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 present study specifically addresses the role of protein kinase C (PKC) activation in human endothelial cell Ca2+ mobilization, a response that is functionally coupled to the production of the potent arachidonate (AA) metabolite, prostacyclin (PGI2). Phorbol 12-myristate 13-acetate (PMA), alpha-thrombin, and sodium fluoride (NaF), a direct G-protein activator, produced a rapid and time-dependent translocation of PKC from the cytosol to the membrane. Activation of PKC by brief pretreatment of human umbilical vein endothelial cell (HUVEC) monolayers with PMA resulted in the inhibition of NaF-induced inositol phosphate increases and attenuation of both alpha-thrombin- and NaF-activated increases in intracellular Ca2+ (Ca2+i). Ca2+ mobilization induced by ionophore A23187 was not affected by PKC preactivation, suggesting PKC-dependent negative feedback inhibition of phosphatidylinositol (PI)-specific phospholipase C (PLC). Agonist-stimulated AA release and PGI2 synthesis in PMA-pretreated cultured human endothelial cells, however, was potentiated, and the enhanced PGI2 synthesis produced by A23187, NaF, and alpha-thrombin was dependent upon the dose of PMA. Treatment of HUVEC monolayers with an intracellular Ca2+ chelator, 1,2-bis(2-aminophenoxy)ethane-N,N,N'N'-tetraacetic acid-acetoxymethylester (BAPTA-AM), dramatically reduced alpha-thrombin-, NaF-, and A23187-induced PGI2 synthesis, demonstrating the importance of Ca2+i availability in PGI2 synthesis. BAPTA pretreatment did not inhibit PMA-induced PKC activation, and BAPTA-mediated inhibition of agonist-stimulated PGI2 synthesis was partially attenuated by prior PMA pretreatment. Staurosporine, a potent PKC inhibitor, at concentrations that inhibited PKC-induced phosphorylation of histone-1, augmented both alpha-thrombin- and NaF-induced production of inositol phosphates but markedly inhibited alpha-thrombin-, NaF-, and A23187-induced PGI2 synthesis. The downregulation of PKC activity by prolonged PMA treatment (18 h) produced similar inhibition of PGI2 synthesis by these agonists (approximately 50% inhibition). These studies indicate that the integrated phospholipase A2 and PLC activities are under complex regulation by factors that include both PKC activation and [Ca2+i]. PKC exerts dual effects on prostaglandin synthesis via negative regulation of Gp-coupled PI-specific PLC and positive feedback regulation of AA release and PGI2 synthesis. PKC is thus a critical determinant in the regulation of human endothelial cell prostaglandin synthesis by both receptor-mediated and G-protein-dependent cellular activation.
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PMID:Role of protein kinase C in the regulation of prostaglandin synthesis in human endothelium. 154 Mar 95

The effects of H2O2 on platelet function were investigated in vitro and ex vivo. H2O2 (0.5 to 5 mumol/L) alone did not influence platelet function, but when it was combined with subthreshold concentrations of arachidonic acid or collagen, it induced platelet aggregation and serotonin release in a dose-dependent fashion. The increase in platelet aggregation was associated with thromboxane A2 production and was prevented by 100 mumol/L aspirin. The amplification of platelet response by H2O2 was also inhibited 2 hours after 300 mg aspirin was given to healthy subjects. H2O2 alone did not affect intraplatelet Ca++ influx or mobilization but, combined with subthreshold concentrations of arachidonic acid, it increased Ca++ mobilization. In platelets prelabeled with tritiated arachidonic acid, H2O2 induced tritium release in a dose-dependent fashion; this effect was prevented by mepacrine, an inhibitor of the phospholipase A2 enzyme. Platelet function was not affected by using H2O2 in combination with other agonists such as thrombin, calcium ionophore, or adenosine diphosphate. This study suggests that H2O2 triggers activation of platelets preexposed to agonists at subthreshold levels by stimulating arachidonic acid metabolism, likely by stimulating the phospholipase A2 enzyme. The stimulation of platelets by concentrations of H2O2 similar to those released by activated leukocytes may give new insights into the functional cooperation between leukocytes and platelets.
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PMID:Hydrogen peroxide triggers activation of human platelets selectively exposed to nonaggregating concentrations of arachidonic acid and collagen. 158 86

1. Peritoneal mast cells from rat were co-incubated in vitro in a platelet aggregometer cuvette with washed rabbit platelets. In response to stimulation with calcium ionophore (A23187; 1-5 microM), the mast cells released a substance which stimulated the platelets to aggregate. These concentrations of ionophore did not stimulate platelet aggregation in the absence of mast cells, nor affect the responsiveness of the platelets to aggregation induced by thrombin or PAF. Release of a PAF-like substance was also observed in response to stimulation of the mast cells with antigen. 2. This pro-aggregatory activity is attributable to the release of PAF by the mast cells, since the activity could be abolished by preincubating the platelets with a specific PAF receptor antagonist (WEB 2086; 10 microM). Furthermore, the platelet-aggregating factor co-migrated with PAF on thin-layer chromatographs and could be abolished by incubation with phospholipase A2 (20 micrograms ml-1) or a specific antibody directed against PAF. 3. The release of PAF by peritoneal mast cells could be inhibited, in a concentration-dependent manner, by PF-5901 (IC50 of 3.9 microM) or Wy-50,295 (IC50 of 1.2 microM), two structurally similar compounds with inhibitory effects on leukotriene synthesis, as well as leukotriene D4 (LTD4) receptor antagonist properties. 4. Inhibition of PAF synthesis was not observed when the mast cells were incubated with a structurally unrelated 5-lipoxygenase inhibitor (A-64077), a structurally dissimilar inhibitor of 5-lipoxygenase activating protein (MK-886) or with a structurally related LTD4 receptor antagonist (MK-571) which lacks inhibitory effects on leukotriene synthesis, each at concentrations of up to 100 microM.5. Neither PF-5901 nor Wy-50,295 (1 or 10 microM) significantly affected histamine release or prostaglandin D2 synthesis by peritoneal mast cells in response to calcium ionophore stimulation.6. These results demonstrate the ability of a class of quinoline-based compounds to inhibit PAF synthesis by peritoneal mast cells. This activity does not appear to be related to effects of these compounds on leukotriene synthesis or LTD4 receptors. The ability of these compounds to inhibit PAF synthesis may contribute to their anti-inflammatory properties.
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PMID:Platelet-activating factor synthesis by peritoneal mast cells and its inhibition by two quinoline-based compounds. 159 92

Certain antiphospholipid antibodies, particularly those associated with arterial thrombosis, reduce vascular prostacyclin production. Studies were conducted to determine whether antibody-mediated inhibition of phospholipase A2 accounts for this effect. In this report we present evidence that purified antiphospholipid antibodies reduce phospholipase A2 activity toward phospholipid substrates, both in vitro and in a defined system. Purified immunoglobulin, obtained from patients at risk for thrombosis who had plasma antiphospholipid antibodies, impaired prostacyclin generation after endothelial stimulation with thrombin or the calcium ionophore A23187. The release of arachidonate in response to A23187 was reduced in endothelial cells pretreated with antibody; the metabolism of exogenous arachidonate to prostacyclin was normal. Thrombin-induced synthesis of platelet-activating factor, which follows phospholipase A2-mediated generation of lysophosphatidylcholine, was also inhibited in parallel with the inhibition of prostacyclin generation. Phospholipase A2 activity was determined in a defined test system with two phospholipases A2. The hydrolysis of fatty acid was less in the presence of patient immunoglobulin than in buffer alone or with normal immunoglobulin. Inhibition by antibody was present at a range of phospholipase concentrations. Antiphospholipid antibodies, purified from patient serum by adsorption to and subsequent elution from immobilized cardiolipin or phosphatidylserine, also inhibited phospholipase A2 activity. The data support our conclusions that purified antiphospholipid antibodies inhibit endothelial phospholipase A2 activity in response to thrombin or ionophore and that phosphatidylcholine in a common metabolic precursor of both prostacyclin and platelet-activating factor. In a defined enzyme assay, inhibition by antiphospholipid antibody of phospholipase A2 activity does not require additional cofactors.
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PMID:Some antiphospholipid antibodies inhibit phospholipase A2 activity. 834 Jul 8

The venom of P. olfersii has high hemorrhagic, edema-inducing and fibrin(ogen)olytic activities. It is devoid of thrombin-like, procoagulant, phospholipase A2 and platelet aggregating enzymes. The main activities are metalloproteinases inhibited by metal chelators (EDTA and 1,10-phenanthroline) and sulfhydryl compounds (DTT and cysteine). The hemorrhagic and fibrinogenolytic enzymes were partially purified by gel filtration on HPLC. The hemorrhagic activity of the venom was neutralized by commercial horse antivenoms to Bothrops species, as well as by rabbit antisera specific for hemorrhagic factors isolated from these Bothrops venoms. No immunoprecipitin reactions were obtained, indicating that the few epitopes of the P. olfersii hemorrhagin are involved in these neutralization reactions. The fibrinogenolytic enzyme cleaves A alpha-chain more quickly than the B beta-chain of human fibrinogen. The venom also solubilizes fibrin. This solubilization appears to occur from the hydrolysis of unpolymerized alpha-chain and cross-linked gamma-gamma dimer. The fibrin peptide products are distinct from those produced by plasmin.
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PMID:Hemorrhagic, fibrinogenolytic and edema-forming activities of the venom of the colubrid snake Philodryas olfersii (green snake). 162 24

Stimulation of human endothelial cells (EC) by thrombin elicits a rapid increase of intracellular free Ca2+ [(Ca2+]i), platelet-activating factor (PAF) production and 1-O-alkyl-2-lyso-sn-glycero-3- phosphocholine (lyso-PAF): acetyl-CoA acetyltransferase (EC 2.3.1.67) activity. The treatment of EC with thrombin leads to a 90% decrease in the cytosolic protein kinase C (PKC) activity; this dramatic decline is accompanied by an increase of the enzymatic activity in the particulate fraction. The role of PKC in thrombin-mediated PAF synthesis has been assessed: (1) by the blockade of PKC activity with partially selective inhibitors (palmitoyl-carnitine, sphingosine and H-7); (2) by chronic exposure of EC to phorbol 12-myristate 13-acetate (PMA), which results in down-regulation of PKC. In both cases, a strong inhibition of thrombin-induced PAF production is observed, suggesting obligatory requirement of PKC activity for PAF synthesis. It is suggested that PKC regulates EC phospholipase A2 (PLA2) activity as thrombin-induced arachidonic acid (AA) release is 90% inhibited in PKC-depleted cells. Brief exposure of EC to PMA strongly inhibits thrombin-induced [Ca2+]i rise, acetyltransferase activation and PAF production, suggesting that, in addition to the positive forward action, PKC provides a negative feedback control over membrane signalling pathways involved in the thrombin effect on EC. Forskolin and iloprost, two agents that increase the level of cellular cAMP in EC, are very effective in inhibiting thrombin-evoked cytosolic Ca2+ rise, acetyltransferase activation and PAF production; this suggests that endogenously generated prostacyclin (PGI2) may modulate the synthesis of PAF in human endothelial cells.
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PMID:Protein kinase C and cyclic AMP modulate thrombin-induced platelet-activating factor synthesis in human endothelial cells. 171 Sep 33

Several workers have described desensitization of endothelial prostacyclin production but conflicting evidence has been published regarding the mechanism of desensitization, whether it is homologous (agonist specific) or heterologous, and whether inactivation of cyclooxygenase is involved. The purpose of the present study was to determine the relation between the intensity of a first thrombin stimulus and the subsequent response to a repeat thrombin, histamine, ionophore A23187 or aluminium fluoride (AlF4) stimulation and to determine possible targets of desensitization. Following thrombin stimulation of confluent cultured human umbilical vein endothelial cells (HUVEC) only homologous desensitization of inositol phosphate production was observed. Both homologous and heterologous desensitization of arachidonic acid release and prostacyclin production occurred, the latter towards both histamine and the ionophore A23187. For any given dose of the first stimulant there was a much greater effect on the homologous response than on the heterologous response. These differences suggest different mechanisms. The homologous desensitization probably involves the receptor whereas the present results suggest that the target of heterologous desensitization is distal to calcium mobilization in the signal transduction pathway. The possibilities include decreased activity of phospholipase A2 or a decreased pool of accessible arachidonic acid.
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PMID:Different mechanisms of homologous and heterologous desensitization of thrombin-induced endothelial prostacyclin production. 176 77

The present study evaluated the effects of the calcium-channel blocking agent diltiazem on platelet aggregation and on synthesis of thromboxane B2 (the stable metabolite of thromboxane A2) from platelet-rich plasma (PRP) and whole blood samples. Our results showed that diltiazem inhibits collagen- and thrombin-induced platelet aggregation and TXB2 production from PRP. Since no significant interference with conversion of arachidonate to thromboxane A2 was demonstrated, inhibition of phospholipase A2 activity may be the prevailing mechanism of the diltiazem effect. The drug demonstrated a dose-related inhibitory activity on TXB2 synthesis from whole blood samples during spontaneous clotting or following stimulation with collagen or thrombin. The present results give further evidences for an antiplatelet activity of diltiazem and support the hypothesis that inhibition of platelet function contributes to the therapeutic efficacy of this drug in the treatment of cardiovascular diseases.
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PMID:Effects of diltiazem on thromboxane B2 production from platelet-rich plasma and whole blood. 180 24

An increase in intracellular calcium level is an important signal in the regulation of cellular responses under normal and pathological conditions. Because two key enzymes in the synthetic pathway of platelet activating factor (PAF), phospholipase A2 and acetyltransferase, are calcium dependent, we hypothesized that calcium channel blockade may inhibit agonist-induced PAF synthesis. Primary cultures of human umbilical vein endothelial cells (EC), pre-incubated with [3H]acetate, were exposed to thrombin (5 U/mL) and PAF production was quantitated by incorporation of radiolabel into the EC lipid fraction co-migrating with exogenous PAF in thin-layer chromatography. The effect of pre-incubation with calcium channel blockers (verapamil, diltiazem, 10(-4) M) or buffer was determined. Results (triplicate experiments, * P less than 0.05 vs buffer, P less than 0.05 vs thrombin) demonstrate that pre-incubation with calcium channel blocker markedly inhibits thrombin-induced PAF production (verapamil:buffer 273 +/- 122, thrombin 10,735 +/- 1524*, thrombin + verapamil 178 +/- 91 cpm/plate; diltiazem:buffer 1097 +/- 581, thrombin 15,283 +/- 2661*, thrombin + diltiazem 280 +/- 56 cpm/plate). The effect of diltiazem was dose-dependent (% inhibition: 10(-7) M, 46%; 10(-5) M, 60%; 10(-4) M, 98%). Diltiazem also inhibited bradykinin (10(-8) M) induced PAF synthesis. In calcium-free medium or in the presence of LaCl3 (10(-3) M), the PAF response of EC to thrombin was blunted (buffer 582 +/- 360, thrombin 5394 +/- 1069, thrombin + calcium free medium 1055 +/- 571, thrombin + LaCl3 1271 +/- 58 cpm/plate). We conclude that calcium channel blockers prevent agonist-induced PAF synthesis, possibly by preventing cellular calcium influx and activation of PAF synthetic enzymes. We speculate that this mechanism may underlie, at least in part, the beneficial effect of calcium channel blockade under various pathological conditions.
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PMID:Calcium channel blockade inhibits platelet activating factor production by human umbilical vein endothelial cells. 181 8

The effect of biscoclaurine (bisbenzylisoquinoline) alkaloids on phospholipase A2 and C activation in signal transduction system of rabbit platelet was studied. Isotetrandrine, cepharanthine and berbamine inhibited the aggregation induced by collagen but not by other stimuli such as thrombin and arachidonic acid, while tetrandrine equally inhibited the aggregation by any of these agonists. All these four alkaloids suppressed arachidonic acid liberation in response to collagen or thrombin, but not diacylglycerol formation and increase in cytoplasmic Ca2+ concentration in response to thrombin or arachidonic acid. In saponin-permeabilized platelets, they also suppressed arachidonic acid liberation induced by an addition of both GTP gamma S and Ca2+, whereas the liberation induced by an addition of Ca2+ alone was not prevented by them. These data suggest that isotetrandrine, cepharanthine and berbamine have a rather specific potency to suppress the phospholipase A2 activation by a mechanism other than direct inhibition of the enzyme or interference with the ligand-receptor interaction. They seem, at least in part, to exert the effect on the GTP-binding protein-phospholipase A2 complex in the platelet signal transduction system. In contrast, tetrandrine appears to inhibit a step following an increase in cytosolic free Ca2+ concentration in the agonist-induced signal transduction system, in addition to suppressing the phospholipase A2 activation.
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PMID:Suppressive effect of biscoclaurine alkaloids on agonist-induced activation of phospholipase A2 in rabbit platelets. 189 90


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