Abstract 78: Extracellular RNA Induces Venous Thrombosis Through TLR3

Abstract

Venous thromboembolism (VTE) afflicts 117 people per 100,000 each year and is an important cause of morbidity and mortality. Vascular and circulating cells may sense ‘pathologic ligands’ accumulating in the circulation via toll-like receptor (TLR) and translate signals into a pro-thrombotic state. TLR3 recognizes self-molecules generated during tissue damage and inflammation such as extracellular RNA (eRNA). We hypothesize that eRNA and TLR3 are involved in the development of venous thrombosis after vessel injury. Injection of RNase1 decreased the size of thrombi after FeCl3-induced inferior vena cava injury (IVC) compared to mice treated with vehicle. Using a specific fluorescent probe for RNA, we found that FeCl3 induced RNA release and thus increased RNA content in the thrombus. WT or TLR3 deficient (-/-) mice received an injection of vehicle, eRNA or poly(I:C) prior to thrombosis induction. In WT mice, thrombus size was increased by eRNA or poly(I:C). Interestingly, no change in thrombus size was observed in TLR3-/- mice regardless of the treatment. Poly(I:C) treatment was associated with higher amount of neutrophils within the thrombus of WT mice compared with vehicle. In TLR3-/- mice, poly(I:C) failed to increase the recruitment of neutrophils. Staining for citrulinated H3, a marker of neutrophil extracellular traps (NETs) formation, was increased by poly(I:C) in WT mice. Interestingly, in TLR3-/- mice, no increase of NET formation was found after poly(I:C) injection as compared to vehicle control. Poly(I:C) also increased the recruitment of monocytes within thrombi in WT but not in TLR3-/- mice. Preliminary results suggest that injection of eRNA may also promote the recruitment of monocytes, neutrophils and the formation of NETs in WT but not in TLR3-/- mice. Human neutrophils were then treated with conditioned media from HUVEC treated with poly(I:C) in presence of control siRNA or TLR3 siRNA. Media from HUVEC transfected with control siRNA and treated with poly(I:C) induced the migration of neutrophils, whereas media from TLR3 siRNA and poly(I:C) treated cells did not. These results suggest that eRNA and TLR3 may participate to thrombus formation by inducing a pro-inflammatory response leading to the recruitment of monocytes and neutrophils.