PB0858 - Effects of neutrophil extracellular traps on blood clot contraction

Background: Neutrophils are a crucial component of the immune system. Platelet-driven contraction or retraction of blood clots is an important pathophysiological mechanism that could be modulated by neutrophils accumulated in inflammatory thrombi. Activation of neutrophils is accompanied by NETosis, that is, the release of neutrophil extracellular traps (NETs) comprising uncondensed chromatin, i.e. a network of DNA with histones and other proteins. Intravascular NET formation is known to enhance blood clotting and promote platelet activation, but the effects of NETs on blood clot contraction are unknown.

Aims: To determine the effects of activated neutrophils on the rate and extent of platelet-mediated contraction of blood clots.

Methods: Isolated human neutrophils were pre-activated with 100 nM PMA (3.5 hours at 37°C) and added to normal human blood or platelet-rich plasma (PRP) followed by thrombin-induced clotting and clot contraction tracked optically as reduction of clot size. Using light (including standard histological H&E staining) and confocal fluorescent microscopy, neutrophils were stained for NETs-specific CitH3 histones and DNA that highlighted neutrophilic nuclei and NETs within isolated PMA-activated neutrophils, supernatant, and fibrin clots. High-resolution scanning electron microscopy was also employed to visualize the presence of NETs with activated neutrophils and within PRP clots.

Results: PMA-activated neutrophils, unlike non-activated cells, caused a significant increase in the rate and extent of clot contraction both in whole blood and in PRP. The promoting effect of the activated neutrophils on clot contraction was likely attributed to the presence of NETs. NETs were visualized both in the supernatant of the PMA-activated isolated neutrophils and within fibrin clots formed in the presence of pre-activated neutrophils. The effect of NETs is presumably mediated by platelet stimulation and/or modulation of fibrin viscoelasticity.

Conclusion(s): Activated neutrophils promote blood clot contraction and this effect is associated with formation of extracellular traps embedded into the fibrin network.