NETs and Deep vein thrombosis

Deep vein thrombosis (DVT) is a major health problem that requires improved prophylaxis and treatment.Inflammatory conditions such as infection, cancer, and autoimmune diseases are risk factors for DVT. We and othershave recently shown that extracellular DNA fibers produced in inflammation and known as neutrophil extracellulartraps (NETs) contribute to experimental DVT. NETs stimulate thrombus formation and coagulation and are abundant inthrombi in animal models of DVT. It appears that, in addition to fibrin and von Willebrand factor, NETs represent a third

thrombus scaffold. Here, we review how NETs stimulate thrombosis and discuss known and potential interactions ofNETs with endothelium, platelets, red blood cells, and coagulation factors and how NETs could influence thrombolysis.It was proposed that drugs that inhibit NET formation or facilitate NET degradation may prevent or treat DVT. 

Deep vein thrombosis (DVT) is a debilitating disease that may be complicated by pulmonary embolism (PE). Together DVT and PE are designated as venous thromboembolism. In the United States, venous thromboembolism develops in an estimated 900000 patients each year, and PE is responsible for ≈300000 deaths, which exceeds the mortality from myocardial infarction or stroke.DVT complications, in addition to PE, include post thrombotic syndrome caused by chronic venous stasis even in the absence of active thrombosis

NETs are produced to allow neutrophils to trap and disarm microbes in the extracellular environment. NETs are scaffolds of intact chromatin fibers with antimicrobial proteins, ideal to retain large quantities of microbes. Therefore, some pathogenic bacteria have evolved to express an extracellular deoxyribonuclease (DNase), which dismantles NETs and promotes virulence. Extracellular traps are formed in humans, animals, and even plants, indicating that NETs provide an evolutionary conserved protective mechanism. 

NETs formation is not restricted to neutrophils, and different cell types use different cellular mechanisms to release extracellular trap. One mechanism used by human neutrophils is NETosis. NETosis is a multistep cell death program . On activation, certain enzymes translocate from the granules to the nucleus. Histones are degraded by neutrophil elastase (NE) and citrullinated by peptidylarginine deiminase 4 to unwind chromatin. Further hallmarks are the breakdown of granular and nuclear membranes and cytolysis as the final step in NETosis.

Implications of NETs in Thrombolysis -To degrade and solubilize thrombi to restore blood flow, fibrin  and VWF as the main scaffolds need to be proteolytically fragmented by the proteases plasmin and a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13,  respectively. NETs are newly recognized third scaffolds that need to be undone during thrombolysis (Figure 1C). NETs were seen to colocalize with fibrin in clots 15 and with VWF in venous thrombi. In vitro, we could show that NETs provide a scaffold for blood clots that is resistant to tPA-induced thrombolysis. We incubated recalcified blood with neutrophils which were prestimulated to release NETs. As shown in  Figure 3, after filtration, blood clots appeared in control samples and tPA- or DNase-treated blood but not in blood treated with the combination of tPA and DNase. Immunostainings revealed that in the presence of tPA, blood clots lacked fibrin and were held together by a scaffold  of extracellular DNA (Figure 3B). DNase1 is the predominant nuclease in plasma. Interestingly, the plasminogen system cooperates with DNase1 during chromatin degradation. DNase1 has only limited activity to degrade chromatin because it preferentially degrades protein-free DNA. Plasminogen, activated by either tPA or urokinase-type plasminogen activator, degrades histones and therefore allows for degradation of DNA by DNase1. Monocytes/macrophages may also support the DNA degradation because their lysosomes contain DNase2, which is important for the removal of apoptotic cells (Figure 1C). NETs and fibrin degradation by plasmin and DNase could result in the simultaneous release of DNA and fibrin fragments.  In baboon DVT, plasma DNA increases with similar kinetics to the fibrin degradation product D-dimers. Recently, in collaboration with Thomas Wakefield’s group, we found increased levels of DNA in plasma from patients with DVT compared with healthy controls and symptomatic patients who did not have DVT. Here also, plasma DNA con-centrations correlated with D-dimers (unpublished data; Diaz and Fuchs, 2012). Therefore, it is plausible that circulating DNA may reflect the degradation of NETs within a thrombus.NETs may also promote thrombolysis. In vitro studies have shown that NE and cathepsin G can degrade fibrin, and these proteases are present on NETs and could enhance fibrinolysis in DVT. In addition, NETs may also recruit plasminogen from the plasma. Histone H2B can serve as a receptor for plasminogen on the surface of human monocytes/macrophages and perhaps could do so in NETs.