P2Y₁₂ purinergic receptor signaling and desensitization in human platelets. (A-B) cAMP accumulation (5 minutes) in the absence (basal; □) or presence of 10 μM forskolin (forskolin; ▨) ± ADP (10 μM; forskolin + ADP; ▪) was determined in (A) 3 different conditions; in the absence of pretreatment (untreated) or after pretreatment with either the P2Y₁₂ receptor antagonist AR-C69931MX (1 μM; 5 minutes) or the P2Y receptor agonist ADPβS (1 mM; 5 minutes). (B) cAMP accumulation was determined in 4 different conditions: (1) in the absence of apyrase (untreated), (2) after pretreatment with apyrase (0.2 U/mL; 180 seconds; + apyrase), (3) after treatment with ADP (1 μM; 300 seconds) and subsequent treatment with apyrase (+ ADP/apyrase), and finally (4) after pretreatment with apyrase (0.2 U/mL; 180 seconds) and AMP (10 μM; 180 seconds) (+ AMP/apyrase). Data represent the mean ± SEM (n = 3) and are expressed as pmol cAMP per 1 × 10⁸ platelets. (C) Platelets were desensitized to ADP, as described in “Materials and methods,” after ADP pretreatment for various time periods, and a percentage of inhibition of forskolin-stimulated cAMP accumulation was plotted as a function of pretreatment time. Data represent mean ± SEM (n = 3) and are expressed as a percentage of inhibition of forskolin-stimulated adenylyl cyclase.