Saulius Butenas

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.

Thromboelastography as a Better Indicator of Hypercoagulable State After Injury Than Prothrombin Time or Activated Partial Thromboplastin Time

OBJECTIVES To investigate the hemostatic status of critically ill, nonbleeding trauma patients. We hypothesized that a hypercoagulable state exists in patients early after severe injury and that the pattern of clotting and fibrinolysis are similar between burned and nonburn trauma patients. MATERIALS Patients admitted to the surgical or burn intensive care unit within 24 hours after injury were enrolled. Blood samples were drawn on days 0 through 7. Laboratory tests included prothrombin time (PT), activated partial thromboplastin time (aPTT), levels of activated factor XI, D-dimer, protein C percent activity, antithrombin III percent activity, and thromboelastography (TEG). RESULTS Study subjects were enrolled from April 1, 2004, to May 31, 2005, and included nonburn trauma patients (n = 33), burned patients (n = 25), and healthy (control) subjects (n = 20). Despite aggressive thromboprophylaxis, three subjects (2 burned and 1 nonburn trauma patients [6%]) had pulmonary embolism during hospitalization. Compared with controls, all patients had prolonged PT and aPTT (p < 0.05). The rate of clot formation (alpha angle) and maximal clot strength were higher for patients compared with those of controls (p < 0.05), indicating a hypercoagulable state. Injured patients also had lower protein C and antithrombin III percent activities and higher fibrinogen levels (p < 0.05 for all). Activated factor XI was elevated in 38% of patients (control subjects had undetectable levels). DISCUSSION Thromboelastography analysis of whole blood showed that patients were in a hypercoagulable state; this was not detected by plasma PT or aPTT. The high incidence of pulmonary embolism indicated that our current prophylaxis regimen could be improved.

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.

Evaluation of the Initiation Phase of Blood Coagulation Using Ultrasensitive Assays for Serine Proteases

The initiation phase of enzyme generation in a reconstituted model of the tissue factor (TF) pathway to thrombin was evaluated. At 1.25 pm added TF, no thrombin generation was observed in the absence of factor V. The substitution of factor Va for factor V increased the rate of thrombin generation. Factor X activation during the initiation phase was not influenced by the absence of factor VIII or thrombin, leading to the conclusion that initially factor Xa is generated exclusively by the factor VIIa-TF complex. When thrombin was eliminated from the system, no contribution of the factor IXa-factor VIIIa complex to factor X activation was observed during the propagation phase. Similarly, factor V activation was also not observed in the absence of thrombin, indicating that thrombin is the only enzyme responsible for factor V and factor VIII activation. Only subnanomolar amounts of factor VII were activated when prothrombin activation was almost complete. In the absence of coagulation inhibitors, factor XI did not influence thrombin generation initiated by 1.25 pm factor VIIa-TF complex. The termination of factor XIa generation by added hirudin in the factor XI experiment indicates that factor XI activation occurs exclusively by thrombin.

Thrombin generation profiles in deep venous thrombosis.

Summary.  Background: Reliable markers and methods to predict risk for thrombosis are essential to clinical management. Objective: Using an integrated approach that defines an individuals comprehensive coagulation phenotype might prove valuable in identifying individuals at risk for experiencing a thrombotic event. Methods: Using a numerical simulation model, we generated tissue factor (TF) initiated thrombin curves using coagulation factor levels from the Leiden Thrombophilia Study population and evaluated thrombotic risk, by sex, age, smoking, alcohol consumption, body mass index (BMI) and oral contraceptive (OC) use. We quantitated the initiation, propagation and termination phases of each individuals’ comprehensive TF‐initiated thrombin generation curve by the parameters: time to 10 nm of thrombin, maximum time, level and rate (MaxR) of thrombin generated and total thrombin. Results: The greatest risk association was obtained using MaxR; with a 2.6‐fold increased risk at MaxR exceeding the 90th percentile. The odds ratio (OR) for MaxR was 3.9 in men, 2.1 in women, and 2.9 in women on OCs. The association of risk with thrombin generation did not differ by age (OR:2.8 ≤ 45 years>OR:2.5), BMI (OR:2.9 ≤ 26 kg m−2>OR:2.3) or alcohol use. In both numerical simulations and empirical synthetic plasma, OC use created extreme shifts in thrombin generation in both control women and women with a prior thrombosis, with a larger shift in thrombin generation in control women. This suggests an interaction of OC use with underlying prothrombotic abnormalities. Conclusions: Thrombin generation based upon the individuals blood composition is associated with the risk for thrombosis and may be useful as a predictive marker for evaluating thrombosis on an individual basis.

Citrate anticoagulation and the dynamics of thrombin generation

Summary.  Background: Sodium citrate has been used as an anticoagulant to stabilize blood and blood products for over 100 years, presumably by sequestering Ca++ ions in vitro. Anticoagulation of blood without chelation can be achieved by inhibition of the contact pathway by corn trypsin inhibitor (CTI). Objective: To evaluate the influence of citrate anticoagulation on the performance of blood, platelet‐rich and platelet‐poor plasma assays. Methods: Blood was anticoagulated in three ways: by collection into citrate, CTI and citrate with CTI. Plasma was prepared using each anticoagulation regimen. Functional analyses included calibrated automated thrombography, thromboelastography, plasma clotting, the synthetic coagulation proteome and platelet aggregation. Coagulation reactions were initiated with tissue factor–phospholipid and Ca++ (when indicated).Results: In all cases, citrate anticoagulation resulted in reaction dynamics significantly altered relative to blood or plasma stabilized with CTI alone. Subsequent experiments showed that calcium citrate itself impairs coagulation dynamics.Conclusion: Coagulation analyses using blood that has been exposed to citrate and recalcified do not yield reliable depictions of the natural dynamics of blood coagulation processes.

The Tissue Factor Requirement in Blood Coagulation

Formation of thrombin is triggered when membrane-localized tissue factor (TF) is exposed to blood. In closed models of this process, thrombin formation displays an initiation phase (low rates of thrombin production cause platelet activation and fibrinogen clotting), a propagation phase (>95% of thrombin production occurs), and a termination phase (prothrombin activation ceases and free thrombin is inactivated). A current controversy centers on whether the TF stimulus requires supplementation from a circulating pool of blood TF to sustain an adequate procoagulant response. We have evaluated the requirement for TF during the progress of the blood coagulation reaction and have extended these analyses to assess the requirement for TF during resupply (“flow replacement”). Elimination of TF activity at various times during the initiation phase indicated: a period of absolute dependence (<10 s); a transitional period in which the dependence on TF is partial and decreases as the reaction proceeds (10–240 s); and a period in which the progress of the reaction is TF independent (>240 s). Resupply of reactions late during the termination phase with fresh reactants, but no TF, yielded immediate bursts of thrombin formation similar in magnitude to the original propagation phases. Our data show that independence from the initial TF stimulus is achieved by the onset of the propagation phase and that the ensemble of coagulation products and intermediates that yield this TF independence maintain their prothrombin activating potential for considerable time. These observations support the hypothesis that the transient, localized expression of TF is sufficient to sustain a TF-independent procoagulant response as long as flow persists.

Active tissue factor in blood

Following mechanical or chemical damage of the vessel or monocyte stimulation, tissue factor is exposed to the blood and binds plasma factor VIIa (FVIIa) forming the FVIIa–tissue factor enzyme complex. During the last several years, a number of studies have reported that physiologically active tissue factor is found circulating in blood of healthy individuals either as a component of blood cells and microparticles or as a soluble plasma protein1. Reports of the presence, source and activity of tissue factor in blood are controversial, with reported concentrations of physiologically active tissue factor varying from undetectable (<60 fM) in whole blood2 to as high as 37 pM in the plasma of healthy individuals3. Blood or plasma activated with (sub)picomolar concentrations of functional tissue factor clots within several minutes (Fig. 1), suggesting that such concentrations of functional tissue factor cannot be present in blood or plasma in vivo.

Tissue Factor in Coagulation. Which? Where? When?

Tissue factor (TF) is an integral membrane protein, normally separated from the blood by the vascular endothelium, which plays a key role in the initiation of blood coagulation. With a perforating vascular injury, TF becomes exposed to blood and binds plasma factor VIIa. The resulting complex initiates a series of enzymatic reactions leading to clot formation and vascular sealing. In some pathological states, circulating blood cells express TF as a result of exposure to an inflammatory stimulus leading to intravascular clotting, vessel occlusion, and thrombotic pathology. Numerous controversies have arisen related to the influence of structural features of TF, its presentation, and its function. There are contradictory reports about the synthesis and presentation of TF on blood cells and the presence (or absence) of functionally active TF circulating in normal blood either on microparticles or as a soluble protein. In this review we discuss TF structure-function relationships and the role of TF during various phases of the blood coagulation process. We also highlight controversies concerning the expression/presence of TF on various cells and in blood in normal and pathological states.