Activated protein C inhibits neutrophil extracellular trap formation in vitro and activation in vivo

• Published in: The Journal of biological chemistry 2017-05, Vol.292 (21), p.8616-8629
• Main Authors: Healy, Laura D., Puy, Cristina, Fernández, José A., Mitrugno, Annachiara, Keshari, Ravi S., Taku, Nyiawung A., Chu, Tiffany T., Xu, Xiao, Gruber, András, Lupu, Florea, Griffin, John H., McCarty, Owen J.T.
• Format: Article
• Language: English
• Subjects: activated protein C; Animals; Antigens, CD - immunology; Antigens, CD - metabolism; Cell Biology; cell signaling; coagulation factor; Disease Models, Animal; Endothelial Protein C Receptor; Escherichia coli; Escherichia coli Infections - blood; Escherichia coli Infections - immunology; Extracellular Traps - immunology; Extracellular Traps - metabolism; Female; hemostasis; Humans; Mac-1; Macrophage-1 Antigen - immunology; Macrophage-1 Antigen - metabolism; Male; neutrophil; Neutrophil Activation - drug effects; Neutrophil Activation - immunology; neutrophil extracellular traps; Neutrophils - immunology; Neutrophils - metabolism; Papio anubis; PAR3; protein C; Protein C - immunology; Protein C - metabolism; Receptors, Cell Surface - immunology; Receptors, Cell Surface - metabolism; Sepsis - blood; Sepsis - immunology; Tetradecanoylphorbol Acetate - pharmacology; thrombosis
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M116.768309

Activated protein C (APC) is a multifunctional serine protease with anticoagulant, cytoprotective, and anti-inflammatory activities. In addition to the cytoprotective effects of APC on endothelial cells, podocytes, and neurons, APC cleaves and detoxifies extracellular histones, a major component of neutrophil extracellular traps (NETs). NETs promote pathogen clearance but also can lead to thrombosis; the pathways that negatively regulate NETosis are largely unknown. Thus, we studied whether APC is capable of directly inhibiting NETosis via receptor-mediated cell signaling mechanisms. Here, by quantifying extracellular DNA or myeloperoxidase, we demonstrate that APC binds human leukocytes and prevents activated platelet supernatant or phorbol 12-myristate 13-acetate (PMA) from inducing NETosis. Of note, APC proteolytic activity was required for inhibiting NETosis. Moreover, antibodies against the neutrophil receptors endothelial protein C receptor (EPCR), protease-activated receptor 3 (PAR3), and macrophage-1 antigen (Mac-1) blocked APC inhibition of NETosis. Select mutations in the Gla and protease domains of recombinant APC caused a loss of NETosis. Interestingly, pretreatment of neutrophils with APC prior to induction of NETosis inhibited platelet adhesion to NETs. Lastly, in a nonhuman primate model of Escherichia coli-induced sepsis, pretreatment of animals with APC abrogated release of myeloperoxidase from neutrophils, a marker of neutrophil activation. These findings suggest that the anti-inflammatory function of APC at therapeutic concentrations may include the inhibition of NETosis in an EPCR-, PAR3-, and Mac-1-dependent manner, providing additional mechanistic insight into the diverse functions of neutrophils and APC in disease states including sepsis.