Figure 6.

P2Y₁₂ receptor signaling in platelets from a subject with a heterozygous P2Y₁₂ P341A substitution. (A-B) P2Y₁₂ receptor signaling was measured in platelets from healthy donors (HDs) or from the subject with a heterozygous P2Y₁₂ P341A mutation (P341A). P2Y₁₂ receptor signaling was assessed by comparing agonist (ADP; 0.1 and 1μM)–dependent inhibition of PGE₂ (10μM; 5 minutes)–stimulated adenylyl cyclase activity. (A) The raw data from the patient and HD1 are expressed as pmol cAMP/1 × 10⁹ platelets. (B) Data from the patient and a range of HDs are expressed as percentage of inhibition of PGE₂-stimulated adenylyl cyclase. (C-D) The result of a partial reduction in P2Y₁₂ receptor availability was further investigated. (C) Displacement of the P2Y-specific ligand [³H]-2MeSADP by ARC69931. Fixed platelets were incubated with [³H]2MeSADP (100nM), and bound ligand was displaced by increasing concentrations of the P2Y₁₂ receptor antagonist AR-C69931MX (0.1nM to 10μM). Data are expressed as total 3H-2MeSADP binding (DPM) and represent means ± SEMs of 3 independent experiments. From this data 2 concentrations of AR-C69931MX were used: 3nM, which displaces ∼ 50% of specific ligand binding, and a maximal concentration of 1μM. (D) Inhibition of ADP-stimulated P2Y₁₂ receptor activity by antagonism with different concentrations of AR-C69931 MX in human platelets. Agonist (ADP; 1nM to 10μM)–dependent inhibition of PGE2 (10μM; 5 minutes)–stimulated adenylyl cyclase activity was assessed in the absence and presence of AR-C69931 MX (3nM and 1μM). Data are expressed as the percentage of inhibition of PGE2-stimulated adenylyl cyclase and represent means ± SEMs of 3 independent experiments. Pretreatment with 3nM AR-C69931 MX shifts the EC₅₀ for ADP from 95 ± 8nM in the absence and 380 ± 13nM in the presence of antagonist (P < .05, Mann-Whitney U test).