Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.21.5 (thrombin)
33,306 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Resveratrol, a polyphenolic compound found in red wines, is believed to be a contributor in decreasing the incidence of coronary heart disease. Although its primary target is unknown, it blocks aggregation of washed platelets by an ill-defined mechanism. We show that resveratrol, at 10-50 microM, blocked aggregation induced by collagen (5 microg/ml), thrombin (0.2 units/ml), and ADP (10 microM). This affect was not overcome by adding exogenous human fibrinogen to the assay, suggesting that an early (wave I) signaling step in the alpha(IIb)beta(3) activation cascade was impaired. To explore this possibility we examined the effect of resveratrol on activation of MAP kinases. In the platelet, MAP kinases become activated as a consequence of agonist binding and not of aggregation, which itself induces signaling events. In fact, we find that collagen-induced activation of MAP kinases is superinduced in the presence of RGDS, an aggregation-blocking peptide. Resveratrol, at concentrations of 10 microM and greater, inhibited MAP kinase activation induced by collagen (in the absence and presence of RGDS peptide), thrombin, and ADP. These data indicate that resveratrol blocks receptor-mediated signaling events in washed platelets. In comparison, resveratrol has poor antiplatelet activity in whole blood. Under these conditions aggregation was not affected by 50-100 microM resveratrol. Concentrations of 200 microM resveratrol were needed to cause a 30-60% decrease in platelet aggregation in whole blood. Together these studies suggest that resveratrol is a potent inhibitor of platelet signaling responses, but its antiplatelet activity is weakened or masked in circulation. Thus, although resveratrol may function as a protective agent of coronary heart disease, its affects are not solely attributed to its effects on platelets in circulation.
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PMID:Resveratrol decreases early signaling events in washed platelets but has little effect on platelet in whole blood. 1100 23

This study investigates the mitogenic effect of the coagulation factor Xa in smooth muscle cells (SMC) from human saphenous vein and the procoagulant activity of these cells. Factor Xa elicited a concentration-dependent increase in [3H]thymidine incorporation. This mitogenic effect of factor Xa was inhibited by DX-9065a and BABCH, indicating the requirement of proteolytic activity of the enzyme. Factor Xa activated the MAP kinases ERK1/2 concentration- and time-dependently. PDGF-neutralizing antibodies neither inhibited the increase in [3H]thymidine incorporation nor ERK-1/2 phosphorylation in factor Xa-stimulated cells, suggesting that factor Xa-induced signaling and mitogenic activity in human venous SMC are independent of PDGF. Exposure of SMC to recalcified plasma resulted in a significant thrombin generation which was inhibited by anti-tissue factor antibody, tissue factor pathway inhibitor, inactivated factor VIIa and DX-9065a. These data indicate that interaction of SMC with the clotting system may contribute to venous graft disease, i.e. thrombus formation and intimal hyperplasia.
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PMID:Factor Xa acts as a PDGF-independent mitogen in human vascular smooth muscle cells. 1101 78

Cytosolic Phospholipase A(2) (cPLA(2)) has been implicated in receptor-mediated release of arachidonic acid from membrane phospholipids, the limiting step in prostacyclin and other eicosanoid production. Its activity is controlled by Ca(++) levels and enzymatically regulated phosphorylation. The purpose of this study was to assess the importance of phosphorylation of cPLA(2) in human umbilical vein endothelial cells and to identify the kinases involved. Inhibitors were used to study the pathways leading to phosphorylation and activation of mitogen activated protein kinases (MAP-kinases) and cPLA(2), as well as release of arachidonic acid and prostacyclin production after stimulation with different agonists. We have found that agonists that release arachidonic acid, including histamine, thrombin, AlF(4)(-), and pervanadate, all activate the MAP kinases ERK, p38 and JNK and cause phosphorylation of cPLA(2). Agonist specific differences in the signal transduction pathways included variable contribution of tyrosine phosphorylation, protein kinase C and ERK activity, and different effects of pertussis toxin. Treatment with PD98059 (inhibitor of ERK-activation) or SB203580 (inhibitor of p38) caused partial decrease in arachidonic acid release and cPLA(2) activity. In contrast the nonspecific protein kinase inhibitor staurosporin completely inhibited cPLA(2) activity. We conclude that in endothelial cells arachidonic acid release is largely mediated by cPLA(2) through agonist-specific pathways. The MAP kinases ERK and p38 both have demonstrable but not major effect on agonist stimulated arachidonic acid release and the data suggest that an additional unidentified kinase also has a role.
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PMID:Involvement of MAP kinases in the control of cPLA(2) and arachidonic acid release in endothelial cells. 1136

1. 3-Hydroxy-3-methylglutaryl co-enzyme A reductase inhibitors (statins) prevent the progression of atherosclerosis by lowering cholesterol. However, the effect of statins on the synthesis of pro-inflammatory cytokines from endothelial cells has not yet been fully investigated. Here, we examined the effect of pravastatin, one of the statins, on IL-8 synthesis induced by thrombin in human aortic endothelial cells (AoEC) cultured with high glucose concentrations. 2. Pravastatin significantly decreased the IL-8 synthesis induced by thrombin. 3. Pravastatin inhibited the p44/42 MAP kinase activity induced by thrombin, but did not inhibit the p38 MAP kinase activity. 4. Translocation of ras protein from the cytosol to plasma membrane was inhibited by pravastatin. 5. Pravastatin inhibit the activator protein-1 activity, but did not inhibit the activation of IkappaB-alpha. 6. Dominant negative ras inhibited the p44/42 MAP kinase activity induced by PMA. 7. Our results suggest that pravastatin inhibits IL-8 synthesis by blocking the ras-MAP (p44/42) kinase pathway rather than nuclear factor-kappaB. Pravastatin may prevent atherosclerosis not only by lowering cholesterol levels, but also by suppressing IL-8 synthesis in AoEC through the inhibition of p44/42 MAP kinase, and this may be more beneficial in diabetic patients than in non-diabetics.
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PMID:Pravastatin suppresses the interleukin-8 production induced by thrombin in human aortic endothelial cells cultured with high glucose by inhibiting the p44/42 mitogen activated protein kinase. 1160 15

In this study, we demonstrated that thrombin activates protein kinase C (PKC), mitogen activated protein kinases (MAP kinases), transcription factor nuclear factor-kappa B (NF-kappa B), and cAMP-dependent protein kinase (PKA) in the human renal carcinoma cell line A-498. In addition, it enhanced the migratory capacity, but had no effect on the proliferation of A-498 cells. The effect of thrombin on migration could be blocked by the PKA inhibitor H-89 but was not influenced by inhibition of PKC, MAP kinases or NF-kappa B. We concluded, that thrombin acts as a regulator on human A-498 renal carcinoma cell migration including PKA.
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PMID:The serine proteinase thrombin promotes migration of human renal carcinoma cells by a PKA-dependent mechanism. 1217 50

Ventilator-induced lung injury syndromes are characterized by profound increases in vascular leakiness and activation of inflammatory processes. To explore whether excessive cyclic stretch (CS) directly causes vascular barrier disruption or enhances endothelial cell sensitivity to edemagenic agents, human pulmonary artery endothelial cells (HPAEC) were exposed to physiologically (5% elongation) or pathologically (18% elongation) relevant levels of strain. CS produced rapid (10 min) increases in myosin light chain (MLC) phosphorylation, activation of p38 and extracellular signal-related kinase 1/2 MAP kinases, and actomyosin remodeling. Acute (15 min) and chronic (48 h) CS markedly enhanced thrombin-induced MLC phosphorylation (2.1-fold and 3.2-fold for 15-min CS at 5 and 18% elongation and 2.1-fold and 3.1-fold for 48-h CS at 5 and 18% elongation, respectively). HPAEC preconditioned at 18% CS, but not at 5% CS, exhibited significantly enhanced thrombin-induced reduction in transendothelial electrical resistance but did not affect barrier protective effect of sphingosine-1-phosphate (0.5 microM). Finally, expression profiling analysis revealed a number of genes, including small GTPase rho, apoptosis mediator ZIP kinase, and proteinase activated receptor-2, to be regulated by CS in an amplitude-dependent manner. Thus our study demonstrates a critical role for the magnitude of CS in regulation of agonist-mediated pulmonary endothelial cell permeability and strongly suggests phenotypic regulation of HPAEC barrier properties by CS.
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PMID:Magnitude-dependent regulation of pulmonary endothelial cell barrier function by cyclic stretch. 1295 24

Thrombin activates human platelets through three different membrane receptors, the protease-activated receptors PAR-1 and PAR-4 and the glycoprotein Ib (GPIb)-IX-V complex. We investigated the contribution of these three receptors to thrombin-induced activation of the small GTPase Rap1B. We found that, similarly to thrombin, selective stimulation of either PAR-1 or PAR-4 by specific activating peptides caused accumulation of GTP-bound Rap1B in a dose-dependent manner. By contrast, in PAR-1- and PAR-4-desensitized platelets, thrombin failed to activate Rap1B. Thrombin, PAR-1-, or PAR-4-activating peptides also induced the increase of intracellular Ca(2+) concentration and the release of serotonin in a dose-dependent manner. We found that activation of Rap1B by selected doses of agonists able to elicit comparable intracellular Ca(2+) increase and serotonin release was differently dependent on secreted ADP. In the presence of the ADP scavengers apyrase or phosphocreatine-phosphocreatine kinase, activation of Rap1B induced by stimulation of either PAR-1 or PAR-4 was totally inhibited. By contrast, thrombin-induced activation of Rap1B was only minimally affected by neutralization of secreted ADP. Concomitant stimulation of both PAR-1 and PAR-4 in the presence of ADP scavengers still resulted in a strongly reduced activation of Rap1B. A similar effect was also observed upon blockade of the P2Y12 receptor for ADP, as well as in P2Y12 receptor-deficient human platelets, but not after blockade of the P2Y1 receptor. Activation of Rap1B induced by thrombin was not affected by preincubation of platelets with the anti-GPIbalpha monoclonal antibody AK2 in the absence of ADP scavengers or a P2Y12 antagonist but was totally abolished when secreted ADP was neutralized or after blockade of the P2Y12 receptor. Similarly, cleavage of the extracellular portion of GPIbalpha by the cobra venom mocarhagin totally prevented Rap1B activation induced by thrombin in the presence of apyrase and in P2Y12 receptor-deficient platelets. By contrast, inhibition of MAP kinases or p160ROCK, which have been shown to be activated upon thrombin binding to GPIb-IX-V, did not affect agonist-induced activation of Rap1B in the presence of ADP scavengers. These results indicate that although both PAR-1 and PAR-4 signal Rap1B activation, the ability of thrombin to activate this GTPase independently of secreted ADP involves costimulation of both receptors as well as binding to GPIb-IX-V.
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PMID:Contribution of protease-activated receptors 1 and 4 and glycoprotein Ib-IX-V in the G(i)-independent activation of platelet Rap1B by thrombin. 1507 82

Using a signal sequence trap for selection of differentially expressed secretory and membrane proteins, we identified a novel member of the adhesion family of G-protein coupled receptors (GPCRs), termed vascular inducible GPCR (VIGR). VIGR contains C1r-C1s, Uegf and Bmp1 (CUB) and pentraxin (PTX)-like modules and a mucin-like spacer, followed by seven transmembrane domains. By surface biotinylation as well as by immunofluorescence analysis we demonstrate that endogenous, highly glycosylated VIGR is expressed on the cell surface of endothelial cells (ECs) upon LPS or thrombin treatment, and inducible expression is mediated by MAP kinases, but not NF-kappaB. We show that VIGR is selectively expressed in ECs derived from larger vessels, but not from microvessels. In summary, VIGR represents a novel GPCR of the adhesion family, which is unique in its long extra-cellular domain comprising CUB and PTX-like modules and in its inducibility by LPS and thrombin in a subset of ECs, suggesting an important function in cell-adhesion and potentially links inflammation and coagulation.
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PMID:VIGR--a novel inducible adhesion family G-protein coupled receptor in endothelial cells. 1522 24

Prolidase [E.C. 3.4.13.9] is a cytosolic imidodipeptidase that plays an important role in collagen biosynthesis. The enzyme contributes to the recovery of proline from protein degradation products (mainly collagen) for collagen resynthesis. Prolidase activity and collagen biosynthesis are supposed to be regulated by beta(1)-integrins, which initiate a signaling pathway in which several kinases and intracellular proteins are involved, including focal adhesion kinase pp125(FAK) (FAK), Src, Shc, growth factor receptor bound protein 2 (Grb-2), son of sevenless protein (SOS), Ras, Raf and mitogen-activated protein kinases (MAPK), extracellular-signal regulated kinase 1 (ERK(1)) and kinase 2 (ERK(2)). We studied the effects of echistatin, a well-known disintegrin and thrombin, a serine protease capable of activation of platelet integrin alpha(2)beta(1) receptor on collagen production, prolidase activity, expression of prolidase, beta(1)-integrin receptor, FAK, SOS-protein and phosphorylated MAP-kinases (ERK(1) and ERK(2)) in confluent human dermal fibroblasts. It has been found that treatment of the cells with 100nM echistatin contributes to inhibition of collagen production, as well as prolidase activity and expression compared to control cells. These phenomena were accompanied by a decrease in the expression of FAK, SOS-protein and phosphorylated MAP-kinases, ERK(1) and ERK(2). An opposite phenomenon was observed in fibroblasts treated with 0.1IU thrombin. In this case, a significant increase in collagen production and prolidase activity, accompanied by a distinct raise in the expression of prolidase, FAK and phosphorylated MAP-kinases and a slight increase in expression of SOS compared to controls were found. The results suggest that regulation of prolidase activity and collagen biosynthesis in human dermal fibroblasts may involve beta(1)-integrin-dependent signaling.
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PMID:Differential effects of echistatin and thrombin on collagen production and prolidase activity in human dermal fibroblasts and their possible implication in beta1-integrin-mediated signaling. 1566 71

We studied the protease activated receptor-1 coupling to a serum response element (SRE)-dependent luciferase activity readout in transfected COS-7 cells. Thrombin, with a pEC50 of 10.5, was 3000-fold more potent than the peptide agonists SFLLR and its derived compound C721-40 in stimulating luciferase activity, although the three agonists exhibited similar efficacy at the maximal concentration tested. Interestingly, SFLLR- and C721-40-induced luciferase activity was biphasic, suggesting that at least two populations of G proteins couple to the receptor. Further pharmacological characterization of this system was performed using selective protease activated receptor-1 antagonists. SCH203099 and ER-112787 blocked SFLLR-induced luciferase activity with similar potencies (pK(B) of 7), slightly higher than that exhibited by an arylisoxazole derivative compound from Merck (pK(B) of 6.1). These values correlated with their affinities established by competition binding experiments using [3H]-C721-40 as radioligand for protease activated receptor-1. Transduction mechanisms of protease activated receptor-1 coupling to SRE-dependent luciferase activity were examined using specific inhibitors. The Ca2+ chelator BAPTA-AM, as well as the calmodulin inhibitors W-7 and ophiobolin A, robustly inhibited SFLLR-induced SRE activation. Overexpression of RGS2 and a dominant negative rhoA protein abolished the SFLLR signal in an additive manner, suggesting a major role of Gq and G12/13 proteins. Furthermore, inhibition of phospholipase C, MAP-kinases, phosphatidyl inositol-3 kinase, rho-kinase and Ca2+/calmodulin-dependent protein kinases, all downstream effectors of Gq and G12/13, partially blocked the SFLLR-induced luciferase signal. Taken together, this SRE-luciferase assay reveals a complex network of transduction pathways of protease activated receptor-1 in accordance with the pleiotrophic action of thrombin.
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PMID:Pharmacological characterization of protease activated receptor-1 by a serum responsive element-dependent reporter gene assay: major role of calmodulin. 1652 61


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