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DNA, histones and neutrophil extracellular traps exert anti-fibrinolytic effects in a plasma environmentFinancial support: This work was supported by the Hungarian Scientific Research Fund OTKA 83023 and 112612.
12 August 2014
Accepted after major revision: 21 January 2015
22 November 2017 (online)
In response to various inflammatory stimuli, neutrophils secrete neutrophil extracellular traps (NETs), web-like meshworks of DNA, histones and granular components forming supplementary scaffolds in venous and arterial thrombi. Isolated DNA and histones are known to promote thrombus formation and render fibrin clots more resistant to mechanical forces and tissue-type plasminogen activator (tPA)-induced enzymatic digestion. The present study extends our earlier observations to a physiologically more relevant environment including plasma clots and NET-forming neutrophils. A range of techniques was employed including imaging (scanning electron microscopy (SEM), confocal laser microscopy, and photoscanning of macroscopic lysis fronts), clot permeability measurements, turbidimetric lysis and enzyme inactivation assays. Addition of DNA and histones increased the median fibre diameter of plasma clots formed with 16 nM thrombin from 108 to 121 and 119 nm, respectively, and decreased their permeability constant from 6.4 to 3.1 and 3.7×10−9 cm2. Histones effectively protected thrombin from antithrombin-induced inactivation, while DNA inhibited plasminogen activation on the surface of plasma clots and their plasmin-induced resolution by 20 and 40 %, respectively. DNA and histones, as well as NETs secreted by phorbol-myristate-acetate-activated neutrophils, slowed down the tPA-driven lysis of plasma clots and the latter effect could be reversed by the addition of DNase (streptodornase). SEM images taken after complete digestion of fibrin in NET-containing plasma clots evidenced retained NET scaffold that was absent in DNase-treated clots. Our results show that DNA and histones alter the fibrin architecture in plasma clots, while NETs contribute to a decreased lytic susceptibility that can be overcome by DNase.
- 1 Brinkmann V, Reichard U, Goosman C. et al. Neutrophil extracellular traps kill bacteria. Science 2004; 303: 1532-1535.
- 2 Branzk N, Papayannopoulos V. Molecular mechanisms regulating NETosis in infection and disease. Semin Immunopathol 2013; 35: 513-530.
- 3 Borissoff JI, Ten Cate H. From neutrophil extracellular traps release to thrombosis: an overshooting host-defense mechanism?: Commentary. J Thromb Haemost 2011; 09: 1791-1794.
- 4 Darbousset R, Thomas GM, Mezouar S. et al. Tissue factor-positive neutrophils bind to injured endothelial wall and initiate thrombus formation. Blood 2012; 120: 2133-2143.
- 5 Fuchs TA, Abed U, Goosmann C. et al. Novel cell death program leads to neutrophil extracellular traps. J Cell Biol 2007; 176: 231-241.
- 6 Urban CF, Ermert D, Schmid M. et al. Neutrophil Extracellular Traps Contain Calprotectin, a Cytosolic Protein Complex Involved in Host Defense against Candida albicans. PLoS Pathog 2009; 05: e1000639.
- 7 Fuchs TA, Bhandari AA, Wagner DD. Histones induce rapid and profound thrombocytopenia in mice. Blood 2011; 118: 3708-3714.
- 8 Saffarzadeh M, Juenemann C, Queisser MA. et al. Neutrophil extracellular traps directly induce epithelial and endothelial cell death: a predominant role of histones. PLoS One 2012; 07: e32366.
- 9 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; 09: 1795-1803.
- 10 Barranco-Medina S, Pozzi N, Vogt AD. et al. Histone H4 Promotes Prothrombin Autoactivation. J Biol Chem 2013; 288: 35749-35757.
- 11 Von Bruhl M-L, Stark K, Steinhart A. et al. Monocytes, neutrophils, and platelets cooperate to initiate and propagate venous thrombosis in mice in vivo. J Exp Med 2012; 209: 819-835.
- 12 Brinkmann V, Zychlinsky A. Neutrophil extracellular traps: Is immunity the second function of chromatin?. J Cell Biol 2012; 198: 773-783.
- 13 Martinod K, Wagner DD. Thrombosis: tangled up in NETs. Blood 2014; 123: 2768-2776.
- 14 Longstaff C, Varjú I, Sόtonyi P. et al. Mechanical Stability and Fibrinolytic Resistance of Clots Containing Fibrin, DNA, and Histones. J Biol Chem 2013; 288: 6946-6956.
- 15 Deutsch D, Mertz E. Plasminogen: purification from human plasma by affinity chromatography. Science 1970; 170: 1095-1096.
- 16 Kolev K, Léránt I, Tenekejiev K. et al. Regulation of fibrinolytic activity of neutrophil leukocyte elastase, plasmin, and miniplasmin by plasma protease inhibitors. J Biol Chem 1994; 269: 17030-17034.
- 17 Varjú I, Sόtonyi P, Machovich R. et al. Hindered dissolution of fibrin formed under mechanical stress. J Thromb Haemost 2011; 09: 979-986.
- 18 Longstaff C, Thelwell C, Williams SC. et al. The interplay between tissue plasminogen activator domains and fibrin structures in the regulation of fibrinolysis: kinetic and microscopic studies. Blood 2011; 117: 661-668.
- 19 Wohner N, Keresztes Z, Sόtonyi P. et al. Neutrophil granulocyte-dependent proteolysis enhances platelet adhesion to the arterial wall under high-shear flow. J Thromb Haemost 2010; 08: 1624-1631.
- 20 Nikolova N, Toneva-Zheynova D, Kolev K. et al. Monte Carlo statistical tests for identity of theoretical and empirical distributions of experimental data. In: Theory and applications of Monte Carlo simulations. InTech 2013; pp. 1-26.
- 21 Brinkmann V, Laube B, Abu Abed U. et al. Neutrophil extracellular traps: how to generate and visualize them. J Vis Exp 2010; 36: e1724.
- 22 Woodhead JL, Nagaswami C, Matsuda M. et al. The ultrastructure of fibrinogen Caracas II molecules, fibres, and clots. J Biol Chem 1996; 271: 4946-4953.
- 23 Kovács A, Szabό L, Longstaff C. et al. Ambivalent roles of carboxypeptidase B in the lytic susceptibility of fibrin. Thromb Res 2014; 133: 80-87.
- 24 Longstaff C, Gaffney PJ. Serpin-serine protease binding kinetics: alpha2-antiplasmin as a model inhibitor. Biochemistry 1991; 30: 979-986.
- 25 Wang Y, Li M, Stadler S. et al. Histone hypercitrullination mediates chromatin decondensation and neutrophil extracellular trap formation. J Cell Biol 2009; 184: 205-213.
- 26 Blombäck B, Carlsson K, Fatah K. et al. Fibrin in human plasma: gel architectures governed by rate and nature of fibrinogen activation. Thromb Res 1994; 75: 521-538.
- 27 Ferry JD, Morrison PR. Preparation and properties of serum and plasma proteins; the conversion of human fibrinogen to fibrin under various conditions. J Am Chem Soc 1947; 69: 388-400.
- 28 Lord ST. Molecular Mechanisms Affecting Fibrin Structure and Stability. Arterioscler Thromb Vasc Biol 2011; 31: 494-499.
- 29 Weisel JW, Litvinov RI. The biochemical and physical process of fibrinolysis and effects of clot structure and stability on the lysis rate. Curr Med Chem-Cardiovasc Hematol Agents 2008; 06: 161-180.
- 30 Xu J, Zhang X, Pelayo R. et al. Extracellular histones are major mediators of death in sepsis. Nat Med 2009; 15: 1318-1321.
- 31 Steinman CR. Free DNA in serum and plasma from normal adults. J Clin Invest 1975; 56: 512-515.
- 32 Leon SA, Shapiro B, Sklaroff DM. et al. Free DNA in the serum of cancer patients and the effect of therapy. Cancer Res 1977; 37: 646-650.
- 33 Saha P, Humphries J, Modarai B. et al. Leukocytes and the Natural History of Deep Vein Thrombosis: Current Concepts and Future Directions. Arterioscler Thromb Vasc Biol 2011; 31: 506-512.
- 34 Liou TG, Campbell EJ. Nonisotropic enzyme-inhibitor interactions: a novel nonoxidative mechanism for quantum proteolysis by human neutrophils. Biochemistry 1995; 34: 16171-16177.
- 35 Esmon CT. Molecular circuits in thrombosis and inflammation. Thromb Haemost 2013; 109: 416-420.
- 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 Wohner N, Sotonyi P, Machovich R. et al. Lytic Resistance of Fibrin Containing Red Blood Cells. Arterioscler Thromb Vasc Biol 2011; 31: 2306-2313.
- 38 Tebbe U, Tanswell P, Seifried E. et al. Single-bolus injection of recombinant tissue-type plasminogen activator in acute myocardial infarction. Am J Cardiol 1989; 64: 448-453.
- 39 Geier B, Grossefeld M, Barbera L. et al. Pharmacokinetics of tissue plasminogen activator in an isolated extracorporeal circuit. J Vasc Surg 2001; 33: 165-169.
- 40 Komissarov AA, Florova G, Idell S. Effects of extracellular DNA on plasminogen activation and fibrinolysis. J Biol Chem 2011; 286: 41949-41962.
- 41 Chang X, Yamada R, Sawada T. et al. The inhibition of antithrombin by peptidylarginine deiminase 4 may contribute to pathogenesis of rheumatoid arthritis. Rheumatology 2005; 44: 293-298.
- 42 Masson-Bessière C, Sebbag M, Girbal-Neuhauser E. et al. The major synovial targets of the rheumatoid arthritis-specific antifilaggrin autoantibodies are deiminated forms of the α-and α-chains of fibrin. J Immunol 2001; 166: 4177-4184.
- 43 Machovich R, Owen WG. An elastase-dependent pathway of plasminogen activation. Biochemistry 1989; 28: 4517-4522.
- 44 Kolev K, Tenekedjiev K, Komorowicz E. et al. Functional evaluation of the structural features of proteases and their substrate in fibrin surface degradation. J Biol Chem 1997; 272: 13666-13675.
- 45 Armstrong PW, Gershlick AH, Goldstein P. et al. Fibrinolysis or Primary PCI in ST-Segment Elevation Myocardial Infarction. N Engl J Med 2013; 368: 1379-1387.
- 46 Adams HP, del Zoppo G, Alberts MJ. et al. Guidelines for the Early Management of Adults With Ischemic Stroke: A Guideline From the American Heart Association/ American Stroke Association Stroke Council, Clinical Cardiology Council, Cardiovascular Radiology and Intervention Council, and the Atherosclerotic Peripheral Vascular Disease and Quality of Care Outcomes in Research Interdisciplinary Working Groups: The American Academy of Neurology affirms the value of this guideline as an educational tool for neurologists. Circulation 2007; 115: e478-e534.
- 47 Marder VJ. Historical perspective and future direction of thrombolysis research: the re-discovery of plasmin: The re-discovery of plasmin. J Thromb Haemost 2011; 09: 364-373.
- 48 Buchanan JT, Simpson AJ, Aziz RK. et al. DNase expression allows the pathogen group A Streptococcus to escape killing in neutrophil extracellular traps. Curr Biol 2006; 16: 396-400.
- 49 Fuchs TA, Brill A, Duerschmied D. et al. Extracellular DNA traps promote thrombosis. Proc Natl Acad Sci U S A 2010; 107: 15880-15885.
- 50 Napirei M, Wulf S, Mannherz HG. Chromatin breakdown during necrosis by serum Dnase1 and the plasminogen system. Arthritis Rheum 2004; 50: 1873-1883.