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Neutralisation of the anti-coagulant effects of heparin by histones in blood plasma and purified systems

Journal: Thrombosis and Haemostasis
ISSN: 0340-6245
Topic:

Theme Issue
Biomechanics in vascular biology and cardiovascular disease

DOI: https://doi.org/10.1160/TH15-03-0214
Issue: 2016: 115/3 (Mar) pp. 465–684
Pages: 591-599
Ahead of Print: 2015-12-03

Neutralisation of the anti-coagulant effects of heparin by histones in blood plasma and purified systems

Online Supplementary Material

C. Longstaff (1), J. Hogwood (1), E. Gray (1), E. Komorowicz (2), I. Varjú (2), Z. Varga (3), K. Kolev (2)

(1) Biotherapeutics, Haemostasis Section, National Institute for Biological Standards and Control, South Mimms, Potters Bar, UK; (2) Department of Medical Biochemistry, Semmelweis University, Budapest, Hungary; (3) Department of Biological Nanochemsitry, Institute of Materials and Environmental Chemistry, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Budapest, Hungary

Keywords

heparin, aPTT, DNA, histones, neutrophil extracellular traps

Summary

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.

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