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)

Human platelets labelled with either [14C]arachidonic acid or [32P]orthophosphate were loaded or not with the Ca2+ fluorescent indicator quin 2. They were then incubated in the presence or in the absence of human thrombin (1 U/ml) in a medium where Ca2+ concentration was adjusted near zero or to 1 mM. Under these conditions, phospholipase A2 activity, as detected by the release of [14C]arachidonate and of its metabolites, or by the hydrolysis of [14C]phosphatidylcholine, was severely impaired in quin 2-loaded platelets upon removal of external Ca2+. However, Ca2+ was not required in non-loaded platelets, where a maximal phospholipase A2 activity was detected in the absence of external Ca2+. In contrast, phospholipase C action, as determined from the amounts of [14C]diacylglycerol, [14C]- or [32P]phosphatidic acid formed, appeared to be much less sensitive to the effects of quin 2 loading and of Ca2+ omission. By using various concentrations of quin 2, it was found that the inhibitory effect exerted against phospholipase A2 could be overcome by external Ca2+ only when the intracellular concentration of the calcium chelator did not exceed 2 mM. At higher concentrations averaging 3.5 mM of quin 2, phospholipase A2 activity was fully suppressed even in the presence of external Ca2+, whereas phospholipase C was still active, although partly inhibited. It is concluded that platelet phospholipase A2 requires higher Ca2+ concentrations than phospholipase C to display a maximal activity. By comparing platelet phospholipase A2 activity under various conditions with the values of cytoplasmic free Ca2+ as detected by quin 2 fluorescence, it is proposed that cytoplasmic free Ca2+ in control platelets stimulated with thrombin can attain concentrations above 1 microM, probably close to 5-10 microM, as recently determined with the photoprotein aequorin (Johnson, P.C., Ware, J.A., Cliveden, P.B., Smith, M., Dvorak, A.M. and Salzman, E.W. (1985) J. Biol. Chem. 260, 2069-2076).
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PMID:Selective inhibition of human platelet phospholipase A2 by buffering cytoplasmic calcium with the fluorescent indicator quin 2. Evidence for different calcium sensitivities of phospholipases A2 and C. 308 24

We tested the effects of calmodulin, two types of calmodulin antagonists, and various phospholipids on the phospholipase A2 activities of intact platelets, platelet membranes, and partially purified enzyme preparations. Trifluoperazine, chlorpromazine (phenothiazines) and N-(6-amino-hexyl)-5-chloro-1-naphthalenesulfonamide (W-7), at concentrations which antagonize the effects of calmodulin, significantly inhibited thrombin- and Ca2+ ionophore-induced production of arachidonic acid metabolites by suspensions of rabbit platelets and Ca2+-induced arachidonic acid release from phospholipids of membrane fractions, but not phospholipase A2 activity in purified enzyme preparations. The addition of acidic phospholipids, but not calmodulin, stimulated phospholipase A2 activity in purified enzyme preparations while decreasing its Km for Ca2+. The dose-response and kinetics of inhibition by calmodulin antagonists of acidic phospholipid-activated phospholipase A2 activity in purified preparations were similar to those of Ca2+-induced arachidonic acid release from membrane fractions. Calmodulin antagonists were also found to inhibit Ca2+ binding to acidic phospholipids in a similar dose-dependent manner. Our results suggest that the platelet phospholipase A2 is the key enzyme involved in arachidonic acid mobilization in platelets and is regulated by acidic phospholipids in a Ca2+-dependent manner and that calmodulin antagonists inhibit phospholipase A2 activity via an action on acidic phospholipids.
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PMID:Calmodulin-independent inhibition of platelet phospholipase A2 by calmodulin antagonists. 308 92

Diverse particulate and soluble stimuli trigger two metabolic bursts in mouse peritoneal macrophages important in the inflammatory and/or cytotoxic actions of the cells: release, oxygenation, and further metabolism of arachidonic acid from endogenous phospholipids and reduction of molecular oxygen to reactive intermediates. We tested the hypothesis that the release of arachidonic acid or formation of its metabolites are obligatory intermediate steps in triggering the NADPH oxidase that reduces O2 to O-2. With phorbol diesters as stimuli, the following inhibitors of phospholipase A2 and lipoxygenase suppressed release of H2O2 at nontoxic concentrations (microM range): p-bromophenacyl bromide, quinacrine, eicosatetraenoic acid, nordihydroguaiaretic acid, and phenidone. Indomethacin and acetylsalicylic acid were ineffective. However, the suppressive effect of the first five agents on H2O2 release could be attributed to their suppression of macrophage glucose uptake at the same concentrations, a previously unrecognized effect of these compounds. Further, concanavalin A, wheat germ agglutinin, and thrombin each stimulated abundant arachidonate release without H2O2 release. Finally, noncytolytic concentrations of cycloheximide and/or emetine suppressed arachidonate release without affecting H2O2 secretion triggered either by phorbol esters or zymosan. Release and metabolism of arachidonic acid and secretion of reactive oxygen intermediates appear to be two frequently coincident but mutually independent metabolic pathways in the mouse peritoneal macrophage.
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PMID:Release of arachidonate and reduction of oxygen. Independent metabolic bursts of the mouse peritoneal macrophage. 309 92

Phospholipase C (PLC) and diacylglycerol lipase (DGL) activities were found in guinea pig platelet microsome preparations. No phospholipase A2 (PLA2) activity was detected. RHC 80267 (1,6-di (0-(carbamoyl) cyclohexanone oxime)hexane) inhibited DGL activity (IC50 = 4 uM) from guinea pig platelet microsomes but had no effect on PLC. RHC 80267 inhibited platelet aggregation (IC50 = 11 uM), release of arachidonic acid (AA), its metabolites, and ATP (IC50 = 4.5 uM) when guinea pig platelets were challenged with a low concentration of thrombin. We propose that PLC-DGL is an important enzymatic pathway for the release of AA in guinea pig platelets.
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PMID:Inhibition of the effects of thrombin on guinea pig platelets by the diacylglycerol lipase inhibitor RHC 80267. 309 68

Cobra venom phospholipase A2 induced a biphasic effect on washed rabbit platelets. The first phase was a reversible aggregation which was dependent on stirring and extracellular calcium. The aggregation and thromboxane B2 formation were inhibited by indomethacin, mepacrine, tetracaine and imipramine, while PGE1 and sodium nitroprusside inhibited only the aggregation, but not the thromboxane B2 formation. The second phase was an inhibitory effect on platelet aggregation induced by arachidonic acid, PAF, ADP or collagen but not that by thrombin or ionophore A23187. The longer the incubation time of cobra venom phospholipase A2 with platelets, the more the inhibitory effect. The aggregating and anti-aggregating effects could be overcome by bovine serum albumin. Lysophosphatidylcholine (Lyso-PC) and arachidonic acid showed synergistic inhibition in platelet aggregation. Lyso-PC decreased thromboxane B2 formation in platelets formed by collagen. The inhibitory effect of Lyso-PC on platelet aggregation was more marked at lower calcium concentrations. It is concluded that the aggregating effect of exogenous addition of venom phospholipase A2 is due to thromboxane formation and the antiplatelet effect is similar to those produced by arachidonic acid and lysophosphatidylcholine.
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PMID:Effect of cobra venom phospholipase A2 on platelet aggregation in comparison with those produced by arachidonic acid and lysophophatidylcholine. 309 24

The serine protease inhibitors diethyl p-nitrophenyl phosphate and phenylmethylsulfonyl fluoride (chemical modifiers of serine residue) and N-acetyl-l-tryptophan ethyl ester (competitive inhibitor of chymotryptic protease) inhibited 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (AGEPC; platelet-activating factor)-induced platelet aggregation and secretion. The inhibition was dependent on the preincubation time with the serine protease inhibitor and on the concentration of AGEPC and inhibitor. The IC50 value of diethyl-p-nitrophenyl phosphate, phenylmethylsulfonyl fluoride, and N-acetyl-l-tryptophan ethyl ester towards 5 X 10(-10) M AGEPC in serotonin release was 2.2 X 10(-4), 8.0 X 10(-4), and 5.0 X 10(-4) M, respectively. In experiments where platelets were incubated with these inhibitors and then washed with buffer, the inhibition of AGEPC stimulation was not observed. Prostaglandin H2 analog U46619 (10(-6) to 10(-5) M)- and thrombin (0.1 unit/ml)-induced platelet activation were also blocked by 1 mM diethyl p-nitrophenyl phosphate and 1 mM N-acetyl-l-tryptophan ethyl ester. The binding of AGEPC (1.5 X 10(-11) to 9.4 X 10(-10) M) to platelets and the platelet cyclic AMP level were not affected by diethyl p-nitrophenyl phosphate, phenylmethylsulfonyl fluoride, and N-acetyl-l-tryptophan ethyl ester. However, 1 mM diethyl p-nitrophenyl phosphate and 1 mM N-acetyl-l-tryptophan ethyl ester suppressed 10(-9) M AGEPC-induced breakdown of phosphatidylinositol 4,5-bisphosphate and formation of phosphatidic acid to 10-12 and 39-42%, 40-kDa protein phosphorylation to 4 and 30%, and arachidonic acid release to 17 and 28% of controls, respectively. On the other hand, 5 mM diethyl p-nitrophenyl phosphate did not inhibit diacylglycerol production and arachidonic acid release initiated by 2.5 mM deoxycholate treatment, suggesting that receptor-mediated phospholipase C and phospholipase A2 activation were inhibited by the serine protease inhibitor, but the deoxycholate (physicochemical stimulant)-initiated activation was not. AGEPC-induced 20-kDa protein phosphorylation and the inhibitory action of AGEPC on cyclic AMP accumulation were abolished in the presence of diethyl p-nitrophenyl phosphate and phenylmethylsulfonyl fluoride. However, a tryptic protease inhibitor, 1 mM p-aminobenzamidine and 1 mM benzoyl-l-arginine methyl ester, did not prevent the AGEPC-induced platelet secretion.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Platelet-activating factor stimulation of rabbit platelets is blocked by serine protease inhibitor (chymotryptic protease inhibitor). 310 43

Cultured human umbilical vein endothelial cells (HUVEC) stimulated with thrombin are known to synthesize prostacyclin at least in part from arachidonate released by phospholipase A2, an enzyme directly activated by calcium. In this study, thrombin stimulation of Quin 2-loaded HUVEC caused rapid and dose-dependent rises in inositol trisphosphate (IP3) and cytosolic free calcium (Ca2+i) levels which preceded a similarly dose-dependent rise in prostacyclin production measured as 6-ketoprostaglandin F1 alpha (6-keto-PGF1 alpha) by radioimmunoassay (ED50 = 0.6-0.7 units/ml for all three effects). Thrombin induced these effects in the absence of extracellular calcium (EGTA) or in the presence of either 8-bromo-cAMP or the calmodulin inhibitor W7. Thrombin inactivated with either diisopropyl fluorophosphate or D-Phe-Pro-Arg-chloromethyl ketone was inactive. In contrast, Quin 2-loaded cultured bovine aortic endothelial cells failed to respond to thrombin, although stimulation with trypsin elevated IP3 and Ca2+i levels and increased 6-keto-PGF1 alpha production. Restimulation of HUVEC with thrombin or histamine 5 min after an initial stimulation with thrombin (2 units/ml for 5 min) failed to induce a second rise in either IP3 or Ca2+i levels or further production of 6-keto-PGF1 alpha, whereas restimulation with ionomycin in the presence or absence of extracellular calcium elevated Ca2+i levels and induced further 6-keto-PGF1 alpha production. However, if the initial stimulation with thrombin was terminated by addition of D-Phe-Pro-Arg-chloromethyl ketone within 10-60 s, restimulation with a second dose of thrombin induced second rises in both IP3 and Ca2+i levels and additional 6-keto-PGF1 alpha production that were greatest when the initial thrombin stimulus was briefest. These results are consistent with the conclusion that IP3 acts as a second messenger by which thrombin elevates Ca2+i levels and initiates prostacyclin synthesis in HUVEC and that in vivo endothelial cells may be stimulated multiple times to synthesize prostacyclin if each period of stimulation is brief.
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PMID:Correlation between thrombin-induced prostacyclin production and inositol trisphosphate and cytosolic free calcium levels in cultured human endothelial cells. 311 Jan 48

The effect of ethanol (10-500 mmol/l) on platelet thromboxane production and on vascular thromboxane and prostacyclin was studied in human fetal tissues. The release of thromboxane B2 (a metabolite of thromboxane A2) during thrombin-induced spontaneous aggregation of fetal platelets was inhibited by ethanol concentrations of 50 mmol/l or higher. Ethanol at concentration from 100 mmol/l also inhibited umbilical artery production of thromboxane B2 and that of 6-keto-prostaglandin F1 alpha (a metabolite of prostacyclin). However, it stimulated the conversion of exogenous arachidonic acid to thromboxane B2 in fetal platelets and to 6-keto-prostaglandin F1 alpha in the umbilical artery. This suggests that ethanol inhibits phospholipase A2, but stimulates the enzymes distal from phospholipase A2 in the prostaglandin-synthesizing enzyme cascade.
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PMID:Effect of ethanol on thromboxane and prostacyclin synthesis by fetal platelets and umbilical artery. 311 May 28

The release of arachidonic acid from thrombin-stimulated platelets can be attributed to the action of phospholipase A2 on membrane phospholipid. Previously, analysis of individual subclasses of phospholipid demonstrated that 1-acyl-2-[3H]arachidonoyl-sn-glycerophosphocholine and to a lesser degree 1-acyl-2-[3H]arachidonoyl-sn-glycerophosphoethanolamine were the main source of [3H]arachidonic acid in thrombin-stimulated cells. In the present work, 1,2-diacyl phospholipid subclasses were analyzed as 1,2-diacylglycerobenzoates by high-pressure liquid chromatography in order to analyze arachidonate release as mass changes in individual molecular species of phospholipid. Following thrombin stimulation (5 U/ml, 5 min, 37 degrees C) all arachidonoyl-containing molecular species of 1,2-diacyl-sn-glycerophosphocholine decreased in mass and [3H]arachidonate content by almost 50%, while those of 1,2-diacyl-sn-glycerophosphoethanolamine decreased by 20%. The mass change was substantial and indicated that these phospholipids are a major source of arachidonate in stimulated cells. No variation was seen in the other non-arachidonate-containing molecular species of either subclass. Thus, deacylation of membrane 1,2-diacylglycerophosphocholine and 1,2-diacylglycerophosphoethanolamine by phospholipase A2 is selective for those molecular species of phospholipid containing arachidonic acid, suggesting that a certain proportion of arachidonoyl-containing molecular species of phospholipid are compartmentalized with the platelet membrane proximal to the site of action of this enzyme. These studies demonstrate that the human platelet is a cell poised and specialized to release rapidly substantial amounts of arachidonic acid upon stimulation.
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PMID:Evidence for the release of arachidonic acid through the selective action of phospholipase A2 in thrombin-stimulated human platelets. 311 41

GTP or GTP gamma S alone caused low but significant liberation of arachidonic acid in saponin-permeabilized human platelets but not in intact platelets. GTP or GTP gamma S also enhanced thrombin-induced [3H]arachidonic acid release in permeabilized platelets. Inhibitors of the phospholipase C (neomycin)/diacylglycerol lipase (RHC 80267) pathway for arachidonate liberation did not reduce the [3H]arachidonic acid release. The loss of [3H]arachidonate radioactivity from phosphatidylcholine was almost equivalent to the increase in released [3H]arachidonic acid, suggesting the hydrolysis of phosphatidylcholine by phospholipase A2. The effect of GTP gamma S was greater at lower Ca2+ concentrations. These data indicate that the release of arachidonic acid by phospholipase A2 in saponin-treated platelets may be linked to a GTP-binding protein.
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PMID:Guanine nucleotides stimulate arachidonic acid release by phospholipase A2 in saponin-permeabilized human platelets. 311 31


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