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

Colin Longstaff; John Hogwood; Elaine Gray; Erzsébet Komorowicz; Imre Varjú; Zoltán Varga; Krasimir Kolev

doi:10.1160/th15-03-0214

Thrombosis and Haemostasis, Table of Contents

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

Authors

Colin Longstaff

¹Biotherapeutics, Haemostasis Section, National Institute for Biological Standards and Control, South Mimms, Potters Bar, UK
John Hogwood

¹Biotherapeutics, Haemostasis Section, National Institute for Biological Standards and Control, South Mimms, Potters Bar, UK
Elaine Gray

¹Biotherapeutics, Haemostasis Section, National Institute for Biological Standards and Control, South Mimms, Potters Bar, UK
Erzsébet Komorowicz

²Department of Medical Biochemistry, Semmelweis University, Budapest, Hungary
Imre Varjú

²Department of Medical Biochemistry, Semmelweis University, Budapest, Hungary
Zoltán Varga

³Department of Biological Nanochemsitry, Institute of Materials and Environmental Chemistry, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Budapest, Hungary
Krasimir Kolev

²Department of Medical Biochemistry, Semmelweis University, Budapest, Hungary

Abstract

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

Full Text

References

References
1 Esmon CT. Molecular circuits in thrombosis and inflammation. Thromb Hae-most 2013; 109: 416-420.
2 Martinod K, Wagner DD. Thrombosis: tangled up in NETs. Blood 2014; 123: 2768-2776.
3 Engelmann B, Massberg S. Thrombosis as an intravascular effector of innate immunity. Nature Rev Immunol 2013; 13: 34-45.
4 Borissoff JI, ten Cate H. From neutrophil extracellular traps release to thrombosis: an overshooting host-defense mechanism?. J Thromb Haemost 2011; 9: 1791-1794.
5 Brill A, Fuchs TA, Savchenko AS. et al. Neutrophil extracellular traps promote deep vein thrombosis in mice. J Thromb Haemost 2012; 10: 136-144.
6 Borissoff JI, Joosen IA, Versteylen MO. et al. Elevated levels of circulating DNA and chromatin are independently associated with severe coronary atherosclerosis and a prothrombotic state. Arterioscler Thromb Vasc Biol 2013; 33: 2032-2040.
7 De Meyer SF, Suidan GL, Fuchs TA. et al. Extracellular chromatin is an important mediator of ischemic stroke in mice. Arterioscler Thromb Vasc Biol 2012; 32: 1884-1891.
8 Demers M, Krause DS, Schatzberg D. et al. Cancers predispose neutrophils to release extracellular DNA traps that contribute to cancer-associated thrombosis. Proc Natl Acad Sci USA 2012; 109: 13076-13081.
9 Tufano A, Di Capua M, Coppola A. et al. The infectious burden in atherothrom-bosis. Semin Thromb Hemost 2012; 38: 515-523.
10 Xu J, Zhang X, Pelayo R. et al. Extracellular histones are major mediators of death in sepsis. Nat Med 2009; 15: 1318-1321.
11 Huang H, Evankovich J, Yan W. et al. Endogenous histones function as alarmins in sterile inflammatory liver injury through Toll-like receptor 9 in mice. Hepa-tology 2011; 54: 999-1008.
12 Thomas GM, Carbo C, Curtis BR. et al. Extracellular DNA traps are associated with the pathogenesis of TRALI in humans and mice. Blood 2012; 119: 6335-6343.
13 Doring Y, Manthey HD, Drechsler M. et al. Auto-antigenic protein-DNA complexes stimulate plasmacytoid dendritic cells to promote atherosclerosis. Circulation 2012; 125: 1673-1683.
14 de Boer OJ, Li X, Teeling P. et al. Neutrophils, neutrophil extracellular traps and interleukin-17 associate with the organisation of thrombi in acute myocardial infarction. Thromb Haemost 2013; 109: 290-297.
15 Longstaff C, Varju I, Sotonyi P. et al. Mechanical stability and fibrinolytic resistance of clots containing fibrin, DNA, and histones. J Biol Chem 2013; 288: 6946-6956.
16 Gansler J, Jaax M, Leiting S. et al. Structural requirements for the procoagulant activity of nucleic acids. PLoS One 2012; 7: e50399.
17 Siri A, Balza E, Carnemolla B. et al. DNA-binding domains of human plasma fi-bronectin. pH and calcium ion modulation of fibronectin binding to DNA and heparin. Eur J Biochem 1986; 154: 533-538.
18 Fuchs TA, Bhandari AA, Wagner DD. Histones induce rapid and profound thrombocytopenia in mice. Blood 2011; 118: 3708-3714.
19 Ammollo CT, Semeraro F, Xu J. et al. Extracellular histones increase plasma thrombin generation by impairing thrombomodulin-dependent protein C activation. J Thromb Haemost 2011; 9: 1795-1803.
20 Ward CM, Tetaz TJ, Andrews RK. et al. Binding of the von Willebrand factor A1 domain to histone. Thromb Res 1997; 86: 469-477.
21 Brinkmann V, Reichard U, Goosmann C. et al. Neutrophil extracellular traps kill bacteria. Science 2004; 303: 1532-1535.
22 Abrams ST, Zhang N, Manson J. et al. Circulating histones are mediators of trauma-associated lung injury. Am J Resp Crit Care Med 2013; 187: 160-169.
23 Saffarzadeh M, Juenemann C, Queisser MA. et al. Neutrophil extracellular traps directly induce epithelial and endothelial cell death: a predominant role of hi-stones. PLoS One 2012; 7: e32366.
24 Fuchs TA, Brill A, Duerschmied D. et al. Extracellular DNA traps promote thrombosis. Proc Natl Acad Sci USA 2010; 107: 15880-15885.
25 Napirei M, Ludwig S, Mezrhab J. et al. Murine serum nucleases--contrasting effects of plasmin and heparin on the activities of DNase1 and DNase1-like 3 (DNase1l3). FEBS J 2009; 276: 1059-1073.
26 Wildhagen KC, Garcia de Frutos P, Reutelingsperger CP. et al. Nonanticoagulant heparin prevents histone-mediated cytotoxicity in vitro and improves survival in sepsis. Blood 2014; 123: 1098-1101.
27 Zeerleder S, Zwart B, Wuillemin WA. et al. Elevated nucleosome levels in systemic inflammation and sepsis. Crit Care Med 2003; 31: 1947-1951.
28 Hirsh J, Fuster V. Guide to anticoagulant therapy. Part 1: Heparin. American Heart Association. Circulation 1994; 89: 1449-1468.
29 Gresele P, Busti C, Paganelli G. Heparin in the prophylaxis and treatment of venous thromboembolism and other thrombotic diseases. In: Heparin-a century of progress. Springer-Verlag; 2012: 179-209.
30 Hirsh J, Bauer KA, Donati MB. et al. Parenteral anticoagulants: American College of Chest Physicians Evidence-Based Clinical Practice Guidelines (8th Edition). Chest 2008; 133: 141S-159S.
31 Nagasawa K, Inoue Y, Kamata T. Solvolytic desulfation of glycosaminoglycuron-an sulfates with dimethyl sulfoxide containing water or methanol. Carbohydrate Res 1977; 58: 47-55.
32 Whitton C, Sands D, Lee T. et al. A reunification of the US (NIH") and International Unit into a single standard for Thrombin. Thromb Haemost 2005; 93: 261-266.
33 Varju I, Longstaff C, Szabo L. et al. DNA, histones and neutrophil extracellular traps exert anti-fibrinolytic effects in a plasma environment. Thromb Haemost 2015; 113: 1289-1298.
34 Pal PK, Starr T, Gertler MM. Neutralization of heparin by histone and its subfrac-tions. Thromb Res 1983; 31: 69-79.
35 Semeraro F, Ammollo CT, Morrissey JH. et al. Extracellular histones promote thrombin generation through platelet-dependent mechanisms: involvement of platelet TLR2 and TLR4. Blood 2011; 118: 1952-1961.
36 Christophorou MA, Castelo-Branco G, Halley-Stott RP. et al. Citrullination regulates pluripotency and histone H1 binding to chromatin. Nature 2014; 507: 104-108.
37 Wang Y, Li M, Stadler S. et al. Histone hypercitrullination mediates chromatin de-condensation and neutrophil extracellular trap formation. J Cell Biol 2009; 184: 205-213.
38 Yipp BG, Kubes P. NETosis: how vital is it?. Blood 2013; 122: 2784-2794.
39 Hogg PJ, Jackson CM, Labanowski JK. et al. Binding of fibrin monomer and heparin to thrombin in a ternary complex alters the environment of the thrombin catalytic site, reduces affinity for hirudin, and inhibits cleavage of fibrinogen. J Biol Chem 1996; 271: 26088-26095.
40 Evans PA, Hawkins K, Morris RH. et al. Gel point and fractal microstructure of incipient blood clots are significant new markers of hemostasis for healthy and anti-coagulated blood. Blood 2010; 116: 3341-3346.
41 Nenci GG, Parise P, Morini M. et al. Fibrin clots obtained from plasma containing heparin show a higher sensitivity to t-PA-induced lysis. Blood Coagul Fibrinolysis 1992; 3: 279-285.
42 Collen A, Smorenburg SM, Peters E. et al. Unfractionated and low molecular weight heparin affect fibrin structure and angiogenesis in vitro. Cancer Res 2000; 60: 6196-6200.
43 Parise P, Morini M, Agnelli G. et al. Effects of low molecular weight heparins on fibrin polymerization and clot sensitivity to t-PA-induced lysis. Blood Coagul Fibri-nolysis 1993; 4: 721-727.
44 Yeromonahos C, Marlu R, Polack B. et al. Antithrombin-independent effects of he-parins on fibrin clot nanostructure. Arterioscler Thromb Vasc Biol 2012; 32: 1320-1324.
45 Smith SA, Morrissey JH. Polyphosphate enhances fibrin clot structure. Blood 2008; 112: 2810-2816.
46 Smith SA, Choi SH, Davis-Harrison R. et al. Polyphosphate exerts differential effects on blood clotting, depending on polymer size. Blood 2010; 116: 4353-4359.

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