Stimulatory role of Akt3 in platelet aggregation and secretion in response to thrombin and PAR4 agonist peptide. (A-D) Washed wild-type (WT) and Akt3^−/− platelets were stimulated with thrombin (A,B) or PAR4 agonist peptide AYPGKF (C,D). Platelet aggregation was monitored using a turbidometric aggregometer at 37°C and 1000 rpm stirring speed (A,C). Platelet secretion of ATP was recorded concomitantly in the presence of luciferin-luciferase agent (B,D). Experiments described in panels A to D were repeated 3 times at a low dose of thrombin (0.02 U/mL) or AYPGKF (60μM) with the results quantified as percentage of light transmission (mean ± SE) or as concentration of secreted ATP (mean ± SE). (E) A low concentration of ADP (1μM), insufficient to induce aggregation on its own, reversed the inhibitory effect of Akt3^−/− platelets on thrombin-stimulated aggregation. (F) Akt3^−/− and WT mouse platelets were treated with vehicle DMSO or SH-6 (15μM) for 2 minutes and aggregation was recorded as shown in panel A.