ACKR3/CXCR7 ligation modulates the platelet lipidome: observation from healthy subjects and CAD patients. (A) Washed 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. Heat map showing significant changes in lipid concentration upon treatment with CXCR7 agonist (100 µg/mL) for a period of (0-60) minutes; linear regression model; the significance level for the corrected (false discovery rate correction [FDR]) P values was 0.05. The data were normalized by z-scores for each donor separately and derived from 5 independent experiments performed with 5 healthy donors. Washed platelets (300 × 10⁶/sample) from n = 11 healthy donors were pretreated with CXCR7 agonist (100 µg/mL) or vehicle control (1% DMSO) for 30 minutes at room temperature. Platelets were then either kept under resting condition or activated with thrombin (0.1 U/mL) for 15 minutes at room temperature. The supernatants were separated from the platelet pellets by centrifugation, and both were used for lipid extraction and subsequent lipidomics analysis. PCA-DA score plots showing distinct grouping or clustering of resting platelets, thrombin-activated platelets, CXCR7 agonist plus thrombin-treated platelets, and CXCR7 agonist–treated platelets as analyzed in (B) platelet pellet and (C) platelet releasate/platelet supernatant. Relative abundance reflecting generation of (D) AA, TxA(B)₂, 12-HHT, and 12-HETE in thrombin (0.1 U/mL)-activated platelet pellet from n = 11 healthy subjects plus CXCR7 agonist (100 µg/mL) or vehicle control and (E) release of AA, TxA(B)₂, 12-HHT, and 12-HETE in thrombin-activated platelet supernatant of healthy subjects plus CXCR7 agonist (100 µg/mL) or vehicle control. Metabolism of (Fi) LPCs, (Fii) diacylglycerol (DGs), and (Fiii) LPIs in thrombin-activated platelets from healthy subjects is significantly reduced by CXCR7 agonist (100 µg/mL) pretreatment with respect to vehicle control. The significance levels for the corrected (FDR) P values are given using nonparametric paired Wilcoxon signed rank test with statistical significance P < .05. Response in the Y-axis of panels D and F refers here to relative signal intensities and were calculated from the raw data (peak heights) after LOWESS normalization. (G) Washed platelets (300 × 10⁶/sample) from CAD patients (Table 2; n = 15 for untargeted analysis of AA and n = 7 for targeted analysis of oxylipins) were pretreated with CXCR7 agonist (100 µg/mL) or vehicle control (1% DMSO) for 30 minutes at room temperature. Platelets were then activated with thrombin (0.1 U/mL) for 15 minutes at room temperature. Generation of platelet-activating prothrombotic lipid mediators AA, TxA(B)₂, 12-HHT, and 12-HETE in thrombin (0.1 U/mL)-activated platelets was significantly reduced upon ex vivo pretreatment with CXCR7 agonist (100 µg/mL) with respect to vehicle control. The significance levels for the corrected (FDR) P values are given using nonparametric paired Wilcoxon signed rank test with statistical significance P < .05. DMSO, dimethyl sulfoxide; LOWESS, locally weighted scatterplot smoothing; PCA-DA, principal components analysis-discriminant analysis.