MEKK3调控血小板活化和动脉血栓形成的机制研究

MAPK/ERK kinase kinase 3 (MEKK3), belongs to MAP3K family, is an important activator of three-tiered classical MAPK pathways. Although MEKK3 plays essential roles in cardiovascular development and immune responses, its functions in platelet activation and thrombosis formation still unknown. Mitogen-activated protein kinases (MAPKs, including ERK1/2, p38 and JNKs), downstream of MEKK3, were shown to regulate platelet activation and function positively. However, the molecular mechanisms by which the MAPKs are regulated in platelets remain largely unknown. In this study, platelet-specific MEKK3-deficient mice were developed to elucidate the platelet-related functions of MEKK3. Ablation of MEKK3 in platelets resulted in the delay of FeCl3-induced carotid artery occlusion in vivo. And also, MEKK3 may regulate ADP-induced platlet aggregation through the phosphrolation of ERK1/2. Therefore, we proposed that MEKK3 had vital roles in platelet activation indeedly. This project presented here would hopefully illuminate the functions and molecular mechanism of MEKK3-MAPKs pathway regulating platelet aggregation, secretion, adhesion, spreading and clot retraction by means of platelet-specific gene knockout mice and kinds of pharmacological inhibitors. Thus, this project could demonstrate the roles of MEKK3 in platelet activation mediated by G-protein-coupled receptors (GPCRs) signaling, adhesive receptors (GPIb-IX-V or GPVI) signaling and integrin αIIbβ3 bidirectional signaling, respectively. On the other hand, the roles of MEKK3 in thrombosis formation were defined by "FeCl3-induced carotid or mesenteric artery thrombosis model" in the presence of MEKK3-MAPKs pathway specific inhibitors or vehicle control. This study is likely to highlight a new path to develop anti-platelet drugs for cardiovascular diseases therapy.

MEKK3是位于MAPK三级激酶级联通路中较上游的调控激酶。而MAPK家族中的ERK、p38及JNKs已被证实可正调控血小板的活化与功能。迄今，血小板中MAPKs受调于何种激酶及其被活化的机制仍不清楚。尽管研究表明，MEKK3在心血管发育及免疫过程中起重要调控作用，而其在血小板活化及动脉血栓形成中的作用尚未见报道。我们首先构建了血小板特异缺失MEKK3的小鼠；初步研究表明，血小板缺失MEKK3的确延缓了氯化铁诱导的颈动脉血栓形成栓塞的时间；且MEKK3可能通过磷酸化ERK来调控ADP引起的血小板聚集。在本项目中，我们期望通过基因敲除小鼠及各种抑制剂来阐明MEKK3在血小板聚集、分泌、粘附、铺展及栓块收缩过程中的调控作用与机制。在体内，我们通过FeCl3诱导的血栓模型并联合各类抑制剂来阐明MEKK3调控血栓形成的功能与机制。本项目有望为研发治疗心血管疾病及其并发症的抗血小板药物提供全新途径。

目的—微栓塞作为心肌梗塞后的严重并发症损害了微脉管环境，增加了心衰，心律不齐以及死亡的发生率。Sin1作为mTORC2复合体的重要组成成分，在细胞增殖，营养，压力和活性氧代谢反应以及AKT和PKC的活化过程中起重要作用。然而，活化和建立Sin1/mTORC2通路在缺血诱导的微栓塞中仍然缺乏研究。.方法和结果—mTORC2靶点AKT-S473的磷酸化在非体外循环冠脉搭桥术病人的左前降支远端血液的血小板中显著升高。与此发现一致，Sin1-T86磷酸化也显著升高。重要的是，我们观察的61位ST-段太高的心肌梗塞术前病人与血管再生后无复流现象中的Sin1磷酸化和AKT磷酸化水平有好的相互关联。我们建立了血小板特异Sin1缺失和Sin1-T86磷酸化缺失的小鼠模型来阐述血小板活化的潜在机制。机制上看来，Sin1-T86磷酸化增强了mTORC2介导的下游信号；Sin1-T86磷酸化调控αIIbβ3-介导的由外向内信号，并在NAD+/Sirt3/SOD2通路介导的低氧/活性氧产生过程中起重要作用。重要的是，在心肌梗塞的小鼠模型中，血小板特异Sin1缺失保护小鼠免受缺血诱导的微脉管栓塞和随后的心脏功能紊乱。.结论—综上所述，我们的研究揭示了Sin1在血小板活化过程中的新功能。因此，Sin1可能作为一个有价值的药物靶点，从而预防缺血诱导的心肌梗塞的恶化。