Neutrophil-derived FXII contributes to local FXIIa formation and enhanced neutrophil integrin activation in SCD. (A) Normal human plasma (NHP) or FXII-deficient plasma (F12^−/− plasma) were incubated with aPTT-R or neutrophils supplemented with 15 μM Zn²⁺. The generation of FXIIa was determined by monitoring the cleavage of S2302 (200 μM) over time. NHP incubated with aPTT-R was used as positive control. n = 3 individual experiments, analyzed in triplicate. (B) Visualization of FXII (green) and 4′,6-diamidino-2-phenylindole (blue) in neutrophils isolated from healthy controls (AA) and patients with SCD. Images shown are representative of 3 individual experiments. Images shown at ×20 original magnification, scale: 10 μm. Mouse HbAA and HbSS neutrophils (n = 3-4, analyzed in duplicate) were assessed for FXIIa activity (S2302 cleavage) over 4 hours. Reaction rate of FXIIa activity was calculated in pM/s (C) per 250 000 neutrophils or (D) multiplied by the total number of circulating neutrophils in each individual mouse before isolation (pM/s × polymorphonuclear leukocyte [PMN]). Data represent mean ± SEM; ∗P < .05 vs HbAA by Student t test. Flow cytometry analysis of (E) uPAR, (F) total αMβ2 integrin, and (G) active αMβ2 integrin surface expression on untreated (Veh) and FXII/Zn²⁺–stimulated neutrophils isolated from healthy controls (Con) and patients with SCD. Data represent mean ± SEM; n = 4 to 9; ∗P < .05, ∗∗P < .01, ∗∗∗P < .001 by Kruskal-Wallis one-way analysis of variance. aPTT-R, aPTT reagent.