Expression of thrombin and collagen receptors, β₃ integrin, and P-selectin, and ATP release. (A-B) Expression of thrombin receptors. Thrombin receptors PAR4 (A) and PAR3 (B) were detected by Western blot. (C) Expression of collagen receptor GPVI. Platelet suspensions were incubated with FITC-labeled anti-GPVI antibody for 15 minutes at room temperature. (D) Expression of β₃ integrin. Gel-filtered platelets were incubated with or without thrombin at 0.5 U/mL for 15 minutes and then fixed. After washing, platelets were incubated with anti-β₃ integrin antibody for 20 minutes followed by FITC-labeled secondary antibody. (E) P-selectin expression. Platelets were stimulated with or without thrombin at 0.05 U/mL or 0.1 U/mL for 5 minutes and then fixed. After washing, platelets were incubated with FITC-labeled P-selectin antibody for 20 minutes. For panels C-E, the samples were analyzed by flow cytometry; 20 000 events were recorded. Bars present mean ± SD from 3 independent experiments. ^*Significant difference between WT and Akt-1-null platelets (P < .05). (F) ATP release. The release of ATP was measured in gel-filtered platelets by a Lumi-Aggregometer type 500 VS using luciferin-luciferase. ATP served as the standard in all experiments. The addition of thrombin at a final concentration of 0.05 U/mL is indicated by the arrow (P < .05).