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)

The aim of this study was the development of a simple chromogenic factor VIII assay for practical clinical use. The criteria that the assay fulfils are: (1) The method is so sensitive that even 1% factor VIII in human plasma is easily detected. (2) The method is linear in the amount of factor VIII from 0 to 200% in plasma. (3) The pipetting scheme is very simple; two reagents are prepared, reagent 1 (factor IXa, thrombin, Ca2+ and phospholipids) and reagent 2 (factor X). Then we pipet at t = 0 s, 100 microliters diluted plasma + 100 microliters reagent 1 in a reaction tube; at t = 30 s, 100 microliters reagent 2 in the same tube and at t = 90 s, 200 microliters of the reaction mixture in a cuvette with 700 microliters EDTA buffer (stop buffer) and the formed factor Xa is measured with a chromogenic substrate. (4) The reaction components are stable during at least a whole working day. Factor VIII was measured in an assay using bovine clotting factors, so one avoids the risk of viral infections, which one might catch by working with clotting factors isolated from human plasma.
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PMID:Development of a simple chromogenic factor VIII assay for clinical use. 250 7

We report a study on the importance of factor IX activation in thromboplastin-dependent coagulation in plasma. Diluted, CaCl2-containing thromboplastin solutions at constant phospholipid concentration were used to trigger the coagulation in plasma from patients with congenital factor IX and factor VIII deficiency in the presence and absence of added factors IX and VIII, and the generation of thrombin activity was monitored. When coagulation is triggered with the high thromboplastin concentrations normally used in clinical routine tests, the generation of thrombin activity in plasma of patients with congenital factor IX deficiency before and after reconstitution with purified factor IX appears identical. When, however, coagulation is triggered with low thromboplastin concentrations, a clear dependency of the generation of thrombin activity on the concentration of factor IX becomes evident at factor IX concentrations lower than 30 nM (about 40% clotting factor activity). Factor VIII is a compulsory cofactor for this factor IX activity because the prothrombinase activity at optimal factor IX concentration is still critically dependent upon the amount of factor VIII present. The lower the amount of thromboplastin, the higher the importance of factor IX and factor VIII activation in thromboplastin-dependent coagulation. This suggests a role of this pathway in pathophysiological thrombin formation.
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PMID:Importance of factor-IX-dependent prothrombinase formation--the Josso pathway--in clotting plasma. 262 Aug 66

The levels of protein C (PC) and other coagulation factors were monitored during endotoxin-induced disseminated intravascular coagulation (DIC) in the dog. Initial evaluation of the effectiveness of intradermal administration of bolus endotoxin quantities into the dog, demonstrated induction of DIC in the canine, without the severe side effects associated with bacterial sepsis. Quantitative determination of canine plasma protein C levels were performed using a multiple step amidolytic assay, that included a specific precipitation of the vitamin K-dependent proteins from citrated plasma, followed by thrombin activation (and neutralization) and subsequent measurement of the activated protein C (APC) by chromogen hydrolysis. This investigation demonstrated, that over a twenty-four hour interval, intradermal administration of endotoxin produces a gradual decrease in the PC activity levels, concomitant with a significant reduction in the Factor V, Factor VIII and fibrinogen levels and platelet count, and a prolongation of the Prothrombin Time and Partial Thromboplastin Time. During the first 6 hours, protein C levels fell below the pre-levels and remained significantly lower in the surviving dogs. Thus, this endotoxin-induced DIC animal model permits evaluation of various hemostatic parameters, yet diminishes the severe clinical findings associated with DIC.
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PMID:Protein C activity levels in endotoxin-induced disseminated intravascular coagulation in a dog model. 278 30

We have proposed previously that the steps in coagulation most sensitive to inhibition by heparin are the thrombin-dependent amplification reactions, and that prothrombinase is formed in heparinized plasma only after Factor Xa activates Factor VIII and Factor V. These propositions were based on the demonstration that both heparin and Phe-Pro-Arg-CH2Cl completely inhibited 125I-prothrombin activation for up to 60 s when contact-activated plasma (CAP) was replenished with Ca2+. Furthermore, the addition of thrombin to CAP before heparin or Phe-Pro-Arg-CH2Cl completely reversed their inhibitory effects. Additional support for the above hypotheses is provided in this study by demonstrating that, when the activity of thrombin is suppressed by heparin (indirectly) or by Phe-Pro-Arg-CH2Cl (directly), exogenous Factor Xa reverses the ability of these two agents to inhibit prothrombin activation. Prothrombin activation was initiated by adding Factor Xa (1 nM) or thrombin (1 or 10 nM) simultaneously with CaCl2 to CAP. In the absence of heparin or Phe-Pro-Arg-CH2Cl, prothrombin activation was seen 15 s later in either case. Heparin failed to delay, and Phe-Pro-Arg-CH2Cl delayed for 15 s, prothrombin activation in CAP supplemented with Factor Xa. In contrast, heparin and Phe-Pro-Arg-CH2Cl completely inhibited prothrombin activation for at least 45 s in CAP supplemented with 1 nM-thrombin. Heparin failed to delay prothrombin activation in CAP supplemented with 10 nM-thrombin, whereas Phe-Pro-Arg-CH2Cl completely inhibited prothrombin activation in this plasma for 45 s. These results suggest that in CAP: (1) Factor Xa can effectively activate Factor VIII and Factor V when the proteolytic activity of thrombin is suppressed; (2) heparin-antithrombin III is less able to inhibit Factor Xa than thrombin; (3) suppression of the thrombin-dependent amplification reactions is the primary anticoagulant effect of heparin.
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PMID:Unfractionated heparin inhibits thrombin-catalysed amplification reactions of coagulation more efficiently than those catalysed by factor Xa. 292 7

Activated protein C (APC) acts as a potent anticoagulant enzyme by inactivating Factor V and Factor VIII. In this study, protein S was shown to increase the inactivation of purified Factor VIII by APC ninefold. The reaction rate was saturated with respect to the concentration of protein S when protein S was present in a 10-fold molar excess over APC. The heavy chain of Factor VIII was cleaved by APC and protein S did not alter the degradation pattern. Factor VIII circulates in a complex with the adhesive protein von Willebrand factor. When purified Factor VIII was recombined with von Willebrand factor, the inactivation of Factor VIII by APC proceeded at a 10-20-fold slower rate as compared with Factor VIII in the absence of von Willebrand factor. Protein S had no effect on the inactivation of the Factor VIII-von Willebrand factor complex by APC. After treatment of this complex with thrombin, however, the actions of APC and protein S towards Factor VIII were completely restored. In hemophilia A plasma, purified Factor VIII associated with endogenous von Willebrand factor, resulting in a complete protection against APC (4 nM). By mixing hemophilic plasma with plasma from a patient with severe von Willebrand's disease, we could vary the amount of von Willebrand factor. 1 U of von Willebrand factor was needed to provide protection of 1 U Factor VIII. Also in plasma from patients with the IIA-type variant of von Willebrand's disease, Factor VIII was protected. In von Willebrand's disease plasma, which was depleted of protein S, APC did not inactivate Factor VIII. These results indicate that protein S serves as a cofactor in the inactivation of Factor VIII and Factor VIIIa by APC and that von Willebrand factor can regulate the action of these two anticoagulant proteins.
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PMID:Inactivation of human factor VIII by activated protein C. Cofactor activity of protein S and protective effect of von Willebrand factor. 297 73

A new method for isolation and culture of endothelial cells from bovine coronary artery (BCoAEC) is presented. This method involves in situ perfusion and digestion of main coronary arteries with a collagenase solution. The isolated cells were cultured and maintained through many cell passages in Dulbecco's Modified Eagle's Medium supplemented with fetal bovine serum derived from either whole blood or plasma. Confirmation of these cells' endothelial origin was obtained by demonstration of typical morphologic and growth characteristics of endothelium, immunofluorescent staining with antibodies to von Willebrand factor (Factor VIII: vWF), and measurement of plasminogen activator (PA). In addition, production of PA was inhibited by enzymatically active thrombin as has been previously described with bovine aortic endothelial cells in culture.
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PMID:Bovine coronary artery endothelium: in vitro culture and production of plasminogen activator. 308 24

Factor VIII activation by thrombin is the result of a proteolytic cleavage of the procoagulant component. These studies examine the effect of human antibody on this activation step in a solid phase immunoadsorbent assay system. Radiolabeled factor VIII antibody: factor VIII protein immune complexes were bound to agarose beads by mouse monoclonal antifactor VIII R:Ag antibody. The incubation of these bound labeled immune complexes with high ionic strength buffers (1 M NaCl, 0.24 M CaCl2), or with acidic buffers (0.01 M glycine-0.1 M NaCl, pH 3.0 or 3.5), or with trypsin (1, 5, and 20 mg per ml) dissociated 14 to 62 percent of the bound radiolabel. Thrombin at a concentration of 0.05 U per ml, however, only dissociated 2.9 percent of the label, an amount not significantly different than borate buffered saline control. It is concluded that inactivation of factor VIII is the result of human antibody inhibition of thrombin-induced proteolysis of factor VIII procoagulant protein.
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PMID:Mechanism of factor VIII inactivation by human antibodies. IV. Antibody binding prevents factor VIII proteolysis by thrombin. 309 25

Cultures of mesothelial cells from bovine pericardium were established and their arachidonate metabolism was characterized. The identification of the cultured cells was based on morphological observations, and by electrophoretic analysis of cytoskeletal proteins, which demonstrated a pattern previously reported for mesothelial cells. Factor VIII-related antigen was present by indirect immunofluorescence, but the cells had no thrombomodulin activity. The cultured pericardial cells metabolized arachidonic acid to 6-ketoprostaglandin F1 alpha and a small amount of prostaglandin E2. The same metabolites were produced by pieces of intact parietal pericardium but not by pieces from which mesothelium had been removed. The cultured mesothelial cells produced 94.6 +/- 60.4 (mean +/- S.D.) ng/mg (n = 3) cell protein of 6-ketoprostaglandin F1 alpha in response to the calcium ionophore A23187, 117.3 +/- 13.6 ng/mg (n = 3) with exogenous arachidonic acid, 18.3 +/- 11.3 ng/mg (n = 5) with bradykinin, 8.4 +/- 4.3 ng/kg (n = 4) with histamine and 11.2 +/- 9.7 ng/mg (n = 5) with thrombin. All of these values were significantly higher (P less than 0.05) than the control (2.1 +/- 1 ng/mg; n = 5). From these results, we conclude that the mesothelial cells account for the arachidonate metabolism in the pericardium. The production of prostaglandin I2 occurs in response to physiological or pathological, agonists, and is substantial. That is, it is approximately the same as endothelial cells. The release of eicosanoids by mesothelial cells into the pericardial space may have a significant role in cardiac physiology and pathology.
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PMID:Culture of mesothelial cells from bovine pericardium and characterization of their arachidonate metabolism. 311 6

Factor VIII functions as a cofactor in the intrinsic coagulation pathway and must first be activated to function optimally in this capacity. Low concentrations of thrombin activate factor VIII, and the presence of stimulated platelets is known to enhance the activation of factor VIII complexed to von Willebrand factor. The current studies show that platelets stimulated by thrombin, collagen, or calcium ionophore will increase the activation of isolated factor VIII by thrombin. Ongoing platelet release is not necessary for the enhanced factor VIII activation, nor is platelet von Willebrand factor or platelet membrane glycoproteins Ib or IIb/IIIa. Platelet membrane phospholipids, on the other hand, are important for the enhanced activation of factor VIII by thrombin because the effect of stimulated platelets is abolished by incubation of the stimulated platelets with phospholipases. These results suggest that the enhanced activation of factor VIII by thrombin in the presence of stimulated platelets may be mediated by factor VIII binding to platelet phospholipid or to a receptor whose functional integrity is dependent on surrounding membrane phospholipid.
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PMID:Phospholipase abolishes the effect of stimulated platelets on the thrombin activation of factor VIII. 312 Aug 21

Proteolytic activation of human Factor VIII (VIIIa) by thrombin was correlated with the generation of a light-chain-derived 73(71) kDa polypeptide plus polypeptides of 51 and 43 kDa derived from the heavy chain(s). Factor VIIIa activity was unstable and decayed to an inactive form (VIIIi) in the absence of additional proteolysis. The subunit structure of Factor VIIIa was studied using two rapid chromatographic methods. Gel filtration of Factor VIIIa showed that coagulant activity was correlated with the 73 and 51 kDa polypeptides which co-eluted with a Stokes radius of 46 A and was separated from the 43 kDa fragment. A similar polypeptide elution pattern was obtained for Factor VIIIi following prolonged incubation with thrombin. Gel filtration of EDTA-inactivated Factor VIIIa showed that the 73 and 51 kDa polypeptides eluted separately with Stokes radii of 32 and 38 A, respectively. Anion-exchange HPLC of Factor VIIIa resolved the coagulant-active 73/51 kDa dimer from the inactive dimer. The labile activity of Factor VIIIa was stabilized by chemical crosslinking reagents, presumably by formation of intra-chain crosslinks. A native Mr of 136,000 for Factor VIIIa, calculated from its Stokes radius (46 A) and sedimentation coefficient (7.1 S), was compatible with a non-covalent dimer composed of 73 and 51 kDa subunits.
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PMID:Subunit structure of thrombin-activated human factor VIIIa. 312 36


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