Kazuya Hosokawa

Role of factor VIII C2 domain in factor VIII binding to factor Xa

Factor VIII (FVIII) is activated by proteolytic cleavages with thrombin and factor Xa (FXa) in the intrinsic blood coagulation pathway. The anti-C2 monoclonal antibody ESH8, which recognizes residues 2248–2285 and does not inhibit FVIII binding to von Willebrand factor or phospholipid, inhibited FVIII activation by FXa in a clotting assay. Furthermore, analysis by SDS-polyacrylamide gel electrophoresis showed that ESH8 inhibited FXa cleavage in the presence or absence of phospholipid. The light chain (LCh) fragments (both 80 and 72 kDa) and the recombinant C2 domain dose-dependently bound to immobilized anhydro-FXa, a catalytically inactive derivative of FXa in which dehydroalanine replaces the active-site serine. The affinity (K d ) values for the 80- and 72-kDa LCh fragments and the C2 domain were 55, 51, and 560 nm, respectively. The heavy chain of FVIII did not bind to anhydro-FXa. Similarly, competitive assays using overlapping synthetic peptides corresponding to ESH8 epitopes (residues 2248–2285) demonstrated that a peptide designated EP-2 (residues 2253–2270; TSMYVKEFLISSSQDGHQ) inhibited the binding of the C2 domain or the 72-kDa LCh to anhydro-FXa by more than 95 and 84%, respectively. Our results provide the first evidence for a direct role of the C2 domain in the association between FVIII and FXa.

Factor VIII C2 domain contains the thrombin-binding site responsible for thrombin-catalyzed cleavage at Arg1689

Thrombin-catalyzed factor VIII activation is an essential positive feedback mechanism regulating intrinsic blood coagulation. A factor VIII human antibody, A-FF, with C2 epitope, exclusively inhibited factor VIII activation and cleavage at Arg1689 by thrombin. The results suggested that A-FF prevented the interaction of thrombin with factor VIII and that the C2 domain was involved in the interaction with thrombin. We performed direct binding assays using anhydro-thrombin, a catalytically inactive derivative of thrombin in which the active-site serine is converted to dehydroalanine. Intact factor VIII, 80-kDa light chain, 72-kDa light chain, and heavy chain fragments bound dose-dependently to anhydro-thrombin, and the K d values were 48, 150, 106, and 180 nm, respectively. The C2 and A2 domains also dose-dependently bound to anhydro-thrombin, and theK d values were 440 and 488 nm, respectively. The A1 domain did not bind to anhydro-thrombin. A-FF completely inhibited C2 domain binding to anhydro-thrombin (IC50, 18 nm), whereas it did not inhibit A2 domain binding. Furthermore, C2-specific affinity purified F(ab)′2 of A-FF, and the recombinant C2 domain inhibited thrombin cleavage at Arg1689. Our results indicate that the C2 domain contains the thrombin-binding site responsible for the cleavage at Arg1689.

A novel automated microchip flow-chamber system to quantitatively evaluate thrombus formation and antithrombotic agents under blood flow conditions

Summary.  Background and aims: In the present study, we describe a newly developed microchip‐based analytical system to evaluate white thrombus formation (WTF). Efficacies of various antithrombotic agents were compared under different flow conditions. Methods: Whole blood containing corn trypsin inhibitor was perfused over a microchip coated with collagen and tissue thromboplastin at the lower and higher shear rates of 240 and 600 s−1, and WTF process inside the microchip was quantified by monitoring a flow pressure. Parameters of T10 (time to 10 kPa), T10–80 (time from 10 to 80 kPa) and OT (occlusion time; time to 80 kPa) were used to evaluate the onset and the growth rate of WTF, and the capillary occlusion, respectively. Results: After perfusion was started, white thrombus composed of activated platelets and fibrin was formed on the coated surface. Thrombus gradually increased in size and eventually occluded the capillary. Among anticoagulants, heparin (0.5–1.0 U mL−1) potently prolonged T10 at both shear rates, whereas low molecular weight heparin (1.0–2.0 IU mL−1) inhibited the growth of WTF at the lower shear rate. Among antiplatelet agents, abciximab (1–2 μg mL−1) significantly reduced the size and number of thrombi, which was additively enhanced in the presence of heparin (0.5 U mL−1). OS‐1 (specific GPIbα‐antagonist) prevented the complete capillary occlusion. Conclusion: The novel monitoring system of WTF may be useful in preclinical and clinical evaluations of different types of antithrombotic strategies, and their effects in combination.

A microchip flow-chamber system for quantitative assessment of the platelet thrombus formation process.

As the pathogenesis of arterial thrombosis often includes platelet thrombus formation (PTF), antiplatelet agents are commonly used for the prevention of thromboembolic events. Here, using a novel microchip flow-chamber system we developed to quantitatively analyze the PTF process, we evaluated the pharmacological efficacies of antiplatelet agents under different arterial shear rates. Hirudin-anticoagulated whole blood was perfused over a collagen-coated microchip at shear rates of 1000, 1500, and 2000s(-1), and PTF in the absence and presence of various antiplatelet agents was observed microscopically and quantified by measuring flow-pressure changes. The onset of PTF was measured as T(10) (time to reach 10 kPa), and AUC(10) (area under the flow pressure curve for the first 10 min) was calculated to quantify the overall stability of the formed thrombus. Aspirin and AR-C66096 (P2Y(12)-antagonist) at high concentrations (50 μM and 1000 nM, respectively) prolonged T(10) only modestly (AR-C66096>aspirin), but effectively decreased AUC(10), resulting in unstable PTF at all examined shear rates. With dual inhibition using both aspirin (25 μM) and ARC-66096 (250 nM), AUC(10) was drastically reduced. Nearly complete suppression of AUC(10) was also observed with abciximab (2 μg ml(-1)) and beraprost (PGI(2)-analog; 4 nM). Although OS-1 (GPIbα-antagonist; 100 nM) prevented complete capillary occlusion, significant amounts of microscopic thrombi were observed on the collagen surface. In contrast to abciximab and beraprost, OS-1 differentially affected PTF under higher shear conditions. Our novel analytical system is capable of distinguishing the pharmacological effects of various antiplatelet agents under physiological shear rates, suggesting that this system may aid in the determination of the appropriate type and dose of antiplatelet agent in the clinical setting.

Analysing responses to aspirin and clopidogrel by measuring platelet thrombus formation under arterial flow conditions

High residual platelet aggregability and circulating platelet-monocyte aggregates in patients administered aspirin and clopidogrel are associated with ischaemic vascular events. To determine the relevance of these factors with residual thrombogenicity, we measured platelet thrombus formation using a microchip-based flow-chamber system in cardiac patients receiving aspirin and/or clopidogrel, and evaluated its correlation with agonist-inducible platelet aggregation and platelet-monocyte aggregates. Platelet thrombus formation was analysed by measuring flow pressure changes due to the occlusion of micro-capillaries and was quantified by calculating AUC 10 (area under the flow pressure curve. The growth and stability of platelet thrombi that formed inside microchips at shear rates of 1000, 1500, and 2000 s⁻¹ were markedly reduced in patients receiving aspirin and/or thienopyridine compared to healthy controls (n=33). AUC 10 values of aspirin therapy patients (n=20) were significantly lower and higher than those of healthy controls and dual antiplatelet therapy patients (n=19), respectively, and showed relatively good correlations with collagen-induced platelet aggregation and platelet-monocyte aggregates at 1000 and 1500 s⁻¹ (rs >0.59, p<0.01). In contrast, AUC 10 in dual antiplatelet therapy patients was significantly correlated with ADP-induced platelet aggregation at all examined shear rates (rs >0.59, p<0.01), but did not correlate with collagen-induced aggregation. Aspirin monotherapy patients with high residual platelet thrombogenicity also exhibited significant elevations in both collagen-induced platelet aggregation and platelet-monocyte aggregates. Our results, although preliminary, suggest that residual platelet thrombogenicity in aspirin-treated patients is associated with either collagen-induced platelet aggregation or circulating platelet-monocyte aggregates, but it is predominantly dependent on ADP-induced platelet aggregation in patients receiving dual antiplatelet therapy.

A comparative study of prothrombin complex concentrates and fresh-frozen plasma for warfarin reversal under static and flow conditions

Prothrombin complex concentrates (PCCs) and fresh-frozen plasma (FFP) have been clinically used for acute warfarin reversal. The recovery of prothrombin time (PT) or international normalised ratio (INR) is often reported as an endpoint, but haemostatic efficacies of PCCs and FFP may not be fully reflected in static clotting test in platelet-poor plasma. Using various in vitro assays, we compared the effects of two PCC preparations (3-factor PCC; Bebulin and 4-factor PCC; Beriplex) and FFP on warfarin reversal under static and flow conditions. First, we added an aliquot of either PCC (0.3 or 0.72 U/ml) or 20% FFP (v/v) to commercial warfarin plasma (INR 3.2, or 10.3), and then measured PT, factor II, factor VII, and thrombin generation. Subsequently, we collected whole blood samples from six consented warfarin-treated patients with mean INR 3.0 ± 0.5 (range 2.5-3.7), and compared clot formation under flow conditions at 280 s-1 before and after addition of either PCC preparation (0.3 and 0.6 U/ml) or 20% of FFP (v/v). PT/INR were restored by either PCC in plasma with INR 3.0, but they were more effectively corrected by 4-factor PCC than 3-factor PCC in plasma with INR 10.3. Effects of FFP were similar to 0.3 U/ml of PCCs in terms of PT, but FFP was less efficacious than PCCs in recovering thrombin generation or factor II levels. In flow experiments, the onset of thrombus formation was shortened by either PCC, but not by FFP, contrary to shortened PT values. For warfarin reversal 20% volume replacement with FFP is inferior to PCCs.

Evaluation of a novel flow chamber system to assess clot formation in factor VIII-deficient mouse and anti-factor IXa-treated human blood

Blood flow properties play important roles in the regulation and formation of thrombus. To evaluate the influence of blood flow on thrombus formation in haemophilia, whole blood samples were obtained from FVIII‐deficient (FVIII−/−) and wild‐type (FVIII+/+) mice (n = 6 respectively), and from six human volunteers. Anti‐FIXa aptamer was added to human blood to model acquired haemophilia B. Recalcified whole blood samples containing corn trypsin inhibitor and danaproid were perfused over the microchip coated with collagen and tissue thromboplastin at shear rates of 1100 and 110 s−1. Thrombus formation in the capillary was quantified by monitoring flow pressure changes. The intervals to 5 kPa (T5) and 40 k Pa (T40) reflect the onset and growth of thrombus formation respectively. Furthermore, fibrin and platelets in thrombi were quantified by immunostaining. T5 at both shear rates were similar in FVIII−/− and FVIII+/+ mice. T40 of FVIII−/− mice (1569 ± 565 s) was significantly delayed compared with FVIII+/+ mice (339 ± 78 s) at 110 s−1 (P < 0.05), but not at 1100 s−1. The delay was normalized by adding human FVIII (2 IU mL−1). Similarly, adding anti‐FIXa aptamer to human blood prolonged T40 at 110 s−1 (P < 0.01), but not at 1100 s−1. Impaired production of fibrin due to anti‐FIXa aptamer at 110 s−1 was shown in the immunostained thrombus. Our perfusion experiments demonstrated that shear rates influence thrombus formation patterns in haemophilia, and that reduced activity of intrinsic tenase (FIXa‐FVIIIa) becomes evident under venous shear rates.

Studies of a microchip flow-chamber system to characterize whole blood thrombogenicity in healthy individuals

INTRODUCTION A whole blood flow-chamber system, the Total Thrombus-formation Analysis System (T-TAS), was developed for quantitative analysis of platelet thrombus formation (PTF) using microchips with thrombogenic surfaces (collagen, PL chip; collagen plus tissue thromboplastin, AR chip) under shear stress conditions. We evaluated the usefulness of the T-TAS for assessing individual thrombogenicity compared with other platelet function tests. MATERIALS AND METHODS Blood samples from 31 healthy volunteers were applied to the T-TAS to measure PTF starting time (T10: time to reach 10 kPa), occlusion time (T60 for PL chip; T80 for AR chip), and area under the curve (AUC10, area under curve until 10 min for PL chip; AUC30, 30 min for AR chip) under various shear rates (1000, 1500, 2000s(-1) for PL chip; 300 s(-1) for AR chip). Platelet functions were also tested using platelet aggregometry, the PFA-100 (collagen and epinephrine [C/EPI], collagen and adenosine diphosphate [C/ADP]), and the VerifyNow P2Y12 assay. RESULTS Individual pressure waveforms, including PTF starting and ending points, varied among healthy subjects. In the PL chip, T10 and AUC10 showed a shear-dependent correlation with C/EPI or C/ADP. VerifyNow P2Y12 values were not significantly associated with the parameters of the T-TAS. Platelet counts were correlated with all AR measurements, and mostly with PL measurements. CONCLUSION The results of the T-TAS were associated with those of the PFA-100 in many respects, indicating that its characteristics are related to shear-induced PTF. The T-TAS showed few correlations with platelet aggregometry and the VerifyNow P2Y12 assay. The T-TAS may allow for the measurement of comprehensive parameters of individual thrombogenicity under whole blood flow conditions.

Preparation of anhydrothrombin and characterization of its interaction with natural thrombin substrates

Thrombin is a serine proteinase that plays a key role in thrombosis and haemostasis through its interaction with several coagulation factors. Anhydrothrombin was prepared from PMSF-inactivated thrombin under alkaline conditions, and the folded anhydrothrombin was successfully recovered after dialysis in the presence of glycerol. Anhydro-derivatives of factor Xa, factor VIIa and activated protein C could also be prepared essentially by the same procedure. Anhydrothrombin retained affinity for various natural substrates of thrombin, including fibrinogen, factor VIII, factor XIII and protein C. In addition, these proteins were bound to anhydrothrombin-agarose in a reversible manner. The K(d) values for factor VIII, fibrinogen, factor XIII and protein C were 1.2x10(-8), 4.4x10(-8), 2.8x10(-7) and 8.1x10(-5) M, respectively. Thus thrombin substrates known to interact with the exosite I of thrombin demonstrated high affinity for anhydrothrombin. Furthermore, in the presence of Na+, substantial enhancement of the association rate constant (k(ass)) was observed for interactions of fibrinogen and factor VIII with anhydrothrombin. These results suggest that anhydrothrombin is useful in the purification of thrombin substrate proteins as well as in the investigation of detailed interactions between thrombin and these substrates in their activation or degradation processes.

Comparative evaluation of direct thrombin and factor Xa inhibitors with antiplatelet agents under flow and static conditions: an in vitro flow chamber model.

Dabigatran and rivaroxaban are novel oral anticoagulants that specifically inhibit thrombin and factor Xa, respectively. The aim of this study is to elucidate antithrombotic properties of these anticoagulant agents under arterial and venous shear conditions. Whole blood samples treated with dabigatran or rivaroxaban at 250, 500, and 1000 nM, with/without aspirin and AR-C66096, a P2Y12 antagonist, were perfused over a microchip coated with collagen and tissue thromboplastin at shear rates of 240 and 600 s−1. Fibrin-rich platelet thrombus formation was quantified by monitoring flow pressure changes. Dabigatran at higher concentrations (500 and 1000 nM) potently inhibited thrombus formation at both shear rates, whereas 1000 nM of rivaroxaban delayed, but did not completely inhibit, thrombus formation. Dual antiplatelet agents weakly suppressed thrombus formation at both shear rates, but intensified the anticoagulant effects of dabigatran and rivaroxaban. The anticoagulant effects of dabigatran and rivaroxaban were also evaluated under static conditions using thrombin generation (TG) assay. In platelet-poor plasma, dabigatran at 250 and 500 nM efficiently prolonged the lag time (LT) and moderately reduce peak height (PH) of TG, whereas rivaroxaban at 250 nM efficiently prolonged LT and reduced PH of TG. In platelet-rich plasma, however, both anticoagulants efficiently delayed LT and reduced PH of TG. Our results suggest that dabigatran and rivaroxaban may exert distinct antithrombotic effects under flow conditions, particularly in combination with dual antiplatelet therapy.