To cite this article: Hosokawa K, Ohnishi T, Kondo T, Fukasawa M, Koide T, Maruyama I, Tanaka KA. A novel automated microchip flow-chamber system to quantitatively evaluate thrombus formation and antithrombotic agents under blood flow conditions. J Thromb Haemost 2011; 9: 2029-37. 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 , and WTF process inside the microchip was quantified by monitoring a flow pressure. Parameters of T 10 (time to 10 kPa), T 10-80 (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 T 10 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 lg 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 GPIba-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.

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.

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 Arg 1689 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. Blood clotting factor VIII (FVIII) 1 is a crucial glycoprotein that accelerates the intrinsic coagulation cascade by acting as a cofactor of factor IXa in the tenase complex (1), and a deficiency of FVIII results in the common hereditary bleeding disorder, hemophilia A. FVIII circulates in plasma as a noncovalent complex with von Willebrand factor (vWF) that stabilizes the synthesis and cofactor activity of FVIII (2-4). Mature FVIII is synthesized as a single chain polypeptide of approximately 300 kDa consisting of 2,332 amino acid residues with a mosaic domain structure arranged in the order of A1-A2-B-A3-C1-C2 (5-7) and secreted into plasma as variable series of heterodimers consisting of the heavy chain (HCh) and the light chain (LCh). The HCh is composed of the A1, A2, and parts of the B domain and exhibits size heterogeneity (90 -210 kDa) due to proteolysis within the B domain. The 90-kDa HCh reflects the absence of the B domain, whereas the 210-kDa species includes the intact B domain. The LCh (80 kDa) corresponds to the A3, C1, and C2 domains. The two chains are noncovalently linked by metal ions between the A1 and A3 domains (6, 7).FVIII is transformed into an active form by limited proteolysis by two essential serine proteases, thrombin and FXa (8, 9). This procoagulant activity is more pronounced after thrombin activation than after FXa activation (10). Cleavage at Arg 740 removes the size-heterogeneous B domain from the HCh, producing a 90-kDa HCh fragment, and further cleavage of the 90-kDa HCh fragment (A1-A2) at Arg 372 between the A1 and A2 domains produces 54-(A1) and 44-kDa (A2) species. Cleavage of the 80-kDa LCh fragment (A3-C1-C2) at Arg 1689 between the B and A3 domains removes 40 amino-terminal acidic peptides from the A3 domain and produces a 72-kDa fragment. A unique cleavage by FXa at Arg 1721 produces a 67-kDa LCh fragment. The active form of FVIII is a metal-linked heterotrimer consisting of the A1/A2/A3-C1-C2 domains, lacking the middle B domain (6). Cleavage at Arg 740 between the A2 and B domains does not contribute to the generation of the FVIII activity. In contrast, cleavages at Arg 372 and Arg 1689 are essential for optimal FVIII activity (11-13). Activation of human FVIII at its physiological concentration and at pH 7.4 is followed by a reduction in activity (14). This loss of activity is not caused by further proteolysis but by dissociation of the A2 domain from the active form of FVIII heterotrime...