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

Abstract

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 T10 (time to reach 10kPa), and AUC10 (area under the flow pressure curve for the first 10min) was calculated to quantify the overall stability of the formed thrombus. Aspirin and AR-C66096 (P2Y12-antagonist) at high concentrations (50μM and 1000nM, respectively) prolonged T10 only modestly (AR-C66096>aspirin), but effectively decreased AUC10, resulting in unstable PTF at all examined shear rates. With dual inhibition using both aspirin (25μM) and ARC-66096 (250 nM), AUC10 was drastically reduced. Nearly complete suppression of AUC10 was also observed with abciximab (2μgml−1) and beraprost (PGI2-analog; 4 nM). Although OS-1 (GPIbα-antagonist; 100nM) 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.