Neutrophil extracellular traps promote deep vein thrombosis in mice

Abstract

Summary Background Upon activation, neutrophils can release nuclear material known as neutrophil extracellular traps (NETs), which were initially described as a part of antimicrobial defense. Extracellular chromatin was recently reported to be pro-thrombotic in vitro and to accumulate in plasma and thrombi of baboons with experimental deep vein thrombosis (DVT). Objective To explore the source and role of extracellular chromatin in DVT. Methods We used an established murine model of DVT induced by flow restriction (stenosis) in the inferior vena cava (IVC). Results We demonstrate that the levels of extracellular DNA increase in plasma after 6 h IVC stenosis, compared to sham-operated mice. Immunohistochemical staining revealed the presence of Gr-1-positive neutrophils in both red (RBC-rich) and white (platelet-rich) parts of thrombi. Citrullinated histone H3 (CitH3), an element of NETs’ structure, was present only in the red part of thrombi and was frequently associated with the Gr-1 antigen. Immunofluorescent staining of thrombi showed proximity of extracellular CitH3 and von Willebrand factor (VWF), a platelet adhesion molecule crucial for thrombus development in this model. Infusion of Deoxyribonuclease 1 (DNase 1) protected mice from DVT after 6 h and also 48 h IVC stenosis. Infusion of an unfractionated mixture of calf thymus histones increased plasma VWF and promoted DVT early after stenosis application. Conclusions Extracellular chromatin, likely originating from neutrophils, is a structural part of a venous thrombus and both the DNA scaffold and histones appear to contribute to the pathogenesis of DVT in mice. NETs may provide new targets for DVT drug development.

Elevated plasma neutrophil elastase concentration is associated with disease activity in patients with thrombotic thrombocytopenic purpura

“…In genetically (rare or common variants in ADAMTS13) or immunologically (ADAMTS13 inhibitors) predisposed individuals, activation of neutrophils and release of NETs may precipitate acute TTP by multiple mechanisms. As shown in mouse model, DNA and histones stimulate thrombosis and promote cytotoxicity [25][26][27]. In addition, reactive oxygen species released by activated neutrophils have prothrombotic effect, mediated in part by inhibition of VWF cleavage by ADAMTS13 [28].…”

Biology, role and therapeutic potential of circulating histones in acute inflammatory disorders

“…The ability of NETs and histones to influence the coagulation cascade and actually initiate venous thrombosis8, 133, 134 is the most recent detail in the emerging field of NETosis research. Clinically, circulating nucleosomes are independent prognostic markers of disseminated intravascular coagulopathy (DIC)135 and some countries, notably Japan, are actively promoting the use of anticoagulants as histone detoxification agents in DIC 136.…”

Coagulation and the Vessel Wall in Pulmonary Embolism

“…1). Treatment of mice with DNase1 21,52 cleaved the NET scaffold and prevented thrombus formation underscoring the importance of NETs for DVT. 50 In vitro, NETs provide a tissue-type plasminogen activator (t-PA)-thrombolysis-resistant scaffold for blood clots.…”

“…NETs are abundant in experimental deep-vein thrombi in baboons 50 and mice, 21,52 where they colocalize with vWF and fibrin. 50,52 NETs are predominantly found in the red, RBC-rich part of the thrombus (Fig.…”