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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.

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.

Whole genome sequencing of an individual completely devoid of plasma- and platelet-derived factor V (FV) identified 167 variants in his F5gene including previously identified and damaging missense mutations at rs6027 and Leu90Ser. Because the administration of fresh frozen plasma (FFP) prevents gastrointestinal bleeding in this individual, its effects on his plasma- and platelet-derived FV concentrations were assessed. The patient's plasma FV levels peaked by 2 hours following FFP administration and were undetectable 96 hours later. In contrast, increased platelet-derived FV/Va concentrations were observed within 6 hours, peaked at 24 hours, decreased slowly over 7 days, and originated from megakaryocyte endocytosis and intracellular processing of plasma FV. Ten days after transfusion, no thrombin was generated in a tissue factor–initiated whole blood clotting assay unless exogenous FV was added, consistent with the complete absence of plasma FV. In marked contrast, release of the patient's platelet-derived FV/Va (7% of normal) following platelet activation resulted in robust thrombin generation, similar to that in an individual with normal plasma- and platelet-derived FV concentrations. Thus, total FV deficiency can be corrected by plasma administration, which partially repletes and sustains the platelet cofactor pool, thereby highlighting the critical role of platelet-derived FV/Va in ensuring hemostatic competence.

Whole genome sequencing of an individual completely devoid of plasma- and platelet-derived factor V (FV) identified 167 variants in his F5 gene including previously identified and damaging missense mutations at rs6027 and Leu90Ser. Because the administration of fresh frozen plasma (FFP) prevents gastrointestinal bleeding in this individual, its effects on his plasma- and platelet-derived FV concentrations were assessed. The patient’s plasma FV levels peaked by 2 hours following FFP administration and were undetectable 96 hours later. In contrast, increased platelet-derived FV/Va concentrations were observed within 6 hours, peaked at 24 hours, decreased slowly over 7 days, and originated from megakaryocyte endocytosis and intracellular processing of plasma FV. Ten days after transfusion, no thrombin was generated in a tissue factor–initiated whole blood clotting assay unless exogenous FV was added, consistent with the complete absence of plasma FV. In marked contrast, release of the patient’s platelet-derived FV/Va (7% of normal) following platelet activation resulted in robust thrombin generation, similar to that in an individual with normal plasma- and platelet-derived FV concentrations. Thus, total FV deficiency can be corrected by plasma administration, which partially repletes and sustains the platelet cofactor pool, thereby highlighting the critical role of platelet-derived FV/Va in ensuring hemostatic competence.

Trauma‐induced coagulopathy (TIC) includes heterogeneous coagulopathic syndromes with different underlying causes, and treatment is challenged by limited diagnostic tests to discriminate between these entities in the acute setting. We provide an overview of progress in understanding the mechanisms of TIC and the context for several of the hypotheses that will be tested in ‘TACTIC’. Although connected to ongoing clinical trials in trauma, TACTIC itself has no intent to conduct clinical trials. We do anticipate that ‘early translation’ of promising results will occur. Functions anticipated at this early translational level include: (i) basic science groundwork for future therapeutic candidates; (ii) development of acute coagulopathy scoring systems; (iii) coagulation factor composition‐based computational analysis; (iv) characterization of novel analytes including tissue factor, polyphosphates, histones, meizothrombin and α‐thrombin–antithrombin complexes, factor XIa, platelet and endothelial markers of activation, signatures of protein C activation and fibrinolysis markers; and (v) assessment of viscoelastic tests and new point‐of‐care methods.

There exists an imbalance between our understanding of the physiology of the blood coagulation process and the translation of this understanding into useful assays for clinical application. As technology advances, the capabilities for merging the two areas have become more attainable. Global assays have advanced our understanding of the dynamics of the blood coagulation process beyond end point assays and are at the forefront of implementation in the clinic.

Thrombosis complicating the rupture of an atherosclerotic plaque can lead to arterial occlusion. Tissue factor, a membrane-bound glycoprotein, is expressed to a greater extent in atherosclerotic plaques and may be a key mediator of microthrombosis and macrothrombosis. This pilot study was designed to determine whether the angiographic presence or the extent of atherosclerosis was correlated with the activity of coagulation factors in blood. A novel computational model was used to predict whether differences in the activity of coagulation factors would alter the generation of thrombin.

We have adapted the corn-trypsin inhibitor whole-blood model to include EA.hy926 as an endothelium surrogate to evaluate the vascular modulation of blood coagulation initiated by relipidated recombinant tissue factor (rTf) and a cellular Tf surrogate, lipopolysaccharide (LPS)-stimulated THP1 cells (LPS-THP-1). Compared with bare tubes, EA.hy926 with rTf decreased the rate of thrombin formation, ITS accumulation, and the production of fibrinopeptide A. These phenomena occurred with increased rates of factor Va (fVa) inactivation by cleavages at R506 and R306. Thus, EA.hy926 provides thrombin-dependent protein C activation and APC fVa inactivation. Comparisons of rTf with LPS-THP-1 showed that the latter gave reduced rates for TAT formation but equivalent fibrinopeptide A, and fV activation/inactivation. In the presence of EA.hy926, the reverse was obtained; with the surrogate endothelium and LPS-THP-1 the rates of TAT generation, fibrinopeptide release, and fV activation were almost doubled, whereas cleavage at R306 was equivalent. These observations suggest cooperativity between the 2 cell surrogates. These data suggest that the use of these 2 cell lines provides a reproducible quasi-endothelial quasi-inflammatory cytokine-stimulated monocyte system that provides a method to evaluate the variations in blood phenotype against the background of stable inflammatory cell activator and a stable vascular endothelial surrogate.

We have adapted the corn-trypsin inhibitor whole-blood model to include EA.hy926 as an endothelium surrogate to evaluate the vascular modulation of blood coagulation initiated by relipidated recombinant tissue factor (rTf) and a cellular Tf surrogate, lipopolysaccharide (LPS)-stimulated THP1 cells (LPS-THP-1). Compared with bare tubes, EA.hy926 with rTf decreased the rate of thrombin formation, ITS accumulation, and the production of fibrinopeptide A. These phenomena occurred with increased rates of factor Va (fVa) inactivation by cleavages at R506and R306. Thus, EA.hy926 provides thrombin-dependent protein C activation and APC fVa inactivation. Comparisons of rTf with LPS-THP-1 showed that the latter gave reduced rates for TAT formation but equivalent fibrinopeptide A, and fV activation/inactivation. In the presence of EA.hy926, the reverse was obtained; with the surrogate endothelium and LPS-THP-1 the rates of TAT generation, fibrinopeptide release, and fV activation were almost doubled, whereas cleavage at R306was equivalent. These observations suggest cooperativity between the 2 cell surrogates. These data suggest that the use of these 2 cell lines provides a reproducible quasi-endothelial quasi-inflammatory cytokine-stimulated monocyte system that provides a method to evaluate the variations in blood phenotype against the background of stable inflammatory cell activator and a stable vascular endothelial surrogate.

The presence of antibodies (Abs) in hemophilia A patients can potentially influence the therapeutic qualities of factor VIII (fVIII) administration. Much work has been focused on the presence of inhibitory antibodies, whereas the quantitation of noninhibitory anti-fVIII antibodies has been largely undetermined. Our objective was to develop a sensitive and specific fluorescence-based immunoassay (FLI) for the quantitation of anti-fVIIIAbs in human plasma. Affinity-purified human anti-fVIIIAb, isolated from a hemophilia A subject, was used as a calibrator with a detectability limit of 40 (±1.5) pM. The calibrator and the human plasma anti-fVIIIAb were captured on recombinant fVIII (rfVIII)– coupled microspheres and probed with mouse anti–human Ig–R-phycoerythrin. Plasma samples from 150 healthy donors and 39 inhibitor-negative hemophilia A subjects were compared with 4 inhibitor-positive hemophilia A plasma samples with inhibitor titers of 1 BU/mL (94.6 ± 0.8 nM), 11 BU/mL (214.3 ± 7.1 nM), 106 BU/mL (2209.4 ± 84.9 nM), 140 BU/mL (2417.7 ± 3.8 nM) as measured by the Nijmegen method. We also describe the validation of a mouse anti–human fVIIIAb as a surrogate calibrator. Four healthy individuals (3%) showed detectable anti-fVIIIAb in the range of 0.6 to 6.2 nM, whereas 13 (33%) of the 39 inhibitor-free hemophilia A subjects were positive for anti-fVIIIAb in the range of 0.5 to 20 nM. The method may be useful for therapeutic management of hemophilia A patients.

The presence of antibodies (Abs) in hemophilia A patients can potentially influence the therapeutic qualities of factor VIII (fVIII) administration. Much work has been focused on the presence of inhibitory antibodies, whereas the quantitation of noninhibitory anti-fVIII antibodies has been largely undetermined. Our objective was to develop a sensitive and specific fluorescence-based immunoassay (FLI) for the quantitation of anti-fVIIIAbs in human plasma. Affinity-purified human anti-fVIIIAb, isolated from a hemophilia A subject, was used as a calibrator with a detectability limit of 40 (±1.5) pM. The calibrator and the human plasma anti-fVIIIAb were captured on recombinant fVIII (rfVIII)– coupled microspheres and probed with mouse anti–human Ig–R-phycoerythrin. Plasma samples from 150 healthy donors and 39 inhibitor-negative hemophilia A subjects were compared with 4 inhibitor-positive hemophilia A plasma samples with inhibitor titers of 1 BU/mL (94.6 ± 0.8 nM), 11 BU/mL (214.3 ± 7.1 nM), 106 BU/mL (2209.4 ± 84.9 nM), 140 BU/mL (2417.7 ± 3.8 nM) as measured by the Nijmegen method. We also describe the validation of a mouse anti–human fVIIIAb as a surrogate calibrator. Four healthy individuals (3%) showed detectable anti-fVIIIAb in the range of 0.6 to 6.2 nM, whereas 13 (33%) of the 39 inhibitor-free hemophilia A subjects were positive for anti-fVIIIAb in the range of 0.5 to 20 nM. The method may be useful for therapeutic management of hemophilia A patients.

We evaluated systemic alterations to the blood coagulation system that occur during a coronary thrombotic event. Peripheral blood coagulation in patients with acute coronary thrombosis was compared with that in people with stable coronary artery disease (CAD). Blood coagulation and platelet activation at the microvascular injury site were assessed using immunochemistry in 28 non-anticoagulated patients with acute myocardial infarction (AMI) versus 28 stable CAD patients matched for age, sex, risk factors, and medications. AMI was associated with increased maximum rates of thrombin-antithrombin complex generation (by 93.8%; P < .001), thrombin B-chain formation (by 57.1%; P < .001), prothrombin consumption (by 27.9%; P = .012), fibrinogen consumption (by 27.0%; P = .02), factor (f) Va light chain generation (by 44.2%; P = .003), and accelerated fVa inactivation (by 76.1%; P < .001), and with enhanced release of platelet-derived soluble CD40 ligand (by 44.4%; P < .001). FVa heavy chain availability was similar in both groups because of enhanced formation and activated protein C (APC)–mediated destruction. The velocity of coagulant reactions in AMI patients showed positive correlations with interleukin-6. Heparin treatment led to dampening of coagulant reactions with profiles similar to those for stable CAD. AMI-induced systemic activation of blood coagulation markedly modifies the pattern of coagulant reactions at the site of injury in peripheral vessels compared with that in stable CAD patients.

We evaluated systemic alterations to the blood coagulation system that occur during a coronary thrombotic event. Peripheral blood coagulation in patients with acute coronary thrombosis was compared with that in people with stable coronary artery disease (CAD). Blood coagulation and platelet activation at the microvascular injury site were assessed using immunochemistry in 28 non-anticoagulated patients with acute myocardial infarction (AMI) versus 28 stable CAD patients matched for age, sex, risk factors, and medications. AMI was associated with increased maximum rates of thrombin-antithrombin complex generation (by 93.8%; P< .001), thrombin B-chain formation (by 57.1%; P< .001), prothrombin consumption (by 27.9%; P= .012), fibrinogen consumption (by 27.0%; P= .02), factor (f) Va light chain generation (by 44.2%; P= .003), and accelerated fVa inactivation (by 76.1%; P< .001), and with enhanced release of platelet-derived soluble CD40 ligand (by 44.4%; P< .001). FVa heavy chain availability was similar in both groups because of enhanced formation and activated protein C (APC)–mediated destruction. The velocity of coagulant reactions in AMI patients showed positive correlations with interleukin-6. Heparin treatment led to dampening of coagulant reactions with profiles similar to those for stable CAD. AMI-induced systemic activation of blood coagulation markedly modifies the pattern of coagulant reactions at the site of injury in peripheral vessels compared with that in stable CAD patients.

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 A2 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 A2 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 β3-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.

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 A2synthesis, 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 A2production. 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 β3-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.

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

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

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.

Introduction: Effective treatment of HIV infection with antiretroviral therapy (ART) has prolonged survival and shifted causes of death to non-AIDS defining illnesses such as cardiovascular disease (CVD). Increased thrombin generation is evident from increased D-dimer levels in HIV; previously we have shown that inflammation and viral replication associate with imbalances in pro and anticoagulant factors resulting in increased thrombin generation profiles when mathematically modeled. We explore for the first time the hypothesis that factor compositional imbalance increases simulated thrombin generation and predicts mortality in HIV.

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.

Abstract 1231

Thrombosis complicating the rupture of atherosclerotic plaques leads to arterial occlusion. Tissue factor (Tf), a membrane-bound glycoprotein, is expressed to a greater extent in atherosclerotic plaques and is likely a key mediator of micro and macro thrombosis. Atherosclerotic vascular disease is a widespread process that can lead to stroke, myocardial infarction and peripheral artery occlusion. Because thrombosis consumes coagulation factors, the goal of the present study was to determine whether the extent of atherosclerosis correlated with the concentration or activity of coagulation factors in blood and to relate, using novel computational approaches, the thrombin generation potential of the arterial blood to the extent of atherosclerosis. Methods. Patients scheduled for diagnostic cardiac catheterization (n=57) for clinical indications were enrolled. The average age was 65 (range 35–92) with a predominance of males (71%). Most patients were treated with aspirin (54/57 – 95%) and a small group had been treated with both aspirin plus clopidogrel (7/57 – 12%). After review of their angiograms, patients were divided into 3 groups based on their maximal extent of stenosis, 1) no coronary artery disease (no CAD, n=10), 2) coronary arterial stenosis <50% (CAD<50%, n=9), and 3) coronary arterial stenosis >50% (CAD>50%, n=38). For patients with CAD>50%, we evaluated those not treated with clopidogrel (n=31) separately from the group taking clopidogrel (n=7). Blood was taken from catheters placed in the femoral artery before administration of anticoagulants. Thrombin and factor Xa generation was assessed computationally based on measurements of plasma coagulation factors (II, V, VII, IX, X) and inhibitors (antithrombin and free tissue factor pathway inhibitor, TFPI) in all patients. Results. Plasma composition was not significantly different between individuals with no CAD versus CAD. In individuals with CAD, the concentrations of fX and prothrombin were significantly less in blood from patients with more than 50% stenosis. This difference was apparent in both those taking clopidogrel and those not taking clopidogrel. Thrombin and fXa generation after a 5 pM Tf initiator was significantly increased in all individuals compared to a physiologic control. Individuals with >50% CAD had significantly lower total thrombin then individuals with CAD<50% (83±16μM.s vs. 96 ±20μM.s, respectively). Kinetic analysis suggested that depletion of fX and prothrombin would decrease the amount of thrombin generated in patients with CAD>50%. To determine whether selected factors influenced the generation of fXa and thrombin, linear regression analysis was performed. The concentrations in blood of TFPI and fVIII were strongly associated (R2=0.5) with the time to the maximum rate of thrombin generation and maximum concentration of fXa generated. In simulations performed with increasing Tf concentrations (12.5 pM and 25 pM) TFPI and fVIII continued to correlate with thrombin and fXa generation. Also, at increasing Tf concentrations, fXa generation had greater (11 fold range) power to resolve each individual's thrombin generation profile (4 fold range). Conclusions. In summary, a reduced concentration of prothrombin and fX was observed in patients with more severe CAD, which could reflect greater consumption of these coagulation factors, reduced production or some combination. The computational analyses indicated important roles for TFPI and fVIII in the thrombin generation potential of individuals with CAD. The kinetics of fXa and thrombin generation suggests that the lower concentrations of fX and prothrombin may have functional implications consistent with clinical observations of patients with severe CAD having a narrower therapeutic window when treated with vitamin K antagonists.No relevant conflicts of interest to declare.

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.

Hemophilia A is a clinically heterogeneous bleeding disorder characterized by the absence of functional factor VIII (FVIII). The most severe complication from exposure to exogenous factor VIII therapy is the development of alloantibodies that neutralize the effect of the therapeutic agent. The standard method for quantitation of FVIII inhibitors in human plasma is by the Nijmegen method, a modification of the Bethesda assay. Our objective was to develop a sensitive and specific fluorescence based immunoassay for the quantitation of anti-FVIII antibodies (FVIIIAb) in human plasma. An affinity purified human anti-FVIIIAb isolated from a hemophilia A patient was used as a calibrator, with a detectability limit of 40±1.5pM. The calibrator and the human plasma anti-FVIIIAb were captured on recombinant FVIII coupled microspheres and probed with mouse anti-human Ig conjugated to R-Phycoerythrin. Citrated human plasma samples from 150 healthy donors and 39 inhibitor-negative hemophilia A subjects were studied and compared to 4 inhibitor-positive hemophilia A plasma samples with inhibitor titers of 1BU (94.6±0.08nM), 11BU (214.3±7.1nM), 106BU (2209.4±84.9nM) and 140BU (2417.7±3.8nM) as measured by the Nijmegen method. Among the 150 normal control plasmas, 4 subjects (3%) were determined as positive (anti-FVIIIAb ≥ 40pM) with concentrations in the range of 0.6nM to 1.9nM, well below that of 1BU (94.6±0.08nM) inhibitor-positive sample. Anti-FVIIIAb was also evaluated in seven commercial inhibitor-free plasma samples reported to contain <1% FVIII activity. Two of the seven plasmas were found to contain anti-FVIIIAb with concentrations of 112.9±1.9nM and 71.1±8.5nM. The concentration of the antibody in both samples was within the range of the measured molar concentration of an inhibitor-positive 1BU titer plasma sample. Thirty two inhibitor-negative hemophilia A subjects were also quantitated for anti-FVIIIAb. Eleven of the 32 samples tested positive for anti-FVIIIAb with molar concentrations in the range of 0.6–20nM. The overall prevalence of the non-inhibitory anti-FVIIIAbs in hemophilia A subjects evaluated by our immunoassay (n=39) is approximately 33% relative to healthy donor population in which 3% were determined as positive (≥40pM) for FVIIIAb.

There are several hypotheses which attempt to explain changes in tissue factor (TF) activity on cell surfaces upon treatment with various agents (“encryption-de-encryption”). However, these hypotheses are mostly based upon conjectural data interpretation. We evaluated the influence of lipopolysaccharide (LPS) stimulation on the surface and total expression of TF antigen on/in THP-1 monocytic cells and the corresponding TF activity. A cell-based immunoassay was used for the quantitation of TF antigen on the surface of monocytic cells, coupled with contact pathway-inhibited (corn trypsin inhibitor; CTI) plasma and blood dilute PT, extrinsic factor (F)Xase and synthetic coagulation proteome assays. Prior to stimulation, the concentration of TF was 240±60 molecules/cell on the cell surface and 510±180 total molecules/cell in cell lysates (n=8). Upon stimulation with LPS (1μg/ml), the TF antigen concentration steadily increased reaching a maximum at 4 hr of stimulation (2400±360 molecules/cell on the cell surface and 8400±360 molecules/cell total). Coincidently, the intact cell FXa generation rate increased from 4.2 pM/s to 46.3 pM/s. The clotting time of multi-donor plasma decreased from 192s to 80s corresponding to a 35.7±4.9-fold increase in TF activity. Thus all assays are consistent with increased surface expression of TF. Kinetic studies showed that the KM of the complex enzyme (TF/FVIIa) formed on the surface of LPS-stimulated monocytes for FX is 0.73±0.07 μM and the kcat 59.4±1.8 s−1. The kinetic properties for the FXase formed in lysates of LPS-stimulated THP-1 cells are similar to those observed for the cell surface TF (see Table 1). However, when LPS-stimulated monocyte TF was purified to homogeneity and relipidated using synthetic phospholipid vesicles (PCPS), the efficiency for FX activation was significantly lower due to both an increased KM and decreased kcat. As a consequence, the second order rate constant for FX activation by the extrinsic FXase on the surface of PCPS was only 8±1% of that observed on stimulated intact cells or lysates. A comparison of purified and relipidated monocyte TF with that cell-bound in situ in the synthetic coagulation proteome with platelets at mean physiologic concentration (2x108/ml) showed that thrombin generation profiles were similar for the reactions initiated with 5 pM relipidated TF and 0.05 pM TF expressed on the cell surface; a difference in specific activity of approximately 100-fold. Similar results were observed in CTI-inhibited blood. CTI blood, triggered with 5 pM relipidated monocyte TF clotted in 310 s, whereas initiation with 1.2 pM monocyte TF in situ led to a 40 s clotting time with maximum rates of thrombin generation of 1.3 and >7.0 nM/s, respectively. These data indicate that the increase in monocyte TF activity upon stimulation with LPS is related to an increased expression of TF protein on/in monocytic cells and that there are components of the cell membrane which substantially enhance TF activity and these accessories maintain their function(s) in cell lysates. These components are not represented by synthetic (PCPS) phospholipid vesicles. Our data emphasize the importance of cell membrane composition on TF activity and are in agreement with observations published by Pendurthi et al. (Blood2007;110:3900). Table 1. Monocytic TF in the extrinsic FXase Preparation of TF KM(μM) kcat(s−1) kcat/KM((μM•s−1) In situ 0.73±0.0 59.4±1.8 81.4 In lysates 0.41±0.0 44.6±0.1 108.8 On PCPS 1.54±0.14 12.0±0.4 7.8

Hemophilia A is a clinically heterogeneous bleeding disorder characterized by the absence of functional factor VIII (FVIII). The most severe complication from exposure to exogenous factor VIII therapy is the development of alloantibodies that neutralize the effect of the therapeutic agent. The standard method for quantitation of FVIII inhibitors in human plasma is by the Nijmegen method, a modification of the Bethesda assay. Our objective was to develop a sensitive and specific fluorescence based immunoassay for the quantitation of anti-FVIII antibodies (FVIIIAb) in human plasma. An affinity purified human anti-FVIIIAb isolated from a hemophilia A patient was used as a calibrator, with a detectability limit of 40±1.5pM. The calibrator and the human plasma anti-FVIIIAb were captured on recombinant FVIII coupled microspheres and probed with mouse anti-human Ig conjugated to R-Phycoerythrin. Citrated human plasma samples from 150 healthy donors and 39 inhibitor-negative hemophilia A subjects were studied and compared to 4 inhibitor-positive hemophilia A plasma samples with inhibitor titers of 1BU (94.6±0.08nM), 11BU (214.3±7.1nM), 106BU (2209.4±84.9nM) and 140BU (2417.7±3.8nM) as measured by the Nijmegen method. Among the 150 normal control plasmas, 4 subjects (3%) were determined as positive (anti-FVIIIAb ≥ 40pM) with concentrations in the range of 0.6nM to 1.9nM, well below that of 1BU (94.6±0.08nM) inhibitor-positive sample. Anti-FVIIIAb was also evaluated in seven commercial inhibitor-free plasma samples reported to contain <1% FVIII activity. Two of the seven plasmas were found to contain anti-FVIIIAb with concentrations of 112.9±1.9nM and 71.1±8.5nM. The concentration of the antibody in both samples was within the range of the measured molar concentration of an inhibitor-positive 1BU titer plasma sample. Thirty two inhibitor-negative hemophilia A subjects were also quantitated for anti-FVIIIAb. Eleven of the 32 samples tested positive for anti-FVIIIAb with molar concentrations in the range of 0.6–20nM. The overall prevalence of the non-inhibitory anti-FVIIIAbs in hemophilia A subjects evaluated by our immunoassay (n=39) is approximately 33% relative to healthy donor population in which 3% were determined as positive (≥40pM) for FVIIIAb.

There are several hypotheses which attempt to explain changes in tissue factor (TF) activity on cell surfaces upon treatment with various agents (“encryption-de-encryption”). However, these hypotheses are mostly based upon conjectural data interpretation. We evaluated the influence of lipopolysaccharide (LPS) stimulation on the surface and total expression of TF antigen on/in THP-1 monocytic cells and the corresponding TF activity. A cell-based immunoassay was used for the quantitation of TF antigen on the surface of monocytic cells, coupled with contact pathway-inhibited (corn trypsin inhibitor; CTI) plasma and blood dilute PT, extrinsic factor (F)Xase and synthetic coagulation proteome assays. Prior to stimulation, the concentration of TF was 240±60 molecules/cell on the cell surface and 510±180 total molecules/cell in cell lysates (n=8). Upon stimulation with LPS (1μg/ml), the TF antigen concentration steadily increased reaching a maximum at 4 hr of stimulation (2400±360 molecules/cell on the cell surface and 8400±360 molecules/cell total). Coincidently, the intact cell FXa generation rate increased from 4.2 pM/s to 46.3 pM/s. The clotting time of multi-donor plasma decreased from 192s to 80s corresponding to a 35.7±4.9-fold increase in TF activity. Thus all assays are consistent with increased surface expression of TF. Kinetic studies showed that the KMof the complex enzyme (TF/FVIIa) formed on the surface of LPS-stimulated monocytes for FX is 0.73±0.07 μM and the kcat59.4±1.8 s−1. The kinetic properties for the FXase formed in lysates of LPS-stimulated THP-1 cells are similar to those observed for the cell surface TF (see Table 1). However, when LPS-stimulated monocyte TF was purified to homogeneity and relipidated using synthetic phospholipid vesicles (PCPS), the efficiency for FX activation was significantly lower due to both an increased KMand decreased kcat. As a consequence, the second order rate constant for FX activation by the extrinsic FXase on the surface of PCPS was only 8±1% of that observed on stimulated intact cells or lysates. A comparison of purified and relipidated monocyte TF with that cell-bound in situin the synthetic coagulation proteome with platelets at mean physiologic concentration (2x108/ml) showed that thrombin generation profiles were similar for the reactions initiated with 5 pM relipidated TF and 0.05 pM TF expressed on the cell surface; a difference in specific activity of approximately 100-fold. Similar results were observed in CTI-inhibited blood. CTI blood, triggered with 5 pM relipidated monocyte TF clotted in 310 s, whereas initiation with 1.2 pM monocyte TF in situled to a 40 s clotting time with maximum rates of thrombin generation of 1.3 and >7.0 nM/s, respectively. These data indicate that the increase in monocyte TF activity upon stimulation with LPS is related to an increased expression of TF protein on/in monocytic cells and that there are components of the cell membrane which substantially enhance TF activity and these accessories maintain their function(s) in cell lysates. These components are not represented by synthetic (PCPS) phospholipid vesicles. Our data emphasize the importance of cell membrane composition on TF activity and are in agreement with observations published by Pendurthi et al. ( Blood 2007; 110:3900).

The conversion of fibrinogen to fibrin and cross-linking to form a stable clot are key processes in effective hemostasis. We investigated fibrinopeptide (FP) A and FPB release, factor XIII (FXIII) activation and fibrin mass in tissue factor initiated coagulation in whole blood from individuals with hemophilia (n=8) and healthy subjects (n=35). For hemophilia whole blood, the rates and patterns of fibrin formation are altered when compared to blood from healthy individuals. The rate of FPA release is decreased from 2.3μ M/min in healthy individuals to 1.5μ M/min in hemophilia and more significantly the rate of FPB release is decreased from 1.3μ M/min to 0.19 μ M/min in hemophilia whole blood. The activated form of FXIII in hemophilia whole blood is formed at a rate of 4nM/min versus healthy individuals where the rate is accelerated to 7nM/min. Prior to clot time in hemophilia whole blood, FPB release is reduced. Whereas more FPA is released in hemophilia whole blood prior to clot time than in whole blood from healthy individuals, 67% versus 47%, respectively. FXIII activation is delayed relative to FPA release. Around clot time in hemophilia whole blood, FPB levels are only ~1μ M and FPA levels are 8 fold higher. In normal whole blood at clot time, FPA release is only 2.5 fold higher than FPB release, potentially suggesting that there is an essential correlation of time dependence between FPA, FPB and FXIII activation to form a stable clot. Whereas, this pattern of fibrin formation is altered in the hemophilia state. Clots from hemophilia whole blood are more soluble as evidenced by a 43% decrease in fibrin mass compared to clots from healthy individuals. Overall, data indicate that hemophilia clots are weaker due to decreased lateral association (FPB release) and are mostly composed of fibrin units that are only partially cross-linked. Thus, suggesting that FXIII activation and FPB release play crucial roles in the pathology of hemophilia and the fragile nature of blood clots in hemophiliacs.

The conversion of fibrinogen to fibrin and cross-linking to form a stable clot are key processes in effective hemostasis. We investigated fibrinopeptide (FP) A and FPB release, factor XIII (FXIII) activation and fibrin mass in tissue factor initiated coagulation in whole blood from individuals with hemophilia (n=8) and healthy subjects (n=35). For hemophilia whole blood, the rates and patterns of fibrin formation are altered when compared to blood from healthy individuals. The rate of FPA release is decreased from 2.3μ M/min in healthy individuals to 1.5μ M/min in hemophilia and more significantly the rate of FPB release is decreased from 1.3μ M/min to 0.19 μ M/min in hemophilia whole blood. The activated form of FXIII in hemophilia whole blood is formed at a rate of 4nM/min versus healthy individuals where the rate is accelerated to 7nM/min. Prior to clot time in hemophilia whole blood, FPB release is reduced. Whereas more FPA is released in hemophilia whole blood prior to clot time than in whole blood from healthy individuals, 67% versus 47%, respectively. FXIII activation is delayed relative to FPA release. Around clot time in hemophilia whole blood, FPB levels are only ~1μ M and FPA levels are 8 fold higher. In normal whole blood at clot time, FPA release is only 2.5 fold higher than FPB release, potentially suggesting that there is an essential correlation of time dependence between FPA, FPB and FXIII activation to form a stable clot. Whereas, this pattern of fibrin formation is altered in the hemophilia state. Clots from hemophilia whole blood are more soluble as evidenced by a 43% decrease in fibrin mass compared to clots from healthy individuals. Overall, data indicate that hemophilia clots are weaker due to decreased lateral association (FPB release) and are mostly composed of fibrin units that are only partially cross-linked. Thus, suggesting that FXIII activation and FPB release play crucial roles in the pathology of hemophilia and the fragile nature of blood clots in hemophiliacs.

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 to be used (dilutions ranging up to 66%) in hemorrhagic shock for restoring hemodynamic stability. Trauma results in massive exposure of tissue factor to the circulation and explosive amounts of thrombin being generated. We studied the effect of various resuscitative formulas to blood coagulation, specifically thrombin generation and fibrin formation, in a controlled setting using corn trypsin inhibited whole blood initiated with a 5pM stimulus of tissue factor. Thrombin generation measured as its complex with antithrombin III (TAT) was evaluated periodically over a time course of 20 min. Fibrin clots were collected and weighed. We investigated four diluents (0.9% NaCl (NS), lactated Ringer’s solution (LR), 6% hydroxyethyl starch (HES) and 3% NaCl (HS)) each at a 0,10, 20, 30, 40, 50 and 60% blood dilution. At a 10% dilution TAT generation was in the order of LR (−4%) < HES (−8%)< NS and HS (−12%). Diluting by 20% resulted in further decreases of TAT formation. The fibrin clot mass decreased dramatically with a 20% dilution for NS (−42%) and HES (−30%). Conversely, HS produced no change in fibrin mass but effected the largest change in thrombin generation rate (−56%). At a 50% dilution, comparable thrombin generation profiles were obtained for LR, HES and NS (~35% decrease). However, the fibrin masses decreased by 27% with LR, 46% with NS and 74% with HES. No clot formation or thrombin generation was seen with HS at > 20% dilution. This in vitro study shows that: 1) LR has the least effect on thrombin generation and gave higher than anticipated clot weights; 2) HES reduced the fibrin clot mass at higher dilutions; 3) HS abolishes coagulation after a 20% dilution. Overall, both the extent and nature of hemodilution cause profound alterations in the hemostatic mechanism.

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”.

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 to be used (dilutions ranging up to 66%) in hemorrhagic shock for restoring hemodynamic stability. Trauma results in massive exposure of tissue factor to the circulation and explosive amounts of thrombin being generated. We studied the effect of various resuscitative formulas to blood coagulation, specifically thrombin generation and fibrin formation, in a controlled setting using corn trypsin inhibited whole blood initiated with a 5pM stimulus of tissue factor. Thrombin generation measured as its complex with antithrombin III (TAT) was evaluated periodically over a time course of 20 min. Fibrin clots were collected and weighed. We investigated four diluents (0.9% NaCl (NS), lactated Ringer's solution (LR), 6% hydroxyethyl starch (HES) and 3% NaCl (HS)) each at a 0,10, 20, 30, 40, 50 and 60% blood dilution. At a 10% dilution TAT generation was in the order of LR (−4%) < HES (−8%)< NS and HS (−12%). Diluting by 20% resulted in further decreases of TAT formation. The fibrin clot mass decreased dramatically with a 20% dilution for NS (−42%) and HES (−30%). Conversely, HS produced no change in fibrin mass but effected the largest change in thrombin generation rate (−56%). At a 50% dilution, comparable thrombin generation profiles were obtained for LR, HES and NS (~35% decrease). However, the fibrin masses decreased by 27% with LR, 46% with NS and 74% with HES. No clot formation or thrombin generation was seen with HS at > 20% dilution. This in vitrostudy shows that: 1) LR has the least effect on thrombin generation and gave higher than anticipated clot weights; 2) HES reduced the fibrin clot mass at higher dilutions; 3) HS abolishes coagulation after a 20% dilution. Overall, both the extent and nature of hemodilution cause profound alterations in the hemostatic mechanism.