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)

The maximal aggregation of platelets induced by alpha-thrombin or by the receptor agonist peptide thrombin-(42-47)-peptide (TRP42/47) rapidly increased the pp60c-src associated with the cytoskeleton fraction. There was good correlation between the tyrosine kinase activity and the mass of pp60c-src. Tyrosine kinase activity associated with the cytoskeleton phosphorylated several endogenous cytoskeleton-associated proteins, as revealed by immunoblotting with anti-phosphotyrosine antibody following incubation with ATP in vitro. However, with the exception of pp60c-src, few phosphotyrosine-containing proteins were retained in the cytoskeleton in intact platelets when compared with total platelet lysates. Translocation of pp60c-src to the cytoskeleton induced by alpha-thrombin and TRP42/47 is dependent on glycoprotein IIb/IIIa (GPIIb/IIIa)-fibrinogen-mediated aggregation, but does not occur when ristocetin/von Willebrand factor produces GPIb-mediated platelet aggregation. The translocation of GPIIb/IIIa and pp60c-src to the cytoskeleton is not necessary for aggregation, as it is not seen when clearly visible small to moderate-sized aggregates are initially formed after exposure to thrombin. The linkage of these proteins to the cytoskeleton occurs only after later extensive formation of large aggregates. Translocation of GPIIa/IIIa to the cytoskeleton is not sufficient for the cytoskeletal association of pp60c-src, as the former occurs independently in platelets stimulated with concanavalin A in the absence of aggregation. Linkage of the integrin GPIIb/IIIa and pp60c-src to the internal cytoskeleton structure, and the corresponding tyrosine phosphorylation of certain proteins upon formation of large aggregates, may be an example of mechanochemical transduction by integrin receptors and may represent a structure with the requisite tensile strength to stabilize large platelet aggregates against high shear stresses.
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PMID:Thrombin and thrombin receptor agonist peptide induce tyrosine phosphorylation and tyrosine kinases in the platelet cytoskeleton. Translocation of pp60c-src and integrin alpha IIb beta 3 (glycoprotein IIb/IIIa) is not required for aggregation, but is dependent on formation of large aggregate structures. 768 31

Human blood platelets contain high levels of non-receptor protein tyrosine kinases of the Src family, particularly pp60c-src, suggesting an important role for these enzymes in platelet physiology. Indeed, in response to various agonists of platelet function, a number of proteins become phosphorylated at tyrosine residues. However, no enzymic activation of an Src-related tyrosine kinase has yet been shown in platelets. In searching for the kinase(s) responsible, we found that all agonists tested that directly or indirectly activate protein kinase C in platelets (phorbol 12-myristate, 13-acetate, thrombin, vasopressin, collagen, calcium ionophore A23187) increased the overall activity of pp60c-src determined by IgG phosphorylation in an immunocomplex assay in the presence of low ATP concentrations. On the other hand, elevation of cyclic AMP directly by forskolin or indirectly by prostaglandin E1, or elevation of cyclic GMP by sodium nitroprusside did not significantly affect the activity of the enzyme. To substantiate the differences in enzyme activity, we determined Km and Vmax, values of pp60c-src from resting and thrombin-stimulated platelets. Thrombin treatment increased substrate affinity of pp60c-src as indicated by a 2- to 3-fold decrease in the Km values for ATP and the exogenous protein substrate casein. Vmax. values were only slightly altered under the assay conditions used. To further rule out modifications of pp60c-src in phosphorylation as a probable cause of the changed substrate affinity, we analysed tryptic phosphopeptides of immunoprecipitated, 32P-labelled pp60c-src of unstimulated and stimulated platelets. The platelet agonists listed above induced an increase in pp60c-src phosphorylation at Ser-12, which is the amino acid phosphorylated by protein kinase C. Surprisingly, we found that elevation of cyclic AMP did not affect 32P labelling of pp60c-src. On the basis of our data, we suggest that phosphorylation at Ser-12 might be one of the signal-triggering events that cause the increase in substrate affinity of pp60c-src.
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PMID:Substrate affinity of the protein tyrosine kinase pp60c-src is increased on thrombin stimulation of human platelets. 769 43

We have demonstrated that the endothelial cell-derived superoxide anion is deeply involved in the endothelial cell injury induced by activated neutrophils (Fujita, H., Morita, I. and Murota, S. (1994) Arch. Biochem. Biophys. 309, 62-69). To clarify the mechanism underlying the increase in the endothelial cell-derived superoxide anion induced by activated neutrophils, the conversion of xanthine dehydrogenase (XD) to xanthine oxidase (XO) in cultured endothelial cells isolated from bovine carotid arteries was investigated. Although the endothelial cells expressed both XD and XO activity, the XO activity of unstimulated cells comprised about 12% of the total (XD + XO) activity. When endothelial cells were exposed to neutrophils activated with phorbol 12-myristate 13-acetate (PMA), XO activity rapidly increased about 3-fold over the control. Whereas treatment of endothelial cells with PMA alone or unstimulated neutrophils alone did not increase the XO activity at all. The increase in XO activity in endothelial cells was also observed on the treatment of the cells with neutrophils activated with leukotriene B4 or thrombin. To determine whether or not proteases released from activated neutrophils are involved in the increased conversion of XD to XO in endothelial cells, the effects of the elastase specific inhibitor, ONO-5046, and protease inhibitors, such as aprotinin, gabexate mesylate and urinastatin, were examined. However, these protease inhibitors did not suppress the conversion of XD to XO induced by PMA-activated neutrophils. Moreover, the treatment of endothelial cells with purified human neutrophil elastase and H2O2 also did not affect the conversion at all. In contrast, monoclonal antibodies against CD11a and CD18 significantly inhibited the increased conversion of XD to XO induced by PMA-activated neutrophils. Moreover, tyrosine kinase inhibitors such as staurosporin and herbimysine also inhibited the increased conversion of XD to XO induced by PMA-activated neutrophils. These results indicate that the adhesion of activated neutrophils to endothelial cells via CD11a/CD18-ICAM-1 is involved in the conversion of XD to XO in endothelial cells induced by activated neutrophils.
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PMID:Conversion of xanthine dehydrogenase to xanthine oxidase in bovine carotid artery endothelial cells induced by activated neutrophils: involvement of adhesion molecules. 769 38

In the present study, the roles of Ca2+ and fibrinogen receptor occupancy in the regulation of phospholipase C by G protein-coupled and tyrosine kinase-linked receptor pathways in human platelets have been investigated. Agonist stimulation of phospholipase C was not altered significantly in the absence of stirring or in the presence of the fibrinogen receptor antagonist arginine-glycine-aspartate-serine, conditions that prevent platelet aggregation. Similarly, elevation of intracellular Ca2+ levels by the ionophores A23187 or ionomycin did not induce formation of inositol phosphates. In contrast, chelation of extracellular Ca2+ by ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) reduced formation of inositol phosphates by G protein receptor (thrombin)- and tyrosine kinase (Fc receptor and peroxovanadate)-regulated pathways. Similarly, short term exposure to Ni2+ ions, which also prevent Ca2+ entry, inhibited thrombin-stimulated formation of inositol phosphates. Loading of platelets with the intracellular Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) markedly suppressed elevation of intracellular Ca2+ and formation of inositol phosphates in platelets stimulated by G protein receptor- and tyrosine kinase-regulated pathways. The greater inhibition of phospholipase C by BAPTA, relative to that induced by EGTA, is consistent with the more pronounced inhibition of intracellular Ca2+ elevation. The tyrphostin tyrosine kinase inhibitor ST271 also reduced intracellular Ca2+ levels and inhibited activation of phospholipase C. The degree of inhibition of phospholipase C by ST271 was slightly greater than that induced by EGTA but was not additive with the effect of EGTA, suggesting a common mode of action. It is concluded that elevation of intracellular Ca2+ regulates agonist-induced activation of phospholipase C and that this contributes to the inhibition of thrombin-induced formation of inositol phosphates by the tyrphostin ST271.
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PMID:Ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) and the tyrphostin ST271 inhibit phospholipase C in human platelets by preventing Ca2+ entry. 772 44

Plasmin caused a modest and gradual increase in platelet cytosolic Ca2+, mediated through both Ca2+ mobilization and external Ca2+ entry. This response was associated with accelerated Ca2+ extrusion and protein tyrosine phosphorylation. Plasmin-enhanced external Ca2+ entry and Ca2+ extrusion (but not Ca2+ mobilization) were attenuated by the tyrosine kinase inhibitor, genistein. Plasmin inhibited the thrombin-evoked increase in cytosolic Ca2+ and also inhibited the Ca2+ response to the tethered peptide TRAP-6 of the thrombin receptor. Furthermore, plasmin inhibited the binding of 125I-labeled alpha-thrombin to platelets. The inhibitory effect of plasmin on the thrombin response shared some characteristics with the effect of protein kinase C stimulators but was not reversed by protein kinase C inhibitors. Plasmin did not change platelet cyclic nucleotides. These results suggest a dual effect of plasmin. Plasmin produces a small rise in platelet cytosolic Ca2+ and a tyrosine kinase-dependent enhancement of Ca2+ turnover (external Ca2+ influx and Ca2+ efflux). However, it also attenuates the thrombin-evoked cytosolic Ca2+ response by blocking Ca2+ mobilization and slowing the rate of external Ca2+ influx. The latter feature would result in a plasmin-induced inhibition of thrombogenesis.
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PMID:Duality of plasmin effect on cytosolic free calcium in human platelets. 773 44

The synthesis of phosphatidylinositol 3',4'-bisphosphate (PtdIns(3,4)P2) in 32P-labeled human platelets induced by the tetrameric lectin concanavalin A and the physiological agonist thrombin were compared. Like thrombin, concanavalin A stimulated a time-dependent accumulation of PtdIns(3,4)P2, which reached maximal levels after 5 min of stimulation. However, while synthesis of PtdIns(3,4)P2 induced by thrombin was dependent on platelet aggregation, the production of PtdIns(3,4)P2 induced by concanavalin A was unchanged when aggregation was prevented by the omission of stirring or when fibrinogen binding to platelets was inhibited by the tetrapeptide RGDS. Accumulation of PtdIns(3,4)P2 was not observed in platelets stimulated with succinyl-concanavalin A, a dimeric derivative of the lectin that binds to the same receptors on the platelet surface but does not promote clustering of membrane glycoproteins. The synthesis of PtdIns(3,4)P2 induced by concanavalin A was also independent of the membrane glycoprotein IIb-IIIa, as normal accumulation of this lipid was observed in platelets from two patients affected by Glanzmann thrombasthenia. In contrast, thrombin showed a strongly reduced ability to stimulate PtdIns(3,4)P2 production in thrombasthenic platelets. Although concanavalin A was able to induce association of the regulatory subunit of the phosphatidylinositol 3-kinase with tyrosine-phosphorylated proteins, the tyrosine kinase inhibitor tyrphostin AG-213 did not inhibit the lectin-induced synthesis of PtdIns(3,4)P2. These results demonstrate the existence of a novel mechanism of PtdIns(3,4)P2 synthesis in human platelets, which is independent of glycoprotein IIb-IIIa and aggregation, but requires clustering of membrane glycoproteins. As clustering events occur during platelet aggregation promoted by physiological agonists, this new mechanism may also be involved in the aggregation-dependent production of PtdIns(3,4)P2 in thrombin-stimulated platelets.
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PMID:Evidence for a glycoprotein IIb-IIIa- and aggregation-independent mechanism of phosphatidylinositol 3',4'-bisphosphate synthesis in human platelets. 776 14

Glial cells normally do not proliferate in the adult retina despite the presence of glial mitogens. In this study, we examined the hypothesis that endogenous antiproliferative molecules inhibit the effects of glial mitogens. Using cultures of glial cells obtained from the adult human retina, we found that transforming growth factor beta 2 (TGF beta 2) and a metabotrophic glutamate agonist (t-ACPD) inhibit the mitogenic effects of basic fibroblast growth factor, platelet-derived growth factor, epidermal growth factor and insulin-like growth factor-1. These antiproliferative effects may involve activation of protein kinase C (PKC) since chelerythine, a specific PKC inhibitor, blocks the antiproliferative effects of TGF beta 2 and t-ACPD. Furthermore, exposure of the glia to a phorbol ester mimics the inhibitory effects of TGF beta 2 or t-ACPD. Although TGF beta 2 and t-ACPD markedly inhibit a number of mitogens, they do not alter the mitogenic response of retinal glia to thrombin and glutamate. A common characteristic of the mitogens sensitive to TGF beta 2 or t-ACPD is activation of tyrosine kinase-linked receptors. In contrast, thrombin acts at a G-protein-linked receptor, and glutamate stimulates retinal glial proliferation via activation of an NMDA receptor. It appears that TGF beta 2 and t-ACPD may selectively inhibit retinal glial mitogenesis mediated by activation of tyrosine kinase-linked receptors. Our experiments support the idea that endogenous antiproliferative molecules play a role in preventing glial proliferation in the retina.
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PMID:Regulation of retinal glial cell proliferation by antiproliferative molecules. 778 23

Phosphatidylinositol-3 kinase becomes activated upon association with stimulated tyrosine kinase coupled receptors, but it is also catalytically active in platelets incubated with the G-protein coupled growth factor receptor agonist, thrombin. Furthermore, phorbol esters have been shown to be growth inhibitory when added to vascular smooth muscle cells simultaneously with thrombin. In order to clarify the role of phosphatidylinositol-3 (PI-3) kinase in thrombin-induced mitogenesis, we asked whether PI-3 kinase activity is decreased in parallel to mitogenesis in cells stimulated with phorbol-12-myristate-13-acetate (PMA) and thrombin. Although PMA inhibits thrombin-stimulated growth by 92% when the two compounds are added simultaneously, the level of PI-3 kinase activity under similar conditions is not decreased. This phenomenon is independent of protein kinase C, since there is no difference in PI-3 kinase activity when similar experiments are performed after protein kinase C is down-regulated by 24 h pre-incubation with PMA. We conclude that either (i) PI-3 kinase is not required for the mitogenic signalling of thrombin, or (ii) PMA is acting downstream of PI-3 kinase in thrombin's signalling pathway.
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PMID:Dissociation of phosphatidylinositol-3 kinase activity and mitogenic inhibition in vascular smooth muscle cells. 779 83

Integrins promote cell-substratum and cell-cell adhesion by acting as transmembrane linker molecules between extracellular adhesion proteins and the actin-rich cytoskeleton. The integrin alpha IIb beta 3 (platelet glycoprotein IIb/IIIa) is essential for platelet spreading, aggregation, fibrin clot retraction, and for the transduction of extracellular signals. We examined the effect of the specific tyrosine kinase inhibitor herbimycin A on integrin and cytoskeletal-mediated events in thrombin-stimulated platelets. Incubation of washed platelets for 24 h with herbimycin A (5 microM) abolished the thrombin-stimulated cytoskeletal enzyme activity of pp60c-src in parallel with a reduction in the tyrosine phosphorylation of multiple platelet proteins, as assessed with anti-phosphotyrosine immunoblots. However, thrombin-induced activation of protein kinase C and the production of thromboxane A2 were not altered by herbimycin A. Despite the absence of cytoskeletal pp60c-src enzyme activity, platelet shape change, aggregation, and serotonin release were unaltered following platelet stimulation with thrombin (0.05-1.0 unit/ml). Herbimycin A-treated platelets also demonstrated normal platelet aggregation in response to collagen (5 micrograms/ml), ionophore A23187 (2 microM), and ADP/adrenaline (10 microM each). However, the ability of herbimycin A-treated platelets to retract fibrin gels was significantly reduced. This defect in clot retraction was associated with reduced incorporation of integrin alpha IIb beta 3 into the cytoskeletal fraction of thrombin-aggregated platelets. Our studies suggest that tyrosine kinases in platelets regulate the cytoskeletal attachment of alpha IIb beta 3, as an essential process for the transmission of cellular contractile forces to fibrin polymers.
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PMID:Tyrosine kinases regulate the cytoskeletal attachment of integrin alpha IIb beta 3 (platelet glycoprotein IIb/IIIa) and the cellular retraction of fibrin polymers. 779 49

Treatment of cultured type-1 astrocytes with thrombin leads to cell proliferation and reversal of stellation. The half-maximal concentrations of thrombin required for each response are 500 and 2 pM, respectively. To test whether they might be mediated by different receptors, we examined the contribution of the G protein-coupled thrombin receptor to these responses in purified rat astrocytes by using the agonist peptide SFLLRNP. In the absence of added growth factors, SFLLRNP fully mimicked the effects of thrombin at half-maximal concentrations of 30 microM for an increase in cell number and DNA synthesis and 100 nM for the reversal of stellation. The role of protein tyrosine phosphorylation in these events was investigated using antiphosphotyrosine antibodies. Thrombin and SFLLRNP at concentrations at least 10-fold greater than those required for half-maximal reversal of stellation but below those required for mitogenesis induced an identical pattern of tyrosine phosphorylation on several proteins of 55-65, 106, 110-115, and 120-130 kDa. The response was rapid (< 1 min) and transient with a peak response after approximately 2 min. The specific tyrosine kinase inhibitor herbimycin A did not affect thrombin- or SFLLRNP-mediated reversal of stellation at concentrations of up to 1 microM. In contrast, 1 microM herbimycin fully inhibited the ability of thrombin and SFLLRNP to increase cell number and stimulate DNA synthesis. Furthermore, this inhibition by 1 microM herbimycin A corresponded to inhibition of receptor-induced tyrosine phosphorylation. Thus, cell proliferation but not reversal of stellation is dependent on thrombin receptor-activated tyrosine kinase activity.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Thrombin receptor activation stimulates astrocyte proliferation and reversal of stellation by distinct pathways: involvement of tyrosine phosphorylation. 783 51


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