The role of P-selectin–PSGL-1 interactions, as well as P2X7 and P2Y2 receptors, in APS-relevant neutrophil-platelet aggregate formation. (A) Cocultures of washed neutrophils and platelets (1:20) from healthy volunteers were treated with various concentrations of control IgG or APS IgG for 1 hour and neutrophil-platelet aggregates (CD15+CD16+CD41+) were quantified by flow cytometry. Thrombin (0.05 U/mL) served as a positive control. (B) Representative ImageStream flow cytometry images using conditions similar to panel A. (C) Representative flow cytometry dot plots of neutrophil-platelet aggregate formation in response to control (50 μg/mL) or APS IgG (50 μg/mL); some samples were additionally pretreated for 15 minutes with anti-CD62P (anti–P-selectin, 2 μg/mL), anti-CD162 (anti–PSGL-1, 2 μg/mL), or GSK-484 (NETosis inhibitor, 10 μM). (D) Neutrophil-platelet cocultures were pretreated with inhibitors of P2X1 (NF 279, 2 μM), P2X7 (AZD9056, 10 μM), P2Y1 (MRS2179, 50 μM), P2Y2 (AR-C 118925XX, 10 μM), or P2Y12 (AR-C 69931, 10 μM) followed by 1 hour of treatment with APS IgG (50 μg/mL). Aggregates were quantified by flow cytometry as above. (E) Neutrophil-platelet cocultures were treated with β2GPI protein and affinity-purified anti-β2GPI IgG from patients with APS, along with blockers of P-selectin, PSGL-1, P2X7, or P2Y2. Aggregates were quantified as above. (F) Neutrophil and platelet cocultures were treated with inhibitors of ectonucleotidases CD39 (ARL 67156, 100 μM) or CD73 (APCP, 100 μM; AB-680, 10 μM; PSB 12379, 100 μM) for 1 hour. (G) Neutrophil and platelet cocultures were pretreated with blockers of P-selectin, PSGL-1, P2X7, or P2Y2 before treating with the CD39 inhibitor for 1 hour. Aggregates were quantified as above. ∗P < .05, ∗∗P < .01, ∗∗∗P < .001, ∗∗∗∗P < .0001, and ns: nonsignificant by one-way ANOVA followed by the Tukey multiple comparisons test.