Activation of PI3Kβ by integrin α2β1 engagement. (A) Human platelets were allowed to adhere to immobilized monomeric type I collagen (i) or GFOGER peptide (ii) for 10, 30, or 60 minutes. Adherent cells and nonadherent cells (NA) from the 60-minute samples were collected and lysed. Akt phosphorylation was analyzed by immunoblotting with anti–phospho-Akt(Ser473) Ab (top rows). Equal loading of the samples was verified by subsequent immunoblotting with anti-tubulin (bottom rows). (B) Human platelets were preincubated with DMSO, wortmannin (100nM, 15 minutes), TGX-221 (0.5μM, 10 minutes), AS252424 (0.5μM, 10 minutes), or PIK-75 (0.5μM, 10 minutes), and Akt phosphorylation (top row) was evaluated by immunoblotting in platelets adherent to monomeric type I collagen for 60 minutes. Subsequent analysis of pleckstrin levels (bottom row) was performed to control loading of the samples. (C) Wild-type murine platelets (WT) and platelets from PI3Kγ^KD or PI3Kβ^KD mice were allowed to adhere to monomeric collagen through integrin α2β1 for 30 or 60 minutes, as indicated. Nonadherent platelets (NA) were also collected after 60 minutes. The top row shows Akt phosphorylation in adherent and nonadherent platelets, and the bottom row shows the comparable expression of tubulin in all samples.