In vitro activation of coagulation by human neutrophil DNA and histone proteins but not neutrophil extracellular traps

Abstract

NETosis is a physiologic process in which neutrophils release their nuclear material in the form of neutrophil extracellular traps (NETs). NETs have been reported to directly promote thrombosis in animal models. Although the effects of purified NET components including DNA, histone proteins, and neutrophil enzymes on coagulation have been characterized, the mechanism by which intact NETs promote thrombosis is largely unknown. In this study, human neutrophils were stimulated to produce NETs in platelet-free plasma (PFP) or in buffer using phorbol myristate actetate or calcium ionophore. DNA and histone proteins were also separately purified from normal human neutrophils and used to reconstitute chromatin using a salt-gradient dialysis method. Neutrophil stimulation resulted in robust NET release. In recalcified PFP, purified DNA triggered contact-dependent thrombin generation (TG) and amplified TG initiated by low concentrations of tissue factor. Similarly, in a buffer milieu, DNA initiated the contact pathway and amplified thrombin-dependent factor XI activation. Recombinant human histones H3 and H4 triggered TG in recalcified human plasma in a platelet-dependent manner. In contrast, neither intact NETs, reconstituted chromatin, individual nucleosome particles, nor octameric core histones reproduced any of these procoagulant effects. We conclude that unlike DNA or individual histone proteins, human intact NETs do not directly initiate or amplify coagulation in vitro. This difference is likely explained by the complex histone-histone and histone-DNA interactions within the nucleosome unit and higher-order supercoiled chromatin leading to neutralization of the negative charges on polyanionic DNA and modification of the binding properties of individual histone proteins.

Acetylsalicylic acid differentially limits the activation and expression of cell death markers in human platelets exposed to Staphylococcus aureus strains

“…However, the detrimental role of NETs should not be underestimated because NETs have been shown to enhance thrombosis 77 , although new evidence tends to distinguish the effects of intact NETs from those of DNA and histones in the induction of coagulation 78 .…”

Amicus or Adversary Revisited: Platelets in Acute Lung Injury and Acute Respiratory Distress Syndrome

“…Intravascular fibrin deposition and formation of platelet-fibrin thrombi are key features of septic ALI in humans (reviewed in [14]), providing clinical relevance. NETs did not directly induce coagulation of human plasma in recent in vitro studies, however, suggesting that there may be species differences or other relevant variables (125). Agents with the ability to inhibit NETosis or dismantle NETs after their formation have been identified (55,59,123,124,126), suggesting potential adjuvant therapeutic approaches in sepsis and septic ARDS.…”

Fibrinogen and Fibrin in Hemostasis and Thrombosis

“…NET components promote thrombin generation by activating the intrinsic pathway of coagulation and by inducing platelet-dependent mechanisms in toll-like receptors-2 and -4-dependent mechanisms. 72–74 Histones also enhance activated protein C generation by thrombin/thrombomodulin in vitro and in mice. 75 By altering these procoagulant and anticoagulant pathways, NETs may alter local thrombin levels and indirectly alter fibrin formation and quality.…”