CXCR7 agonist–induced generation of antiplatelet lipid 12-HETrE and platelet inhibitory mediator cAMP. Washed platelets (200 × 106/sample) from n = 5 healthy donors were treated with CXCR7 agonist (100 µg/mL) for 0, 5, 15, and 30 minutes at room temperature. Thereafter platelet pellets were collected by centrifugation and used for detection of cAMP levels by LC-ESI-MS/MS analysis. (A) Gradual elevation in intraplatelet cAMP in response to CXCR7 agonist (100 µg/mL); *P < .05, **P < .01 vs 0 minute by Wilcoxon matched-pairs signed rank test, n = 5 donors. Washed human platelets (200 × 106/sample) from n = 5 healthy donors were treated with CXCR7 agonist (100 µg/mL) for 0, 5, 15, 30, and 60 minutes at room temperature. Intraplatelet levels of (Bi) linoleic acid (right y-axis), AA, and DGLA (left y-axis); (Bii) DGLA, 12-HETrE, and TxA(B)2; (Biii) linoleic acid (left y-axis) and 13-HODE (right y-axis); (Biv) EPA (left y-axis) and 12-HEPE (right y-axis); and (Bv) 12-HETrE, 12-HEPE, and 13-HODE as compared with TxA(B)2. #P < .05 and ##P < .01 vs AA; *P < .05, **P < .01 vs 0 minute using Welch’s t test. (C) Washed platelets (300 × 106/sample) from n = 9 healthy donors were treated with CXCR7 agonist (100 µg/mL) or vehicle control (1% DMSO) for 30 and 60 minutes at room temperature. IP receptor antagonist (RO-1138452 10 µM) was given as a pretreatment before CXCR7 agonist for 15 minutes at room temperature. At the end of incubation period, the supernatants were separated from the platelet pellets by centrifugation, and both were used for lipid extraction and subsequent targeted lipidomics analysis for 12-HETrE. Data are mean ± SEM. The significance levels for the corrected (FDR) P values are given using nonparametric paired Wilcoxon signed rank test with statistical significance *P < .05, **P < .01. Experiments done with blood from healthy donors (n = 5) showing inhibitory effect of CXCR7 agonist (100 µg/mL) on CRP (5 µg/mL) and TRAP (25 µM)-induced platelet (Di) PAC-1 binding, (Dii) CD62P surface expression (flow cytometry), (E) collagen and TRAP-induced aggregation (whole blood impedance aggregometry), and (F) thrombus formation was counteracted by IP receptor antagonist (RO-1138452 10 µM) given as a pretreatment before CXCR7 agonist for 15 minutes at room temperature. Data are mean ± SEM from 5 experiments with healthy donors. *P < .05, **P < .01, ***P < .001, ****P < .0001 with ANOVA followed by Sidak’s multiple comparison test. (Gi) Washed platelets from CAD patients (Table 2; n = 12) were treated with vehicle control (1% DMSO) or CXCR7 agonist (100 μg/mL) for 30 minutes at room temperature and processed for targeted lipidomics analysis for 12-HETrE. CXCR7 agonist treatment to CAD patient platelets ex vivo significantly increased 12-HETrE levels. Data are mean ± SEM. The significance levels for the FDR corrected P values are given using nonparametric paired Wilcoxon signed rank test with statistical significance *P < .05. (Gii-Giii) Inhibitory effect of CXCR7 agonist (100 µg/mL) observed in CAD patients on (Gii) platelet CD62P surface expression, PAC-1 binding (n = 5), and (Giii) TRAP induced aggregation (n = 6) was counteracted in presence of IP receptor antagonist (RO-1138452 10 µM) given as a pretreatment before CXCR7 agonist for 15 minutes at room temperature. In panels Gii through Giii, data are mean ± SEM; ####P < .0001 vs resting; §§§§P < .0001 vs CRP, *P < .02, ***P < .001; &&P < .002, &P < .02 with ANOVA followed by Sidak’s multiple comparison test. DMSO, dimethyl sulfoxide; Supernt., supernatant.