Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.21.5 (thrombin)
 33,306 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have studied the interaction between thrombin and washed, human platelets using prostacyclin, a reversible inhibitor of platelet secretion. The effect of thrombin is limited to those reactions that are not inhibited by an increased concentration of platelet cyclic adenosine 3',5'-monophosphate, because prostacyclin is a potent inducer of the latter. Prostacyclin-treated platelets were briefly (15-30 s) exposed to low concentrations of human thrombin (0.01-0.2 U/ml). After removal of the prostacyclin and thrombin, the platelets were incubated with fresh thrombin. Although they had not undergone the release reaction after the first thrombin incubation, these platelets had a diminished capacity to secrete [(3)H]serotonin when exposed to thrombin the second time. Refractoriness was concentration dependent: the higher the initial thrombin concentration, the greater the degree of inhibition of serotonin secretion on subsequent thrombin exposure. Inhibition was closely related to the ability of thrombin to induce platelet secretion and not to its esterase or fibrinogen clotting activity. Diisopropyl fluorophosphate-inactive thrombin did not induce refractoriness. Refractoriness to thrombin did not increase when the time of the initial incubation with thrombin was lengthened, nor was it reversible.INHIBITION WAS THROMBIN SPECIFIC: serotonin secretion induced by collagen, wheat germ agglutinin, and the ionophore A23187 was minimally affected. For an equivalent amount of thrombin bound, a decrease was observed in serotonin secretion by thrombin-pretreated platelets compared to control platelets. Thus, there is at least one step in the secretory pathway between thrombin binding and regulation of adenylate cyclase. This step appears to transmit the signal that leads to extrusion of intracellular granular contents.
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PMID:Thrombin-induced platelet secretion. Further evidence for a specific pathway. 37 55

The binding ability of low molecular weight heparin (FR-860), and conventional unfractionated heparin (UF-heparin) to factor Xa (F.Xa), thrombin and AT III was investigated using FR-860- and UF-heparin-Sepharoses. FR-860 could not bind directly to F.Xa. FR-860 bound to thrombin and AT III with stronger affinity to AT III than to thrombin. On the other hand, UF-heparin bound to F.Xa, thrombin and AT III with the strongest affinity to AT III followed by thrombin and F.Xa. AT III mediated the binding between F.Xa and FR-860 and accelerated the reaction between F.Xa and UF-heparin. On the other hand, AT III did not affect the binding between thrombin and FR-860 or UF-heparin. Diisopropyl fluorophosphate-treated thrombin inhibited the binding between AT III and FR-860, but not that between AT III and UF-heparin. These results suggest that the anti-F.Xa activity of FR-860 is mediated by AT III. Furthermore, the difference of antithrombin activity between FR-860 and UF-heparin depends on the capability to form ternary complex of FR-860 or UF-heparin, AT III and thrombin.
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PMID:Study of anticoagulant mechanism of low molecular weight heparin. 132 95

Indian green pit viper venom was studied for its coagulant activity. It was observed that the venom contained a significant amount of coagulant activity, which was similar to thrombin in its action on plasma as well as on fibrinogen. The physicochemical properties studied suggested that the venom coagulant activity lacked both platelet aggregating and factor XIII activating properties. Unlike thrombin, the activity was retained in the presence of heparin and at high temperatures. The activity was inhibited by Diisopropyl fluorophosphate and phenyl methyl sulphonyl fluoride, indicating that it was a serine esterase.
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PMID:A study of the coagulant activity of Indian green pit viper venom. 313 59

Bovine testicular hyaluronidase from various commercial sources showed the presence of an inhibitor of human plasma prothrombin time (PT). A testicular anticoagulant protein (TAP) was isolated from it by a 3-step procedure. The material was first passed through conconavalin-A-sepharose affinity chromatography where the anticoagulant material was separated from the hyaluronidase and protease which were retained by the column. In the second step the lower molecular weight proteins were removed by ultrafiltration. The supernatant which contained the anticoagulant protein was passed through the carboxymethyl cellulose column and the active material was eluted by 0.4 M NaCl solution. Sodium dodecyl sulfate (SDS) gel electrophoresis gave a molecular weight of approximately 35000. Unlike many small molecular weight proteins from bovine testes, TAP looses its anticoagulant property by heating for 30 minutes at 55 degrees C or by storage at pH 3.0 for 2 hours and it does not inhibit trypsin or thrombin. Its isoelectric pH was 9.7. Diisopropyl fluorophosphate (DFP) treated TAP was not effective as an anticoagulant.
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PMID:Isolation and properties of a new anticoagulant protein from commercial bovine testicular hyaluronidase. 661 84

A fibrinogen-clotting enzyme (bothrombin) was purified from the venom of Bothrops jararaca. Bothrombin showed M(r) values of 33,000 under nonreducing and 35,000 under reducing conditions on SDS polyacrylamide gel electrophoresis and specific fibrinogen-clotting activity equivalent to 814-904 NIH alpha-thrombin units/mg. Diisopropyl fluorophosphate totally abolished its activity, but hirudin, a specific alpha-thrombin inhibitor, had negligible effect on bothrombin activity. Unlike alpha-thrombin, bothrombin split off fibrinopeptide A without releasing fibrinopeptide B. Bothrombin activated blood coagulation factor VIII, but its activity was about 950 times less than that of alpha-thrombin. Bothrombin did not induce aggregation or serotonin release of washed normal platelets by itself, but did aggregate platelets in the presence of exogenous fibrinogen. This latter activity was completely inhibited by either anti-glycoprotein (GP) IIb/IIIa monoclonal antibody (which blocks fibrinogen binding to GP IIb/IIIa) or anti-GP Ib monoclonal antibody (which specifically inhibits alpha-thrombin binding to GP Ib). Prostaglandin E1 (1 microM) and EDTA (10 mM) also abolished platelet aggregation without affecting clotting activity. Washed platelets from a patient with Bernard-Soulier syndrome did not respond to bothrombin even in the presence of exogenous fibrinogen, suggesting that the initial binding of bothrombin on platelets is GP Ib, but not a recently cloned thrombin receptor. The complete amino acid sequence of bothrombin was determined by analysis of (S)-pyridylethylated protein and peptides generated by digestion with cyanogen bromide and Achromobacter protease I, respectively. Bothrombin is composed of 232 amino acid residues and contains three Asn-linked oligosaccharide chains.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Purification and characterization of bothrombin, a fibrinogen-clotting serine protease from the venom of Bothrops jararaca. 811 Jul 87

A thrombin-like enzyme, grambin, was purified to homogeneity by gel filtration, affinity and ion-exchange chromatography from the venom of Trimeresurus gramineus. Its mol. wt was estimated to be 45,400 by SDS-PAGE under reduced conditions. The mass of neutral sugars in grambin is estimated to be 20.7% of total mass. Grambin's NH2-terminal ten amino acid residues show a high homology to other venom thrombin-like enzymes. It clots human fibrinogen with a specific activity of 220-250 NIH thrombin-equivalent units/mg protein. It preferentially releases fibrinopeptide A accompanied by a slow release of trace amounts of fibrinopeptide B as monitored by HPLC following enzyme treatment of fibrinogen. EDTA, aprotinin, hirudin and heparin did not affect the fibrinogen-clotting activity of grambin in purified human fibrinogen solution. Diisopropyl fluorophosphate, phenylmethylsulfonyl fluoride and leupeptin inhibited the clotting activity of grambin whereas iodoacetamide did not affect its activity, indicating that grambin is a serine protease rather than a cysteine protease. In addition, it caused defibrinogenation and showed a marked antiplatelet effect when administered intravenously to mice. It also significantly prolonged the time lapse of platelet-rich thrombus formation in the irradiated mesenteric venules of fluorescein sodium-treated mice. Therefore, grambin may be used as a therapeutic agent not only in treatment of venous thrombosis but also in prevention of arterial thrombosis.
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PMID:Characterization of a thrombin-like enzyme, grambin, from the venom of Trimeresurus gramineus and its in vivo antithrombotic effect. 853 42

Trimeresurus stejnegeri venom which contains TSV-PA (a specific plasminogen activator sharing 60-70% sequence homology with venom fibrinogen-clotting enzymes), also possesses fibrinogen-clotting activity in vitro. A fibrinogen-clotting enzyme (stejnobin) has been purified to homogeneity by gel filtration and ion-exchange chromatography on a Mono-Q column. It is a single-chain glycoprotein with a mol. wt of 44,000. The NH2-terminal amino acid sequence of stejnobin shows great homology with venom fibrinogen-clotting enzymes and TSV-PA. Like TSV-PA, stejnobin was able to hydrolyse several chromogenic substrates. Comparative study of substrate specificities of stejnobin and other venom proteases purified in our laboratory was carried out on five chromogenic substrates. Stejnobin clotted human fibrinogen with a specific activity of 122 NIH thrombin-equivalent units/mg protein. However, stejnobin did not act on other blood coagulation factors, such as factor X, prothrombin and plasminogen. Diisopropyl fluorophosphate and phenylmethanesulfonyl fluoride inhibited its activity, whereas ethylenediamine tetracetic acid had no effect on it, indicating that it is a serine protease. Although stejnobin showed strong immunological cross-reaction with polyclonal antibodies raised against TSV-PA, it was interesting to observe that, unlike the case of TSV-PA, these antibodies did not inhibit the amidolytic and fibrinogen-clotting activities of stejnobin.
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PMID:Characterization of a fibrinogen-clotting enzyme from Trimeresurus stejnegeri venom, and comparative study with other venom proteases. 960 87