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

Laura D. Healy, Cristina Puy, José A. Fernández, Annachiara Mitrugno, Ravi S. Keshari, Nyiawung A. Taku, Tiffany T. Chu, Xiao Xu, Andras Gruber, Florea Lupu, John H. Griffin, Owen McCarty

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29 Scopus citations

Abstract

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 whetherAPCis 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.

Original languageEnglish (US)
Pages (from-to)8616-8629
Number of pages14
JournalJournal of Biological Chemistry
Volume292
Issue number21
DOIs
StatePublished - May 26 2017

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ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Healy, L. D., Puy, C., Fernández, J. A., Mitrugno, A., Keshari, R. S., Taku, N. A., Chu, T. T., Xu, X., Gruber, A., Lupu, F., Griffin, J. H., & McCarty, O. (2017). Activated protein C inhibits neutrophil extracellular trap formation in vitro and activation in vivo. Journal of Biological Chemistry, 292(21), 8616-8629. https://doi.org/10.1074/jbc.M116.768309