Abstract
Cell-free DNA (cfDNA) is the major structural component of neutrophil extracellular traps (NETs), an innate immune response to infection. Antimicrobial proteins and peptides bound to cfDNA play a critical role in the bactericidal property of NETs. Recent studies have shown that NETs have procoagulant activity, wherein cfDNA triggers thrombin generation through activation of the intrinsic pathway of coagulation. We have recently shown that thrombin binds to NETs in vitro and consequently can alter the proteome of NETs. However, the effect of NETs on thrombin is still unknown. In this study, we report that DNA binding leads to thrombin autolysis and generation of multiple thrombin-derived C-terminal peptides (TCPs) in vitro. Employing a 25-residue prototypic TCP, GKY25 (GKYGFYTHVFRLKKWIQKVIDQFGE), we show that TCPs bind NETs, thus conferring mutual protection against nuclease and protease degradation. Together, our results demonstrate the complex interplay between coagulation, NET formation, and thrombin cleavage and identify a previously undisclosed mechanism for formation of TCPs.
Highlights
Neutrophil extracellular traps (NETs) are web-like structures exuded by activated neutrophils as first line of host defense, in response to pathogens and other stimuli [1]
The key novel finding of the present study is that binding of thrombin to cell-free DNA triggers its autoproteolysis, generating thrombin Cterminal peptides (TCPs)
The findings are of relevance in the context of prothrombin activation and thrombin generation in various inflammatory conditions involving neutrophil chemotaxis, activation and NETs release
Summary
Neutrophil extracellular traps (NETs) are web-like structures exuded by activated neutrophils as first line of host defense, in response to pathogens and other stimuli [1]. NET fibers comprise majorly of cell-free DNA (cfDNA) as the structural scaffold, decorated with histones, granule derived enzymes (such as neutrophil elastase and cathepsin G) and antimicrobial proteins and peptides (lactoferrin, lysozyme, a-defensins and LL-37). Such a complex structure with a high local concentration of antimicrobial enables NETs to effectively trap pathogens, contain microbial spread and kill microbial invaders, playing a critical role in innate immune defense [1]. Several studies have reported on the procoagulant activity of NETs. cfDNA triggers thrombin generation via activation of the contact pathway and elevated levels of circulating cfDNA is a hallmark feature in patients with deep vein thrombosis and sepsis [4,5,6,7]
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