Mechanical Stability and Fibrinolytic Resistance of Clots Containing Fibrin, DNA, and Histones (original) (raw)

2013, Journal of Biological Chemistry

Background: Neutrophil extracellular traps (NETs) composed of DNA and proteins form a scaffold in thrombi, supplementing the fibrin matrix. Results: DNA and histones modify the structure of fibrin and render it resistant to mechanical and enzymatic destruction. Conclusion: NET components are essential factors in thrombus stability. Significance: Therapeutic strategies could be optimized to enhance fibrinolysis in clots containing DNA and histones. Neutrophil extracellular traps are networks of DNA and associated proteins produced by nucleosome release from activated neutrophils in response to infection stimuli and have recently been identified as key mediators between innate immunity, inflammation, and hemostasis. The interaction of DNA and histones with a number of hemostatic factors has been shown to promote clotting and is associated with increased thrombosis, but little is known about the effects of DNA and histones on the regulation of fibrin stability and fibrinolysis. Here we demonstrate that the addition of histone-DNA complexes to fibrin results in thicker fibers (increase in median diameter from 84 to 123 nm according to scanning electron microscopy data) accompanied by improved stability and rigidity (the critical shear stress causing loss of fibrin viscosity increases from 150 to 376 Pa whereas the storage modulus of the gel increases from 62 to 82 pascals according to oscillation rheometric data). The effects of DNA and histones alone are subtle and suggest that histones affect clot structure whereas DNA changes the way clots are lysed. The combination of histones ؉ DNA significantly prolongs clot lysis. Isothermal titration and confocal microscopy studies suggest that histones and DNA bind large fibrin degradation products with 191 and 136 nM dissociation constants, respectively, interactions that inhibit clot lysis. Heparin, which is known to interfere with the formation of neutrophil extracellular traps, appears to prolong lysis time at a concentration favoring ternary histone-DNA-heparin complex formation, and DNase effectively promotes clot lysis in combination with tissue plasminogen activator. Neutrophil extracellular traps (NETs) 2 are networks of DNA decorated with histones and other proteins, proteases, and other antimicrobial factors (1). They are produced when neutrophils, basophils, or mast cells release nucleosomes after stimulation by inflammatory cytokines or LPS for example (1) or by interaction with platelets after stimulation of platelet tolllike receptors 4 and 2 by microbial structures (2, 3). It is proposed that NET formation is the first line of defense of the innate immune system, providing an effective way of trapping pathogenic microbes and removing them from the circulation (4). The DNA and histones of NETs provide a scaffold for cell localization (including neutrophils and red blood cells), platelet aggregation, and activation that serves to promote coagulation and thrombosis (5). Thus, NETs are a focus of significant crosstalk between innate immunity, inflammation, and hemostasis, and there are many points of contact including bidirectional interactions with platelets, inflammatory cytokines (IL-8), fibrinogen, fibronectin, von Willebrand factor, tissue factor pathway inhibitor, protein C, thrombomodulin, factor XII, and neutrophil-derived serine proteases (3-6). Histones H3 and H4 have been identified as critical players and mediators of damage able to stimulate phosphatidylserine exposure and factor Va expression on platelets and interact with polyphosphates to promote clotting even in the absence of factor XII (3). Histones may come from NETs or dying cells and are implicated in the progression of sepsis. Stimulation of coagulation by NETs can result in unwanted thrombosis (7), and infection is a common event in the development of deep vein thrombosis (8, 9). Targeting the release of nucleosomes, development of NETs, and availability of circulating histones could be a strategy for pre-* This work was supported by Wellcome Trust Grant 083174, Hungarian Scientific Research Fund Grants OTKA 75430 and 83023, and German Academic Exchange Service Grant DAAD A/12/01760.