Figure 2.
CXCR7 agonist modulates thrombotic and thromboinflammatory platelet response without compromising plasma dependent coagulation. CXCR7 agonist (300 µg/mouse) or vehicle control (2% DMSO) was administered IV 1 hour prior to the surgical procedure. Surface expression of (Ai) ACKR3/CXCR7 (anti-human/mouse CXCR7-PE) and CXCR4 (anti-mouse CXCR4-Fluorescein) on circulating murine platelets. (Aii) Platelet JON/A response (JON/A-PE) and CD62P (anti-mouse CD62P-FITC) surface expression and (Aiii) surface expression of CXCL12/SDF-1α (anti human/mouse CXCL12/SDF-1α- Fluorescein) (denoting degranulation) in basal and CRP-activated (incubated ex vivo for 30 minutes at room temperature) state were evaluated by flow cytometry using blood collected pre-MI (before beginning the surgical procedure) and 24 hours post-MI/RI. (Aiv) Thrombus coverage ex vivo. (B) Levels of proinflammatory mediators in plasma (violin plot with median line) measured with cytometric bead arrays (Legendplex murine 13-plex inflammation panel) 24 hours post-MI/RI. (C) Percentage of CD42b+ (anti-mouse CD42b-FITC)-CD45+ (anti-mouse CD45-APC) platelet-leukocyte aggregates in blood collected pre-MI and 24 hours post-MI/RI. In panels A through C, *P < .05, **P < .01, ***P < .001, ****P < .0001 using Mann-Whitney U test. (Di-Dii) Platelet count and mean platelet volume (MPV) 24 hours post-MI/RI. CXCR7 agonist (300 µg/mouse) or vehicle control (2% DMSO) was administered IV, and experiments depicted in (E-G) performed 1 hour post-administration. (E) Surface availability of receptors glycoprotein Ibα, glycoprotein V (GPV), glycoprotein VI (GPVI), glycoprotein IX (GPIX), αv-integrin, and β3-integrin on murine platelets detected by whole blood flow cytometry (using anti-mouse CD42b/GPIbα-Dylight 649, anti-mouse GPV-FITC, anti-mouse GPVI-FITC, anti-mouse GPIX-FITC, anti-mouse β3-FITC, and anti-mouse αv-FITC). Data in the graphs are presented as mean ± SEM derived from 6 mice per group. (F) Tail bleeding time and (G) plasma coagulation profile (PT, APTT) was evaluated using the START4 platform. Data are mean ± SEM from 5 mice per group. (H) Phosphatidylserine exposure (annexin V-FITC MFI) on human platelets and percentage of annexin V+ platelets under basal resting condition and following CRP (5 µg/mL) stimulation for 1 hour 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 of 4 experiments with healthy donors. I. Thrombin generation under basal condition and in the presence of platelet-activating CRP in platelet-rich plasma (PRP) and platelet-poor plasma (PPP) evaluated by calibrated automated thrombinoscopy (Stago) 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 of independent experiments performed with n = 4 healthy donors. (H-I) **P < .01, ***P < .001, ****P < .0001 using ANOVA followed by Sidak’s post hoc test. (J) Thromboelastographic analysis (Whole Blood Hemostasis System) using the TEG6s PlateletMapping cartridges (Haemonetics, Germany) evaluating (Ji) the time to initiation of the appearance of first clot (R) and clot strength deciphered as maximum amplitude (MA). (Jii) Percentage of ADP aggregation; percentage of (extent of) inhibition imposed on ADP-induced aggregation, ascertained separately in the HKH (kaolin with heparinase); ActF (ActivatorF); and ADP assays using blood from healthy subjects in presence/absence of CXCR7 agonist (100 µg/mL)/vehicle control (1% DMSO). Data are shown as mean ± SEM of 5 independent experiments. ##P= .002, **P = .003 using a paired Student t test. APC, apocyanin; DMSO, dimethyl sulfoxide; FITC, fluorescein isothiocyanate; MFI, mean fluorescence intensity; PE, phosphatidylethanolamine.

CXCR7 agonist modulates thrombotic and thromboinflammatory platelet response without compromising plasma dependent coagulation. CXCR7 agonist (300 µg/mouse) or vehicle control (2% DMSO) was administered IV 1 hour prior to the surgical procedure. Surface expression of (Ai) ACKR3/CXCR7 (anti-human/mouse CXCR7-PE) and CXCR4 (anti-mouse CXCR4-Fluorescein) on circulating murine platelets. (Aii) Platelet JON/A response (JON/A-PE) and CD62P (anti-mouse CD62P-FITC) surface expression and (Aiii) surface expression of CXCL12/SDF-1α (anti human/mouse CXCL12/SDF-1α- Fluorescein) (denoting degranulation) in basal and CRP-activated (incubated ex vivo for 30 minutes at room temperature) state were evaluated by flow cytometry using blood collected pre-MI (before beginning the surgical procedure) and 24 hours post-MI/RI. (Aiv) Thrombus coverage ex vivo. (B) Levels of proinflammatory mediators in plasma (violin plot with median line) measured with cytometric bead arrays (Legendplex murine 13-plex inflammation panel) 24 hours post-MI/RI. (C) Percentage of CD42b+ (anti-mouse CD42b-FITC)-CD45+ (anti-mouse CD45-APC) platelet-leukocyte aggregates in blood collected pre-MI and 24 hours post-MI/RI. In panels A through C, *P < .05, **P < .01, ***P < .001, ****P < .0001 using Mann-Whitney U test. (Di-Dii) Platelet count and mean platelet volume (MPV) 24 hours post-MI/RI. CXCR7 agonist (300 µg/mouse) or vehicle control (2% DMSO) was administered IV, and experiments depicted in (E-G) performed 1 hour post-administration. (E) Surface availability of receptors glycoprotein Ibα, glycoprotein V (GPV), glycoprotein VI (GPVI), glycoprotein IX (GPIX), αv-integrin, and β3-integrin on murine platelets detected by whole blood flow cytometry (using anti-mouse CD42b/GPIbα-Dylight 649, anti-mouse GPV-FITC, anti-mouse GPVI-FITC, anti-mouse GPIX-FITC, anti-mouse β3-FITC, and anti-mouse αv-FITC). Data in the graphs are presented as mean ± SEM derived from 6 mice per group. (F) Tail bleeding time and (G) plasma coagulation profile (PT, APTT) was evaluated using the START4 platform. Data are mean ± SEM from 5 mice per group. (H) Phosphatidylserine exposure (annexin V-FITC MFI) on human platelets and percentage of annexin V+ platelets under basal resting condition and following CRP (5 µg/mL) stimulation for 1 hour 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 of 4 experiments with healthy donors. I. Thrombin generation under basal condition and in the presence of platelet-activating CRP in platelet-rich plasma (PRP) and platelet-poor plasma (PPP) evaluated by calibrated automated thrombinoscopy (Stago) 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 of independent experiments performed with n = 4 healthy donors. (H-I) **P < .01, ***P < .001, ****P < .0001 using ANOVA followed by Sidak’s post hoc test. (J) Thromboelastographic analysis (Whole Blood Hemostasis System) using the TEG6s PlateletMapping cartridges (Haemonetics, Germany) evaluating (Ji) the time to initiation of the appearance of first clot (R) and clot strength deciphered as maximum amplitude (MA). (Jii) Percentage of ADP aggregation; percentage of (extent of) inhibition imposed on ADP-induced aggregation, ascertained separately in the HKH (kaolin with heparinase); ActF (ActivatorF); and ADP assays using blood from healthy subjects in presence/absence of CXCR7 agonist (100 µg/mL)/vehicle control (1% DMSO). Data are shown as mean ± SEM of 5 independent experiments. ##P= .002, **P = .003 using a paired Student t test. APC, apocyanin; DMSO, dimethyl sulfoxide; FITC, fluorescein isothiocyanate; MFI, mean fluorescence intensity; PE, phosphatidylethanolamine.

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