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)

Quiescent cultures of chick embryo fibroblasts incubated with human alpha-thrombin (14-219 pM) incorporated [methyl-3H]thymidine proportional to concentration. Inactivated forms of this protease (e.g. active-site-conjugated alpha-thrombin or its hirudin complex) had no mitogenic activity and did not compete with 124I-alpha-thrombin for binding to specific plasma membrane receptors. The noncoagulant but esterolytic active forms, gamma- and nitro-alpha-thrombins, were weakly mitogenic and correspondingly competed weakly for binding. Trypsin competed equally as well as native thrombin for binding, whereas chymotrypsin, elastase, and human urokinase competed with 80-fold less affinity. Plasma, arginine-specific proteases associated with nerve or epidermal growth factors, insulin, and insulin-like growth factors did not compete for binding. These data demonstrate that (a) functional catalytic residues of the thrombin active site are necessary for mitogenic activity and for specific binding; (b) regions adjacent to the active site, i.e. the high affinity protein recognition site, appear to enhance binding; and (c) the receptor can discriminate between other proteases and binds those which are also mitogens for the avian cells. The characteristics of 125I-alpha-thrombin binding were determined, and it was found to be (i) proportional to cell number; (ii) optimal at pH 6.8; (iii) 70-90% specific; (iv) at equilibrium after 60 min of incubation at 22-24 degrees C or 180 min at 0-4 degrees C (the rate constants for association, i.e. ka, at 22 and 4 degrees C were 18 and 1.1 x 10(7) M-1 min-1, respectively); and (v) essentially nondissociable. Nondissociable thrombin that bound during incubation at 0-4 degrees C was distributed equally between trypsin-sensitive and insensitive compartments. Thrombin associated with the former was released into the media when the cells were incubated at 0-4 degrees C with hirudin or hydroxylamine, or transferred to the insensitive compartment when incubated at 22 degrees C. Finally, confluent cultures of fibroblasts bind 2-3 x 10(4) 125I-alpha-thrombin molecules/cell with an apparent binding constant, i.e. Kd, of 0.7 nM (a true Kd could not be determined because of the irreversible nature of thrombin binding). The binding capacity per cell and the apparent Kd value increased proportionally to an increase in culture density.
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PMID:Protease mitogenic response of chick embryo fibroblasts and receptor binding/processing of human alpha-thrombin. 625 43

Recently we presented evidence that normal human foreskin fibroblasts (HF cells) limit the activity of secreted urokinase by secreting it as a proenzyme and by secreting protease nexin , an inhibitor of urokinase and certain other serine proteases (Scott, R.W., Eaton, D. L. Duran , N., and Baker, J.B. (1983) J. Biol. Chem. 258, 4397-4403). Using immunoaffinity chromatography we have now purified the HF cell urokinase proenzyme. It is a single 52-kDa polypeptide chain that is inactive toward both plasminogen and low molecular weight substrates. After proteolytic activation, this material (specific activity of 3 X 10(4) Committee on Thrombolytic Agents units/mg) is composed of two disulfide-bridged 33- and 19-kDa chains, and is thus similar to the predominant form of urokinase found in urine. Plasmin at 2 X 10(-10) M causes 50% activation of the proenzyme (1 X 10(-9) M) in 30 min at 37 degrees C. Thrombin and trypsin are one-twentieth as effective as plasmin. Activated HF cell 125I-urokinase forms sodium dodecyl sulfate stable complexes with purified protease nexin or protease nexin present in medium conditioned by HF cells. Purified protease nexin inhibits purified HF cell urokinase action on both plasminogen and low molecular weight substrates. The association rate constant for the reaction between protease nexin and HF cell urokinase is approximately 1.7 X 10(5) M-1 S-1. In contrast, the association rate constants for reactions between protease nexin and the one- and two-chain forms of tissue-type plasminogen activator are approximately 2 X 10(3) and approximately 3 X 10(4) M-1 S-1, respectively. The importance of protease nexin as a regulator of HF cell urokinase is supported by the finding that anti-protease nexin antibody potentiates the fibrinolytic activity of HF cell-conditioned medium incubated with plasminogen.
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PMID:Purification of human fibroblast urokinase proenzyme and analysis of its regulation by proteases and protease nexin. 637 53

Presence of chronic DIC (disseminated intravascular coagulation) eliciting an impeded blood coagulation has been postulated of late as one of the etiology causing toxemia of pregnancy, for which studies have been immunologically made. These theories remain unestablished. In this regard, the role of complement in blood coagulation has been noted, and their correlation is being elucidated. The author introduced a concept of complement to etiological theory of an impeded blood coagulation origin, by which toxemia of pregnancy was studied with emphasis placed on their correlation. The results obtained are as follow: 1) Thrombin and thromboplastin allowed in vitro to decreases the potency of complement, and the lowering also was seen even in the case of simultaneous supplement of urokinase and plasminogen. 2) The decrease also was periodically seen in rabbit's DIC experimentally made. 3) An increase in CH50, C3, C4, and factor B of normal pregnancy were of significance when compared with those of the control (p less than 0.001), while C1 inactivator decreased significantly (p less than 0.001). 4) CH50 was 52.2 +/- 2.4U/ml in severe toxemia, a decrease being of significance (p less than 0.01) as compared with that in third trimester of normal pregnancy. Those other parameters which tended to decrease included hemolytic activity of alternative pathway (AP-CH50), C4, and factor B except C1 inactivator with a trend being high.
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PMID:[Studies on relationship between complement and blood coagulation system in toxemia of pregnancy (author's transl)]. 706 48

Fibrin contains a factor which promotes growth of the mesenchymal cells and such may be related tissue repair. Effects of fibrin and fibrinogen degradation products (FDP) on the growth of smooth muscle cells (SMC) of rabbit aortas in culture were investigated, in relation to atherogenesis. Fibrin, free from plasminogen enhanced the proliferation of SMC during the experimental period of 48 h. Fibrin, rich in plasminogen also stimulated the proliferation of SMC within 24 h, but inhibited it after 48 h. FDP (fragments D and E) inhibited the proliferation of SMC. SMC of rabbit aortas demonstrated plasminogen activator activity. Thrombin and urokinase exhibited no promoting effects on the growth of SMC. These results support the hypothesis that the proliferation of SMC is stimulated by fibrin and later inhibited by FDP, as produced by the fibrinolytic activity of SMC. It is proposed that the metabolism of fibrin in the arterial wall may be of importance in the regulation of SMC proliferation and that the coagulation-fibrinolysis system may play a significant role in atherogenesis.
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PMID:Effects of fibrin and fibrinogen-degradation products on the growth of rabbit aortic smooth muscle cells in culture. 713 18

We examined in patients with acute myocardial infarction (AMI) the pharmacokinetics of saruplase, an unglycosylated, single chain, urokinase-type plasminogen activator (rscu-PA) by measuring urokinase-type plasminogen activator (u-PA) antigen and total u-PA activity, its conversion to active two-chain urokinase-type plasminogen activator (tcu-PA) and evaluated its effect on haemostatic parameters. Twelve patients were studied during and after administration of 20 mg bolus plus 60 mg continuous 1 h i.v. infusion of saruplase. For u-PA antigen and total u-PA activity (expressed as protein equivalents), where 234 U corresponds to 1 microgram, respectively, steady state plasma concentrations were 2.75 +/- 8.3 and 2.50 +/- 7.0 micrograms/ml (mean +/- standard deviation) and were reached within 20 min, t1/2 lambda 1 was 9.1 +/- 1.8 and 7.8 +/- 1.3 min, t1/2 lambda 2 1.2 +/- 0.2 and 1.9 +/- 0.5 h, and the total clearance was 393 +/- 110 and 427 +/- 113 ml/min. Inactivation of saruplase in plasma was negligible. After 15 min, tcu-PA was detected in plasma. From the ratio of the areas under the curve of tcu-PA and total u-PA activities it was calculated that 28 +/- 9.3% of the saruplase dose is converted into active tcu-PA. Systemic plasminaemia occurs as shown by a decrease in alpha 2-antiplasmin and fibrinogen and an increase in fibrinogen degradation products. Thrombin-antithrombin complex formation indicated activation of the clotting system. Saruplase is eliminated rapidly from plasma in AMI patients.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Pharmacokinetics and pharmacodynamics of saruplase, an unglycosylated single-chain urokinase-type plasminogen activator, in patients with acute myocardial infarction. 753 46

Endothelial cells are central in fibrinolysis because of their high production of both activators (t-PA, uPA) and inhibitors (PAI-1). The t-PA and PAI-1 synthesis could be regulated by signals transduction at several cellular levels. The purpose of this in vitro study, on cultured endothelial cells, was to explore the receptor/second messenger regulation of the t-PA and PAI-1 synthesis. Quiescent confluent human umbilical vein endothelial cells, cultured in passage 1, were exposed to different test substances. Samples from the conditioned medium were collected after 16 and 24 h and analysed for t-PA and PAI-1 antigen. All data presented were related to the data from control dishes (= 100%), in the same experiment. The results from the present study (mean +/- 95% confidence interval) demonstrated the following. (1) Forskolin, with a documented direct cAMP-inducing effect, decreased the basal PAI-1 production to 61 +/- 15%, and Na-nitroprusside, with a documented cGMP-inducing effect, increased the basal PAI-1 production to 141 +/- 38% without affecting the basal t-PA production. The surface receptor agonists isoprenalin or ephedrine, which indirectly affect adenylate cyclase, had no effect on t-PA or PAI-1 production. (2) Phorbolester (PMA), which directly activates proteinkinase C (PKC), increased the basal t-PA and PAI-1 production to 350 +/- 71%, and 163 +/- 35% respectively. (3) Thrombin, but not endothelin-1 (ET-1), increased the basal t-PA and PAI-1 production to 195 +/- 34% and 136 +/- 18%, respectively, indicating an PKC-mediated thrombin effect.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Complex intracellular signal transduction regulates tissue plasminogen activator (t-PA) and plasminogen activator inhibitor type-1 (PAI-1) synthesis in cultured human umbilical vein endothelium. 756 35

Fibrin deposition is characteristic of inflammatory diseases. The monocytes is central to the inflammatory response and can affect fibrinolysis by expression of urokinase (u-PA) and plasminogen activator inhibitor types 1 and 2 (PAI-1 and PAI-2, respectively). This study examines whether thrombin, which promotes fibrin deposition, can contribute to fibrin persistence by modulating expression of proteins of the fibrinolytic system. Monocytes were isolated from human peripheral blood and analyzed for PAI-2, PAI-1, and u-PA antigens by enzyme-linked immunosorbent assay (ELISA). Monocytes responded to thrombin by increased expression of PAI-2 in a dose- and time-dependent manner, with maximal synthesis at a concentration of 1 U/mL to 10 U/mL. This trend was also evident for PAI-1, which was present at much lower levels. Thrombin and lipopolysaccharide (LPS) stimulated comparable levels of PAI-2, studied at the antigen and mRNA level. The dose effet of LPS on PAI-2 and PAI-1 was found to differ from that of thrombin. The level of u-PA was undetectable by ELISA and zymography in all samples. Thrombin stimulates PAI-2 synthesis by human monocytes, therefore creating an imbalance in the fibrinolytic system. This may contribute to persistence of fibrin, deposited during inflammation.
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PMID:Thrombin modulates synthesis of plasminogen activator inhibitor type 2 by human peripheral blood monocytes. 757 47

alpha-Thrombin regulation of endothelial cell (EC) fibrinolysis has been documented by using endothelia derived from a number of anatomic locations but not with those derived from the human cerebral vasculature. In the present study, the fibrinolytic properties of human cerebral microvascular ECs and their regulation by alpha-thrombin are delineated and contrasted with those of human umbilical vein and foreskin microvascular ECs. In cerebral ECs, alpha-thrombin elicited a unique dose-dependent increase in urokinase production and DNA synthesis. Maximal stimulation, observed with 10 nmol/L alpha-thrombin, resulted in a 30- to 50-fold increase in urokinase production and a concomitant fourfold increase in DNA synthesis; the increase in urokinase was reflected in higher steady-state levels of urokinase mRNA. The major urokinase product secreted is the single-chain form of the enzyme. No effect was observed with the addition of other proteases or catalytically inactive variants of alpha-thrombin. A thrombin receptor agonist peptide upregulated urokinase production but had no effect on DNA synthesis, suggesting that fibrinolysis is mediated by the thrombin receptor but that proliferation is regulated by a different pathway. These findings suggest the possibility that the cerebral microvasculature may be a specialized region of the vascular system in which urokinase-type plasminogen activator, not tissue-type plasminogen activator, is the key catalyst of fibrin lysis when the brain responds to thrombotic events and that alpha-thrombin may regulate repair of the cerebral microvascular system.
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PMID:Alpha-thrombin stimulates urokinase production and DNA synthesis in cultured human cerebral microvascular endothelial cells. 760 Jan 22

Plasminogen activator inhibitor-1 (PAI-1) is the major inhibitor for plasmin formation promoted by tissue and urokinase plasminogen activators. The present study demonstrates that thrombin increase PAI-1 antigen, biological activity, and gene expression in cultured baboon aortic smooth muscle cells (BASMC). Thrombin elevates PAI-1 antigen in conditioned medium of BASMC within 10 min of the treatment, with the peak increase after 30 min of the treatment. Overexpression of PAI-1 gene was detected in the cultures exposed to thrombin for at least 60 min. PAI activity in conditioned medium increased in the cultures treated with thrombin for at least 4 h. The thrombin-induced early increase of PAI-1 antigen (up to 30 min of the stimulation) was blocked by hirudin (a specific inhibitor of thrombin), mimicked by trypsin and not suppressed by cycloheximide (a protein synthesis inhibitor). The majority of metabolically labeled PAI-1 associated with BASMC was present in extracellular matrix. The level of extracellular matrix-associated PAI-1 was reduced 40% by 30 min of thrombin treatment. Our results suggest that thrombin not only increases PAI-1 transcription but also proteolytically cleaves PAI-1 from the extracellular matrix of vascular SMC. PAI-1 released by thrombin from the extracellular matrix may not alter PAI activity in extracellular fluid but may reduce the storage of PAI-1 in the extracellular matrix of vascular smooth muscle cells.
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PMID:Effect of thrombin on release of plasminogen activator inhibitor-1 from cultured primate arterial smooth muscle cells. 774 May 4

The endothelial cell (EC) urokinase receptor plays an important role in the localization and receptor-mediated activation of EC-bound plasminogen and hence surface-localized fibrinolysis. Thrombin induced a rapid (< 5 minute), time- (0 to 30 minutes) and dose- (0.1 to 8 U/mL) dependent decrease in the specific binding of 125I-labeled two-chain urokinase-type plasminogen activator (tcu-PA) or diisopropylfluoro-phosphate-tcu-PA to urokinase-type plasminogen activator receptor (u-PAR) in cultured ECs from various sources (range, 21% to 50%). The thrombin receptor activation peptide but not control peptide showed a similar but reduced decrease in the specific binding of 125I-labeled tcu-PA to u-PAR. Incubation of thrombin-treated cultures (10 to 12 hours) in complete medium restored 125I-labeled tcu-PA ligand binding to normal levels. u-PAR mRNA levels rapidly (1 hour) increased and peaked 10 to 12 hours after thrombin treatment as analyzed by reverse transcriptase-polymerase chain reaction. Decreased thrombin-induced 125I-labeled tcu-PA binding correlated with the time-dependent decrease in surface-localized plasmin generation, as measured by the direct activation of 125I-labeled Glu-plasminogen and quantification of the 20-kD light chains of 125I-labeled plasmin. After incubation with thrombin, plasmin generation was decreased 50% to 56% (125 to 152 fmol/3 to 3.5 x 10(4) cells). Isolation of metabolically labeled 35S-labeled u-PAR from the media of thrombin and phospholipase C-treated human aortic cultures yielded approximately 10- and approximately 12-fold more 55-kD M(r) and approximately 6-fold more 35-kD M(r) 35S-labeled u-PAR forms than control cultures, respectively. The u-PAR antigen forms (M(r), 54 kD) and the glycosyl-phosphatidylinositol-anchored protein CD59 (M(r), 20 kD) were also simultaneously identified by immunoprecipitation in the media of thrombin-treated cultures. This suggests that thrombin may release u-PAR and decrease u-PA ligand binding through a common pathway involving phospholipase C. These results establish a novel interrelation between thrombin and EC fibrinolysis and suggest that thrombin may also have an additional regulatory role in the net expression of surface-localized EC fibrinolytic activity.
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PMID:Thrombin decreases the urokinase receptor and surface-localized fibrinolysis in cultured endothelial cells. 774 51


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