Effects of PI3K and Akt inhibitors and Akt knockout on platelet spreading on immobilized vWF. Microtiter chamber slides were coated with 30 μg/mL vWF. Washed WT mouse platelets (2 × 10⁷/mL) were preincubated with or without vehicle (DMSO), SH-6 (15 μM), or wortmannin (100 nM) for 5 minutes. WT, Akt1^−/−, or Akt2^−/− platelets were allowed to adhere and spread on vWF for 90 minutes in the presence of botrocetin (1 μg/mL). Adherent platelets were stained with fluorescein-labeled phalloidin and photographed under a fluorescence microscope as described in “Platelet spreading on immobilized vWF and fibrinogen.” Platelets were also treated with the integrin inhibitor, RGDS (1 mM), to indicate the integrin–dependent platelet spreading. Shown in the figure are representative pictures from one of 3 experiments with similar results and a bar graph of the quantitative analysis of platelet surface area as an indicator of spreading. The data are from 3 randomly selected fields from 3 experiments. The bars in the graph represent the average surface area (± SD) of individual platelets. ^###P < .001 versus WT platelets. ***P < .001 versus DMSO–treated WT platelets. Numbers of platelets analyzed for each group are indicated above the bars.