α_Mβ₂- And GPIbα-mediated Akt phosphorylation is crucial for platelet activation. (A) Gel-filtered human platelets (4 × 10⁷ in 200 μL) were incubated in Tyrode buffer containing 1 mM Ca²⁺ with increasing concentrations (0-100 μg/mL) of resting or PMA MPs for 30 minutes at 37°C and lysed by the addition of 4× Laemmli buffer. Lysates were resolved on 10% SDS-PAGE and immunoblotted with Abs to activated Akt (PSer473; top) or to total Akt (bottom). (B) The experiments were performed as described in panel A, but platelets and MPs (final concentration, 50 μg/mL) were pretreated with respective blocking reagents for 20 minutes at 37°C and then combined. The observed Akt activation status is representative of 3 independent experiments. (C) Human platelets (2 × 10⁷ in 150 μL) were incubated with or without GPIbα mAb (VM16d; 10 μg/mL) for 15 minutes at room temperature followed by incubation in the presence or absence of F(ab′)₂ fragments of goat anti–mouse IgG (20 μg/mL) for 15 minutes at 37°C. Lysates were analyzed by Western blots using biotinylated Abs to activated or total Akt and streptavidin-HRPO conjugate. (D,E) Human platelets were pretreated with increasing concentrations (0-100 μM) of Akt inhibitor II for 20 minutes at 37°C followed by incubation in the presence or absence of PMA MPs (25 μg/mL) and anti–P-selectin-PE (D,E, left panels), PAC-1-FITC (D,E, right panels) or mouse isotype control antibodies for 1 hour at 37°C. After incubation, platelets were fixed and analyzed by FACS. The data are MFI (± SEM) and are representative from 2 independent experiments.