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

Abstract

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 NLRP3^−/− mice exhibited severely impaired thrombosis and hemostasis, whereas apoptosis-associated speck-like protein containing a CARD (ASC)^−/−, caspase-1^−/−, and Nlrp3 ^A350V/+ CrePF4 mice did not exhibit such changes. NLRP3^−/− 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.

Keywords

platelets - NLRP3 - inflammasome-independent - GPIb-IX - thrombotic diseases

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