NLRP3 regulates platelet integrin αIIbβ3 outside-in signaling, hemostasis and arterial thrombosis

In addition to hemostatic function, platelets play an important role in regulating the inflammatory response. The platelet NLRP3 inflammasome not only promotes IL-1β secretion, but was upregulated during platelet activation and thrombus formation in vitro. However, the role of NLRP3 in platelet function and thrombus formation in vivo remains unclear. In this study, we aimed to investigate the role of NLRP3 in platelet integrin αIIbβ3 signaling transduction. Using NLRP3-/- mice, we showed NLRP3-deficient platelets exhibit no significant differences in expression of the platelet-specific adhesive receptors αIIbβ3 integrin, GPIbα or GPVI, however NLRP3-/- platelets transfused into wild-type mice resulted in prolonged tail-bleeding time and delayed arterial thrombus formation, as well as exhibiting impaired spreading on immobilized fibrinogen and defective clot retraction, concomitant with decreased phosphorylation of c-Src, Syk and PLCγ2 in response to thrombin stimulation. Interestingly, addition of exogenous recombinant IL-1β reversed the defect in NLRP3-/- platelet spreading and clot retraction, and restored thrombin-induced phosphorylation of c-Src/Syk/PLCγ2, whereas an anti-IL-1β antibody blocked spreading and clot retraction mediated by wild-type platelets. Using direct NLRP3 inhibitor CY-09, we demonstrated significantly reduced human platelet aggregation in response to threshold concentration of collagen and ADP, as well as impaired clot retraction in CY-09-treated human platelets, supporting a role for NLRP3 in also regulating human platelet αIIbβ3 outside-in signaling. This study identifies a novel role for NLRP3 and IL-1β in platelet function, and provides a new potential link between thrombosis and inflammation, suggesting therapeutic targeting NLRP3 or IL-1β might be beneficial for treating inflammation-associated thrombosis.