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Thromb Haemost 2016; 115(03): 591-599
DOI: 10.1160/th15-03-0214
Coagulation and Fibrinolysis
Schattauer GmbH

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

Colin Longstaff
1   Biotherapeutics, Haemostasis Section, National Institute for Biological Standards and Control, South Mimms, Potters Bar, UK
,
John Hogwood
1   Biotherapeutics, Haemostasis Section, National Institute for Biological Standards and Control, South Mimms, Potters Bar, UK
,
Elaine Gray
1   Biotherapeutics, Haemostasis Section, National Institute for Biological Standards and Control, South Mimms, Potters Bar, UK
,
Erzsébet Komorowicz
2   Department of Medical Biochemistry, Semmelweis University, Budapest, Hungary
,
Imre Varjú
2   Department of Medical Biochemistry, Semmelweis University, Budapest, Hungary
,
Zoltán Varga
3   Department of Biological Nanochemsitry, Institute of Materials and Environmental Chemistry, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Budapest, Hungary
,
Krasimir Kolev
2   Department of Medical Biochemistry, Semmelweis University, Budapest, Hungary
› Author Affiliations Financial support: The work was supported in part by grants OTKA 83023 and 112612 to KK. IV was supported in part by a short-term fellowship granted by the European Molecular Biology Organisation, as well as a postdoctoral fellowship by the Hungarian Academy of Sciences. Part of this work was commissioned and funded as independent research by the UK Department of Health Policy Research Programme (NIBSC Regulatory Science Research Unit, 044/0069). The views expressed in this publication are those of the author(s) and not necessarily those of the Department of Health.
Further Information

Publication History

Received: 10 March 2015

Accepted after major revision: 22 October 2015

Publication Date:
20 March 2018 (online)

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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.

Keywords

Neutrophil extracellular traps - DNA - histones - heparin - APTT

Supplementary Material

 

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