The CXCL12–CXCR4 axis functions as a positive feedback loop in human platelet activation. Platelet aggregation was assessed by MEA in human blood activated by collagen (0.2 μg/mL) (A) or human plaque homogenate (B). CXCR4 was inhibited by 100 nM AMD3465 (n = 5-8). (C) Thrombus formation was induced by perfusion (600 s−1) of human blood, preincubated with PBS or 1 μM AMD3465, in a plaque-coated flow chamber and thrombus volume determined by confocal microscopy (n = 7). (D) CXCL12 was visualized in resting human platelets that were permeabilized and double-stained with antibodies against CXCL12 (purple) and CXCL4 antibody (green) by stimulated emission depletion (STED) microscopy (Leica SP8; scale bar, 2 μm). (E) CXCL12 release from isolated human platelets after activation with collagen (5 μg/mL) was determined by enzyme-linked immunosorbent assay (n = 3). (F-G) Platelet aggregation was assessed by MEA of human blood incubated with different concentrations of recombinant CXCL12 (n = 5-10) (F) or combinations of collagen (0.1 μg/mL), recombinant CXCL12 (0.1 μg/mL), and AMD3465 (100 nM) as indicated (n = 6-10) (G). Data represent mean ± standard deviation from the indicated numbers of independent experiments. *P ≤ .05, ***P ≤ .001, ****P ≤ .0001, as analyzed by paired t test (panels A and B), unpaired t test (panels C and E), and one-way analysis of variance with Tukey’s multiple comparison test (panels F and G). AU, arbitrary units.