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 effect of gamma-irradiation on purified prothrombin and thrombin in aqueous solution has been assessed with reference to bifunctional activities, e.g., clotting and esterase functions, physico-chemical changes in structure, and kinetics. The inactivation curves indicated that the clotting activity was more susceptible to gamma-radiation than the esterolytic function in both the proteins. Prothrombin was comparatively more sensitive to radiation than thrombin. The irradiation of prothrombin (100 kR) caused modifications in the protein resulting in reduced formation of thrombin after activation by Factor Xa. The modifications caused by irradiation were assessed in these proteins by changes in spectral characteristics, levels of tryptophan and disulphides, electrophoretic mobility and amino acid composition. Radiation-induced changes in thrombin were reflected in its kinetic behaviour. The clotting activity of thrombin was almost completely lost at 100 kR, while esterolysis was relatively less affected. The modification of tyrosine and tryptophan residues in thrombin influenced the clotting activity, while these were not involved for esterolysis. Histidine had involvement in both these activities.
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PMID:Radiation-induced changes in purified prothrombin and thrombin. 712 90

Some characteristics of the blood coagulation system were analyzed after injection to rats of hirudin-thrombin and phenylmethylsulfonyl-thrombin devoid of clotting and esterase activities because of the blocking of the substrate binding or catalytic site of the active center. Unlike native thrombin, both forms of modified thrombin did not activate the anticoagulation system.
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PMID:[Effect of hirudin-thrombin and phenylmethylsulfonyl-thrombin on various blood clotting indices]. 724 3

A procedure is described for isolation of prethrombin I using biospecific chromatography of the thrombin hydrolysate of prothrombin complex on heparin-Sepharose. The prothrombin complex was activated in 0.05 M Tris-HCl buffer, pH 7.4, containing 0.15 M NaCl, ImM EDTA-Na salt at 37 degrees within 15-20 min; the ratio of the enzyme and substrate was equal to 1 : 1300 (evaluated by the enzymatic activity). Then prethrombin I was isolated by the affinity chromatography. Prethrombin I was specially adsorbed on the immobilized heparin and eluted with 0.45 M NaCl. The substance was free from factor X and alpha-thrombin, which were eluted with 0.64 M and 0.8-1.0 M NaCl, respectively. Fragment I of prothrombin was not adsorbed on heparin-Sepharose and was found in the free volume of column. Preparation of prethrombin I (molecular mass 67,000 +/- 3,000) did not exhibit the coagulating, esterase and prothrombin activities but produced thrombin in presence of factor Xa. The abilities of prethrombin I to interact with blood vessel chemoreceptors and to activate the anticoagulation system were shown, when physiological activity of prethrombin I was studied using perfusion of rabbit carotid sinus, which was insulated from humoral action but retained the nervous connections with a body.
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PMID:[Use of affinity chromatography for isolating prethrombin 1]. 733 61

Indole was shown to stimulate TAME hydrolysis by alpha-thrombin with KA=7,7 mM, alpha=0,55, beta=1,5 and that by beta/gamma-thrombin with KA=9,5 mM, alpha=0,47 and beta=2,08. Indole promotes both the formation and transformation of the enzyme-substrate complex. No effect of substrate activation was observed in the presence of indole, which suggests the identity of the binding sites of indole and the added TAME molecule. Heparin was shown to form an equimolar stoicheometric complex with both alpha- and beta/gamma-thrombin, which results in 40% inhibition of the TAME-esterase, clotting and prothrombinolytic activities of alpha-thrombin and 25% and 40% inhibition of the TAME-esterase and prothrombinolytic activity of beta/gamma-thrombin, respectively. The fact that alpha- and beta/gamma-thrombin partially retain their catalytic properties even at a 5-fold molar excess of the inhibitor indicates that heparin binds to the thrombin molecule at a site other than the active center. Heparin does not prevent the effect of substrate activation at high TAME concentrations. This finding suggests that the localization of binding sites of heparin and the added TAME molecule (and, therefore, indole) in the thrombin molecule is different.
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PMID:[Regulation of alpha and beta/gamma-thrombin activity by heparin and indole]. 737 98

Human gamma-thrombin is a three (noncovalently linked)-domain enzyme which contains the known serine protease catalytic triad, Asp-His-Ser, one on each of the three noncovalent domains (Asp 99 on the A-B3 chain). While protein-folding dogma does not necessarily predict that the denatured form of this enzyme could refold to the correct conformation, a monitor of the esterase activity (Tos-Arg-OMe) shows complete recovery of native catalytically active conformation. When compared with the covalently intact alpha form which refolds from urea in less than 2 min with complete return of both clotting and esterase activity, gamma-thrombin requires up to 90 min to regain full esterase activity. The gamma-thrombin reactivation data best fit a single first order rate constant, k = 0.03 +/- 0.005 min-1. It was suggested that the gamma-thrombin renaturation process might represent first the rapid refolding, then subsequent reassociation and reisomerization of the three noncovalent domains to yield a lower energy, fully active, conformation. This study represents the only example known of the refolding (reconstitution) of a three (noncovalent)-domain protein.
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PMID:Refolding of a three (noncovalently linked)-domain enzyme. Human gamma-thrombin. 738 Aug 43

beta/gamma-thrombin containing 25% of beta-form and 75% of gamma-form was obtained from alpha-thrombin by limited trypsin proteolysis. As compared to the parent alpha-thrombin, these preparations preserved less than 0.02% of fibrinogen-clotting activity, the entire esterase activity and 27% of the amidolytic activity toward N-benzoyl-Phe-Val-Arg-p-nitroanilide. Like alpha-thrombin, beta/gamma-thrombin splits prothrombin to prethrombin I (Mr 58000) and fragment I of prothrombin (Mr 25000) possessing antithrombin activity. Kinetics of prothrombin hydrolysis was studied by different concentrations of enzymes. It was shown that alpha-form is more specific than beta/gamma-form that preserved approximately 5% of prothrombinolytic activity of alpha-thrombin. It is emphasized that beta/gamma-form devoid of clotting activity retains regulatory functions.
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PMID:[Proteolysis of prothrombin by beta/gamma thrombin]. 738 59

Serine esterase inhibitors (phenylmethanesulfonyl fluoride, 5-dimethylaminonaphthalene-1-sulfonyl (dansyl) fluoride, 2-nitro-4-carboxyphenyl-N,N-diphenylcarbamate, or p-nitrophenyl anthranilate) blocked the production of malonyldialdehyde by platelets induced with a variety of stimuli (including thrombin, trypsin, collagen, and A23187). These inhibitors did not block malonyldialdehyde production by platelets from exogenous arachidonic acid. Those inhibitors studied in greater detail (phenylmethanesulfonyl fluoride and 2-nitro-4-carboxyphenyl-N,N-diphenylcarbamate) were shown to inhibit the release of [1-14C]arachidonic acid from phosphatidylinositol and phosphatidylcholine in intact platelets but not the conversion of arachidonic acid to thromboxanes, prostaglandins, or hydroxyfatty acids. These inhibitors also blocked the stimulus-induced production of [32P]phosphatidic acid in intact platelets. Both arachidonic acid release from phosphatidylinositol and phosphatidic acid production have been reported to depend on the production of diglyceride by the action on the phosphatidylinositol-specific phospholipase C.f a phosphatidylinositol-specific phospholipase C. That enzyme in the soluble fraction from disrupted platelets was inhibited at concentrations of serine esterase inhibitors which block arachidonic acid release in intact platelets. These results indicate that serine esterase inhibitors block the stimulus-induced mobilization of arachidonic acid in platelets at least in part by their action o
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PMID:Serine esterase inhibitors block stimulus-induced mobilization of arachidonic acid and phosphatidylinositide-specific phospholipase C activity in platelets. 739 Oct 2

A comparative study of leukocyte adhesion to the endothelium of the thoracic aorta and left carotid artery in rats has been performed after administration of two hyperlipidemic diets for 15 days, proinflammatory agents (thrombin, lipopolysaccharide and zymosan activated serum) and plasma expanders [dextran, polyvinylpyrrolidone (PVP), rat albumin and several bovine albumins from different sources]. Leukocytes adhered to the endothelium were demonstrated in surface preparations by esterase activity. Activation of circulating leukocytes was measured by nitroblue tetrazolium reduction and luminol enhanced chemiluminescence. Both hyperlipidemic diets produced, in all rats, more leukocyte adhesion in the aorta than in the carotid artery. All proinflammatory agents produced at 1 h, increases in leukocyte adhesion--which in all rats were greater in the carotid artery than in the aorta--and leukocyte activation, which was higher at 3 h than at 1 h. Dextran, PVP, bovine albumins 103700 and A-4503 at 18 h produced slight increases in leukocyte adhesion in the aorta but not in the carotid artery. Rat albumin and bovine albumin A-7906 determined an intense leukocyte adhesion at 18 h which was not preferential to either vessel. Adhesion produced by A-7906 was maximal at 12 h and partially inhibited by dexamethasone. This last albumin produced leukocyte activation at 3 h and was sequestered 5 min after administration, reaching normal values at 1 h. Albumins 103700 and A-4503 neither activated leukocytes nor were sequestered after administration.
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PMID:Effect of hyperlipidemic diets, proinflammatory agents and plasma expanders on leukocyte adhesion to the endothelium of aorta and carotid artery of rats. 753 42

A potent platelet aggregation inducer, aggretin, was purified from Malayan-pit-viper (Calloselasma rhodostoma) venom by ionic-exchange chromatography, gel-filtration chromatography and HPLC. It is a heterodimeric protein (29 kDa) devoid of esterase, phospholipase A and thrombin-like activity. Aggretin (> 5 nM) elicited platelet aggregation with a lag period in both human platelet-rich plasma and washed platelet suspension. EDTA (5 mM), prostaglandin E1 (1 microM) and 3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester ('TMB-8'; 100 microM) abolished its aggregating activity, indicating that exogenous bivalent cations and intracellular Ca2+ mobilization are essential for aggretin-induced platelet aggregation. Neomycin (4 mM) and mepacrine (50 microM) completely inhibited aggretin (33 nM)-induced aggregation; however, creatine phosphate/creatine phosphokinase (5 mM, 5 units/ml) and indomethacin (50 microM) did not significantly affect its aggregating activity. Aggretin caused a significant increase of [3H]InsP formation in [3H]Ins-loaded platelets, intracellular Ca2+ mobilization and thromboxane B2 formation. Neomycin, a phospholipase C inhibitor, completely inhibited both the increase of [3H]InsP and intracellular Ca2+ mobilization of platelets stimulated by aggretin. A monoclonal antibody (6F1) directed against glycoprotein Ia/IIa inhibited platelet shape change and aggregation induced by aggretin. 125I-aggretin bound to platelets with a high affinity (Kd = 4.0 +/- 1.1 nM), and the number of binding sites was estimated to be 2119 +/- 203 per platelet. It is concluded that aggretin may act as a glycoprotein Ia/IIa agonist to elicit platelet aggregation through the activation of endogenous phospholipase C, leading to hydrolysis of phosphoinositides and subsequent intracellular Ca2+ mobilization.
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PMID:Aggretin, a novel platelet-aggregation inducer from snake (Calloselasma rhodostoma) venom, activates phospholipase C by acting as a glycoprotein Ia/IIa agonist. 763 79

A coagulant enzyme, okinaxobin I, which was purified from Trimeresurus okinavenis (himehabu snake) venom, released specifically fibrinopeptide B from fibrinogen to form fibrin clots. In the present study, its isozyme denoted as okinaxobin II has been purified to homogeneity from the same venom by chromatographies on Sephadex G-100, CM-Toyopearl 650M, and FPLC Mono-Q columns. Differently from okinaxobin I, okinaxobin II specifically cleaved fibrinopeptides A and B from fibrinogen similarly as found for alpha-thrombin. The enzyme acted on fibrinogen with specific activity of 42 NIH units/mg at optimum pH of 8.0. Okinaxobin II was a monomeric glycoprotein with a mol. wt of 37,500 on SDS-PAGE, which was reduced to 29,500 after treatment with N-glycanase. Okinaxobin II was much more basic (pI = 8.1) than okinaxobin I (pI = 5.4). The N-terminal sequence was highly similar to those of okinaxobin I and some other snake venom coagulant enzymes such as flavoxobin (Trimeresurus flavoviridis), batroxobin (Bothrops atrox and Bothrops moojeni), and catroxobin (Crotalus atrox). Okinaxobin II hydrolyzed tosyl-L-arginine methyl ester and benzoyl-L-arginine p-nitroanilide. The esterase activity was strongly inhibited by diisopropylfluorophosphate and to a lesser extent by tosyl-L-lysine chloromethyl ketone, indicating that the enzyme is a serine protease like alpha-thrombin. In terms of amino acid composition, okinaxobin II was similar to okinaxobin I and dissimilar to alpha-thrombin.
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PMID:Purification and characterization of a coagulant enzyme, okinaxobin II, from Trimeresurus okinavensis (himehabu snake) venom which release fibrinopeptides A and B. 772 19


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