Characterization of integrin α2β1–induced PI3Kβ activation. (A) Role of secreted ADP, TxA2, and integrin αIIbβ3 in integrin α2β1–triggered Akt phosphorylation. Platelets were incubated with 0.5mM ASA for 15 minutes, with 2 U/mL of apyrase, 0.5mM RGDS, or a mixture of 100μM MRS2179 and 0.5μM AR-C69931MX for 2 minutes, and then allowed to adhere to monomeric collagen for 60 minutes. Nonadherent platelets from untreated samples (NA) were also collected. In panel i, the top row shows a typical immunoblot with anti–phospho-Akt(Ser 473) Ab, and the bottom row shows the level of tubulin in the different samples. Panel ii shows a quantitative evaluation of Akt phosphorylation performed by densitometric analysis of immunoblots. Data are the means ± SD of 3 different experiments. (B) Analysis of Akt phosphorylation in platelets after 60 minutes of adhesion after treatment with BAPTA-AM (20μM, 30 minutes), RO318220 (10μM, 5 minutes), PP2 (20μM, 15 minutes), or 2-APB (100μM, 10 minutes), as indicated. As a negative control, nonadherent platelets (NA) from untreated samples (none) were also analyzed. Subsequent immunoblotting with anti-tubulin (bottom row) was performed as control for equal loading. (C) Analysis of Akt phosphorylation in murine platelets from wild-type (WT) and PLCγ2–knockout (PLCγ2 KO) mice. Adherent platelets were recovered after 30 and 60 minutes, as indicated on the top. Nonadherent cells were analyzed after 60 minutes.
