Increased RAP1 activation, Akt phosphorylation, and reduced cAMP formation downstream of P2Y₁₂-dependent signaling in GRK2^−/− platelets. (A) RAP1 activation. Ai: RAP1 protein expression in WT and GRK2^−/− platelets. Aii: RAP1-GTP levels were measured in platelets that were either unstimulated or stimulated with ADP for 5 minutes (mean ± standard error of the mean; n = 4). (B) Akt phosphorylation. Gel-filtered platelets from GRK2^−/− mice or matched WT controls were lysed directly or incubated with 10 μM ADP for 5 minutes in the presence or absence of the P2Y₁ antagonist MRS2500 (50 mM) or the P2Y₁₂ antagonist cangrelor (100 nM), as indicated. (Bi) Lysates were probed with anti–p-Akt (S473) and reprobed with anti-Akt antibody. (Bii) The p-Akt signal was normalized to the Akt loading control and is represented as signal relative to that of the WT with or without antagonist MRS2500 (mean ± standard error of the mean; n = 6). (C) cAMP formation. cAMP levels in resting platelets and platelets stimulated with PGI₂ in the presence or absence of ADP at the final concentrations indicated (mean ± standard error of the mean; n = 3). ns, not significant.