The role of PI 3–kinase in regulating platelet calcium responses under static conditions.

Calcium indicator dye-loaded platelets were incubated with vehicle alone (control), LY294002 (25 μM), or wortmannin (100 nM) for 15 minutes prior to adhesion on immobilized VWF (10 μg/mL). Adherent platelets were allowed to spread in the presence of extracellular Ca⁺⁺ (1 mM) for up to 60 minutes under static conditions. Changes in the cytosolic calcium concentration of adherent cells were monitored at the indicated time points by confocal microscopy (× 63W objective) and fluorescence ratios quantified. The results presented in panel A demonstrate the percentage of platelets undergoing oscillatory calcium transients at the indicated time points. Results represent mean ± SEM from 3 to 5 independent experiments. The results presented in panel B show a representative calcium oscillation profile of individual vehicle-treated (control) and wortmannin-treated platelets at 0 minute (thin line) and after 15 minutes of spreading (bold line). Statistical analysis was performed using a ttest comparing control versus LY294002- or wortmannin-treated platelets (P < .05*; P < .01**).