P2Y₁ purinergic receptor desensitization in human platelets. (A) Platelets were not pretreated (i; □) or were pretreated with (ii) P2Y₁ receptor antagonist A3P5P 1 mM for 180 seconds (▪), (iii) apyrase alone 0.2 U/mL for 180 seconds (▨ ), (iv) ADP 10 μM for 30 seconds then apyrase 0.2 U/mL for 180 seconds (▩), or (v) AMP 10 μM for 30 seconds then apyrase 0.2 U/mL for 180 seconds (▦). ADP (10 μM)–induced calcium responses were subsequently measured, and peak responses were compared with those in nonpretreated control platelets. ADP (10 μM) premixed with apyrase for 3 minutes was also added to control platelets (vi; ▥), and subsequent calcium responses were measured. Data are expressed as peak calcium response (percentage of 10 μM ADP alone) and represent the mean ± SEM (n = 3). (B) Platelets were pretreated with ADP 1 μM for 0 to 300 seconds, and then apyrase 0.2 U/mL was added for 180 seconds. ADP (10 μM)–induced calcium responses were subsequently measured and compared with those of control platelets incubated with apyrase 0.2 U/mL only. Data are expressed as peak calcium response relative to control responses (percentage of 10 μM ADP alone) and represent the mean ± SEM (n = 6). (C) Platelets were pretreated with ADP 10 μM for 30 seconds, and then apyrase 0.2 U/mL was added for 180 seconds. Collagen (30 μg/mL)–induced calcium responses were subsequently measured, and peak responses (▪) were compared with those of nonpretreated control platelets (□). ADP/apyrase pretreatment did not affect peak calcium responses before collagen addition compared with nonpretreated controls (data not shown). Data are expressed as peak change in fluorescence ratio (340:380 nM) and represent the mean ± SEM (n = 3).