Inflammasome-Independent Mechanism of NLRP3 is Critical for Platelet GPIb-IX Function and Thrombosis

INTRODUCTION: Platelets link thrombosis and inflammation, but how platelets handle the endogenous intraplatelet inflammatory machinery is less well understood. NACHT, LRR, and PYD domain-containing protein 3 (NLRP3) is the central component of the interleukin (IL)-1-producing inflammasome. Elucidating the cell type-specific mechanism of NLRP3 in platelets may improve our understanding of thrombotic diseases. METHODS: Ferric chloride-induced mesenteric arteriole thrombosis models, tail bleeding models, and microfluidic whole-blood perfusion were used to study thrombosis and hemostasis. Additionally, we utilized aggregometry, flow cytometry, immunoprecipitation, and western blotting to investigate glycoprotein (GP)Ib-IX-mediated platelet function and signaling. RESULTS: platelets exhibited reduced adhesion to injured vessel walls and collagen and impaired von Willebrand factor (vWF)-dependent translocation and rolling behavior. NLRP3 deficiency decreased botrocetin-induced platelet aggregation and the phosphorylation of key signaling molecules in the GPIb-IX pathway. Mechanistically, decreased cAMP/PKA activity led to reduced phosphorylation of NLRP3, thereby enabling the interaction between NLRP3 and filamin A. This interaction accelerated the dissociation of filamin A from GPIbα, which allowed a 14-3-3ζ-dependent increase in GPIb-IX affinity to vWF. Finally, platelet NLRP3 was found to largely regulate thrombotic disease models, such as models of stroke and deep vein thrombosis. CONCLUSION: NLRP3 promoted the function of the major platelet adhesion receptor GPIb-IX without involving NLRP3 inflammasome assembly or IL-1β production.