Induction of A_2AR reduces platelet activation by enhancing cAMP. (A) Healthy platelets were pretreated with either vehicle or CGS21680 (A_2AR-a, 5μM) or BAY 60-6583 (A_2BR-a, 5μM), followed by stimulation with thrombin (0.05 U/mL) for 15 minutes. CD62P was assessed using flow cytometry. (B-C) Flow cytometric analysis of surface CD62P in platelets preincubated with vehicle or A_2AR inhibitor (SCH-442416, 5μM) for 20 minutes, followed by A_2AR-a and thrombin (0.05 U/mL) stimulation. (D) Quantification of released ATP in A_2AR-a and thrombin-treated platelets. (E-F) A_2AR-a (1μM and 5μM) pretreated platelets were stimulated with either convulxin (20 ng/mL) or (G-H) U46619 (5μM) and assessed for surface CD62P using a flow cytometer. (I) Purified platelets were pretreated with vehicle or A_2AR-a for 15 minutes before stimulation with thrombin (0.05 U/ml), or convulxin (20 ng/mL), or U46619 (5μM) for 15 minutes. The representative immunoblots show the phosphorylated-Akt (pAkt, Ser 473), total Akt, (pGSK3β, Ser9), and total GSK3β. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was also used as an internal loading control for all the samples. Data represent mean ± SD. ∗∗∗P < .001, ∗∗∗∗P < .0001, and ns by a 1-way ANOVA with the Tukey multiple comparisons correction (n = 4).