THU0051 Tissue Factor Expression in Neutrophil Extracellular Traps in Patients with Active ANCA Associated Vasculitis

Background ANCA-associated vasculitis (AAV) is a group of disorders characterized by neutrophil activation [1], an exuberant inflammatory response and hypercoagulability associated with a significant increase in the prevalence of venous thromboembolism [2]. Neutrophils may contribute to thromboinflammation through the release of neutrophil extracellular traps (NETs) and the expression of tissue factor (TF) [3]. Objectives To study the role of neutrophils in thromboinflammation in AAV. Methods Neutrophils from three patients and sera from 16 patients with ANCA-associated vasculitis with active disease and remission were collected. TF expression was assessed by immunoblotting in cell lysates and confocal microscopy in NETs. Serum DNA levels were also evaluated after staining with propidium iodide and fluorescence measurement with fluorometer [4]. Results Peripheral blood neutrophils from patients with active disease released NETs expressing TF visualized by confocal microscopy after staining with TF, elastase and DAPI. The activation of autophagy and the inclusion of TF in autophagosomes were further demonstrated with confocal microscopy, using the autophagy markers LC3, LysoTracker and p62-SQSTM1. Neutrophils isolated from the bronchoalveolar lavage from two patients also released TF-expressing NETs. Similar structures were also detected in nasal biopsy specimens from two patients, as indicated by the co-staining with elastase, TF and DAPI. Elevated levels of circulating DNA were further observed in sera from patients with active disease, indicating the in vivo release of NETs. TF expression, assessed by immunoblotting and release of TF expressing NETs were attenuated after successful induction of remission. Treatment of control neutrophils with sera from patients with active disease resulted in the formation of TF expressing NETs. This effect was abolished after IgG depletion. Conclusions This study demonstrates, for the first time, the expression of TF, the principal in vivo inducer of coagulation, in NETs during active AAV. TF-induced thrombin generation may activate protease activated receptor (PAR)-signaling and promote thrombosis and inflammation in active AAV. References Lane SE, et al. Primary systemic vasculitis: clinical features and mortality. QJM 2005;98:97–111. Allenbach Y, et al. High frequency of venous thromboembolic events in Churg-Strauss syndrome, Wegener’s granulomatosis and microscopic polyangiitis but not polyarteritis nodosa: a system