i[VREY]₄ inhibits the prothrombotic activity of CXCL12. (A-D) The effects of i[VREY]₄ (5 µM) on platelet aggregation in human blood activated with collagen and recombinant CXCL12 (0.1 µg/mL; n = 15) (A), recombinant CXCL12 alone (0.1 µg/mL) (B), collagen alone (0.2 µg/mL; n = 5) (C), or homogenized human plaque (833 µg/mL, n = 4) (D) were measured by using MEA. (E) Thrombus formation was induced by perfusion of human blood through a plaque-coated flow chamber at 600 s⁻¹. Thrombus volume in the absence and presence of i[VREY]₄ (5 µM) was analyzed by confocal microscopy (n = 6-7). (F) Time to occlusion as in Figure 1A. i[VREY]₄ (100 µg, n = 10) or saline control (n = 9) was injected intraperitoneally 1 hour before induction of thrombosis. (G) Mouse blood from the indicated genotypes was mixed with 1 µg/mL collagen in the presence or absence of 5 µM i[VREY]₄, and platelet aggregation was measured by using MEA (n = 6-8). (H) i[VREY]₄-biot plasma levels were detected at various time points after intraperitoneal (i.p.) injection of 75 µg by enzyme-linked immunosorbent assay. (I) Neutrophil mobilization from the bone marrow of C57BL/6 mice was assessed 1 hour and 2 hours after i.p. injection of PBS with 100 μg i[VREY]₄ or 100 μg AMD3465 by using an automated blood counter (n = 3-7). Data represent mean ± standard deviation from the indicated numbers of independent experiments or mice. *P ≤ .05, **P ≤ .01, ***P ≤ .001, ****P ≤ .0001 as analyzed by using one-way analysis of variance with Tukey’s multiple comparison test (panels A, G, and I), unpaired t test (panels B and E), paired t test (panels C and D), and Mann-Whitney U test (panel F). AU, arbitrary units; ns, not significant.