TY - JOUR
T1 - Activated protein C inhibits neutrophil extracellular trap formation in vitro and activation in vivo
AU - Healy, Laura D.
AU - Puy, Cristina
AU - Fernández, José A.
AU - Mitrugno, Annachiara
AU - Keshari, Ravi S.
AU - Taku, Nyiawung A.
AU - Chu, Tiffany T.
AU - Xu, Xiao
AU - Gruber, András
AU - Lupu, Florea
AU - Griffin, John H.
AU - McCarty, Owen J.T.
N1 - Funding Information:
This work was supported by National Institutes of Health Grants R01HL101972 (to O. J. T. M) and R01GM116184 (to O. J. T. M. and F. L.); R01HL052246 (to J. H. G.); R44HL117589 (to A. G.); T32AI007472 (to L. D. H); and R01GM097746, U19AI062629, and P30GM114731 (to F. L.). The authors declare that they have no conflicts of interest with the contents of this article.The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Publisher Copyright:
© 2017 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.
PY - 2017/5/26
Y1 - 2017/5/26
N2 - 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.
AB - 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.
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U2 - 10.1074/jbc.M116.768309
DO - 10.1074/jbc.M116.768309
M3 - Article
C2 - 28408624
AN - SCOPUS:85019709759
VL - 292
SP - 8616
EP - 8629
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 21
ER -