Figure 1.
ACKR3/CXCR7 exerts antithrombotic effects. (A) Aggregation response to ADP (ADPTest, Roche) and TRAP-6 (TRAPTest, Roche) evaluated by impedance aggregometry in whole blood samples acquired in hirudinized tubes (Sarstedt) from CAD (n = 230; ACS, n = 142; CCS, n = 88) patients. P values using Mann-Whitney U test (line in box plots denote median). Flow cytometric detection of ACKR3/CXCR7 (mouse anti-human/mouse ACKR3/CXCR7-PE) internalization from the platelet surface following CXCR7 agonist treatment in a (Bi) time and (Bii) dose-dependent manner, gating for platelet-specific marker CD42b (anti-human CD42b-FITC) in whole blood. Data in the graphs are mean ± SEM from 4 healthy donors. T-TAS (Fujimori Kogyo Co Ltd, Shinjuku, Japan) data showing (Ci) thrombus coverage (AUC) and time to attainment of 10 kPa (T10) pressure over collagen (PL-chip) using 320 µL of hirudin anticoagulated blood and (Cii) thrombus coverage (AUC) and time to occlusion over collagen + tissue factor (AR-chip) using 450 µL of recalcified citrated human blood at arterial shear rates in presence/absence of CXCR7 agonist (100 µg/mL) or vehicle control incubated for 30 minutes at room temperature before perfusion. Data in the graphs are mean ± SEM from 5 experiments with healthy donors. *P < .05, **P < .01 using Mann-Whitney test. (Di) Surface expression of ACKR3/CXCR7 on platelets in n = 11 ACS patients as compared with healthy subjects (n = 11). Data in the graphs are mean ± SEM. **** P < .0001 using Mann-Whitney U test. Ex vivo whole blood functional assays performed with blood collected from ACS patients (n = 11) showing (Dii) surface expression of CD62P (anti-human CD62P-FITC) and PAC-1 (PAC-1–FITC) binding detected by flow cytometry gating for CD42b+-platelet (anti-human CD42b-PE) population; (Diii) aggregation response to ADP (ADPTest), TRAP-6(TRAPTest), and collagen(ColTest) in hirudinized blood; and (Div) thrombus formation over collagen coated (100 µg/mL) surface in parallel plate flow chamber assay in presence/absence of CXCR7 agonist (100 µg/mL)/vehicle control preincubated for 30 minutes at room temperature. *P < .05, **** P < .0001 using Mann-Whitney U test between 2 groups and ANOVA followed by Sidak’s multiple comparison test for >2 groups. Ex vivo analysis of murine platelet functions performed 1 hour after administration of CXCR7 agonist (100 µg/mL)/vehicle control (1% DMSO) showing (Ei) JON/A (JON/A-PE) response, (Eii) CD62P (anti-mouse CD62P-FITC) surface expression by whole blood flow cytometry gating for platelet-specific marker CD42b (anti-mouse CD42b-Dylight-649), and (Eiii-Eiv) thrombus coverage over collagen-coated surface (100 µg/mL) in ex vivo parallel plate flow chamber assay. Data in the graphs are mean ± SEM from 5 mice per group. CXCR7 agonist (100 µg per mouse) or vehicle control (1% DMSO) along with in vivo platelet-labeling antibody (GPIbβ-Dylight 488, 0.1 µg/gm by body weight) was administered IV 1 hour prior to surgical procedures to inflict carotid artery injury. Intravital microscopy (IVM) with NIS-Elements (Nikon) microscope was carried out using a 10x objective following carotid artery injury inflicted by application of a filter paper soaked in 15% FeCl3 for 1 minute. (Fi) Reduced thrombus formation (green fluorescence from platelets in circulation stained in vivo with GPIbβ-Dylight 488) and (Fii) time to vessel occlusion (*P = .034). Following IVM analysis, blood was collected to perform further ex vivo analysis of platelet functions. Flow cytometric detection of (Fiii) JON/A response, (Fiv) platelet (anti-mouse GPIbβ-Dylight 488) aggregate formation with leukocytes (anti-mouse CD45-APC), lymphocytes (anti-mouse CD3-APC), monocytes, and neutrophils (anti-mouse Ly6C-APC, anti-mouse Ly6G-PE, anti-mouse CD11b-APC) following carotid artery injury in CXCR7 agonist/vehicle control–administered mice. In panels Ei through Fiii, *P < .05, **P < .01, ***P < .001 using an unpaired Student t test with Welch’s correction; in panel Fiv, *P < .05, ****P < .0001 using Mann-Whitney U test for each surface marker. Data in the graphs are mean ± SEM from 14 mice per group. AUC, area under the curve; APC, apocyanin; DMSO, dimethyl sulfoxide; FITC, fluorescein isothiocyanate.

ACKR3/CXCR7 exerts antithrombotic effects. (A) Aggregation response to ADP (ADPTest, Roche) and TRAP-6 (TRAPTest, Roche) evaluated by impedance aggregometry in whole blood samples acquired in hirudinized tubes (Sarstedt) from CAD (n = 230; ACS, n = 142; CCS, n = 88) patients. P values using Mann-Whitney U test (line in box plots denote median). Flow cytometric detection of ACKR3/CXCR7 (mouse anti-human/mouse ACKR3/CXCR7-PE) internalization from the platelet surface following CXCR7 agonist treatment in a (Bi) time and (Bii) dose-dependent manner, gating for platelet-specific marker CD42b (anti-human CD42b-FITC) in whole blood. Data in the graphs are mean ± SEM from 4 healthy donors. T-TAS (Fujimori Kogyo Co Ltd, Shinjuku, Japan) data showing (Ci) thrombus coverage (AUC) and time to attainment of 10 kPa (T10) pressure over collagen (PL-chip) using 320 µL of hirudin anticoagulated blood and (Cii) thrombus coverage (AUC) and time to occlusion over collagen + tissue factor (AR-chip) using 450 µL of recalcified citrated human blood at arterial shear rates in presence/absence of CXCR7 agonist (100 µg/mL) or vehicle control incubated for 30 minutes at room temperature before perfusion. Data in the graphs are mean ± SEM from 5 experiments with healthy donors. *P < .05, **P < .01 using Mann-Whitney test. (Di) Surface expression of ACKR3/CXCR7 on platelets in n = 11 ACS patients as compared with healthy subjects (n = 11). Data in the graphs are mean ± SEM. **** P < .0001 using Mann-Whitney U test. Ex vivo whole blood functional assays performed with blood collected from ACS patients (n = 11) showing (Dii) surface expression of CD62P (anti-human CD62P-FITC) and PAC-1 (PAC-1–FITC) binding detected by flow cytometry gating for CD42b+-platelet (anti-human CD42b-PE) population; (Diii) aggregation response to ADP (ADPTest), TRAP-6(TRAPTest), and collagen(ColTest) in hirudinized blood; and (Div) thrombus formation over collagen coated (100 µg/mL) surface in parallel plate flow chamber assay in presence/absence of CXCR7 agonist (100 µg/mL)/vehicle control preincubated for 30 minutes at room temperature. *P < .05, **** P < .0001 using Mann-Whitney U test between 2 groups and ANOVA followed by Sidak’s multiple comparison test for >2 groups. Ex vivo analysis of murine platelet functions performed 1 hour after administration of CXCR7 agonist (100 µg/mL)/vehicle control (1% DMSO) showing (Ei) JON/A (JON/A-PE) response, (Eii) CD62P (anti-mouse CD62P-FITC) surface expression by whole blood flow cytometry gating for platelet-specific marker CD42b (anti-mouse CD42b-Dylight-649), and (Eiii-Eiv) thrombus coverage over collagen-coated surface (100 µg/mL) in ex vivo parallel plate flow chamber assay. Data in the graphs are mean ± SEM from 5 mice per group. CXCR7 agonist (100 µg per mouse) or vehicle control (1% DMSO) along with in vivo platelet-labeling antibody (GPIbβ-Dylight 488, 0.1 µg/gm by body weight) was administered IV 1 hour prior to surgical procedures to inflict carotid artery injury. Intravital microscopy (IVM) with NIS-Elements (Nikon) microscope was carried out using a 10x objective following carotid artery injury inflicted by application of a filter paper soaked in 15% FeCl3 for 1 minute. (Fi) Reduced thrombus formation (green fluorescence from platelets in circulation stained in vivo with GPIbβ-Dylight 488) and (Fii) time to vessel occlusion (*P = .034). Following IVM analysis, blood was collected to perform further ex vivo analysis of platelet functions. Flow cytometric detection of (Fiii) JON/A response, (Fiv) platelet (anti-mouse GPIbβ-Dylight 488) aggregate formation with leukocytes (anti-mouse CD45-APC), lymphocytes (anti-mouse CD3-APC), monocytes, and neutrophils (anti-mouse Ly6C-APC, anti-mouse Ly6G-PE, anti-mouse CD11b-APC) following carotid artery injury in CXCR7 agonist/vehicle control–administered mice. In panels Ei through Fiii, *P < .05, **P < .01, ***P < .001 using an unpaired Student t test with Welch’s correction; in panel Fiv, *P < .05, ****P < .0001 using Mann-Whitney U test for each surface marker. Data in the graphs are mean ± SEM from 14 mice per group. AUC, area under the curve; APC, apocyanin; DMSO, dimethyl sulfoxide; FITC, fluorescein isothiocyanate.

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