SCD-associated enhanced H3Cit⁺ neutrophils can be inhibited by AnxA1_Ac2-26. (A) Neutrophil isolation and NET analysis. (B) Images of NETs: H3Cit (green/Alexa Fluor 488), NE (red/Alexa Fluor 568), and nucleus (4′,6-diamidino-2-phenylindole). Bars in main images represent 100 µm; in insets, 10 µm. (C) Percentage of NETs hypercitrullinated at histone H3 (H3Cit⁺) quantified from unstimulated and ionomycin-stimulated neutrophils from control volunteers (unstimulated [n = 10], 1 outlier removed; and stimulated [n = 10], 1 outlier removed) and from patients with SCD (unstimulated [n = 10]; and stimulated [n = 14]). (D) Plasma levels of circulating annexin A1 (n = 5, 6 respectively) were determined in control volunteers and patients with SCD. Statistical significance was determined by unpaired Student t test and presented as *P < .05 vs control volunteers. (E) Percentage of H3Cit⁺ ionomycin-stimulated neutrophils from control volunteers (n = 8 vehicle and n = 9 AnxA1_Ac2-26 pretreatment) and patients with SCD (n = 14 vehicle, n = 9 AnxA1_Ac2-26, n = 10 AnxA1_Ac2-26+Boc-2, and n = 9 AnxA1_Ac2-26+WRW4). Data are means ± SEM from independent experiments. *P < .05; **P < .01 vs control unstimulated neutrophils. ^####P < .0001 vs SCD unstimulated neutrophils. ^{$ $ $ $}P < .001 vs ionomycin-stimulated SCD neutrophils. ^ΔP < .05; ^ΔΔP < .01 vs SCD+AnxA1_Ac2-26–treated SCD neutrophils. ^φφφP < .001 vs stimulated control neutrophils.