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

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. Unfraction-ated 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 lU/ml UFH was 1.8 |jg/ml hi­stones in APTT and 4.6 |jg/ml against 0.6 lU/ml UFH in a purified sys­tem. Histones also inhibited the activities of LMWHs with thrombin (IC₅₀ 6.1 and 11.0 |jg/ml histones, for different LMWHs) or FXa (IC₅₀ 7.8 and 7.0 |jg/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 anti-coagulation 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.