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)

Clinical and laboratory evaluation of severe bleeding can detect the presence of an intrinsic or acquired coagulation disorder. The three most common inherited coagulation disorders are factor VIII deficiency (hemophilia A), factor IX deficiency (hemophilia B), and von Willebrand's disease. Vitamin K deficiency, liver disease, and disseminated intravascular coagulation are the most common acquired disorders. A thorough clinical history is crucial to diagnosis. Screening tests that measure prothrombin time, partial thromboplastin time, thrombin time, and platelet count permit initial classification and guide selection of more specific tests. Results can then be used to determine appropriate therapy.
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PMID:Potentially catastrophic bleeding disorders. Approach to diagnosis and management. 267 67

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

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

A chromogenic substrate kit for the determination of factor VIII activity (COATEST Factor VIII) has been evaluated in five different laboratories, one of them using a semi-automated procedure. This chromogenic method was compared to one-stage clotting assays for factor VIII determination in plasmas from healthy subjects, carriers of hemophilia A, severe, mild and moderate hemophilia A as well as von Willebrand's patients. In all these cases, a high correlation between these two methods was obtained (r = 0.96-0.99, n = 385) with a good agreement of the assigned potencies at all levels of factor VIII. A good correlation (r = 0.94) was also obtained for the levels of factor VIII after infusion of concentrates in six severe hemophiliacs or after administration of DDAVP to von Willebrand's patients. The chromogenic method is insensitive to preactivation of factor VIII by thrombin, thus yielding valid potency assignments also in these situations. The precision was higher with the chromogenic method than with the one-stage clotting assays (C.V. = 2-5% vs 4-15%). Altogether, the new chromogenic substrate method has proven itself suitable for determination of factor VIII in plasma and concentrates.
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PMID:Clinical application of a chromogenic substrate method for determination of factor VIII activity. 393 77

Factor VIII (antihemophilic factor) is the protein that is deficient or defective in patients with classical hemophilia and Von Willebrand syndrome. Factor VIII in plasma is thought to be associated in a complex with the highest molecular weight multimers of another glycoprotein, Von Willebrand protein. Highly purified human factor VIII appears to have an Mr of between 200,000 and 300,000 and to consist of several polypeptide chains. The concentration of factor VIII in plasma is around 100-200 ng/ml, equivalent to around 1 nM. The purified proteins retain one or more of the known properties of factor VIII, including the acceleration of factor IXa-mediated activation of factor X, ability to be activated by thrombin and factor Xa, inactivation by activated protein C, and by human antibodies to factor VIII. Among the known clotting factors, factors VIII and V are exceptional in not possessing enzymatic activity. Factors IXa and VIII and X appear to form a functional complex, all of which need to be present and active simultaneously for optimal activation of factor X. The mechanism by which factor VIII promotes activation of factor X by factor IXa is not known, but the major effect is to increase the rate of the reaction. Following treatment of factor VIII with thrombin, a new and smaller polypeptide Mr around 70,000 +/- 5,000 is produced. Factors IXa and Xa also have been reported to activate factor VIII. It is not known whether limited proteolytic cleavage is required absolutely for the expression of factor VIII activity or if it only increases an activity already expressed by the uncleaved protein. Factor VIII is inactivated by thrombin and by activated protein C. Thus, factor VIII can be modulated by at least four of the serine proteases in the clotting system. A major goal for future research is to increase our understanding of the role in blood clotting played by factor VIII, and to apply this information to clinical problems which result from inherited abnormalities of factor VIII.
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PMID:Factor VIII: structure and function in blood clotting. 642 37

Antihemophilic factor concentrates were surveyed for amidolytic activity on the chromogenic substrates S2238, S2302, S2222, and S2251, which are sensitive to thrombin, kallikrein, factor Xa, and plasmin, respectively. For antihemophilic factor concentrates from two manufacturers, the rates of amidolysis of S2238 and S2302 were approximately an order of magnitude greater than the rates of amidolysis of S2222 and S2251. The S2238 and S2302 activities were characterized by quantitating their interactions with specific substrates or inhibitors. The Km for amidolysis of S2238 was 558 mumol/L, which is 80 times higher than for thrombin but in close agreement to the reported value for activated protein C. The S2238 activity was not inhibited by the thrombin-specific inhibitor dansylarginine N-(3-ethyl-1,5-pentanediyl)amide, nor by soybean trypsin inhibitor or micromolar concentrations of antithrombin III in the presence of heparin. The S2238 activity was inhibited by D-Phe-Pro-Arg-CH2Cl, but with an estimated second-order rate constant of 3 X 10(5) mol/L-1 minute-1, approximately 1000 times less than for thrombin. These data are consistent with the identity of the S2238 activity as activated protein C. On the other hand, the S2302 activity in antihemophilic factor concentrates was most likely attributable to kallikrein. This was based on the agreement with authentic kallikrein of the Km for S2302 of 154 mumol/L as well as by the rapid inactivation by nanomolar concentrations of the kallikrein-specific inhibitor D-Phe-Phe-Arg-CH2Cl. However, the relative resistance of the S2302 activity to inhibition by soybean trypsin inhibitor or antithrombin III and the partial inhibition by aprotinin suggested that a large proportion of the kallikrein was bound to alpha 2-macroglobulin. This was confirmed by immunoprecipitation using specific anti alpha 2-macroglobulin IgG. The potential for proteolysis of factor VIII:von Willebrand protein during its purification from antihemophilic factor concentrates was demonstrated, and the proteolyzed factor VIII coagulant species was characterized. High-pressure gel permeation chromatography of purified factor VIII:von Willebrand protein at high ionic strength resulted in two sharp peaks of factor VIII procoagulant activity. The earlier eluting peak corresponded with the void volume, and the later peak eluted with an apparent molecular weight of 53,000 daltons. Immediately after separation, the 53,000-dalton factor VIII coagulant had at least a 100-fold higher specific activity than the factor VIII coagulant present in the void volume. However, the 53,000-dalton factor VIII coagulant was labile, with a half-life of 80 minutes.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Characterization of proteases in AHF concentrates: effect on factor VIII:von Willebrand protein as assessed by high-pressure gel permeation chromatography. 643 16

Prothrombin complex concentrates (PCC) are known to carry a risk of thromboembolism. We have chosen to reassess the problem of detecting in vivo signs of activation of the clotting mechanism by assaying fibrinopeptide A (FpA) after PCC administration in hemophilic patients during bleeding episodes. FpA was significantly increased above baseline levels 15 to 60 min after the infusion of 19 doses of 5 different types of commercial PCC in 14 hemophilia B patients treated for bleeding episodes or dental extractions. A more marked increase followed 16 infusions of the activated PCC FEIBA and Auto IX in 4 hemophilia A patients with F. VIII inhibitors. There was no significant FpA change after F. VIII concentrates administered to a control group of 7 patients with hemophilia A. These findings suggest that circulation of thrombin occurs frequently after PCC administration, even though clinical manifestations of thromboembolism appear to be relatively rare.
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PMID:Increased fibrinopeptide A after prothrombin complex concentrates. 677

Partially purified (approx. 5000-fold), low molecular weight human antihemophilic factor, free of detectable Von Willebrand factor (ristocetin cofactor activity or Von Willebrand antigen), was prepared from fresh citrated plasma by limited reduction with 1 mM dithiothreitol and chromatography on Sepharose CL-4B, Sephadex G-100, and polyelectrolyte E-5. The ratio of antihemophilic factor activity to Von Willebrand factor activity or antigen was greater than 27 000 : 1. The antihemophilic factor activity could be neutralized with homologous antibody and could be further increased with thrombin. The Mr (approx. 116 000) was determined by calibrated gel permeation chromatography, electrophoresis in 5% polyacrylamide gels with sodium dodecyl sulfate and by electrophoresis in large-pore acrylamide gels without it. Since the low Mr antihemophilic factor could be prepared by treating fresh rather than fresh-frozen plasma with dithiothreitol, it was concluded that partial reduction of the antihemophilic factor with this reagent helped to maintain the antihemophilic factor in a low Mr form. When iodo[l-14C]acetamide was used to alkylate the reduced plasma proteins prior to purification, the molecular weight of the purified antihemophilic factor remained low despite numerous purification steps. By this means, one of four radioactive proteins (Mr 116 000) in the final preparation was bound specifically to homologous antihemophilic factor antibody and attributed to 14C-labeled antihemophilic factor. While the data suggest that antihemophilic factor in fresh plasma contains one or more dithiothreitol-sensitive intramolecular disulfide bonds, the possibility of disulfide linkages with other proteins(s) cannot be excluded.
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PMID:Partial purification of biologically active, low molecular weight, human antihemophilic factor free of Von Willebrand factor. I. Partial characterization and evidence for disulfide bond(s) susceptible to limited reduction. 678 56

The relationship between Factor VIII coagulant antigen (VIII:CAg) and Factor VIII-associated von Willebrand factor (VIII:vWF), and the effect of thrombin on VIII:CAg have been determined in plasma by using complexes of VIII:CAg and 125I-labeled human anti-VIII:CAg-Fab. Antibody-treated plasma samples were electrophoresed on NaDodSO4/polyacrylamide agarose gels and analyzed by autoradiography. The major VIII:CAg-125I-labeled Fab complex that persisted in NaDodSO4 had Mr 3.2 x 10(5). This Mr value was confirmed by column chromatography and sucrose density centrifugation and is presumed to reflect a free VIII:CAg of Mr 2.7 x 10(5). Minor bands were also present on autoradiograms of normal plasma corresponding to Mr values of 2.5, 1.85, and 1.7 x 10(5) (free VIII:CAg related proteins with Mr values of 2.0, 1.35, and 1.2 x 10(5), respectively). None of the VIII:CAg bands was present in plasma samples from five patients with severe hemophilia A. No radioactivity was associated with VIII:vWF multimers on NaDodSO4 gels. Thrombin treatment of normal plasma eliminated the radioactive band at 3.2 x 10(5) and increased the intensity of a band of Mr 1.7 x 10(5). Generation of this presumed VIII:CAg fragment of Mr is approximately equal to 1.2 x 10(5) coincided with a thrombin-induced increase in Factor VIII coagulant activity. These data demonstrate that the form of VIII:CAg detected in normal plasma is not covalently linked to VIII:vWF multimers and is absent in plasma from five hemophilia A patients. Thrombin-induced proteolysis of VIII:CAg can be detected in microliter quantities of normal plasma.
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PMID:Analysis of factor VIII coagulant antigen in normal, thrombin-treated, and hemophilic plasma. 679 28

Several enzymes can activate factor VII in vitro, but the protease responsible for generating factor VIIa in vivo has not been determined. Using recombinant tissue factor that has undergone a COOH-terminal truncation, a sensitive functional assay has been established for measuring plasma factor VIIa levels. To evaluate the mechanism responsible for the generation of factor VIIa in vivo, we measured the levels of this enzyme after administering purified concentrates of factor IX and factor VIII to patients with severe deficiencies of these clotting factors. In patients with hemophilia B, factor VIIa levels were initially reduced to 0.5 +/- 0.1 ng/mL and gradually increased to normal after infusing 100 U/kg of body weight (BW) of factor IX. Despite these increases, there were no significant changes in the generation of factor Xa or thrombin. In patients with hemophilia A, only a slight reduction in factor VIIa levels (2.5 +/- 1.3 ng/mL) was observed as compared with controls (3.3 +/- 1.1 ng/mL) and no significant changes were observed after factor VIII levels were normalized. The administration of recombinant factor VIIa (10 micrograms/kg BW) to patients with factor VII deficiency increased the mean circulating level of the enzyme to 118 ng/mL, but this only resulted in normalization of the levels of the activation peptides of factor IX and factor X. The above data indicate that factor IXa is primarily responsible for the basal levels of free factor VIIa generated in vivo (ie, in the absence of thrombosis or provocative stimuli) and that changes in the plasma concentrations of free factor VIIa in the blood do not necessarily lead to alterations in the extent of factor X activation.
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PMID:Determinants of plasma factor VIIa levels in humans. 757 95


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