跨膜型二硫键异构酶TMX3调控血栓形成的作用与机制

Thrombosis is the clot formation inside blood vessel, resulting from the activation of platelets and coagulation system. The mechanisms underlying the initiation of thrombosis remain largely unknown. Thiol isomerases are enzymes catalyzing the oxidation, reduction and isomerization of thiol-disulfide exchange, thus they are important in normal protein folding and function. There are two groups of thiol isomerases, secretion-type and transmembrane-type. Recently we and others have reported that the secretion-type thiol isomerases, PDI and ERp57, are important in thrombosis. However, the role of transmembrane-type thiol isomerases in thrombosis has never been characterized. Our new preliminary studies have suggested that transmembrane-type thiol isomerase TMX3 was expressed in platelets and endothelial cells, and regulated platelet function and coagulation system. In contrast to secretion-type thiol isomerases, the expression of TMX3 on membrane surface was constitutive and increased upon cell activation, we thus hypothesize that TMX3 plays an important role in the maintenance of homeostasis of haemostatic proteins and the regulation of their dynamic activation. This proposal is innovative, because this is the first study of the role of transmembrane-type thiol isomerases in thrombosis. In this proposal, to test our hypothesis, we have generated tissue-specific gene knockout mouse model lacking TMX3 and monoclonal anti-TMX3 antibodies, and plan to utilize in vivo thrombosis assays, cell biological, biochemical and molecular approaches. In Aim 1 we will determine the specific role of TMX3 in platelet accumulation and fibrin deposition, and will define the cellular sources of TMX3 for the initiation of thrombosis. In Aim 2 we will dissect the molecular mechanisms by which TMX3 regulates the functions of platelet receptors and the activities of coagulation factors. Our proposed investigations will provide fundamental knowledge of the physiological role of transmembrane-type thiol isomerase in thrombosis, and novel insight into the integration mechanisms of platelet function and coagulation activation. Thus, the results to be produced in this proposal will not only improve in depth our understanding of the molecular and cellular mechanisms of thrombosis, but will also identify new therapeutic target for anti-thrombotic treatment.

血栓形成是由血小板活化和凝血系统激活所致，其机制非常复杂。二硫键异构酶催化巯基-二硫键的氧化还原，包括分泌型和跨膜型两种类型。近来研究表明分泌型二硫键异构酶在血栓形成中起重要作用，但是跨膜型二硫键异构酶是否参与血栓形成不清楚。我们的前期研究提示，跨膜型二硫键异构酶TMX3介导血小板活化和凝血系统激活，但与分泌型二硫键异构酶不同的是，TMX3在静息血小板和内皮细胞膜表面呈构成性表达，并随细胞活化而增加。基于这些新的发现，我们提出了TMX3在血小板和凝血系统的稳态维持和活化中起重要调控作用的假说。为验证该假说，在本项目中我们建立了TMX3基因敲除小鼠和抗TMX3单克隆抗体，将判定TMX3调控血小板活化和凝血系统激活的特异性作用，剖析其细胞来源及作用的分子机制，并检测抗TMX3抗体的抗血栓效果。预期结果将为阐明血栓形成的细胞与分子机制带来突破性认识，并推动以二硫键异构酶为靶向的抗血栓药物的研发。

当前我们当前对跨膜型二硫键异构酶在血栓形成中作用的认识存在空白，本项目这对这一研究背景，从血栓形成、血小板活化和凝血途径激活等多个层面，应用基因敲除小鼠，结合巯基质谱，血栓形成模型，细胞及分子等实验技术，对跨膜型二硫键异构酶TMX3在血栓形成中的特异性生理作用进行了全面而系统的研究，确定了在血栓形成中起双重调控血小板积聚和纤维蛋白生成的关键作用，及在血栓形成早期TMX3的细胞来源，剖析了TMX3在血小板和凝血系统活化中作用靶点及分子机制，探索了TMX3的靶向抑制剂防治血栓形成的效果。 这些研究结果为阐明整个二硫键异构酶家族如何调控血小板活化和凝血系统激活的协同作用和整合机制提供了必要的基础资料，为揭示血栓形成启动的细胞和分子机制提供了创新性认识，为充分认识血栓形成的氧化还原调控网络提供了新的资料。本项目通过对TMX3调控血栓形成的作用和机制的认识，将为血栓性疾病的预测、评估及干预提供新的研究途径。在机体正常情况下，血液处于氧化还原内稳态，血液与血管系统的细胞表面TMX3的表达水平较低。但是当血管损伤时，随着氧化还原内稳态的破坏，TMX3在活化的血小板和内皮细胞的表达和酶活性增加而达到阈值浓度，构成启动和加速血小板活化和凝血系统激活的重要因素。因此，本项目通过解析TMX3的动态变化和活化机制，有助于判定TMX3表达水平和酶活性能否作为预测血栓倾向和评估血栓危险程度的指标。此外，对于跨膜型二硫键异构酶调控血小板活化和凝血系统激活共性机制的深入认识，为开发有效而特异的跨膜型二硫键异构酶抑制剂奠定必要的理论基础，从而推动以跨膜型二硫键异构酶为靶向，兼具阻断血小板和凝血系统活化双重效应的抗血栓药物的研发，同时为如何避免出血并发症提供必要的资料。