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Although use of thromboelastography (TEG) to diagnose coagulopathy and guide clinical decision-making is increasing, relative performance of different TEG methods has not been well-defined. Rapid-TEG (rTEG), kaolin-TEG (kTEG), and native-TEG (nTEG) were performed on blood samples from burn patients presenting to a regional center from admission to 21 days. Patients were categorized by burn severity, mortality, and fibrinolytic phenotypes (Shutdown [SD], Physiologic [PHYS], and Hyperfibrinolytic [HF]). Manufacturer ranges and published TEG cutoffs were examined. Concordance correlations (Rc) of TEG parameters (R, α-angle, maximum amplitude [MA], LY30) measured agreement and Cohen’s Kappa (κ) determined interclass reliability. Patients (n= 121) were mostly male (n= 84; 69.4%), with median age 40 years, median TBSA burn 13%, and mortality 17% (n= 21). Severe burns (≥40% TBSA) were associated with lower admission α-angle for rTEG (P= .03) and lower MA for rTEG (P= .02) and kTEG (P= .01). MA was lower in patients who died (nTEG, P= .04; kTEG, P= .02; rTEG, P= .003). Admission HF was associated with increased mortality (OR, 10.45; 95% CI, 2.54–43.31, P= .001) on rTEG only. Delayed SD was associated with mortality using rTEG and nTEG (OR 9.46; 95% CI, 1.96–45.73; P= .005 and OR, 6.91; 95% CI, 1.35–35.48; P= .02). Admission TEGs showed poor agreement on R-time (Rc, 0.00–0.56) and α-angle (0.40 to 0.55), and moderate agreement on MA (0.67–0.81) and LY30 (0.72–0.93). Interclass reliability was lowest for R-time (κ, −0.07 to 0.01) and α-angle (−0.06 to 0.17) and highest for MA (0.22-0.51) and LY30 (0.29-0.49). Choice of TEG method may impact clinical decision-making. rTEG appeared most sensitive in parameter-specific associations with injury severity, abnormal fibrinolysis, and mortality.

Burn injury is associated with endothelial dysfunction and coagulopathy and concomitant inhalation injury (IHI) increases morbidity and mortality. The aim of this work is to identify associations between IHI, coagulation homeostasis, vascular endothelium, and clinical outcomes in burn patients. One hundred and twelve patients presenting to a regional burn center were included in this retrospective cohort study. Whole blood was collected at set intervals from admission through 24 hours and underwent viscoelastic assay with rapid thromboelastography (rTEG). Syndecan-1 (SDC-1) on admission was quantified by ELISA. Patients were grouped by the presence (n= 28) or absence (n= 84) of concomitant IHI and rTEG parameters, fibrinolytic phenotypes, SDC-1, and clinical outcomes were compared. Of the 112 thermally injured patients, 28 (25%) had IHI. Most patients were male (68.8%) with a median age of 40 (interquartile range, 29–57) years. Patients with IHI had higher overall mortality (42.68% vs 8.3%; P< .0001). rTEG LY30 was lower in patients with IHI at hours 4 and 12 (P< .05). There was a pattern of increased abnormal fibrinolytic phenotypes among IHI patients. There was a greater proportion of IHI patients with endotheliopathy (SDC-1 > 34 ng/ml) (64.7% vs 26.4%; P= .008). There was a pattern of increased mortality among patients with IHI and endotheliopathy (0% vs 72.7%; P= .004). Significant differences between patients with and without IHI were found in measures assessing fibrinolytic potential and endotheliopathy. Mortality was associated with abnormal fibrinolysis, endotheliopathy, and IHI. However, the extent to which IHI-associated dysfunction is independent of TBSA burn size remains to be elucidated.

Thromboelastic testing provides an assessment of a patient’s coagulation and fibrinolytic systems. In recent years, thromboelastic testing has become an important point of care technique. However, its direct connection with the underlying biochemistry of coagulation and clot formation is not obvious. Toward this issue, we describe a validated reduced order mathematical model of coagulation and fibrinolysis, consisting of 22 ordinary differential equations, which described clot formation from initiation of the coagulation cascade through the degradation of polymerized fibrin by plasmin. We trained the model via leave one out cross validation on ROTEM measurements, a common thromboelastic test, on four patients, and then predicted ROTEM trajectories on four unseen patients, in whole blood and whole blood with the addition of 2 nM tissue plasminogen activator. Following model validation, sensitivity analysis suggested which biochemical interactions and species controlled the system response. Lastly, we investigated if we could estimate protein concentrations from commonly reported thromboelastic metrics. These estimation studies suggested we could (on average) relearn the initial fibrinogen concentration to within 20% of its true value. Taken together, this work presents a model which connects the underlying biochemistry of coagulation and clot formation in patients to a common point of care thrombelastographic test.

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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 (Kd = 2.8 μM) and ∼0.3 molecules of thrombin at high-affinity binding sites (Kd = 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 (Re < 1) and reaction rates are driven by enzyme kinetics (Pe = 100, Da = 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.

Viscoelastic measurements are frequently being used in clinical and research settings for a rapid assessment of the hemostatic processes of blood clot formation and degradation. These measurements are being performed on either of two instruments (TEG and ROTEM) using their proprietary reagents. Standardization between the instruments and the reagents has been lacking but is necessary to compare results across instruments. In this study, we perform a crossover analysis between the TEG and ROTEM instruments using proprietary reagents from each manufacturer.

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.

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.

The generation of proteolyzed prothrombin species by preassembled prothrombinase in phospholipid-coated glass capillaries was studied at physiologic shear rates (100–1000 s−1). The concentration of active thrombin species (α-thrombin and meizothrombin) reaches a steady state, which varies inversely with shear rate. When corrected for shear rate, steady-state levels of active thrombin species exhibit no variation and a Michaelis-Menten analysis reveals that chemistry of this reaction is invariant between open and closed systems; collectively, these data imply that variations with shear rate arise from dilutional effects. Significantly, the major products observed include nonreactive species arising from the loss of prothrombin's phospholipid binding domain (des F1 species). A numerical model developed to investigate the spatial and temporal distribution of active thrombin species within the capillary reasonably approximates the observed output of total thrombin species at different shears; it also predicts concentrations of active thrombin species in the wall region sufficient to account for observed levels of des FI species. The predominant feedback formation of nonreactive species and high levels of the primarily anticoagulant intermediate meizothrombin (∼40% of total active thrombin species) may provide a mechanism to prevent thrombus propagation downstream of a site of thrombosis or hemorrhage.

We report the effect of homocysteine on the inactivation of factor Va by activated protein C (APC) using clotting assays, immunoblotting, and radiolabeling experiments. Homocysteine, cysteine, or homocysteine thiolactone have no effect on factor V activation by α-thrombin. Factor Va derived from homocysteine-treated factor V was inactivated by APC at a reduced rate. The inactivation impairment increased with increasing homocysteine concentration (pseudo first order rate k= 1.2, 0.9, 0.7, 0.4 min−1at 0, 0.03, 0.1, 1 mmhomocysteine, respectively). Neither cysteine nor homocysteine thiolactone treatment of factor V affected APC inactivation of derived factor Va. Western blot analyses of APC inactivation of homocysteine-modified factor Va are consistent with the results of clotting assays. Factor Va, derived from factor V treated with 1 mmβ-mercaptoethanol was inactivated more rapidly than the untreated protein sample. Factor V incubated with [35S]homocysteine (10–450 μm) incorporated label within 5 min, which was found only in those fragments that contained free sulfhydryl groups: the light chain (Cys-1960, Cys-2113), the B region (Cys-1085), and the 26/28-kDa (residues 507–709) APC cleavage products of the heavy chain (Cys-539, Cys-585). Treatment with β-mercaptoethanol removed all radiolabel. Plasma of patients assessed to be hyperhomocysteinemic showed APC resistance in a clot-based assay. Our results indicate that homocysteine rapidly incorporates into factor V and that the prothrombotic tendency in hyperhomocysteinemia may be related to impaired inactivation of factor Va by APC due to homocysteinylation of the cofactor by modification of free cysteine(s).

No relevant conflicts of interest to declare.

Background: Pregnancy has been described as a hypercoagulable state and is characterized by an increased risk of thrombotic events; yet the absolute incidence is low. The glycocalyx is a network of proteoglycans and glycoproteins projecting from the endothelial cell surface that plays a key role in maintaining endothelial integrity. Syndecan-1 is a component of the endothelial glycocalyx and is substituted with heparan sulfate chains which promote its interactions with key anticoagulants such as tissue factor pathway inhibitor and antithrombin. Proteolytic release of components of the glycocalyx (i.e.shedding) has been shown to occur in response to a number of physiologic challenges. Thrombomodulin, another endothelial cell transmembrane protein, is a key component of the primary dynamic anticoagulant mechanism limiting thrombus growth and is also subject to proteolytic release. In this study, we longitudinally evaluate hemostatic activation (D-dimer), glycocalyx shedding (syndecan-1) and thrombomodulin release in healthy women who became pregnant.

No relevant conflicts of interest to declare.

No relevant conflicts of interest to declare.

Trauma is the leading cause of death in individuals under the age of 44. Acute traumatic coagulopathy (ATC) occurs when severe injury is combined with shock and results in a hypocoagulable state resulting in increased bleeding, increased resuscitation requirements and worsened outcomes including a 4-fold increase in mortality. Our group has implicated protein C activation as a likely mechanism for ATC. We have evaluated whether activated protein C (APC) induces coagulopathy through cleavage of factor (f)V/Va by systematically evaluating 4 groups of increasing level of traumatic coagulopathy.

IntroductionThe rapid coagulation response to vascular injury is mediated by the formation of the extrinsictenase, intrinsictenase, and prothrombinasecomplexes. The prothrombotic response to injury is down-regulated by the presence of circulating active protease inhibitors as well as the protein C pathway. Protein C is activated by the thrombin-thrombomodulin complex; activated protein C (APC) then regulates thrombin generation by proteolytically inactivating factors Va and VIIIa, cofactors of the procoagulant prothrombinaseand intrinsic tenasecomplexes, respectively. Previous reports have analyzed the biochemistry of the protein C system in closed systems. Our goal is to characterize the behavior of the protein C system under flow as well as the impact of circulating cells on the activation of protein C.

The rapid coagulation response to vascular injury is mediated by the formation of three enzyme cofactor complexes (extrinsic tenase, intrinsic tenase and prothrombinase) on membrane surfaces. A common structural feature of these proteases is their GLA domains, each of which requires the binding of divalent metal ions at multiple sites to achieve the conformation necessary for optimal membrane and cofactor binding. Both Ca2+ and Mg2+ ions have been reported to bind to GLA domain sites. However almost all studies kinetically characterizing these complexes have been done in the presence of Ca2+ (2-5 mM) as the sole metal ion, despite the relatively equivalent availability in plasma of both free Ca2+ (∼1.1 mM) and Mg2+ (∼0.6 mM) (Ca2+/Mg2+). A recent study has systematically examined the effects of various Ca2+ concentrations with and without Mg2+ on the membrane binding of activated protein C (APC) and FVIIa and enzymatic activity of APC and the extrinsic tenase complex which were enhanced in Ca2+/Mg2+ relative to Ca2+ alone (Vadivel, K., et al, 2013 JMB). In the current study we compare the effects of plasma levels of Ca2+ and Mg2+ versus Ca2+alone on the catalytic performances of the extrinsic tenase, intrinsic tenase and prothrombinase complexes individually and collectively.All experiments were conducted in Hepes buffered saline pH 7.4 containing 0.1% PEG and either 2 mM Ca2+ or 1.1 mM Ca2+/0.6 mM Mg2+ (Ca2+/Mg2+). In closed system experiments, enzyme-cofactor complexes were assembled on phospholipid vesicles composed of a 3:1 ratio of synthetic phosphatidylcholine and phosphatidylserine (PCPS), and zymogen activation monitored via sampling into assay mixtures containing the appropriate chromogenic substrate. In open system experiments complexes were preassembled on PCPS coated capillaries, the zymogen delivered in the flowing phase and the extent of zymogen activation monitored in the effluent as described previously (Haynes, LM., et al, 2011 Biophys J). The combined interaction of the procoagulant enzyme cofactor complexes under both metal ion conditions was studied in a synthetic coagulation proteome monitoring thrombin (IIa) generation as previously described (van’t Veer, C., and Mann, KG, 1997 JBC).Extrinsic tenase The extrinsic tenase complex had an approximately two-fold higher rate of FXa generation in the presence of Ca2+/Mg2+ (1.78 ±0.05 pM/s) versus Ca2+ alone (0.88 ± 0.02 pM/s) (N=3, p<0.001). Experiments were also conducted in the absence or presence of tissue factor pathway inhibitor (TFPI); in the presence of TFPI and Ca2+/Mg2+ ions the extrinsic tenase complex was three times as catalytically active compared to TFPI and Ca2+ only (N=3, p<0.05). Direct FXa inhibition by TFPI was not significantly different between the Ca2+ and Ca2+/Mg2+ containing buffers. Intrinsic tenase In closed system experiments the intrinsic tenase complex showed impaired FXa generation in the presence of Ca2+/Mg2+ (29.3 ± 3.4 pM/s) compared to Ca2+ alone (51.7 ± 3.0 pM/s) (N=3, p<0.001). Thrombin activation of FVIII was not statistically different between both buffers, however the presence of Ca2+/Mg2+ resulted in a more rapid loss of cofactor activity over Ca2+ alone. Prothrombinase Under flow, IIa generation measured from prothrombinase was not statistically different between both buffers when measured at five different shear rates (100-1000s-1, N≥2). Plasma proteome In the synthetic coagulation proteome (N=4) the presence of Ca2+/Mg2+ led to an average 31% increase in maximum IIa levels compared to Ca2+alone and an average decrease of 1 minute to reach maximum levels.In summary, the presence of plasma levels of Ca2+ and Mg2+, compared to Ca2+ alone, enhances the initiation phase of the extrinsic pathway of coagulation by enhancing the rate of FXa generation from the extrinsic tenase complex while also impairing the TFPI inhibition of the extrinsic tenase complex. These procoagulant effects are potentially abrogated by the suppressive effects of Ca2+/Mg2+ on the intrinsic tenase complex, potentially caused by the increased rate of spontaneous inactivation of FVIIIa. These results highlight the importance of complementing the assessments of individual enzyme complex systems with studies of the larger complex systems in which they function to identify the net physiologic impact of metal ions that target various components in the coagulation cascade.Mann: Haematologic Technologies, Inc: Equity Ownership, Membership on an entity’s Board of Directors or advisory committees.

The rapid coagulation response to vascular injury is mediated by the formation of three enzyme cofactor complexes (extrinsic tenase, intrinsic tenase and prothrombinase) on membrane surfaces. A common structural feature of these proteases is their GLA domains, each of which requires the binding of divalent metal ions at multiple sites to achieve the conformation necessary for optimal membrane and cofactor binding. Both Ca2+and Mg2+ions have been reported to bind to GLA domain sites. However almost all studies kinetically characterizing these complexes have been done in the presence of Ca2+(2-5 mM) as the sole metal ion, despite the relatively equivalent availability in plasma of both free Ca2+(∼1.1 mM) and Mg2+(∼0.6 mM) (Ca2+/Mg2+). A recent study has systematically examined the effects of various Ca2+concentrations with and without Mg2+on the membrane binding of activated protein C (APC) and FVIIa and enzymatic activity of APC and the extrinsic tenase complex which were enhanced in Ca2+/Mg2+relative to Ca2+alone (Vadivel, K., et al, 2013 JMB). In the current study we compare the effects of plasma levels of Ca2+and Mg2+versus Ca2+alone on the catalytic performances of the extrinsic tenase, intrinsic tenase and prothrombinase complexes individually and collectively.

In in vitro models of tissue factor (TF)-initiated coagulation, FXI activation has been linked to increased thrombin generation. However the effects of FXI in experimental models of normal hemostasis have often been subtle, prompting ongoing investigations to define contributing cofactors, potential collaborating activators and/or reaction conditions (e.g. low TF concentrations). In this study, predictions from a computational model of TF-initiated thrombin generation that includes thrombin dependent FXI activation are used to direct an investigation of the role of FXI in two empirical models of TF-initiated coagulation. Methods. FXI activation and FXIa interactions were computationally modeled by adding the appropriate sets of equations describing thrombin activation of FXI, FXIa activation of FIX, antithrombin inhibition of FXIa, and high molecular weight kininogen binding to FXI to the existing framework of differential equations. The efficacy of FXIa in promoting thrombin generation (α- thrombin-antithrombin, αTAT) was assessed via titration in contact pathway inhibited whole blood and compared to activation by TF, FIXa, FXa and α-thrombin. TF-initiated reactions and their resupply were performed as described previously (Orfeo T et al. J. Biol. Chem., 2008) in either contact pathway inhibited blood ± an inhibitory anti-FXI antibody) or in synthetic coagulation proteome (SCP) mixtures (± FXI), allowing FXI effects on both the TF dependent phase and the resulting procoagulant pool of catalysts to be evaluated. Results. The computational model (± FXI pathway) was validated by showing congruence between computational thrombin generation profiles initiated with 5 pM TF and corresponding SCP reconstructions, which showed the lag phase shortened by 30 to 60 s and maximum thrombin levels increased when FXI was present. Similarly there was good correspondence between computational and SCP thrombin generation when FXIa (4 pM by active site) was the initiator. A computational analysis provided the following ranking of effectiveness in initiating thrombin generation: FXIa>TF>FIXa>FXa>α-thrombin. When tested in contact pathway inhibited blood, addition of 5 pM FXIa (by active site) resulted in clot times and 20 min αTAT levels similar to those observed with 5 pM TF, while similar outcomes required 25 pM FIXa, 100 pM FXa or between 10–100 nM α-thrombin. The computational modeling made clear two consequences of FXI feedback activation. The first was mechanistic, demonstrating that the amplification of thrombin generation was achieved by better coordinating the initial activations of FIX and FVIII; limited early initiation phase activation of FXI (<0.01% zymogen activation) increases the amount of FIXa available during the activation of FVIII, yielding higher concentrations of intrinsic tenase earlier in the initiation phase. The second consequence was increased FIXa accumulation as the reaction proceeds. This prediction was confirmed in two ways: 1) Western blot analysis of SCP reactions containing FXI showed ∼20% consumption of FIX over 20 min compared to no detectable FIX consumption in reactions without FXI; and 2) Resupply of TF-initiated SCP reactions (± 30 nM FXI) demonstrated that both the FVIII dependence and the long-term stability of the observed re-initiation of thrombin generation depended on the presence of FXI. Resupply studies were then performed in contact pathway inhibited blood (N=4 individuals) by using an inhibitory anti-FXI antibody to negate FXI contributions to both the TF-initiated and resupply dynamics. No effect of blocking FXI function on the time courses of αTAT formation was observed in the TF initiated phase. Two of the four individuals showed attenuated αTAT formation upon resupply when FXI function was blocked from the onset of TF initiation. Conclusions. FXIa is a potent initiator of coagulation, and the computational and SCP analyses predict the potential for feedback activation by thrombin after TF initiation to somewhat enhance the propagation phase of thrombin generation and more dramatically enhance the resupply response. However in contact pathway inhibited blood, FXI contributions to Tf-initiated thrombin generation are not discernible and the effect on the resupply response is variable between individuals suggesting additional mechanisms suppressing FXI activation in blood.Mann: Haematologic Technologies: Chairman of the Board, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; corn trypsin inhibitor: Patents & Royalties; NIH, DOD, Baxter: Research Funding; Merck, Daiichi Sankyo, Baxter, GTI: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

Abstract 2239

Factor VLeiden (G1691A;R506Q) is an autosomal dominant allele displaying high prevalence (3–7%) in the United States Caucasian population and a high incidence of venous thrombosis in homozygotes (50% lifetime risk) but reduced penetrance in heterozygote carriers (<10% lifetime risk). Factors that precipitate or suppress the expression of the thrombotic phenotype in factor VLeiden heterozygotes are not well defined except for relatively infrequent instances of coexpression of other thrombophilic risk factors, e.g. the prothrombin G20210A mutant. Our goal is to mathematically evaluate the effect of factor VLeiden on tissue factor-initiated thrombin generation and determine whether concentration variations within the normal range of antithrombin (86–128%), prothrombin (60–140%) or protein C (77–183%) modulate the factor VLeiden effect when all other factor levels are at mean physiologic concentrations. Our mathematical model (Hockin et al. (2002) JBC 277:18322) was extended to incorporate the protein C pathway by including descriptions of thrombin binding to thrombomodulin, activation of protein C and factor Va inactivation by activated protein C. Simulations were conducted with 100% factor V, 50% factor V/50% factor VLeiden (heterozygote), and 100% factor VLeiden (homozygote). Heterozygous expression of factor VLeiden increases the maximum level of thrombin by 25% and the maximum rate of thrombin generation by a factor of 1.6 over that seen with 100% factor V. Homozygous factor VLeiden yields a 2.6-fold increase in maximum rate and a 60% increase in maximum thrombin level compared to 100% factor V. Decreasing the protein C concentration to 77% in a factor VLeiden heterozygote results in a thrombin generation profile with maximum levels and rates comparable to the factor VLeiden homozygote. At 150% protein C, thrombin generation is suppressed to levels similar to those seen with 100% factor V. When the effect of variable prothrombin levels in a factor VLeiden heterozygote is examined, a 60% level of prothrombin decreases the maximum level of thrombin generation below that seen with 100% factor V. When prothrombin levels are at 140% in the factor VLeiden heterozygote, the thrombin generation profile is similar to that observed with 100% factor VLeiden . When the effect of variable antithrombin levels was tested in a factor VLeiden heterozygote, a 14% decrease in antithrombin resulted in maximum thrombin levels approaching those of the factor VLeiden homozygote. In contrast, 128% antithrombin in a factor VLeiden heterozygote resulted in the suppression of thrombin generation to levels consistent with those seen with 100% factor V. Our results predict that the thrombotic effect of heterogenous expression of factor VLeiden can be reduced when prothrombin levels are at the low extreme of the normal range or when antithrombin or protein C levels are at the high extreme of the normal range. Conversely, the thrombotic effect of the heterozygous expression of factor VLeiden is intensified when prothrombin levels are at the high extreme of normal values or when antithrombin or protein C levels at the low extreme of normal. These results may contribute to the understanding of phenotypic variation within factor VLeiden heterozygotes.

Factor VLeiden(G1691A;R506Q) is an autosomal dominant allele displaying high prevalence (3–7%) in the United States Caucasian population and a high incidence of venous thrombosis in homozygotes (50% lifetime risk) but reduced penetrance in heterozygote carriers (<10% lifetime risk). Factors that precipitate or suppress the expression of the thrombotic phenotype in factor VLeidenheterozygotes are not well defined except for relatively infrequent instances of coexpression of other thrombophilic risk factors, e.g.the prothrombin G20210A mutant. Our goal is to mathematically evaluate the effect of factor VLeidenon tissue factor-initiated thrombin generation and determine whether concentration variations within the normal range of antithrombin (86–128%), prothrombin (60–140%) or protein C (77–183%) modulate the factor VLeideneffect when all other factor levels are at mean physiologic concentrations. Our mathematical model (Hockin et al.(2002) JBC 277:18322) was extended to incorporate the protein C pathway by including descriptions of thrombin binding to thrombomodulin, activation of protein C and factor Va inactivation by activated protein C. Simulations were conducted with 100% factor V, 50% factor V/50% factor VLeiden(heterozygote), and 100% factor VLeiden(homozygote). Heterozygous expression of factor VLeidenincreases the maximum level of thrombin by 25% and the maximum rate of thrombin generation by a factor of 1.6 over that seen with 100% factor V. Homozygous factor VLeidenyields a 2.6-fold increase in maximum rate and a 60% increase in maximum thrombin level compared to 100% factor V. Decreasing the protein C concentration to 77% in a factor VLeidenheterozygote results in a thrombin generation profile with maximum levels and rates comparable to the factor VLeidenhomozygote. At 150% protein C, thrombin generation is suppressed to levels similar to those seen with 100% factor V. When the effect of variable prothrombin levels in a factor VLeidenheterozygote is examined, a 60% level of prothrombin decreases the maximum level of thrombin generation below that seen with 100% factor V. When prothrombin levels are at 140% in the factor VLeidenheterozygote, the thrombin generation profile is similar to that observed with 100% factor VLeiden. When the effect of variable antithrombin levels was tested in a factor VLeidenheterozygote, a 14% decrease in antithrombin resulted in maximum thrombin levels approaching those of the factor VLeidenhomozygote. In contrast, 128% antithrombin in a factor VLeidenheterozygote resulted in the suppression of thrombin generation to levels consistent with those seen with 100% factor V. Our results predict that the thrombotic effect of heterogenous expression of factor VLeidencan be reduced when prothrombin levels are at the low extreme of the normal range or when antithrombin or protein C levels are at the high extreme of the normal range. Conversely, the thrombotic effect of the heterozygous expression of factor VLeidenis intensified when prothrombin levels are at the high extreme of normal values or when antithrombin or protein C levels at the low extreme of normal. These results may contribute to the understanding of phenotypic variation within factor VLeidenheterozygotes.

Sodium citrate has been used as an anticoagulant to prepare blood and fractions for over 100 years. Its use is sufficiently common that many investigators do not differentiate anticoagulated blood from blood per se. To evaluate the influence of citrate anticoagulation on the dynamics of the blood coagulation process, we compared the properties of blood, platelet rich plasma (PRP) and platelet poor plasma (PPP) prepared and stimulated using standard citrate/Ca++ reconstitution methodologies with matching samples in which transient anticoagulation without disruption of Ca++ homeostasis was achieved by blockade of the contact pathway (using corn trypsin inhibitor (CTI) or inhibitory antibodies to factor XIa). Phlebotomy blood was collected into citrate, CTI or CTI supplemented with citrate, the latter combination establishing conditions in which the effects of citrate anticoagulation in the absence of contact pathway contributions could be studied. Coagulation reactions in citrated milieus were initiated in typical fashion by Ca++ addition or following preincubation with Ca++ prior to tissue factor (Tf) addition in CTI citrate plasma; blood/products stabilized by CTI alone were initiated via Tf addition. Reaction dynamics were analysed using either calibrated automated thrombography (CAT) or thromboelastography (TEG). Platelet aggregation studies compared the reactivity of a panel of agonists. Significant differences were seen in the response of samples exposed to citrate. These results included: CTI plasma and blood displayed a more potent response to Tf when compared to their respective CTI citrate products. Specifically shorter lag phases (~120 s) and higher thrombin levels (1.5–2 fold higher maximum thrombin levels) are observed in CTI plasma with CAT and similarly shorter r (lag) times [(mean ± SD)CTI blood, r = 7.1 ± 0.9 min (n=21, 9 individuals) vs CTI citrate blood, r = 9.1 ± 1.5 min (n=10, 5 individuals)] with TEG. Reconstitution of prechelation Ca++ in CTI citrate plasma or blood prior to Tf addition only partially restored thrombin generation to that of its prechelation product. For example preincubation with Ca++ resulted in altered TEG profiles with prolonged “K” (rate) values (mean ± SD) for CTI citrate blood (7.5 ± 0.6 min (n=5)) compared to CTI blood (3.3 ± 0.5 min (n=5)) from the same individuals. Vitamin K dependent protein conformational transitions in response to chelation/Ca++ reconstitution were important contributors to the differences observed between CTI and citrated blood/plasmas, including the process of protein S polymerization and depolymerization Platelet function studies in citrate containing PRP showed significant enhancement in reactivity toward epinephrine and ristocetin with lesser effects with collagen; in 3 of 4 donors CTI PRP showed no sensitivity to epinephrine. The magnitude of alterations induced by citrate chelation were in the order epinephrine> ristocetin> ADP > collagen. Historically, the regimen of citrate chelation followed by Ca++ addition has proven useful in the interpretation of the integrity of the coagulation system and thereby in clinical diagnosis. However, our results suggest that the procedure of Ca++ chelation/recalcification results in artifactual representations of the dynamics and biology of the blood coagulation process.

Prothrombinase, the enzyme complex required to activate prothrombin, is composed of the serine protease factor Xa and the cofactor factor Va, associated in 1:1 stoichiometry on a phospholipid surface in the presence of Ca2+. Incorporation of factor Va in prothrombinase is required for any meaningful rate of thrombin generation and the arrest of hemorrhage. Factor Va inactivation down-regulates thrombin production resulting in the termination of the hemostatic response. The principal enzyme involved in this down-regulation is activated protein C (APC). Factor Va is formed following enzymatic cleavage of the single chain procofactor, factor V (Mr 330,000) by thrombin. Thrombin cleaves and activates the procofactor sequentially at Arg709, Arg1018, and Arg1545. The active cofactor, factor Va, is composed of heavy (HC105, Mr 105,000) and light (Mr 74,000) chains non-covalently associated in the presence of divalent ions. Previous studies of factor Va inactivation on human umbilical vein endothelial cells (HUVEC) have shown that thrombin cleaves the heavy chain at the COOH-terminus to produce a Mr 97,000 fragment containing the NH2-terminal portion of the heavy chain and a Mr 8,000 peptide representing the COOH-terminus of the molecule which remains attached to the heavy chain by a disulfide bond. The thrombin cleavage appeared to occur between residues 586 and 654. This region contains a consensus sequence for cleavage by thrombin located between residues 640–643 (S-P-R). To evaluate the functional importance of thrombin cleavage at Arg643 for factor Va inactivation, site-directed mutagenesis was used to create recombinant factor V molecules with mutations R643?Q (factor VR643Q) and R643?A (factor VR643A). All recombinant molecules were purified to homogeneity and assayed for activity following extended activation with thrombin. Under similar experimental conditions, cleavage of HC105 and appearance of the Mr 97,000 heavy chain fragment in the wild type molecule correlated with partial loss of cofactor activity, while following incubation of factor VR643Q and factor VR643A with thrombin no cleavage of HC105 at Arg643 was observed and no presence of the Mr 97,000 heavy chain fragment was noticed. Further, no loss in cofactor activity was observed using these mutant recombinant factor Va molecules following extended incubation with thrombin. The endothelial cell surface has been presumed to be the site of PC activation and factor Va inactivation in vivo. The relative phospholipid composition of endothelial membranes has been suggested to be consistent with their ability to support factor Va inactivation in a manner analogous to the commonly used phospholipid system composed of 25% phosphatidylserine and 75% phosphatidylcholine. In the experiments conducted on the HUVEC surface incubation of 20 nM plasma factor V with 0.1 nM thrombin resulted in almost complete cleavage of HC105 over a 60 minute thrombin treatment. In the experiments presented herein much higher concentrations of thrombin were necessary to obtain a similar effect. The combined data suggest the presence of a cofactor for thrombin on the surface of endothelial cells that would facilitate cleavage of factor Va heavy chain at Arg643. Collectively, the data demonstrate that cleavage of HC105 at Arg643 by thrombin results in a partially inactive cofactor molecule and provide for an APC-independent anticoagulant effect of thrombin.

Prothrombinase, the enzyme complex required to activate prothrombin, is composed of the serine protease factor Xa and the cofactor factor Va, associated in 1:1 stoichiometry on a phospholipid surface in the presence of Ca2+. Incorporation of factor Va in prothrombinase is required for any meaningful rate of thrombin generation and the arrest of hemorrhage. Factor Va inactivation down-regulates thrombin production resulting in the termination of the hemostatic response. The principal enzyme involved in this down-regulation is activated protein C (APC). Factor Va is formed following enzymatic cleavage of the single chain procofactor, factor V (Mr330,000) by thrombin. Thrombin cleaves and activates the procofactor sequentially at Arg709, Arg1018, and Arg1545. The active cofactor, factor Va, is composed of heavy (HC105, Mr105,000) and light (Mr74,000) chains non-covalently associated in the presence of divalent ions. Previous studies of factor Va inactivation on human umbilical vein endothelial cells (HUVEC) have shown that thrombin cleaves the heavy chain at the COOH-terminus to produce a Mr97,000 fragment containing the NH2-terminal portion of the heavy chain and a Mr8,000 peptide representing the COOH-terminus of the molecule which remains attached to the heavy chain by a disulfide bond. The thrombin cleavage appeared to occur between residues 586 and 654. This region contains a consensus sequence for cleavage by thrombin located between residues 640–643 (S-P-R). To evaluate the functional importance of thrombin cleavage at Arg643for factor Va inactivation, site-directed mutagenesis was used to create recombinant factor V molecules with mutations R643→Q (factor VR643Q) and R643→A (factor VR643A). All recombinant molecules were purified to homogeneity and assayed for activity following extended activation with thrombin. Under similar experimental conditions, cleavage of HC105and appearance of the Mr97,000 heavy chain fragment in the wild type molecule correlated with partial loss of cofactor activity, while following incubation of factor VR643Qand factor VR643Awith thrombin no cleavage of HC105at Arg643was observed and no presence of the Mr97,000 heavy chain fragment was noticed. Further, no loss in cofactor activity was observed using these mutant recombinant factor Va molecules following extended incubation with thrombin. The endothelial cell surface has been presumed to be the site of PC activation and factor Va inactivation in vivo. The relative phospholipid composition of endothelial membranes has been suggested to be consistent with their ability to support factor Va inactivation in a manner analogous to the commonly used phospholipid system composed of 25% phosphatidylserine and 75% phosphatidylcholine. In the experiments conducted on the HUVEC surface incubation of 20 nM plasma factor V with 0.1 nM thrombin resulted in almost complete cleavage of HC105over a 60 minute thrombin treatment. In the experiments presented herein much higher concentrations of thrombin were necessary to obtain a similar effect. The combined data suggest the presence of a cofactor for thrombin on the surface of endothelial cells that would facilitate cleavage of factor Va heavy chain at Arg643. Collectively, the data demonstrate that cleavage of HC105at Arg643by thrombin results in a partially inactive cofactor molecule and provide for an APC-independent anticoagulant effect of thrombin.

Sodium citrate has been used as an anticoagulant to prepare blood and fractions for over 100 years. Its use is sufficiently common that many investigators do not differentiate anticoagulated blood from blood per se. To evaluate the influence of citrate anticoagulation on the dynamics of the blood coagulation process, we compared the properties of blood, platelet rich plasma (PRP) and platelet poor plasma (PPP) prepared and stimulated using standard citrate/Ca++ reconstitution methodologies with matching samples in which transient anticoagulation without disruption of Ca++ homeostasis was achieved by blockade of the contact pathway (using corn trypsin inhibitor (CTI) or inhibitory antibodies to factor XIa). Phlebotomy blood was collected into citrate, CTI or CTI supplemented with citrate, the latter combination establishing conditions in which the effects of citrate anticoagulation in the absence of contact pathway contributions could be studied. Coagulation reactions in citrated milieus were initiated in typical fashion by Ca++ addition or following preincubation with Ca++ prior to tissue factor (Tf) addition in CTI citrate plasma; blood/products stabilized by CTI alone were initiated via Tf addition. Reaction dynamics were analysed using either calibrated automated thrombography (CAT) or thromboelastography (TEG). Platelet aggregation studies compared the reactivity of a panel of agonists. Significant differences were seen in the response of samples exposed to citrate. These results included:

A circulating form of tissue factor (TF) has been suggested to have an important role in clot growth, and a soluble TF protein lacking the transmembrane domain (sTF) has been described. We have evaluated the potential role of TF in contributing to clot growth using numerical, synthetic plasma and whole blood models. In these studies we differentiate the influence of membrane-bound (mbTF) and sTF218 on thrombin generation. In previous studies we have shown that thrombin generation consists of an initiation phase (IP), a propagation phase and a termination phase. During the IP, complex enzymes are assembled leading to the robust thrombin generation. Initiation with 5 pM mbTF in numerical simulations yields an IP of thrombin generation spanning ~240 s, a maximum rate of thrombin generation (vmax) of 2 nM/sec and a maximum level of active thrombin (IIamax) of 275 nM. No change in the IP and limited decreases in the vmax and IIamax are observed when the mbTF is electronically nullified 240 s after the initiation of the reaction. No thrombin generation is observed when mbTF is removed at 0 s or 10 s post initiation. In synthetic plasma −2 μM phospholipid and 5 pM mbTF- the IP is 240 s, vmax is 2.6 nM/s and IIamax is 300 nM. Quenching of TF activity at 240 s post initiation, accomplished using a mixture of monoclonal anti-TF and anti-factor VII(a) antibodies, has no effect on these parameters. No thrombin generation is observed over 840 s when antibodies are added to the reaction mixture at 0 s. Contact pathway-inhibited whole blood clotted in 253 s after the addition of 5 pM mbTF. This clotting time was only slightly extended by the addition of antibodies at 60–240 s after the initiation of the reaction. No clot was observed in 1000 s when antibodies were added to blood at 0 s. These data indicate that a functional, mbTF is essential for the initiation of thrombin generation but is not required for the propagation of the process during which factor X activation is largely governed by the factor VIIIa/factor IXa complex. We hypothesized that sTF218 should act as a competitive inhibitor by binding factor VIIa in an inactive complex and effectively decreasing the concentration of the mbTF/factor VIIa complex. In synthetic plasma −2 μM phospholipid and 5 pM mbTF-, the addition of 10 nM TF218 extends the IP by 120 s. A further increase in TF218 to 40 nM prolongs the IP by 220 s. No thrombin generation is observed in the absence of mbTF, with or without 100 nM TF218. The vmax and IIamax during the propagation phase are barely affected by the presence of 10–40 nM TF218 consistent with primary factor Xa generation by the factor VIIIa/factor IXa complex during this phase of the reaction. Similar results were obtained in numerical simulations using TF218 as a competitive inhibitor of factor VIIa/mbTF complex formation. These data indicate that sTF acts as an inhibitor of thrombin generation by inhibiting the formation of the mbTF/factor VIIa complex. Collectively our data identify mbTF as the “fuse” and sTF as the “extinguisher” of the “coagulation explosion”.

A circulating form of tissue factor (TF) has been suggested to have an important role in clot growth, and a soluble TF protein lacking the transmembrane domain (sTF) has been described. We have evaluated the potential role of TF in contributing to clot growth using numerical, synthetic plasma and whole blood models. In these studies we differentiate the influence of membrane-bound (mbTF) and sTF218on thrombin generation. In previous studies we have shown that thrombin generation consists of an initiation phase (IP), a propagation phase and a termination phase. During the IP, complex enzymes are assembled leading to the robust thrombin generation. Initiation with 5 pM mbTF in numerical simulations yields an IP of thrombin generation spanning ~240 s, a maximum rate of thrombin generation (vmax) of 2 nM/sec and a maximum level of active thrombin (IIamax) of 275 nM. No change in the IP and limited decreases in the vmaxand IIamaxare observed when the mbTF is electronically nullified 240 s after the initiation of the reaction. No thrombin generation is observed when mbTF is removed at 0 s or 10 s post initiation. In synthetic plasma −2 μM phospholipid and 5 pM mbTF- the IP is 240 s, vmaxis 2.6 nM/s and IIamaxis 300 nM. Quenching of TF activity at 240 s post initiation, accomplished using a mixture of monoclonal anti-TF and anti-factor VII(a) antibodies, has no effect on these parameters. No thrombin generation is observed over 840 s when antibodies are added to the reaction mixture at 0 s. Contact pathway-inhibited whole blood clotted in 253 s after the addition of 5 pM mbTF. This clotting time was only slightly extended by the addition of antibodies at 60–240 s after the initiation of the reaction. No clot was observed in 1000 s when antibodies were added to blood at 0 s. These data indicate that a functional, mbTF is essential for the initiation of thrombin generation but is not required for the propagation of the process during which factor X activation is largely governed by the factor VIIIa/factor IXa complex. We hypothesized that sTF218should act as a competitive inhibitor by binding factor VIIa in an inactive complex and effectively decreasing the concentration of the mbTF/factor VIIa complex. In synthetic plasma −2 μM phospholipid and 5 pM mbTF-, the addition of 10 nM TF218extends the IP by 120 s. A further increase in TF218to 40 nM prolongs the IP by 220 s. No thrombin generation is observed in the absence of mbTF, with or without 100 nM TF218. The vmaxand IIamaxduring the propagation phase are barely affected by the presence of 10–40 nM TF218consistent with primary factor Xa generation by the factor VIIIa/factor IXa complex during this phase of the reaction. Similar results were obtained in numerical simulations using TF218as a competitive inhibitor of factor VIIa/mbTF complex formation. These data indicate that sTF acts as an inhibitor of thrombin generation by inhibiting the formation of the mbTF/factor VIIa complex. Collectively our data identify mbTF as the “fuse” and sTF as the “extinguisher” of the “coagulation explosion”.