Abstract 17883: Neutrophil Extracellular Traps as an Interface Between Inflammation and Thrombosis in DVT

Abstract

Objective: Neutrophil extracellular traps (NET) have mainly been implicated in antimicrobial host defense, but recently their contribution to arterial and microcirculatory thrombosis has been identified. These extracellular DNA structures also have been found in deep venous thrombosis (DVT), but how they participate in thrombus formation in vivo is unclear. In this study we wanted to assess the dynamics of NET formation and their impact on DVT development in vivo in a murine flow reduction model of the inferior vena cava (IVC). Methods: Thrombosis was induced in C57Bl6 mice by placing a narrowing ligature around the IVC, resulting in a reduction of blood flow velocity (n=16). NET formation in vivo was visualized by intravital epifluorescence microscopy. The IVC was harvested 48h after flow reduction, weighed and stained by immunofluorescence. Thrombogenesis and NET formation were quantified in DNase, anti-H2A-H2B-DNA antibody, and heparin treated animals as well as in GPIb-/- mice. Results: Extracellular DNA could be detected in vivo as early as 3h after flow reduction (n=3). These DNA structures were positive for neutrophil elastase, myeloperoxidase, and histones, confirming that they are indeed NETs. In the IVC harvested 48 hours after the flow restricting procedure, we found that NETs were binding platelets, tissue factor, and fibrinogen. The functional impact of NETs for DVT formation is indicated by the finding that disruption of NETs by DNase (n=5) or anti-H2A-H2B-DNA antibody (n=5) treatment resulted not only in a reduced number of NETs, but also in a significantly reduced thrombus weight compared to wt control. Surprisingly, injection of heparin resulted in a diminished number of NETs inside the IVC, which could add to its antithrombotic effect (n=3). In addition, in the IVC of GPIb-/- mice only very few NETs could be identified, suggesting that platelets trigger NET formation during DVT (n=3). Conclusion: Here we show, that neutrophils contribute to DVT by NET formation. This provides a platform for platelet adhesion and concentration of procoagulatory factors on their surface, linking inflammation and thrombosis at the cellular level. Thus, disruption of NETs could be an interesting new therapeutic approach for prophylaxis and treatment of DVT.