Inflammasome-independent mechanism of NLRP3 is critical for platelet GPIb-IX function and thrombosis

Introduction Platelets bridge thrombosis and inflammation, but how platelets use the endogenous intraplatelet inflammatory machinery is less well understood. NACHT, LRR, and PYD domain-containing protein 3 (NLRP3) is best known as the central component of the interleukin (IL)-1-producing inflammasome. Unveiling a cell type-specific mechanism of NLRP3 in platelets may improve our understanding of thrombotic diseases. Methods Ferric chloride-induced mesenteric arteriole thrombosis, tail bleeding models and microfluidic whole-blood perfusion were used to assess thrombosis and hemostasis. Additionally, we utilized aggregometry, flow cytometry, immunoprecipitation and western blot to investigate glycoprotein (GP) Ib-IX mediated platelet function and signaling. Results Our findings revealed that NLRP3-/- mice displayed severely impaired thrombosis and hemostasis, whereas apoptosis-associated speck-like protein containing a CARD (ASC)-/-, caspase-1-/-, or Nlrp3A350V/+CrePF4 mice did not exhibit such impairment. Subsequently, NLRP3-/- platelets exhibit reduced adhesion to injured vessel walls and collagen and impaired von Willebrand factor (vWF)-dependent translocation and rolling behavior. NLRP3 deficiency decreased botrocetin-induced aggregation and the phosphorylation of key signaling molecules in the GPIb-IX pathway. Mechanistically, diminished cAMP/PKA activity led to reduced phosphorylation of the Ser291 site on NLRP3, thereby enabling an NLRP3-filamin A interaction. This interaction accelerated the dissociation of filamin A from GPIbα, which allowed the 14-3-3ζ-dependent upregulation of GPIb-IX affinity to vWF. Finally, platelet NLRP3 largely regulated thrombotic disease models such as 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.