EC:2.7.11.13 - FACTA Search

Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.11.13 (protein kinase C)
49,245 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Zinc deficiency has been linked to a bleeding tendency and impaired wound healing in several disease states. A number of investigators have suggested that zinc ions play a role in platelet aggregation in vitro as well as in in vivo studies. The purpose of the present study was to explore the mechanism by which adenosine diphosphate (ADP) and Zn2+ may act cooperatively during activation of blood platelets. We demonstrate that Zn2+ alone does not affect either formation of thromboxane A2 or intracellular calcium mobilization in platelets. On the other hand, we show that ADP and Zn2+ exert a cooperative effect on the phosphorylation of P-47 protein (pleckstrin), a substrate of protein kinase C in platelets. The inhibitory effect of this reaction by the compound Ro31, a specific inhibitor of the regulatory domain of protein kinase C, was compatible with our contention that Zn2+ may act directly on protein kinase C. Our study provides evidence that zinc ions present in plasma or platelets may modulate ADP-induced platelet aggregation. N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), a zinc chelator, blocked ADP-induced platelet aggregation. This aggregation was restored by 10 mumol/L of Zn2+ but not by other ions. Also, a Zn2+ ionophore, pyrithione, potentiated the ADP-induced platelet aggregation and this potentiation was blocked by TPEN. Experiments with the zinc ionophore suggest that intracellular zinc ions play an important role in activation of platelets, and in the absence of other platelet agonists it appears that it may be a requirement for ADP-induced platelet aggregation to occur.
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PMID:Zinc ions potentiate adenosine diphosphate-induced platelet aggregation by activation of protein kinase C. 828 49

4,4'-Diisothiocyanato-stilbene-2,2'-disulfonic acid (DIDS) stimulates human platelets via alpha 2A-adrenergic receptor-mediated activation of protein kinase C (PKC) independent of the phospholipase C pathway. Here we show, that in permeabilized platelets activation of PKC by DIDS (20 microM), measured as 32P incorporation in pleckstrin, is completely inhibited by guanosine 5'-(2-O-thio)diphosphate (200 microM), an inhibitor of heterotrimeric G-proteins. Also pertussin toxin (4 micrograms/ml), which ADP-ribosylates the alpha-subunits of Gi's and Go, prevents pleckstrin phosphorylation by DIDS. N-Ethylmaleimide (50 microM), which uncouples Gi from alpha 2A-adrenoceptors, inhibits pleckstrin phosphorylation by DIDS in intact platelets. Activation of PKC by 55 nM phorbol 12-myristate 13-acetate and 500 nM platelet-activating factor are not disturbed by NEM. DIDS inhibits by 40 +/- 5% (n = 4) the pertussis toxin-catalyzed [32P]ADP-ribosylation of a 41 kDa protein fraction previously shown to contain the alpha-subunits of Gi alpha-1, Gi alpha-2 and Gi alpha-3. Thus, the alpha 2A-adrenergic receptor activates PKC via a G-protein of the Gi-family.
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PMID:Alpha 2A-adrenergic receptors activate protein kinase C in human platelets via a pertussis toxin-sensitive G-protein. 831 82

Recent experiments have shown that the stimulation of animal cells in vitro by direct protein kinase C (PKC) activators is significantly reduced under microgravity (micro g). Platelets undergo protein phosphorylation and morphological changes a few seconds after stimulation by agonists such as phorbol esters which activate PKC. Therefore, taking advantage of parabolic plane flight to obtain short periods of microgravity, we studied phosphorylation of myosin light chain (20K), specific PKC-dependent phosphorylation of a 40,000 M(r) protein, pleckstrin (40K) and platelet shape change. SDS-PAGE analysis and electron microscopy were performed on platelets subjected to 20 seconds microgravity as compared to normal gravity (1 g) conditions. These investigations showed that neither Ca(2+)-calmodulin-mediated activation nor the PKC-dependent pathways are inhibited during short periods of microgravity.
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PMID:Platelet shape change and protein phosphorylation induced by ADP and thrombin are not sensitive to short periods of microgravity. 831 74

Although several previous studies have indicated a role for tyrosine phosphorylated proteins in platelet function, their precise function and relationship to other biochemical processes remains elusive. In the present study genistein, an inhibitor of tyrosine kinase activity, was used to address this latter question. Genistein inhibited aggregation of washed human platelets in response to the thromboxane analogue U46619, to the phorbol ester phorbol myristate acetate, and to the calcium ionophore A23187. Only in the case of U46619, however, did the concentration of genistein required (IC50 of 10 micrograms/ml) correlate to that reported to inhibit tyrosine kinases. Likewise, genistein also inhibited U46619-induced serotonin secretion, elevation of cytosolic calcium, [32P]-phosphatidic acid production (an index of phospholipase C activity) and the phosphorylation of pleckstrin (an index of protein kinase C activity) at similar concentrations (IC50 of 4-9 micrograms/ml). U46619 caused the phosphorylation of a phosphoprotein which was insensitive to KOH digestion and therefore presumably a phosphotyrosine. This phosphorylation was also inhibited by genistein (IC50 of 3 micrograms/ml. However genistein also inhibited [3H]-U46619 binding to platelets with an IC50 of 3 micrograms/ml. These data suggest that the inhibitory effects of genistein on platelet activation occurs as a result of antagonism of the thromboxane receptor.
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PMID:The effects of genistein on platelet function are due to thromboxane receptor antagonism rather than inhibition of tyrosine kinase. 832 74

The effects of the Ca(2+)-ATPase inhibitors thapsigargin (Tg) and 2,5-di-(t-butyl)-1,4-benzohydroquinone (tBuBHQ) were examined by using Ca(2+)-regulatory systems of platelet mixed membranes, saponin-permeabilized and intact platelets. Both agents inhibit Ca(2+)-ATPase activities of platelet mixed membranes, without any effect on the basal Mg(2+)-ATPase activity. Tg is more effective (EC50 = 35 nM) than tBuBHQ (EC50 = 580 nM). The effect of the two inhibitors on 45Ca2+ release from saponin-permeabilized platelets has also been characterized. 45Ca2+ uptake into non-mitochondrial intracellular stores occurs via an ATP-dependent mechanism, and if added at equilibrium the second messenger Ins(1,4,5)P3 releases 50% of the accumulated 45Ca2+. Maximally effective concentrations of Tg (1 microM) and tBuBHQ (50 microM) release 77% and 68% of the accumulated 45Ca2+. Addition of Ins(1,4,5)P3 together with either Tg or tBuBHQ resulted in a non-additive release which was the same as with either Tg or tBuBHQ alone, indicating that the Ins(1,4,5)P3-sensitive Ca2+ pool was a subset of the pool that is sensitive to the Ca(2+)-ATPase inhibitors. Release of 45Ca2+ by either Tg or tBuBHQ was not affected by heparin, which totally blocked Ins(1,4,5)P3-induced Ca2+ release, and Tg was found not to affect [32P]Ins(1,4,5)P3 binding to its receptor on mixed membranes. Thus both Tg and tBuBHQ release Ca2+ from a pool that totally overlaps the Ins(1,4,5)P3-sensitive pool without affecting Ins(1,4,5)P3 function. In intact indomethacin-treated Fura 2-loaded platelets, Tg and tBuBHQ cause Ca2+ elevation, arising from release from intracellular stores and influx from the outside. Both Tg and tBuBHQ elevated Ca2+ to similar levels, which were less and slower than those observed with thrombin. Addition of thrombin to cells already treated with Tg or tBuBHQ produced further elevation of Ca2+, indicating agonist utilization of a Ca(2+)-ATPase inhibitor-insensitive pool. In aggregation experiments Tg and tBuBHQ showed different functional effects. In indomethacin-treated cells Tg induces slow aggregation and secretion responses, whereas tBuBHQ only induces shape change. Both agents show synergistic secretory responses with the protein kinase C activator dioctanoylglycerol (DiC8). Tg also showed greater ability than tBuBHQ to release [3H]arachidonic acid (AA) from [3H]AA-labelled platelets. Additionally, in [32P]Pi-labelled platelets both Tg and tBuBHQ induced phosphorylation of myosin light chain, a 27 kDa protein and the 45 kDa protein pleckstrin, but Tg showed a greater ability than tBuBHQ to cause phosphorylation of pleckstrin. These studies indicate that Tg and tBuBHQ are effective in releasing the Ins(1,4,5)P3-sensitive Ca2+ pool in platelets.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Ca2+ release from platelet intracellular stores by thapsigargin and 2,5-di-(t-butyl)-1,4-benzohydroquinone: relationship to Ca2+ pools and relevance in platelet activation. 836 62

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 the present study, we investigated whether stimulation of platelets by epinephrine affects the Na+/H+ exchanger, an antiport that regulates the cytosolic pH (pHi). Epinephrine alone failed to modulate Na+/H+ exchange, as reflected by a constant fluorescence of the pHi indicator BCECF. In contrast, epinephrine accelerated Na+/H+ exchange upon stimulation with a threshold concentration of platelet activating factor (PAF). The extra Na+/H+ exchange was not caused by a better binding of PAF to platelets and occurred also in the presence of indomethacin, excluding a role for cyclooxygenase products. Epinephrine failed to mobilize Ca2+i (measured by fura-2 fluorescence) and did not activate protein kinase C ([32P]pleckstrin) or phospholipase C ([32P]phosphatidic acid). In combination with PAF, epinephrine left the PAF-induced mobilization of Ca2+i and accumulation of [32P]phosphatidic acid unchanged, but induced a 1.3-fold increase in the phosphorylation of pleckstrin. These data indicate that epinephrine enhances Na+/H+ exchange via a direct effect of alpha 2A-adrenergic receptors on protein kinase C.
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PMID:Effect of epinephrine on the regulation of Na+/H+ exchange in human platelets. 838 95

The peptide YFLLRNP antagonizes the aggregation of human platelets when induced by low concentrations of alpha-thrombin or the thrombin receptor agonist peptide (SFLLRNP), demonstrating that it interacts specifically with the thrombin receptor. Platelets exposed to YFLLRNP show immediate shape change (pseudopod formation) and potentiation of the ADP and platelet-activating factor response, but no Ca2+ mobilization, P47 (pleckstrin) phosphorylation, secretion, or aggregation. Thus, YFLLRNP induces a state of partial activation of the platelets. Furthermore, with platelets prestimulated with adrenalin (10 microM), YFLLRNP induces aggregation, but no secretion, and only in the presence of added fibrinogen. We also found that prostacyclin inhibits the YFLLRNP-induced shape change; but EDTA, aspirin, and apyrase (ADP scavenger) do not. Thus, the thrombin receptor in platelets may communicate, independently of Ca2+ mobilization and P47 phosphorylation (protein kinase C activation), with intracellular signaling mechanisms that 1) modulate the cytoskeleton structure, 2) potentiate other platelet responses, and 3) stimulate coupling between the thrombin receptor and fibrinogen binding (the glycoprotein IIb-IIIa complex). YFLLRNP may be useful for differentiating between several possible activation states of the platelet thrombin receptor.
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PMID:A peptide ligand of the human thrombin receptor antagonizes alpha-thrombin and partially activates platelets. 839 Sep 90

The aim of this study was to establish further the role of protein kinase C in aggregation and secretion of 5-hydroxytryptamine (5-HT) from human platelets by using the selective inhibitor Ro 31-8220. Ro 31-8220 (3 microM) inhibited completely phosphorylation of pleckstrin, the major protein kinase C substrate, induced by thrombin, A23187 or phorbol dibutyrate (PDBu). Myosin light-chain phosphorylation induced by PDBu was also inhibited completely, but that induced by thrombin or A23187 was only inhibited partially. As myosin light chain is a substrate for both myosin light-chain kinase and protein kinase C, these results suggest that Ro 31-8220 is inhibiting only the protein kinase C-induced phosphorylation and that Ro 31-8220 has a greater selectivity to protein kinase C than does its structural analogue staurosporine. The stimulation of secretion of 5-HT by maximally effective concentrations of thrombin and A23187 was decreased significantly by 3 microM Ro 31-8220, but not inhibited completely. These results indicate a major role for protein kinase C in the stimulation of secretion by agonist- and ionophore-induced activation. On its own, a maximal concentration of PDBu induced a small degree of secretion (3.3 +/- 1.0%), but potentiated markedly the response to a submaximal concentration of A23187 (300 nM) to a level greater than seen with a maximal concentration of A23187. A similar set of results was also seen with aggregation, but not with shape change. We interpret these results to mean that the signalling event for secretion and aggregation is Ca2+, and this is potentiated markedly by protein kinase C. In the case of secretion, it appears that it is the synergy which is the major determining factor in influencing the extent.
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PMID:Synergy between Ca2+ and protein kinase C is the major factor in determining the level of secretion from human platelets. 842 66

High levels of fluid shear stress at the blood vessel wall directly stimulate von Willebrand factor (vWF)-mediated platelet adhesion and aggregation and thereby contribute to the pathogenesis of arterial thrombosis. We have found that a pathological level of arterial wall shear stress (90 dynes/cm2) induces platelet aggregation that is associated with the phosphorylation of pleckstrin, a M(r) 47,000 protein kinase C substrate (p47). Shear-induced p47 phosphorylation depends entirely on vWF binding to platelet glycoprotein (Gp) Ib and GpIIb-IIIa, and the specific inhibition of protein kinase C with the staurosporine analogue Ro 31-7549 inhibits the full aggregation response to shear. Shear stress-induced platelet p47 phosphorylation occurs independent of any measurable change in diacylglycerol mass or hydrolysis of phosphatidylinositol 4,5-bisphosphate. These results indicate that mechanical shear stress induces vWF to bind to platelet GpIb and GpIIb-IIIa, stimulating a diacylglycerol-independent pathway of protein kinase C activation that contributes to platelet aggregation in response to shear.
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PMID:Protein kinase C is activated in platelets subjected to pathological shear stress. 842 27

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