Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.73 (urokinase-type plasminogen activator)
 10,685 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Extracellular proteolysis is required for matrix degradation and the regulation of cytokine activity during angiogenesis, and this is dependent on a cohort of proteases and protease inhibitors produced by endothelial and nonendothelial cells. The plasminogen activator (PA)/plasmin system has been extensively investigated in these processes, and descriptive studies have demonstrated that urokinase-type PA (uPA), uPA receptor (uPAR) and PA inhibitor-1 (PAI-1) are expressed by endothelial cells during angiogenesis in vivo. In vitro studies have led to the notion that normal capillary morphogenesis is dependent on a protease-antiprotease equilibrium. These findings are discussed in the context of recent observations on uPA-, uPAR-, PAI-1 and plaminogen-deficient mice, in which developmental and physiological angiogenesis appear to occur normally. This has led to a reevaluation of the role of the PA/plasmin system during angiogenesis. In particular, these observations raise the possibility that the role of this system may be limited to situations in which endothelial cells encounter and must degrade fibrin in order to form new capillary sprouts.
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PMID:Angiogenesis: a paradigm for balanced extracellular proteolysis during cell migration and morphogenesis. 879 3

Human endothelial cells express antithrombotic properties by producing prostacyclin, heparan sulfate and plasminogen activator (PA). Using an established cell line, TKM-33, from human umbilical vein endothelial cells, the pericellular urokinase-type PA (u-PA) activity and expression of u-PA receptor (u-PAR) were investigated. The endothelial cells produced and secreted large amounts of u-PA and low levels of tissue-type PA (t-PA) and of PA inhibitor-1 (PAI-1), which were identified by immunohistochemical study and electrophoretic enzymography. Diisopropylfluoro-phosphate-treated 125I-u-PA bound specifically to acid-treated monolayered endothelial cells with a Kd of 3.46 +/- 1.17 nM, and Bmax of (0.09 +/- 0.04) x 10(6) sites/cell. mRNA of u-PAR was detected by using Northern blot analysis. Thus, these endothelial cells express u-PAR which bounds u-PA specifically. Phorbol myristate acetate (PMA) stimulation to the endothelial cells altered the Kd value to 3.18 +/- 0.64 nM, and Bmax value to (0.19 +/- 0.10) x 10(6) sites/cell, respectively. PMA treatment of endothelial cells increased u-PAR mRNA. Similarly, H7-treated endothelial cells showed a dose-dependent increase of u-PAR mRNA. However, PMA and H7 did not stimulate the expression of u-PA and t-PA mRNAs significantly. The expression of PAI-1 mRNA was increased by both PMA and H7. These findings suggest that the established endothelial cell line, TKM-33, possesses the character of endothelial cells and expresses u-PAR on their cell surface which is occupied by intrinsic u-PA secreted from the cells. The pericellular u-PA activity and the expression of u-PAR were regulated by protein kinase pathway.
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PMID:Identification of urokinase-type plasminogen activator receptor in human endothelial cells and its modulation by phorbol myristate acetate. 882 63

Induction of the urokinase type plasminogen activator receptor (uPAR) promotes cell adhesion through its interaction with vitronectin (VN) in the extracellular matrix, and facilitates cell migration and invasion by localizing uPA to the cell surface. We provide evidence that this balance between cell adhesion and cell detachment is governed by PA inhibitor-1 (PAI-1). First, we demonstrate that uPAR and PAI-1 bind to the same site in VN (i.e., the amino-terminal somatomedin B domain; SMB), and that PAI-1 competes with uPAR for binding to SMB. Domain swapping and mutagenesis studies indicate that the uPAR-binding sequence is located within the central region of the SMB domain, a region previously shown to contain the PAI-1-binding motif. Second, we show that PAI-1 dissociates bound VN from uPAR and detaches U937 cells from their VN substratum. This PAI-1 mediated release of cells from VN appears to occur independently of its ability to function as a protease inhibitor, and may help to explain why high PAI-1 levels indicate a poor prognosis for many cancers. Finally, we show that uPA can rapidly reverse this effect of PAI-1. Taken together, these results suggest a dynamic regulatory role for PAI-1 and uPA in uPAR-mediated cell adhesion and release.
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PMID:Is plasminogen activator inhibitor-1 the molecular switch that governs urokinase receptor-mediated cell adhesion and release? 883 Jul 83

Rat endometrial stromal cells undergoing decidualization in vitro secrete urokinase-type plasminogen activator (uPA), and this secretion is regulated by prostaglandin E2. The present study was undertaken to determine whether uPA and plasminogen activator inhibitor-1 (PAI-1) mRNAs are expressed in vivo in the decidua of pregnant rats and in the deciduoma of "pseudopregnant" rats. Total RNA was prepared from nondecidualized and decidualized endometrial tissues at various stages of early pregnancy and examined by Northern blot analysis using specific cDNA probes for rat uPA and PAI-1. There was little uPA mRNA in the endometrium during the first 5 days of pregnancy (Day 1 = the presence of sperm in the vagina). A high level of uPA mRNA was detected on Day 7, and it declined thereafter. There was a gradual increase in PAI-1 mRNA in the decidua from Day 7 of pregnancy, reaching a peak level on Day 15 when the decidua was transformed into the maternal placenta. (RNA was not analyzed beyond Day 15 of pregnancy in this study.) In situ hybridization studies verified that uPA mRNA was present in the decidua adjacent to the implanting embryo on Day 7. Plasminogen activator inhibitor-1 mRNA was scattered in the decidualized endometrium, but greater amounts of PAI-1 mRNA were found in the fetal tissue on Day 10 of pregnancy. Northern blot analysis of RNA from the deciduoma produced in ovariectomized, steroid-treated rats by intrauterine injection of oil demonstrated a similar temporal pattern of expression of uPA mRNA; i.e., the level of uPA mRNA was highest on Day 7 and decreased thereafter. The level of PAI-1 mRNA in deciduoma was not detectable by Northern blot technique during the first 10 days of pseudopregnancy. These findings confirm that uPA mRNA is present in vivo in rat decidual cells, independent of the presence of a conceptus. By contrast, the level of PAI-1 mRNA in the uterus is probably influenced by the presence of the conceptus.
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PMID:Presence of urokinase plasminogen activator and plasminogen activator inhibitor-1 messenger ribonucleic acids in rat endometrium during decidualization in vivo. 886 64

The murine plasminogen/urokinase-type plasminogen-activator (u-PA) system was studied using purified proteins, plasma and endothelioma cells. Recombinant murine u-PA was obtained as a single-chain molecule of 45 kDa which was converted to two-chain u-PA with plasmin by cleavage of the Lys159-Ile160 peptide bond. Murine plasminogen, purified from plasma as a single-chain protein of 95 kDa, was resistant to quantitative activation with murine recombinant two-chain u-PA: only 15% activation within 1 h at 37 degrees C was obtained in mixtures of 1 microM plasminogen and 5 nM recombinant two-chain u-PA, whereas quantitative activation was observed in the autologous human system. Addition of 6-aminohexanoic acid to native murine plasminogen resulted in quantitative activation within 1 h. In murine plasma in vitro, plasminogen was also resistant to quantitative activation with u-PA (50% activation within 1 h at 37 degrees C with 50 nM recombinant two-chain u-PA, whereas in the human system nearly quantitative activation was obtained). Murine plasma clots submerged in murine plasma were resistant to lysis with u-PA; < or = 2% clot lysis in 2 h was obtained with 80 nM recombinant two-chain u-PA in the autologous murine system whereas 50% clot lysis in 2 h required only 15 nM recombinant two-chain u-PA in the autologous human system. Saturable binding of murine recombinant two-chain u-PA was observed to murine endothelioma cells that are genetically deficient in u-PA (u-PA-/- End cells). Binding was characterized by a Kd of 5.5 nM and 800000 binding sites/cell. However, u-PA-/- End cells did not significantly stimulate the activation rate of murine plasminogen by murine recombinant two-chain u-PA and did not enhance the plasmin-mediated conversion rate of murine recombinant single-chain u-PA to its two-chain derivative. Murine recombinant two-chain u-PA bound to murine endothelioma cells was quantitatively inhibited by murine plasminogen-activator inhibitor-1 (PAI-1). Thus, the interactions between murine plasminogen, u-PA and PAI-1 are qualitatively similar to those between their human counterparts. However, quantitative differences were observed both in the presence of cells and in plasma which may contribute to a reduced u-PA-mediated fibrinolytic activity in the murine systems.
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PMID:Characterization of the murine plasminogen/urokinase-type plasminogen-activator system. 894 73

Plasminogen activator inhibitor-1 (PAI-1), the primary inhibitor of tissue-type plasminogen activator and urokinase, is known to convert readily to a latent form by insertion of the reactive center loop into a central beta-sheet. Interaction with vitronectin stabilizes PAI-1 and decreases the rate of conversion to the latent form, but conformational effects of vitronectin on the reactive center loop of PAI-1 have not been documented. Mutant forms of PAI-1 were designed with a cysteine substitution at either position P1' or P9 of the reactive center loop. Labeling of the unique cysteine with a sulfhydryl-reactive fluorophore provides a probe that is sensitive to vitronectin binding. Results indicate that the scissile P1-P1' bond of PAI-1 is more solvent exposed upon interaction with vitronectin, whereas the N-terminal portion of the reactive loop does not experience a significant change in its environment. These results were complemented by labeling vitronectin with an arginine-specific coumarin probe which compromises heparin binding but does not interfere with PAI-1 binding to the protein. Dissociation constants of approximately 100 nM are calculated for the vitronectin/PAI-1 interaction from titrations using both fluorescent probes. Furthermore, experiments in which PAI-1 failed to compete with heparin for binding to vitronectin argue for separate binding sites for the two ligands on vitronectin.
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PMID:The use of fluorescent probes to characterize conformational changes in the interaction between vitronectin and plasminogen activator inhibitor-1. 903 May 77

Plasminogen activator inhibitor-1 (PAI-1), a unique member of the serpin superfamily, plays an important role in fibrinolysis and is an established risk factor for cardiovascular diseases. PAI-1 can occur in three interconvertible conformations: an active, a latent and a substrate form. To study conformational and functional relationships in PAI-1, a wide variety of monoclonal antibodies were evaluated for their influence on PAI-1 activity. Out of 77 monoclonal antibodies, directed against human PAI-1, six were selected for their strong inhibitory effect towards PAI-1 activity, i.e., 80 to 100% inhibition in the presence of a 1- to 16-fold molar excess of monoclonal antibody. Detailed analysis of the reaction products formed during the interaction between PAI-1 and its target proteinases tissue-type plasminogen activator (t-PA) or urokinase-type plasminogen activator (u-PA), in the presence of these monoclonal antibodies, revealed two distinct mechanisms of PAI-1 inactivation. Incubation of PAI-1 with one series of monoclonal antibodies resulted in the absence of any reaction indicative for direct interaction with the reactive-site loop or a facilitated conversion to the latent conformation. The loss of PAI-1 activity in the presence of the other group of monoclonal antibodies was associated with the concomitant formation of a 41 kDa cleavage product after interaction with the target proteinase. The latter observation demonstrates that binding of these antibodies induced a conformational change thereby converting the inhibitory, active conformation to the non-inhibitory substrate conformation. No conformational changes could be observed in latent PAI-1 under these conditions. Analysis of cross-reactivity revealed that some of these functionally important epitopes were conserved throughout PAI-1 obtained from various species including rabbit mouse and/or pig, resulting in similar functional and conformational effects induced by these antibodies. Thus, we have demonstrated the occurrence of two distinct mechanisms by which the inhibitory activity of PAI-1 can be neutralized. This may have implications for the design of therapeutic or preventive strategies to interfere with PAI-1 activity. Cross-reactivity of these inhibitory antibodies with PAI-1 from various species may also allow their application in experimental animal models studying the in vivo role of PAI-1 in various diseases (e.g. atherosclerosis, thrombosis, angiogenesis,...).
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PMID:Neutralization of plasminogen activator inhibitor-1 inhibitory properties: identification of two different mechanisms. 904 3

Plasminogen activator inhibitor-1 (PAI-1), the major physiologic inhibitor of tissue-type plasminogen activator and urokinase, is abundantly expressed in atherosclerotic vascular wall. To determine the role of PAI-1 in vascular wall, we have used a novel inhibitor of PAI-1, (3E, 4E)-3-benzylidene-4-(3,4,5-trimethoxy-benzylidene) -pyrrolidine-2,5-dione (T-686). T-686 was given to human vascular endothelial cells in vitro and to rabbits subjected to high cholesterol diet and mechanical injury in vivo. T-686 attenuated the augmentation of PAI-1 antigen accumulation induced by transforming growth factor beta in conditioned medium from the human umbilical vein endothelial cells. In rabbits with aortic atherosclerosis induced by hypercholesterolemia and implantation of indwelling plastic tubing, oral administration of T-686 (30mg/kg body weight/day) for 8 weeks attenuated the increase in plasma PAI-1 activity induced by vascular injury without decreasing blood triglyceride and cholesterol. This was accompanied by the reduction in aortic PAI-1 mRNA expression and the inhibition of development of atherosclerosis lesions. Thus, T-686 not only decreased PAI-1 synthesis in vascular cells in vitro but also protected against the development of vascular lesions in vivo. This compound may be useful in defining the role of PAI-1 in atherothrombotic states.
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PMID:A new butadiene derivative, T-686, inhibits plasminogen activator inhibitor type-1 production in vitro by cultured human vascular endothelial cells and development of atherosclerotic lesions in vivo in rabbits. 906 54

The plasminogen activator (PA)-plasmin proteolytic system has recently received considerable attention because of its participation in a wide variety of biological activities and in pathological conditions involving tissue destruction. Excessive mechanical stress such as occlusal trauma is associated with alveolar bone loss in severe periodontitis. Therefore, mechanical stress may involve degradation of the extracellular matrix by occlusal trauma through activation of the PA-plasmin proteolytic system. We examined the effects of mechanical stress on PA activity, gene expressions of tissue type (t) PA, urokinase type (u) PA and PA inhibitor-1 (PAI-1) in human PDL cells. Human PDL cells were cultured on flexible-bottomed culture plates and placed on a Flexercell Strain Unit. The cells were flexed at 6 cycles (5 s strain, 5 s relaxation) at 9% and 18% elongation for 5 d. Application of tension-force induced significantly higher PA activity in stressed PDL cells than in non-stressed controls, and did so in a time- and magnitude-dependent manner (p < 0.001, ANOVA). Western-blot analysis revealed that the high level of activity was due to tPA and not uPA. Gene expression of tPA mRNA in stressed PDL cells, as examined by RT-PCR, increased on d 5. These findings suggest that tPA may be involved in periodontal metabolism in response to mechanical stress.
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PMID:Effect of tension-force on plasminogen activator activity from human periodontal ligament cells. 913 97

Plasminogen activator inhibitor-1 (PAI-1) is a unique member of the serpin superfamily. The alternative behavior of PAI-1 as an inhibitor, a non-inhibitory substrate, or a non-reactive latent form has been shown to be dependent on the initial conformation. In this study, we have evaluated the effect of a substitution outside the reactive site loop (P18) or in the reactive site loop (P6 and P10) on proteinase specificity and conformational transitions in PAI-1. Wild-type PAI-1 (wtPAI-1) revealed the same conformational distribution pattern toward tissue-type plasminogen activator (t-PA) as toward urokinase-type plasminogen activator (u-PA) (i.e. 53 +/- 6. 9% active, 36 +/- 6.8% latent, and 12 +/- 1.9% substrate). Inactivation of wtPAI-1 resulted in the conversion of the labile active form into the latent form while the stable substrate form remained unchanged. PAI-1-P6 (Val --> Pro at P6) revealed a target specificity for t-PA (39 +/- 7% versus 3 +/- 2% of the theoretical maximal value toward t-PA and u-PA, respectively), PAI-1-P10 (Ser --> Pro at P10) was 4-fold more active toward u-PA than toward t-PA, and PAI-1-P18 (Asn --> Pro at P18) exhibited inhibitory properties exclusively toward u-PA (41 +/- 10%). Surprisingly, inactivation of these mutants revealed functional and conformational transitions distinct from those observed for wtPAI-1. Inactivation of PAI-1-P6(Val --> Pro) resulted in a total conversion of the active form into the latent form and in a partial conversion of the substrate form into the latent form. The active forms of both PAI-1-P10(Ser --> Pro) and PAI-1-P18(Asn --> Pro) are also labile but, in contrast to the active form of wtPAI-1, convert into substrate forms. Based on the existence of various conformations of PAI-1, we propose an alternative reaction scheme describing the putative interactions between serpins and their target proteinases. The unusual conformational and functional flexibility of PAI-1 that, according to the current study, appears not to be restricted to the reactive site loop further underlines the importance of potential structural rearrangements (e.g. upon binding to cofactors) in PAI-1 (or serpins in general) for its functional behavior at particular biological sites.
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PMID:Proteinase specificity and functional diversity in point mutants of plasminogen activator inhibitor 1. 913 22


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