The role of PI 3–kinase in regulating platelet adhesion, spreading, and integrin αIIbβ3activation under flow conditions.
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. Pretreated platelets were either perfused immediately over immobilized VWF (100 μg/mL) at 150 s-1 or reconstituted with red blood cells prior to perfusion at 1800 s-1. In some experiments, tethered platelets were incubated with PAC-1 antibody prior to fixation and staining with a FITC-conjugated secondary antibody. These results demonstrate the effect of the PI 3–kinase inhibitors on (A) the level of stationary platelet adhesion and (B) the mean surface area of adherent platelets following perfusion at 150 s-1 or 1800 s-1; (C) the morphology of adherent platelets as visualized by scanning electron microscopy (bar = 2 μm); and (D) platelet spreading and PAC-1 binding (bar = 10 μm). (A,B) Results represent mean ± SEM from 4 independent experiments. Statistical analysis was performed using a t test comparing control versus LY294002- or wortmannin-treated platelets (P < .05*;P < .01**; P < .001***). (C,D) Images are from a single experiment representative of 3.
