Total and phosphorylated Akt. Gel-filtered platelets (1 × 10⁸/mL) were resuspended in the presence of integrin inhibitors to completely block fibrinogen binding. Platelet lysates were subjected to SDS-PAGE and Western blotting using antibodies against all Akt isoforms (Akt^pan), phosphorylated Akt (phospho-Akt^pan), or actin to ensure equal protein loading. (A) Total Akt levels (top) and their densitometric analysis (bottom) in 2 separate samples of unstimulated platelets. (B) Akt phosphorylation in response to different concentrations of thrombin (top) and densitometric analysis of phosphorylated Akt (bottom). (C) Akt phosphorylation in response to collagen (20 μg/mL) at different time points.