of apoli protein E2 (ApoE2) and recombinant tissue plasminogen activator (rt-PA) treatment with 120 kHz ultrasound in an in-vitro human clot model

    Science.gov (United States)

    Meunier, Jason M.; Cheng, Jason Y.; Clark, Joseph F.; Shaw, George J.

    2005-04-01

    Currently, the only FDA approved therapy for acute ischemic stroke is recombinant tissue plasminogen activator (rt-PA). However rt-PA has substantial side effects such as hemorrhage. This has led to interest in other potential therapies. For example, ultrasound (US) increases the lytic efficacy of rt-PA. Also, apolipoprotein E2 (ApoE2) increases rt-PA activity. This suggests combining US, ApoE2 and rt-PA to improve thrombolysis, but the efficacy is not known. Here, the lytic efficacy of apoE2, rt-PA and 120 kHz US is measured in a human clot model. Whole blood was obtained from volunteers, after local institutional approval. Clots were formed in 1.7 mm micropipettes, and placed in a water tank that allowed microscopic video imaging during US and thrombolytic exposure. Clots were treated with rt-PA ([rt-PA]=3.15 ?g/ml), rt-PA and apoE2 ([apoE2]=9.8 ?g/ml), or rt-PA, apoE2 and 120 kHz US (0.35 MPa, PRF=1667 Hz, 80% duty cycle) for 15 min at 37°C in human plasma. Clot lysis was visually recorded and the lysis depth (LD) determined from these data using an image analysis algorithm. LD was linear with time for all treatments (R2>=0.81), allowing the determination of a lytic rate (LR). LR was found to be 0.35+/-0.03, 1.55+/-0.11, and 0.75+/-0.04 ?m/min for the rt-PA, rt-PA and apoE2, and US treated groups respectively. The thrombolytic efficacy of rt-PA is enhanced by ApoE2. The interaction of 120 kHz with apoE2 and rt-PA showed a reduced lytic efficacy compared with rt-PA and apoE2 treatment alone. It is possible that US interferes with the ApoE2-mediated activation of rt-PA.

  11. Marker genes for activation of the RNA interference (RNAi) pathway in the free-living nematode Caenorhabditis elegans and RNAi development in the ovine nematode Teladorsagia circumcincta.

    Science.gov (United States)

    Tzelos, T; Matthews, J B; Whitelaw, B; Knox, D P

    2015-03-01

    The nematode Teladorsagia circumcincta is a major cause of parasitic gastroenteritis in sheep in temperate regions. The development of resistance to the major anthelmintic classes used for its control is a threat to small ruminant farming sustainability. Vaccination is a potential alternative control method for this nematode. Gene datasets can be exploited to identify potential vaccine candidates and these validated further by methods such as RNA interference (RNAi) prior to vaccine trials. Previous reports indicate that RNAi in parasitic nematodes is inconsistent and, to date, there are no internal controls that indicate activation of the RNAi pathway in response to double-stranded RNA (dsRNA). The present aims were to determine whether or not the transcription levels of potential marker genes in the RNAi pathway could indicate activation of the pathway in Caenorhabditis elegans and to develop an RNAi platform in T. circumcincta. In C. elegans, transcript levels of three candidate marker genes, Ce-dcr-1 (Dicer), Ce-ego-1 (Enhancer of Glp-One family member) and Ce-rsd-3 (RNAi Spreading Defective), were analysed and results indicated that activation of the pathway had no effect on transcript levels of these genes. In T. circumcincta, two vaccine candidate genes from the Activation-associated Secreted Protein (ASP) family were targets for knockdown. RNAi experiments showed successful silencing of both targets, although inconsistencies in efficacy were observed. After testing a number of parameters that might affect variability, it was found that the length of the storage period of the larvae plays an important role in the consistency of the RNAi results. PMID:24345514

  12. Identification of a peroxisome proliferator responsive element (PPRE)-like cis-element in mouse plasminogen activator inhibitor-1 gene promoter

    International Nuclear Information System (INIS)

    PAI-1 is expressed and secreted by adipose tissue which may mediate the pathogenesis of obesity-associated cardiovascular complications. Evidence is presented in this report that PAI-1 is not expressed by preadipocyte, but significantly induced during 3T3-L1 adipocyte differentiation and the PAI-1 expression correlates with the induction of peroxisome proliferator-activated receptor ? (PPAR?). A peroxisome proliferator responsive element (PPRE)-like cis-element (-206TCCCCCATGCCCT-194) is identified in the mouse PAI-1 gene promoter by electrophoretic mobility shift assay (EMSA) combined with transient transfection experiments; the PPRE-like cis-element forms a specific DNA-protein complex only with adipocyte nuclear extracts, not with preadipocyte nuclear extracts; the DNA-protein complex can be totally competed away by non-labeled consensus PPRE, and can be supershifted with PPAR? antibody. Mutation of this PPRE-like cis-element can abolish the transactivation of mouse PAI-1 promoter mediated by PPAR?. Specific PPAR? ligand Pioglitazone can significantly induce the PAI-1 expression, and stimulate the secretion of PAI-1 into medium

  13. Valor diagnóstico de los niveles séricos del receptor soluble de la uroquinasa en adultos con síndrome nefrótico idiopático / Diagnostic value of soluble urokinase-type plasminogen activator receptor serum levels in adults with idiopathic nephrotic syndrome

    Scientific Electronic Library Online (English)

    Alfons, Segarra; Elías, Jatem; M. Teresa, Quiles; M. Antonia, Arbós; Helena, Ostos; Naiara, Valtierra; Clara, Carnicer; Irene, Agraz; M. Teresa, Salcedo.

    Full Text Available Introducción: Estudios recientes sugieren que los niveles del receptor soluble de la uroquinasa (suPAR) podrían ser útiles para diferenciar la glomeruloesclerosis focal y segmentaria (GFS) idiopática de otras glomerulopatías causantes de síndrome nefrótico, pero estos datos no han sido confirmados e [...] n estudios independientes. El objetivo de nuestro estudio es analizar si los niveles circulantes de suPAR son útiles para identificar la enfermedad renal primaria en enfermos afectos de síndrome nefrótico secundario a GFS, enfermedad por cambios mínimos o nefropatía membranosa (NM) idiopática. Métodos: Se realizaron mediciones de niveles de suPAR circulante en el momento del diagnóstico en 60 pacientes con síndrome nefrótico secundario a GFS, enfermedad por cambios mínimos (ECM) y NM. Se analizaron las correlaciones entre niveles de suPAR y variables demográficas, clínicas y bioquímicas. La sensibilidad y la especificidad de suPAR para diferenciar a los enfermos con GFS se analizaron mediante curvas ROC. Resultados: Tras ajustar por edad y función renal, los niveles de suPAR fueron significativamente más elevados en enfermos con GFS que en ECM (p 3531 pg/ml podría tener una elevada especificidad (pero baja sensibilidad) para el diagnóstico de GFS. Abstract in english Introduction: Recent studies suggest that soluble urokinase-type plasminogen activator receptor (suPAR) levels could be useful for distinguishing idiopathic focal segmental glomerulosclerosis (FSGS) from other glomerulopathies that cause nephrotic syndrome, but these data have not been confirmed in [...] independent studies. The objective of our study is to analyse whether circulating levels of suPAR are useful for identifying primary kidney disease in patients with nephrotic syndrome secondary to FSGS, minimal change disease or idiopathic membranous nephropathy (MN). Methods: We measured circulating suPAR at diagnosis in 60 patients with nephrotic syndrome secondary to FSGS, minimal change disease (MCD) and membranous nephropathy (MN). The correlations between suPAR levels and demographic, clinical and biochemical variables were analysed. The sensitivity and specificity of suPAR in distinguishing FSGS patients were analysed by ROC curves. Results: After adjusting for age and renal function, suPAR levels were significantly higher in patients with FSGS than in those with MCD (p3531pg/ml could have a high specificity (but a low sensitivity) in the diagnosis of FSGS.

  14. Plasminogen interacts with human platelets through two distinct mechanisms.

    OpenAIRE

    Miles, L A; Ginsberg, M H; White, J G; Plow, E. F.

    1986-01-01

    Glu-plasminogen, the native form of plasminogen, interacts in a specific and saturable manner with unstimulated human platelets, and the binding is enhanced fivefold by thrombin stimulation (Miles and Plow, 1985. J. Biol. Chem. 260:4303). This study characterizes the nature of the Glu-plasminogen binding sites by analyzing platelets deficient in selected proteins and functions. Platelets from patients with afibrinogenemia, Gray platelet syndrome, and the Cam Variant of thrombasthenia, a form ...

  15. 78 FR 68327 - Importation of Ovine Meat From Uruguay

    Science.gov (United States)

    2013-11-14

    ... effective and published in the Federal Register on May 29, 2003 (68 FR 31940-31949, Docket No. 02-109-3... FR 10266-10269, Docket No. APHIS-2008-0085), we proposed to also allow the importation of fresh ovine... Health Inspection Service 9 CFR Part 94 RIN 0579-AD17 Importation of Ovine Meat From Uruguay...

  16. Translation Elongation Factor Tuf of Acinetobacter baumannii Is a Plasminogen-Binding Protein

    Science.gov (United States)

    Koenigs, Arno; Zipfel, Peter F.; Kraiczy, Peter

    2015-01-01

    Acinetobacter baumannii is an important nosocomial pathogen, causing a variety of opportunistic infections of the skin, soft tissues and wounds, urinary tract infections, secondary meningitis, pneumonia and bacteremia. Over 63% of A. baumannii infections occurring in the United States are caused by multidrug resistant isolates, and pan-resistant isolates have begun to emerge that are resistant to all clinically relevant antibiotics. The complement system represents the first line of defense against invading pathogens. However, many A. baumannii isolates, especially those causing severe bacteremia are resistant to complement-mediated killing, though the underlying mechanisms remain poorly understood. Here we show for the first time that A. baumannii binds host-derived plasminogen and we identify the translation elongation factor Tuf as a moonlighting plasminogen-binding protein that is exposed on the outer surface of A. baumannii. Binding of plasminogen to Tuf is at least partly dependent on lysine residues and ionic interactions. Plasminogen, once bound to Tuf can be converted to active plasmin and proteolytically degrade fibrinogen as well as the key complement component C3b. Thus, Tuf acts as a multifunctional protein that may contribute to virulence of A. baumannii by aiding in dissemination and evasion of the complement system. PMID:26230848

  17. Genetic architecture of gene expression in ovine skeletal muscle

    DEFF Research Database (Denmark)

    Kogelman, Lisette Johanna Antonia; Byrne, Keren; Vuocolo, Tony; Watson-Haigh, Nathan S.; Kadarmideen, Haja; Kijas, James W.; Oddy, Hutton V.; Gardiner, Graham E.; Gondro, Cedric; Tellam, Ross L.

    2011-01-01

    -based gene expression data we directly tested the hypothesis that there is genetic structure in the gene expression program in ovine skeletal muscle.Results: The genetic performance of six sires for a well defined muscling trait, longissimus lumborum muscle depth, was measured using extensive progeny testing...... has potential, amongst other mechanisms, to alter gene expression via cis- or trans-acting mechanisms in a manner that impacts the functional activities of specific pathways that contribute to muscling traits. By integrating sire-based genetic merit information for a muscling trait with progeny...... and expressed as an Estimated Breeding Value by comparison with contemporary sires. Microarray gene expression data were obtained for longissimus lumborum samples taken from forty progeny of the six sires (4-8 progeny/sire). Initial unsupervised hierarchical clustering analysis revealed strong genetic...

  18. A unique method to produce transgenic embryos in ovine, porcine, feline, bovine and equine species.

    Science.gov (United States)

    Pereyra-Bonnet, F; Fernández-Martín, R; Olivera, R; Jarazo, J; Vichera, G; Gibbons, A; Salamone, D

    2008-01-01

    Transgenesis is an essential tool in many biotechnological applications. Intracytoplasmic sperm injection (ICSI)-mediated gene transfer is a powerful technique to obtain transgenic pups; however, most domestic animal embryos do not develop properly after ICSI. An additional step in the protocol, namely assistance by haploid chemical activation, permits the use of ICSI-mediated gene transfer to generate transgenic preimplantation embryos in a wide range of domestic species, including ovine, porcine, feline, equine and bovine. In the present study, spermatozoa from five species were coincubated with pCX-EGFP plasmid and injected into metaphase II oocytes. The chemical activation protocol consisted of ionomycin plus 6-dimethylaminopurine. We detected high proportions of fluorescent EGFP embryos for all five species (23-60%), but with a high frequency of mosaic expression (range 60-85%). To our knowledge, this is the first study to produce exogenous DNA expression in feline and equine embryos. Chemical activation reduces the lag phase of egfp expression in ovine embryos. Our results show that this unique method could be used to obtain ovine, porcine, feline, bovine and equine transgenic preimplantation embryos. PMID:18842176

  19. Structure of uPAR, plasminogen, and sugar-binding sites of the 300 kDa mannose 6-phosphate receptor

    OpenAIRE

    Olson, Linda J.; Yammani, Rama D.; Dahms, Nancy M; Kim, Jung-Ja P

    2004-01-01

    The 300 kDa cation-independent mannose 6-phosphate receptor (CI-MPR) mediates the intracellular transport of newly synthesized lysosomal enzymes containing mannose 6-phosphate on their N-linked oligosaccharides. In addition to its role in lysosome biogenesis, the CI-MPR interacts with a number of different extracellular ligands at the cell surface, including latent transforming growth factor-?, insulin-like growth factor-II, plasminogen, and urokinase-type plasminogen activator receptor (uPAR...

  20. Substantial differences between human and ovine mesenchymal stem cells in response to osteogenic media: how to explain and how to manage?

    Science.gov (United States)

    Kalaszczynska, Ilona; Ruminski, Slawomir; Platek, Anna E; Bissenik, Igor; Zakrzewski, Piotr; Noszczyk, Maria; Lewandowska-Szumiel, Malgorzata

    2013-10-01

    It is expected that use of adult multipotential mesenchymal stem cells (MSCs) for bone tissue engineering (TE) will lead to improvement of TE products. Prior to clinical application, biocompatibility of bone TE products need to be tested in vitro and in vivo. In orthopedic research, sheep are a well-accepted model due to similarities with humans and are assumed to be predictive of human outcomes. In this study we uncover differences between human and ovine bone marrow-derived MSCs (BMSCs) and adipose tissue-derived MSCs (ADSCs) in response to osteogenic media. Osteogenic differentiation of BMSCs and ADSCs was monitored by alkaline phosphatase (ALP) activity and calcium deposition. Mineralization of ovine BMSC was achieved in medium containing NaH2PO4 as a source of phosphate ions (Pi), but not in medium containing ?-glycerophosphate (?-GP), which is most often used. In a detailed study we found no induction of ALP activity in ovine BMSCs and ADSCs upon osteogenic stimulation, which makes ?-GP an unsuitable source of phosphate ions for ovine cells. Moreover, mineralization of human ADSCs was more efficient in osteogenic medium containing NaH2PO4. These results indicate major differences between ovine and human MSCs and suggest that standard in vitro osteogenic differentiation techniques may not be suitable for all types of cells used in cell-based therapies. Since mineralization is a widely accepted marker of the osteogenic differentiation and maturation of cells in culture, it may lead to potentially misleading results and should be taken into account at the stage of planning and interpreting preclinical observations performed in animal models. We also present a cell culture protocol for ovine ADSCs, which do not express ALP activity and do not mineralize under routine pro-osteogenic conditions in vitro. We plan to apply it in preclinical experiments of bone tissue-engineered products performed in an ovine model. PMID:24083091

  1. Effects of antibacterial agents on in vitro ovine ruminal biotransformation of the hepatotoxic pyrrolizidine alkaloid jacobine.

    Science.gov (United States)

    Wachenheim, D E; Blythe, L L; Craig, A M

    1992-08-01

    Ingestion of pyrrolizidine alkaloids, naturally occurring plant toxins, causes illness and death in a number of animal species. Senecio jacobaea pyrrolizidine alkaloids cause significant economic losses due to livestock poisoning, particularly in the Pacific Northwest. Some sheep are resistant to pyrrolizidine alkaloid poisoning, because ovine ruminal biotransformation detoxifies free pyrrolizidine alkaloids in digesta. Antibacterial agents modify ruminal fermentation. Pretreatment with antibacterial agents may account for some animal variability in resistance to pyrrolizidine alkaloid toxicosis, and antibacterial agents can also be used for characterizing ruminal pyrrolizidine alkaloid-biotransforming microflora. The objective of this study was to evaluate the effects of antibacterial agents on biotransformation of a predominant S. jacobaea pyrrolizidine alkaloid, jacobine, in ovine ruminal contents. Ovine ruminal jacobine biotransformation was tested in vitro with 20 independent antibacterial agents. Low amounts of rifampin and erythromycin prevented jacobine biotransformation. Chlortetracycline, lasalocid, monensin, penicillin G, and tetracycline were slightly less effective at inhibiting jacobine biotransformation. Bacitracin, crystal violet, kanamycin, and neomycin were moderately inhibitory against jacobine biotransformation. Brilliant green, chloramphenicol, gramicidin, nalidixic acid, polymyxin B SO4, sodium azide, streptomycin, sulfisoxazole, and vancomycin had little to no effect on jacobine biotransformation. The antibiotics that were most effective at inhibiting biotransformation were those that are active against gram-positive bacteria. Therefore, gram-positive bacteria are most likely critical members of the jacobine-biotransforming consortia. PMID:1514802

  2. Enrichment and Short Term Culture of the Ovine Gonocyte

    Directory of Open Access Journals (Sweden)

    Rongfeng Li

    2011-01-01

    Full Text Available The aim of this study was to investigate the effects of two types of ovine testis cells population as feeder cell on in vitro culture of the enriched ovine gonocytes. The feeder cell populations were prepared from 5-6 months old ovine testis. The 1-2 months old neonatal rams were used to isolate germ cells through a two step enzymatic digestion followed by differential plating for SSCs enrichment. Isolated and enriched cells were characterized by using PLZF and VASA antibody. During the 1st week of culture, gonocyte formed pairs and chains of type A spermatogonia. After 1 week, colonies started to increase in size. About 2 weeks later, more colonies in type II feeder group kept undifferentiated and looks more effective regarding colony formation of spermatogonia compared with type I feeder group in the ovine spermatogonial stem cell culture system.

  3. The ovine mammary gland as an experimental model to determine the virulence of animal ureaplasmas.

    OpenAIRE

    Ball, H. J.; Mackie, D. P.

    1985-01-01

    As an estimate of their virulence, the ability of ovine, bovine, canine, feline and simian ureaplasma strains to cause mastitis in the ovine mammary gland was investigated. Five ovine ureaplasmas produced a clinical mastitis. Broth cultures of seven bovine ureaplasmas were unable to infect the ovine gland, but two of these strains plus one other were able to do so following passage through the bovine udder. One of two canine strains and a feline strain both caused mastitis, but the simian str...

  4. Digestion and absorption of protein along ovine gastrointestinal tract

    International Nuclear Information System (INIS)

    Digestion and absorption of protein were determined in ovine gastrointestinal tract with cerium-141 as an unabsorbed reference substance. Nitrogen flows changed little in rumen and reticulum, but in the proximal small intestine flows increased because of secretion of .9 g nitrogen per day per kg body weight. This secretion included trypsin, chymotrypsin, elastase, and carboxypeptidases A and B; maximal activity was in proximal segments of the small intestine and decreased with distance from the pylorus. Activity of chymotrypsin decreased more rapidly than that of trypsin. Amino acid flows reflected the influx of protein in the duodenum; absorption was approximately 55% in the terminal ileum. No major changes of proportions of individual amino acids were observed. Overall nitrogen absorption was 72.6% of which 6% was in the large intestine. The major soluble protein fraction in the gastrointestinal tract consisted of peptides with molecular weight 7,000 to 14,000 daltons. Soluble high molecular weight protein was observed only in rumen and duodenum. Low molecular weight peptides and amino acids accumulated only in the proximal small intestine. Solubilization of protein and breakdown of peptides of 7,000 to 14,000 molecular weight appear to be rate limiting for protein absorption in sheep

  5. Genetic architecture of gene expression in ovine skeletal muscle

    DEFF Research Database (Denmark)

    Kogelman, Lisette Johanna Antonia; Byrne, Keren

    2011-01-01

    Background: In livestock populations the genetic contribution to muscling is intensively monitored in the progeny of industry sires and used as a tool in selective breeding programs. The genes and pathways conferring this genetic merit are largely undefined. Genetic variation within a population has potential, amongst other mechanisms, to alter gene expression via cis- or trans-acting mechanisms in a manner that impacts the functional activities of specific pathways that contribute to muscling traits. By integrating sire-based genetic merit information for a muscling trait with progeny-based gene expression data we directly tested the hypothesis that there is genetic structure in the gene expression program in ovine skeletal muscle.Results: The genetic performance of six sires for a well defined muscling trait, longissimus lumborum muscle depth, was measured using extensive progeny testing and expressed as an Estimated Breeding Value by comparison with contemporary sires. Microarray gene expression data were obtained for longissimus lumborum samples taken from forty progeny of the six sires (4-8 progeny/sire). Initial unsupervised hierarchical clustering analysis revealed strong genetic architecture to the gene expression data, which also discriminated the sire-based Estimated Breeding Value for the trait. An integrated systems biology approach was then used to identify the major functional pathways contributing to the genetics of enhanced muscling by using both Estimated Breeding Value weighted gene co-expression network analysis and a differential gene co-expression network analysis. The modules of genes revealed by these analyses were enriched for a number of functional terms summarised as muscle sarcomere organisation and development, protein catabolism (proteosome), RNA processing, mitochondrial function and transcriptional regulation.Conclusions: This study has revealed strong genetic structure in the gene expression program within ovine longissimus lumborum muscle. The balance between muscle protein synthesis, at the levels of both transcription and translation control, and protein catabolism mediated by regulated proteolysis is likely to be the primary determinant of the genetic merit for the muscling trait in this sheep population. There is also evidence that high genetic merit for muscling is associated with a fibre type shift toward fast glycolytic fibres. This study provides insight into mechanisms, presumably subject to strong artificial selection, that underpin enhanced muscling in sheep populations. © 2011 Kogelman et al; licensee BioMed Central Ltd.

  6. Magnetic resonance imaging (MRI) anatomy of the ovine lumbar spine.

    Science.gov (United States)

    Nisolle, J F; Wang, X Q; Squélart, M; Hontoir, F; Kirschvink, N; Clegg, P; Vandeweerd, J M

    2014-06-01

    Although the ovine spine is a useful research model for intervertebral disc pathology and vertebral surgery, there is little peer-reviewed information regarding the MRI anatomy of the ovine spine. To describe the lumbar spine MRI anatomy, 10 lumbar segments of cadaver ewes were imaged by 1.5-Tesla MR. Sagittal and transverse sequences were performed in T1 and T2 weighting (T1W, T2W), and the images were compared to gross anatomic sagittal and transverse sections performed through frozen spines. MRI was able to define most anatomic structures of the ovine spine in a similar way as can be imaged in humans. In both T1W and T2W, the signals of ovine IVDs were similar to those observed in humans. Salient anatomic features were identified: (1) a 2- to 3-mm linear zone of hypersignal was noticed on both extremities of the vertebral body parallel to the vertebral plates in sagittal planes; (2) the tendon of the crura of the diaphragm appeared as a hypointense circular structure between hypaxial muscles and the aorta and caudal vena cava; (3) dorsal and ventral longitudinal ligaments and ligamentum flavum were poorly imaged; (4) no ilio-lumbar ligament was present; (5) the spinal cord ended between S1-S2 level, and the peripheral white matter and central grey matter were easily distinguished on T1W and T2W images. This study provides useful reference images to researchers working with ovine models. PMID:23668479

  7. 77Se NMR studies on ovine erythrocyte glutathione peroxidase

    International Nuclear Information System (INIS)

    To facilitate 77Se NMR observation of the endogenous active site selenium in ovine erythrocyte glutathione peroxidase (GSHPx), lambs have been maintained on an artificial diet deficient in selenium and supplemented with 0.2 ppm 92atom% 77Se , as selenite. After 5 months, preparations of GSHPx showed that incorporation of selenium from the artificial diet represented 88% of the GSHPx selenium. Each monthly bleeding of two sheep routinely yielded 20mg of pure 77Se-enriched GSHPx. Limitations on the solubility of the enzyme have so far prevented observation of 77Se resonances from the intact enzyme. Upon denaturation, a broad resonance is observed at 277 ppm, indicating that the selenium is involved in mixed selenide sulfide bonds both inter and intramolecularly. Reduction of the SeS bonds with dithiothreitol resulted in an upfield shift of the 77Se resonance to -212 ppm at pH 8 and -55ppm at pH4.2, consistent with formation of Se- and SeH respectively. It is concluded that the selenium is most probably in the SeS or Se- form in the intact enzyme. Relaxation time measurements were made at field strengths of 4.7 and 9.4T, which demonstrated the dominance of chemical shift anisotropy (CSA) relaxation for the selenium in GSHPx. A value of ? 262 ppm was determined for the CSA of the iodoacetamide derivative of GSHPx

  8. Characterization and Isolation of Ovine Spermatogonial Stem Cells

    Directory of Open Access Journals (Sweden)

    Muren Herrid

    2012-01-01

    Full Text Available Identification and isolation of Spermatogonial Stem Cells (SSCs are a prerequisite for culture, genetic manipulation and transplantation research. In this study, the appropriate age of the donor ram for SSCs isolation was investigated. Spermatogonial stem cell specific marker protein Promyelocytic Leukaemia Zinc-Finger (PLZF and male germ cell marker VASA double staining were checked on the paraffin sections of ovine testis. Type A spermatogonial marker PGP9.5 and sertoli cell marker Vimentin double staining were also used to confirm testes development. Results indicated that testes from neonatal rams had only one type of undifferentiated germ cell gonocyte and should be suitable for ovine SSCs isolation. VASA and Vimentin double staining of the isolated testis cells confirm the efficiency of ovine SSCs isolation.

  9. Acquisition of Host Plasmin Activity by the Swine Pathogen Streptococcus suis Serotype 2

    OpenAIRE

    Jobin, Marie-Claude; Brassard, Julie; Quessy, Sylvain; Gottschalk, Marcelo; Grenier, Daniel

    2004-01-01

    In this study, the plasminogen-binding activity of Streptococcus suis serotype 2 was investigated. Bound human plasminogen was activated by purified streptokinase, urokinase, or Streptococcus dysgalactiae subsp. equisimilis culture supernatant. Both human and porcine plasminogen were bound by S. suis. Binding was inhibited by ?-aminocaproic acid, and the plasminogen receptor was heat and sodium dodecyl sulfate resistant. One of the receptors was identified as glyceraldehyde-3-phosphate dehydr...

  10. Global distribution and diversity of ovine-associated Staphylococcus aureus.

    Science.gov (United States)

    Smith, Edward M; Needs, Polly F; Manley, Grace; Green, Laura E

    2014-03-01

    Staphylococcus aureus is an important pathogen of many species, including sheep, and impacts on both human and animal health, animal welfare, and farm productivity. Here we present the widest global diversity study of ovine-associated S. aureus to date. We analysed 97 S. aureus isolates from sheep and sheep products from the UK, Turkey, France, Norway, Australia, Canada and the USA using multilocus sequence typing (MLST) and spa typing. These were compared with 196 sheep isolates from Europe (n=153), Africa (n=28), South America (n=14) and Australia (n=1); 172 bovine, 68 caprine and 433 human S. aureus profiles. Overall there were 59 STs and 87 spa types in the 293 ovine isolates; in the 97 new ovine isolates there were 22 STs and 37 spa types, including three novel MLST alleles, four novel STs and eight novel spa types. Three main CCs (CC133, CC522 and CC700) were detected in sheep and these contained 61% of all isolates. Four spa types (t002, t1534, t2678 and t3576) contained 31% of all isolates and were associated with CC5, CC522, CC133 and CC522 respectively. spa types were consistent with MLST CCs, only one spa type (t1403) was present in multiple CCs. The three main ovine CCs have different but overlapping patterns of geographical dissemination that appear to match the location and timing of sheep domestication and selection for meat and wool production. CC133, CC522 and CC700 remained ovine-associated following the inclusion of additional host species. Ovine isolates clustered separately from human and bovine isolates and those from sheep cheeses, but closely with caprine isolates. As with cattle isolates, patterns of clonal diversification of sheep isolates differ from humans, indicative of their relatively recent host-jump. PMID:24035790

  11. Leptin Matures Aspects of Lung Structure and Function in the Ovine Fetus.

    Science.gov (United States)

    De Blasio, Miles J; Boije, Maria; Kempster, Sarah L; Smith, Gordon C S; Charnock-Jones, D Stephen; Denyer, Alice; Hughes, Alexandra; Wooding, F B Peter; Blache, Dominique; Fowden, Abigail L; Forhead, Alison J

    2016-01-01

    In human and ovine fetuses, glucocorticoids stimulate leptin secretion, although the extent to which leptin mediates the maturational effects of glucocorticoids on pulmonary development is unclear. This study investigated the effects of leptin administration on indices of lung structure and function before birth. Chronically catheterized singleton sheep fetuses were infused iv for 5 days with either saline or recombinant ovine leptin (0.5 mg/kg · d leptin (LEP), 0.5 LEP or 1.0 mg/kg · d, 1.0 LEP) from 125 days of gestation (term ?145 d). Over the infusion, leptin administration increased plasma leptin, but not cortisol, concentrations. On the fifth day of infusion, 0.5 LEP reduced alveolar wall thickness and increased the volume at closing pressure of the pressure-volume deflation curve, interalveolar septal elastin content, secondary septal crest density, and the mRNA abundance of the leptin receptor (Ob-R) and surfactant protein (SP) B. Neither treatment influenced static lung compliance, maximal lung volume at 40 cmH2O, lung compartment volumes, alveolar surface area, pulmonary glycogen, protein content of the long form signaling Ob-Rb or phosphorylated signal transducers and activators of transcription-3, or mRNA levels of SP-A, C, or D, elastin, vascular endothelial growth factor-A, the vascular endothelial growth factor receptor 2, angiotensin-converting enzyme, peroxisome proliferator-activated receptor ?, or parathyroid hormone-related peptide. Leptin administration in the ovine fetus during late gestation promotes aspects of lung maturation, including up-regulation of SP-B. PMID:26479186

  12. Regulation of DNA synthesis and the cell cycle in human prostate cancer cells and lymphocytes by ovine uterine serpin

    OpenAIRE

    Hansen Peter J; Padua Maria B

    2008-01-01

    Abstract Background Uterine serpins are members of the serine proteinase inhibitor superfamily. Like some other serpins, these proteins do not appear to be functional proteinase inhibitors. The most studied member of the group, ovine uterine serpin (OvUS), inhibits proliferation of several cell types including activated lymphocytes, bovine preimplantation embryos, and cell lines for lymphoma, canine primary osteosarcoma and human prostate cancer (PC-3) cells. The goal for the present study wa...

  13. Plasminogen and fibrinogen plasma levels in coronary artery disease

    Scientific Electronic Library Online (English)

    Luciana Moreira, Lima; Maria das Graças, Carvalho; Marinez de Oliveira, Sousa.

    Full Text Available OBJECTIVE: The formation of thrombi at the site of atherosclerotic lesions plays a central role in atherothrombosis. Impaired fibrinolysis may exacerbate pre-existing coronary artery disease and potentiate its evolution. While the fibrinogen plasma level has been strongly associated with the severit [...] y of coronary artery disease, its relevance in the evaluation of plasminogen in coronary artery disease patients remains unclear. This study evaluated fibrinogen and plasminogen levels in subjects with coronary artery disease as diagnosed by angiography. METHODS: This is a cross-sectional study. Blood samples obtained from 17 subjects with angiographically normal coronary arteries (controls), 12 with mild/moderate atheromatosis and 28 with severe atheromatosis were evaluated. Plasma plasminogen and fibrinogen levels were measured by chromogenic and coagulometric methods, respectively. RESULTS: Fibrinogen levels were significantly higher in the severe atheromatosis group compared to the other groups(p-value

  14. Development of a Cost-effective Ovine Polyclonal Antibody-Based Product, EBOTAb, to Treat Ebola Virus Infection.

    Science.gov (United States)

    Dowall, Stuart David; Callan, Jo; Zeltina, Antra; Al-Abdulla, Ibrahim; Strecker, Thomas; Fehling, Sarah K; Krähling, Verena; Bosworth, Andrew; Rayner, Emma; Taylor, Irene; Charlton, Sue; Landon, John; Cameron, Ian; Hewson, Roger; Nasidi, Abdulsalami; Bowden, Thomas A; Carroll, Miles W

    2016-04-01

    The highly glycosylated glycoprotein spike of Ebola virus (EBOV-GP1,2) is the primary target of the humoral host response. Recombinant EBOV-GP ectodomain (EBOV-GP1,2ecto) expressed in mammalian cells was used to immunize sheep and elicited a robust immune response and produced high titers of high avidity polyclonal antibodies. Investigation of the neutralizing activity of the ovine antisera in vitro revealed that it neutralized EBOV. A pool of intact ovine immunoglobulin G, herein termed EBOTAb, was prepared from the antisera and used for an in vivo guinea pig study. When EBOTAb was delivered 6 hours after challenge, all animals survived without experiencing fever or other clinical manifestations. In a second series of guinea pig studies, the administration of EBOTAb dosing was delayed for 48 or 72 hours after challenge, resulting in 100% and 75% survival, respectively. These studies illustrate the usefulness of EBOTAb in protecting against EBOV-induced disease. PMID:26715676

  15. Experimental infection of cattle with ovine Dichelobacter nodosus isolates

    DEFF Research Database (Denmark)

    Knappe-Poindecker, Maren; Jørgensen, Hannah Joan; Jensen, Tim Kåre; Tesfamichael, Bereket; Ulvund, Martha Jakobsen; Hektoen, Lisbeth; Fjeldaas, Terje

    2015-01-01

    Dichelobacter nodosus is the main causative agent of ovine footrot, and there are strong indications that the bacterium can be transferred to cattle grazing on the same pasture as sheep. The aim of this study was to investigate if benign and virulent D. nodosus strains isolated from sheep can be ...

  16. Revealing the structural and mechanical characteristics of ovine teeth.

    Science.gov (United States)

    O'Brien, Simona; Keown, Amanda J; Constantino, Paul; Xie, Zonghan; Bush, Mark B

    2014-02-01

    The survival and function of dentition over the lifetime of an animal depends upon the ability of the teeth to resist wear and chemical erosion, and to withstand occlusal loading conditions without suffering debilitating fracture. Understanding how geometrical factors (radius, height, enamel thickness) and mechanical properties of the dental tissues (Young's modulus E, hardness H and toughness KIC of enamel and dentin) combine to ensure the survival of an animal's teeth can provide great insight into the evolutionary history of the animal and its dietary adaptation. While the geometrical factors are beginning to be understood, the range of animals for which measurements of dental tissue properties are available is very narrow, being restricted almost entirely to humans and other primates. The absence of comparative data across a broader range of species makes it impossible to draw conclusions with any certainty. The present study expands knowledge of mammalian dental tissue properties by reporting the Young's modulus and hardness of ovine (sheep) enamel and dentin measured using nano-indentation. We found that sheep molar enamel Young's modulus and hardness are both lower than those of human enamel, by approximately 30%, and 9% respectively, while the properties of dentin are similar. The combination of E and H makes the ovine enamel approximately 30% more resistant to wear than human enamel, which is an imperative in ruminant dentition. The results of this study are interpreted in terms of the ovine feeding ecology, and the structure of the ovine molar and its occlusal surface. PMID:24316873

  17. Isolation and characterization of bifidobacteria from ovine cheese.

    Czech Academy of Sciences Publication Activity Database

    Bunešová, V.; Killer, Ji?í; Vlková, E.; Musilová, S.; Tomáška, M.; Rada, V.; Kme?, V.

    2014-01-01

    Ro?. 188, ?. 1 (2014), s. 26-30. ISSN 0168-1605 R&D Projects: GA ?R GA13-08803S Institutional support: RVO:67985904 Keywords : Bifidobacterium sp. * ovine cheese * cultivation Subject RIV: EE - Microbiology, Virology Impact factor: 3.082, year: 2014

  18. DISTANCE TRANSMISSION OF OVINE HERPESVIRUS 2 FROM SHEEP TO BISON

    Science.gov (United States)

    Malignant catarrhal fever (MCF) is potentially devastating to American bison. Virtually all bison MCF cases in North America are caused by ovine herpesvirus 2 (OvHV-2), a member of the gammaherpesvirus subfamily, which is carried almost exclusively by sheep. In this communication, we report transm...

  19. Schistosoma bovis: Plasminogen binding in adults and the identification of plasminogen-binding proteins from the worm tegument

    OpenAIRE

    Ramajo Hernández, Alicia; Pérez Sánchez, Ricardo; Ramajo Martín, Vicente; Oleaga, Ana

    2007-01-01

    Schistosoma bovis is a ruminant haematic parasite that lives for years in the mesenteric vessels of the host. The aim of this work was to investigate the ability of adult S. bovis worms to interact with plasminogen, a central component in the host fibrinolytic system. Confocal microscopy analysis revealed that plasminogen bound to the tegument surface of the male—but not female—S. bovis worms and that this binding was strongly dependent on lysine residues. It was also observed that a protein ...

  20. Effects of chlordecone and chlordecone alcohol on isolated ovine erythrocytes

    International Nuclear Information System (INIS)

    Chlordecone (CHLO, 1-30 uM) and chlordecone alcohol (CHLO ALC, 1-23 uM) altered the permeability of isolated ovine erythrocytes (OE) as evidenced by a concentration- and time-dependent induction of K+ efflux and hemolysis. Hemolysis, but no K+ efflux, was markedly delayed when OE were suspended in isotonic sucrose. Low concentrations of both compounds (1-4 uM) protected OE against hypotonic hemolysis. Neither CHLO (30 uM) nor CHLO ALC (23 uM) induced the release of trapped K+ from KSCN-loaded, OE-lipid, unilamellar liposomes. CHLO- and CHLO ACL-induced hemolysis and K+ efflux were dependent upon the pH of the external media. CHLO ALC-induced K+ efflux and hemolysis showed a slight pH dependence, with increased potency of the compound detected over the pH range 8.3-9.4 CHLO ALC-induced protection against hypotonic hemolysis was pH independent. The potency of CHLO in all three assays decreased as the pH was raised from 6.4 to 9.4. [14C]-CHLO and [14C]-CHLO ALC binding to OE and OE membranes was pH independent. However, the binding of [14C]-CHLO to polypropylene and glass was pH dependent. [14C]-CHLO binding to polypropylene and glass decreased from pH 6.4 to pH 10.4. The pKa of CHLO was estimated to be 8.9. After the CHLO results were corrected for the fraction of CHLO present in the unionized form, it was estimated the ionized CHLO possessed 1/3 to 1/20 of the activity of the unionized species

  1. Prostaglandin E2 Acts via Multiple Receptors to Regulate Plasminogen-Dependent Proteolysis in the Primate Periovulatory Follicle

    OpenAIRE

    Markosyan, Nune; Duffy, Diane M

    2008-01-01

    The ovulatory gonadotropin surge regulates expression of plasminogen activator (PA) family members within the ovarian follicle, which are implicated in follicle wall degradation at ovulation. Gonadotropin also stimulates follicular prostaglandin E2 (PGE2) production, which is required for follicle rupture. To determine whether the ovulatory gonadotropin surge regulates PA-mediated proteolysis via PGE2 in the primate follicle, monkeys received gonadotropins to stimulate follicle development. F...

  2. Ovine HSP90AA1 gene promoter: functional study and epigenetic modifications.

    Science.gov (United States)

    Salces-Ortiz, Judit; González, Carmen; Bolado-Carrancio, Alfonso; Rodríguez-Rey, Jose Carlos; Calvo, Jorge H; Muñoz, Rubén; Serrano, M Magdalena

    2015-11-01

    When environmental temperatures exceed a certain threshold, the upregulation of the ovine HSP90AA1 gene is produced to cope with cellular injuries caused by heat stress. It has been previously pointed out that several polymorphisms located at the promoter region of this gene seem to be the main responsible for the differences in the heat stress response observed among alternative genotypes in terms of gene expression rate. The present study, focused on the functional study of those candidate polymorphisms by electrophoretic mobility shift assay (EMSA) and in vitro luciferase expression assays, has revealed that the observed differences in the transcriptional activity of the HSP90AA1 gene as response to heat stress are caused by the presence of a cytosine insertion (rs397514115) and a C to G transversion (rs397514116) at the promoter region. Next, we discovered the presence of epigenetic marks at the promoter and along the gene body founding an allele-specific methylation of the rs397514116 mutation in DNA extrated from blood samples. This regulatory mechanism interacts synergistically to modulate gene expression depending on environmental circumstances. Taking into account the results obtained, it is suggested that the transcription of the HSP90AA1 ovine gene is regulated by a cooperative action of transcription factors (TFs) whose binding sites are polymorphic and where the influence of epigenetic events should be also taken into account. PMID:26253285

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

    Science.gov (United States)

    Brzoska, Tomasz; Tanaka-Murakami, Aki; Suzuki, Yuko; Sano, Hideto; Kanayama, Naohiro; Urano, Tetsumei

    2015-01-01

    The fibrinolytic system plays a pivotal role in the regulation of hemostasis; however, it remains unclear how and when the system is triggered to induce thrombolysis. Using intra-vital confocal fluorescence microscopy, we investigated the process of plasminogen binding to laser-induced platelet-rich microthrombi generated in the mesenteric vein of transgenic mice expressing green fluorescent protein (GFP). The accumulation of GFP-expressing platelets as well as exogenously infused Alexa Fluor 568-labeled Glu-plasminogen (Glu-plg) on the injured vessel wall was assessed by measuring the increase in the corresponding fluorescence intensities. Glu-plg accumulated in a time-dependent manner in the center of the microthrombus, where phosphatidylserine is exposed on platelet surfaces and fibrin formation takes place. The rates of binding of Glu-plg in the presence of ?-aminocaproic acid and carboxypeptidase B, as well as the rates of binding of mini-plasminogen lacking kringle domains 1-4 and lysine binding sites, were significantly lower than that of Glu-plg alone, suggesting that the binding was dependent on lysine binding sites. Furthermore, aprotinin significantly suppressed the accumulation of Glu-plg, suggesting that endogenously generated plasmin activity is a prerequisite for the accumulation. In spite of the endogenous generation of plasmin and accumulation of Glu-plg in the center of microthrombi, the microthrombi did not change in size during the 2-hour observation period. When human tissue plasminogen activator was administered intravenously, Glu-plg further accumulated and the microthrombi were lysed. Glu-plg appeared to accumulate in the center of microthrombi in the early phase of microthrombus formation, and plasmin activity and lysine binding sites were required for this accumulation. PMID:25806939

  4. Glycosaminoglycans affect the interaction of human plasma kallikrein with plasminogen, factor XII and inhibitors

    Scientific Electronic Library Online (English)

    A.J., Gozzo; V.A., Nunes; H.B., Nader; C.P., Dietrich; A.K., Carmona; M.U., Sampaio; C.A.M., Sampaio; M.S., Araújo.

    2003-08-01

    Full Text Available Human plasma kallikrein, a serine proteinase, plays a key role in intrinsic blood clotting, in the kallikrein-kinin system, and in fibrinolysis. The proteolytic enzymes involved in these processes are usually controlled by specific inhibitors and may be influenced by several factors including glycos [...] aminoglycans, as recently demonstrated by our group. The aim of the present study was to investigate the effect of glycosaminoglycans (30 to 250 µg/ml) on kallikrein activity on plasminogen and factor XII and on the inhibition of kallikrein by the plasma proteins C1-inhibitor and antithrombin. Almost all available glycosaminoglycans (heparin, heparan sulfate, bovine and tuna dermatan sulfate, chondroitin 4- and 6-sulfates) reduced (1.2 to 3.0 times) the catalytic efficiency of kallikrein (in a nanomolar range) on the hydrolysis of plasminogen (0.3 to 1.8 µM) and increased (1.9 to 7.7 times) the enzyme efficiency in factor XII (0.1 to 10 µM) activation. On the other hand, heparin, heparan sulfate, and bovine and tuna dermatan sulfate improved (1.2 to 3.4 times) kallikrein inhibition by antithrombin (1.4 µM), while chondroitin 4- and 6-sulfates reduced it (1.3 times). Heparin and heparan sulfate increased (1.4 times) the enzyme inhibition by the C1-inhibitor (150 nM).

  5. Glycosaminoglycans affect the interaction of human plasma kallikrein with plasminogen, factor XII and inhibitors

    Directory of Open Access Journals (Sweden)

    Gozzo A.J.

    2003-01-01

    Full Text Available Human plasma kallikrein, a serine proteinase, plays a key role in intrinsic blood clotting, in the kallikrein-kinin system, and in fibrinolysis. The proteolytic enzymes involved in these processes are usually controlled by specific inhibitors and may be influenced by several factors including glycosaminoglycans, as recently demonstrated by our group. The aim of the present study was to investigate the effect of glycosaminoglycans (30 to 250 µg/ml on kallikrein activity on plasminogen and factor XII and on the inhibition of kallikrein by the plasma proteins C1-inhibitor and antithrombin. Almost all available glycosaminoglycans (heparin, heparan sulfate, bovine and tuna dermatan sulfate, chondroitin 4- and 6-sulfates reduced (1.2 to 3.0 times the catalytic efficiency of kallikrein (in a nanomolar range on the hydrolysis of plasminogen (0.3 to 1.8 µM and increased (1.9 to 7.7 times the enzyme efficiency in factor XII (0.1 to 10 µM activation. On the other hand, heparin, heparan sulfate, and bovine and tuna dermatan sulfate improved (1.2 to 3.4 times kallikrein inhibition by antithrombin (1.4 µM, while chondroitin 4- and 6-sulfates reduced it (1.3 times. Heparin and heparan sulfate increased (1.4 times the enzyme inhibition by the C1-inhibitor (150 nM.

  6. A monoclonal antibody to pestviruses in bovine and ovine sera

    International Nuclear Information System (INIS)

    An enzyme-linked immunoabsorbent assay (ELISA) has been developed to defeat antibodies to pestviruses in bovine and ovine sera. Single sera from 211 cattle and 22 sheep from 7 different farms were tested using ELISA and Serum Neutralisation Test (SNT). 17 Monoclonal antibodies (Mabs) directed against P80, gp48 and gp53 were tested for ability to coat ELISA plates and capture the bovine viral diarrhea antigen. 5 mabs(WB 103, WB, 105, WB 112 against P80 kDa protein, WB 210 and WB 214 directed against gp48 and gp 53 kDa protein. Specific antibody to BVDV was detected by rabbit anti-bovine and anti-ovine IgG antisera. The quantitative correlation between two tests was good

  7. Optimizing aerosol gene delivery and expression in the ovine lung.

    OpenAIRE

    MCLACHLAN, G; Baker, A.; Tennant, P; Gordon, C; Vrettou, C; Renwick, L.; Blundell, R.; Cheng, SH; Scheule, RK; Davies, L; Painter, H; Coles, RL; Lawton, AE; Marriott, C.; Gill, DR

    2007-01-01

    We have developed the sheep as a large animal model for optimizing cystic fibrosis gene therapy protocols. We administered aerosolized gene transfer agents (GTAs) to the ovine lung in order to test the delivery, efficacy, and safety of GTAs using a clinically relevant nebulizer. A preliminary study demonstrated GTA distribution and reporter gene expression throughout the lung after aerosol administration of plasmid DNA (pDNA):GL67 and pDNA:PEI complexes. A more comprehensive study examined th...

  8. Activin promotes oocyte development in ovine preantral follicles in vitro

    OpenAIRE

    Telfer Evelyn E; Armstrong David G; Thomas Fiona H

    2003-01-01

    Abstract Activins have been implicated as important regulating factors for many reproductive processes. The aim of this study was to determine the effect of activin A on the development of ovine preantral follicles in vitro. Mechanically isolated preantral follicles (161 ± 2 microm) were cultured for 6 days in the presence of human recombinant activin A (0, 10 and 100 ng/ml). Half of the medium was replaced every second day and follicle diameters were measured. Conditioned medium was subseque...

  9. Study of wool characteristics in the Aranese ovine breed

    OpenAIRE

    Parés Casanova, Pere-Miquel; Jordana i Vidal, Jordi; Perezgrovas Garza, Raúl

    2011-01-01

    To date, no ethnological study on the wool characteristics of the Spanish Aranese ovine breed has been published. Fifty three animals belonging to this breed are tested as fleece samples. Each sample is analyzed for fleece type and length, yield by isoalcohol scouring, fiber length for each kind of fiber, variation in fiber diameter, and proportions of non-medullated and medullated or kemp fibers. Fiber length appears shorter than that previously reported for the breed by other authors. Fleec...

  10. Treatment of ulcerated necrobiosis lipoidica with ovine forestomach matrix.

    Science.gov (United States)

    Simcock, J W; Than, M; Ward, B R; May, B C H

    2013-07-01

    Necrobiosis lipoidica (NL) results from degradation of the collagen extracellular matrix; these recurring ulcerated lesions are an especially challenging condition to treat. Ovine forestomach matrix (OFM) is a decellularised extracellular matrix and was used to successfully close a pretibial ulcer resulting from NL. Complete closure of the wound was achieved in 22 weeks, after four applications of OFM. This suggests OFM may be considered for the treatment of these challenging wounds. PMID:24159661

  11. Role of platelet activating factor on the fibrinolytic activation in the pathogenesis of gastric mucosal damage induced by endothelin-1.

    OpenAIRE

    Kurose, I; Miura, S.; Fukumura, D.; TASHIRO, H.; Imaeda, H; Shiozaki, H; Suematsu, M; Nagata, H.; Sekizuka, E; Tsuchiya, M.

    1992-01-01

    We have examined the hypothesis that the release of tissue type plasminogen activator may play a prominent role in endothelin induced gastric mucosal injury. We determined tissue type plasminogen activator activity in the regional blood sample and the concentration of platelet activating factor in the gastric mucosa after the administration of endothelin-1 in a range of 50-500 pmol/kg into the left gastric artery of male Wistar rats. Endothelin-1 increased the tissue type plasminogen activato...

  12. Ovine fetal immune response to Cache Valley virus infection.

    Science.gov (United States)

    Rodrigues Hoffmann, Aline; Dorniak, Piotr; Filant, Justyna; Dunlap, Kathrin A; Bazer, Fuller W; de la Concha-Bermejillo, Andres; Welsh, Christabel Jane; Varner, Patricia; Edwards, John Francis

    2013-05-01

    Cache Valley virus (CVV)-induced malformations have been previously reproduced in ovine fetuses. To evaluate the development of the antiviral response by the early, infected fetus, before the development of immunocompetency, ovine fetuses at 35 days of gestation were inoculated in utero with CVV and euthanized at 7, 10, 14, 21, and 28 days postinfection. The antiviral immune response in immature fetuses infected with CVV was evaluated. Gene expression associated with an innate, immune response was quantified by real-time quantitative PCR. The upregulated genes in infected fetuses included ISG15, Mx1, Mx2, IL-1, IL-6, TNF-?, TLR-7, and TLR-8. The amount of Mx1 protein, an interferon-stimulated GTPase capable of restricting growth of bunyaviruses, was elevated in the allantoic and amniotic fluid in infected fetuses. ISG15 protein expression was significantly increased in target tissues of infected animals. B lymphocytes and immunoglobulin-positive cells were detected in lymphoid tissues and in the meninges of infected animals. These results demonstrated that the infected ovine fetus is able to initiate an innate and adaptive immune response much earlier than previously known, which presumably contributes to viral clearance in infected animals. PMID:23468505

  13. Production of bacteriocin-like substances by lactic acid bacteria isolated from regional ovine cheese

    Scientific Electronic Library Online (English)

    Cássia Regina, Nespolo; Adriano, Brandelli.

    2010-12-01

    Full Text Available Lactic acid bacteria (LAB) were isolated from ovine milk and cheeses manufactured in the South Region of Brazil. Among 112 bacterial isolates investigated, 59 were chosen through a screening for LAB. Among these 59 strains of LAB, 21% showed antimicrobial, proteolytic and lipolytic activities. Based [...] on this screening, Lactobacillus plantarum LCN 17 and Lactobacillus rhamnosus LCN 43 were selected and tested for the production of bacteriocin-like substances (BLS). The BLS produced by both isolates showed antimicrobial activity against Listeria monocytogenes, whereas that produced by L. plantarum LCN 17 presented higher stability to different temperature, pH and enzyme treatments. These strains present potential for production of BLS, and for use as starter cultures.

  14. The bacteriological quality of goat and ovine milk

    Directory of Open Access Journals (Sweden)

    Kateřina Bogdanovičová

    2015-05-01

    Full Text Available This study concentrates on information concerning the microbiological hazards that can be present in raw milk from animal species other than cows. A total of 54 (23 of ovine and 31 of goat bulk tank milk samples from 10 farms in the Czech Republic were collected in years 2013 - 2014. The sampling was done at regular time intervals during the whole year, with five to eight samples collected from each of the 10 dairy farms involved in the study. All milk samples were collected into sterile sampling bottles and transported in a cooler sampling case to the laboratory for immediate examination. Farms were randomly selected to cover the whole area of the Czech Republic. The prevalence and characteristic of Escherichia coli, Staphylococcus aureus, Salmonella spp., Campylobacter spp. and Listeria monocytogenes was studied. Raw cow's milk can be contaminated by E. coli intramammarily during clinical or subclinical mastitis and either directly through animal feces or indirectly during milk collection through farm employees or the milking equipment. E. coli was detected in 90.3% of the goat milk and 95.7% of the ovine milk samples. The genes encoding Shiga toxins 1 and 2- (stx1, stx2 were not detected and no STEC was identified. The Eae was the detected in 3 (4.6% isolates. S. aureus was detected in 9 (29.0% samples of goat milk and 8 (34.8% samples of ovine milk. A total 12 (57.1% enterotoxin positive S. aureus were obtained; 6 (28.6% were positive for the production of sec encoding enterotoxin SEC; in 4 (19.0% isolates the gene seh was detected; 2 (9.5% isolates were proven positive for seg (4.8% and combination seg and sei (4.8%. The presence of MRSA was not detected in the tested samples in our study. L. monocytogenes was detected in 1 (3.2% samples of goat milk and 1 (4.3% samples of ovine milk. The serotype (1/2a, 1/2b was detected in our study. Campylobacter spp. and Salmonella spp. were not isolated from any of the samples. These results form the basis for determining the microbiological quality standards for goat and ovine milk.

  15. Anti-Plasminogen Antibodies Compromise Fibrinolysis and Associate with Renal Histology in ANCA-Associated Vasculitis

    OpenAIRE

    Berden, Annelies E.; Nolan, Sarah L.; Morris, Hannah L.; Bertina, Rogier M.; Erasmus, Dianhdra D.; Hagen, E. Christiaan; Hayes, Donal P.; van Tilburg, Nico H.; Bruijn, Jan A; Savage, Caroline O.S.; Bajema, Ingeborg M.; Hewins, Peter

    2010-01-01

    Antibodies recognizing plasminogen, a key component of the fibrinolytic system, associate with venous thrombotic events in PR3-ANCA vasculitis. Here, we investigated the prevalence and function of anti-plasminogen antibodies in independent UK and Dutch cohorts of patients with ANCA-associated vasculitis (AAV). We screened Ig isolated from patients (AAV-IgG) and healthy controls by ELISA. Eighteen of 74 (24%) UK and 10/38 (26%) Dutch patients with AAV had anti-plasminogen antibodies compared w...

  16. Potent antitumor activity of a urokinase-activated engineered anthrax toxin

    Science.gov (United States)

    Liu, Shihui; Aaronson, Hannah; Mitola, David J.; Leppla, Stephen H.; Bugge, Thomas H.

    2003-01-01

    The acquisition of cell-surface urokinase plasminogen activator activity is a hallmark of malignancy. We generated an engineered anthrax toxin that is activated by cell-surface urokinase in vivo and displays limited toxicity to normal tissue but broad and potent tumoricidal activity. Native anthrax toxin protective antigen, when administered with a chimeric anthrax toxin lethal factor, Pseudomonas exotoxin fusion protein, was extremely toxic to mice, causing rapid and fatal organ damage. Replacing the furin activation sequence in anthrax toxin protective antigen with an artificial peptide sequence efficiently activated by urokinase greatly attenuated toxicity to mice. In addition, the mutation conferred cell-surface urokinase-dependent toxin activation in vivo, as determined by using a panel of plasminogen, plasminogen activator, plasminogen activator receptor, and plasminogen activator inhibitor-deficient mice. Surprisingly, toxin activation critically depended on both urokinase plasminogen activator receptor and plasminogen in vivo, showing that both proteins are essential cofactors for the generation of cell-surface urokinase. The engineered toxin displayed potent tumor cell cytotoxicity to a spectrum of transplanted tumors of diverse origin and could eradicate established solid tumors. This tumoricidal activity depended strictly on tumor cell-surface plasminogen activation. The data show that a simple change of protease activation specificity converts anthrax toxin from a highly lethal to a potent tumoricidal agent.

  17. Hexamethylenebisacetamide (HMBA) is a growth factor for human, ovine and porcine thyroid cells.

    Science.gov (United States)

    Fayet, G; Amphoux-Fazekas, T; Aouani, A; Hovsépian, S

    1996-03-01

    Hexamethylenebisacetamide (HMBA) provokes in murine erythroleukemia cells (MELC) a commitment to terminal differentiation leading to the activation of the expression of hemoglobin. HMBA has been tested also in other cells from colon cancer, melanoma or lung cancer. However it has not yet been tested in the thyroid. We demonstrate in this paper that HMBA in kinetics and concentration-response experiments increases the proliferation of human thyroid cells isolated from Graves'-Basedow patients. It also acts like a growth factor for ovine and porcine thyroid cells, respectively, from the OVNIS line and the ATHOS line. This molecule which is a differentiating factor in the MELC system and a growth factor in human thyroid cell cultures represents a potential to get human thyroid cell lines expressing specialized functions. PMID:8734479

  18. Electrically evoked potentials in an ovine model for the evaluation of visual prosthesis efficacy.

    Science.gov (United States)

    Barriga-Rivera, Alejandro; Eiber, Calvin D; Dodds, Christopher W D; Fung, Adrian T; Tatarinoff, Veronica; Lovell, Nigel H; Suaning, Gregg J

    2015-08-01

    Visual prostheses are becoming a reality as a therapy to restore functional vision to the blind. New stimulation strategies and novel electrode designs are contributing to accelerate the development of such devices triggering the interest of scientists, clinicians and the blind community worldwide. In this scenario, there is a need for large animal models that are suitable for preclinical testing of retinal neuroprostheses. This study presents an electrophysiology assessment of an ovine model for single and simultaneous electrode stimulation from the suprachoroidal space, using symmetric biphasic current pulses with a monopolar return configuration. Visually and electrically evoked potentials were recorded using supradural surface electrodes, showing charge thresholds comparable to those in humans. This model represents an alternative to feline or canine models with analogous activation levels and an eye anatomy similar to that of humans. PMID:26737012

  19. Fibre Characterization of the fat tailed zambian ovine breed

    OpenAIRE

    Parés Casanova, Pere-Miquel; R Pérezgrovas; Mwaanga, Edwell S.

    2014-01-01

    To-date, no ethnological study of the Fat-tailed Zambian ovine breed has been published. In order to contribute to the knowledge of its wool, ten fleece samples were studied. Each sample was analysed for yield by isoalcohol scouring, fibre length for each kind of fibre, variation in fibre diameter, and proportions of non-medullated and medulla ted fibres. Fleeces of the Fat-tailed Zambian sheep breed can be described as pen-brush, 'closed' and relatively long. The high yield by isoalcohol sco...

  20. Lower apoptosis rate in ovine preantral follicles from ovaries stored in supplemented preservation media.

    Science.gov (United States)

    Gonçalves, R J S; Cavalcante, A Y P; Gouveia, B B; Lins, T L B; Barberino, R S; Menezes, V G; Barros, V R P; Macedo, T J S; Figueiredo, J R; Matos, M H T

    2015-12-01

    The aim of this study was to investigate the effect of ovarian tissue transportation conditions (medium and period of time) on the morphology, apoptosis and development of ovine preantral follicles cultured in vitro. Each ovarian pair was cut into nine slices, with one fragment being fixed immediately (fresh control). The remaining fragments were placed individually in cryotubes containing conservation medium (minimal essential medium (MEM) without supplementation or MEM+ - with supplementation) and stored at 35ºC for 6 or 12 h without (non-cultured) or with subsequent culture for 5 days. Then, the fragments were processed for histological and terminal deoxynucleotidyl transferase (TdT) mediated dUTP nick-end labelling (TUNEL) examination. Preservation of ovarian slices in MEM or MEM+ (non-cultured) resulted in similar percentages of normal follicles when compared with the fresh control. Nevertheless, compared with the fresh control, a decrease in the percentage of normal follicles was observed in tissues cultured for 5 days. Only for tissues preserved in supplemented medium (MEM+) for 6 h, the percentage of TUNEL positive cells was similar between non-cultured tissues and tissues cultured for 5 days. Follicular activation and growth (follicular and oocyte diameter) were higher in cultured tissues than in fresh control or non-cultured tissues, except those from fragments preserved for 6 h in MEM and then cultured for 5 days in which no growth was observed. In conclusion, ovine ovarian tissue was successfully preserved in supplemented medium (MEM+) at a temperature close to physiological values (35°C) for up to 6 h without affecting apoptosis in the ovarian follicles and their ability to develop in vitro. PMID:25626913

  1. Efficiency of sperm-mediated gene transfer in the ovine by laparoscopic insemination, in vitro fertilization and ICSI.

    Science.gov (United States)

    Pereyra-Bonnet, Federico; Gibbons, Alejandro; Cueto, Marcela; Sipowicz, Pablo; Fernández-Martín, Rafael; Salamone, Daniel

    2011-04-01

    Transgenesis constitutes an important tool for pharmacological protein production and livestock improvement. We evaluated the potential of laparoscopic insemination (LI), in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) to produce egfp-expressing ovine embryos, using spermatozoa previously exposed to pCX-EGFP plasmid in two different sperm/DNA incubation treatments: "Long Incubation" (2 h at 17 C) and "Short Incubation" (5 min at 5 C). For LI, Merino sheep were superovulated and inseminated with treated fresh semen from Merino rams. The embryos were recovered by flushing the uterine horns. For IVF and ICSI, slaughterhouse oocytes were fertilized with DNA-treated frozen/thawed sperm. All recovered embryos were exposed to blue light (488 nm) to determine green fluorescent morulae and blastocysts rates. High cleavage and morulae/blastocysts rates accompanied the LI and IVF procedures, but no egfp-expressing embryos resulted. In contrast, regardless of the sperm/plasmid incubation treatment, egfp-expressing morulae and blastocysts were always obtained by ICSI, and the highest transgenesis rate (91.6%) was achieved with Short Incubation. In addition, following the incubation of labeled plasmid DNA, after Long or Short exposure treatments, with fresh or frozen/thawed spermatozoa, only non-motile fresh spermatozoa could maintain an attached plasmid after washing procedures. No amplification product could be detected following PCR treatment of LI embryos whose zonae pellucidae (ZP) had been removed. In order to establish conditions for transgenic ICSI in the ovine, we compared three different activation treatments, and over 60% of the obtained blastocysts expressed the transgene. For ICSI embryos, FISH analysis found possible signals compatible with integration events. In conclusion, our results show that in the ovine, under the conditions studied, ICSI is the only method capable of producing exogenous gene-expressing embryos using spermatozoa as vectors. PMID:21079375

  2. Does the result of thrombolysis with recombinant tissue-type plasminogen activator (rt-PA) in rabbits depend on the erythrocyte- and fibrin-content of a thrombus?; Haengt das Ergebnis e