Neutrophil extracellular traps induce aggregation of washed human platelets independently of extracellular DNA and histones

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.Electronic supplementary materialThe online version of this article (10.1186/s12964-018-0235-0) contains supplementary material, which is available to authorized users.

Extracellular histones are clinically associated with primary graft dysfunction in human liver transplantation

“…6,20 MPO activity is an index of neutrophil, monocyte/macrophage activation, whereas S100A8/ A9, which is abundantly stored in neutrophil cytoplasm, is another classical marker for neutrophil activation. 21,22 Therefore, we assayed both MPO and S100A8/A9 levels to assess activation of innate immune cells particularly neutrophils, in the sera of patients undergoing liver transplantation using commercially available kits.…”

COVID-19 update: Covid-19-associated coagulopathy

“…NETs represent part of a continuum of sterile inflammation and thrombosis that can involve all vascular beds, including the microvascular circulation [55][56][57][58][59][60][61][62][63][64][65][66].…”

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“…Finally, the recent XANTHIPPE study demonstrated a reduction in PLAs in the ticagrelor group compared to placebo [113]. To date, no study reported a significant modulation of NETosis by P2Y 12 inhibitors, although ticagrelor was recently shown to reduce in vitro NET-induced platelet aggregation, secretion and expression of P-selectin [113,121].…”

“…NETs are large, web-like extracellular chromatin structures that are released by neutrophils upon various activating stimuli, including exogenous microorganisms (bacteria, fungi, virus, and parasites), immune complexes and activated platelets [120]. NETs can trap, neutralize and kill microorganisms, but also activate platelets and promote coagulation [121]. They are considered as critical players in many inflammatory conditions, especially sepsis-induced organ injuries, autoimmunity, tumorigenesis and metastasis [120].…”