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)

Staurosporine in the micromolar range raised inositol trisphosphate in intact human platelets to levels comparable to that mediated by thrombin. This response was inhibited by neomycin, a phospholipase C antagonist. Staurosporine alone induced a weak, transient rise in cytosolic free calcium levels ([Ca2+]i) from release of internal Ca2+ stores but potentiated the effect induced by thrombin. Therefore, it is unlikely that this alkaloid suppressed inositol trisphosphate mobilization of Ca2+. Additional studies show that staurosporine, 0.5-5 microM, stimulated GTPase activity in platelet membranes while 2 microM K252a and 20 microM H7 were inactive. Present results suggest that staurosporine may activate platelet phospholipase C at the level of G proteins or receptors.
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PMID:Staurosporine induces hydrolysis of phosphatidyl inositol 4,5-bisphosphate in human platelets. 816 13

Although the importance of protein kinases in platelet activation, particularly protein kinase C (PKC), is well established there remain many problems regarding the various phosphorylation cascades, the role of phosphatases and the importance of other serine/threonine and tyrosine kinases. A particular problem is the mechanism of activation of the fibrinogen receptor, GPIIb/IIIa, a critical step in aggregation. Although GPIIIa is phosphorylated (on threonine) neither the stoichiometry nor the minor changes on activation seem adequate to explain the response. Relatively unspecific inhibitors of PKC such as staurosporine prevent PO4 incorporation into most kinase substrates but only inhibit platelet aggregation partially. However, staurosporine does induce activation and then inhibits several renaturable serine/threonine kinases, probably via phosphatases. Staurosporine did not, however, inhibit the platelet Ca2+ signal in response to thrombin but rather enhanced it. 17-Hydroxywortmannin (HWT), a fungal metabolite, has been shown to inhibit respiratory burst in neutrophils and causes haemorrhages. It was recently reported to be a myosin light chain kinase (MLCK) inhibitor and to inhibit PKC only at much higher concentrations. In platelets, HWT inhibits aggregation and partially inhibits phosphorylation of myosin light chain and P47 in thrombin-activated platelets. It also allows the discrimination of an early and a late phase in the cytoplasmic Ca2+ signal since at lower concentrations it only inhibits the late phase. The late phase of ATP release was also inhibited in a dose-dependent manner. The activation of most of the renaturable serine/threonine kinases was also inhibited by HWT. These results support earlier conclusions that the early phase of the Ca2+ signal is phospholipase C dependent but indicate that other mechanisms must be responsible for the late phase. The relative specificity of HWT for MLCK might indicate that this has an unexpected major role in controlling these late phase reactions including activation of GPIIb/IIIa or its clustering. However, staurosporine completely inhibits phosphorylation of myosin light chain by its kinase (as well as other kinases) and has the opposite effect on Ca2+ signals. Clearly, the interactions and feed-back mechanisms between these kinases are very complex but the results suggest that phosphatases acting together with their complementary kinases should also be considered as important platelet activation regulators. P47, long considered a major PKC substrate, may also be phosphorylated by MLCK.
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PMID:Serine/threonine kinases in signal transduction in response to thrombin in human platelets. Use of 17-hydroxywortmannin to discriminate signals. 820 81

The primary phase of ADP-induced aggregation of human platelets does not involve appreciable formation of thromboxane A2 or release of granule contents; lack of formation of inositol trisphosphate has also been noted. Because these responses of platelets to ADP differ so markedly from their responses to other aggregating agents, the roles in ADP-induced aggregation of diacylglycerol, protein kinase C, increases in cytosolic [Ca2+], phosphorylation of pleckstrin (47 kDa) and phosphatases 1 and 2a were investigated. Washed human platelets, prelabelled with [14C]5-hydroxytryptamine and suspended in Tyrode solution (2 mM Ca2+, 1 mM Mg2+), were used for comparisons between the aggregation induced by 2-4 microM ADP, in the presence of fibrinogen, and that induced by 0.05 units/ml thrombin. The diacylglycerol kinase inhibitor 6-(2-[(4-fluorophenyl)phenyl-methylene]-1-piperidinylethyl)-7-meth yl-5H-thiazolo[3,2-a]-pyrimidin-5-one (R59022; 25 microM) had no, or only a slight, enhancing effect on ADP-induced aggregation, but potentiated thrombin-induced responses to a much greater extent. 1,2-Dihexanoyl-sn-glycerol or 1-oleoyl-2-acetyl-sn-glycerol (25 microM) added with or 30-90 s before ADP greatly potentiated aggregation without formation of thromboxane; staurosporine, an inhibitor of protein kinase C, reduced this potentiation. Staurosporine (25 nM) did not inhibit ADP-induced aggregation, although it strongly inhibited thrombin-induced aggregation and release of [14C]5-hydroxytryptamine. All these observations indicate little or no dependence of primary ADP-induced aggregation on the formation of diacylglycerol or on the activation of protein kinase C. At 2-4 microM, ADP did not significantly increase the phosphorylation of pleckstrin (studied with platelets prelabelled with [32P]orthophosphate), but 1,2-dihexanoyl-sn-glycerol- induced phosphorylation of pleckstrin was increased by ADP. Surprisingly, the diacylglycerols strongly inhibited the ADP-induced rise in cytosolic [Ca2+] concurrently with potentiation of ADP-induced aggregation; thus the extent of primary aggregation is independent of the level to which cytosolic [Ca2+] rises. Incubation of platelets with 1,2-dihexanoyl-sn-glycerol or 1-oleoyl-2-acetyl-sn-glycerol for several minutes reversed their potentiating effects on aggregation, and inhibition was observed. Incubation of platelets with okadaic acid, an inhibitor of phosphatases 1 and 2a, inhibited ADP- and thrombin-induced aggregation; although the reason for this effect is unknown, it is unlikely to involve inhibition of phospholipase C, since formation of diacylglycerol appears to have little involvement in the primary phase of ADP-induced aggregation.
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PMID:Activation of phospholipase C and protein kinase C has little involvement in ADP-induced primary aggregation of human platelets: effects of diacylglycerols, the diacylglycerols, the diacylglycerol kinase inhibitor R59022, staurosporine and okadaic acid. 838 48

In human platelets stimulated with thrombin (40 mU/ml), Na+/H+ exchange activity [the ethylisopropylamiloride (EIPA)-sensitive increase of cytoplasmic pH (pHc)] and protein kinase C (PKC) activity [phosphorylation of 47 kDa protein (P47), a substrate for PKC] were determined in the presence of protein kinase inhibitors, staurosporine (0.05-1 microM), K-252a (0.5-10 microM), H-7 (100 microM) and sphingosine (20-40 microM). Staurosporine and K-252a completely blocked PKC activity. H-7 and sphingosine reduced the P47 phosphorylation to 64% and 35%, respectively. On the contrary, the thrombin-induced pHc increase was not inhibited by staurosporine, K-252a or H-7. Sphingosine elevated the resting pHc by 0.26-0.42 independently of the Na+/H+ exchanger and inhibited the thrombin-induced pHc increase. However, after the resting pHc elevated by sphingosine had been reduced to the initial level by adding sodium propionate, the thrombin-induced pHc increase was observed again. These results suggested that sphingosine inhibited the thrombin-induced pHc increase by elevating the resting pHc. Thus, we concluded that the Na+/H+ exchanger was activated by thrombin through a pathway independent of PKC.
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PMID:Na+/H+ exchange activity induced by thrombin is not inhibited by protein kinase inhibitors, staurosporine, K-252a, H-7 and sphingosine, in human platelets. 839 29

Changes in the shape of human platelets and the biochemical mechanism responsible were evaluated, using the shape-change parameter. Neither the Na+/H+ exchanger, nor intracellular or extracellular calcium ions (Ca2+) affected disc-sphere changes induced by low doses of thrombin. Treatment with inhibitors of Ca2+ mobilization, calmodulin or mysoin light-chain kinase had no significant effect on the shape change of platelets. Staurosporine and H-7, both of which are inhibitors of protein kinase C, inhibited disc-sphere changes at low concentrations. Moreover, calphostin C, a specific inhibitor of protein kinase C, effectively inhibited thrombin-induced shape change, as assessed by the shape-change parameter, in a dose-dependent manner. 1-Oleoyl-2-acetyl-glycerol and 1,2-dioctanoyl-glycerol, which are synthetic protein kinase C activators, induced shape changes similar to those induced by thrombin. A decrease in the surface area of platelet images on scanning electron micrographs was used to quantify the disc-sphere transformation. The mean platelet areas was significantly decreased after stimulation with thrombin or 1-oleoyl-2-acetyl-glycerol. Pretreatment with H-7 inhibited the thrombin-induced disc-sphere change, as assessed by changes in the platelet surface area. Our results obtained with various inhibitors suggest that thrombin-induced platelet change, as assessed by the shape-change parameter, are associated with activation of protein kinase C.
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PMID:Protein kinase C inhibitors suppress disc-sphere changes of human platelets, as assessed with the shape-change parameter. 850 6

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

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

The regulatory mechanism of thrombin receptor responsiveness in in situ endothelial cells was investigated by evaluating elevations of cytosolic Ca(2+) concentration ([Ca(2+)](i)) in fura-2-loaded porcine aortic valvular strips. Once stimulated with thrombin, endothelial cells did not respond to the second thrombin stimulation within 90 min. However, applying thrombin receptor activating peptide (TRAP7) at 15 min after the thrombin stimulation caused [Ca(2+)](i) elevation, which was smaller than that seen without preceding stimulation. After 90 min, response to TRAP7 recovered to the control level. When stimulated with TRAP7, the subsequent responses to thrombin and TRAP7 were attenuated at 15 min, and fully recovered after 90 min. Staurosporine partially prevented the TRAP7-induced desensitization. The recovery of responsiveness was inhibited completely by calyculin-A and partially by okadaic acid. Proteolysis and phosphorylation thus play an important role in thrombin receptor desensitization in in situ endothelial cells. Both cleaved and uncleaved receptors were desensitized through phosphorylation in part by staurosporine-sensitive kinase, and restored the responsiveness through dephosphorylation by type 1 phosphatase. The mechanism of regulation of thrombin receptor activity in in situ endothelial cells differed from those reported in cultured endothelial cells. We suggest that the cell-specific regulatory mechanism may be altered by culture conditions.
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PMID:Proteolysis and phosphorylation-mediated regulation of thrombin receptor activity in in situ endothelial cells. 1068 91

The thrombin receptor on human platelets is activated by thrombin to stimulate platelet aggregation through the tethered ligand SFLLRN. This study examined the effects of thrombin and SFLLRN on aggregation and calcium mobilization ([Ca2+]i) in rat, guinea pig, rabbit, dog, monkey, and human platelets, and the role of protein kinases in regulating these functions. Thrombin induced platelet aggregation and [Ca2+]i in all species studied; however, only guinea pig, monkey and human platelets were responsive to SFLLRN. Similar species specific effects were obtained with [Ca2+]i studies. The kinetic profile for [Ca2+]i differed among species, suggesting that regulatory mechanisms for calcium differed between agonists and among species. Staurosporine, a non-selective inhibitor of protein kinases, inhibited platelet aggregation induced by thrombin or SFLLRN in all species. Staurosporine inhibited thrombin-induced [Ca2+]i in guinea pigs, had no effect in rat, and increased [Ca2+]i in all other species. Staurosporine inhibited SFLLRN-induced [Ca2+]i in guinea pig, yet had no effect in monkey or human. Tyrphostin 23, a specific inhibitor of tyrosine protein kinases, inhibited thrombin-induced aggregation of rabbit, monkey, dog and human platelets. SFLLRN-induced aggregation was also inhibited by tyrphostin 23. Tyrphostin 23 inhibited [Ca2+]i induced by either thrombin or SFLLRN in all species. Based on the differential response to agonist stimulation, we propose that thrombin can activate platelets via SFLLRN-dependent and independent mechanisms, which could involve yet unrecognized subtypes of the thrombin receptor or distinct cellular activating mechanisms. Furthermore, differential regulation of calcium mobilization and aggregation was observed in those platelets responding to either thrombin or SFLLRN.
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PMID:Species differences in platelet responses to thrombin and SFLLRN. receptor-mediated calcium mobilization and aggregation, and regulation by protein kinases. 1571 52


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