Coengagement of PECAM-1 with FcγRIIa cross-linking with IV.3 results in dephosphorylation of serine/threonine kinase, Akt. (A) Washed platelets were stimulated by FcγRIIa cross-linking with and without coengagement of PECAM-1 over a time course of 0 to 5 minutes at 37°C with stirring. Platelet lysate (200 μg) was electrophoresed on a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel, followed by immunoblotting with a mixture of antiphosphoSer473 Akt and antiphosphoThr308 Akt (1/2000) followed by HRP-conjugated antirabbit (1/10 000) and ECL development. (B) Blot was stripped and reprobed for Akt antigen content with goat anti-Akt antibody (0.3 μg/mL) and HRP-conjugated antigoat (1/4000) and ECL development.