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)

Thrombin induces a number of physiological responses in several types of cells. To determine the action of thrombin in the vein, the electrophysiological and mechanical effects of thrombin were studied in rat portal vein smooth muscle cells. Ca2+ channel currents were recorded using the whole-cell patch-clamp technique. Thrombin had both inhibitory and stimulatory effects on the Ca2+ channel current. The inhibitory effect was reversed on washout of thrombin, whereas the stimulatory effect was maintained after thrombin was removed. Thrombin (1 unit/ml) produced a reversible decrease of 27.3 +/- 3.3% (n = 12) in the current amplitude and a sustained increase of 71.2 +/- 12.9% (n = 20). The thrombin-induced inhibition of Ca2+ channel current was blocked by the thrombin inhibitor hirudin and by the protease inhibitor leupeptin. The stimulatory effect of thrombin was inhibited by hirudin, by intracellular application of guanosine 5'-O-(beta-thio)diphosphate, and by antiphophatidylinositide antibodies but not by pertussis toxin. The thrombin-induced enhancement of the Ca2+ channel current amplitude was not observed when the current was previously stimulated by phorbol 12,13-dibutyrate. This suggests that the inhibitory effect of thrombin was related to its proteolytic activity and that the stimulatory effect involved activation of a pertussis toxin-insensitive GTP-binding protein, phosphatidylinositide hydrolysis, and protein kinase C activation. Both thrombin effects occurred in the same concentration range (0.001-10 units/ml). The thrombin-induced contraction of portal vein strips was completely inhibited by isradipine, and thrombin did not produce an increase in cytosolic [Ca2+], measured by indo-1 fluorescence in cells clamped at -50 mV, sufficient to activate Ca(2+)-dependent chloride current.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Dual effect of thrombin on voltage-dependent Ca2+ channels of portal vein smooth muscle cells. 838 25

We examined the effect of thrombin on phosphatidylcholine-hydrolyzing phospholipase D activity in osteoblast-like MC3T3-E1 cells. Thrombin stimulated the formation of choline dose dependently in the range between 0.01 and 1 U/ml, but not the phosphocholine formation. Diisopropylfluorophosphate (DFP)- inactivated thrombin had little effect on the choline formation. The combined effects of thrombin and 12-O-tetradecanoylphorbol-13-acetate, a protein kinase C-activating phorbol ester, on the choline formation were additive. Staurosporine, an inhibitor of protein kinases, had little effect on the thrombin-induced formation of choline. Combined addition of thrombin and NaF, an activator of heterotrimeric GTP-binding protein, did not stimulate the formation of choline further. Pertussis toxin had little effect on the thrombin-induced formation of choline. Thrombin stimulated Ca2+ influx from extracellular space time and dose dependently. The depletion of extracellular Ca2+ by EGTA exclusively reduced the thrombin-induced choline formation. Thrombin had only a slight effect on phosphoinositide-hydrolyzing phospholipase C activity. Thrombin induced diacylglycerol formation and DNA synthesis, and increased the number of MC3T3-E1 cells, but DFP-inactivated thrombin did not. Thrombin suppressed both basal and fetal calf serum-induced alkaline phosphatase activity in these cells. Propranolol, an inhibitor of phosphatidic acid phosphohydrolase, inhibited both the thrombin-induced diacylglycerol formation and DNA synthesis. These results suggest that thrombin stimulates phosphatidylcholine-hydrolyzing phospholipase D due to self-induced Ca2+ influx independently of protein kinase C activation in osteoblast-like cells and that its proliferative effect depends on phospholipase D activation.
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PMID:Thrombin induces proliferation of osteoblast-like cells through phosphatidylcholine hydrolysis. 864 17

The effect of berbamine, a biscoclaurine alkaloid, on cytosolic phospholipase A2 activation in rabbit platelets was investigated. Berbamine inhibited arachidonic acid liberation induced by thrombin but not that by ionomycin. The alkaloid did not affect thrombin-stimulated Ca2+ mobilization. Ca(2+)-dependent translocation of cytosolic phospholipase A2 to membranes, or the activity of partially purified cytosolic phospholipase A2. Furthermore, berbamine had no effect on the thrombin-elicited increase in cytosolic phospholipase A2 activity. However, berbamine suppressed arachidonic acid liberation in platelets stimulated with GTP-binding protein activators. Although incubation of platelet membranes with a GTP analogue decreased the islet-activating protein-catalyzed ADP-ribosylation of an approximately 40 kDa protein in the membranes, pretreatment of the membranes with berbamine did not influence the decrease in ADP-ribosylation. These results suggest that berbamine may impair GTP-binding protein-mediated activation of cytosolic phospholipase A2, probably without influencing the enzyme translocation to membranes or the increase in the enzyme activity, and thus may cause the suppression of thrombin-induced arachidonic acid liberation.
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PMID:Effect of berbamine on cytosolic phospholipase A2 activation in rabbit platelets. 871 19

Polycationic mast cell activators, such as compound 48/80 and substance P, have been reported to activate connective tissue-type mast cells specifically by interacting directly with the Gi family of trimeric GTP-binding protein. We now demonstrate that mouse bone marrow-derived mast cells (BMMC) developed in IL-3, an immature mast cell population lacking responsiveness to the Gi-coupled polycationic mast cell activators, underwent maturation toward a connective tissue-type mast cells-like phenotype that responded to polycationic compounds after only 4 to 6 days of coculture with Swiss 3T3 fibroblasts in concert with recombinant soluble c-kit ligand (KL), whereas 3T3 or KL alone was insufficient to mediate this process. Under optimal conditions, cocultured BMMC released approximately 30% beta-hexosaminidase and generated approximately 1 ng of PGD2/10(6) cells within a few minutes in response to compound 48/80 or substance P. Furthermore, these cells expressed cytokines, such as IL-1beta and IL-6, and PG endoperoxide synthase-2 1 to 4 h after stimulation with compound 48/80 or substance P. All these responses were suppressed effectively by pertussis toxin, implicating functional Gi coupling. Regardless of the remarkable change in polycationic compound sensitivity, there was only a minimal change in the constitutive expression of Gi3 alpha after coculture. These results together with the observation that before coculture BMMC responded to thrombin through its Gi-coupled receptor suggest that the alteration in a certain step(s) distinct from the level of Gi3 alpha protein expression is important for the acquisition of responsiveness to the polycationic compounds by the synergistic action of KL and 3T3 fibroblast-derived factor. Several lines of evidence have revealed that 3T3-derived factor appears to differ from the known cytokines, prostanoids, and adhesion molecules and is a labile soluble substance.
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PMID:Mouse bone marrow-derived mast cells undergo exocytosis, prostanoid generation, and cytokine expression in response to G protein-activating polybasic compounds after coculture with fibroblasts in the presence of c-kit ligand. 897 15

The aim of our study was to evaluate the effect of ADP and the role of cytoskeleton reorganization during reversible and irreversible platelet aggregation induced by ADP and thrombin, respectively, on the heterodimeric (p85alpha-p110) phosphoinositide 3-kinase translocation to the cytoskeleton and its activation. Reversible ADP-induced aggregation was accompanied by a reversible reorganization of the cytoskeleton and an increase in levels of the regulatory subunit p85alpha in this cytoskeleton similar to the increase observed in thrombin-activated platelets. This translocation followed a course parallel to the amplitude of aggregation. No increase in levels of both phosphatidylinositol (3, 4)-bisphosphate (PtdIns(3,4)P2) and phosphatidylinositol-(3,4,5)P3 could, however, be detected even at the maximum aggregation and PI 3-kinase alpha translocation. Moreover, in contrast to the situation for thrombin stimulation, the GTP-binding protein RhoA was hardly translocated to the cytoskeleton when platelets were stimulated with ADP, whereas translocation of pp60(c-)src and focal adhesion kinase did occur. These results suggest (i) translocation of signaling enzymes does not necessarily imply their activation, (ii) the reversibility of ADP-induced platelet aggregation may be the cause or the result of a lack of PI 3-kinase activation and hence of PtdIns(3,4)P2 production, and (iii) RhoA does not seem to be involved in the ADP activation pathway of platelets. Whether PtdIns(3,4)P2 or RhoA may contribute to the stabilization of platelet aggregates remains to be established.
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PMID:Reversible translocation of phosphoinositide 3-kinase to the cytoskeleton of ADP-aggregated human platelets occurs independently of Rho A and without synthesis of phosphatidylinositol (3,4)-bisphosphate. 903 May 42

We examined the effects of platelet activators and inhibitors of platelet function on the voltage-gated delayed rectifier K+ current of human megakaryocytes. We found that both the activators such as thrombin, the thrombin receptor peptide (TRP42-47) and ADP and the inhibitors such as prostacyclin suppressed the delayed rectifier current through two different mechanisms. The cAMP dependent protein kinase (A-kinase) inhibitor IP20 blocked the suppression of the delayed rectifier current by prostacyclin and failed to block the suppression by thrombin, TRP42-47 and ADP. The effects of IP20 suggest that the action of prostacyclin is mediated by A-kinase and the action of the three activators is not mediated by A-kinase. Pertussis toxin (PTX) an inhibitor of the inhibitory GTP-binding proteins (Gi) blocked the suppression of the delayed rectifier current by thrombin, TRP42-47 and ADP and failed to block the suppression by prostacyclin. The effects of PTX suggests that the action of the three activators is mediated by Gi or some other PTX-sensitive GTP-binding protein. We speculate that thrombin and other platelet activators that activate Gi may be suppressing the delayed rectifier current via a direct interaction of Gi or a subunit of it with the delayed rectifier potassium channel itself.
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PMID:Suppression of the voltage-gated K+ current of human megakaryocytes by thrombin and prostacyclin. 906 Oct 4

The signal transduction systems that mediate growth factor receptor-induced cellular shape change have not been fully elucidated, but are known to involve alterations in the state of actin filaments, termed stress fibres. It now appears from several studies that the GTP-binding protein, Rho, is involved. However, the mechanisms by which Rho is activated, and what effectors Rho in turn stimulates are largely matters of conjecture. The present work shows that thrombin is an effective stimulant of stress fibre formation in Swiss 3T3 cells. In addition, we show the 70 kDa form of S6 kinase (p70s6k) to colocalise with stress fibres in both unstimulated and thrombin-activated cells. Coincident with the thrombin-induced formation of stress fibres is the elevated association p70s6k with the fibres. Pretreatment of cells with rapamycin, to inhibit p70s6k activation, inhibits thrombin-induced stress fibre formation and the associated presence of p70s6k on the fibres, supporting a role for p70s6k in thrombin-stimulated stress fibre formation. Thrombin is also shown to stimulate p70s6k activity and that this is inhibited by rapamycin. Thus, the data presented show that thrombin activates stress fibre formation through stimulation of p70s6k via a non-Gi pathway.
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PMID:Regulation of thrombin-induced stress fibre formation in Swiss 3T3 cells by the 70-kDa S6 kinase. 914 21

Vasoactive GTP-binding protein-coupled receptor agonists (e.g., angiotensin II [AII] and alpha-thrombin) stimulate the production of mitogenic factors from vascular smooth muscle cells. In experiments to identify mitogens secreted from AII- or alpha-thrombin-stimulated rat aortic smooth muscle (RASM) cells, neutralizing antibodies directed against several growth factors (e.g., PDGF and basic fibroblast growth factor [basic FGF]) failed to inhibit the mitogenic activity of conditioned media samples derived from the cells. In this report, we found that polyclonal neutralizing antibodies directed against purified human placental basic FGF reduced the mitogenic activity of AII-stimulated RASM cell-conditioned media and in immunoblot experiments identified a 26-kD protein (14 kD under reducing conditions) that was distinct from basic FGF. After purification from RASM cell-conditioned medium, amino acid sequence analysis identified the protein as activin A, a member of the TGF-beta superfamily. Increased activin A expression was observed after treatment of the RASM cells with AII, alpha-thrombin, and the protein kinase C agonist PMA. In contrast, PDGF-BB or serum caused only a minor induction of this protein. Although activin A alone only weakly stimulated RASM cell DNA synthesis, it demonstrated a potent comitogenic effect in combination with either EGF or heparin-binding EGF-like growth factor in the RASM cells, increasing DNA synthesis by up to fourfold. Furthermore, in a rat carotid injury model, activin A mRNA was upregulated within 6 h after injury followed by increases in immunoreactive protein detected in the expanding neointima 7 and 14 d later. Taken together, these results indicate that activin A is a vascular smooth muscle cell-derived factor induced by vasoactive agonists that may, either alone or in combination with other vascular derived growth factors, have a role in neointimal formation after arterial injury.
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PMID:Stimulation of activin A expression in rat aortic smooth muscle cells by thrombin and angiotensin II correlates with neointimal formation in vivo. 923 11

Stimulation of platelet thrombin receptors or protein kinase C causes fibrinogen-dependent aggregation that is a function of integrin alphaIIb beta3 activation. Such platelets rapidly and transiently form phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) and a small amount of phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P2). After aggregation, a larger amount of PtdIns(3,4)P2 is generated. We report that this latter PtdIns(3,4)P2 arises largely through wortmannin-inhibitable generation of PtdIns3P and then phosphorylation by PtdIns3P 4-kinase (PtdIns3P 4-K), a novel pathway apparently contingent upon the activation of the Ca2+-dependent protease calpain. Elevation of cytosolic Ca2+ by ionophore, without integrin/ligand binding, is insufficient to activate the pathway. PtdIns3P 4-K is not the recently described "PIP5KIIalpha." Cytoskeletal activities of phosphatidylinositol 3-kinase and PtdIns3P 4-K increase after aggregation. Prior to aggregation, PtdIns3P 4-K can be regulated negatively by the beta gamma subunit of heterotrimeric GTP-binding protein. After aggregation, PtdIns3P 4-K calpain-dependently loses its susceptibility to Gbeta gamma and is, in addition, activated. Both PtdIns(3,4,5)P3 and PtdIns(3,4)P2 have been shown to stimulate PKBalpha/Akt phosphorylation and activation by phosphoinositide-dependent kinase 1. We find that activation of PKBalpha/Akt in platelets is phosphorylation-dependent and biphasic; the initial phase is PtdIns(3,4,5)P3-dependent and more efficient, whereas the second phase depends upon PtdIns(3,4)P2 generated after aggregation. There is thus potential for both pre- and post-aggregation-dependent signaling by PKBalpha/Akt.
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PMID:Biphasic activation of PKBalpha/Akt in platelets. Evidence for stimulation both by phosphatidylinositol 3,4-bisphosphate, produced via a novel pathway, and by phosphatidylinositol 3,4,5-trisphosphate. 956 82

Thrombin can regulate the-fibrinolytic system by increasing the endothelial production of both tissue plasminogen activator (t-PA) and plasminogen activator inhibitor type-1 (PAI-1). The thrombin receptor transducts signals through the GTP-binding protein system, the classical pathway being the Galpha q-protein. The purpose of the present study was to examine the roles of Galpha i-protein and tyrosine kinases in the thrombin signal transduction of t-PA and PAI-1 production from human adult vein endothelial cells (HAVEC). t-PA and PAI-1 antigen were analysed in conditioned medium from cultured HAVEC after 16 h incubation. Data are expressed as percentages of basal release (100%), means +/- 95% confidence intervals. Thrombin increased t-PA and PAI-1 production (234 +/- 42% and 211 +/- 42%, respectively). Pertussis toxin (PTX) (inhibiting Galpha i-pathway) reduced basal PAI-1 (66 +/- 8%), but had only a weak influence on basal t-PA production. Pertussis toxin and genistein (inhibiting tyrosine kinase) significantly reduced the thrombin induction of both t-PA and PAI-1 (PTX: 142 +/- 23% and 146 +/- 19%, respectively, genistein: 156 +/- 42% and 76 +/- 24%, respectively). The present study demonstrated that thrombin can increase the production of t-PA and PAI-1 by transducting signals through the Galpha i and tyrosine kinase pathway, in addition to the Galpha q/protein kinase C pathway as has been found previously.
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PMID:Thrombin signal transduction of the fibrinolytic system in human adult venous endothelium in vitro. 974 23


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