PAR agonist causes a more robust Akt phosphorylation and translocation in platelets than ADP. Washed human platelets (A) and murine platelets (B) were stimulated with 2MeSADP (100 nM) or AYPGKF (500 μM) for 2 minutes, and phosphorylation of Ser473 (S473) residue on Akt was evaluated by western blot analysis of whole cell lysates. Densitometric analysis of the western blot shows the percentage of phosphorylated Akt Ser473 normalized to β-actin. Phosphorylation (Ser473) and translocation of Akt was evaluated by western blot and densitometric analysis of membrane fractions prepared by ultracentrifugation of human (C) or mouse (D) platelets stimulated either with 100 nM 2MeSADP or 500 μM AYPGKF. β₃-integrin was used as the lane-loading control as well as a membrane marker. Western blot shows Akt phosphorylation and translocation in whole cell lysate (E) and platelet membrane fraction (F) after human platelets were stimulated with different concentrations of 2MeSADP (30, 50, and 100 nM) or AYPGKF (50, 100, 250, and 500 μM) for 2 minutes in an aggregometer. Densitometric analysis is represented as a percentage of phospho-Akt S473 normalized to β-actin or Akt translocated to the membrane normalized with β₃-integrin at different concentrations of 2MeSADP and AYPGKF. The western blot analysis shown is representative of 3 independent experiments using anti-Akt mouse mAb (Cell Signaling Technology).