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Hemostatic tests have been utilized to clarify the blood coagulation potential. The novel thrombin generation (TG) assay of this study provides explicit information and is the most physiologically-relevant hemostatic test ex vivo. We describe how this assay allows for TG under a number of relevant circumstances. First, whole blood (WB) from healthy individuals was analyzed ± 5 pM tissue factor (TF) and ± contact pathway inhibition. Without an exogenous initiator TG was decreased and delayed, but addition of 5 pM TF shortened the lag phase and increased peak thrombin. Additional experiments included fresh WB from a trauma patient analyzed for endogenous activity and TG from healthy donors subjected to TG antagonists which prolonged the lag phase whereas TG agonists consistently shortened the lag phase in a dose dependent manner. Lastly, platelet-poor plasma was reconstituted with packed red blood cells and TG was monitored in the presence and absence of both TF as an activator and PCPS as a phospholipid surface. Our data illustrate the potential that this continuous TG assay has in the evaluation of disorders relevant to blood coagulation and in the monitoring of treatments administered in response to these disorders., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Background: Intact red blood cells (RBCs) appear to support thrombin generation in in vitro models of blood coagulation. During storage of RBC units, biochemical, structural, and physiological changes occur including alterations to RBC membranes and release of microparticles, which are collectively known as storage lesion. The clinical consequences of microparticle formation in RBC units are unclear. This study was performed to assess thrombin generation via the prothrombinase complex by washed RBCs and RBC-derived microparticles as a function of RBC unit age., Methods: Well-characterized kinetic and flow cytometric assays were used to quantify and characterize microparticles isolated from leukocyte-reduced RBC units during storage for 42 days under standard blood banking conditions., Results: Stored RBCs exhibited known features of storage lesion including decreasing pH, cell lysis, and release of microparticles demonstrated by scanning electron microscopy. The rate of thrombin formation by RBC units linearly increased during storage, with the microparticle fraction accounting for approximately 70% of the prothrombinase activity after 35 days. High-resolution flow cytometric analyses of microparticle isolates identified phosphatidylserine-positive RBC-derived microparticles; however, their numbers over time did not correlate with thrombin formation in that fraction., Conclusion: Red blood cell-derived microparticles capable of supporting prothrombinase function accumulate during storage, suggesting an increased potential of transfused units as they age to interact in unplanned ways with ongoing hemostatic processes in injured individuals, especially given the standard blood bank practice of using the oldest units available.

In closed system models of fibrin formation, exosite-mediated thrombin binding to fibrin contributes to clot stability and is resistant to inhibition by antithrombin/heparin while still susceptible to small, active-site inhibitors. Each molecule of fibrin can bind ∼1.6 thrombin molecules at low-affinity binding sites (K d  = 2.8 μM) and ∼0.3 molecules of thrombin at high-affinity binding sites (K d  = 0.15 μM). The goal of this study is to assess the stability of fibrin-bound thrombin under venous flow conditions and to determine both its accessibility and susceptibility to inhibition. A parallel-plate flow chamber (7 × 50 × 0.25 mm) for studying the stability of thrombin (0-1400 nM) adhered to a fibrin matrix (0.1-0.4 mg/mL fibrinogen, 10 nM thrombin) under a variety of venous flow conditions was developed using the thrombin-specific, fluorogenic substrate SN-59 (100 μM). The flow within this system is laminar (R e  < 1) and reaction rates are driven by enzyme kinetics (P e  = 100, D a  = 7000). A subpopulation of active thrombin remains stably adhered to a fibrin matrix over a range of venous shear rates (46-184 s -1 ) for upwards of 30 min, and this population is saturable at loads >500 nM and sensitive to the initial fibrinogen concentration. These observations were also supported by a mathematical model of thrombin adhesion to fibrin, which demonstrates that thrombin initially binds to the low-affinity thrombin binding sites before preferentially equilibrating to higher affinity sites. Antithrombin (2.6 μM) plus heparin (4 U/mL) inhibits 72% of the active clot-bound thrombin after ∼10 min at 92 s -1 , while no inhibition is observed in the absence of heparin. Dabigatran (20 and 200 nM) inhibits (50 and 93%) clot-bound thrombin reversibly (87 and 66% recovery). This model illustrates that clot-bound thrombin stability is the result of a constant rearrangement of thrombin molecules within a dense matrix of binding sites., (Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Thrombin has multiple functions in blood coagulation and its regulation is central to maintaining the balance between hemorrhage and thrombosis. Empirical and computational methods that capture thrombin generation can provide advancements to current clinical screening of the hemostatic balance at the level of the individual. In any individual, procoagulant and anticoagulant factor levels together act to generate a unique coagulation phenotype (net balance) that is reflective of the sum of its developmental, environmental, genetic, nutritional and pharmacological influences. Defining such thrombin phenotypes may provide a means to track disease progression pre-crisis. In this review we briefly describe thrombin function, methods for assessing thrombin dynamics as a phenotypic marker, computationally derived thrombin phenotypes versus determined clinical phenotypes, the boundaries of normal range thrombin generation using plasma composition based approaches and the feasibility of these approaches for predicting risk.

Red blood cells have historically been viewed as innocent bystanders in the process of blood coagulation and thrombin generation; however a century of clinical evidence linking red blood cells to thrombosis suggests the contrary. In this brief review, the biochemical evidence for red blood cell involvement in thrombin generation is evaluated. It is concluded that in addition to platelets, red blood cells actively participate in thrombin generation. A sub-fraction of red blood cells express phosphatidylserine on their surface and unlike platelets, red blood cells produce thrombin through the meizothrombin pathway, which has interesting consequences in the context of clot formation and stabilization., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The standard clinical coagulation assays, activated partial thromboplastin time (aPTT) and prothrombin time (PT) cannot predict thrombotic or bleeding risk. Since thrombin generation is central to haemorrhage control and when unregulated, is the likely cause of thrombosis, thrombin generation assays (TGA) have gained acceptance as "global assays" of haemostasis. These assays generate an enormous amount of data including four key thrombin parameters (lag time, maximum rate, peak and total thrombin) that may change to varying degrees over time in longitudinal studies. Currently, each thrombin parameter is averaged and presented individually in a table, bar graph or box plot; no method exists to visualize comprehensive thrombin generation data over time. To address this need, we have created a method that visualizes all four thrombin parameters simultaneously and can be animated to evaluate how thrombin generation changes over time. This method uses all thrombin parameters to intrinsically rank individuals based on their haemostatic status. The thrombin generation parameters can be derived empirically using TGA or simulated using computational models (CM). To establish the utility and diverse applicability of our method we demonstrate how warfarin therapy (CM), factor VIII prophylaxis for haemophilia A (CM), and pregnancy (TGA) affects thrombin generation over time. The method is especially suited to evaluate an individual's thrombotic and bleeding risk during "normal" processes (e.g pregnancy or aging) or during therapeutic challenges to the haemostatic system. Ultimately, our method is designed to visualize individualized patient profiles which are becoming evermore important as personalized medicine strategies become routine clinical practice.

Prothrombin activation can proceed through the intermediates meizothrombin or prethrombin-2. To assess the contributions that these 2 intermediates make to prothrombin activation in tissue factor (Tf)-activated blood, immunoassays were developed that measure the meizothrombin antithrombin (mTAT) and α-thrombin antithrombin (αTAT) complexes. We determined that Tf-activated blood produced both αTAT and mTAT. The presence of mTAT suggested that nonplatelet surfaces were contributing to approximately 35% of prothrombin activation. Corn trypsin inhibitor-treated blood was fractionated to yield red blood cells (RBCs), platelet-rich plasma (PRP), platelet-poor plasma (PPP), and buffy coat. Compared with blood, PRP reconstituted with PPP to a physiologic platelet concentration showed a 2-fold prolongation in the initiation phase and a marked decrease in the rate and extent of αTAT formation. Only the addition of RBCs to PRP was capable of normalizing αTAT generation. FACS on glycophorin A-positive cells showed that approximately 0.6% of the RBC population expresses phosphatidylserine and binds prothrombinase (FITC Xa·factor Va). These data indicate that RBCs participate in thrombin generation in Tf-activated blood, producing a membrane that supports prothrombin activation through the meizothrombin pathway.

Factor (f)Xa is a critical enzyme in blood coagulation that is responsible for the initiation and propagation of thrombin generation. Previously we have shown that analysis of computationally generated thrombin profiles is a tool to investigate hemostasis in various populations. In this study, we evaluate the potential of computationally derived time courses of fXa generation as another approach for investigating thrombotic risk. Utilizing the case (n = 473) and control (n = 426) population from the Leiden Thrombophilia Study and each individual's plasma protein factor composition for fII, fV, fVII, fVIII, fIX, fX, antithrombin and tissue factor pathway inhibitor, tissue factor-initiated total active fXa generation was assessed using a mathematical model. FXa generation was evaluated by the area under the curve (AUC), the maximum rate (MaxR) and level (MaxL) and the time to reach these, TMaxR and TMaxL, respectively. FXa generation was analyzed in the entire populations and in defined subgroups (by sex, age, body mass index, oral contraceptive use). The maximum rates and levels of fXa generation occur over a 10- to 12- fold range in both cases and controls. This variation is larger than that observed with thrombin (3-6 fold) in the same population. The greatest risk association was obtained using either MaxR or MaxL of fXa generation; with an ∼2.2 fold increased risk for individuals exceeding the 90(th) percentile. This risk was similar to that of thrombin generation(MaxR OR 2.6). Grouping defined by oral contraceptive (OC) use in the control population showed the biggest differences in fXa generation; a >60% increase in the MaxR upon OC use. FXa generation can distinguish between a subset of individuals characterized by overlapping thrombin generation profiles. Analysis of fXa generation is a phenotypic characteristic which may prove to be a more sensitive discriminator than thrombin generation among all individuals.

In terms of its soluble precursors, the coagulation proteome varies quantitatively among apparently healthy individuals. The significance of this variability remains obscure, in part because it is the backdrop against which the hemostatic consequences of more dramatic composition differences are studied. In this study we have defined the consequences of normal range variation of components of the coagulation proteome by using a mechanism-based computational approach that translates coagulation factor concentration data into a representation of an individual's thrombin generation potential. A novel graphical method is used to integrate standard measures that characterize thrombin generation in both empirical and computational models (e.g max rate, max level, total thrombin, time to 2 nM thrombin ("clot time")) to visualize how normal range variation in coagulation factors results in unique thrombin generation phenotypes. Unique ensembles of the 8 coagulation factors encompassing the limits of normal range variation were used as initial conditions for the computational modeling, each ensemble representing "an individual" in a theoretical healthy population. These "individuals" with unremarkable proteome composition was then compared to actual normal and "abnormal" individuals, i.e. factor ensembles measured in apparently healthy individuals, actual coagulopathic individuals or artificially constructed factor ensembles representing individuals with specific factor deficiencies. A sensitivity analysis was performed to rank either individual factors or all possible pairs of factors in terms of their contribution to the overall distribution of thrombin generation phenotypes. Key findings of these analyses include: normal range variation of coagulation factors yields thrombin generation phenotypes indistinguishable from individuals with some, but not all, coagulopathies examined; coordinate variation of certain pairs of factors within their normal ranges disproportionately results in extreme thrombin generation phenotypes, implying that measurement of a smaller set of factors may be sufficient to identify individuals with aberrant thrombin generation potential despite normal coagulation proteome composition.

The underlying cause of thrombosis in a large protein C (PC) deficient Vermont kindred appears to be multicausal and not explained by PC deficiency alone. We evaluated the contribution of coagulation factors to thrombin generation in this population utilizing a mathematical model that incorporates a mechanistic description of the PC pathway. Thrombin generation profiles for each individual were generated with and without the contribution of the PC pathway. Parameters that describe thrombin generation: maximum level (MaxL) and rate (MaxR), their respective times (TMaxL, TMaxR), area under the curve (AUC) and clotting time (CT) were examined in individuals ± PC mutation, ± prothrombin G20210A polymorphism and ± thrombosis history (DVT or PE). This family (n = 364) is shifted towards greater thrombin generation relative to the mean physiologic control. When this family was analyzed with the PC pathway, our results showed that: carriers of the PC mutation (n = 81) had higher MaxL and MaxR and greater AUC (all p<0.001) than non-carriers (n = 283); and individuals with a DVT and/or PE history (n = 13) had higher MaxL (p = 0.005) and greater AUC (p<0.001) than individuals without a thrombosis history (n = 351). These differences were further stratified by gender, with women in all categories generating more thrombin than males. These results show that all individuals within this family with or without PC deficiency have an increased baseline procoagulant potential reflective of increased thrombin generation. In addition, variations within the plasma composition of each individual can further segregate out increased procoagulant phenotypes, with gender-associated plasma compositional differences playing a large role.

The view that clot time-based assays do not provide a sufficient assessment of an individual's hemostatic competence, especially in the context of anticoagulant therapy, has provoked a search for new metrics, with significant focus directed at techniques that define the propagation phase of thrombin generation. Here we use our deterministic mathematical model of tissue-factor initiated thrombin generation in combination with reconstructions using purified protein components to characterize how the interplay between anticoagulant mechanisms and variable composition of the coagulation proteome result in differential regulation of the propagation phase of thrombin generation. Thrombin parameters were extracted from computationally derived thrombin generation profiles generated using coagulation proteome factor data from warfarin-treated individuals (N = 54) and matching groups of control individuals (N = 37). A computational clot time prolongation value (cINR) was devised that correlated with their actual International Normalized Ratio (INR) values, with differences between individual INR and cINR values shown to derive from the insensitivity of the INR to tissue factor pathway inhibitor (TFPI). The analysis suggests that normal range variation in TFPI levels could be an important contributor to the failure of the INR to adequately reflect the anticoagulated state in some individuals. Warfarin-induced changes in thrombin propagation phase parameters were then compared to those induced by unfractionated heparin, fondaparinux, rivaroxaban, and a reversible thrombin inhibitor. Anticoagulants were assessed at concentrations yielding equivalent cINR values, with each anticoagulant evaluated using 32 unique coagulation proteome compositions. The analyses showed that no anticoagulant recapitulated all features of warfarin propagation phase dynamics; differences in propagation phase effects suggest that anticoagulants that selectively target fXa or thrombin may provoke fewer bleeding episodes. More generally, the study shows that computational modeling of the response of core elements of the coagulation proteome to a physiologically relevant tissue factor stimulus may improve the monitoring of a broad range of anticoagulants.

Introduction: More than 80% of cerebrovascular events are ischemic and largely thromboembolic by nature. We evaluated whether plasma factor composition and thrombin generation dynamics might be a contributor to the thrombotic phenotype of ischemic cerebrovascular events., Materials and Methods: We studied (1) 100 patients with acute ischemic stroke (n=50) or transient ischemic attack (TIA) (n=50) within the first 24 hours from symptom onset, and (2) 100 individuals 1 to 4 years following ischemic stroke (n=50) or TIA (n=50). The tissue factor pathway to thrombin generation was simulated with a mathematical model using plasma levels of clotting factors (F)II, V, VII, VIII, IX, X, antithrombin and free tissue factor pathway inhibitor (TFPI)., Results: The plasma levels of free TFPI, FII, FVIII, and FX were higher, while antithrombin was lower, in the acute patients compared to the previous event group (all p≤0.02). Thrombin generation during acute events was enhanced, with an 11% faster maximum rate, a 15% higher maximum level and a 26% larger total production (all p<0.01). The increased thrombin generation in acute patients was determined by higher FII and lower antithrombin, while increased free TFPI mediated this effect. When the groups are classified by etiology, all stroke sub-types except cardioembolic have increased TFPI and decreased AT and total thrombin produced., Conclusion: Augmented thrombin generation in acute stroke/TIA is to some extent determined by altered plasma levels of coagulation factors., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Growing evidence indicates that rheumatoid arthritis (RA) is associated with an increased risk for thromboembolic cardiovascular events. We investigated thrombin generation profiles in RA patients and their dependence on plasma factor/inhibitor composition. Plasma factor (F) compositions (II, V, VII, VIII, IX, X), antithrombin and free tissue factor pathway inhibitor (TFPI) from 46 consecutive RA patients with no cardiovascular events (39 female, 7 male, aged 57 [range, 23-75] years; DAS28 [Disease Activity Score] 5.2 +/- 1.1) were compared with those obtained in age- and sex-matched apparently healthy controls. Using each individual's plasma coagulation protein composition, tissue factor-initiated thrombin generation was assessed both computationally and empirically. RA patients had higher fibrinogen (4.18 [IQR 1.09] vs. 2.56 [0.41] g/l, p<0.0001), FVIII (226 +/- 40 vs. 113 +/- 15%, p<0.001), PC (107 [16] vs. 100 [14]%, p<0.001), and free TFPI levels (22.3 [2.2] vs. 14.7 [2.1] ng/ml, p<0.001). DAS28, but not age, RA duration, or C-reactive protein, was associated with FV, FVIII, FIX, FX, antithrombin, and free TFPI (r from 0.27 to 0.48, p<0.05). Intergroup comparison of computational thrombin generation profiles showed that in RA patients, maximum thrombin levels (p=0.01) and the rate of thrombin formation (p<0.0001) were higher, whereas the initiation phase of thrombin generation (p<0.0001) and the time to maximum thrombin levels (p<0.0001) were longer. Empirical reconstructions of the populations reproduced the thrombin generation profiles generated by the computational model. Simulations of thrombin formation suggest that blood plasma composition, i.e. a marked increase in FVIII, somewhat counterbalanced by free TFPI, contributes to the prothrombotic phenotype in RA patients.

The proverb that probably best exemplifies my career in research is attributable to Yogi Berra (http://www.yogiberra.com/), ie, "when you come to a fork in the road ... take it." My career is a consequence of chance interactions with great mentors and talented students and the opportunities provided by a succession of ground-breaking improvements in technology.

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.

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.

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.

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.

Thrombin activated factor Va (factor V[sub IIa], residues 1-709 and 1546-2196) has an apparent dissociation constant (K[sub d,app]) for factor Xa within prothrombinase of ∼0.5 nM. A protease (NN) purified from the venom of the snake Naja nigricollis nigricollis, cleaves human factor V at Asp[sup 697], Asp[sup 1509], and Asp[sup 1514] to produce a molecule (factor V[sub NN]) that is composed of a M[sub r] 100,000 heavy chain (amino acid residues 1-696) and a M[sub r] 80,000 light chain (amino acid residues 1509/1514-2196). Factor V[sub NN], has a K[sub d,app] for factor Xa of 4 nM and reduced clotting activity. Cleavage of factor V[sub IIa] by NN at Asp[sup 697] results in a cofactor that loses ∼60-80% of its clotting activity. An enzyme from Russell's viper venom (RVV) cleaves human factor V at Arg[sup 1018] and Arg[sup 1545] to produce a M[sub r] 150,000 heavy chain and M[sub r] 74,000 light chain (factor V[sub RVV], residues 1-1018 and 1546-2196). The RVV species has affinity for factor Xa and clotting activity similar to the thrombin-activated factor Va. Cleavage of factor V[sub NN] at Arg[sup 1545] by α-thrombin (factor V[sub NN/IIa]) or RVV (factor V[sub NN/RVV]) leads to enhanced affinity of the cofactor for factor Xa (K[sub d,app] ∼ 0.5 nM). A synthetic peptide containing the last 13 residues from the heavy chain of factor Va (amino acid sequence 697-709, D13R) was found to be a competitive inhibitor of prothrombinase with respect to prothrombin. The peptide was also found to specifically interact with thrombin-agarose. These data demonstrate that 1) cleavage at Arg[sup 1545] and formation of the light chain of factor V[sub IIa] is essential for high affinity binding and function of factor Xa within prothrombinase and 2) a binding site for prothrombin is contributed by amino acid residues 697-709 of the heavy chain of the cofactor. [ABSTRACT FROM AUTHOR]

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 (<10s); 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 (>240s). 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. [ABSTRACT FROM AUTHOR]

The prothrombinase complex consists of the protease factor Xa, Ca2+, and factor Va assembled on an anionic membrane. Factor Va functions both as a receptor for factor Xa and a positive effector of factor Xa catalytic efficiency and thus is key to efficient conversion of prothrombin to thrombin. The activation of the procofactor, factor V, to factor Va is an essential reaction that occurs early in the process of tissue factor-initiated blood coagulation; however, the catalytic sequence leading to formation of factor Va is a subject of disagreement. We have used biophysical and biochemical approaches to establish the second order rate constants and reaction pathways for the activation of phospholipid-bound human factor V by native and recombinant thrombin and meizothrombin, by mixtures of prothrombin activation products, and by factor Xa. We have also reassessed the activation of phospholipid-bound human prothrombin by factor Xa. Numerical simulations were performed incorporating the various pathways of factor V activation including the presence or absence of the pathway of factor V-independent prothrombin activation by factor Xa. Reaction pathways for factor V actiration are similar for all thrombin forms. Empirical rate constants and the simulations are consistent with the following mechanism for factor Va formation, α-Thrombin, derived from factor Xa cleavage of phospholipidbound prothrombin via the prothrombin 2 pathway, catalyzes the initial activation of factor V; generation of factor Va in a milieu already containing factor Xa enables prothrombinase formation with consequent meizothrombin formation; and meizothrombin functions as an amplifier of the process of factor V activation and thus has an important procoagulant role. Direct activation of factor V by factor Xa at physiologically relevant concentrations does not appear to be a significant contributor to factor Va formation. [ABSTRACT FROM AUTHOR]