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.

Deep vein thrombosis (DVT) and its complication, pulmonary embolism, are frequent causes of disability and mortality. Although blood flow disturbance is considered an important triggering factor, the mechanism of DVT initiation remains elusive. Here IntroductionAnnually, approximately 900 000 people in the United States suffer from deep vein thrombosis (DVT) and its life-threatening complication, pulmonary embolism. 1 Although various risk factors predisposing to DVT have been clinically identified, mechanisms of thrombus initiation remain unclear. The classic Virchow triad attributes an important role in thrombosis to the combination of blood flow restriction, hypercoagulable state of the blood, and prothrombotic changes in the vessel wall. In contrast to arterial thrombosis, which usually starts from platelet adhesion to the exposed subendothelial matrix or ruptured atherosclerotic lesion, in DVT, the endothelial layer shows no major damage. 2 In accordance with this triad, disturbances of blood flow and stasis in veins are considered major pathogenic factors of DVT, 3 and several hereditary procoagulant conditions, such as prothrombin G20210A mutation, 4 deficiency in protein C, 5 or resistance to activated protein C 6 due to Factor V Leiden mutation, 7 are associated with an increased risk of DVT. However, disturbed blood flow may be a dominant factor as it can provoke DVT as a result of long-term immobilization without marked blood hypercoagulability. 8,9 It is known that hypoxia activates endothelium, promotes Weibel-Palade body (WPB) release, and facilitates blood coagulation. 10,11 It has been hypothesized that hypoxia-induced expression of P-selectin, an adhesion receptor stored in WPBs, on the endothelial surface may recruit tissue factor-rich microparticles, thus initiating thrombus development. 12 Indeed, endothelial cells in the inferior vena cava (IVC) rapidly express P-selectin from WPBs and recruit tissue factor in stasis-induced DVT in rats. 13 Whether flow disturbance per se without complete occlusion can induce release of WPB constituents remains unknown.WPBs also contain von Willebrand factor (VWF), a multimeric protein mediating platelet adhesion to the vessel wall and platelet recruitment to the growing thrombus. 14 VWF is released as a large multimer, 15 which is then cleaved by a plasma enzyme, 16,17 now designated as a disintegrin-like and metalloproteinase with thrombospondin type-1 motifs 13 (ADAMTS13), 18,19 to smaller molecules with lower prothrombotic activity. The A1 domain of VWF binds the platelet glycoprotein Ib␣ promoting platelet adhesion and incorporation in a developing thrombus. The key impact of VWF in arterial thrombosis has been repeatedly demonstrated, and more recently the role of VWF in platelet plug formation in injured venules was reported. 20 However, whether VWF plays a role in platelet recruitment in DVT remains elusive.In the present work, we investigated whether platelet interaction with VWF expressed on the endothelium as a result of flow restriction plays ...