Myeloperoxidase (MPO) Inhibits the Procoagulant Activity (PCA) of Extracellular DNA

Scientific Research Question: MPO and DNA are main constituents of neutrophil extracellular traps (NETs), which promote (thrombotic) vessel occlusions in various pathological scenarios. At sites of NETosis, extracellular DNA activates the contact pathway of coagulation via its negative surface charge. Recently, we have shown that MPO, a highly cationic enzyme under physiological pH, is a negative regulator of phospholipid-dependent coagulation involving both, the electrostatic binding and chemical modification of negatively charged phospholipids (PLs), i.e., phosphatidylserine. The aim of our current study was to investigate whether MPO also exerts direct effects on the PCA of extracellular DNA. Methods: We used a fluorogenic thrombin generation assay to analyze the PCA of polymorphonuclear leukocyte (PMN) DNA in relipidated PL-free plasma in the presence or absence of leukocyte-derived MPO and its substrate, hydrogen peroxide. PMNs were separated from heparin-anticoagulated whole blood by density-gradient centrifugation, and DNA was subsequently purified with a commercially available silica membrane-based isolation kit. Findings: PMN-derived extracellular DNA amplified thrombin generation in relipidated PL-free plasma in a concentration- and FXII-dependent manner, as evidenced by the inhibitory effects of CTI and FXII depletion. Under these conditions, MPO alone or together with its substrate, which allows for generation of highly reactive hypochlorous acid, did not affect thrombin generation. However, MPO potently inhibited DNA-induced thrombin generation in a concentration-dependent manner, an effect that was independent of the enzyme´s catalytic activity and could be fully abolished by heat denaturation, indicating that electrostatic interactions between the cationic MPO and anionic DNA were involved in this process. Conclusions: Our findings indicate that PMN-derived MPO inhibits the PCA of extracellular DNA through electrostatic complex formation and thus provide novel insights into NET biology.