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)

Treatment of platelets (10(9) cells/ml) with thrombin (1 U/ml) resulted in rapid disappearance of fibrinogen from the system as measured by the tanned red cell hemagglutination inhibition immunoassay (TRCHII). Plasmin digestion of individual pellet and supernatant fractions that had been previously separated from thrombin-treated platelet suspensions by centrifugation resulted in recovery of TRCHII-detectable material in platelet pellets. To elucidate the specific association of fibrin to platelet membranes, control and thrombin-treated platelets were homogenized by a modified glycerol-loading and nitrogen decompression technique. Ultracentrifugation of homogenates through 27% sucrose cushions yielded three subcellular fractions: supernatant, small membrane vesicles, and a particulate fraction for controls; and supernatant membrane vesicles, and aggregated membrane "ghosts" for thrombin preparations. Ultrastructurally identifiable fibrin was noted only in the thrombin fraction containing membrane ghosts. Fibrinogen recovered from 3 thrombin fractions was markedly decreased (3% of the control). Plasmin digestion produced 23% and 46-fold increase in TRCHII-detectable material from 3 subcellular fractions of control and thrombin preparations, respectively. More than 97% of TRCHII material recovered from thrombin preparations was in the fraction containing aggregated membrane fractions. Results suggest that platelet plasma membranes function as surfaces for fibrin deposition.
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PMID:Localizaton of fibrin converted by thrombin from released platelet fibrinogen. 644 58

An increase in cytoplasmic free calcium, [Ca2+]i, is thought to be the trigger for secretory exocytosis in many cells. In blood platelets, large rises in [Ca2+]i can cause secretion and calcium has been regarded as the final common activator not only for secretion but also for shape-change and aggregation. We have shown that while thrombin and platelet-activating factor (PAF) normally elevate [Ca2+]i, they can also stimulate shape-change and secretion even when the [Ca2+]i rise is suppressed. The present results strongly implicate diacylglycerol, produced by stimulus-dependent breakdown of phosphoinositide, in this calcium-independent activation. Exogenous diacylglycerol activates a protein kinase (C-kinase) in platelets as do PAF, thrombin and collagen. 12-O-tetradecanoyl phorbol-13-acetate (TPA) also activates C-kinase and is a potent stimulus for secretion and aggregation. It is shown here that the exogenous diacylglycerol 1-oleoyl-2-acetyl-glycerol (OAG) and TPA evoke similar secretion and aggregation without elevating [Ca2+]i above the basal level of 0.1 microM. The pattern of secretion resembles that produced by collagen and thrombin when [Ca2+]i remains at basal levels. Modest increases in [Ca2+]i, insufficient to stimulate secretion, markedly accelerate the responses to TPA and OAG.
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PMID:Diacylglycerol and phorbol ester stimulate secretion without raising cytoplasmic free calcium in human platelets. 662 85

The relative degradation of the various molecular species of [3H]phosphatidylcholine in response to thrombin was studied in human platelets following prelabeling with [3H]glycerol and compared to results obtained following labeling with [14C]oleic, [14C]linoleic, or [14C]arachidonic acids. This was of interest since previous work using radioactive fatty acids had led to the conclusion that the 1-acyl-2-arachidonoyl species of phosphatidylcholine is exclusively hydrolyzed in thrombin-stimulated platelets. Within 90 s, the thrombin-dependent release of [14C]arachidonic acid from phosphatidylcholine amounted to 25% but only 3 and 6% for oleic and linoleic acids, respectively, in general agreement with previous work. However, for [3H]glycerol-labeled phosphatidylcholine, all molecular species (saturates, monoenes, dienes, trienes, tetraenes, and greater than tetraenes) were subject to significant hydrolysis in the presence of thrombin within 90 s, ranging from 12-24% across the various classes. Furthermore, the degradation of the tetraenoic species (1-acyl-2-arachidonoyl) of [3H]phosphatidylcholine was found to be only 1.5 and 1.4 times that for the monoenoic (predominantly 1-acyl-2-oleoyl) and dienoic (predominantly 1-acyl-2-linoleoyl) species, respectively. A much heavier proportional labeling of plasma membrane relative to whole platelet phosphatidylcholine was observed with [3H]glycerol as compared to [14C] oleate or [14C]arachidonate. These results indicate that the 1-acyl-2-arachidonoyl species of phosphatidylcholine are not exclusively degraded by phospholipase A2 activity in thrombin-stimulated platelets and suggest that the differential compartmentation of molecular species of phosphatidylcholine according to their metabolic origins can influence their apparent susceptibility to hydrolysis.
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PMID:Relative degradation of different molecular species of phosphatidylcholine in thrombin-stimulated human platelets. 674 52

Human platelets prelabeled with [3H]glycerol exhibited a trasient increase in radioactivity (1.5-fold gain) in 1,2-diacylglycerol when they were exposed to thrombin. An alteration in radioactivity in monoacylglycerol which is derived from diacylglycerol by diacylglycerol lipase, however, was not observed during the whole period of incubation with thrombin. Lysophosphatidylcholine and lysophosphatidylethanolamine gained radioactivity. By contrast, the level of lysophosphatidylinositol plus lysophosphatidylserine did not show any change. When the effects of thrombin on platelet lipids were examined for [3H]arachidonate-labeled platelets, thrombin-activation induced a 15-fold increase in radioactivity in 1,2-diacylglycerol, a subsequent decrease of which was accompanied by accumulation of radioactivity in phosphatidic acid. There was a concurrent release of free arachidonic acid. These findings, taken together with phospholipid alteration analyzed by phosphorus assay upon thrombin-activation, indicate evidence than newly produced diacyglycerol in thrombin-activated platelets may be immediately converted to phophatidic acid by a diacylglycerol kinase rather than metabolized to monoacylglycerol or arachidonic acid by diacylglycerol lipase, and also that arachidonic acid would be mainly released from phosphatidylcholine and phosphatidylethanolamine by a phospholipase A2 activity.
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PMID:Evidence for predominance of phospholipase A2 in release of arachidonic acid in thrombin-activated platelets: phosphatidylinositol-specific phospholipase C may play a minor role in arachidonate liberation. 681 81

The relative degradation of the various molecular species of phosphatidylinositol in response to thrombin was studied in human platelets. For this purpose, platelets were prelabeled with [2-3H]glycerol and the loss of radioactivity from the saturated, monoenoic, dienoic, trienoic, tetraenoic, and greater than tetraenoic [3H]phosphatidylinositols was determined after conversion to their 1,2-diacylglycerol acetate derivatives and fractionation by argentation thin layer chromatography. Within 90 s, when the thrombin-dependent degradation of total [3H] phosphatidylinositol amounted to 49.5%, the percentage loss of radioactivity from the tetraenoic (1-stearoyl 2-arachidonoyl) species and greater than tetraenes was significantly greater than that for the other molecular classes and approximately twice that for the monoenes. Furthermore, the extent of degradation tended to decrease with decreasing unsaturation of the phosphatidylinositols in intact platelets. These results indicate that the thrombin-dependent hydrolysis of phosphatidylinositol in intact human platelets exhibits a preferential degradation of 1-acyl (predominantly stearoyl plus oleoyl) 2-arachidonoyl species relative to other molecular species which may possibly be of importance in platelet aggregation.
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PMID:Degradation of different molecular species of phosphatidylinositol in thrombin-stimulated human platelets. 685 17

Microsomes isolated from human platelets synthesize phosphatidylinositol by the action of acyl-CoA: 1-acyl-sn-glycerol-3-phosphorylinositol(1-acyl-GPI) acyltransferase. The properties of 1-acyl-GPI acyltransferase were compared with those of 1-acyl-glycerophosphorylcholine (1-acyl-GPC) acyltransferase. Apparent Km values of 1-acyl-GPI and 1-acyl-GPC acyltransferases for the corresponding acyl acceptor (lysophospholipid) were 22 and 20 microM, respectively, in the presence of arachidonoyl-CoA as fatty acyl donor. However, the Km value (1.3 microM) of 1-acyl-GPI acyltransferase for arachidonoyl-CoA was much lower than that (5.0 microM) of 1-acyl-GPC acyltransferase. Under optimal conditions, the acylation rate of 1-acyl-GPI with arachidonoyl-CoA was 2-6 times higher than with oleoyl-CoA and linoleoyl-CoA, and was very low with saturated fatty acyl-CoAs. The acylation rates with various acyl-CoAs for 1-acyl-GPI were different from those for 1-acyl-GPC. These results suggest that the reacylation pathway of 1-acyl-GPI participates in the incorporation of arachidonic acid to phosphatidylinositol in platelet microsomes. Furthermore, there were no significant effects of thrombin-activation on acyl-CoA specificity for 1-acyl-GPI and 1-acyl-GPC acyltransferase in human platelets.
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PMID:Possible involvement of 1-acyl-glycerophosphorylinositol acyltransferase in arachidonate enrichment of phosphatidylinositol in human platelets. 686 Jul

Purified human and bovine thrombin produced comparable tonic contractions in isolated canine basilar arteries. The magnitude of the contractions was closely related to the number of thrombin Units studied rather than to the amount of protein added to the isolation bath. Thrombin had a much slower onset of action, but was more potent in generating sustained contractions than either serotonin or prostaglandin F2 alpha. Moreover, in contrast to serotonin and prostaglandin F2 alpha, the contractions caused by thrombin were not terminated by equivalent washing. The thrombin-induced contractions were significantly inhibited by prostacyclin, meclofenamic acid, phenoxybenzamine and glycerol. Prostacyclin was the most potent of these inhibitors. The results suggest that thrombin in a "free" form may cause vasoconstriction, in addition to platelet aggregation, in hemostasis and could contribute to the genesis of cerebral vasospasm associated with subarachnoid hemorrhage.
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PMID:Cerebral arterial contractions induced by human and bovine thrombin. 699 61

Two to five units of platelet concentrate were frozen together in single bags using glycerol-glucose as cryoprotective agents and a special freezing plate which produced a reproducible, rapid freezing rate when immersed in liquid nitrogen. After thawing there was no loss in platelets compared to prefreezing controls. Phase microscopic evaluation after thawing, however, demonstrated large numbers of severely damaged platelets and a significant decrease in morphology score. Total ATP levels fell to 37-52% of controls, and thawed platelets did not aggregate with adenosine diphosphate or collagen or undergo release of nucleotides following incubation with thrombin. In vivo recovery one hour after transfusion of autologous platelets administered to patients with leukemia was significantly inferior (p less than 0.025) to results achieved in the same patients with autologous platelets frozen with dimethylsulfoxide. These results indicate that significant damage occurs following freezing and thawing utilizing glycerol-glucose as cryoprotective agents for frozen platelets and that further investigation is required prior to their clinical use. Blood banks currently interested in platelet cryopreservation for clinical use should utilize dimethylsulfoxide as a cryoprotective agent.
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PMID:Cryopreservation of platelet concentrates using glycerol-glucose. 707 14

The conformations of human alpha-thrombin and gamma-thrombin have been compared by circular dichroism, solvent perturbation different spectroscopy, and chemical modification. Circular dichroism studies indicate that proteolytic conversion of alpha-thrombin to gamma-thrombin is accompanied by considerable conformational changes which include a decrease in alpha-helical content from 5-7% to 0-1%. Solvent perturbation at pH 6.0 obtained with 20% ethylene glycol, 20% glycerol, and 20% dimethyl sulfoxide indicates an apparent exposure of 3.5 +2- 0.2 tryptophan and 7.8 +/- 0.1 tyrosine residues in alpha-thrombin and 4.6 +/- 0.2 tryptophan and 9.2 +/0 0.3 tyrosine residues in gamma-thrombin. This increased exposure is substantiated by the greater reactivity of tryptophan residues in gamma-thrombin toward dimethyl (2-hydroxy-5-nitrobenzyl) sulfonium bromide. It suggests that gamma-thrombin is a less compact molecule than the parent alpha-thrombin. Solvent perturbation studies of alpha-thrombin and gamma=thrombin inhibited by phenyl-methanesulfonyl fluoride showed that 0.3 +/- tryptophan and 0.9 +/- 0.3 tyrosine residues in alpha-thrombin and 0.6 +/- 0.3 tryptophan and 1.3 +/- 0.4 tyrosine residues in gamma-thrombin were blocked by the inhibitor. These subtle differences in the extent of blocking of tyrosine and tryptophan suggest a tighter conformation in the catalytic site of gamma-thrombin compared to that of alpha-thrombin.
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PMID:Conformational differences between high clotting human alpha-thrombin and nonclotting gamma-thrombin. 730 21

Poliovirus protein 3B (also known as VPg) is covalently linked to the 5' ends of both genomic and antigenomic viral RNA. Genetic and biochemical studies have implicated protein 3AB, the membrane-bound precursor to VPg, in the initiation of genomic RNA synthesis. We have purified 3AB to near homogeneity following thrombin cleavage of purified glutathione S-transferase-3AB. When added to transcription reaction mixtures catalyzed by poliovirus RNA polymerase (3Dpol), 3AB stimulated RNA synthesis up to 75-fold with oligo(U)-primed virion RNA, globin mRNA, and unprimed synthetic, full-length minus-strand viral RNA as the templates. Synthetic VPg also stimulated RNA synthesis but was only 1 to 2% as effective as 3AB on a molar basis. The increased level of transcription was not the result of enhancing the elongation rate of the polymerase. No evidence was found for uridylylation of 3AB or for covalent linkage to RNA transcription products. 3AB sedimented as a multimer in glycerol gradients. In the presence of the polymerase, the sedimentation rate of both proteins increased, suggesting the formation of a complex. Detergent prevented both multimerization and complex formation. The polymerase also bound to immobilized glutathione S-transferase-3AB; this procedure was used to purify the polymerase to near homogeneity. These results suggest a mechanism for bringing together 3AB, 3Dpol (or its precursor 3CD), and viral RNA in host cell membranous vesicles in which all viral RNA synthesis occurs.
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PMID:Poliovirus protein 3AB forms a complex with and stimulates the activity of the viral RNA polymerase, 3Dpol. 747 38


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