Abstract 369: Muscle Ischemia Induces Nlrp3 Inflammasome Activation in Platelets via Tlr4, Promoting Platelet Aggregation and Interfering With Perfusion Recovery

Abstract

Introduction: Angiogenesis is an adaptive response to chronic ischemia, but is deficient in peripheral arterial disease (PAD). We have recently shown that the pattern recognition receptors toll-like receptor 4 (TLR4) and nucleotide-binding domain leucine rich repeat containing protein 3 (NLRP3) expressed by platelets control aggregation and thrombosis. The role of platelet TLR4 and NLRP3 in PAD is unexplored. Methods: Unilateral femoral artery ligation (FAL) was performed in transgenic mice with platelet-specific ablation of TLR4 (TLR4 PF4) and in global NLRP3 knockout (NLRP3 KO) mice. Platelet NLRP3 inflammasome activation was monitored by caspase-1 activation (fluorescent labeled inhibitor of caspase-1, FLICA) and cleavage of IL1β (Western blot). Platelet aggregation was evaluated with impedance aggregometry. Laser Doppler perfusion imaging (LDPI) verified perfusion in the ischemic (right) and non-ischemic (left) limb over time. Angiogenesis and myoblast regeneration were measured histologically. Results: Platelet NLRP3 inflammasome activity was significantly upregulated following FAL (p<0.001), and reversed with TAK242, a TLR4 inhibitor. FAL significantly increased aggregation of circulating platelets, which was significantly suppressed in TLR4 PF4 (p<0.01) and NLRP3 KO mice (p<0.001). Down-regulation of platelet aggregation and caspase-1 activity in TLR4 PF4 mice was nearly completely reversed by nigericin, a NLRP3 activator. Ischemic limb perfusion (Fig 1A) was significantly higher in TLR4 PF4 (Fig1B, p<0.05) and NLRP3 KO mice (Fig1C, p<0.001) than in controls 14d after FAL. Angiogenesis and regeneration were significantly improved in TLR4 PF4 mice (p<0.05). Conclusion: We show that the platelet NLRP3 inflammasome is activated in muscle ischemia via platelet TLR4, which upregulates platelet aggregation and deters recovery from FAL. Thus, platelet TRL4/NLRP3 activation may be a therapeutic target to improve limb salvage in PAD.