Neutrophil extracellular trap (NET) impact on deep vein thrombosis

Abstract

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 others have recently shown that extracellular DNA fibers produced in inflammation and known as neutrophil extracellular traps (NETs) contribute to experimental DVT. NETs stimulate thrombus formation and coagulation and are abundant in thrombi 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 of NETs with endothelium, platelets, red blood cells, and coagulation factors and how NETs could influence thrombolysis. We propose that drugs that inhibit NET formation or facilitate NET degradation may prevent or treat DVT.

Figure 1. NETs in the timeline of DVT: a model

(A) DVT is initiated by local hypoxia and activation of endothelial cells (EC) as a result of flow restriction/disturbances. Activated endothelium releases ultra-large von Willebrand factor (ULVWF) and P-selectin from Weibel-Palade bodies (WPB) which mediate platelet and neutrophil adhesion. Activated platelets recruit tissue factor (TF)- containing microparticles that enhance thrombin generation in the growing thrombus. (B) Activated platelets and endothelium or other stimulus induce NET formation in adherent neutrophils. NETs provide an additional scaffold for platelet and RBC adhesion, promote fibrin formation, and exacerbate platelet and endothelial activation. (C) Plasmin, ADAMTS13 and DNase mediate thrombolysis by degrading fibrin, ULVWF and DNA, respectively. Monocytes/macrophages (MØ) release an additional source of DNase and generate plasmin and promote restoration of blood flow.

Figure 2. NET formation and function

A) Electron micrograph of NETs with trapped Salmonella typhimurium; Bar: 1µm. Courtesy of Dr. Volker Brinkmann, Max Planck Institute for Infection Biology, Berlin, Germany. (B) Electron micrograph of NETs with adherent platelets. Bar: 1µm. Reproduced and modified from reference . (C) Scheme of NET formation (NETosis). Enzymes from granules (red) translocate to the nucleus (blue) and facilitate chromatin decondensation. Internal membranes break down and cytolysis releases NETs.

Figure 3. NETs are a tPA-resistant scaffold of blood clots

(A) Photographs of blood clots. Citrated blood is mixed with neutrophils, which are pre-stimulated to release NETs. Blood is supplemented with DNase and/or tPA as indicated and clotting is induced by re-calcification. Controls (Ctrl) do not receive DNase or tPA. tPA in combination with DNase dissolves blood clot. Bar: 1cm. (B) Fluorescence images of DNA (light blue), fibrinogen (green) and VWF (red) in blood clots shown in panel A. Extracellular DNA co-localizes with fibrin in untreated blood clots (Ctrl). DNase degrades extracellular DNA but not nuclei and fibrin in blood clots. tPA induces degradation of fibrin but not extracellular DNA. The remaining extracellular DNA scaffold is sufficient to retain RBCs and platelets (indicated by punctuate VWF staining). In this in vitro experiment, ultra large VWF multimers were not present and their role in the thrombus scaffold could not be evaluated. Bar: 50µm. Reproduced and modified from reference .