TREM2受体在血小板中的表达及功能

Coronary artery disease (CAD), also known as ischemic heart disease, is the No.1 killer worldwide. The underlying cause of CAD is atherosclerosis—a slow, progressive buildup of deposits called plaques within the walls of coronary arteries. The pathological basis of CAD is coronary artery narrowing and obstruction. Plague rupture will trigger excessive platelet activation, thrombus formation in coronary arteries, leads to acute coronary syndrome (ACS), the most serious form of CAD; therefore, antiplatelet drugs have proven benefits to CAD patients. However, the current antiplatelet drugs are still not satisfactory due to limited efficacy and bleeding adverse effect, and the improvement in efficacy is often counterbalanced by an increase of bleeding. We still need novel antiplatelet agents with improved efficacy and lower bleeding risk. Intensive study on the mechanism underlying platelet activation will identify novel targets for new antiplatelet agents with better balance between antithrombotic efficacy and bleeding. TREM2 (triggering receptor expressed on myeloid cells 2), a pattern recognition receptor belonging to immunoglobulin variable (IgV) receptor superfamily, is predominantly expressed on myeloid cells including macrophages, dendritic cells, microglia, and osteoclasts. In our pursuit to identify novel antiplatelet targets, we found that 1) platelets also express TREM2 and TREM2 deficiency enhances mouse platelet aggregation; 2) deficiency of DAP12, a downstream adaptor of TREM2, increases mouse platelet aggregation and ATP release; 3) anti-TREM2, an activation antibody, abolishes human platelet aggregation and significantly inhibits Syk phosphorylation. Our preliminary data also shows dramatically impaired TREM2 expression in platelets from ACS patients. All these results clearly indicate that platelets express functional TREM2 receptor and TREM2 negatively regulate platelet activation. We hypothesize that TREM2 in platelets negatively regulates platelet activation, thrombosis, and impaired platelet TREM2 expression contributes to the pathogenesis of ACS. We also propose that TREM2 activation antibody has antiplatelet and antithrombotic effect. To test our hypothesis, we will first assay in vitro platelet activation (aggregation, ATP release, spreading, and clot retraction) using platelets from TREM2-/-, DAP12-/- mice, and ACS patients. Secondly, we will explore the role of TREM2 in thrombosis and hemostasis using TREM2-/- and DAP12 -/- mice (FeCl3-injured mesenteric arterial thrombosis model, laser-injured cremaster arteriole thrombosis model). Thirdly, we will explore the antiplatelet and antithrombotic effects of the TREM2 activation antibody by assaying in vitro (aggregation, ATP release, spreading, and clot retraction), ex vivo (flow chamber) platelet function, and in vivo thrombus formation. Platelets, whole blood will be pretreated with the TREM2 activation antibody for in vitro and ex vivo study, mice will be intravenously or intraperitoneally administered TREM2 activation antibody for in vivo thrombus formation study. Finally, we will study the mechanism underlying the negative regulation of TREM2 on platelet activation and thrombosis. We will detect p-Syk, p-ERK, p-Tyr of ITAM in DAP12, DAP12 binding to Syk and SHIP-1 in platelets stimulated with regular platelet agonists (collagen, CRP, thrombin, AYPGKF, ADP) and TREM2 activation antibody. Platelets from TREM2 knockout mice and ACS patients will be studied and compared with wild type mice and healthy donors, respectively. These studies will clarify the role of platelet TREM2 in platelet activation, thrombosis, hemostasis, and the underlying mechanism. It will also shed novel insight into the role of TREM2 in ACS as well as the use of anti-TREM2 antibody as a potential antiplatelet agent targeting TREM2 receptor.

研究血小板激活机制、发现抗血小板的新靶点有助于研发抗血小板新药。我们发现血小板表达TREM2受体(triggering receptor expressed on myeloid cells 2),TREM2敲除小鼠血小板聚集增强，TREM2接头蛋白DAP12敲除小鼠血小板聚集、ATP释放均增强,激活TREM2的抗体显著抑制人血小板聚集、Syk磷酸化，急性冠脉综合征(ACS)病人血小板TREM2表达降低。拟用TREM2、DAP12基因敲除小鼠、激活TREM2的抗体、正常和ACS病人的血小板研究：1)TREM2在血小板激活、血栓形成中的作用及TREM2负调控血小板激活的机制；2)激活TREM2的抗体的抗血小板、抗血栓作用；3)TREM2在ACS中的作用。本研究将明确TREM2在血小板激活、血栓形成、ACS中的作用和机制,并为以TREM2为靶点研发抗血小板新药提供重要的理论和实验依据。

动脉血栓性疾病是人类健康的首位杀手，抗血小板治疗效果肯定，但不尽如人意。明确血小板激活机制有助于研发新的抗血小板药。TREM2 (髓系细胞触发受体2, triggering receptor expressed on myeloid cells 2）为近年来新发现的模式识别受体，在炎症与先天免疫中发挥重要作用，在阿尔茨海默方面研究很多，针对TREM2的激活性抗体已经进入临床试验。.我们发现血小板表达TREM2后，1) 研究了其在血小板激活、动脉血栓形成、实验性心梗中的作用；2) 分析了冠心病人血小板TREM2的表达水平、与血小板反应性的关系; 3) 发现冠心病人血小板TREM2降低后，研究了血小板TREM2下调的机制；4）初步探索了TREM2负向调控血小板激活的机制；5)筛选了市面上的TREM2抗体，希望找到一种激活性抗体抑制TREM2的生物学效应，通过抗炎、抗血小板、抗血栓，防治动脉粥样硬化相关的动脉血栓性疾病。.主要发现: (1) 血小板表达TREM2, 冠心病人的血小板TREM2表达降低，而且与病人的血小板反应性增高有关; (2) 冠心病人血小板内质网应激增加，通过CHOP-C/EBPα通路下调血小板TREM2；(3) TREM2缺失增加血小板激活、炎症反应、动脉血栓形成，加重实验性心梗；(4) 筛选到一种新的、可激活TREM2的抗体, 该抗体有抗血小板、抗炎、抑制小鼠肠系膜动脉血栓和肺栓塞、减轻实验性心梗的作用；(5) TREM2/DAP12/SHIP1通路通过抑制PIP2/Akt，负向调控血小板激活。 .我们的工作进一步明确了血小板激活、血栓形成、冠心病的发病机制，丰富了模式识别受体TREM2的生物学功能，为冠心病等动脉血栓性疾病的防治提供了新的思路。.该项目已发表相关研究论文2篇，主要工作“ER stress-induced TREM2 downregulation exacerbates platelet activation and myocardial infarction in coronary artery disease.”投稿Circ Res (manuscript number: CIRCRES/2022/321824; 目前状态为“Invited de novo resubmission”.