Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.86 (clotting enzyme)
 176 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Bovine prothrombin was activated, in both the absence and presence of dissopropyphosphofluoridate (DEP) and benzamidine, by an activator which was highly purified from the venom of Echis carinatus (saw-scaled viper, ECV). The process of activation was monitored by sodium dodecysulfate (SDS)-polyacrylamide gel electrophoresis, and the reaction products were isolated and chemically characterized. In the absence of the inhibitors, prothrombin yielded two fragments with molecular weights of 28,000 and 57,000, of which the former was the N-terminal fragment of the zymogen and the latter was intermediate 1, consisting of a single polypeptide chain. Intermediate 1 was subsequently converted to an active intermediate, named intermediate ECV, without decrease of molecular weight. This new intermediate ECV, which showed little clotting activity but a strong alpha-N-tosyl-L-arginine methyl ester (TAME)-esterolytic activity and which bound with hirudin or antithrombin III, consisted of two polypeptide chains with molecular weights of 35,000 of 27,000 daltons. The former was indentified as the thrombin B chain with the N-terminal sequence Ile-Val-Glu-Gly and C-terminal serine, and the latter was a fragment with N-terminal Ser-Gly-Gly, linked to the thrombin A chain. On prolonged incubation, intermediate ECV autocaralytically yielded a fragment (inner fragment) of 14,000 daltons with N-terminal serine and the clotting enzyme alpha-thrombin [EC 3.4.21.5], which consists of A and B chains. In the presence of the inhibitors, intermediate ECV and the N-terminal fragment were accumulated in the activation mixture. On the other hand, when prothrombin was activated by the venom activator in the presence of hirudin, antithrombin III, or p-nitrophenyl p'-guanidinobenzoate, it did not yield any fragments but was converted to a derivative with two polypeptide chains having molecular weights of 51,000 and 34,000 daltons, of which the former consisted of N-terminal fragment, the inner fragment, and thrombin A chain, and the latter was thrombin B chain. This new prothrombin derivative, named prothrombin ECV, formed a high-molecular-weight complex, associating with antithrombin III. The complex was not dissociable even in the presence of SDS. Moreover, prothrombin ECV reacted with p-nitrophenyl p'-guanidinobenzoate. On the basis of the results described above, the mechanism of activaton of prothrombin by Echis carinatus venom activator can be summarized as follows: The venom activator first cleaves an Arg-Ile bond liniking thrombin A and B chains in the zymogen molecule, forming an active derivative, prothrombin ECV. This active derivative converts autocatalytically to intermediate ECV, liberating the N-terminal fragment, and active intermediate ECV generates alpha-thrombin, releasing the inner fragment. Thus, only a single peptide bond cleavage along the polypeptide chain of prothrombin is associated with activation by the venom activator...
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PMID:The mechanism of activation of bovine prothrombin by an activator isolated from Echis carinatus venon and characterization of the new active intermediates. 95 36

The effect of the aromatic diamidine derivative 2,6-bis (4-amidinobenzyl)-cyclohexanon-(1) on blood coagulation and fibrinolysis in vitro and in vivo was compared with that of the benzamidine derivative 4-amidinophenyl pyruvic acid and the aromatic diamidine derivative 4,4'-diamidinophenoxypentane. 2,6-Bis(4-amidinobenzyl)-cyclohexanon-(1) was found to be a strong inhibitor of the clotting enzyme thrombin. Because of the toxic side effects and pharmacokinetic properties of both diamidine derivatives their in vivo use as anticoagulants is limited.
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PMID:[Effects of aromatic bisamidines on blood coagulation and fibrinolysis]. 98 14

A turbidimetric assay for clottable plasma fibrinogen which is not sensitive to heparin or Pyran inhibition is described. The basis of the assay is the substitution of Reptilase-R for the thrombin usually employed. The assay correlates very well with a thrombin turbidimetric method and also has other advantages, including better stability of the clotting enzyme and more rapid attainment of equilibrium.
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PMID:A rapid, quantitative determination of clottable fibrinogen unaffected by heparin. 111 33

Clots formed upon the addition of thrombin to human platelet-rich plasma (PRP) retracted readily but the clotting enzyme from Bothrops atrox venom did not cause retraction in PRP unless ADP, collagen, epinephrine, or low concentrations of thrombin (0.1 U) were added. The latter type of retraction was inhibited by apyrase and creatine phosphate kinase in the presence of creatine phosphate, but that induced with higher concentration of thrombin (2 U) was not. In a system composed of washed human platelets and purified fibrinogen, Bothrops marajoensis (BM) thrombinlike enzyme (highly purified preparations of viper venom) did not cause clot retraction. Addition of ADP to the platelet-fibrinogen mixture prior to BM enzyme resulted in stimulation of clot retraction that could be dissociated from the release of platelet constituents. Addition of low concentrations of thrombin (0.1 U/ml) caused retraction associated with a considerable release of adenine nucleotides that was inhibited by potato apyrase. Electron micrographs showed platelet-fibrin aggregates in all types of retracted clots. Nonretracted clots formed in the presence of potato apyrase contained discoidal platelets that were not in close association with fibrin. It has been postulated that platelet-dependent fibrin clot retraction induced by collagen, epinephrine, and low concentration of thrombin is mediated by ADP. High concentrations of thormbin may possibly promote clot retraction independently of ADP.
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PMID:ADP, thrombin, and Bothrops atrox thrombinlike enzyme in platelet-dependent fibrin retraction. 121 67

The action of the blood clotting enzyme thrombin on single channel and whole cell Ca(2+)-currents was studied in isolated mammalian cardiac myocytes. Thrombin, at a concentration of 10(-8) mol/l, increased the Ca(2+)-channel activity in cell-attached patches. The mean open probability of the channel was enhanced, while the number of sweeps without openings, which reflects the availability of the channel, was significantly reduced. Neither the single channel conductance nor the activation curve were affected by thrombin. Thrombin was added to the bath solution, and its effect is therefore indirect and probably mediated via a second messenger. However, thrombin did not affect whole-cell Ca(2+)-currents, whereas a beta-adrenergic stimulation in the same cell increased the Ca(2+)-current. It is concluded that thrombin affects an intracellular mechanism for Ca2+ channel current regulation, which is still unknown and which is rapidly lost during conventional whole-cell Ca2+ current measurements.
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PMID:Thrombin stimulates L-type calcium channels of guinea pig cardiomyocytes in cell-attached patches but not after intracellular dialysis. 131 2

Prothrombin contains two kringle domains that are removed during activation to the blood clotting enzyme alpha-thrombin. By analogy with other kringle-containing proteins the prothrombin kringles may play a role in the protein-protein interactions necessary for prothrombin activation. Four monoclonal antibodies to prothrombin kringle 2 have been produced against human prothrombin, and a fifth monoclonal antibody was produced against a synthetic peptide consisting of amino acid residues 216-231 of kringle 2. Each antibody was tested for its ability to block prothrombin activation by factor Xa. In the presence of phosphatidylcholine/phosphatidylserine vesicles and factor Va, two of the antibodies, alpha HII-3 and alpha HII-4, inhibited prothrombin activation at a 90 and 50% level, respectively. Two other monoclonal antibodies (alpha HII-6 and alpha HII-7) and the antipeptide antibody (alpha HII-5) had no effect on prothrombin activation. When factor Xa was the catalyst alone, antibody alpha HII-3 lost the ability to inhibit prothrombin activation whereas antibody alpha HII-4 again partially inhibited the reaction. When human platelets were the reaction surface, the patterns of inhibition by the anti-fragment 2 antibodies were identical to that observed with phospholipid vesicles. These data suggest a role for prothrombin fragment 2 in activation, possibly by mediating the interaction of substrate prothrombin with factor Xa or factor Va on the phospholipid surface.
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PMID:Modulation of human prothrombin activation on phospholipid vesicles and platelets using monoclonal antibodies to prothrombin fragment 2. 202 54

Three recombinant variants of hirudin with the most evident difference in amino acid position 47 (Lys-47, Arg-47, Asn-47) were studied for their selectivity and affinity for the target enzyme thrombin in comparison to native hirudin. Native hirudin and the recombinant hirudins inhibit selectively the clotting enzyme thrombin. The affinity of native hirudin does not differ significantly from that of recombinant hirudin Lys-47 whereas a distinctly lower affinity for thrombin is found for recombinant hirudin Arg-47 and recombinant hirudin Asn-47. All hirudins investigated have the same potency to inhibit thrombin-induced coagulation. Changes in the affinity of hirudins for thrombin become evident from the inhibition of thrombin-induced platelet aggregation only.
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PMID:Antithrombin effects of native and recombinant hirudins. 227 Oct 10

Fibrinogen Aarhus was found in a woman with slightly prolonged whole blood clotting time. The thrombin induced clotting of plasma and purified fibrinogen was much prolonged. Kinetic analysis of FPA and FPB release revealed larger apparent Km and Vmax values for fibrinogen Aarhus than for normal fibrinogen. No clot formation of fibrinogen Aarhus was demonstrated in the presence of Batroxobin and the release of FPA was slower than normal. Upon addition of the clotting enzyme from Agkistrodon contortrix contortrix clotting did occur but the clotting time was much prolonged in comparison with normal fibrinogen. The turbidity of fibrin gels obtained from fibrinogen Aarhus was similar to normal fibrin gels at low thrombin concentrations. Increasing thrombin concentration resulted in appearance of degradation products in the fibrin gels from fibrinogen Aarhus and at the same time a relative increase in turbidity of the gels was observed. Possibly reasons for the slow release of fibrinopeptides, the delayed gelation, and susceptibility to degradation by thrombin are discussed.
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PMID:Fibrinogen Aarhus--a new case of dysfibrinogenemia. 293 91

The clotting enzyme thrombin induces not only blood coagulation but it also receptor-mediated cellular events. In our studies, thrombin at nanomolar concentrations caused irreversible aggregation of washed human platelets which was inhibited by the naturally occurring tight-binding inhibitor hirudin. Thrombin within the same concentration range caused concentration-dependent transient relaxation of PGF2 alpha-precontracted pig coronary artery ring segments with intact endothelium. The relaxant response was neither affected by indomethacin nor by verapamil and was only slightly inhibited by exposure to calcium-free medium. Methylene blue enhanced the PGF2 alpha-induced contraction and diminished the thrombin-induced relaxation. Hirudin inhibited the relaxant effect of thrombin in a concentration-dependent manner. After removal of the endothelium by mechanical rubbing the thrombin-induced relaxation was absent. The present studies suggest that thrombin generated during coagulation is able to modify the vascular smooth muscle tone.
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PMID:Studies on thrombin-induced endothelium-dependent vascular effects. 320 49

In pig coronary arteries with intact endothelium the clotting enzyme thrombin at physiologically relevant concentrations caused a reversible transient and concentration-dependent relaxation of PGF2 alpha-precontracted vessels which was absent in case of mechanically removed endothelium. The thrombin-induced relaxation was not affected by indomethacin. Exposure of the coronary arteries to a calcium-free medium or preincubation with verapamil led to the reduction of the PGF2 alpha-induced contraction while the relaxant effect of thrombin was only slightly inhibited. However, the guanylate cyclase inhibitor methylene blue diminished the relaxant response to thrombin. The specific tight-binding thrombin inhibitor hirudin prevented the relaxant effect of thrombin in a concentration-dependent manner.
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PMID:Endothelium-dependent relaxant effect of thrombin on isolated pig coronary arteries. 324 18


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