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Abstract: 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 &y& Elsevier]

Presents concise descriptions on the four steps in the blood coagulation process. Initiation of the process in response to vascular injury; Propagation; Constitutive and reactive termination reactions of the process; Elimination; Repair; Integration of the processes at a conceptual level; Lipid involvement in the processes of hemostasis and thrombosis.

The article focuses on abnormal thrombotic responses in the clotting system of people with atherosclerosis and discusses whether the responses have a genetic basis or are simply adaptations to the disease state. Topics discussed include blood-coagulation mechanism as a collection of zymogen-to-enzyme transformations, cellular adhesive reactions and association of thrombosis with occlusion of a blood vessels, and antithrombin Ill-heparin anticoagulant and the activated protein C pathway.

Abstract: Tissue factor plays a primary role in both hemorrhage control and thrombosis depending upon whether its presentation is extravascular or intravascular. The molecular architecture and function of the tissue factor molecule and its role in the activations of factor IX and factor X have been elegantly elucidated but controversies prevail with respect to distinctions between tissue factor sources and tissue factor “activity.” This presentation will review data on the architecture and functions of the tissue factor-factor VIIa complex and discuss the elements of the controversies associated with tissue factor presentation in both normal and pathologic milieu. [Copyright &y& Elsevier]

The research aims of our laboratory are to provide a realistic description of biologic processes involved in protection from hemorrhage and the evolution of thrombosis. To evaluate these processes, we use 4 models of coagulation ranging from 1) studies of blood exiting from microvascular wounds in humans through 2) minimally altered whole blood induced to clot by tissue factor (TF) to 3) reconstitution of the blood coagulation proteome with purified components and to 4) mathematical descriptions of the chemical processes and dynamics that occur. The integration of these 4 models permits comprehensive analyses of the blood coagulation system and predictions of its behavior under normal and pathologic conditions. Data accumulated thus far have led to advances in our understanding of 1) the processes occurring during the initiation and propagation phases of thrombin generation, 2) the roles for individual proteins involved in blood coagulation and its regulation, 3) defects in thrombin generation and clot formation in hemophilia, 4) actions and limitations of pharmacologic agents used to control hemorrhage, thrombosis, and chronic cardiovascular disease, and 5) the relationship between genotypic and phenotypic features of an individual’s plasma proteome and his/her immediate and long-term thrombotic risk. [Copyright &y& Elsevier]

The article focuses on research related to thrombosis, and states that its goal is to improve longevity and reduce morbidity, either by dietary modification or drug intervention. Studies discussed include alterations in blood vessels that occur in the process of lesion development, the role of hemostasis in thrombosis in relation to blood coagulation and its regulation, and effects of nutrients or drugs in thrombotic disease.

This study investigates the dynamics of zymogen activation when both extrinsic tenase and prothrombinase are assembled on an appropriate membrane. Although the activation of prothrombin by surface-localized prothrombinase is clearly mediated by flow-induced dilutional effects, we find that when factor X is activated in isolation by surface-localized extrinsic tenase, it exhibits characteristics of diffusion-mediated activation in which diffusion of substrate to the catalytically active region is rate-limiting. When prothrombin and factor X are activated coincident with each other, competition for available membrane binding sites masks the diffusion-limiting effects of factor X activation. To verify the role of membrane binding in the activation of factor X by extrinsic tenase under flow conditions, we demonstrate that bovine lactadherin competes for both factor X and Xa binding sites, limiting factor X activation and forcing the release of bound factor Xa from the membrane at a venous shear rate (100 s-1). Finally, we present steady-state models of prothrombin and factor X activation under flow showing that zymogen and enzyme membrane binding events further regulate the coagulation process in an open system representative of the vasculature geometry. [ABSTRACT FROM AUTHOR]

The effect of glycosylation on tissue factor (TF) activity was evaluated, and site-specific glycosylation of full-length recombinant TF (rTF) and that of natural TF from human placenta (pTF) were studied by liquid chromatography-tandem mass spectrometry. The amidolytic activity of the TFfactor Vila (FVIIa) complex toward a fluorogenic substrate showed that the catalytic efficiency (Vmax) of the complex increased in the order rTF1-243 (Escherichia coli) < rTF1-263 (Sf9 insect cells) < pTF for the glycosylated and deglycosylated forms. Substrate hydrolysis was unaltered by deglycosylation. In FXase, the Km of FX for rTF1-263-FVIIa remained unchanged after deglycosylation, whereas the kcat decreased slightly. A pronounced decrease, 4-fold, in kcat was observed for pTFFVIIa upon deglycosylation, whereas the Km was minimally altered. The parameters of FX activation by both rTF1-263D-FVIIa and pTFD-FVIIa were identical and similar to those for rTF1-243-FVIIa. In conclusion, carbohydrates significantly influence the activity of TF proteins. Carbohydrate analysis revealed glycosylation on asparagines 11, 124, and 137 in both rTF1-263 and pTF. The carbohydrates of rTF1-263 contain high mannose, hybrid, and fucosylated glycans. Natural pTF contains no high mannose glycans but is modified with hybrid, highly fucosylated, and sialylated sugars. [ABSTRACT FROM AUTHOR]

Studies focused on patient responsiveness to antiplatelet therapies, particularly aspirin and clopidogrel, have increased in recent years. However, the relations of in vivo platelet function and adverse clinical events to results of ex vivo platelet function tests remain largely unknown. This article describes current methods of measuring platelet function in various clinical and research situations and their advantages and disadvantages, reviews evidence for antiplatelet response variability and resistance, discusses the potential pitfalls of monitoring platelet function, and demonstrates emerging data supporting the positive clinical and treatment implications of platelet function testing. [Copyright &y& Elsevier]

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]

Abstract We previously showed that personalizing prophylaxis on the basis of an individual’s pharmacokinetic (PK) response to factor VIII (FVIII) infusion reduces joint and other bleeding events in patients with hemophilia A. We theorized that the FVIII assay used, FVIII product selected, and interpatient differences impact PK assessment and the ability to precisely dose prophylaxis. A comprehensive search of the literature for articles published from January 2004 to September 2017 was performed to identify the variables associated with these three domains. Collectively, product- and patient-related assay discrepancies, variability among plasma-derived and unmodified and modified recombinant FVIII products, and interpatient differences in the response to FVIII infusions are obstacles to precision prophylactic dosing. Stringent laboratory quality assurance programs and proficiency testing to improve the accuracy of FVIII measurement, the widespread use of PK assessment to fine-tune FVIII dosing, and new research to identify patient characteristics and other contributors to bleeding risk and complication development are essential to optimizing outcomes for patients with hemophilia A receiving FVIII prophylaxis. [ABSTRACT FROM AUTHOR]

Introduction: An elevated procoagulant activity observed in trauma patients is, in part, related to tissue factor (TF) located on blood cells and microparticles. However, analysis of trauma patient plasma indicates that there are other contributor(s) to the procoagulant activity. We hypothesize that factor (F)XIa and FIXa are responsible for an additional procoagulant activity in burn patients.Methods: Multiple time-point plasma samples from 56 burn patients (total number of samples was 471; up to 20 time-points/patient collected in 3 weeks following admission) were evaluated in a thrombin generation assay using inhibitory antibodies to TF, FIXa and FXIa.Results: Due to the limited volume of some samples, not all were analyzed for all three proteins. At admission, 10 of 53 patients (19%) had active TF, 53 of 55 (96%) had FXIa and 48 of 55 (87%) had FIXa in their plasma. 34 patients of 56 enrolled (61%) showed TF activity at one or more time-points. All patients had FXIa and 96% had FIXa at one or more time-points. Overall, TF was observed in 99 of 455 samples analyzed (22%), FXIa in 424 of 471 (90%) and FIXa in 244 of 471 (52%). The concentration of TF was relatively low and varied between 0 and 2.1pM, whereas that of FXIa was higher, exceeding 100pM in some samples. The majority of samples with FIXa had it at sub-nanomolar concentrations. No TF, FXIa and FIXa activity was detected in plasma from healthy individuals.Conclusions: For the first time reported, the majority of plasma samples from burn patients have active FXIa and FIXa, with a significant fraction of them having active TF. The concentration of all three proteins varies in a wide range. [ABSTRACT FROM AUTHOR]

Abstract: Background: Expression of tissue factor (TF) antigen and activity in platelets is controversial and dependent upon the laboratory and reagents used. Two forms of TF were described: an oxidized functional form and a reduced nonfunctional form that is converted to the active form through the formation of an allosteric disulfide. This study tests the hypothesis that the discrepancies regarding platelet TF expression are due to differential expression of the two forms. Methods: Specific reagents that recognize both oxidized and reduced TF were used in flow cytometry of unactivated and activated platelets and western blotting of whole platelet lysates. TF-dependent activity measurements were used to confirm the results. Results: Western blotting analyses of placental TF demonstrated that, in contrast to anti-TF#5, which is directed against the oxidized form of TF, a sheep anti-human TF polyclonal antibody recognizes both the reduced and oxidized forms. Flow cytometric analyses demonstrated that the sheep antibody did not react with the surface of unactivated platelets or platelets activated with thrombin receptor agonist peptide, PAR-1. This observation was confirmed using biotinylated active site-blocked factor (F)VIIa: no binding was observed. Likewise, neither form of TF was detected by western blotting of whole platelet lysates with sheep anti-hTF. Consistent with these observations, no FXa or FIXa generation by FVIIa was detected at the surface of these platelets. Similarly, no TF-related activity was observed in whole blood using thromboelastography. Conclusion and significance: Platelets from healthy donors do not express either oxidized (functional) or reduced (nonfunctional) forms of TF. [Copyright &y& Elsevier]

Abstract: Background: Tissue factor (TF), an in vivo initiator of blood coagulation, is a transmembrane protein and has two disulfides in the extracellular domain. The integrity of one cysteine pair, Cys186–Cys209, has been hypothesized to be essential for an allosteric “decryption” phenomenon, presumably regulating TF procoagulant function, which has been the subject of a lengthy debate. The conclusions of published studies on this subject are based on indirect evidences obtained by the use of reagents with potentially oxidizing/reducing properties. Methods: The status of disulfides in recombinant TF1–263 and natural placental TF in their non-reduced native and reduced forms was determined by mass-spectrometry. Functional assays were performed to assess TF cofactor function. Results: In native proteins, all four cysteines of the extracellular domain of TF are oxidized. Reduced TF retains factor VIIa binding capacity but completely loses the cofactor function. Conclusion: The reduction of TF disulfides (with or without alkylation) eliminates TF regulation of factor VIIa catalytic function in both membrane dependent FX activation and membrane independent synthetic substrate hydrolysis. General significance: Results of this study advance our knowledge on TF structure/function relationships. [Copyright &y& Elsevier]

The protease α-thrombin is a key enzyme of the coagulation process as it is at the cross-roads of both the pro- and anticoagulant pathways. The main source of α-thrombin in vivo is the activation of prothrombin by the prothrombinase complex assembled on either an activated cell membrane or cell fragment, the most relevant of which is the activated platelet surface. When prothrombinase is assembled on synthetic phospholipid vesicles, prothrombin activation proceeds with an initial cleavage at Arg-320 yielding the catalytically active, yet effectively anticoagulant intermediate meizothrombin, which is released from the enzyme complex ∼30-40% of the time. Prothrombinase assembled on the surface of activated platelets has been shown to proceed through the inactive intermediate prethrombin-2 via an initial cleavage at Arg-271 followed by cleavage at Arg-320. The current work tests whether or not platelet-associated prothrombinase proceeds via a concerted mechanism through a study of prothrombinase assembly and function on collagen-adhered, thrombin-activated, washed human platelets in a flow chamber. Prothrombinase assembly was demonstrated through visualization of bound factor Xa by confocal microscopy using a fluorophore-labeled anti-factor Xa antibody, which demonstrated the presence of distinct platelet subpopulations capable of binding factor Xa. When prothrombin activation was monitored at a typical venous shear rate over preassembled platelet-associated prothrombinase neither potential intermediate, meizothrombin or prethrombin-2, was observed in the effluent. Collectively, these findings suggest that platelet-associated prothrombinase activates prothrombin via an efficient concerted mechanism in which neither intermediate is released. [ABSTRACT FROM AUTHOR]

Abstract: Tissue factor is a membrane protein, which in a complex with factor VIIa initiates in vivo blood coagulation. Due to the scarcity of natural tissue factor protein, most studies have relied upon recombinant tissue factor forms. However, there have been only cursory experimental comparisons of natural and recombinant tissue factor proteins. Our preliminary data suggested that placental tissue factor in a complex with factor VIIa was more efficient activator of factor X than the recombinant protein. After deglycosylation, both forms of tissue factor showed almost an identical activity in the extrinsic factor Xase. Analyses using tryptic digestion and mass-spectrometry revealed that the levels of glycosylation and the composition of carbohydrates present in natural placental tissue factor were different than those in its recombinant counterpart. These data indicate that natural and recombinant tissue factor proteins differ in their posttranslational modifications and that these differences translate into different cofactor activity. Thus the use of recombinant tissue factor proteins for the quantitation of natural tissue factor is misleading. [Copyright &y& Elsevier]

Abstract: Tissue factor (TF) is the major physiological initiator of blood coagulation. It exists as an integral membrane protein that upon injury to the blood vessel becomes exposed to the blood stream and initiates a series of enzymatic reactions that cause blood to clot. TF is also found within circulating blood cells, requiring specific signaling events to promote its expression. In this review we will discuss current controversies concerning the structure–activity relationships of TF and contributions of TF to the hemostatic process, the potential roles of intravascular TF, including non-cell-bound TF and cell-expressed TF and the overall relationship between TF function and hemorrhage control. [Copyright &y& Elsevier]

Abstract: Background: Tissue factor (TF) is a single polypeptide integral membrane glycoprotein composed of 263 residues and is essential to life in its role as the initiator of blood coagulation. Previously we have shown that the activity of the natural placental TF (pTF) and the recombinant TF (rTF) from Sf9 insect cells is different (Krudysz-Amblo, J. et al (2010) J. Biol. Chem. 285, 3371–3382). Methods: In this study, using mass spectrometry, we show by quantitative analysis that the extent of glycosylation varies on each protein. Results and conclusions: Fractional abundance of each glycan composition at each of the three glycosylation sites reveals the most pronounced difference to be at asparagine (Asn) 11. This residue is located in the region of extensive TF-factor VIIa (FVIIa) interaction. Carbohydrate fractional abundance at Asn11 revealed that glycosylation in the natural placental TF is much more prevalent (~76%) than in the recombinant protein (~20%). The extent of glycosylation on Asn124 and Asn137 is similar in the two proteins, despite the pronounced differences in the carbohydrate composition. Additionally, 77% of rTF exists as TF des-1, 2 (missing the first two amino acids from the N-terminus). In contrast, only 31% of pTF is found in the des-1, 2 form. Conclusion: These observations may attribute to the difference in the ability of TF–FVIIa complex to activate factor X (FX). General significance: Structural and functional comparison of the recombinant and natural protein advances our understanding and knowledge on the biological activity of TF. [Copyright &y& Elsevier]

Abstract: 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 24hours from symptom onset, and (2) 100 individuals 1 to 4years 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. [ABSTRACT FROM AUTHOR]

Abstract: Introduction: The quantitation of factor (F)VIII by activity-based assays is influenced by the method, procedure, the quality and properties of reagents used and concentrations of other plasma proteins, including von Willebrand factor (VWF). Objective: To compare FVIII concentrations measured by activity-based assays with those obtained by an immunoassay and to establish the influence of plasma dilution on the FVIII clotting activity (FVIIIc). Methods: The APTT, a chromogenic assay (Coatest) and two in-house immunoassays were used. Albumin-free recombinant FVIII was used as the calibrator in all assays. Results: For a group of 44 healthy individuals (HI), the mean value observed for FVIII antigen (FVIIIag; 1.22±0.56nM; S.D.) is substantially higher than that for FVIIIc (0.65±0.29nM) and the chromogenic assay (FVIIIch; 0.50±0.23nM). A positive correlation between FVIIIag and VWFag with R2 =0.20 was observed. Since plasma VWF has an inhibitory effect on FVIIIc, we evaluated the influence of plasma dilutions on FVIIIc in HI (n=105). At a 4-fold dilution, estimates of FVIIIc by clotting assay were much lower than FVIIIag (0.77±0.31 vs. 1.14±0.48nM). At 10- and 25-fold dilutions, the estimated FVIIIc increased to 0.87±0.36 and 0.94±0.44nM, respectively. Conclusions: 1) In plasma, FVIIIag is higher than FVIIIc and FVIIIch; and 2) Real FVIII concentrations in plasma can be estimated by measuring FVIIIag. [Copyright &y& Elsevier]

The function of tissue factor (Tf)-initiated coagulation is hemorrhage control through the formation and maintenance of an impermeable platelet-fibrin barrier. The catalytic processes involved in the clot maintenance function are not well defined, although the rebleeding problems characteristic of individuals with hemophilias A and B suggest a link between specific defects in the Tf-initiated process and defects in the maintenance function. We have previously demonstrated, using a methodology of "flow replacement" (or resupply) of ongoing Tf-initiated reactions with fresh reactants, that procoagulant complexes are produced during Tf-initiated coagulation, which are capable of reinitiating coagulation without input from extrinsic factor Xase activity (Orfeo, T., Butenas, S., Brummel-Ziedins, K. E., and Mann, K. G. (2005)1. Biol. Chem. 280, 42887-42896). Here we used Tf-initiated reactions in normal and hemophilia blood or in their corresponding proteome mixtures as sources of pro-coagulant end products and then varied the resupplying mate- rial to determine the identity of the catalysts that drive the new cycle of thrombin formation. The central findings are as follows: 1) the prothrombinase complex (fVa-fXa-Ca2+-membrane) accumulated during the episode of Tf-initiated coagulation is the primary catalyst responsible for the observed pattern of prothrombin activation after resupply; 2) impairments in intrinsic factor Xase function, i.e. hemophilias A and B, result in an impaired capacity to mount a resupply response; and 3) in normal hemostasis the intrinsic factor Xase function contributes to the durability of the resupply response. [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 presence of activation peptides (AP) of the vitamin K-dependent proteins in the phlebotomy blood of human subjects suggests that active serine proteases may circulate in blood as well. The goal of the current study was to evaluate the influence of trace amounts of key coagulation proteases on tissue factor-independent thrombin generation using three models of coagulation. With procoagulants and select coagulation inhibitors at mean physiological concentrations, concentrations of factor IXa, factor Xa, and thrombin were set either equal to those of their AP or to values that would result based upon the rates of AP/enzyme generation and steady state enzyme inhibition. In the latter case, numerical simulation predicts that sufficient thrombin to produce a solid clot would be generated in ∼2 min. Empirical data from the synthetic plasma suggest clotting times of 3-5 min, which are similar to that observed in contact pathway-inhibited whole blood (4.3 min) initiated with the same concentrations of factors IXa and Xa and thrombin. Numerical simulations performed with the concentrations of two of the enzymes held constant and one varied suggest that the presence of any pair of enzymes is sufficient to yield rapid clot formation. Modeling of states (numerical simulation and whole blood) where only one circulating protease is present at steady state concentration shows significant thrombin generation only for factor IX. The addition of factor Xa and thrombin has little effect (if any) on thrombin generation induced by factor IXa alone. These data indicate that 1) concentrations of active coagulation enzymes circulating in vivo are significantly lower than can be predicted from the concentrations of their AP, and 2) expected trace amounts of factor IXa can trigger thrombin generation in the absence of tissue factor. [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]

Abstract: Background: Elevated factor (F)XI is associated with an increased risk for ischemic stroke. Activated FXI (FXIa) and tissue factor (TF) have not been studied following stroke. The aim of the current study was to evaluate circulating FXIa and TF in patients with prior cerebrovascular events. Patients/Methods: We studied 241 patients, including 162 after ischemic stroke and 79 after transient ischemic attack (TIA), recruited 6months to 4years (median, 36months) after the events. Plasma TF and FXIa activity following the index event were determined in clotting assays by measuring the response to inhibitory monoclonal antibodies. Results: Active TF was detected in 25 (10.4%) of the patients, while FXIa activity (median, 37.5 [IQR 397] pM) was found in 64 (26.7%) of the patients (p<0.01). The prevalence of active TF and FXIa was higher in subjects with previous stroke compared with those with a history of TIA (13% vs 5.1%, p=0.05, and 34% vs 11.4%, p<0.0001, respectively). Patients with circulating FXIa were younger and had higher fibrinogen and interleukin-6 compared to the remainder. Patients with detectable TF or FXIa activity had higher NIHSS score, higher modified Rankin scale and lower Barthel Index than the remaining subjects (all p<0.05). Conclusion: Circulating active TF and FXIa can occur in patients with cerebrovascular ischemic events ≥6months after the events. The presence of these factors is associated with worse functional outcomes, which highlights the role of persistent hypercoagulable state in cerebrovascular disease. [Copyright &y& Elsevier]