Abstract
BackgroundSepsis is associated with systemic inflammatory responses and induction of coagulation system. Neutrophil extracellular traps (NETs) constitute an antimicrobial mechanism, recently implicated in thrombosis via platelet entrapment and aggregation.Methodology/Principal FindingsIn this study, we demonstrate for the first time the localization of thrombogenic tissue factor (TF) in NETs released by neutrophils derived from patients with gram-negative sepsis and normal neutrophils treated with either serum from septic patients or inflammatory mediators involved in the pathogenesis of sepsis. Localization of TF in acidified autophagosomes was observed during this process, as indicated by positive LC3B and LysoTracker staining. Moreover, phosphatidylinositol 3-kinase inhibition with 3-MA or inhibition of endosomal acidification with bafilomycin A1 hindered the release of TF-bearing NETs. TF present in NETs induced thrombin generation in culture supernatants, which further resulted in protease activated receptor-1 signaling.Conclusions/SignificanceThis study demonstrates the involvement of autophagic machinery in the extracellular delivery of TF in NETs and the subsequent activation of coagulation cascade, providing evidence for the implication of this process in coagulopathy and inflammatory response in sepsis.
Highlights
Systemic activation of coagulation cascade and formation of thrombi in the microvasculature contribute to organ dysfunction that characterizes sepsis [1,2]
The previously described implication of autophagy in Neutrophil extracellular traps (NETs) release [20,21] prompted us to investigate whether autophagy inhibition attenuates the release of NETs from neutrophils derived from patients hospitalized with gram-negative sepsis
Treatment of sepsis neutrophils with 3 methyl-adenine (3-MA), a modulator of class III phosphatidylinositol 3kinase (PI3K) that inhibits autophagy, or bafilomycin A1, which impairs the formation of acidified autophagosomes, abrogated NET release (Fig. 1A, Fig. S1)
Summary
Systemic activation of coagulation cascade and formation of thrombi in the microvasculature contribute to organ dysfunction that characterizes sepsis [1,2]. Thrombin and TF/factor VIIa complex signaling through protease activated receptor-1 (PAR-1) and PAR-2 respectively was implicated in the induction of inflammation in experimental models of sepsis, linking coagulation to inflammation [6,7]. NETs are extracellular chromatin structures that entrap microbes and are composed of nuclear and granule constituents of neutrophils [10,11]. They are formed after phagocytosis of pathogens or treatment with inflammatory stimuli [10,11] and are implicated in the pathogenesis of sepsis [12]. Neutrophil extracellular traps (NETs) constitute an antimicrobial mechanism, recently implicated in thrombosis via platelet entrapment and aggregation
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