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)

p-Nitrobenzyl p-toluenesulfonyl-L-arginine is hydrolyzed by thrombin, plasmin, and trypsin to p-nitrobenzyl alcohol and tosyl-L-arginine. The absorption of p-nitrobenzyl alcohol formed is measured at 271 nm (AmM 8.89). With 0.10 mM of the ester in 0.1 M Tris-HCl at pH 8.4 and 30 degrees C, the hydrolysis catalyzed by thrombin, plasmin, and trypsin is linearly proportional to time up to consumption of 60% of the substrate. Km is 14 micron and Vmax is 0.037 mumol/min/NIH unit for bovine thrombin, Km is 78 micron and Vmax is 0.31 mumol/min/CTA unit/ml for human plasmin, and Km is 12 micron and Vmax is 138 mumol/min/mg protein/ml for bovine trypsin. Samples of bovine and human thrombin ranging in specific clotting activity from 59 to 2,133 NIH units/mg protein showed esterase activities ranging from 0.15 to 0.4 mumol p-nitrobenzyl alcohol formed/10 min/NIH unit. Useful ranges for assay of enzymes were (per milliliter): 0.05-0.2 NIH units (thrombin), 0.005-0.02 CTA units (plasmin), and 0.01-0.04 microgram (trypsin).
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PMID:p-Nitrobenzyl p-toluenesulfonyl-L-arginine: a chromogenic substrate for thrombin, plasmin and trypsin. 2 27

Equal volumes of plasma and 0.3 M K2HPO4, pH 7.4, were mixed, diluted 20-fold, and adjusted to pH 5.2. After incubation at 37 degrees C for 30 min, the euglobulin percipitate, redissolved in 0.1 M K2HPO4, pH 7.4, developed caseinolytic activity (0.05 CTA U/ml). Na2HPO4 or NaCl of similar ionic strength could replace K2HPO4. The pH optimum of the protease was 6.5, activity falling off sharply below pH 6.0 and above 7.4. The proteolytic activity was inhibited by diisopropylphosphofluoridate and by pancreatic trypsin inhibitor, but was not inhibited by soybean trypsin inhibitor. The activity was not due to plasmin, contact activation, or coagulation factors, since it was fully generated in plasminogen-depleted, factors XII, XI, VII deficient, and prekallikrein-deficient plasmas. Purified Cl-esterase was not caseinolytic in our system. Redissolved euglobulin precipitate prepared from normal plasma without salt addition could serve as starting material for the generation of caseinolytic activity, as could serum, indicating that the Hageman factor cofactor and thrombin are not required. The protease had no detectable procoagulant or fibrinolytic activity.
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PMID:Nonplasminogen-dependent protease in human plasma. 3 47

The effects of plasmin treatment upon washed human platelets were studied in an attempt to elucidate the mechanisms underlying thrombin-induced platelet aggregation. At calcium concentrations of 10-20 muM, PLASMIN (0.2 CTA U/ml) inhibited thrombin-induced aggregation almost completely, but did not diminish the thrombin-induced release of adenine nucleotides, 5-hydroxytryptamine, or calcium. Increasing the calcium concentration partially antagonized plasmin's inhibition of aggregation. Studies utilizing calcium chelators and the Kunitz soybean trypsin inhibitor (SBTI) as a plasmin inhibitor indicated that in order to achieve maximal block of aggregation, plasmin must act upon a substrate made fully available only after an initial thrombin-platelet interaction has taken place. Moreover, the time course of this inhibition parallels the time course of the thrombin-induced release reaction. Plasmin inhibition of aggregation could not be mimicked by exposing the platelets to proteolytic digests of fibrinogen at concentrations as high as 17% total platelet protein. Nor could inhibitory activity be recovered from supernatants of plasmin-treated platelets, upon centrifugation and treatment with SBTI. With the use of a "cold initiation" technique, the release by thrombin of 46.7 plus or minus 6.7 (mean plus or minus SEM) mu-g of fibrinogen immunological equivalents per mg platelet protein could be demonstrated. Platelets in which thrombin-induced aggregation was abolished by plasmin treatment (and the plasmin subsequently inactivated by STBI) aggregated normally upon addition of as little as 10 mu-g human plasma fibrinogen per mg platelet protein. It is concluded that plasmin inhibition of aggregation most likely results from its attack upon a protein that is released or becomes fully available subsequent to interaction of thrombin with a platelet receptor mediating release. The results of this study are consistent with a cofactor role for fibrinogen in the aggregation of human platelets by thrombin.
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PMID:Plasmin inhibition of thrombin-induced platelet aggregation. 12 75

125I-fibrinogen, adsorbed to polystyrene tubes at low ionic strength and treated with thrombin, serves as a substrate for a rapid, convenient, and sensitive test tube assay for plasmin and activators and inhibitors of this enzyme. 125I-labeled digestion products released from the 125I-fibrin-polystyrene matrix are readily separated and quantitated and behave, on gel permeation, in the same manner as plasmin-generated degradation products from an unlabeled conventional fibrin clot. The 125I-fibrin, in probable non-cross-linked form, is firmly bound to the polystyrene and is resistant to nonspecific release, with control (no enzyme) values equivalent to 15.2 ng +/- 1.2 (SD) fibrin (1% of the total bound 125I-fibrin). This fact permits consistent detection of lysis of 30-50 ng 125I-fibrin, which exceeds published sensitivities (1000-5000 ng) using 125I- or fluorochrome-labeled fibrin clots as substrate. The sensitivity for plasmin (0.2 mug/ml) is tenfold greater than that of the fibrin-plate method (2.0-2.5 mug/ml), while sensitivities for streptokinase and urokinase activation of plasmin are 0.02 U/ml and 0.04 CTA U/ml, respectively (sensitivity of fibrin-plate method, 0.5 U/ml for both). The method provides a reasonable analogue of the solid-phase nature of fibrin under physiologic conditions, and the ease of preparation of large batches of tubes makes the method suitable for large-scale screening of factors modulating the plasminogen-plasmin system.
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PMID:A rapid and sensitive 125I-fibrin solid-phase fibrinolytic assay for plasmin. 12 94

A new method is presented for estimating the activator (plasminogen-streptokinase complex) concentration in native plasma of patients undergoing streptokinase infusion. The principle of the method is based on clot lysis time as recorded by the thromboelastograph. The test clot constituents were bovine fibrinogen, bovine plasminogen, EDTA, human plasma (with unknown activator concentrations), and thrombin. In order to obtain a standardization line, urokinase dissolved in NaCl solution was substituted for patients' plasma. Thus, each lysis time could easily be converted into urokinase equivalent (CTA-u/ml). Streptokinase and plasminogen molecules in undiluted patients' plasma were found to exist both in an activator-bound (equimolar plasminogen-streptokinase complex) and in a freely circulating form. This result is in agreement with earlier findings where the activator complex was demonstrated to be a widely dissociated complex in highly diluted plasma of patients, thus displaying an ample proportion of free streptokinase and plasminogen and molecules. Streptokinase treatment using dosage schemes of 100,000 u SK/h, and 200,000 u/h were monitored by quantitative activator, streptokinase, and plasminogen measurements. An average activator concentration of 50-100 CTA-u/ml and a SK-concentration of 7-16 u/ml were recorded during streptokinase infusion. Plasminogen values averaged 0.25%, independent of the amount of streptokinase infused. Each drop in streptokinase was accompanied by a drop in activator during the infusion, and each rise in streptokinase by a rise in activator. There was a strong correlation between streptokinase and activator concentrations in that, on the average, 1 u streptokinase equalled 8.4 CTA-u/ml activator (correlation coefficient r = 0.9) It is concluded that the activator concentration in the plasma of patients undergoing fibrinolytic treatment can easily be adjusted by regulating the hourly streptokinase influx.
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PMID:Studies on activator formation in human plasma with streptokinase. III. Investigation of activator kinetics in undiluted plasma in terms of urokinase equivalents. 13 62

p-Nitrobenzyl p-toluenesulfonyl-L-arginine has been synthesized. A number of trypsin-like enzymes can catalyze the hydrolysis of this ester leading to formation of p-nitrobenzyl alcohol. After separation from the ester and p-toluenesulfonylarginine by extraction into chloroform, the p-nitrobenzyl alcohol liberated can be measured spectrophotometrically at 271 nm. Under the conditions of the assay, the hydrolysis of 1 micronmol/ml of the ester is equivalent to an absorbance change of 4.45 cm-1 at 271 nm. With 0.10 mM p-nitrobenzyl p-toluenesulfonyl-L-arginine in 0.1 M Tris-HCl at pH 8.4 and 30 degrees, the enzymatic hydrolysis is linearly proportional to time up to consumption of 60% of the ester. Product formation is proportional to enzyme concentration with 0.05 to 0.2 NIH clotting units/ml for bovine or human thrombin, 0.005 to 0.02 CTA units/ml for human plasmin, and 0.01 to 0.04 microgram/ml protein for bovine pancreatic trypsin. In 0.1 M Tris-HCl at pH 8.4 and 30 degrees, Km is 14 micrometer and Vmax is 0.037 micronmol/min/NIH unit/ml for bovine thrombin, Km is 78 micrometer and Vmax is 0.31 micronmol/min/CTA unit/ml for human plasmin, and Km is 12 micrometer and Vmax is 138 micronmol/min/mg protein/ml for bovine trypsin. With bovine thrombin, activities at pH 7.3 and at pH 9.2 were 30% lower and 40-50% higher than the rate at pH 8.4. Samples of bovine and human thrombin ranging in specific clotting activity from 59 to 2133 NIH units/mg protein showed esterase activities varying from 0.15 to 0.4 micronmol p-nitrobenzyl alcohol formed/10 min/NIH unit.
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PMID:Assay of the esterase activity of thrombin, plasmin and trypsin with a chromogenic substrate p-nitrobenzyl p-toluenesulfonyl-l-arginine. 14 54

A new fluorogenic peptide substrate for plasmin, 7-(N-succinoylalanylphenylalanyl-lysylamido)-4-methylcoumarin trifluoroacetate salt, was prepared that can be used in a simple and direct assay. The results obtained by the assay method are linear over a wide range of enzyme concentrations and sensitive enough to detect as little as 10(-5) CTA units of plasmin. By making use of the inhibitor Trasylol and the differences in kinetic constants, plasmin can be specifically assayed even in the presence of the plasminogen activator thrombin, as well as in culture fluids from HeLa cells.
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PMID:A new fluorogenic substrate for plasmin. 16 7

We have previously reported paradoxical prothrombotic effects manifest by elevations of fibrinopeptide A (FPA) after administration of streptokinase to patients with acute myocardial infarction. To characterize mechanisms responsible and their dependence on streptokinase (SK) as opposed to other activators of the fibrinolytic system, the present study was performed to compare effects of streptokinase, tissue-type plasminogen activator (t-PA), and urokinase on plasma and on purified prothrombin in concentrations similar to those achieved pharmacologically. The effects of plasmin were assessed to determine the extent to which the elevations of FPA seen could be attributed to activation of plasminogen. Elevations of FPA were observed after incubation of each of the activators with citrated plasma at 37 degrees C for 60 minutes. However, they were most marked with streptokinase (64.5 +/- 4.6 pmol FPA/ml (mean +/- SE) with 100 IU SK/ml, and 77.6 +/- 5.0 pmol/ml with 500 IU SK/ml). Elevations of FPA induced by streptokinase were attenuated by 100 IU/ml heparin [15.2 +/- 1.9 pmol/ml after 100 IU of SK (p less than 0.001 compared with results with streptokinase without heparin)]. Human plasmin, 2.5 CTA/ml, caused changes similar to those induced by streptokinase. The minimal elevations of FPA induced by t-PA or urokinase (less than 10 pmol/ml without heparin) were not significantly attenuated by heparin. Incubation of barium citrate adsorbed plasma (vitamin K factor depleted) with streptokinase markedly attenuated elevation of FPA. Addition of prothrombin (1.5 microM) and streptokinase (100 IU/ml) to the barium citrate adsorbed plasma elicited elevations of FPA similar to those induced by streptokinase in citrated plasma. Amidolytic activity with the "thrombin" substrate H-D-phenylalanyl-L-pipecolyl-L-arginine-p-nitroanilide dihydrochloride (S-2238) was evident when streptokinase, plasminogen (0.24 microM), and prothrombin (1.5 microM) were incubated in buffer. Thus, concentrations of streptokinase that are low in terms of therapeutic blood levels activate prothrombin in plasma, likely due to activation of plasminogen. Neither tissue-type plasminogen activator nor urokinase in pharmacologically comparable concentrations increase thrombin activity appreciably perhaps because of less intense activation of plasminogen. Consideration of the prothrombotic effects observed may be relevant to selection of specific agents for therapeutic thrombolysis, to appropriate titration of dose, and to the need for the use of heparin conjointly with particular activators of the fibrinolytic system such as streptokinase.
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PMID:Differential effects of activation of prothrombin by streptokinase compared with urokinase and tissue-type plasminogen activator (t-PA). 313 87

A family is described in which venous thrombosis developed in five members as early as 14 years of age. Routine coagulation studies, plasma antithrombin III, factor V, plasminogen, beta-thromboglobulin, fibrinopeptide A, prothrombin fragment F1+2, and thrombin-antithrombin III complex were all within normal limits. However, defective release of vascular plasminogen activator was observed on several occasions in all five subjects as compared with a control population of 125 persons (0.04 Committee on Thrombolytic Agents [CTA] units/ml plasma as compared with 0.21 CTS units/ml). In addition, levels of factor VII/von Willebrand's factor were significantly elevated above the normal range in this pedigree.
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PMID:Venous thrombosis in a family with defective release of vascular plasminogen activator and elevated plasma factor VIII/von Willebrand's factor. 640 91

Prolonged clotting times and reduced levels of clotting factors have been reported in hematin-treated patients. This effect persists for up to 5 hr after hematin infusion, associated with plasma levels ranging from 0.01 to 0.04 mg/ml. Therefore we performed in vitro studies to investigate the effects of hematin on fibrinogen, thrombin, factor VIII:C, and plasmin. Hematin in a final concentration of 0.01 mg/ml inhibited the clotting of bovine fibrinogen (1.3 to 2.6 mg/ml) by bovine thrombin (0.12 U/ml) and inhibited the hydrolysis of a synthetic substrate by human thrombin. However, if the hematin was first mixed with albumin (25 mg/ml), fourfold higher concentrations were required to prolong the thrombin clotting time. Hematin, 0.035 mg/ml, reduced VIII:C activity from 0.88 to 0.40 U/ml as measured by two-stage assay. Hematin (0.05 mg/ml) also inhibited the activation of VIII:C by thrombin (0.04 U/ml): baseline activity, 0.84 U/ml; thrombin-activated, 2.94 U/ml; with hematin added, 1.33 U/ml. Hematin also inhibited clot lysis. The inclusion of hematin (0.03 mg/ml) in the diluting buffer reduced the lysis of whole blood clots from 86% +/- 5 to 23% +/- 5 (p less than 0.001, mean +/- S.D. of four determinations) and decreased the lysis of 125I-fibrin clots induced by plasmin (0.02 CTA U/ml) from 100% to 27%. In concentrations as low as 0.09 microgram/ml, hematin inhibited the hydrolysis of a synthetic substrate by plasmin. Hematin was mixed with fibrinogen, albumin, or thrombin, and the mixtures applied to Sephadex G-200 columns. Adherence of the hematin to Sephadex was prevented by either prerinsing the column with albumin or using borate buffer at pH 9.2. Hematin co-eluted with each protein applied to the column and, in the case of fibrinogen, altered its electrophoretic mobility and markedly prolonged the thrombin clotting time of the eluted fibrinogen. We conclude that hematin binds to a variety of hemostatic proteins, inhibiting their biologic activity.
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PMID:The inactivation of hemostatic factors by hematin. 641 43


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