Abstract
BackgroundThe release of neutrophil extracellular traps (NETs), a mesh of DNA, histones and neutrophil proteases from neutrophils, was first demonstrated as a host defence against pathogens. Recently it became clear that NETs are also released in pathological conditions. NETs released in the blood can activate thrombosis and initiate a cascade of platelet responses. However, it is not well understood if these responses are mediated through direct or indirect interactions. We investigated whether cell-free NETs can induce aggregation of washed human platelets in vitro and the contribution of NET-derived extracellular DNA and histones to platelet activation response.MethodsIsolated human neutrophils were stimulated with PMA to produce robust and consistent NETs. Cell-free NETs were isolated and characterised by examining DNA-histone complexes and quantification of neutrophil elastase with ELISA. NETs were incubated with washed human platelets to assess several platelet activation responses. Using pharmacological inhibitors, we explored the role of different NET components, as well as main platelet receptors, and downstream signalling pathways involved in NET-induced platelet aggregation.ResultsCell-free NETs directly induced dose-dependent platelet aggregation, dense granule secretion and procoagulant phosphatidyl serine exposure on platelets. Surprisingly, we found that inhibition of NET-derived DNA and histones did not affect NET-induced platelet aggregation or activation. We further identified the molecular pathways involved in NET-activated platelets. The most potent single modulator of NET-induced platelet responses included NET-bound cathepsin G, platelet Syk kinase, and P2Y12 and αIIbβ3 receptors.ConclusionsIn vitro-generated NETs can directly induce marked aggregation of washed human platelets. Pre-treatment of NETs with DNase or heparin did not reduce NET-induced activation or aggregation of human washed platelets. We further identified the molecular pathways activated in platelets in response to NETs. Taken together, we conclude that targeting certain platelet activation pathways, rather than the NET scaffold, has a more profound reduction on NET-induced platelet aggregation.
Highlights
The release of neutrophil extracellular traps (NETs), a mesh of DNA, histones and neutrophil proteases from neutrophils, was first demonstrated as a host defence against pathogens
Histones are capable of generating thrombin in the presence of plasma and activating platelet aggregation which has been suggested to be mediated through toll-like receptor (TLR) 2 and TLR 4 [18]
NETs induce aggregation, secretion and activation of washed human platelets We first examined the ability of cell-free NETs to directly induce aggregation of washed human platelets independently of the coagulation pathway
Summary
The release of neutrophil extracellular traps (NETs), a mesh of DNA, histones and neutrophil proteases from neutrophils, was first demonstrated as a host defence against pathogens. NETs can serve as more than just a host defence mechanism, as studies have implicated the role of NETs in inflammatory and autoimmune diseases and pathological conditions including thrombosis [4]. The second most abundant constituent and protein found on NETs, extracellular histones, have been studied extensively and known to activate platelets and promote coagulation through multiple mechanisms [9, 17,18,19]. The involvement of TLRs in platelet aggregation is not clear as Clark et al have shown that LPS induced platelet activation but not aggregation [20] neutrophil granular enzymes that are bound to NETs such as neutrophil elastase (NE) and cathepsin G (Cat G), can separately promote coagulation and thrombus growth by facilitating intravascular fibrin formation and degrading tissue factor pathway inhibitor [21], while myeloperoxidase (MPO) can prime platelets [22]
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