The role of Akt in GPIb-IX–mediated platelet aggregation and the PI3K-dependent Akt activation induced by vWF. (A) Washed wild-type (WT) and Akt1^−/− mouse platelets were stimulated with botrocetin (2 μg/mL) and vWF (7.5 μg/mL). Platelet aggregation was monitored using a turbidometric aggregometer at 37°C and 1000 rpm stirring speed. (B) WT and Akt1^−/− platelets were treated with DMSO or SH-6 (15 μM) for 2 minutes and then stimulated with botrocetin (1.2 μg/mL) and vWF (10 μg/mL). (C) WT and Akt2^−/− platelets were stimulated with botrocetin and vWF as in panel B. (D) Washed human platelets were preincubated with Akt inhibitor SH-6 (15 μM) or the vehicle (DMSO) for 2 minutes, and then stimulated with botrocetin and vWF as in panel B. (E) Washed human platelets (5 × 10⁸/mL) were preincubated with DMSO, PI3K inhibitors, wortmannin (100 nM), or LY294002 (20 μM), then stimulated with ristocetin (0.5 mg/mL) in the absence or presence of 25 μg/mL vWF for 1 minute in a platelet aggregometer. Platelets were solubilized and immunoblotted with a rabbit antibody specifically recognizing Akt phosphorylated at serine-473 (p-Akt) or with a rabbit anti-Akt antibody to indicate loading levels (Akt).