Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.21.4 (trypsin)
 42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A critical component in the regulation of thrombus formation and clearance is the balance between tissue plasminogen activator (tPA) and plasminogen activator inhibitor type-1 (PAI-1). An increase in the plasma concentration of PAI-1 has been proposed as a risk factor in thrombotic disease. Inhibition of PAI-1 activity may have utility in the treatment of thromboembolic disease. We report here the evaluation of three diketopiperazine-based low molecular weight inhibitors of PAI-1 activity (XR334, XR1853 and XR5082). In vitro these compounds reversed the inhibitory effects of PAI-1 against both tPA and urokinase (UK) (IC50: 5 to 80 muM). In contrast, other serpin-serine protease interactions, including alpha 1-antitrypsin-trypsin, alpha 2-antiplasmin- plasmin and antithrombin-thrombin, were not affected, neither did these inhibitors affect global tests of haemostasis. In the light of this promising in vitro profile these compounds were evaluated in a standard radioisotopic assay of clot lysis in whole rat blood following intravenous administration. In this assay these compounds dose-dependently enhanced fibrinolysis ex vivo. After intravenous bolus administration XR334, XR1853 and XR5082 at 5 mg/kg increased clot lysis by 32.0 +/- 5.1% SEM (n = 25, p < 0.01), 36.7 +/- 3.5% SEM (n = 36, p < 0.01) and 60.0 +/- 2.8% SEM (n = 17, p < 0.01) respectively compared to vehicle. Intravenous infusion of these compounds (1 mg/kg/min for 20 min) significantly prolonged (approximately twofold) the time to blood vessel occlusion in the rat electrically-stimulated carotid artery thrombosis model. Thus, these low molecular weight inhibitors of PAI-1 activity enhanced fibrinolysis ex vivo and protected against thrombus formation in the rat.
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PMID:Evaluation of a low molecular weight modulator of human plasminogen activator inhibitor-1 activity. 872 28

Artificially induced rat decidual tissue expresses plasminogen activator inhibitor (PAI). This PAI, isolated and purified employing chromatographic techniques, is a low molecular weight protein unlike the known PAIs. The final purified preparation resolves into a single band following SDS-PAGE and has an approximate molecular weight of 29 kDa. The properties studied include specificity for urokinase-type (uPA) and tissue-type (tPA) plasminogen activators, binding to conA and heparin, inhibition of thrombin, plasmin and trypsin. Decidual PAI is immunogenic in rabbit and a monospecific antiserum raised against the decidual inhibitor cross reacts with an extract of human placenta.
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PMID:A molecular variant of plasminogen activator inhibitor of rat decidual tissue. 874 33

Serpin inhibitors are believed to form an acyl enzyme intermediate with their target proteinases which is stabilized through insertion of the enzyme-linked part of the reactive center loop (RCL) as strand 4 in beta-sheet A of the inhibitor. To test critically the role and timing of these steps in the reaction of the plasminogen activator inhibitor PAI-1, we blocked the vacant position 4 in beta-sheet A of this serpin with an octapeptide. The peptide-blocked PAI-1 was a substrate for both tissue-type plasminogen activator (tPA) and trypsin and was hydrolyzed at the scissile bond. The reactivity of the peptide-blocked substrate PAI-1 was compared to that of the unmodified inhibitor by rapid acid quenching as well as photometric techniques. With trypsin as target, the limiting rate constants for enzyme acylation were essentially the same with inhibitor and substrate PAI-1 (21-23 s-1), as were also the associated apparent second-order rate constants (2.8-2.9 microM-1 s-1). With tPA, inhibitor and substrate PAI-1 reacted identically to form a tightly bound Michaelis complex (Kd approximately Km approximately 20 nM). The limiting rate constant for acylation of tPA, however, was 57 times faster with inhibitor PAI-1 (3.3 s-1) than with the substrate form (0.059 s-1), resulting in a 5-fold difference in the corresponding second-order rate constants (13 vs 2.5 microM-1 s-1). We attribute the ability of tPA to discriminate between the two PAI-1 forms to exosite bonds that cannot occur with trypsin. The exosite bonds retain specifically the distal part of the PAI-1 RCL in the substrate pocket, which favors a reversal of the acylation step. Acylation of tPA becomes effective only by separating the products of the acylation step. With substrate PAI-1, this depends on passive displacement of bonds, whereas with inhibitor PAI-1, separation is accomplished by loop insertion that pulls tPA from its docking site on PAI-1, resulting in faster acylation than with substrate PAI-1.
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PMID:Inhibitory mechanism of serpins: loop insertion forces acylation of plasminogen activator by plasminogen activator inhibitor-1. 979 12

Human kallikrein (hK) 2 is an arginine-selective serine protease expressed predominantly in the prostate that has an 80% sequence identity with prostate-specific antigen. Expression of hK2 is elevated in the tumor epithelium compared to benign prostate tissue. We have purified, sequenced, and identified a novel hK2 complex in prostate tissue consisting of hK2 and a serine protease inhibitor known as protease inhibitor-6 (PI-6). This 64-kDa SDS-PAGE stable complex is elevated in the tumor and is approximately 10% of total hK2. No comparable complex of prostate-specific antigen was detected. PI-6, also known as cytoplasmic antiprotease, has been characterized as an intracellular inhibitor of trypsin and chymotrypsin-like proteases, which has high homology to plasminogen activator inhibitor 1 and 2. The physiological role of PI-6 in the prostate and its relationship to hK2 and prostate cancer are under investigation.
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PMID:Identification of a novel complex between human kallikrein 2 and protease inhibitor-6 in prostate cancer tissue. 1046 85

The plasminogen activator isolated from the venom of the snake Trimeresurus stejnegeri (TSV-PA) triggers plasmin production, along with tissue-type plasminogen activators (t-PA) and urokinase (u-PA). The half-life of TSV-PA in plasma is remarkable. We unveil in this paper two of the molecular mechanisms allowing TSV-PA to escape inhibition by plasma serpins. The first involves a phenylalanine at position 193 (chymotrypsinogen numbering system). Phe(193) distinguishes TSV-PA from nearly all trypsin-like proteinases, having glycine at this position. A mutant of TSV-PA (F193G), in which Phe(193) had been replaced by a glycine, was inactivated by plasminogen activator inhibitor 1 (PAI-1) and alpha(2)-antiplasmin 100-fold more rapidly than the wild-type enzyme. The second mechanism originates from the 37-loop of TSV-PA. Swapping the 37-loop of TSV-PA for either that of t-PA or that of u-PA also increased dramatically the rate of inactivation by PAI-1. Loop swapping and F193G mutations were additive, resulting in a rate of inactivation by PAI-1 that was 4 orders of magnitude higher than for the wild-type enzyme. The potential role of Phe(193) and of the 37-loop in the immunity of TSV-PA toward alpha(1)-antitrypsin and antithrombin is also discussed.
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PMID:The stratagem utilized by the plasminogen activator from the snake Trimeresurus stejnegeri to escape serpins. 1208 98

The importance of proteoglycans for secretion of proteolytic enzymes was studied in the murine macrophage cell line J774. Untreated or 4beta-phorbol 12-myristate 13-acetate (PMA)-stimulated macrophages were treated with hexyl-beta-d-thioxyloside to interfere with the attachment of glycosaminoglycan chains to their respective protein cores. Activation of the J774 macrophages with PMA resulted in increased secretion of trypsin-like serine proteinase activity. This activity was completely inhibited by plasminogen activator inhibitor 1 and by amiloride, identifying the activity as urokinase plasminogen activator (uPA). Treatment of both the unstimulated or PMA-stimulated macrophages with xyloside resulted in decreased uPA activity and Western blotting analysis revealed an almost complete absence of secreted uPA protein after xyloside treatment of either control- or PMA-treated cells. Zymography analyses with gels containing both gelatin and plasminogen confirmed these findings. The xyloside treatment did not reduce the mRNA levels for uPA, indicating that the effect was at the post-translational level. Treatment of the macrophages with xylosides did also reduce the levels of secreted matrix metalloproteinase 9. Taken together, these findings indicate a role for proteoglycans in the secretion of uPA and MMP-9.
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PMID:Secretion of macrophage urokinase plasminogen activator is dependent on proteoglycans. 1451 79

Human kallikrein 5 (KLK5) is a member of the human kallikrein gene family of serine proteases. Preliminary results indicate that the protein, hK5, may be a potential serological marker for breast and ovarian cancer. Other studies implicate hK5 with skin desquamation and skin diseases. To gain further insights on hK5 physiological functions, we studied its substrate specificity, the regulation of its activity by various inhibitors, and identified candidate physiological substrates. After producing and purifying recombinant hK5 in yeast, we determined the k(cat)/K(m) ratio of the fluorogenic substrates Gly-Pro-Arg-AMC and Gly-Pro-Lys-AMC, and showed that it has trypsin-like activity with strong preference for Arg over Lys in the P1 position. The serpins alpha(2)-antiplasmin and antithrombin were able to inhibit hK5 with an inhibition constant (k(+2)/K(i)) of 1.0 x 10(-) (2)and 4.2 x 10(-4) m(-1) min(-1), respectively. No inhibition was observed with the serpins alpha(1)-antitrypsin and alpha(1)-antichymotrypsin, although alpha(2)-macroglobulin partially inhibited hK5 at high concentrations. We also demonstrated that hK5 can efficiently digest the extracellular matrix components, collagens type I, II, III, and IV, fibronectin, and laminin. Furthermore, our results suggest that hK5 can potentially release (a) angiostatin 4.5 from plasminogen, (b) "cystatin-like domain 3" from low molecular weight kininogen, and (c) fibrinopeptide B and peptide beta15-42 from the Bbeta chain of fibrinogen. hK5 could also play a role in the regulation of the binding of plasminogen activator inhibitor 1 to vitronectin. Our findings suggest that hK5 may be implicated in tumor progression, particularly in invasion and angiogenesis, and may represent a novel therapeutic target.
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PMID:Biochemical and enzymatic characterization of human kallikrein 5 (hK5), a novel serine protease potentially involved in cancer progression. 1571 79

Polyserase-1 (polyserine protease-1)/TMPRSS9 (transmembrane serine protease 9) is a type II transmembrane serine protease (TTSP) that possesses unique three tandem serine protease domains. However, the physiological function of each protease domain remains poorly understood. We discovered a new splice variant of polyserase-1, termed Serase-1B, which contains 34 extra amino acids consisting a SEA module (a domain found in sea urchin sperm protein, enterokinase and agrin) adjacent to the transmembrane domain and the first protease domain with a mucin-like box at the C-terminus. The tissue distribution of this enzyme by RT (reverse transcription)-PCR analysis revealed high expression in the liver, small intestine, pancreas, testis and peripheral blood CD14+ and CD8+ cells. To investigate the role of Serase-1B, a full-length form recombinant protein was produced. Interestingly, recombinant Serase-1B was partly secreted as a soluble inactive precursor and it was also activated by trypsin. This activated enzyme selectively cleaved synthetic peptides for trypsin and activated protein C, and it was inhibited by several natural serine protease inhibitors, such as aprotinin, alpha2-antiplasmin and plasminogen activator inhibitor 1. In addition, Serase-1B efficiently converted pro-uPA (urokinase-type plasminogen activator) into active uPA and this activation was strongly inhibited by these natural inhibitors. Furthermore, this activation was also negatively regulated by glycosaminoglycans. Our results indicate that Serase-1B is a novel member of TTSPs that might be involved in uPA/plasmin-mediated proteolysis and possibly implicated in biological events such as fibrinolysis and tumour progression.
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PMID:Serase-1B, a new splice variant of polyserase-1/TMPRSS9, activates urokinase-type plasminogen activator and the proteolytic activation is negatively regulated by glycosaminoglycans. 1687 79

Protease-activated receptor-2 (PAR-2) is a cellular receptor expressed prominently on epithelial, mesangial, and endothelial cells in the kidney and on macrophages. PAR-2 is activated by serine proteases such as trypsin, tryptase, and coagulation factors VIIa and Xa. It induces pleiotropic effects including vasodilatation, increasing plasminogen activator inhibitor (PAI-1) expression, mesangial cell proliferation, and cytokine production by macrophages. The role of PAR-2 in renal inflammation was studied in antiglomerular basement membrane antibody-induced crescentic glomerulonephritis (CGN) using PAR-2-deficient (PAR-2(-/-)) mice and wild-type littermate controls. PAR-2(-/-) mice had reduced crescent formation, proteinuria, and serum creatinine compared with wild-type mice 21 days after initiation of CGN. Glomerular accumulation of CD4(+) T cells and macrophages and the number of proliferating cells in glomeruli were similar in both groups. Glomerular fibrin deposition was significantly reduced in PAR-2(-/-) mice, and this was associated with reduced renal plasminogen activator inhibitor expression and increased renal matrix-metalloprotinase-9 activity. These results demonstrate a proinflammatory role for PAR-2 in CGN that is independent of effects on glomerular leukocyte recruitment and mesangial cell proliferation. PAR-2-mediated augmentation of renal plasminogen activator inhibitor expression and inhibition of matrix-metalloprotinase-9 activity may contribute to increased glomerular fibrin accumulation and glomerular injury in CGN.
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PMID:Protease-activated receptor-2 augments experimental crescentic glomerulonephritis. 1764 Sep 68

Protease nexin-1 (PN-1) is a serine protease inhibitor (SERPIN) protein with functional roles in growth, development, patho-physiology and injury. Here, we report our work to clone, analyze the expression profile and characterize the properties of the PN-1 gene in rock bream (Rb), Oplegnathus fasciatus. RbPN-1 encodes a peptide of 397 amino acids (AA) with a predicted molecular mass of 44 kDa and a 23 AA signal peptide. RbPN-1 protein was found to harbor a characteristic SERPIN domain comprised of a SERPIN signature and having sequence homology to vertebrate PN-1s. The greatest identity (85%) was observed with PN-1 from the three-spined stickleback fish, Gasterosteus aculeatus. The functional domains, including a heparin binding site and reactive centre loop were conserved between RbPN-1 and other fish PN-1s; in particular, they were found to correspond to components of the human plasminogen activator inhibitor 1, PAI-1. Phylogenetic analysis indicated that RbPN-1 was closer to homologues of green spotted pufferfish and Japanese pufferfish. Recombinant RbPN-1 demonstrated antiprotease activity against trypsin (48%) and thrombin (89%) in a dose-dependent manner, and its antithrombotic activity was potentiated by heparin. The anticoagulant function prolonged clotting time by 3.7-fold, as compared to the control in an activated partial thromboplastin time assay. Quantitative real-time PCR results indicated that RbPN-1 is transcribed in many endogenous tissues at different levels. Lipopolysaccharide (LPS) stimulated a prolonged transcriptional response in hematic cells, and Rb iridovirus up-regulated the RbPN-1 mRNA level in hematic cells to a maximum of 3.4-fold at 12 h post-infection. Interestingly, LPS and Edwardsiella tarda significantly induced the RbPN-1 transcription at the late phase of infection. In vivo studies indicated that injury response caused a temporal suppression in RbPN-1 transcription, in conjunction with that of another SERPIN, rock bream heparin cofactor II, RbHCII. Taken together, our findings suggest that PN-1 functions as an antiprotease and anticoagulant and that SERPINs (PN-1 and HCII) are likely to contribute to immunity and post-injury responses.
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PMID:Rock bream (Oplegnathus fasciatus) serpin, protease nexin-1: transcriptional analysis and characterization of its antiprotease and anticoagulant activities. 2141 93


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