Kathleen E. Brummel

The dynamics of thrombin formation.

The central event of the hemostatic process is the generation of thrombin through the tissue factor pathway. This is a highly regulated, dynamic process in which thrombin itself plays many roles, positively and negatively its production and destruction. The hemostatic process is essential to normal physiology and is also the Achilles heel of our aging population. The inappropriate generation of thrombin may lead to vascular occlusion with the consequence of myocardial infarction, stroke, pulmonary embolism, or venous thrombosis. In this review, we summarize our present views regarding the tissue factor pathway by which thrombin is generated and the roles played by extrinsic and intrinsic factor Xa generating complexes in hemostasis and the roles of the stoichiometric and dynamic inhibitors that regulate thrombin generation.

What is all that thrombin for

Summary.  The hemostatic process initiated by the exposure of tissue factor to blood is a threshold limited reaction which occurs in two distinct phases. During an initiationphase, small amounts of factor (F)Xa, FIXa and thrombin are generated. The latter activates the procofactors FV and FVIII to the activated cofactors which together with their companion serine proteases form the intrinsic FX activator (FVIIIa‐FIXa) and prothrombinase (FVa‐FXa) which generate the bulk of FXa and thrombin during a propagation phase. The clotting process (fibrin formation) occurs at the inception of the propagation phase when only 5‐10 nM thrombin has been produced. Consequently, the vast majority (greater than 95%) of thrombin is produced after clotting during the propagation phase of thrombin generation. The blood of individuals with either hemophilia A or hemophilia B has no ability to generate the intrinsic FXase, and hence is unable to support the propagation phase of the reaction. Since clot based assays conclude before the propagation phase they are not sensitive to hemophilia A and B. The inception and magnitude of the propagation phase of thrombin generation is influenced by genetic polymorphisms associated with thrombotic and hemorrhagic disease, by the natural abundance of pro‐ and anticoagulants in healthy individuals and by pharmacologic interventions which influence thrombotic pathology. Therefore, it is our suspicion that the performance of the entire process of thrombin generation from initiation through propagation and termination phases of the reaction are relevant with respect to both hemorrhagic and thrombotic pathology.

Simvastatin Depresses Blood Clotting by Inhibiting Activation of Prothrombin, Factor V, and Factor XIII and by Enhancing Factor Va Inactivation

BackgroundThe 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 ResultsTissue 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 &agr;-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. ConclusionsSimvastatin 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.

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 γ-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 β-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 ε-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 γ-γ cross-linking prior to the formation of Fn II with Bβ-chain remaining mostly intact followed by the selective degradation of FPB to des-Arg FPB.

Aspirin Alters the Cardioprotective Effects of the Factor XIII Val34Leu Polymorphism

Background—The mechanism underlying decreased risk for myocardial infarction in carriers of the Leu34 polymorphism of the factor (F) XIII A-subunit is unclear. Given that acetylation of fibrinogen by aspirin can alter its clotting properties and the presence of fibrin stimulates thrombin-mediated activation of FXIII, we have tested the hypothesis that treatment with aspirin differentially modulates the influence of the FXIII Val34Leu polymorphism on its activation in vivo. Methods and Results—The rates of the disappearance of FXIIIA chain and the appearance of its activated form (FXIIIAa) in sequential 30-second blood samples collected at the site of microvascular injury were compared in 14 healthy carriers of the Leu34 allele and 23 Val34 homozygotes both before and after a 7-day aspirin ingestion (75 mg/d), with the use of quantitative Western blotting. The presence of the Leu34 allele was associated with a significant increase in the maximum rate of FXIII activation by thrombin. Although the Leu34-positive and -negative subjects were similar with respect to aspirin-related impairment of thrombin generation, aspirin led to a more pronounced inhibition of the activation of FXIII in the Leu34 carriers as compared with the Val34 homozygotes. Conclusions—Inhibition of FXIII activation by aspirin is enhanced in the Leu34 carriers in vivo, suggesting that these subjects might benefit more than the Leu34-negative subjects from the reduction in risk for myocardial infarction with low-dose aspirin.

How factor VIIa works in hemophilia.

Summary.  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 5× normal platelets (10 × 108/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 × 108/mL activated platelets (5× normal); (d) with TF, 10 nM rFVIIa and 3–5× normal nonactivated platelets (6–10 × 108/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.

Influence of Factor VIIa and Phospholipids on Coagulation in “Acquired” Hemophilia

Objective—This study was performed to evaluate the influences of phospholipids and recombinant factor VIIa (rFVIIa) on thrombin generation and clot formation in “acquired” hemophilia B. Methods and Results—A synthetic mixture corresponding to hemophilia A (SHA) and “acquired” hemophilia B blood (AHBB) manufactured in vitro by an anti-FIX antibody were used in this study. With 10 pmol/L tissue factor (TF), 10 nmol/L rFVIIa, and saturating phospholipid, established thrombin generation in SHA was similar to that observed in the presence of factor VIII and rFVIIa at physiological concentrations. At lower phospholipid concentrations, thrombin generation was delayed and reduced. With 5 pmol/L TF, contact pathway-inhibited AHBB clotted later than normal blood and showed reduced clot stability and thrombin generation. These parameters of effectiveness were increased by the addition of phospholipids to AHBB, which restored clot stability and increased thrombin generation. No correction of clot formation or thrombin generation was observed when rFVIIa and phospholipids were added to AHBB in the absence of TF. Conclusions—The influence of rFVIIa is dependent on TF, and phospholipids substantially increase the hemostatic (or thrombotic) potential of rFVIIa/TF.

Molecular and Cellular Hemostasis and Fibrinolysis

Vascular damage triggers a combination of tightly integrated processes that may be subdivided into initiation of coagulation, propagation of thrombin formation, termination of the procoagulant reactions, elimination of the resulting clot, and tissue repair and regeneration [1, 2]. This collection of processes is subject to precise control, which localizes the response to the injured area, provides a level of response appropriate to the extent of injury, and maintains vascular hydraulic integrity until tissue repair is complete. The terms initiation, propagation, termination, elimination, and regeneration are useful to describe these discrete events, but all of these processes are highly intertwined and occur concomitantly (Fig. 1).