CXCR7 agonist–induced changes in platelet lipidome affect activatory mediators and functional response. (A) Western blot analysis showing phosphorylation of PLCγ and Src family kinases in response to CRP (5 µg/mL) stimulation for 10 minutes at room temperature in presence/absence of CXCR7 agonist (100 µg/mL) or vehicle control (1% DMSO) given as a pretreatment of 15 minutes at room temperature. (B) Flow cytometric histogram overlays and data showing CRP (5 µg/mL)-induced intraplatelet calcium mobilization detected with Fluo-3 am (5 µM) in presence/absence of CXCR7 agonist (100 µg/mL)/vehicle control in whole blood assay gating for platelet-specific marker CD42b (anti-human CD42b-PE). Data are mean ± SEM from 5 experiments; **P < .0077, ****P < .0001 vs CRP using ANOVA followed by Bonferroni’s multiple comparison test. (C) Phosphorylation of PKC, PI3K, Akt, and p38MAPK triggered by CRP (5 µg/mL) in presence/absence of CXCR7 agonist (100 µg/mL)/vehicle control (1% DMSO). In panels A and C, data are representative of 3 western blots. (D) SICM imaging showed reduced initial spreading rate on collagen (100 µg/mL)-coated surfaces in presence/absence of CXCR7 agonist (50 µg/mL) or vehicle control (0.1% DMSO). (E) Decreased final spreading area of CXCR7 agonist (50 µg/mL)-treated platelets on collagen- and fibrinogen (100 µg/mL)-coated surfaces with respect to vehicle control. Data are mean ± SEM from 3 experiments. Student t test; *P < .05, ***P < .001. (F) SICM stiffness mapping showing reduced elastic modulus of platelets pretreated with CXCR7 agonist (50 µg/mL) while interacting with fibrinogen-coated surfaces. Plots show geometric mean ± SEM from 3 experiments. Student t test, ***P < .001. Number of platelets: 5 to 6 in panel D, 18 to 24 in panel E, and 25 to 31 in panel F. (G) Surface expression of CD63 (δ-granule release, anti-human CD63-FITC) detected by flow cytometry following CRP (5 µg/mL)-induced activation for 30 minutes at room temperature in presence/absence of CXCR7 agonist (100 µg/mL)/vehicle control (1% DMSO) given as a pretreatment of 30 minutes at room temperature. Data are mean ± SEM from 4 experiments; **P < .002, ****P < .0001 using ANOVA followed by Sidak’s multiple comparison text. (H) CRP (2.5 µg/mL) and TRAP (10 µM) induced release of ATP from δ-granules in presence/absence of CXCR7 agonist (100 µg/mL)/vehicle control ascertained by lumi-aggregometry. Extracellular ATP released from activated platelets was assessed employing the luciferase bioluminescent assay, and the amount of ATP release was calculated using the exogenously added ATP standard with aggrolink-8 software (ChronoLog). (I) Corresponding aggregatory response to CRP (2.5 µg/mL) and TRAP (10 µM) for a duration of 10 minutes at 37°C and under a stirring speed of 1000 revolutions per minute (RPM) in presence/absence of CXCR7 agonist (100 µg/mL)/vehicle control. In panels H through I, data are mean ± SEM from 6 experiments with healthy donors; ***P = .0002, ****P < .0001 using ANOVA followed by Sidak’s multiple comparison text. Washed human (200 × 10⁶/sample) and murine (100 × 10⁶/sample) were preincubated with CXCR7 agonist (100 µg/mL) or vehicle control (1% DMSO) for 30 minutes and thereafter stimulated with platelet agonists as specified below for 30 and 15 minutes, respectively, at room temperature. Samples were then centrifuged to collect activated platelet supernatant. Violin plots (line denotes median) showing release of inflammatory mediators from (J) CRP (5 µg/mL)-activated human (Legendplex human 13-plex inflammation panel) and (K) thrombin (0.1 U/mL)-activated murine platelets (Legendplex murine 13-plex inflammation panel) evaluated by cytometric bead arrays. In panels J and K, *P < .05, **P < .01, ****P < .0001 using Mann-Whitney U test for each analyte. Data are plotted as mean ± SEM from 5 healthy donors in panel J and from 10 mice in panel K. DMSO, dimethyl sulfoxide.