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.

简介 血小板将血栓形成和炎症联系起来，但血小板如何处理内源性血小板内炎症机制尚不清楚。含 NAHT、LRR 和 PYD 结构域的蛋白 3 (NLRP3) 是产生白细胞介素 (IL)-1 的炎症小体的核心成分。阐明血小板中 NLRP3 的细胞类型特异性机制可能会提高我们对血栓性疾病的认识。

方法 采用氯化铁致肠系膜小动脉血栓模型、尾部出血模型、微流控全血灌注模型研究血栓形成及止血作用。此外，我们利用聚集分析、流式细胞术、免疫沉淀和蛋白质印迹来研究糖蛋白 (GP)Ib-IX 介导的血小板功能和信号传导。

结果 NLRP3 ^{−/−小鼠表现出严重受损的血栓形成和止血功能，而含有 CARD (ASC) −/−}、 caspase-1 ^−/−和Nlrp3 ^A350V/+ CrePF4小鼠的凋亡相关斑点样蛋白则没有表现出此类变化。 NLRP3 ^-/−血小板对受损血管壁和胶原蛋白的粘附力降低，血管性血友病因子 (vWF) 依赖性易位和滚动行为受损。 NLRP3 缺乏会降低 Botrocetin 诱导的血小板聚集和 GPIb-IX 通路中关键信号分子的磷酸化。从机制上讲，cAMP/PKA 活性降低导致 NLRP3 磷酸化减少，从而实现 NLRP3 和细丝蛋白 A 之间的相互作用。这种相互作用加速了细丝蛋白 A 从 GPIbα 的解离，从而使 GPIb-IX 出现 14-3-3ze 依赖性增加与 vWF 的亲和力。最后，发现血小板NLRP3在很大程度上调节血栓性疾病模型，例如中风和深静脉血栓形成模型。

结论 NLRP3 促进主要血小板粘附受体 GPIb-IX 的功能，而不涉及 NLRP3 炎性体组装或 IL-1β 产生。