Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.21.5 (thrombin)
 33,306 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Binding of a coagulation factor VIII to phosphatidylserine-containing membranes is critical for exerting its cofactor activity. The use of surface plasmon resonance allows studying factor VIII interaction with immobilized phospholipids. In the present study we compared factor VIII-binding properties of phospholipid surfaces immobilized on L1 and HPA Biacore chips in the form of a flexible bilayer and rigid monolayer, respectively. We demonstrated that immobilized phospholipid surfaces with physiological contents of PS and PE formed on L1 but not on HPA chip closely mimic intact phospholipid vesicles in their factor VIII and thrombin-activated factor VIII (factor VIIIa) binding properties.
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PMID:Comparison of the properties of phospholipid surfaces formed on HPA and L1 biosensor chips for the binding of the coagulation factor VIII. 1146 Oct 13

TAFI (thrombin activatable fibrinolysis inhibitor) down regulates fibrinolysis after activation by relatively high concentrations of thrombin generated during coagulation via thrombin mediated factor XI activation and subsequent activation of the intrinsic pathway. It is this secondary burst of thrombin that is severely diminished in haemophilia A, a deficiency of coagulation factor VIII. We therefore investigated the role of TAFI in haemophilia A by measuring the clot lysis times of tissue factor induced fibrin formation and tPA mediated fibrinolysis. In haemophilia A plasma clot lysis times were normal at relatively high tissue factor concentrations but severely decreased at moderate to low tissue factor concentrations, indicating that the thrombin generation via the extrinsic pathway was insufficient to activate TAFI. Addition of factor VIII, TAFI or thrombomodulin restored the clot lysis times at low tissue factor concentrations. This confirms the hypothesis that the bleeding disorder in haemophilia A is not merely a defect in the initial clot formation but is in fact a triple defect: reduced thrombin formation via the extrinsic pathway at low tissue factor concentrations, a reduced secondary burst of thrombin generation via the intrinsic pathway and a defective down regulation of the fibrinolytic system by the intrinsic pathway.
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PMID:The defective down regulation of fibrinolysis in haemophilia A can be restored by increasing the TAFI plasma concentration. 1168 21

Inhibitor antibodies of blood coagulation factor VIII (FVIII) impair FVIII replacement therapy, constituting a serious complication in haemophilic patients. anti-FVIII antibodies may also develop in a variety of disease-associated autoimmunity. Mapping of human FVIII inhibitors in haemophilia A or autoantibody origin have delineated three major clusters of B-cell inhibitory epitopes (domain A2, A3 and C2). Inhibitory and non-inhibitory FVIII antibodies have also been described in plasma of healthy donors and pools of immunoglobulins. The purpose of this study was to use synthetic FVIII-peptides to more closely define regions of the molecule targeted by natural anti-FVIII antibodies. Predictive algorithms were used for defining the positions of potential continuous epitopes. To investigate the presence of peptide-reactive antibodies in normal plasma pools of healthy donors, a plasma fraction (Cohn fraction II+III) containing all IgG subclasses was purified by affinity chromatography on peptide-Sepharose columns. The results of ELISAs and Western blotting experiments (with the selected peptides and well-defined recombinant FVIII thrombin fragments) confirmed the reaction specificities of the affinity-purified human antibodies. For each IgG preparation, the isotopic subclass was also determined. In the clotting assay, several IgG preparations showed neutralising activity in a dose-dependent manner. Our observations support the recent hypothesis that FVIII inhibitors in haemophilia A and autoimmune disease may originate from the proliferation of natural FVIII-specific B-cell clones.
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PMID:Mapping of natural anti-factor VIII antibodies in plasma pools from healthy donors: use of rationally designed synthetic peptides. 1185 20

Bothrombin, a snake-venom serine protease, specifically cleaves fibrinogen, releasing fibrinopeptide A to form non-crosslinked soft clots, aggregates platelets in the presence of exogenous fibrinogen and activates blood coagulation factor VIII. Bothrombin shares high sequence homology with other snake-venom proteases such as batroxobin (94% identity), but only 30 and 34% identity with human alpha-thrombin and trypsin, respectively. Single crystals of bothrombin have been obtained and X-ray diffraction data have been collected at the Laboratorio Nacional de Luz Sincrotron to a resolution of 2.8 A. The crystals belong to the space group P2(1)2(1)2(1), with unit-cell parameters a = 94.81, b = 115.68, c = 155.97 A.
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PMID:Crystallization of bothrombin, a fibrinogen-converting serine protease isolated from the venom of Bothrops jararaca. 1203 9

The interdomain acidic region a1 is a unique structural feature of coagulation factor VIII (FVIII) and may mediate the proteolytic activation of FVIII and the inactivation of FVIIIa. We report an individual with a Tyr346-->Cys substitution within region a1, who presented with a one-stage FVIII activity (FVIII:C) of 0.34 iu/ml (normal range 0.5-2.0) but normal two-stage FVIII:C and FVIII antigen values. In a factor Xa (FXa)-generation assay for FVIII in which the activation time with thrombin was varied, the variant plasma showed normal FVIII:C at both short and long activation times. However, at intermediate activation times the FXa generation of the variant plasma was less than that of normal pooled plasma. In a modified one-stage FVIII:C assay in which partially purified FVIII was activated with thrombin at low concentrations, the variant FVIII showed less activation than wild-type FVIII, although this defect corrected with increasing concentrations of thrombin. When partially purified variant FVIII was activated with a large molar excess of thrombin, the subsequent rate of decay of FVIII:C was greater for variant FVIII. The complex defects in activation and inactivation displayed by FVIII Tyr346-->Cys support the hypothesis that the a1 sequence is a key regulator of FVIII activity.
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PMID:A Tyr346-->Cys substitution in the interdomain acidic region a1 of factor VIII in an individual with factor VIII:C assay discrepancy. 1213 51

Hemophilia A is the inherited bleeding disorder that results from mutation of blood coagulation factor VIII (fVIII). Described here is the generation of a regulated expression system producing recombinant murine fVIII. Murine B-domainless fVIII was expressed at a peak level of 4 units/106 cells/24 h in serum-free media. Subsequently, a two-step purification procedure resulted in 5,300-fold enrichment and a 70% yield. Highly purified recombinant murine fVIII had a specific coagulant activity of 660 units per nanomole. It underwent proteolytic processing by thrombin to yield an activated heterotrimer that demonstrated significantly greater stability than activated human fVIII. Recombinant murine fVIII was utilized to generate an anti-fVIII polyclonal antibody. Intravenous injection of recombinant murine fVIII into hemophilia A mice failed to induce a significant anti-fVIII immune response using a schedule that yielded high titer inhibitory antibodies to human fVIII. This may provide an important model for the study of immune tolerance to fVIII.
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PMID:Expression and characterization of recombinant murine factor VIII. 1235 75

There is now convincing evidence that a high level of coagulation factor VIII is an important risk factor for venous thromboembolism. A factor VIII plasma concentration above 1500 IU/l is associated with an almost 5-fold risk for a first episode of venous thrombosis. In thrombosis patients high factor VIII has been shown to persist over time and is not related to an acute phase reaction. High factor VIII is also an important risk factor for recurrence of venous thrombosis. In a prospective cohort study factor VIII levels exceeding the 90th percentile of the patient population conferred an almost 7-fold risk of recurrent venous thrombosis. The pathomechanisms leading to venous thrombosis in patients with high factor VIII are still unclear. In many patients, however, a biochemically detectable hypercoagulable state (as represented by elevated levels of the coagulation activation marker prothrombin thrombin fragment F1.2) was demonstrated. The optimal duration of secondary thromboprophylaxis for patients with high factor VIII levels is uncertain. We currently perform an interventional trial comparing conventional to extended anticoagulation. Reduction of factor VIII by administration of a non-selective ss-receptor blocker might be a promising therapeutic concept which is currently under investigation.
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PMID:High factor VIII and the risk of venous thromboembolism. 1256 99

Hyperactivity of coagulation factor VIII (fVIII) marks hypercoagulation. FVIII enhances activity of factor IX and their combination activates factor X, which is of primary importance in prothrombin transformation into thrombin, on the phospholipid membrane. The activity of fVIII was studied in 28 patients (26 women, 2 men, mean age 49.6 +/- 7.8 years) with Sneddon's syndrome (SS). SS manifests clinically similarly to primary antiphospholipid syndrome (PAS). The leading of them are ischemic disorders of cerebral circulation (IDCC) and advanced livedo present in all the examinees. Hyperactivity of fVIII was registered in 21 (75%) of 28 patients. Most of thrombosis-related symptoms occurred more frequently in patients with high than normal activity of fVIII: ischemic strokes (91% vs 57%, p > 0.05), repeated strokes (71% vs 0%, p = 0.0014), transient IDCC (76% vs 57%, p > 0.05), vascular dementia (43% vs 0%, p > 0.05), ischemic heart disease (43% vs 0%, p > 0.05), thickening of heart valves according to echocardiography (91% vs 57%, p > 0.05), peripheral venous thromboses (24% vs 0%, p > 0.05). In high fVIII activity cardiolipin antibodies occurred more rarely (24% vs 43%, p > 0.05) but lupus anticoagulant was seen more often (47% vs 14%, p > 0.05). High fVIII activity was in 8 of 12 aPL-negative patients. It is demonstrated that elevated fVIII activity is an essential mechanism of thrombosis development in SS. The cause of this enhanced activity is suggested to be special aPL in interaction with which fVIII becomes insensitive to inactivation with protein C. The activity of protein C was normal in all the cases.
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PMID:[Clotting factor VIII in Sneddon syndrome]. 1459 91

Peptides that mimic protein epitopes are interesting drug candidates. However, the design of effective peptidic drugs is difficult for several reasons, such as the fast degradation of peptides, their high flexibility, and thus high entropy loss on binding to the target. We therefore propose an in silico method for the automated design of peptides that are optimal with respect to several objectives. We present a Pareto-based multiobjective evolutionary algorithm for in silico peptide design. Using a simple molecular model, we apply the method to the design of peptides that (a) mimic antibody epitopes of the proteins thrombin and blood coagulation factor VIII, respectively, that (b) are short, and (c) are conformationally stable.
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PMID:A multiobjective evolutionary method for the design of peptidic mimotopes. 1647 25

The chronic and immediate post-exercise responses in the hemostatic and fibrinolytic systems have been shown to be variable and reflect differing adaptations with ageing and responses to exercise protocols. This study investigated the effects of acute and exhaustive exercise on the amplitude and duration of hemostatic and fibrinolytic responses in young adolescent males. The sample comprised 10 sedentary boys (13.2+/-0.5 years, 55.8+/-11.3kg, 165.7+/-7.4cm), who had not exercised or received any medication for at least 2 weeks before the experiments. The subjects performed exhaustive stepping exercise, consisting of 1s up and down cycles to fatigue. When the subjects were unable to maintain the required stepping rhythm, they were given a 30s recovery period. Following each 30s recovery participants recommenced the stepping cadence until fatigue prevented them continuing. Venous blood samples were drawn before and immediately, 1 and 24h after exercise to assess the following coagulation and fibrinolytic parameters: Platelet counts, activated partial thromboplastin time (aPTT), prothrombin time (PT), coagulation factor VIII (FVIII:C), von Willebrand factor (vWF), fibrinogen concentration, thrombin-antithrombin complex (TAT), D-dimer, plasminogen activator inhibitor (PAI-1), and tissue-type plasminogen activator (t-PA). Immediately following exercise, platelet counts, aPTT, FVIII, vWF and t-PA were significantly elevated in contrast to PAI-1, which decreased significantly until 1h after exercise. FVIII and platelet counts were elevated at 1 and 24h after exercise, respectively. Only the parameters FVIII and PAI-1 did not return to baseline values during the first hour after physical exercise. When compared to adults the results revealed different rates and ranges of coagulation and fibrinolysis parameters being activated by exhaustive exercise in this group of adolescents.
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PMID:Hemostatic response to acute physical exercise in healthy adolescents. 1684 9


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