title: Neutralisation of the anti-coagulant effects of heparin by histones in blood plasma and purified systems.
abstract: Neutrophil extracellular traps (NETs) composed primarily of DNA and histones are a link between infection, inflammation and coagulation. NETs promote coagulation and approaches to destabilise NETs have been explored to reduce thrombosis and treat sepsis. Heparinoids bind histones and we report quantitative studies in plasma and purified systems to better understand physiological consequences. Unfractionated heparin (UFH) was investigated by activated partial thromboplastin time (APTT) and alongside low-molecular-weight heparins (LMWH) in purified systems with thrombin or factor Xa (FXa) and antithrombin (AT) to measure the sensitivity of UFH or LMWH to histones. A method was developed to assess the effectiveness of DNA and non-anticoagulant heparinoids as anti-histones. Histones effectively neutralised UFH, the IC50 value for neutralisation of 0.2 IU/ml UFH was 1.8 µg/ml histones in APTT and 4.6 µg/ml against 0.6 IU/ml UFH in a purified system. Histones also inhibited the activities of LMWHs with thrombin (IC50 6.1 and 11.0 µg/ml histones, for different LMWHs) or FXa (IC50 7.8 and 7.0 µg/ml histones). Direct interactions of UFH and LMWH with DNA and histones were explored by surface plasmon resonance, while rheology studies showed complex effects of histones, UFH and LMWH on clot resilience. A conclusion from these studies is that anticoagulation by UFH and LMWH will be compromised by high affinity binding to circulating histones even in the presence of DNA. A complete understanding of the effects of histones, DNA and heparins on the haemostatic system must include an appreciation of direct effects on fibrin and clot structure.
authors.affiliation: Colin Longstaff, Biotherapeutics Group, National Institute for Biological Standards and Control, S Mimms, Herts, EN6 3QG, UK, Tel.: +44 1707 641253, Fax: +44 1707 641050, E-Mail: colin.longstaff@nibsc.org.
authors.forename: Colin
authors.initials: C
authors.lastname: Longstaff
authors.forename: John
authors.initials: J
authors.lastname: Hogwood
authors.forename: Elaine
authors.initials: E
authors.lastname: Gray
authors.forename: Erzsebet
authors.initials: E
authors.lastname: Komorowicz
authors.forename: Imre
authors.initials: I
authors.lastname: Varjú
authors.forename: Zoltán
authors.initials: Z
authors.lastname: Varga
authors.forename: Krasimir
authors.initials: K
authors.lastname: Kolev
mesh_terms: D000925:Anticoagulants / Q000737:chemistry*; D000991:Antithrombins / Q000737:chemistry; D001777:Blood Coagulation; D003029:Coagulants / Q000737:chemistry; D004247:DNA / Q000737:chemistry; D004548:Elasticity; D005337:Fibrin / Q000737:chemistry; D006487:Hemostasis; D006493:Heparin / Q000097:blood / Q000737:chemistry*; D006495:Heparin, Low-Molecular-Weight / Q000737:chemistry; D006657:Histones / Q000097:blood / Q000737:chemistry*; D006801:Humans; D020128:Inhibitory Concentration 50; D010314:Partial Thromboplastin Time; D010949:Plasma / Q000737:chemistry*; D011485:Protein Binding; D018805:Sepsis / Q000628:therapy; D020349:Surface Plasmon Resonance; D013927:Thrombosis / Q000097:blood; D014783:Viscosity
chemical_list: D000925:Anticoagulants; D000991:Antithrombins; D003029:Coagulants; D006495:Heparin, Low-Molecular-Weight; D006657:Histones; D005337:Fibrin; D006493:Heparin; D004247:DNA
keywords: APTT; DNA; Neutrophil extracellular traps; heparin; histones