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)

Generation of coagulation factor Xa by the intrinsic pathway protease complex is essential for normal activation of the coagulation cascade in vivo. Monocytes and platelets provide membrane sites for assembly of components of this protease complex, factors IXa and VIII. Under biologically relevant conditions, expression of functional activity by this complex is associated with activation of factor VIII to VIIIa. In the present studies, autocatalytic regulatory pathways operating on monocyte and platelet membranes were investigated by comparing the cofactor function of thrombin-activated factor VIII to that of factor Xa-activated factor VIII. Reciprocal functional titrations with purified human factor VIII and factor IXa were performed at fixed concentrations of human monocytes, CaCl2, factor X, and either factor IXa or factor VIII. Factor VIII was preactivated with either thrombin or factor Xa, and reactions were initiated by addition of factor X. Rates of factor X activation were measured using chromogenic substrate specific for factor Xa. The K1/2 values, i.e., concentration of factor VIIIa at which rates were half maximal, were 0.96 nM with thrombin-activated factor VIII and 1.1 nM with factor Xa-activated factor VIII. These values are close to factor VIII concentration in plasma. The Vsat, i.e., rates at saturating concentrations of factor VIII, were 33.3 and 13.6 nM factor Xa/min, respectively. The K1/2 and Vsat values obtained in titrations with factor IXa were not significantly different from those obtained with factor VIII. In titrations with factor X, the values of Michaelis-Menten coefficients (Km) were 31.7 nM with thrombin-activated factor VIII, and 14.2 nM with factor Xa-activated factor VIII. Maximal rates were 23.4 and 4.9 nM factor Xa/min, respectively. The apparent catalytic efficiency was similar with either form of factor VIIIa. Kinetic profiles obtained with platelets as a source of membrane were comparable to those obtained with monocytes. These kinetic profiles are consistent with a 1:1 stoichiometry for the functional interaction between cofactor and enzyme on the surface of monocytes and platelets. Taken together, these results indicate that autocatalytic pathways connecting the extrinsic, intrinsic, and common coagulation pathways can operate efficiently on the monocyte membrane.
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PMID:Functional assembly of intrinsic coagulation proteases on monocytes and platelets. Comparison between cofactor activities induced by thrombin and factor Xa. 161 61

Platelet activity is controlled, in part, by cytosolic free ionized calcium concentration ([Ca++]i). Regulation of platelet thromboxane (TXB2) synthesis may be by regulation of [Ca++]i. Dietary linoleate is a regulator of TXB2 synthesis, therefore, it may act by influencing [Ca++]i. Aspirin is a regulator of TXB2 synthesis by inhibition of cyclooxygenase; ouabain and nifedipine are regulators of [Ca++]i. This study was conducted to determine whether these affectors of TXB2 synthesis and [Ca++]i cause associated responses. Male nonobese Zucker rats were fed diets supplying 30% of energy (en%) as fat. Dietary fat was a mixture of corn oil and beef tallow to provide 3.0, 4.5, 6.0, or 7.5 en% linoleic acid, with cholesterol added to provide equal cholesterol in all diets. Rats were fed for 30 days with 6 rats/diet. Isolated rat platelets were assayed for FA composition; the percentage of linoleic acid in platelet FA rose linearly with increasing dietary linoleate (r = 0.76, P less than 0.0001). Resting and thrombin-stimulated platelet [Ca++]i and TXB2 synthesis were measured in the presence or absence of extracellular calcium and aspirin, ouabain, or nifedipine. Aspirin caused reductions in both parameters; nifedipine blocked [Ca++]i, but did not affect TXB2; ouabain increased both. Changes induced by those modifiers of TXB2 and platelet [Ca++]i caused changes that were in the same direction for both. CaCl2 caused an increase in both and the [Ca++]i was correlated with the square root of the TXB2; without CaCl2 the two were negatively correlated; aspirin, ouabain, and nifedipine treatments resulted in no significant correlations. The results suggest that there is a common modifier of [Ca++]i and TXB2 synthesis.
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PMID:The relationship between thromboxane and cytosolic calcium modifiers in rat platelets. 165 70

Low molecular weight (LMW) heparin preparations have unknown distributions of ATIII-binding material, so mean molecular weights as such might bear little information on their anti-factor Xa and anti-thrombin activities, and on the neutralization of these activities by platelet factor 4 (PF4). These properties were investigated in pure systems with proteins of human origin. Pseudo-first order rate constants of inactivation of factor Xa and thrombin by antithrombin III were determined as function of heparin concentration, in the presence of 4.0 mM CaCl2. Despite a large variation in the mean molecular weights, the ratios of the anti-factor Xa over the anti-thrombin activities were essentially the same for the 4th International Standard for heparin (0.46), the 1st International Standard for LMW heparin (0.32), CY216 (0.42) and enoxaparin (0.50). The ultra LMW heparin CY222 had only a 2-times higher ratio (0.98). Analysis of CY216 subfractions, obtained by gel filtration, showed that the heparin molecules of the upper region of the molecular weight distribution are responsible for the anti-thrombin, but also to a large extent for the anti-factor Xa activities. The results indicate that depolymerization of unfractionated heparin does not result in an increased anti-factor Xa/anti-thrombin ratio, because in the presence of Ca(2+)-ions the rate constants of inactivation of factor Xa are lowered as compared to those of native heparin. PF4-dependent neutralization of anti-factor Xa and anti-thrombin activities of fixed concentrations of the LMW heparins was studied by measuring rate constants as function of PF4 concentration.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Low molecular weight heparin-catalyzed inactivation of factor Xa and thrombin by antithrombin III--effect of platelet factor 4. 166 94

When the global coagulation tests Quick's test, aPTT and thrombin time are carried out on dogs using reagents from the human medical sector according to the manufacturer's instructions, they react insensitively to deficiencies of the corresponding coagulation factors or to the presence of fibrin degradation products. In the present study it was attempted to develop sensitive global tests for the dog by modifying these tests. The Quick's test reacted apparently more sensitively after a 1:4 dilution of the test plasma with physiological saline solution. The sensitivity of the aPTT was demonstrated by the addition of BaSO4-adsorbed plasma to normal plasma. It seems to be particularly advantageous to combine a 1:3 plasma dilution with a reduction in the CaCl2-concentration to 5 mmol/l. Likewise, plasma dilution allows the thrombin time to react more sensitively in the presence of increased concentrations of fibrin degradation products. The aPTT and thrombin time are, as in human beings, suited for the control of a heparin therapy. However, since prolongation of the coagulation times is considerably dependent on the test chosen, the heparin sensitivity of the test should be determined in vitro prior to use.
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PMID:[Modification of the global coagulation tests for the dog. Significance for the monitoring of heparin therapy]. 175 73

Fibrin glue is a topical biological adhesive, the effect of which imitates the final stages of coagulation. The glue consists of a solution of concentrated human fibrinogen which is activated by the addition of bovine thrombin and calcium chloride. The resultant clot aids haemostasis and tissue sealing and is completely absorbed during wound healing without foreign body reaction or extensive fibrosis. The fibrinogen component of fibrin glue can be produced from fresh frozen plasma obtained from single unit donations thereby reducing the risks of transfusion transmitted infections encountered by exposure to pools from large numbers of donors. Methods involving precipitation of fibrinogen by cryoprecipitation, polyethylene glycol or ammonium sulphate have been described and evaluated. The risk of transmission of infection can be further reduced by using plasma from 'accredited donors' who are plasma donors regularly tested for ALT and markers of viral infection or by use of fibrinogen prepared in advance of surgery from autologous blood. The second component, a mixture of thrombin and CaCl2, is quantitatively and qualitatively well defined and commercially available (Armour Pharmaceutical Co., Thrombinar (bovine thrombin]. Thrombin is applied to the operation site simultaneously and in equal volume to the fibrinogen but from a separate syringe. In the UK a commercial heat treated fibrin glue prepared from pooled plasma is available on a doctor/named patient basis (Tisseel, Immuno, Vienna). The haemostatic and adhesive properties of fibrin glue can be employed in virtually every surgical specialty. The usefulness of the glue is particularly well documented in the fields of cardiovascular surgery, ENT and neurosurgery.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Fibrin glue. 178 83

The influence of Extrinsic pathway inhibitor (EPI) on global clotting times of plasma was studied using activity-blocking IgG antibodies. Dilute tissue thromboplastin (TP) clotting times in plasma collected after intravenous injection of heparin were dramatically shortened by the addition of anti-EPI IgG. Anti-EPI IgG shortened the TP times to a lesser degree in plasma heparinized in vitro. Compared to plasma heparinized in vitro, the TP clotting times were markedly prolonged in post-heparin plasma of equal heparin concentration. Addition of anti-antithrombin IgG reduced the clotting times somewhat more than did anti-EPI IgG, particularly in normal plasma. In plasma from patients with cancer, about equal effect was obtained by blocking either EPI or antithrombin. These clotting time studies suggested that much of the anticoagulant effect caused by injection of heparin depended on EPI. This was confirmed by recording the release of fibrinopeptide A (FPA), as marker of thrombin generation, following addition of TP and CaCl2 to citrated blood. Thrombin generation was delayed and markedly reduced in post-heparin blood compared to that in normal blood. After incubating post-heparin citrated blood with anti-EPI IgG, the generation of FPA was more rapid; the amounts released 30 seconds after addition of TP were 6 times greater (36 vs 6 ng/ml) than in post-heparin blood without anti-EPI IgG. The subsequent FPA values were midway between pre-injection and post-heparin values. In conclusion, between one third and one half of the inhibition of TP-initiated coagulation in post-heparin plasma depends on EPI. This inhibition is mainly due to inactivation of the factor VIIa-TP complex. A small, but distinct contributing effect observed in the APTT assay (and hence no TP) indicates that even increased inactivation of activated factor X contributes. In cancer patients, these EPI-heparin interactions contribute even more to the anticoagulant effects of heparin.
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PMID:The anticoagulant effect in heparinized blood and plasma resulting from interactions with extrinsic pathway inhibitor. 181 36

Binding of iodine-125-labeled thrombin to fibrin clots from two siblings with juvenile stroke was 30% of normal, and abnormally high amounts of the radioligand (not adsorbed by fibrin) were found in the supernatant. In concordance with this finding, supernatants from the patients' fibrin clots caused abnormal enhancement of platelet aggregation, ATP secretion, and binding of 125I-fibrinogen to platelets exposed to subthreshold concentrations of ADP or epinephrine. Hirudin suppressed the enhancing effect of the patients' supernatants, and substitution of gamma-thrombin for alpha-thrombin led to normalization of platelet responses. Under some experimental conditions, degradation of the patients' fibrinogen by plasmin was impaired. However, the euglobulin lysis time, the rate of fibrin degradation by plasmin, and the lysis of the patients' plasma clots by human melanoma tissue-type plasminogen activator were normal. Patients' plasmas, as well as purified fibrinogen, showed a prolonged thrombin time (partially corrected by 10 mM CaCl2) and an impaired release of fibrinopeptide A in response to thrombin. However, the release in response to reptilase was normal, and the reptilase, ancrod, and thrombin coagulase times were within control (normal) values. In addition, the patients' fibrinogen showed normal polymerization of preformed fibrin monomers, normal sialic acid content, and normal binding to ADP or epinephrine-stimulated platelets. Our studies support the concept that thrombin and platelets play an important role in the occurrence of stroke in these patients and suggest a direction to be followed to identify the mechanism(s) contributing to thrombosis in subjects with abnormal fibrinopeptide release.
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PMID:A role for platelets and thrombin in the juvenile stroke of two siblings with defective thrombin-adsorbing capacity of fibrin(ogen). 182 31

Among the vitamin K-dependent plasma proteins, only protein S contains the post-translationally modified amino acid erythro-beta-hydroxyasparagine (Hyn). Protein S also contains erythro-beta-hydroxyaspartic acid (Hya). The function of these unusual amino acids, located in the epidermal growth factor-like domains, is unknown. To determine if these post-translational modifications contribute to the functional integrity of human protein S (HPS), recombinant human protein S lacking Hya and Hyn (rHPSdesHya/Hyn) was purified from the medium of human kidney 293 cells that were transfected with HPS cDNA and grown in the presence of the hydroxylase inhibitor 2,2'-dipyridyl. Solution-phase equilibrium binding studies revealed that rHPSdesHya/Hyn binds C4b-binding protein (C4BP) in a manner indistinguishable from recombinant HPS and plasma-derived HPS, exhibiting a Kd in the presence of 2 mM CaCl2 of approximately 0.7 nM and a Kd in the presence of 4 mM EDTA approximately 10-fold higher. In a purified component system, rHPSdesHya/Hyn displayed normal anticoagulant cofactor activity in the activated protein C-catalyzed inactivation of coagulation factor Va bound in the prothrombinase complex. In addition, digestion of rHPSdesHya/Hyn with thrombin in the presence of EDTA appeared normal, and 2 mM CaCl2 prevented the cleavage. Together these results suggest that the post-translational modifications of Asn and Asp residues are not necessary for the macromolecular or Ca2+ interactions associated with the anticoagulant and C4BP binding characteristics of HPS.
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PMID:beta-Hydroxyaspartic acid and beta-hydroxyasparagine residues in recombinant human protein S are not required for anticoagulant cofactor activity or for binding to C4b-binding protein. 183 48

The effect of alpha-thrombin and ADP on calcium mobilization and alpha-granule release in individual platelets was investigated by flow cytometry. alpha-Thrombin (4.5 nM) caused a uniform rise of free cytosolic calcium ([Ca2+]i) among indo-1-loaded human platelets. Despite the uniformity of this effect, approximately 20% of the cells failed to secrete alpha-granule content, as shown by binding of fluorescein isothiocyanate (FITC)-conjugated S12 monoclonal antibody. ADP (10 microM) caused a similar brisk and uniform rise of calcium but did not increase S12 binding to any platelets. On the other hand, with alpha-thrombin (0.5 nM), calcium mobilization was heterogeneous and paralleled granule release. [Ca2+]i increased rapidly in some platelets, while only slowly in others. When an electronic gate was set according to FITC-S12 fluorescence, cells with a greater secretory response proved to be those with a higher calcium level. With both alpha-thrombin and ADP, chelation of external calcium by EGTA (2 mM) reduced calcium response of individual cells. NiCl2 (1 mM) also inhibited calcium rise of individual platelets to the same extent as EGTA (2 mM) in spite of the presence of 1 mM CaCl2 in the extracellular media. The effects of EGTA and NiCl2 were not limited to a particular subpopulation of cells. These data suggest that the putative Ni2(+)-inhibitable divalent cation channel(s) may be responsible for the increased influx of calcium that occurs during platelet activation by alpha-thrombin and ADP. It appears that these calcium channels contribute to the elevation of [Ca2+]i among virtually all the platelets.
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PMID:Quasi-simultaneous measurement of ionized calcium and alpha-granule release in individual platelets. 184 85

Diets containing high levels of monounsaturated, n-6 polyunsaturated and n-3 polyunsaturated fatty acids were fed to Wistar rats. This resulted in decreases in the arachidonate content in platelet phospholipids to 91%, 79% and 51% respectively of the level found after feeding a diet rich in saturated fatty acids. In the presence of CaCl2, collagen- and thrombin-induced aggregation of washed platelets from the saturated-fat dietary group (with highest level of arachidonate) was low compared with that of platelets from the other dietary groups, despite a relatively high production of thromboxane B2. On the other hand, n-3 polyunsaturated fatty acids in the diet resulted in platelets aggregating actively, but producing low levels of levels of thromboxane B2. When indomethacin-treated rat platelets were activated with the thromboxane A2 analogue U46619, the presence of a second agonist such as collagen. ADP or thrombin was necessary for aggregate formation. U46619-induced aggregation in combination with either co-activator was relatively low in arachidonate-rich platelets, and was higher in platelets with a low arachidonate content. Similarly, phospholipase C-catalysed formation of L-myo-inositol phosphates was higher in platelets with a low arachidonate content. We conclude that the ability of platelets to react with thromboxane A2 is modified by diet in such a way that a decreased substrate-limited generation of thromboxane A2 is compensated for by an increased response to thromboxane, and vice versa. No significant differences were detected in the binding of U46619 or SQ29548 to platelets from the various dietary groups. Therefore the changed response seems not to be caused by modified properties of the thromboxane A2/prostaglandin H2 receptors, but by altered transduction of the thromboxane signal.
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PMID:Dietary fat modifies thromboxane A2-induced stimulation of rat platelets. 189 33


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