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

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 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 -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.

Regulation of immune cell signaling by activated protein C

“…APC’s multiple cytoprotective activities [20–22] have been demonstrated on a variety of cell types including but not limited to endothelial cells [55,64], monocytes [62,65–67], macrophages [65,68] and neutrophils [58,60,69–72]. The beneficial effects of APC on immune system cells are summarized in Table 1 [20,73,74].…”

“…Notably, APC inhibits induction of NETs by phorbol 12-myristate 13-acetate (PMA) or by autologous platelet secretome, and this effect of APC requires the neutrophil receptors EPCR, PAR3, and Mac-1 (α M β 2 ; CD11b/CD18) [69]. The signaling-selective APC mutant 3K3A-APC demonstrated a similar degree of inhibition of NETs generation as did wt-APC, further supporting the immuno-regulatory potential of cytoprotective-selective APC mutants.…”

“…The signaling-selective APC mutant 3K3A-APC demonstrated a similar degree of inhibition of NETs generation as did wt-APC, further supporting the immuno-regulatory potential of cytoprotective-selective APC mutants. Evidence for APC’s in vivo beneficial effects on neutrophils in sepsis came from a baboon model of E. coli -induced sepsis in which infusion of APC reduced levels of myeloperoxidase, a marker for neutrophil activation linked to NETs formation [69]. Currently, much remains to be clarified regarding receptor interactions and intracellular signaling pathways that mediate APC’s anti-inflammatory activities involving neutrophils.…”

“…APC inhibits leukocyte inflammatory processes in a receptor-dependent manner, requiring Mac-1 (CD11b/CD18), endothelial protein C receptor (EPCR), PAR1 and PAR3, inducing intracellular cytoprotective signaling. Figure adapted from [69]. …”

Platelets: an outlook from biology through evidence-based achievements in critical care

“…The nuclear and granular membranes disintegrate and elastase enters into the nucleus, followed by hypercitrullination of histones, chromatin decondensation into the cytoplasm, rupture of the plasma membrane, and extrusion of nuclear material from the cell into the extracellular space. NETs have been shown to promote activation of select coagulation factors and platelets, thereby exacerbating several disease states including thrombosis (69). The term "immunothrombosis" was recently coined (70) to describe an innate immune response induced by the formation of thrombi in microvessels or other vascular beds.…”