Mechanistic Studies of DNase I Activity: Impact of Heparin Variants and PAD4

Background: Excessive production of neutrophil extracellular traps (NETs) in sepsis contributes to vascular occlusion by acting as a scaffold and stimulus for thrombus formation. Removal of extracellular DNA, the major structural component of NETs, by DNase I may reduce host injury. Objectives: (1) To determine how heparin variants (unfractionated heparin, enoxaparin, Vasoflux, and fondaparinux) affect DNase I activity, (2) to measure temporal changes in circulating DNA and DNase I in septic patients. Methods: DNA-histone complexes were treated with DNase I +/- heparin variants and visualized via agarose gels. We compared the ability of DNase I +/- heparin variants to digest NETs released by phorbol 12-myristate 13-acetate-stimulated neutrophils versus DNA-histone complexes released by necrotic HEK293 cells. Plasma DNA and DNase I levels were measured longitudinally in 76 septic patients. Results: Heparin enhances DNase I-mediated digestion of DNA-histone complexes in a size-dependent manner that does not require the antithrombin-binding region. In contrast, DNase I alone was able to degrade the DNA-histone component of NETs presumably due to peptidylarginine deiminase 4 (PAD4)-mediated histone citrullination that weakens DNA-histone interactions. In purified systems, PAD4 treatment of DNA-histone complexes enhanced the ability of DNase I to degrade histone-bound DNA. In septic patients, endogenous DNase I levels remained persistently low over 28 days, and there were no significant correlations between DNA and DNase I levels. Conclusion: Heparin enhances DNA-mediated digestion of DNA-histone complexes in a size-dependent manner that is independent of its anticoagulant properties. Citrullination of histones by PAD4 renders DNA-histone complexes susceptible to DNase I digestion. Endogenous DNase I levels are persistently decreased in septic patients, which supports the potential utility of DNase I as a therapy for sepsis.