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There is little knowledge regarding the in-vivo course of coagulation reactions in subjects with factor (F)V Leiden (G1691A, R506Q). The aim of the current study was to evaluate the effect of FV Leiden on coagulant reactions triggered by vascular injury. At the site of microvascular injury, prothrombin activation and FVa formation and inactivation have been evaluated in 16 apparently healthy subjects and 16 patients on long-term anticoagulation with acenocoumarol, with eight heterogenous carriers of FV Leiden in the healthy and in the anticoagulated group. Thrombin formation, measured by levels of thrombin-antithrombin complexes (TAT) and thrombin B-chain, proceeded at similar rates in the bleeding-time blood in carriers and non-carriers of FV Leiden. The onset and rate of FV activation did not differ significantly among the four groups. When healthy carriers of FV Leiden were compared to healthy non-carriers, the onset of FVa inactivation by activated protein C (APC) was delayed by 60 to 90 seconds (p=0.01). Anticoagulated individuals also showed the same pattern of FV Leiden associated differences in the time of occurrence of the 30 kD FVa heavy-chain fragment (p=0.021). Our results indicate that when blood clotting is triggered by microvascular injury, the heterozygous expression of FV Leiden has no effect on thrombin generation in healthy or coumadin-treated subjects. Inactivation of FVa by the APC mechanism is attenuated significantly in carriers of FV Leiden but the magnitude of this effect is smaller than that observed in most purified systems.

Previous studies of factor (F)Va inactivation on human umbilical vein endothelial cells have shown that alpha-thrombin cleaves the heavy chain near the COOH-terminus to produce a M(r) 97,000 fragment containing the NH(2)-terminal portion of the heavy chain and a M(r) 8,000 peptide containing the rest of the molecule. The alpha-thrombin cleavage appeared to occur between amino acid residues 586 and 654 of FV. This region contains a consensus sequence for alpha-thrombin cleavage located at residues 640-644 (S-S-P-R-S). To test the hypothesis that alpha-thrombin cleaves the FVa heavy chain at Arg(643) and to evaluate the functional importance of this cleavage for FVa inactivation, site-directed mutagenesis was used to create recombinant FV molecules with mutations R(643) --> Q (FV(R643Q)) and R(643) --> A (FV(R643A)). All recombinant molecules were purified to homogeneity and assayed for activity following extended activation with alpha-thrombin. Under similar experimental conditions, appearance of the M(r) 97,000 heavy chain fragment in the plasma and wild-type FVa molecules correlated with partial loss of cofactor activity, while following extended incubation of FV(R643Q) and FV(R643A) with alpha-thrombin no cleavage of the heavy chain at Arg(643) was detected and no presence of the M(r) 97,000 heavy-chain fragment was noticed. Further, no loss in cofactor activity was observed using these mutant recombinant FVa molecules. Our data demonstrate that cleavage of FVa at Arg(643) by alpha-thrombin results in a partially inactive cofactor molecule and provides for an activated protein C (APC)-independent anticoagulant effect of alpha-thrombin.

Bivalirudin, a synthetic analog of the carboxy-terminus of hirudin, is a reversible thrombin inhibitor used during coronary balloon angioplasty. The objective of this study was to evaluate the influence of bivalirudin on thrombin generation. Three in-vitro models (numerical simulations, synthetic coagulation proteome and whole blood) of contact pathway-independent blood coagulation triggered with tissue factor were used in this study. Increasing concentrations of bivalirudin prolong the initiation phase of thrombin generation in a concentration-dependent manner. At subpharmacologic bivalirudin concentrations (0.5-2 micromol/l), total thrombin generation was significantly increased. At a pharmacologic concentration (5 micromol/l), bivalirudin suppressed thrombin generation in the synthetic coagulation proteome; in numerical simulations and contact pathway-inhibited whole blood, no thrombin generation was detected over 1200-2000 s and platelet activation was inhibited by 80%. The addition of a pharmacologic concentration (9 micromol/l) of a naturally occurring protease inhibitor aprotinin in the presence of at least 0.5 micromol/l bivalirudin provided limited enhancement of the bivalirudin inhibitory effect. In conclusion, bivalirudin at pharmacologic concentrations is an efficient inhibitor of thrombin generation, platelet activation and clot formation, which acts not as a modulator but as an 'on-off' switch of blood coagulation.

Aspirin is effective in the prevention of cardiovascular events in high-risk patients. The primary established effect of aspirin on hemostasis is to impair platelet aggregation via inhibition of platelet thromboxane A(2) synthesis, thus reducing thrombus formation on the surface of the damaged arterial wall. Growing evidence also indicates that aspirin exerts additional antithrombotic effects, which appear to some extent unrelated to platelet thromboxane A(2) production. Aspirin can reduce thrombin generation with the subsequent attenuation of thrombin-mediated coagulant reactions such as factor XIII activation. Aspirin also acetylates lysine residues in fibrinogen resulting in increased fibrin clot permeability and enhanced clot lysis as well as directly promoting fibrinolysis with high-dose aspirin. The variable effectiveness of aspirin in terms of clinical outcomes and laboratory findings, which has been termed aspirin resistance, may be related to these additional antithrombotic effects that are altered when associated with common genetic polymorphisms such as the Leu33Pro beta(3)-integrin or Val34Leu factor XIII mutations. However, the clinical relevance of these observations is still unclear. Elucidation of the actual impacts of aspirin other than antiaggregation effects could be important in view of the widespread use of this drug in the prevention of thrombotic manifestations of atherosclerosis.

Background: Trauma is the leading cause of death in the younger population in the United States, frequently from the development of hemorrhagic shock. Controversy exists over the type of volume resuscitation for restoring hemodynamic stability that should be used in hemorrhagic shock. Little is known about how various resuscitative paradigms affect the coagulation cascade, which is essential to controlling hemorrhagic shock., Methods: We studied the effect of various resuscitative formulas on blood coagulation using a new model of whole blood in a controlled setting with corn trypsin inhibitor and a 5-pM stimulus of tissue factor. We investigated thrombin generation, fibrin formation, and platelet activation with four diluents: 0.9% NaCl (NS), lactated Ringer's solution (LR), 6% hydroxyethyl starch (HES), and 3% NaCl (HS), each from 0% to 75% blood dilution. Thrombin generation was measured periodically during a time course of 20 minutes in its complex with antithrombin III. Platelet activation and fibrinopeptide A (FPA) release were monitored in serum at a 20-minute time point. Fibrin clots were collected and weighed., Results: The coagulation markers (thrombin generation, platelet activation, and FPA release) were significantly different by dilution (p < 0.001 in all) and diluent by dilution (p < 0.001 in all). Thrombin generation, platelet activation, and FPA release decreased the least with the diluents NS and LR. LR caused the least amount of variation in thrombin generation over the dilution course. HS produced the most dramatic change in all of the markers; no coagulation was seen between 30% to 75% dilution (p < 0.05). HES produced greater decreases in thrombin generation and FPA release than NS and LR. Fibrin clot mass decreased with a 10% to 20% dilution for NS and LR, whereas stable fibrin mass did not decrease with the diluents HES and HS at 10% to 20% dilutions. At >30% dilutions, HS produced no stable clots and HES dramatically decreased clot formation by 61% and maintained this level., Conclusions: LR and NS had the least effect on thrombin generation, clot formation, and platelet activation at various concentrations compared with HES and HS. This observational data suggests that volume expanders such as HES and HS may be detrimental in treatment of hemorrhagic shock.

Our research aims to provide quantitatively transparent, biologically realistic descriptions of the processes involved in hemostasis which will permit predictions of the behavior of the coagulation system in normal and pathologic states. We use four models of coagulation: (1) numerical approximations of the tissue factor (Tf) pathway of thrombin generation based upon mechanism and dynamics; (2) Tf activation of the "blood coagulation proteome" from isolated cells and proteins; (3) Tf activated contact pathway inhibited whole blood in vitro; and (4) blood shed from standardized microvascular wounds in vivo. The results from these models are integrated in interactive assessments aimed at achieving convergence of biochemical rigor and biological authenticity. Microvascular injury is the most biologically secure but least accessible to mechanistic study. Numerical models while quantitatively transparent are biologically limited. By the integrated analyses of all four models, we establish observations which require inclusion or discovery of new parameters to achieve mechanistically interpretable biological reality. Discoveries made in this fashion have included thrombin's role in the initiation phase, TFPI/ATIII/APC synergy interactions, rfVIIa in fVII deficiency, the roles of fVIII and fIX in the Tf reaction, and the cleavage of fIX by fXa membrane. Ideally, our results will provide descriptions which predict the behavior of the biological blood coagulation system under normal and pathologic conditions.

Objective: The high and low responder phenomenon describes individual differences in lipopolysaccharide (LPS)-induced monocyte tissue factor (TF) activity. We characterized patterns of intracellular accumulation, externalization, and shedding of TF in response to LPS in mononuclear cells (MNCs) from high responders (HRs) and low responders (LRs)., Methods and Results: After 2 hours of LPS stimulation of whole blood, flow cytometry analyses revealed a larger population of TF-positive monocytes in HRs (32.0+/-3.5%) versus LRs (11.2+/-1.2%; P< or =0.05), along with a stronger mean fluorescence intensity of TF signal in HRs (7.1+/-0.5 arbitrary units [AU]) compared with LRs (5.4+/-0.4 AU; P< or =0.05). The LPS-treated blood of the HR group contained 2-fold more TF-positive microparticles than LRs. In-cell Western assay demonstrated higher intracellular accumulation of TF in mononuclear cells (MNCs) from LRs because LPS induced a 3.7-fold increase of total TF levels in LRs versus a 1.5-fold increase in HRs. In contrast, in response to LPS stimulation, MNCs from HRs exhibited a 4-fold induction of surface TF, whereas MNCs from LRs only had a minor increase in surface TF levels., Conclusions: The higher availability of surface TF antigen on MNCs from HRs and TF-containing microparticles might make these individuals more susceptible to hypercoagulation.

Tissue factor (TF) is an integral membrane protein essential for hemostasis. During the past several years, a number of studies have suggested that physiologically active TF circulates in blood at concentrations greater than 30 pM either as a component of blood cells and microparticles or as a soluble plasma protein. In our studies using contact pathway-inhibited blood or plasma containing activated platelets, typically no clot is observed for 20 minutes in the absence of exogenous TF. An inhibitory anti-TF antibody also has no effect on the clotting time in the absence of exogenous TF. The addition of TF to whole blood at a concentration as low as 16 to 20 fM results in pronounced acceleration of clot formation. The presence of potential platelet TF activity was evaluated using ionophore-treated platelets and employing functional and immunoassays. No detectable TF activity or antigen was observed on quiescent or ionophore-stimulated platelets. Similarly, no TF antigen was detected on mononuclear cells in nonstimulated whole blood, whereas in lipopolysaccharide (LPS)-stimulated blood a significant fraction of monocytes express TF. Our data indicate that the concentration of physiologically active TF in non-cytokine-stimulated blood from healthy individuals cannot exceed and is probably lower than 20 fM.

The 3-hydroxy-3-methylglutaryl (HMG)-coenzyme A (CoA) reductase inhibitors (statins) have been shown to exhibit several vascular protective effects, including antithrombotic properties, that are not related to changes in lipid profile. There is growing evidence that treatment with statins can lead to a significant downregulation of the blood coagulation cascade, most probably as a result of decreased tissue factor expression, which leads to reduced thrombin generation. Accordingly, statin use has been associated with impairment of several coagulant reactions catalyzed by this enzyme. Moreover, evidence indicates that statins, via increased thrombomodulin expression on endothelial cells, may enhance the activity of the protein C anticoagulant pathway. Most of the antithrombotic effects of statins are attributed to the inhibition of isoprenylation of signaling proteins. These novel properties of statins, suggesting that these drugs might act as mild anticoagulants, may explain, at least in part, the therapeutic benefits observed in a wide spectrum of patients with varying cholesterol levels, including subjects with acute coronary events. The HMG-CoA reductase inhibitors (statins) have been shown to exhibit several vascular protective effects, including antithrombotic properties, that are not related to changes in lipid profile. Treatment with statins can lead to a significant downregulation of the blood coagulation cascade, most probably as a result of decreased tissue factor expression, which leads to reduced thrombin generation.

Thrombin is the product of the hemostatic response essential to the conversion of fibrinogen to fibrin. In addition, it is also responsible for the aggregation of blood platelets in the formation of the "platelet plug" as well as the activation of factor VIII, factor V, factor XI, factor XIII and protein C. The action of thrombin is not confined to the hemostatic response as it also has a critical function in the wound healing process by stimulating 'mitogenic' events through interaction with cell surface receptors. In this review, we consider the various biological activities of thrombin as they relate to current therapeutic use. While there has been considerable interest in the development of fibrin sealant products, there has been considerably less interest in documenting the continuing use of thrombin as a therapeutic. The use of thrombin for topical hemostasis and the treatment of pseudoaneurysms will be discussed in detail. It is concluded that the use of thrombin as a drug will not only continue but also will significantly increase. However, the availability of a safe human thrombin preparation will be critical for the continued use of thrombin as a therapeutic.

The generation of the enzyme thrombin from its precursor prothrombin is the central event of the blood coagulation process, which is essential to hemostasis and the culprit in thrombosis. Thrombin is produced by a complex series of proteolytic events that are initiated when cryptic tissue factor interacts with plasma factor VIIa to initiate the complex series of events leading to the formation of the blood coagulation enzyme complexes that lead to the efficient generation of the enzyme. During these processes, thrombin contributes to both the generation of the catalysts involved in its ultimate production and to the catalysts that lead to attenuation of its production. Thrombin-catalyzed events both enhance and diminish the process of thrombin generation, which is down-regulated by stoichiometric and dynamic inhibitory processes. The combinations of intensities of activation and inhibition processes provide tight regulation of the hemostatic process, establishing reaction thresholds, essentially leading to an "on/off" switch. This review provides a brief summary of the evolution of knowledge with respect to present-day concepts of thrombin generation via the tissue factor pathway and its regulation.

Expression and purification of proteins in recombinant DNA systems is a powerful and widely used technique. Frequently there is the need to express the protein of interest as a fusion protein or chimeric protein. Fusion protein technology is frequently used to attach a "signal" which can be used for subsequent localization of the protein or a "carrier" which can be used to deliver a "therapeutic" such as a radioactive molecule to a specific site. In addition to these applications, fusion protein technology can be employed for several other useful purposes. Of these, the most frequent reason is to provide a 'tag' or 'handle' which will aid in the purification of the protein. Another useful purpose is to improve the expression or folding of the protein of interest. In these latter two situations, it is often necessary to remove the fusion partner before the recombinant protein of interest can be used for further studies. This removal process involves the insertion of a unique amino acid sequence that is susceptible to cleavage by a highly specific protease. Thrombin and factor Xa are the most frequently used proteases for this application. The purpose of this review is to discuss the application of thrombin and factor Xa for the cleavage of fusion proteins. It is emphasized that while these enzymes are quite specific for cleavage at the inserted cleavage site, proteolysis can frequently occur at other site(s) in the protein of interest. It is necessary to characterize the protein of interest after cleavage from the affinity label to assure that there are no changes in the covalent structure of the protein of interest. Examples are presented which describe the proteolysis of the protein of interest by either factor Xa or thrombin.

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.

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.

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.

Tissue factor-induced blood coagulation was studied in 20 individuals, for varying periods of time during 54 months, in contact pathway-inhibited whole blood at 37 degrees C and evaluated in terms of the activation of various substrates. After quenching over time with inhibitors, the soluble phases were analyzed for thrombin-antithrombin III (TAT) complex formation, prothrombin fragments, platelet activation (osteonectin release), factor Va generation, fibrinopeptide (FP) A and FPB release, and factor XIII activation. TAT complex formation, for 35 experiments, showed an initiation phase (up to 4.6 +/- 0.6 minutes) in which thrombin was generated at an average rate of 0.93 +/- 0.3 nM/min catalyzed by about 1.3 pM prothrombinase yielding approximately 26 nM thrombin. During a subsequent propagation phase, thrombin was generated at a rate of 83.9 +/- 3.8 nM/min by about 120 pM prothrombinase, reaching ultimate levels of 851 +/- 53 nM. Clot time, determined subjectively, occurred at 4.7 +/- 0.2 minutes and correlated with the inception of the propagation phase. The thrombin concentrations associated with the transitions to rapid product formation are 510 +/- 180 pM for platelet activation (1.9 +/- 0.2 minutes), 840 +/- 280 pM for factor XIII activation and factor Va generation (2.2 +/- 0.6 minutes), 1.3 +/- 0.4 nM for FPA release (2.5 +/- 0.7 minutes), 1.7 +/- 0.5 nM for FPB release and prethrombin 2 (2.8 +/- 0.8 minutes), 7.0 +/- 2.2 nM for thrombin B chain (3.6 +/- 0.2 minutes), and 26 +/- 6.2 nM for the propagation phase of TAT formation (4.6 +/- 0.6 minutes). These results illustrate that the initial activation of thrombin substrates occurs during the initiation phase at less than 2 nM thrombin (0.2%). Most thrombin (96%) is formed well after clotting occurs.

We have developed a model of the extrinsic blood coagulation system that includes the stoichiometric anticoagulants. The model accounts for the formation, expression, and propagation of the vitamin K-dependent procoagulant complexes and extends our previous model by including: (a) the tissue factor pathway inhibitor (TFPI)-mediated inactivation of tissue factor (TF).VIIa and its product complexes; (b) the antithrombin-III (AT-III)-mediated inactivation of IIa, mIIa, factor VIIa, factor IXa, and factor Xa; (c) the initial activation of factor V and factor VIII by thrombin generated by factor Xa-membrane; (d) factor VIIIa dissociation/activity loss; (e) the binding competition and kinetic activation steps that exist between TF and factors VII and VIIa; and (f) the activation of factor VII by IIa, factor Xa, and factor IXa. These additions to our earlier model generate a model consisting of 34 differential equations with 42 rate constants that together describe the 27 independent equilibrium expressions, which describe the fates of 34 species. Simulations are initiated by "exposing" picomolar concentrations of TF to an electronic milieu consisting of factors II, IX, X, VII, VIIa, V, and VIIII, and the anticoagulants TFPI and AT-III at concentrations found in normal plasma or associated with coagulation pathology. The reaction followed in terms of thrombin generation, proceeds through phases that can be operationally defined as initiation, propagation, and termination. The generation of thrombin displays a nonlinear dependence upon TF, AT-III, and TFPI and the combination of these latter inhibitors displays kinetic thresholds. At subthreshold TF, thrombin production/expression is suppressed by the combination of TFPI and AT-III; for concentrations above the TF threshold, the bolus of thrombin produced is quantitatively equivalent. A comparison of the model with empirical laboratory data illustrates that most experimentally observable parameters are captured, and the pathology that results in enhanced or deficient thrombin generation is accurately described.

The ability of factor VIIa to initiate thrombin generation and clot formation in blood from healthy donors, blood from patients with hemophilia A, and in anti-factor IX antibody-induced ("acquired") hemophilia B blood was investigated. In normal blood, both factor VIIa-tissue factor (TF) complex and factor VIIa alone initiated thrombin generation. The efficiency of factor VIIa was about 0.0001 that of the factor VIIa-TF complex. In congenital hemophilia A blood and "acquired" hemophilia B blood in vitro, addition of 10 to 50 nM factor VIIa (pharmacologic concentrations) corrected the clotting time at all TF concentrations tested (0-100 pM) but had little effect on thrombin generation. Fibrinopeptide release and insoluble clot formation were only marginally influenced by addition of factor VIIa. TF alone had a more pronounced effect on thrombin generation; an increase in TF from 0 to 100 pM increased the maximum thrombin level in "acquired" hemophilia B blood from 120 to 480 nM. Platelet activation was considerably enhanced by addition of factor VIIa to both hemophilia A blood and "acquired" hemophilia B blood. Thus, pharmacologic concentrations of factor VIIa cannot restore normal thrombin generation in hemophilia A and hemophilia B blood in vitro. The efficacy of factor VIIa (10-50 nM) in hemophilia blood is dependent on TF.