The role of PI 3–kinase in regulating platelet adhesion and integrin α_IIbβ₃ activation under static conditions and the effect of apyrase on VWF-induced integrin α_IIbβ₃ activation.

Washed platelets (1.5 × 10⁸/mL) were incubated with vehicle alone (control) or the indicated concentrations of LY294002 (0-25 μM) or wortmannin (0-100 nM) for 15 minutes or with apyrase (16 U/mL) for 30 minutes. Platelets were allowed to adhere and spread on immobilized human VWF (10 μg/mL) for 60 minutes under static conditions. In panel B, pretreated platelets were incubated with PAC-1 antibody, prior to adhesion and spreading. These results demonstrate (A) the effect of LY294002 or wortmannin on the level of platelet adhesion, and (B) the effect of LY294002, wortmannin, or apyrase on PAC-1 binding as visualized by confocal microscopy (× 63 W objective; bar = 10 μm). Statistical analysis of the results was performed using a t test and the P values are indicated where appropriate (P < .01**). Results are the mean ± SEM of 3 experiments, and images are from a single experiment representative of 3.