P-selectin is a stimulant of NET formation. (A) Neutrophils were incubated with unstimulated or thrombin (Thr)-stimulated platelets (Plt) in the presence of P-selectin aptamer inhibitor ARC5690 (P-sel ap) or ARC5694 as control aptamer (Control ap). H3Cit⁺ cells and NET formation were evaluated (n = 5; ***P < .001). (B) H3Cit⁺ cells and NET formation were quantified in neutrophils incubated with unstimulated or thrombin-stimulated platelets from WT or P-selectin^−/− mice (P-sel^−/−) (n = 5; **P < .01, ***P < .001). (C) The behavior of platelets in panels A and B was visualized by differential interference contrast microscopy. The images are representative from 5 experiments (scale bar represents 5 μm). (D) NET formation was examined after incubation with unstimulated or thrombin-stimulated WT platelets in the presence of anti-PSGL-1–blocking antibody or its immunoglobulin control (Ig). The percentage of H3Cit⁺ neutrophils was quantified (n = 4; *P < .05). (E) Left: WT neutrophils were stimulated with P-sel-Ig or with the same class of immunoglobulin as a control (n = 4; **P < .01, ***P < .001). Right: Representative fluorescence microscopy images of control immunoglobulin (Control-Ig) or P-sel-Ig treatments showing H3Cit-negative neutrophils (yellow arrow), H3Cit⁺ neutrophils without NET release (red arrow), and H3Cit⁺ neutrophils releasing NETs (white arrowhead). Scale bar represents 20 μm.