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)

Most platelet agonists activate and elevate the cytosolic free calcium concentration in human platelets through receptor-dependent mechanisms that are antagonized by cAMP- and cGMP-elevating agents. Nitrovasodilators such as nitroprusside and endothelium-derived relaxing factor are potent cGMP-elevating platelet inhibitors. In the present study, the role of cGMP and cGMP-dependent protein kinase in nitrovasodilator inhibition of ADP- and thrombin-evoked calcium elevation and activation of human platelets was investigated. Preincubation of platelets with 8-(4-chlorophenylthio)guanosine 3',5'-cyclic monophosphate (8-pCPT-cGMP; a membrane-permeant selective activator of the cGMP-dependent protein kinase that does not significantly affect cGMP-regulated phosphodiesterases) inhibited the thrombin-induced phosphorylation mediated by myosin light chain kinase and protein kinase C. Nitrovasodilator-induced protein phosphorylation in human platelets was distinct from that induced by cAMP-elevating prostaglandins and could be mimicked by 8-pCPT-cGMP. Preincubation of human platelets with nitrovasodilators or 8-pCPT-cGMP inhibited the ADP- and thrombin-evoked calcium elevation in the presence and absence of external calcium. Nitrovasodilators and 8-pCPT-cGMP also inhibited the agonist-induced Mn2+ influx, but stopped-flow experiments indicated that the ADP receptor-operated cation channel was not significantly inhibited. These results suggest that in human platelets nitrovasodilators inhibit the agonist-induced calcium mobilization from intracellular stores and the secondary store-related calcium influx but not the ADP receptor-operated cation channel. The results also suggest that these nitrovasodilator effects are mediated by cGMP and the cGMP-dependent protein kinase.
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PMID:Role of cGMP and cGMP-dependent protein kinase in nitrovasodilator inhibition of agonist-evoked calcium elevation in human platelets. 131 May 37

The regulation of extracellular Ca2+ entry into fura-2-loaded human platelets was examined following stimulation with thrombin. In the presence of external Ca2+, stimulation of platelets with thrombin resulted in a rapid increase, followed by a plateau, in intracellular Ca2+ concentration ([Ca2+]i). Pretreatment with wortmannin, a specific inhibitor of myosin light chain kinase, suppressed only the plateau phase and had no effect on the initial rapid increase in [Ca2+]i. In Ca(2+)-free EGTA buffer, thrombin induced a transient and relatively small increase in [Ca2+]i caused by Ca2+ release from internal stores. When Ca2+ was added subsequently to the Ca(2+)-free medium within 10 min after thrombin activation, a marked increase in [Ca2+]i was seen, reflecting thrombin-stimulated external Ca2+ entry. With the Ca(2+)-free medium, wortmannin did not affect either the Ca2+ mobilization from the internal stores or the rapid external Ca2+ entry at early time points (within 5 s) after thrombin stimulation, whereas it significantly inhibited Ca2+ entry when Ca2+ was added later (at 3 min). Wortmannin inhibition of this late Ca2+ entry and that of 20-kDa myosin light chain phosphorylation after thrombin stimulation were dose- and preincubation time-dependent and correlated well with each other. These results suggest that two different channels are responsible for Ca2+ entry in human platelets at the early and late phases of thrombin stimulation and that the channel responsible for the late phase of Ca2+ entry may be activated by a mechanism involving myosin light chain kinase.
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PMID:Two thrombin-activated Ca2+ channels in human platelets. 132 22

Protein tyrosine kinase (PTK) blockers (tyrphostins) inhibit in a dose-dependent fashion thrombin-induced aggregation and serotonin release with IC50 values in the 10-35 microM concentration range. The inhibition of thrombin-induced aggregation correlates with their potency in inhibiting phosphorylation of proteins on tyrosine residues. Using metabolically 32P-labelled human platelets, it was found that the tyrphostins have no effect on the decrease in [32P]phosphatidylinositol bisphosphate but prevent the replenishment of [32P]polyphosphoinositide. Tyrphostins decreased [32P]phosphatidic acid production induced by thrombin, although never by more than 50%, and only delayed the peak of diacylglycerol, suggesting that phospholipase C was still activated. Tyrphostins inhibited the thrombin-elicited early phosphorylation of p43 and p20, substrates for protein kinase C (PKC) and myosin light chain kinase, respectively, at short times of activation. This inhibition, however, was overcome after 1 min of stimulation with thrombin. Tyrphostin AG213 also inhibited platelet aggregation and tyrosine protein phosphorylation induced by phorbol myristate acetate (PMA), but did not inhibit pleckstrin phosphorylation. These results suggest that thrombin induces the phosphorylation of proteins on tyrosine residues which most probably results in the activation of phosphoinositide kinases. The ability of tyrphostins to inhibit phosphorylation of p43 and p20 when induced by thrombin but not when induced by PMA confirms that PTKs may be involved subsequent to PKC activation.
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PMID:Inhibition of platelet activation by tyrosine kinase inhibitors. 138 25

A dose-dependent effect of magnesium on the inhibition of platelet aggregation and release of ATP from dense granules was observed in human platelets (in whole blood, platelet-rich plasma, or washed platelets) against various aggregation agents (ADP, U46619, collagen, or thrombin). The synthesis and release of the proaggregatory cyclooxygenase (CO) and lipoxygenase (LO) products, thromboxane A2 (TXA2) and 12-hydroxyeicosatetraenoic acid (12-HETE), respectively, in platelets were also inhibited by Mg in a dose-dependent manner (IC50 4 to 6 mmol/L). These Mg-mediated activities were further enhanced when platelets were preincubated with insulin (100 microU/mL). The effect of extracellular Mg on the change of intracellular calcium concentration ([Ca2+]i) was assessed using Fura-2/AM loaded cells in the presence or absence of extracellular Ca. Thrombin-stimulated influx of Ca ions decreased from 194 +/- 30 nmol/L to 156 +/- 21 nmol/L in the presence of 5 mmol/L Mg and to 111 +/- 16 nmol/L in 10 mmol/L Mg. However, the intracellular Ca release (as determined in the presence of 5 mmol/L EGTA) was not affected by Mg. The intracellular Ca-dependent protein kinase C and myosin light chain kinase activities on the phosphorylation of endogenous p47 and p20 proteins studied after 2 min of thrombin addition decreased only 10 to 25% in the presence of 5 to 10 mmol/L Mg. Similar results were obtained when EGTA was added prior to the initiation of protein phosphorylation. We conclude that Mg can dose dependently inhibit a wide variety of agonists on platelet aggregation. Furthermore, insulin can potentiate the inhibitory effects of Mg on platelet activation.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Effect of extracellular magnesium on platelet activation and intracellular calcium mobilization. 141 32

To clarify further the activity of rT3, we examined the effect of rT3 on collagen-induced platelet activation as reflected by aggregation, serotonin release, and protein phosphorylation. rT3, T4, T3, and triiodothyroacetic acid inhibited collagen-induced platelet aggregation and serotonin release from platelets in a dose-dependent manner. However, thyronine did not inhibit collagen-induced platelet aggregation. The concentration at which rT3 inhibited by 50% collagen-induced platelet aggregation was 30 +/- 4 (mean +/- SE) mumol/L. rT3, T4, and T3 did not differ significantly in their abilities to inhibit platelet aggregation. Moreover, rT3 inhibited collagen-induced phosphorylation of the 20-kilodalton protein (myosin light chain) in platelets. In contrast, rT3 did not inhibit 12-O-tetradecanoylphorbol 13-acetate (TPA)- or thrombin-induced platelet aggregation and inhibited only minimally TPA-induced 40-kilodalton protein phosphorylation. These results suggest that rT3 inhibits collagen-induced platelet activation by inhibiting the activity of myosin light chain kinase and that it may be interesting to investigate some kinds of activity of rT3.
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PMID:3,3',5'-Triiodothyronine inhibits collagen-induced human platelet aggregation. 151 61

Recently, one of the authors (K.I.) and other investigators reported that myosin light chain (MLC) of smooth muscle (gizzard, arterial and tracheal) was diphosphorylated by myosin light chain kinase (MLCK) and that diphosphorylated myosin showed a marked increase in the actin-activated myosin ATPase activity in vitro and ex vivo. In this study, we prepared myosin, actin, tropomyosin (human platelet), MLCK (chicken gizzard) and calmodulin (bovine brain) and demonstrated diphosphorylation of MLC of platelet by MLCK in vitro. Our results are as follows. (1) Platelet MLC was diphosphorylated by a relatively high concentration (greater than 20 micrograms/ml) of MLCK in vitro. As a result of diphosphorylation, the actin-activated myosin ATPase activity was increased 3 to 4-fold as compared to the monophosphorylation. (2) Both di- and monophosphorylation reactions showed similar Ca2+, KCl, MgCl2-dependence. Maximal reaction was seen at [Ca2+] greater than 10(-6) M, 60 mM KCl and 2 mM MgCl2. This condition was physiological in activated platelets. (3) Di- and monophosphorylated myosin showed similar Ca2+, KCl-dependence of ATPase activity but distinct MgCl2-dependence. Diphosphorylated myosin showed maximal ATPase activity at 2 mM MgCl2 and monophosphorylated myosin showed a maximum at 10 mM MgCl2. (4) The addition of tropomyosin stimulated actin-activated ATPase activity in both di- and monophosphorylated myosin to the same degree. (5) ML-9, a relatively specific inhibitor of MLCK, inhibited the aggregation of human platelets induced by thrombin ex vivo in a dose-dependent manner. Moreover, this drug also partially inhibited both di- and monophosphorylation reactions and actin-activated ATPase activity. On the other hand, H-7, a synthetic inhibitor of protein kinase C, had little effect on the aggregation of human platelets induced by thrombin ex vivo. From these results, we conclude that diphosphorylation of platelet myosin by MLCK may play an important role in activated platelets in vivo.
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PMID:Diphosphorylation of platelet myosin by myosin light chain kinase. 153 1

We investigated the intracellular processes of the shape change in the human megakaryoblastic leukemia cell, MEG-01, by platelet agonists. Thrombin induced the formation of many pseudopods. This shape change was also induced by TPA and A23187, but not by ADP, collagen, or epinephrine. Electron microscopy and FITC-labeled phalloidin staining revealed thick submembranous microfilament bundles in the pseudopods of the shape-changed cells induced by thrombin. Shape change was inhibited by cytochalasin B. Protein kinase C (RKC) inhibitor, H-7, markedly inhibited thrombin-induced shape change, while the myosin light chain kinase (MLCK) inhibitor, ML-9 did not. These results suggest that thrombin-induced reorganization of microfilaments and shape change of MEG-01 cells are mediated by PKC but not by MLCK.
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PMID:[Shape change in human megakaryoblastic leukemia cells, MEG-01]. 161 74

The formation of membrane microparticles through vesiculation of the platelet plasma membrane is known to provide catalytic surface for several enzyme complexes of the coagulation system, and to underlie the procoagulant responses elicited with platelet activation. This induced shedding of vesicles from the plasma membrane is most prominent when platelets are activated by the terminal complement proteins, C5b-9, by a Ca2+ ionophore, or by the combination of thrombin plus collagen. Although shown to require elevated [Ca2+], the cellular events that initiate plasma membrane evagination and fusion to form the shed vesicles remain unresolved. To gain additional insight into the cellular events that regulate membrane microparticle formation, we have examined how this process is influenced by the activity of cellular protein kinases. Cytoplasmic [Ca2+] of gel-filtered platelets was increased by membrane assembly of the terminal complement proteins C5b-9 in the presence of selective inhibitors of protein kinase or phosphatase reactions, and resulting microparticle formation was quantitated by fluorescence-gated flow cytometry. Pre-equilibration of the phosphatase inhibitor vanadate into the platelet cytosol increased microparticle formation by as much as 40%, suggesting that vesiculation of the platelet plasma membrane is influenced by the state of phosphorylation of a cellular constituent. By contrast to the stimulatory effects of vanadate, microparticle formation was partially inhibited in platelets treated with the protein kinase inhibitor sphingosine, the myosin light chain kinase inhibitor ML-7, the calmodulin-antagonist W-7, and under conditions of elevated cytosolic concentration of cyclic adenosine monophosphate. These results indicate that complement-induced platelet microparticle formation is influenced by one or more protein kinase(s) as well as by calmodulin, and suggest a role for the platelet myosin light chain kinase or another Ca(2+)-pluscalmodulin-regulated membrane component.
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PMID:Participation of protein kinases in complement C5b-9-induced shedding of platelet plasma membrane vesicles. 165 68

In primary hypertension, phospholipase C (PLC) is hypersensitive in several target tissues (platelets, vascular smooth muscle cells, aortic fibroblasts). Protein kinase C (PKC) and myosin light chain kinase (MLCK), which are physiologically activated by PLC-triggered second messengers (diacylglycerol and Ca2+ ions, respectively), phosphorylate specific proteins closely involved in the cell functional responses. In this study, we have examined and compared between platelets of spontaneously hypertensive rats (SHR) and their normotensive controls Wistar-Kyoto (WKY), the patterns of protein phosphorylation obtained either with the receptor-mediated agonist thrombin (i.e. which acts via PLC) or with direct activators of the protein kinases, PKC and MLCK. Activation by thrombin of 32P-prelabeled platelets induced incorporation of radioactivity into two proteins, P20 (myosin light chain) and P47. The curves obtained when platelets were challenged with either increasing doses of thrombin (0.025-0.3 U/ml) for 20 sec or with a low dose of the agent (0.1 U/ml) for up to 1 min, revealed that phosphorylation of the target proteins of PKC (P47) and of MLCK (P20) were significantly enhanced in platelets of SHR compared to WKY. In contrast, direct activation of PKC by phorbol ester and of MLCK by the calcium ionophore A23187 evoked the selective phosphorylation of the respective target proteins, P47 and P20, to a similar extent in platelets of SHR and WKY. Taken together, these results demonstrate that a physiological agonist (thrombin) induces an enhanced phosphorylation of intracellular proteins in platelets of SHR.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:[Increased phosphorylations of proteins involved in the expression of the physiologic response of platelets in SHR rats]. 212 75

This study investigates the role of protein kinase C and of myosin light chain kinase in mediating platelet hyperresponsiveness in spontaneously hypertensive rats (SHR). For this purpose, 32P-labeled washed platelets of both SHR and normotensive controls Wistar-Kyoto (WKY) were challenged either with a receptor-mediated agonist (thrombin) or with direct activators of myosin light chain kinase and protein kinase C. Such enzymatic activities were assessed by measuring changes in 32P-labeling of their respective target proteins, namely myosin light chain (20 KDa) and the 47 KDa protein. In resting platelets, the patterns of protein phosphorylation were similar between SHR and WKY, suggesting that the two cell types were in a comparable quiescent status. By contrast, in both dose-response and time-course studies, thrombin promoted a significantly greater phosphorylation of the 20- and 47 KDa proteins in platelets of SHR compared with that for WKY. Sensitivity of myosin light chain kinase to the calcium ionophore A23187 and of protein kinase C to both phorbol ester and dioctanoylglycerol was apparently not different between the two cell types. The data indicate that the exaggerated thrombin-induced protein phosphorylation observed for platelets of SHR is not linked to alterations in protein kinase C and/or myosin light chain kinase per se. These results therefore suggest that platelet hyperresponsiveness in SHR is likely to be related, at least in part, to abnormalities in receptor-mediated transmembrane signalling.
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PMID:Receptor-dependent and -independent protein phosphorylation in platelets of spontaneously hypertensive rats. 217 66


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