PS1494 REDUCED ANTIOXIDANT CAPACITY INDUCED PYROPTOSIS IN PLATELETS IN IMMUNE THROMBOCYTOPENIA PATIENTS

Abstract

Background:The pathophysiology of primary immune thrombocytopenia (ITP) is not fully understood. Antioxidant capacitie (AOC) is reduced in ITP platelets. Mitochondrial reactive oxygen species (ROS) generation can lead to NLRP3 inflammasome activation and pyroptosis. However, the process has not been studied in ITP platelets.Aims:To assess whether the reduced AOC of platelets in ITP can affect the NLRP3 inflammasome signaling pathways and to further explore the pathophysiology of ITP.Methods:We measured mitochondrial membrane potential (MMP) of platelets from patients and healthy donors using JC‐1 by flow cytometry. Next, we investigated AOC of platelets by supplementation of 300 μM H2O2following loading with 50 μM 2’,7’‐dichlorodihydrofluorescein diacetate. ROS level as well as expression of NLRP3, ASC, pro‐caspase‐1 and p20 in platelets after stimulation with H2O2was detected. Besides, co‐localization of NLRP3 and ASC in platelets after stimulation was assessed by immunofluorescence. Blocking experiments were performed using antioxidant N‐Acetyl‐L‐cysteine, NLRP3 inhibitorMCC950 (CP‐456773) and caspase‐1 inhibitorZ‐VAD‐FMK. Furthermore, we studied the pyroptosis of platelets in passive ITP mouse model using NLRP3‐/‐ mice, Caspase‐1‐/‐ mice and wild type mice.Results:The platelets from ITP patients showed significantly reduced antioxidant capacity compared to healthy donors. A significant lower MMP and higher ROS level were detected in ITP platelets. Expression of NLRP3, ASC, pro‐caspase‐1 and p20 was obviously increased and co‐localization of NLRP3 and ASC was more evident in ITP platelets. An increased level of IL‐1β and IL‐18 were observed in culture supernatant. The application of antioxidant N‐Acetyl‐L‐cysteine (NAC), NLRP3 inhibitor MCC950(CP‐456773) and caspase‐1 inhibitor Z‐VAD‐FMK to H2O2‐stimulated ITP platelets significantly inhibit pyroptosis. The indicators of pyroptosis were down‐regulated in NLRP3‐/‐ and Caspase‐1‐/‐ mice.Summary/Conclusion:The reduced AOC of platelets in ITP results in accumulation of intracellular ROS, which contributes to the activation of NLRP3 inflammasome signaling pathways and leads to pyroptosis, which accelerates thrombocytopenia in ITP.image