title: 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.
authors.affiliation: Division of Hematology/Oncology, Department of Medicine, University of North Carolina, Chapel Hill, NC; and.
authors.forename: Denis F
authors.initials: DF
authors.lastname: Noubouossie
authors.affiliation: Division of Hematology/Oncology, Department of Medicine, University of North Carolina, Chapel Hill, NC; and.
authors.forename: Matthew F
authors.initials: MF
authors.lastname: Whelihan
authors.affiliation: Division of Hematology, Department of Medicine, Taipei Veterans General Hospital, Taipei, Taiwan.
authors.forename: Yuan-Bin
authors.initials: YB
authors.lastname: Yu
authors.affiliation: Division of Hematology/Oncology, Department of Medicine, University of North Carolina, Chapel Hill, NC; and.
authors.forename: Erica
authors.initials: E
authors.lastname: Sparkenbaugh
authors.affiliation: Division of Hematology/Oncology, Department of Medicine, University of North Carolina, Chapel Hill, NC; and.
authors.forename: Rafal
authors.initials: R
authors.lastname: Pawlinski
authors.affiliation: Division of Hematology/Oncology, Department of Medicine, University of North Carolina, Chapel Hill, NC; and.
authors.forename: Dougald M
authors.initials: DM
authors.lastname: Monroe
authors.affiliation: Division of Hematology/Oncology, Department of Medicine, University of North Carolina, Chapel Hill, NC; and.
authors.forename: Nigel S
authors.identifier: 0000-0002-8930-4304
authors.initials: NS
authors.lastname: Key
mesh_terms: D001777:Blood Coagulation; D002469:Cell Separation; D004247:DNA / Q000302:isolation & purification / Q000378:metabolism*; D065206:Extracellular Traps / Q000378:metabolism*; D006657:Histones / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D009504:Neutrophils / Q000166:cytology / Q000378:metabolism*; D009707:Nucleosomes / Q000378:metabolism
chemical_list: D006657:Histones; D009707:Nucleosomes; D004247:DNA