Functional Significance of Adenosine 5′-Diphosphate Receptor (P2Y 12 ) in Platelet Activation Initiated by Binding of von Willebrand Factor to Platelet GP Ibα Induced by Conditions of High Shear Rate

Abstract

Background — The role of the adenosine 5′-diphosphate receptor P2Y 12 in platelet activation initiated by the von Willebrand factor (VWF)–GP Ibα interaction under high shear rate was investigated. Methods and Results — Blood samples were obtained from 11 donors. Shear-induced platelet aggregation was detected by optically modified cone-plate viscometer. Shear-induced VWF binding, P-selectin expression, and microparticle release were detected by flow cytometry. Platelet interaction with immobilized VWF was also investigated by parallel-plate flow chamber equipped with epifluorescent videomicroscopy. Effects of a selective P2Y 12 antagonist AR-C69931 MX were tested. AR-C69931 MX inhibited shear-induced platelet aggregation in a dose-dependent manner, achieving the maximum inhibition at 100 nmol/L. The extent of aggregation after exposure to a shear rate of 10 800 s −1 for 6 minutes in the presence of 100 nmol/L AR-C69931 MX was 32.4±8.2% (mean±SD), which was significantly lower than the value in the controls of 69.7±9.6% ( P <0.01). The inhibiting effects of AR-C69931 MX were reversed by exogenous addition of adenosine 5′-diphosphate. Shear-induced VWF binding and P-selectin surface translocation, which occurred in 4696±911 and 5964±784, respectively, of 10 000 measured platelets, was also inhibited by AR-C69931 MX (100 nmol/L) to 1948±528 and 2797±718, respectively ( P =0.0018 and P =0.0009). Microparticle release was similarly inhibited. In a flow chamber experiment, firm platelet attachment on immobilized VWF was inhibited by AR-C69931 MX, whereas transient interaction was not influenced. All the above reactions were completely inhibited by blocking VWF–GP Ibα interaction. Conclusions — We have demonstrated that the stimulation of P2Y 12 is involved in platelet activation initiated by the binding of VWF to GP Ibα induced by a high shear rate.