Abstract 305: Genetic Deficiency in Nox2-containing NADPH Oxidase Decreases Susceptibility to Venous Thrombosis in Mice

Abstract

Activated neutrophils release neutrophil extracellular traps (NETs) via a pathway dependent on reactive oxygen species (ROS). NETs are enriched in extracellular DNA and histones, and contribute to venous thrombosis. Nox2-containing NADPH oxidase is a major upstream mediator of ROS-dependent release of NETs. Neutrophils from mice deficient in Nox2 or patients with X-linked chronic granulomatous disease due to Nox2 deficiency do not produce NETs, suggesting a role for Nox2 in venous thrombosis. We hypothesized that mice genetically deficient in Nox2 are protected from venous thrombosis. We studied male and female mice deficient in Nox2 (Nox2-KO) and their wild type (WT) littermates at 10-14 weeks of age (n=7-13/group). Using electron spin resonance and H2C-DCFH-DA staining, we confirmed that neutrophils from Nox2-KO mice do not produce ROS at baseline or upon PMA-activation. Neutrophils from WT mice displayed a dose-dependent release of NETs upon PMA-stimulation (SYTOX green staining using fluorescence microscopy), whereas Nox2-KO mice did not produce NETs. We next examined susceptibility to venous thrombosis in a stasis model of inferior vena cava (IVC) ligation. After 48 hours of ligation, both male and female Nox2-KO mice developed significantly smaller thrombi compared with WT mice (P<0.05). Markers for NETs were measured in plasma (nucleosome levels using ELISA) and venous thrombi (staining for citrullinated histones, Cit-H3). Levels of nucleosomes in the mice that did not develop thrombi were similar to those in baseline samples collected before IVC ligation. All of the mice that developed thrombi had significant elevation of plasma nucleosome levels compared with mice that did not develop thrombi (P<0.05), but nucleosome levels did not differ between WT and Nox2-KO mice. Likewise, the heterogeneous pattern of NET formation within thrombi detected by Cit-H3 staining was similar in WT and Nox2-KO mice. These findings suggest that Nox2 is an important determinant of thrombus size during IVC ligation. Interestingly, our study also provides evidence for Nox2-independent pathways for NET release during venous thrombosis.