Abstract 358: Factor Xa Deteriorates Atherosclerosis by Facilitating Inflammasome Formation via PAR-2-mediated Autophagy Suppression

Abstract

Background: It is known that an endogenous blood coagulation factor Xa (FXa) plays a critical role in facilitating atherosclerosis by activating protease-activated receptor-2 (PAR-2). However, the precise mechanism how FXa-mediated PAR-2 activation promotes atherogenesis remains to be elucidated. Purpose: The aim of this study is to explore how FXa promotes atherosclerosis through PAR-2-associated signaling pathway. Methods & Results: Administration of direct FXa inhibitor rivaroxaban (Riv; 120 mg/kg/day) to the mice significantly suppressed the plasma FXa activity compared with untreated mice. Administration of Riv to ApoE knockout mice fed with high fat diet (ApoE-KO-HFD) significantly reduced atherosclerotic area in the aorta compared with those in the untreated ApoE-KO-HFD. The plaque size of ApoE-KO mice crossed with PAR-2 knockout mice fed with HFD was similar to those of Riv-treated ApoE-KO-HFD. Ultrastructural examinations of atherosclerotic lesions revealed that the number of autophagosomes in the plaque-resident macrophages of Riv-treated ApoE-KO-HFD was significantly smaller than those of the untreated ApoE-KO-HFD. Immunostaining of NLRP3 revealed that Riv attenuated the inflammasome formation in the atherosclerotic lesion in ApoE-KO-HFD. In vitro experiments demonstrated that treatment of 7-ketocholesterol (7KC) markedly enhanced autophagy activity in the murine macrophages. The addition of FXa significantly promoted mTOR (Ser 2448 ) phosphorylation and blocked autophagy activity induced by 7KC, which was reversed in the presence of Riv (1 μM). Furthermore, immunoblot analyses demonstrated that FXa administration significantly accelerated inflammasome formation induced by 7KC, which was blocked in the presence of Riv. On the other hand, treatment with FXa failed to inhibit 7KC-induced autophagy and inflammasome activation in PAR-2-KO mice-derived macrophages. Conclusion: These results suggest that FXa worsens atherogenesis through PAR-2-mediated pathway by inhibiting macrophage autophagy which, in turn, promoting inflammasome activation.