Abstract 14079: Outside-in Signaling of Integrin α IIb β 3 Mediates Shear-dependent Platelet Microvesicle Formation and Phosphatidylserine Exposure

Introduction and Hypothesis: Platelets promote coagulation mainly by exposing membrane phosphatidylserine (PS) and releasing PS-expressing microvesicles (MV). We have recently shown that PS exposure and MV release induced by platelet agonists requires shear stress. To identify the receptor responsible for the shear-dependent signaling leading to PS exposure and MV release, we investigated the hypothesis that Integrin αIIbβ 3 may play a role in shear-dependent platelet MV release and PS exposure. Methods and Results: To determine the role of integrin αIIbβ 3 in shear-dependent PS exposure and MV release, we compared platelets from β 3 -/- mice and wild-type mice in MV release and PS exposure under shear introduced using a cone-plate rheometer. MV release and PS exposure were determined by flow cytometry. Shear-dependent PS exposure and MV release were significantly suppressed in β 3 -/- platelets. Similarly, Wild type platelets treated with integrin antagonists also showed defective PS exposure and MV release. These data indicate an important role for the ligand binding function of integrin αIIbβ 3 in shear-dependent MV release and PS exposure. To determine whether the role of integrin αIIbβ 3 is due to its outside-in signaling, β 3 -/- platelets were transplanted with wild type β 3 or a mutant β 3 with the critical Gα13 binding site of the β 3 cytoplasmic domain (EEE) changed to alanines (AAA), which selectively abolish outside-in signaling of αIIbβ 3 . Transplantation of wild type β 3 rescued the defective MVs release and PS exposure of β 3 -/- platelets. In contrast, AAA mutant failed to rescue these defects. Consistently, wild type platelets treated with the selective inhibitor of Gα13-integrin interaction, inhibited integrin outside-in signaling and also PS exposure and MV release under shear stress. Furthermore, inhibition of Src, which is important in outside-in signaling downtream of Gα13, also abolished shear-dependent MV release and PS exposure. Conclusions: These data suggest that integrin outside-in signaling mediated by the Gα13-β3 interaction and Src-dependent signaling pathway plays an important role in transmitting shear-induced mechanical signals leading to MV release and PS exposure in activated platelets.