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.
