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)

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

We have developed a radioimmunoassay for human thrombin using rabbit anti-human thrombin IgG. The assay can measure 2 ng thrombin/ml plasma, 500-fold more sensitive than clotting assays. Human prothrombin is less reactive in the assay than thrombin by at least four orders of magnitude, and there is no demonstrable cross-reactivity with human factor Xa, the clotting factor structurally most similar to thrombin. The assay does not detect thrombin bound to anthithrombin III. Using the assay, we have demonstrated that plasma from 20 normal subjects does not contain detectable thrombin. We measured thrombin generation in clotting blood in polypropylene tubes and observed that thrombin appears (approximately equal to 3 ng/ml) within 45 S-5 min after venipuncture. This material is thrombin, not intermediates of prothrombin activation, since it disappears after addition of heparin, which promotes thrombin antithrombin III complex formation. After a plateau of 2-10 min, there is further thrombin generation, which results in clotting after 15-27 min at a level of 40-50 ng thrombin/ml. The thrombin generated 9-25 min before clotting may activate factors V and VIII and stimulate platelet aggregation and release. In contrast, the cascade hypothesis assigns a role for thrombin only late in blood clotting. Radioimmunoassay of thrombin and other clotting factors will be useful for clinical and physiological studies of blood clotting especially since the assay seems specific for thrombin and is independent of other activities that affect bioassays.
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PMID:The measurement of thrombin in clotting blood by radioimmunoassay. 99 43

Methods for the assay of antifactor Xa activity in the presence and absence of heparin are described. Diluted plasma is incubated with bovine, activated factor X (Xa) in stage I, and remaining Xa is measured with the chromogene substrate Bz-Ile-Glu-Gly-Arg-pNA in stage II. In the presence of heparin, the inactivation is completed in 30 sec, and this method measures total Xa-inactivating capacity in diluted plasma (Method I). In a clincal material, this capacity showed a strong positive correlation (r=0.85) to the thrombin-inactivating capacity of diluted heparinized plasma (heparin cofactor activity) and apparently reflects antithrombin III (At-III) concentration. In the absence of heparin, the inactivation of factor xa occurs slowly. With an incubation of 5 min, about 25% of Xa is inactivated, and this assay reflects initial inactivation of Xa (Method II). With this method, a positive, but less strong correlation to the thrombin-inactivating capacity was found (r=0.58), indicating that inhibitors different from At-III accounts for a minor part of the initial inactivation. Determinations in plasma, in which At-III was removed by immunoadsorption, indicated that At-III accounts for about 80% of the initial inactivation. The results of the assays are not significantly influenced by varying concentrations of fibrinogen, fibrinogen degradation products or heparin in the test plasma.
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PMID:Antifactor Xa activity measured with amidolytic methods. 101 22

In experiments with rats, antithrombin III (a natural inhibitor of fibrinolysis) was shown to decrease the activation of the anticoagulation system either in prophylactic administration or during the development of thrombogenesis, caused by intravenous administration of tissue thromboplastin. The phenomenon led to the maintaining of the more high content of fibrinogen in blood of experimental animals, to the shortening of the thrombin time and to the increase in content of alpha2-macroglobulin and alpha1-antitrypsin (in-hibitors of fibrinolysis) as compard with control animals, administered with the only thrombolastin. Effect of antithrombin III depended on periods of administration of the prearation and on injection of tissue thromboplastin.
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PMID:[Effect of antithrombin III on anticoagulating system function in animals intravenously injected with tissue thromboplastin]. 102 86

Hemorrhagic diathesis was observed in patients with renal insufficiency after carbenicillin at serum levels greater than 300 mug/ml. Normal coagulation factors (F. I, II, V, VII, VIII, X), normal PTT, normal platelet counts, negative ethanol gelation test (fibrin monomers) were found as well as a prolongation of thromboplastin time (Quick), thrombin time, reptilase time and thrombin coagulase time. Platelet function was disturbed. In addition, the plasmatic system was involved: inhibition of fibrinogen-fibrin conversion (Belitser assay) and enhanced antithrombin III activity; in vivo the latter was ascribed to a heparin-like activity. In vitro, abnormal III was seen: however an enhanced antithrombin III activity in vitro was not found with carbenicillin and various penicillin derivatives. This study demonstrates that carbenicillin, in addition to its known effect on platelet function, also disturbs the plasmatic coagulation system. This additional effect of carbenicillin is clinically important since protamin chloride effectively blocks bleeding without interfering with antibacterial activity. Both penicillin and penicillin derivatives have been shown to interfere with hemostasis and to cause clinically manifest hemorrhagic diathesis (Fleming and Fish 1947, Lurie et al. 1970a, b, McClure et al. 1970, Yudis et al. 1972, Demos 1971, Waisbren et al. 1971). Carbenicillin interferes with ADP-, collagen- or thrombin-induced platelet aggregation and with the release reaction both in vivo (McClure et al. 1970, Cazenae et al. 1973) and in vitro (McClure et al. 1970, Cazenave et al. 1973). In addition Lurie and colleagues (1970b) concluded that an inhibition of the conversion of fibrinogen to fibrin is involved although no experimental details were given. Later Brown and colleagues (1974) concluded that carbenicillin at usual dose levels "only affects the platelet component of hemostasis and has little effect on fibrin formation or other phases of coagulation in patients with normal renal function".
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PMID:Bleeding in uremic patients after carbenicillin. 103

Twenty-seven patients with peripheral vascular disease had studies of thrombin generation, antithrombin III -alues, factor VIII values, platelet adhesitivity, and activated partial thromboplastin time. Of the studies performed, the thrombin generation index, antithrombin III values, and, to a lesser extent, activated partial thromboplastin time were reliable in confirming clinically suspected hypercoagulability. When patients were placed into groups with and without operative complications, it was noted that the group with operative complications had higher average values of thrombin generation index and lower average values of antithrombin III and activated partial thromboplastin time than did the group without operative complications. It is thought that these tests may be useful in selecting those patients with a high risk of thrombotic complications from vascular surgery and who may benefit from anticoagulant management while undergoing necessary vascular reconstructive procedures.
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PMID:Hypercoagulability in patients with peripheral vascular disease. 115 13

When intermediate-strength thromboplastin was continuously infused into dogs for 10 days or more, platelet counts decreased sharply and factor VIII concentrations decreased by more than 50%. There was little change in plasma fibrinogen, prothrombin, factor V, antithrombin III, plasminogen, prothrombin time, and thrombin time values. When heparin was infused (25-50 U/kg per h) along with the same thromboplastin, there was no change in onset or degree of thrombocytopenia. However, the decrease in factor VIII was abolished and there were significant increases in fibrinogen, prothrombin, and factor V. The absolute concentrations of the various clotting factors seemed to give no indication of their turnover rates. Unexplained is the remarkable heparin tolerance that developed in these dogs.
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PMID:Effect of heparin on chronically induced intravascular coagulation in dogs. 116 71

A simple assay procedure for antithrombin III is described. The synthetic product benzoyl-Phe-Val-Arg-p-nitroanilide (S-2160) is used as substrate. By thrombin p-nitroaniline is split from the tripeptide molecule. The yellowish color of this split product can be measured in a photometer. Inactivation of thrombin by antithrombin III results in inhibition of this reaction. The percentage of thrombin inhibited is in direct relation to the activity of antithrombin III. Various modifications were tested to optimate the method. Comparable results were obtained between the coagulation method of HENSEN and LOELIGER and the photometric method.
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PMID:Photometric assay of antithrombin III with a chromogenic substrate. 120 36

Thrombin, while reacting in the presence of hepatin, impairs the inhibitory capacity of antithrombin III so that subsequent inhibition of thrombin or factor Xa is decreased or abolished. This adverse effect of hepatin has been observed directly with at least 1.5 Iowa units of thrombin per each unit of purified human antithrombin III participating in the reaction. The inhibitory capacity was then totally destroyed and some residual thrombin remained in the active form. With a lower enzyme/inhibitor ratio inactivation of thrombin in the presence of hepatin was fast and complete, however, a significant decrease of inhibitory capacity below that found in reaction without heparin, has been established by measuring the residual antithrombin III activity. In defibrinated human plasma at least 2 units of thrombin per each antithrombin III unit were required to demonstrate directly the adverse effect of heparin but a fast depletion of inhibitory capacity has been also observed after repeated additions of small thrombin portions into plasma heparinized in vitro or in vivo. Portions of enzyme initially added disappeared with great velocity; subsequent portions, however, accumulated building up a high thrombin level not seen in the absence of heparin. The accumulation of residual enzyme was more extensive in plasma containing about 1 heparin unit per ml than anticoagulant at lower concentrations and was particularly noticeable in antithrombin III deficient plasma. These results may have some bearings on the approach to heparin therapy in the event when thrombin continuously generates or when a marked deficiency of antithrombin III exists.
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PMID:Adverse effect of heparin in thrombin-antithrombin III interaction. 120 44

A series of in vitro studies designed to ascertain the potential in vivo thrombogenicity of human factor IX-containing concentrates is described. Using concentrates obtained from several different Centres the fibrinogen clotting time with some preparations was less than 6 h and/or the recalcification time of normal plasma was shortened. In some preparations, however, the plasma recalcification time was lengthened. Further studies revealed that all diluted factor-IX concentrates generated thrombin after recalcification, and that the rate of thrombin generation appeared to be characteristic of a particular preparation. This characteristic has been designated the TGt50, which is the incubation period in minutes, after recalcification, required to obtain a 50 s clotting time of a fibrinogen substrate. The TGt50 was found to correlate most strongly with recalcification time of celite exhausted plasma (P less than 0.001), but no correlation was observed between it and the immunological antithrombin III or factor-VIII antigen levels. Evidence is presented which suggests that the thrombin generation test and recalcification time of celite exhausted plasma may represent suitable in vitro quality control assays for factor-IX concentrates.
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PMID:In vitro spontaneous thrombin generation in human factor-IX concentrates. 121 34


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