Matthew Gissel

Factor XIa and tissue factor activity in patients with coronary artery disease

It has been established that inflammation and enhanced pro-coagulant activity are associated with the pathogenesis of atherosclerotic vascular disease. We evaluated and compared the contributions of the factor (F)XIa and tissue factor (TF) activity in plasma of patients with coronary artery disease (CAD). Citrate plasma was obtained prior to therapy from 53 patients with stable angina (29 with a history of previous myocardial infarction; CAD-MI) and 30 with acute coronary syndrome (ACS) within 12 hours from pain onset. Four ACS patients treated with heparin were excluded. FXIa and TF activity were determined in clotting assays based upon the prolongation of clotting time by inhibitory monoclonal antibodies. Twenty-five of 26ACS patients (96%) and 22 of 29 CAD-MI patients (76%) had quantifiable FXIa (50 +/- 33 and 42 +/- 45pM, respectively). Ten of 26 (38%) ACS patients and only three of 53 (6%) stable CAD patients showed TF activity (<0.4pM). No FXIa or TF activity was observed in age-matched healthy controls (n = 12). For both CAD-MI and ACS patients, there were correlations (p < 0.05) between FXIa and interleukin-6 (R(2) = 0.59 and 0.39, respectively) and between FXIa and TAT (R(2) = 0.64 and 0.63, respectively). In conclusion, the majority of ACS and CAD-MI patients have circulating FXIa that correlates with markers of coagulation and inflammation.

Thrombin generation in acute coronary syndrome and stable coronary artery disease: dependence on plasma factor composition

Summary.  Background: Acute coronary syndrome (ACS) is associated with thrombin formation, triggered by ruptured or eroded coronary atheroma. We investigated whether thrombin generation based on circulating coagulation protein levels, could distinguish between acute and stable coronary artery disease (CAD). Methods and results: Plasma coagulation factor (F) compositions from 28 patients with ACS were obtained after onset of chest pain. Similar data were obtained from 25 age‐ and sex‐matched patients with stable CAD. All individuals took aspirin. Patients on anticoagulant therapy were excluded. The groups were similar in demographic characteristics, comorbidities and concomitant treatment. Using each individual’s coagulation protein composition, tissue factor (TF) initiated thrombin generation was assessed both computationally and empirically. TF pathway inhibitor (TFPI), antithrombin (AT), factor II (FII) and FVIII differed significantly (P < 0.01) between the groups, with levels of FII, FVIII and TFPI higher and AT lower in ACS patients. When thrombin generation profiles from individuals in each group were compared, simulated maximum thrombin levels (P < 0.01) and rates (P < 0.01) were 50% higher with ACS while the initiation phases of thrombin generation were shorter. Empirical reconstructions of the populations reproduced the thrombin generation profiles generated by the computational model. The differences between the thrombin generation profiles for each population were primarily dependent upon the collective contribution of AT, FII and FVIII. Conclusion: Simulations of thrombin formation based on plasma composition can discriminate between acute and stable CAD.

Factor VIIa replacement therapy in factor VII deficiency1

Summary.  Factor (F)VII deficiency is an autosomal recessive disorder for which a replacement therapy is not universally available; recombinant FVIIa has been utilized as a therapeutic substitute. As FVII competes with FVIIa for binding to tissue factor in initiating the extrinsic pathway of blood coagulation, a lower dose of FVIIa replacement in cross‐reacting material‐negative (CRM–) individuals can achieve hemostasis. Three coagulation models (computational, synthetic and in vitro whole blood) were used to predict the FVIIa levels needed to provide apparent hemostasis in a non‐bleeding state. Our whole blood results show that a ‘normalized’ coagulation profile for FVII‐deficient individuals has an initiation phase that ends at 5.8 ± 0.5 min (clot time) and the propagation phase of thrombin generation (thrombin–antithrombin III) yields a maximum concentration of 380 ± 29 nmol L−1. When CRM– FVII‐deficient subjects were infused with a prophylactic dose of 23 µg kg−1 of recombinant FVIIa, 6–8 h postinfusion resulted in a comparable normalized whole blood profile. This FVIIa concentration (0.3–0.7 nmol L−1/equivalent dose: 0.8–1.8 µg kg−1) is approximately 1/10 that currently used in treating FVII‐deficient individuals and suggests that therapies should be altered relative to the concentration of the FVII zymogen.

The Nature of the Stable Blood Clot Procoagulant Activities

The function of tissue factor (Tf)-initiated coagulation is hemorrhage control through the formation and maintenance of an impermeable platelet-fibrin barrier. The catalytic processes involved in the clot maintenance function are not well defined, although the rebleeding problems characteristic of individuals with hemophilias A and B suggest a link between specific defects in the Tf-initiated process and defects in the maintenance function. We have previously demonstrated, using a methodology of “flow replacement” (or resupply) of ongoing Tf-initiated reactions with fresh reactants, that procoagulant complexes are produced during Tf-initiated coagulation, which are capable of reinitiating coagulation without input from extrinsic factor Xase activity (Orfeo, T., Butenas, S., Brummel-Ziedins, K. E., and Mann, K. G. (2005) J. Biol. Chem. 280, 42887–42896). Here we used Tf-initiated reactions in normal and hemophilia blood or in their corresponding proteome mixtures as sources of procoagulant end products and then varied the resupplying material to determine the identity of the catalysts that drive the new cycle of thrombin formation. The central findings are as follows: 1) the prothrombinase complex (fVa-fXa-Ca2+-membrane) accumulated during the episode of Tf-initiated coagulation is the primary catalyst responsible for the observed pattern of prothrombin activation after resupply; 2) impairments in intrinsic factor Xase function, i.e. hemophilias A and B, result in an impaired capacity to mount a resupply response; and 3) in normal hemostasis the intrinsic factor Xase function contributes to the durability of the resupply response.

Coagulation procofactor activation by factor XIa.

Summary.  Background: In the extrinsic pathway, the essential procofactors factor (F) V and FVIII are activated to FVa and FVIIIa by thrombin. In the contact pathway and its clinical diagnostic test, the activated partial thromboplastin time (APTT) assay, the sources of procofactor activation are unknown. In the APTT assay, FXII is activated on a negatively charged surface and proceeds to activate FXI, which activates FIX upon the addition of Ca2+. FIXa feeds thrombin generation through activation of FX. FIXa is an extremely poor catalyst in the absence of its FVIIIa cofactor, which, in the intrinsic FXase complex, increases FXa generation by ∼ 107. One potential APTT procofactor activator in this setting is FXIa. Objective: To test the hypothesis that FXIa can activate FVIII and FV. Methods: Recombinant FVIII and plasma FV were treated with FXIa, and the activities and integrities of each procofactor were measured using commercial clotting assays and sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS‐PAGE). Results: Kinetic analyses of FXIa‐catalyzed activation and inactivation of FV and FVIII are reported, and the the timing and sites of cleavage are defined. Conclusions: FXIa activates both procofactors at plasma protein concentrations, and computational modeling suggests that procofactor activation during the preincubation phase of the APTT assay is critical to the performance of the assay. As the APTT assay is the primary tool for the diagnosis and management of hemophilias A and B, as well as in the determination of FVIII inhibitors, these findings have potential implications in the clinical setting.

Thrombin generation and bleeding in haemophilia A.

Summary.  Haemophilia A displays phenotypic heterogeneity with respect to clinical severity. The aim of this study was to determine if tissue factor (TF)‐initiated thrombin generation profiles in whole blood in the presence of corn trypsin inhibitor (CTI) are predictive of bleeding risk in haemophilia A. We studied factor(F) VIII deficient individuals (11 mild, 4 moderate and 12 severe) with a well‐characterized 5‐year bleeding history that included haemarthrosis, soft tissue haematoma and annual FVIII concentrate usage. This clinical information was used to generate a bleeding score. The bleeding scores (range 0–32) were separated into three groups (bleeding score groupings: 0, 0 and ≤9.6, >9.6), with the higher bleeding tendency having a higher score. Whole blood collected by phlebotomy and contact pathway suppressed by 100 μg mL−1 CTI was stimulated to react by the addition of 5 pm TF. Reactions were quenched at 20 min by inhibitors. Thrombin generation, determined by enzyme‐linked immunosorbent assay for thrombin–antithrombin was evaluated in terms of clot time (CT), maximum level (MaxL) and maximum rate (MaxR) and compared to the bleeding score. Data are shown as the mean±SD. MaxL was significantly different (P < 0.001) between the groups: 504 ± 114, 315 ± 117 and 194 ± 91 nm; with higher thrombin concentrations in the groups with lower bleeding scores. MaxR was higher in the groups with a lower bleeding score; 97 ± 51, 86 ± 60 and 39 ± 16 nm min−1 (P = 0.09). No significant difference was detected in CT among the groups, 5.6 ± 1.3, 4.7 ± 0.7 and 5.6 ± 1.3 min. Our empirical study in CTI‐inhibited whole blood shows that the MaxL of thrombin generation appears to correlate with the bleeding phenotype of haemophilia A.

Anticoagulation by factor Xa inhibitors

Summary.  Background: Therapeutic agents that regulate blood coagulation are critical to the management of thrombotic disorders, with the selective targeting of factor (F) Xa emerging as a promising approach. Objective: To assess anticoagulant strategies targeting FXa. Methods: A deterministic computational model of tissue factor (Tf)‐initiated thrombin generation and two empirical experimental systems (a synthetic coagulation proteome reconstruction using purified proteins and a whole blood model) were used to evaluate clinically relevant examples of the two available types of FXa‐directed anticoagulants [an antithrombin (AT)‐dependent agent, fondaparinux, and an AT‐independent inhibitor, Rivaroxaban] in experimental regimens relevant to long‐term (suppression of new Tf‐initiated events) and acute (suppression of ongoing coagulation processes) clinical applications. Results: Computational representations of each anticoagulant’s efficacy in suppressing thrombin generation over a range of anticoagulant concentrations in both anticoagulation regimens were validated by results from corresponding empirical reconstructions and were consistent with those recommended for long‐term and acute clinical applications, respectively. All three model systems suggested that Rivaroxaban would prove more effective in the suppression of an ongoing coagulation process than fondaparinux, reflecting its much higher reactivity toward the prothrombinase complex. Conclusion: The success of fondaparinux in acute settings in vivo is not explained solely by its properties as an FXa inhibitor. We have reported that FIXa contributes to the long‐term capacity of clot‐associated catalysts to restart a coagulation process, suggesting that the enhanced anti‐FIXa activity of fondaparinux‐AT may be critical to its success in acute settings in vivo.

Thrombin generation in rheumatoid arthritis: Dependence on plasma factor composition

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

Effect of BAX499 aptamer on tissue factor pathway inhibitor function and thrombin generation in models of hemophilia.

INTRODUCTION In hemophilia, thrombin generation is significantly suppressed due to decreased factor (F)X activation. Clinical studies and experiments with transgenic mice have suggested that the severity of hemophilia is substantially reduced by tissue factor pathway inhibitor (TFPI) deficiency. METHODS We evaluated the effect of TFPI antagonist aptamer BAX499 (formerly ARC19499) on TFPI function in purified systems and on thrombin generation and clot formation in plasma and blood. RESULTS BAX499 effectively neutralized TFPI inhibition of FXa and FXa dependent inhibition of TF/FVIIa by TFPI. BAX499 did not inhibit FXa or TF/FVIIa when used up to 500 nM. In the synthetic coagulation proteome with TFPI at its mean physiologic concentration, BAX499 at 1 - 10nM increased thrombin generation triggered with 5 pM relipidated TF in a concentration-dependent manner. In severe hemophilia A or B models using the synthetic coagulation proteome, the addition of BAX499 at 5 nM increased thrombin generation to the levels observed in normal control. Thrombin generation measured in induced hemophilia B plasma required ~100nM BAX499 to restore thrombin levels to those seen in untreated plasma. In induced hemophilia B whole blood, BAX499 repaired the clotting time but failed to appreciably impact the propagation phase of thrombin generation. CONCLUSION These data suggest that inhibition of TFPI by BAX499 may have potential for hemophilia treatment but requires further study in blood-based hemophilia systems.

Potency and mass of factor VIII in FVIII products.

Summary.  Several factor (F) VIII products of different origin and structure are being used for haemophilia A treatment worldwide. The assessment of FVIII concentration in these products is done using activity assays, which are dependent upon the assay and its modifications. To evaluate FVIII products for potency and for FVIII concentration and specific activity, three activity‐based assays [activated partial thromboplastin time (APTT), intrinsic FXase and synthetic coagulation proteome] and two immunoassays (ELISA and western blotting) were used in this study with albumin‐free full‐length recombinant (r) FVIII as a standard. In all activity assays, products A and B (both contain full‐length rFVIII) at 1 U mL−1 showed potency similar to that of the 0.7 nm (1 U mL−1) rFVIII standard. Product E (contains truncated rFVIII) was less potent in the APTT (83% of standard) and product C (contains plasma FVIII) was less potent in FXase assays (66%). The ELISA immunoassay revealed that the specific activity of FVIII proteins in products A–C and E varied over a wide range (3900–13 200 U mg−1) and was higher for most lots when compared with the standard (5000 U mg−1), whereas the specific activity of product D (contains plasma FVIII) was lower than expected (3200–4800 U mg−1). (i) FVIII potency estimated in different assays gives dissimilar results; (ii) the specific activity of FVIII in various FVIII products is different and inconsistent. Thus, the administration of an equal FVIII potency in units means the administration of different amounts of FVIII protein, which may partly explain apparent discrepancies in product performance.