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)

A colorimetric assay for plasma antithrombin III (ATIII) has been developed, using a new synthetic peptide substrate, PS-915 (H-D-Phe-Pro-Arg-CHA). PS-915 is freely water-soluble and its aqueous solution is stable. It is highly specific for thrombin and its Km value is about the same as that of S-2238. By enzyme hydrolysis, the new substrate liberates 3-carboxy-4-hydroxyaniline (CHA), which turns blue in color due to the complex formation with added alkaline-pentacyanoammine ferroate. The assay is a three-stage kinetic, one-point procedure, based on the method of antithrombin (heparin cofactor activity) determination. Since absorption measurements of the CHA are usually obtained at 700 nm, no blank correction is necessary even when hyperbilirubinemic, lipemic and/or hemolyzed plasma are used. The calibration curve for ATIII is linear over the range of 0-175% of normal and has a high degree of reproducibility. The results obtained by this method are well correlated with data obtained by the commonly used chromogenic assay or by radial immunodiffusion.
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PMID:A colorimetric assay for plasma antithrombin III using a new synthetic peptide substrate (PS-915). 639 81

Thrombin-activated bovine factor V (factor Va), an essential component of the blood clotting cascade pro thrombinase complex, is composed of two nonidentical subunits (Vl and Vh) and Ca2+ in tight association. We have examined Vl, Vh, and factor Va using analytical ultracentrifugation. At pH 7.65 in 50 mM tris(hydroxymethyl)aminomethane, 0.1 M NaCl, 1 mM benzamidine, and 10 mM Ca2+, the Vl subunit has a molecular weight (Mr) of 82 500, an S0(20) ,w = 5. 0(2)S , and, assuming a model of a prolate ellipsoid with 0.3 g of H2O/g of protein, an axial ratio of 5:1. The corresponding values for the Vh subunit are an Mr of 92 300, an S0(20) ,w = 5.2(9) S, and an axial ratio of 5:1. We found these same values for Vl and for Vh in a buffer that contained 2 mM ethylenediaminetetraacetate (EDTA) rather than the 10 mM Ca2+. The Vl subunit undergoes a weak, reversible self-association at 9 degrees C with an apparent monomer-dimer association constant of 5.6 X 10(3) M-1 in the presence of 2 mM EDTA and 2.3 X 10(3) M-1 in the presence of 10 mM Ca2+. Our data indicate that the Vl self-association includes dimer and higher oligomers. Factor Va, examined in the presence of 10 mM Ca2+ and at 20 degrees C, has an Mr of 174 000, and S0(20) ,w = 8.1(8)S, an axial ratio of 5:1, and an apparent Vl-Vh association constant of at least 2.7 X 10(8) M-1. Our results suggest that factor Va self-associates to form higher multimers. When solutions of Va are dialyzed against a buffer that contains no Ca2+ and 2 mM EDTA, the apparent Vl-Vh subunit association constant is reduced to 9.4 X 10(3) M-1. Our hydrodynamic data indicate that there is a substantial decrease in molecular asymmetry when factor V is proteolytically activated by thrombin to form factor Va and that Vl and Vh are arranged "side by side" rather than "end to end" in factor Va.
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PMID:Structure of bovine blood coagulation factor Va. Determination of the subunit associations, molecular weights, and asymmetries by analytical ultracentrifugation. 642 3

Bleeding time in rats was markedly prolonged after the administration of the water extract of Hsien-Ho-T'sao. This antihemostatic effect was more marked in the group of i.p. injection of the drug than in the group of p.o. administration for 2 or 7 consecutive days. Blood coagulation studies showed that plasma prothrombin time, activated partial thromboplastin time and stypven time were prolonged, while thrombin time and fibrinogen level were not changed. The thromboelastographic recording showed that reaction time was prolonged and maximal elasticity of clot was decreased. In addition, ADP- and collagen- induced aggregations of platelet-rich plasma were suppressed. In conclusion, the prolongation of the bleeding time might be due to both anticoagulant and antiplatelet action of the drug.
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PMID:Antihemostatic effect of Hsien-Ho-T'sao (Agrimonia pilosa). 649 95

Experiments were performed to determine whether activation of the coagulation cascade was required for pulmonary vascular permeability to increase during microembolization of the lung. For 30-45 min air microemboli were intravenously infused (0.05-0.10 ml X kg-1 X min-1) into awake sheep with chronic lung-lymph fistulas and anesthetized mongrel dogs. During embolization the pulmonary arterial pressure increased, and O2 partial pressure (PaO2) fell by more than 20 Torr (P less than 0.01). Subsequently lymph flow nearly tripled without a change in the lymph-to-plasma protein concentration ratio. Partial thromboplastin and prothrombin times, biological activity of antithrombin III, and circulating concentration of 125I-labeled dog or sheep fibrinogen did not change during or following air infusion. In two additional sheep an intravenous infusion of thrombin at 0.6 U X kg-1 X min-1 for 15 min resulted in a 20% decrease in 125I-labeled sheep fibrinogen concentration without a change in pulmonary arterial pressure or PaO2. We conclude that air microembolization can increase permeability to water and protein without a detectable activation of the coagulation cascade in the sheep or dog.
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PMID:Assessment of coagulation cascade during air microembolization of the lung. 666 65

The binding of folic acid as a model compound and methotrexate as a representative of antifolates to bovine fibrinogen with the aid of 1-ethyl-3-(3-dimethylamino-propyl)-carbodiimide was investigated in order to study the possibility of using fibrinogen as a drug carrier. Soluble modified fibrinogen derivatives containing 0.03-0.1 mg of folic acid or methotrexate per mg of protein were obtained under optimal conditions. These derivatives retained the ability to form fibrin clot by the action of thrombin and to copolymerize with native fibrinogen to the three dimensional fibrin network. At higher concentrations of water soluble carbodiimide, higher temperature and low pH highly cross-linked derivatives of fibrinogen and folic acid (or methotrexate) were formed which were insoluble in water and salt solutions (pseudofibrin). The modified fibrin was extensively proteolytically cleaved by plasmin, pepsin, trypsin and cathepsin D, whereas the proteolysis of insoluble pseudofibrin was very slow.
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PMID:Chemical binding of folic acid and methotrexate to bovine fibrinogen. 668 28

Epidemiologic studies have shown that several environmental factors are associated with coronary heart disease (CHD). Most of them are predisposing factors known also as risk factors. Other factors appear to have preventive effects. Blood lipids have been considered the main blood mediator between most of these factors and CHD. In recent years, this concept has been challenged since many of these factors did not affect serum lipids. By contrast blood platelets, involved in both thrombosis and atherosclerosis, appear to have their functions markedly changed by most of the factors associated with CHD. To determine whether saturated fats would affect platelet functions as shown in animals and in pilot studies in man, groups of male farmers (40-45 years) from 2 regions of France (Var and Moselle) in which the mortality rate from CHD differed markedly were studied, particularly regarding their platelet functions in relation to the intake of saturated fats. No difference could be observed in blood between the 2 regions concerning total cholesterol, HDL cholesterol, or triglycerides, the coagulation was markedly accelerated, as well as the platelet clotting activity in farmers from Moselle. The response of platelets mostly to thrombin but also to adenosine diphosphate (ADP), epinephrine, and collagen was more elevated in Moselle farmers. In Moselle farmers, there was significantly higher intake of saturated fats (16% of the calories) as compared to Var (12%). To determine whether the abnormal platelet response in Moselle farmers was really due to the diet or whether a genetic factor might be involved, a group of 50 Moselle farmers were persuaded to change their dietary habits in order to lower their intake of saturated fats to 10% of the calories and that of polyunsaturated to approximately 12%. 1 year after diet modification, the clotting time (PCT) and clotting activity of platelets were considerably prolonged and the response to thrombin drastically reduced. These results confirmed that the platelet functions were largely dependent upon the intake of dietary lipids. Studies in rats fed saturated fats demonstrated that addition of alcohol (6%) to the drinking water was markedly inhibiting the response of platelets to thrombin and ADP aggregation and prolonged the clotting time, despite inducing a significant hypertriglyceridemia. Several investigators have shown that platelet functions were markedly increased in diabetic patients while serum lipids were similar in the diabetic and nondiabetic subjects. Studies peformed more than 10 years ago indicated that women taking oral contraceptives (OCs) presented mostly an increase in the clotting activity of their platelets. In female rats it has been found that the administration of an OC was able, in addition to the effect of clotting, to increase the response fo platelets to thrombin and ADP induced aggregation.
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PMID:Risk factors for coronary heart disease and platelet functions. 669 70

Double-indicator dilution methods can be used for measurement of lung water. The thermal conductivity method is based on heat as a diffusible and conductivity as a non-diffusible indicator. In the present study we correlated lung thermal volume with gravimetrical measurement of extravascular lung water after thrombin-induced microembolization in dog lungs. The embolization was accompanied by significantly increased vascular permeability and accumulation of interstitial water. Under these conditions there was a close correlation between the two methods of measuring lung water (r = 0.78, p less than 0.01).
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PMID:Lung thermal volume as an indicator of pulmonary extravascular water. 711 30

Solute and fluid compartments in the lungs were investigated following thrombin-induced intravascular coagulation in rats treated with the fibrinolysis inhibitor, Trans-4-(amino-methyl) cyclohexanecarboxalic acid. The lung weight was increased to almost three times normal due to accumulation of extravascular water with albumin and chloride concentrations similar to those in plasma. The blood content and dry weight were doubled. Microscopic sections were characterized by widespread fibrin-rich microemboli, thickened alveolar walls, distension of peribronchiolar and perivascular spaces with fluid, dilated lymph vessels and protein-rich alveolar oedema. An increased microvascular permeability to protein explains the findings. When the dose of thrombin was decreased to a point where no pulmonary oedema developed, supplementary infusion of low molecular weight fibrinogen degradation products induced oedema formation as verified microscopically.
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PMID:Pulmonary insufficiency in the rat after intravascular coagulation and inhibition of fibrinolysis. II. Investigations on oedema formation and morphology. 711 31

Pulmonary microembolization was induced by infusion of thrombin during inhibition of the fibrinolytic system. After embolization cardiac output decreased, pulmonary vascular resistance and pulmonary arterial pressure increased. There was a transient decrease in PaO2. Immediately after embolization there was an increased permeability indicated by tremendous increase in lymph flow with a constant lymph/plasma protein ratio. The lymph/plasma ratio for hemoglobin and for FITC-Dextran (mw 150 000) also increased indicating leakage of large molecules. The increased permeability was accompanied by a significant increase in extra-vascular lung-water as measured both with the thermal conductivity and the dry/wet weight method.
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PMID:Studies on the pulmonary capillary permeability after induced microembolism. 715 6

The experiments were carried out in order to study the mechanism of thrombin--induced stimulation of glycolysis in washed human platelets. The platelets, isolated from human blood and resuspended in Krebs-Ringer-phosphate buffer (pH 7.4), were incubated in water bath (37 degrees C) either without (control) or with thrombin (1 NIH U/ml platelet suspension). Lactate, glycolytic intermediates and adenine nucleotides were measured in the neutralized perchloric acid extracts of platelets by enzymatic analysis and using the fluorimetric methods. The results of this study show that thrombin significantly increases the rate of lactate production in washed human platelets. At the same time thrombin induces the profound changes in the levels of platelet glycolytic intermediates with the "crossover" point at phosphofructokinase step of glycolysis. The results are interpreted as indicating that thrombin stimulates platelet glycolysis through allosteric phosphofructokinase activation due to the changes in adenine nucleotides energy charge.
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PMID:[The effect of thrombin on glycolysis in human thrombocytes]. 734 56


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