The impact of deleting GRK6 on Akt phosphorylation. (A) Gel-filtered platelets from GRK6^−/− 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 μM) or the P2Y₁₂ antagonist cangrelor (100 nM), as indicated. (Ai) Lysates were probed with anti–p-Akt (S473) and reprobed with anti-Akt antibody. (Aii) The p-Akt signal was normalized to the Akt loading control and is represented as signal relative to that of the WT without antagonist and (Aiii) relative to that of the WT under the same conditions in the presence of the P2Y₁ antagonist MRS2500. Data are mean ± SEM (N = 3). (B). There is no difference of cAMP formation in WT vs GRK6^−/−. (Left) Basal cAMP concentration in GRK6^−/− platelets (N = 4) and matched controls (N = 4). (Right) cAMP levels in platelets incubated with PGI₂ at the final concentrations 15 μM (mean ± SEM, N = 4). Basal cAMP levels were normal in GRK6^−/− platelets, and there was no difference in PGI₂-stimulated cAMP formation in WT vs GRK6^−/− platelets.