The role of PI 3–kinase in regulating calcium mobilization under flow conditions.

Calcium indicator dye-loaded platelets were incubated with vehicle alone (control) or wortmannin (100 nM) for 15 minutes prior to reconstitution with red blood cells and perfusion over immobilized VWF (100 μg/mL) at 1800 s^-1. Changes in the cytosolic calcium concentration of adherent cells were monitored by confocal microscopy (× 63W objective) and fluorescence ratios quantified. The results presented in panel A demonstrate the effect of wortmannin on the percentage of cells undergoing oscillatory calcium transients relative to untreated (control) platelets. The images and results presented in panels B and C show a representative calcium oscillation response (solid line) and the displacement (dotted line) of individual vehicle- (control) and wortmannin-treated platelets under shear conditions. Results and images are from a single experiment representative of 3. Bar = 10 μm.