How factor VIIa works in hemophilia.

The influence of elevated platelet concentration and recombinant factor VIIa (rFVIIa) on thrombin generation at 5 pM tissue factor (TF) in a synthetic mixture corresponding to hemophilia B (SHB) and "acquired" hemophilia B blood (AHBB) produced in vitro by an antifactor IX antibody was evaluated. (a) Thrombin generation in SHB and AHBB was delayed and reduced; (b) with 10 nM rFVIIa or 5x normal platelets (10 x 10(8)/mL) SHB and AHBB showed a slight increase in thrombin generation; (c) in the absence of TF, almost no thrombin generation was detected in SHB and AHBB in the presence of 10 nM rFVIIa and 10 x 10(8)/mL activated platelets (5x normal); (d) with TF, 10 nM rFVIIa and 3-5x normal nonactivated platelets (6-10 x 10(8)/mL), thrombin levels approaching normal values were attained. FVIIa appears to function effectively and locally by the combined effect of TF expression and platelet accumulation at the site of a vascular lesion.

Oral anticoagulation thresholds.

BACKGROUND: Monitoring patients on oral anticoagulation is essential to prevent hemorrhage and recurrent thrombosis. We studied tissue factor-induced whole-blood coagulation in patients on warfarin therapy with similar international normalized ratios (INRs). METHODS AND RESULTS: Contact pathway-suppressed whole-blood coagulation initiated with tissue factor was studied in 8 male subjects (group W) and in 1 individual multiple times (subject A). Coagulation profiles for group W showed that subjects with similar INRs had widely varying clot times (6.2 to 23 minutes) and thrombin-antithrombin III (TAT) profiles with rates of 25 to 40 nmol. L(-1). min(-1) and maximum levels varying from 192 to 349 nmol/L. The normal control group exhibited clot times of 5.7+/-0.3 minutes and TAT rates of 57+/-13 nmol. L(-1). min(-1), reaching maximum levels of 742+/-91 nmol/L. Subject A, who was stably anticoagulated at an INR of 2.1+/-0.4 for 6 months, had widely ranging profiles with clot times of 9.0 to 22.7 minutes, TAT maximums varying from 141 to 345 nmol/L, and TAT formation rates of 10 to 57 nmol. L(-1). min(-1). INR did not correlate with TAT formation. Platelet activation was decreased by anticoagulants but also displayed variability. Fibrinopeptide A generation showed threshold variability independent of the INR. Factor VIII levels were increased (P=0.03) in group W (204+/-34.4%) compared with normal control subjects (149.4+/-37.4%). A significant correlation was identified between increasing factor VIII levels and years on warfarin therapy (r=0.78, P=0.01), suggesting a possible factor VIII compensatory mechanism. CONCLUSIONS: These results suggest that control of anticoagulation in patients to a set INR therapeutic range may be less secure than anticipated. Patients with similar INRs show significant individual variability in their tissue factor coagulation response, suggesting different risks to anticoagulation when confronted with underlying vascular anomalies.

Simvastatin depresses blood clotting by inhibiting activation of prothrombin, factor V, and factor XIII and by enhancing factor Va inactivation.

BACKGROUND: The mechanism of the antithrombotic action of statins is unclear. The aim of this study was to evaluate the effects of simvastatin on the coagulation process at sites of microvascular injury. METHODS AND RESULTS: Tissue factor-initiated coagulation was assessed in blood samples collected every 30 seconds from bleeding-time wounds of 17 patients who had advanced coronary artery disease and total cholesterol levels of 224.6+/-11.8 mg/dL (mean+/-SEM). Quantitative Western blotting for time courses of fibrinogen depletion and activation of prothrombin, factor V, and factor XIII was performed before and after 3 months of simvastatin treatment (20 mg/d). Simvastatin induced reductions in total cholesterol (23%) and LDL-cholesterol (36%), which were accompanied by significant decreases in the rates of prothrombin activation (16.2+/-2.1%; P=0.004), formation of alpha-thrombin B-chain (27.4+/-1.8%; P=0.001), generation of factor Va heavy chain (29.7+/-3.1%; P=0.007) and factor Va light chain (18.9+/-1.2%; P=0.02), factor XIII activation (19.8+/-1.3%; P=0.001), and fibrinogen conversion to fibrin (72.2+/-3%; P=0.002). Posttreatment fibrinopeptides A and B concentrations, determined by using high-performance liquid chromatography, were reduced within the last 30 seconds of bleeding. The 30-kDa fragment of the factor Va heavy chain (residues 307 to 506), produced by activated protein C, and the 97-kDa fragment of the factor Va heavy chain (residues 1 to 643) were released more rapidly after simvastatin treatment. The antithrombotic actions of simvastatin showed no relationship to its cholesterol-lowering action. CONCLUSIONS: Simvastatin treatment depresses blood clotting, which leads to reduced rates of prothrombin activation, factor Va generation, fibrinogen cleavage, factor XIII activation, and an increased rate of factor Va inactivation. These effects are not related to cholesterol reduction.

Models of blood coagulation.

We have used three models to study the process of tissue factor-initiated blood coagulation. These are: synthetic 'plasma' mixtures prepared with the proteins and membranes involved in the reaction and its regulation; mathematical models based on the reaction kinetics, binding constants and stoichiometries of individual procoagulant and inhibitor reactions, and contact pathway-inhibited coagulation of minimally altered whole blood in vitro. In all of these models, the procoagulant process may be divided into two phases: an initiation phase and a propagation phase. The initiation phase is characterized by the appearance of thrombin and other coagulation enzymes, and the activation of pro-cofactors V and VIII. The propagation phase is characterized by explosive and extensive prothrombin activation. During normal blood coagulation, the bulk of thrombin generation occurs after clot formation, while most release of fibrinopeptide A is observed just at the conclusion of the initiation phase. In the case of haemophilia A and B, the initiation phase is slightly extended, while thrombin generation during the propagation phase is significantly suppressed. The clot time, as well as fibrinopeptide release, is delayed in these patients. Data obtained in our laboratory, employing the above models, indicate that they are efficient tools for blood coagulation studies.

An integrated study of fibrinogen during blood coagulation.

The rate of conversion of fibrinogen (Fg) to the insoluble product fibrin (Fn) is a key factor in hemostasis. We have developed methods to quantitate fibrinopeptides (FPs) and soluble and insoluble Fg/Fn products during the tissue factor induced clotting of whole blood. Significant FPA generation (>50%) occurs prior to visible clotting (4 +/- 0.2 min) coincident with factor XIII activation. At this time Fg is mostly in solution along with high molecular weight cross-linked products. Cross-linking of gamma-chains is virtually complete (5 min) prior to the release of FPB, a process that does not occur until after clot formation. FPB is detected still attached to the beta-chain throughout the time course demonstrating release of only low levels of FPB from the clot. After release of FPB a carboxypeptidase-B-like enzyme removes the carboxyl-terminal arginine resulting exclusively in des-Arg FPB by the 20-min time point. This process is inhibited by epsilon-aminocaproic acid. These results demonstrate that transglutaminase and carboxypeptidase enzymes are activated simultaneously with Fn formation. The initial clot is a composite of Fn I and Fg already displaying gamma-gamma cross-linking prior to the formation of Fn II with Bbeta-chain remaining mostly intact followed by the selective degradation of FPB to des-Arg FPB.