HTRA1 generates A1ATM₃₈₃S-CF in vivo, which decreases early NET formation in neonatal mouse pups. (A) We tested plasma isolated from mouse pups, both HTRA1^+/+ littermate controls and HTRA1^−/−, for low-molecular-weight HTRA1 C-terminus cleavage fragments via western blotting between days 4 to 6 and 7 to 10 following birth. The western blots shown are representative of 3 separate experiments using 3 different mouse pups per group. (B) Plasma “switch” experiments were performed by using plasma isolated 1 to 3, 4 to 6, or 7 to 10 days following birth from both HTRA1^+/+ (filled circles) and HTRA1^−/− (open circles) mouse pups. Mature C57BL/6 mouse plasma served as a negative control (open and filled squares). We preincubated PMNs isolated from mature mouse bone marrow with mouse pup plasma or mature mouse control plasma for 1 hour before stimulation with LPS (100 ng/mL; 1 hour). NET formation was quantified by using a standardized grid system. The y-axis depicts NET formation with NETs crossing standardized grid lines/high-power field (hpf) ± SEM. We next isolated PMNs from mouse pups, both HTRA1^+/+ (filled circles) and HTRA1^−/− (open circles), at the indicated times and stimulated them with LPS (100 ng/mL; 1 hour). NET formation was assessed qualitatively via live cell imaging (C) and semiquantitatively by using a standardized grid system (D). Yellow arrows indicate NET formation. Images are representative of 3 to 12 separate experiments using between 3 and 12 different mouse pups per group. Scale bars, 50 μm. *P < .05, **P < .01. MW, molecular weight.