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

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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 1 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, PtdIns(3,4)P2. Under static conditions, inhibiting PI 3–kinase with LY294002 or wortmannin did not prevent platelet adhesion, integrin αIIbβ3activation, 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. Real-time 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.
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