Essential role for phosphoinositide 3-kinase in shear-dependent signaling between platelet glycoprotein Ib/V/IX and integrin αIIbβ3

Abstract

Platelet adhesion and aggregation at sites of vascular injury are critically dependent on the interaction between von Willebrand factor (VWF) and 2 major platelet adhesion receptors, glycoprotein (GP) Ib/V/IX and integrin αIIbβ3. GP Ib/V/IX binding to VWF mediates platelet tethering and translocation, whereas activation of integrin αIIbβ3 promotes cell arrest. To date, the signaling pathways used by the VWF-GP Ib/V/IX interaction to promote activation of integrin αIIbβ3, particularly under shear, have remained poorly defined. In this study, the potential involvement of type I phosphoinositide (PI) 3-kinases in this process was investigated. Results show that platelet adhesion and spreading on immobilized VWF results in a specific increase in the PI 3-kinase lipid product, Ptdlns(3,4)P2. Under static conditions, inhibiting PI 3-kinase with LY294002 or wortmannin did not prevent platelet adhesion, integrin αIIbβ3 activation, or platelet spreading although it significantly delayed the onset of these events. In contrast, PI 3-kinase inhibition under shear dramatically reduced both platelet adhesion and spreading. Realtime analysis of intracellular calcium demonstrated that under static conditions inhibiting PI 3-kinase delayed the onset of intracellular fluxes in adherent platelets, but did not affect the final magnitude of the calcium response. However, under shear, inhibiting PI 3-kinase dramatically reduced intracellular calcium mobilization and integrin αIIbβ3 activation, resulting in impaired thrombus growth. The studies demonstrate a shear-dependent role for PI 3-kinase in promoting platelet adhesion on immobilized VWF. Under static conditions, platelets appear to mobilize intracellular calcium through both PI 3-kinase-dependent and -independent mechanisms, whereas under shear PI 3-kinase is indispensable for VWF-induced calcium release. © 2002 by The American Society of Hematology.