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)

Pleckstrin is the major substrate phosphorylated on serine and threonine in response to stimulation of human platelets by thrombin (Abrams, C. S., Zhao, W., Belmonte, E., and Brass, L. F. (1995) J. Biol. Chem. 270, 23317-23321). We now show that pleckstrin in platelets is in a complex with inositol polyphosphate 5-phosphatase I (5-phosphatase I). This enzyme hydrolyzes the 5-phosphate from inositol 1,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate and thus serves as a calcium signal-terminating enzyme, since the substrates but not the products mobilize intracellular calcium. Pleckstrin co-immunoprecipitates with 5-phosphatase I in homogenates of platelets. Platelet homogenates fractionated by anion exchange chromatography show co-elution of pleckstrin and 5-phosphatase I. Fractions containing phosphorylated pleckstrin have 7-fold greater 5-phosphatase activity than those containing unphosphorylated pleckstrin. Mixing experiments with recombinant 5-phosphatase I and pleckstrin in vitro show that they form a stoichiometric complex. A mutant form of pleckstrin, in which the serine and threonine residues that are phosphorylated by protein kinase C are substituted with glutamic acid (pseudophosphorylated pleckstrin), activates recombinant 5-phosphatase I 2-3-fold while native unphosphorylated pleckstrin does not stimulate the enzyme. Thus pleckstrin functions to terminate calcium signaling in platelets when it is phosphorylated by binding to and activating 5-phosphatase I.
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PMID:Phosphorylation of platelet pleckstrin activates inositol polyphosphate 5-phosphatase I. 899 61

The strong inhibition of thrombin-induced platelet functions induced by okadaic acid is not correlated with the partial modification of pleckstrin phosphorylation, which remains still phosphorylated two min after stimulation, indicating that protein kinase C is not affected by okadaic acid. We then investigated the effect of okadaic acid on platelet lipid metabolism. Our data indicate that inhibition indeed strongly affects phosphatidic acid as well as phosphatidylinositol 3,4-bisphosphate synthesis at low concentrations of okadaic acid, and phosphatidylinositol 4,5-bisphosphate at higher concentrations. Since thrombin-induced tyrosine phosphorylations were completely inhibited in the presence of okadaic acid, as a consequence, phosphatidylinositol 3-kinase was no longer detected in antiphosphotyrosine immunoprecipitates, thus explaining the absence of phosphatidylinositol, 3,4-bisphosphate synthesis. Finally, okadaic acid inhibited thrombin-induced fibrinogen binding, indicating that serine/threonine phosphatases may affect the inside-out signalling which regulates the alpha 11bb3 integrin, downstream protein kinase C activation.
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PMID:Total inhibition of phospholipase C and phosphatidylinositol 3-kinase by okadaic acid in thrombin-stimulated platelets. 906 40

Effects of myosin light chain (MLC) kinase inhibitor ML-7 [1-(5-iodonaphthalene-1-sulphonyl)-1H-hexahydro-1,4-diazepine hydrochloride] and protein kinase C inhibitor H-7 [1-(5-isoquinolinesulphonyl)-2-methylpiperazine dihydro-chloride] on collagen-induced platelet activation were investigated in washed rabbit platelets. Upon stimulation with collagen (1 microg/mL), H-7 decreased protein kinase C-mediated pleckstrin phosphorylation, but had no inhibitory effect on thromboxane (TX) A2 formation or platelet aggregation. In contrast, ML-7 produced a concentration-dependent inhibition of the collagen-induced platelet aggregation and TXA2 formation by preventing arachidonic acid (AA) liberation from membrane phospholipids. However, ML-7 had little effect on AA liberation induced by thrombin, Ca2+ ionophore A-23187 or melittin, suggesting that ML-7 may affect the signal transduction pathway specific for collagen-induced AA liberation, without direct inhibition of phospholipase A2 activity. In indomethacin-treated platelets, collagen caused MLC phosphorylation and AA liberation in the absence of a significant increase in intracellular Ca2+ concentration ([Ca2+]i) or protein tyrosine phosphorylation. ML-7 inhibited both MLC phosphorylation and AA liberation induced by collagen in indomethacin-treated platelets. These results demonstrate that MLC phosphorylation and AA liberation are early events detectable in collagen-stimulated platelets, and suggest that ML-7 inhibits these early steps of collagen-induced signal transduction pathway in rabbit platelets.
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PMID:Selective inhibition of collagen-induced arachidonic acid liberation by 1-(5-iodonaphthalene-1-sulphonyl)-1H-hexahydro-1,4-diazepine hydrochloride (ML-7), a myosin light chain kinase inhibitor, in washed rabbit platelets. 937 23

It has been shown that platelets from patients suffering from eclampsia are hyporesponsive to stimulation by agonists like thrombin and ADP. Although platelet hyporeactivity contributes to the pathogenesis of the disease process, the cause for this is still not known. Platelet aggregation and secretion are membrane-based phenomena initiated by the processes of cell signalling. Hence, to understand the mechanisms underlying platelet hyporeactivity in eclampsia, membrane microviscosity and activities of the signalling enzymes were measured in human platelets stimulated with thrombin. Membrane fluidity was determined from the steady-state fluorescence anisotropy of diphenylhexatriene incorporated in cell membranes. Activities of phospholipase C and protein kinase C in stimulated platelets were assessed from the extents of phosphatidic acid generation and pleckstrin phosphorylation, respectively. Platelet membrane microviscosity in eclampsia (2.3 +/- 0.2 SEM, n = 5) was significantly lower (P < 0.05) than that in the matched gravid control subjects (3.1 +/- 0.2, n = 4). In eclampsia, generation of phosphatidic acid and phosphorylation of pleckstrin were decreased by 25% (P < 0.05, n = 3) and 35% (P < 0.05, n = 3), respectively, after 60 sec of platelet stimulation. It was concluded that the hyporeactive platelets obtained from eclampsia have more fluid membranes and diminished activities of phospholipase C and protein kinase C. In summary, this study shows that alterations in membrane fluidity and activities of the signalling enzymes (phospholipase C and protein kinase C) may contribute to the diminished platelet responsiveness observed in the eclamptic condition.
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PMID:Platelets from eclampsia patients have reduced membrane microviscosity and lower activities of the signalling enzymes. 959 60

This report presents a comparison of the effects of cis- and trans-diamminedichloroplatinum complexes on in vitro platelet functions. Pretreatment of platelets with cis-platinum (cisplatin) induced a slow, dose-dependent (0.1-0.45 mM), increase in the cytosolic Ca2+ concentration, pleckstrin (47 kDa) phosphorylation and serotonin secretion, as well as a slight shape modification with emission of a few pseudopodia. All these effects were remarkably increased in platelets exposed to trans-platinum (transplatin). The rise in cytosolic Ca2+ concentration and serotonin secretion evoked by stimulation of platelets with thrombin were not significantly influenced by cellular exposure to cis-platinum, whereas they were enhanced and inhibited, respectively, by exposure to trans-platinum. Trans-platinum also inhibited thrombin-promoted platelet aggregation to a greater extent than the cis-isomer. While the viscosity of platelet rich-plasma tended to decrease in the presence of cis-platinum, it tended to increase in the presence of trans-platinum. Taken together, these results indicate that the effects on platelet functions of the efficacious antitumor complex cis-platinum is rather different from that of the inactive complex trans-platinum. Therefore, the in vitro tests of platelet functions employed in this study might provide an index of antitumor drug toxicity and serve as a preliminary indicator of therapeutic efficacy.
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PMID:Action of antitumoral platinum complexes on in vitro platelet functions. 960 87

The platelet integrin IIbbeta3 has become a new target for the treatment of pathological thrombosis. It becomes apparent that the affinity of IIbbeta3 for its ligands is dynamically regulated by inside-out signaling. However, the components that couple diverse intracellular signals to the cytoplasmic domains of IIbbeta3 remain obscure. Employing a chymotrypsin-induced IIbbeta3 activation model, we previously proposed the hypothesis that Na+/Ca2 + exchanger (NCX) may be involved in inside-out signaling (Shiraga et al: Blood 88:2594, 1996). In the present study, employing two unrelated Na+/Ca2+ exchange inhibitors, 3',4'-dichlorobenzamil (DCB) and bepridil, we investigated the role of NCX in platelet activation induced by various agonists in detail. Both inhibitors abolished platelet aggregation induced by all agonists examined via the inhibition of IIbbeta3 activation. Moreover, these inhibitors abolished IIbbeta3 activation induced by phorbol 12-myristate 13-acetate or A23187. On the other hand, neither of these inhibitors showed apparent inhibitory effects on protein phosphorylation of pleckstrin or myosin light chain, or an increase in intracellular calcium ion concentrations evoked by 0.1 U/mL thrombin. These effects of the NCX inhibitors are in striking contrast to those of protein kinase C inhibitor, Ro31-8220. Biochemical and ultrastructural analyses showed that NCX inhibitors, particularly DCB, made platelets "thrombasthenic". These findings suggest that the NCX is involved in the common steps of inside-out signaling through integrin IIbbeta3.
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PMID:Involvement of Na+/Ca2+ exchanger in inside-out signaling through the platelet integrin IIbbeta3. 980 65

Activation of the platelet integrin alpha(IIb)beta3, an essential step in platelet aggregation, is regulated by intracellular signal pathways (inside-out signaling). In this study, we characterize a 35-year-old Japanese female, HM, with a life-long history of mucocutaneous bleeding. HM showed a Glanzmann thrombasthenia-like phenotype with normal expression of alpha(IIb)beta3, and failure of platelet aggregation induced by various agonists. An activation-independent ligand mimic monoclonal antibody (mAb), OP-G2, and RGDS peptides bound normally to the patient's alpha(IIb)beta3, while an activating anti-beta3 mAb, AP5, induced normal aggregation of HM platelets. The nucleotide sequence of the entire coding region of the patient's alphaIIb and beta3, including the cytoplasmic domains of each subunit, revealed no abnormalities. Agonist-induced phosphorylation of platelet pleckstrin and myosin light chain was not impaired. Recently, we proposed that a Na+/Ca2+ exchanger is involved in inside-out signaling, especially in the case of chymotrypsin-induced alpha(IIb)beta3 activation (Blood 88: 2594, 1996). However, chymotrypsin-induced platelet aggregation occurred normally in patient HM. Measurement of changes in cytosolic free calcium concentration ([Ca2+]i) revealed that the plateau level of [Ca2+]i after thrombin stimulation was significantly inhibited in patient HM. Our data suggest that patient HM exhibits a Glanzmann thrombasthenia-like phenotype associated with an abnormality in inside-out signaling which would otherwise activate alpha(IIb)beta3.
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PMID:A Glanzmann thrombasthenia-like phenotype caused by a defect in inside-out signaling through the integrin alpha(IIb)beta3. 984 64

Thrombin and other agonists that induce secretion and aggregation in human platelets also activate phospholipase D (PLD), but the signaling cascade leading to activation of PLD in human platelets is not yet clear. We have determined that apyrase, which scavenges ADP secreted during platelet activation, is able to block or reduce the PLD activation stimulated by low (0.1 U/ml or less) or high (0.3- 1.0 U/ml) concentrations of thrombin, respectively. Neither ADP (up to 100 microM) nor its more potent analogue 2-methylthio-ADP (up to 100 microM), however, are able to stimulate PLD alone, and even the addition of fibrinogen, which results in platelet aggregation, is not sufficient for PLD activation. In contrast, ADP is able to stimulate PLD in the presence of low concentrations of thrombin that alone have little or no effect, suggesting ADP may play an amplifying role in platelet PLD activation. This hypothesis is supported by the finding that the purinergic receptor antagonist ARL 66096, an ATP analogue, reduces in a concentration-dependent fashion the PLD response to thrombin (IC50=28 nM with 0.1 U/ml thrombin). ARL 66096 also abolishes the PLD activation by ADP observed in the presence of low concentrations of thrombin, confirming that the antagonist inhibits an ADP-dependent component of the response. In addition, the thromboxane A2 receptor agonist U46619 activates PLD, and this response is markedly reduced by ARL 66096. Concomitantly, phosphorylation of the protein kinase C substrate pleckstrin in response to thrombin or U46619 is partially or totally inhibited by ARL 66096, respectively, consistent with ADP stimulation of protein kinase C being involved in the PLD response to these agonists. Based on these findings, we conclude that ADP secretion and activation of purinergic ADP receptors is an important amplification mechanism in the signal transduction pathways leading to PLD activation in human platelets.
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PMID:Secreted ADP plays a central role in thrombin-induced phospholipase D activation in human platelets. 986 70

We compared several responses in thrombin-stimulated and collagen (type I)-stimulated platelets with and without forskolin and inhibitors of autocrine stimulation (IAS: an ADP-removing system of creatine phosphate/creatine phosphokinase, Arg-Gly-Asp-Ser peptide to prevent fibrinogen/fibronectin binding to GPIIb/IIIa, SQ 29.548 as a thromboxane A2 receptor antagonist, cyproheptadine as a serotonin receptor antagonist, BN 52021 as a platelet-activating factor receptor antagonist). The pattern of tyrosine-phosphorylated proteins, the phosphorylation of lipids in the polyphosphoinositide cycle and phosphorylation of pleckstrin (P47) were studied as markers for signal-transducing responses, exposure of CD62 (P-selectin) and CD63 (Glycoprotein 53), as well as secretion of ADP + ATP and beta-N-acetyl-glycosaminidase were studied as final activation responses. Clear differences between thrombin-stimulated and collagen-stimulated platelets were observed. First, practically all protein-tyrosine phosphorylation induced by thrombin was inhibited by IAS, while a partial inhibition was observed for collagen; the phosphorylation due to collagen alone was apparently stimulated by elevation of cAMP. Secondly, the other responses to thrombin were inhibited by increased levels of cAMP, independent of autocrine stimulation. In contrast, only the autocrine part of the collagen-induced responses was inhibited by elevation of cAMP. Thus, the inhibition by elevated cAMP seen in collagen-stimulated platelets seems to be due to removal of the G-protein-mediated activation from secreted autocrine stimulators either by IAS or forskolin. The remaining activity is a pure collagen effect which is not affected by elevated levels of cAMP.
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PMID:Role of autocrine stimulation on the effects of cyclic AMP on protein and lipid phosphorylation in collagen-activated and thrombin-activated platelets. 1009 87

Changes in shape, and aggregation that accompanies platelet activation, are dependent on the assembly and reorganization of the cytoskeleton. To assess the changes in cytoskeleton induced by thrombin and PMA, suspensions of aspirin-treated,32P-prelabeled, washed pig platelets in Hepes buffer containing ADP scavengers were activated with thrombin, and with PMA, an activator of protein kinase C. The cytoskeletal fraction was prepared by adding Triton extraction buffer. The Triton-insoluble (cytoskeletal) fraction isolated by centrifugation was analysed by SDS-PAGE and autoradiography. Incorporation of actin into the Triton-insoluble fraction was used to quantify the formation of F-actin. Thrombin-stimulated platelet cytoskeletal composition was different from PMA-stimulated cytoskeletal composition. Thrombin-stimulated platelets contained not only the three major proteins: actin (43 kDa), myosin (200 kDa) and an actin-binding protein (250 kDa), but three additional proteins of Mr56 kDa, 80 kDa and 85 kDa in the cytoskeleton, which were induced in by thrombin dose-response relationship. In contrast, PMA-stimulated platelets only induced actin assembly, and the 56 kDa, 80 kDa and 85 kDa proteins were not found in the cytoskeletal fraction. Exposure of platelets to thrombin or PMA induced phosphorylation of pleckstrin parallel to actin assembly. Staurosporine, an inhibitor of protein kinase C, inhibited actin assembly and platelet aggregation induced by thrombin or PMA, but did not inhibit the incorporation of 56 kDa, 80 kDa and 85 kDa into the cytoskeletal fraction induced by thrombin. These three extra proteins seem to be unrelated to the induction of protein kinase C. We conclude that actin polymerization and platelet aggregation were induced by a mechanism dependent on protein kinase C, and suggest that thrombin-activated platelets aggregation could involve additional cytoskeletal components (56 kDa, 80 kDa, 85 kDa) of the cytoskeleton, which made stronger actin polymerization and platelet aggregation more.
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PMID:Cytoskeletal changes in platelets induced by thrombin and phorbol myristate acetate (PMA). 1032 51


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