Effect of PI3K and Akt inhibitors and Akt knockout on platelet spreading on immobilized fibrinogen. Microtiter chamber slides were coated with 30 μg/mL fibrinogen. Washed WT mouse platelets (2 × 107/mL) were preincubated with 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 immobilized fibrinogen for 90 minutes. Platelets were also treated with the integrin inhibitor, RGDS (1 mM), to indicate the integrin-dependent platelet adhesion and spreading. Adherent platelets were stained with fluorescein-labeled phalloidin and photographed under a fluorescence microscope as described in “Platelet spreading on immobilized vWF and fibrinogen.” Shown in the figure are the 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 DMSO–treated WT platelet. Numbers of platelets analyzed for each group are indicated above the bars.
