PAD4^−/− mice release less extracellular DNA and are better protected than PAD4^+/+ mice in an LPS-induced endotoxemic shock model. (A) Mice were injected IV with a lethal dose of LPS (25 mg/kg) and monitored every 2 hours beginning at hour 12 for moribundity. (B-C) Mice injected IV with a less lethal LPS dose (10 mg/kg) were measured for signs of hypothermia and thrombocytopenia. PAD4^−/− mice had a milder temperature drop (B) and had higher platelet counts than PAD4^+/+ mice (C). (D) Using a sublethal dose (2.5 mg/kg), which allowed us to follow mice surviving for 24 hours, similar differences in hypothermia and thrombocytopenia were observed. Total leukocyte and neutrophil levels were also higher in PAD4^−/− mice. (E-F) Plasma was collected at the time of sacrifice and analyzed for NET biomarkers. (E) Twelve hours after LPS infusion, DNA levels were lower in PAD4^−/− mice. (F) Both histone H3 and H3Cit were identified in the plasma of PAD4^+/+ mice at 24 hours and were no longer detected at 48 hours postinjection. H3Cit was not found in PAD4^−/− mouse plasma, whereas histone H3 was detected. Representative of n = 7 (24 hours) and n = 6 (48 hours). (G-H) IL-6 levels (G) were similar in PAD4^+/+ and PAD4^−/− mice at 12 hours, whereas IL-10 levels (H) were higher in PAD4^−/− mice. PAD4^+/+, n = 13. PAD4^−/− n = 12. (I-J) sPsel levels (I) and TAT complexes (J) in plasma were significantly elevated in PAD4^+/+ mice compared with PAD4^−/− mice, indicating that the presence of NETs activates platelets (increases P-selectin shedding) and induces the generation of thrombin. Results are expressed as fold increase over control, untreated mice. PAD4^+/+, black, n = 13; PAD4^−/−, red, n = 11. *P < .05, **P < .01, ***P < .001.