血小板膜受体蛋白GPIb-IX复合物结构和功能的进化机制研究

Platelets are anuclear circulating cells derived from megakaryocytes, which can form thrombi to sustain hemostasis after vessel injury. This well-known cell type has extremely high blood coagulation capability than any other coagulation-associated cells in evolution, and exists exclusively in mammals. In lower vertebrates, a group of nucleated thrombocytes are believed to express many platelet proteins and function analogously to platelets. Recent study showed that avian thrombocytes responded to many of platelets stimuli, but didn't form occlusive thrombi. This phenomenon indicates that the existence of platelets enables mammals to avoid blood loss and essentially obtain evolutionary advantages. However, the excessive coagulation capability of platelets also can trap humans into high risk of cardiovascular diseases. .The glycoprotein (GP) Ib-IX complex is one of the major adhesion receptors expressed on the surface of circulating platelets. The GPIb-IX complex contains 3 subunits, GPIbα，GPIbβ and GPIX, with a 1:2:1 stoichiometry. Covalent and noncovalent interactions are important to the quaternary stabilization of the receptor. GPIbα links to 2 GPIbβ subunits through membrane-proximal disulfide. GPIX tightly associates with GPIb through noncovalent interactions. The platelet glycoprotein (GP) Ib-IX complex is an adhesion receptor complex that helps to initiate platelet activation and aggregation under hemostatic and thrombotic conditions. .Our previous results showed the regulation of GPIbα shedding by GPIbβ intracellular domain and the quaternary organization of GPIb-IX complex by the structures of GPIbβ and a GPIbβ /GPIX chimera. However, compared to GPIb-IX complex in mammals, less is known about the structure and function of the complex in lower vertebrates. In order to increase our understanding of GPIb-IX complex during evolution, study will be carried out to identify and characterize each subunit of the complex in chicken and zebra fish. The assembly and function of the complex will also be studied. In addition, the structure and function of the complex in platelets will compared to that of in thrombocytes to explore the evolutional mechanism of the complex. The goal of this project is to establish a solid basis for the evolution of GPIb-IX complex which can provide insight into the prevention and treatment of cardiovascular diseases.

血小板是一类仅存在于哺乳动物体内没有细胞核的小型循环细胞，它能在伤口处快速凝结。低等脊椎动物的凝血细胞能具有与血小板相似的凝血功能。最近研究显示，血小板的进化极大的提高了外伤的愈合能力，但也将人类暴露于心血管疾病的威胁之下。GPIb-IX复合物是血小板凝血及血栓形成中的关键蛋白复合体，其表达也广泛分布在低等脊椎动物中。我们前期对人类GPIb-IX复合物的结构与功能进行了深入研究，了解人GPIb-IX复合物组装及参与凝血的机制。然而，低等脊椎动物中GPIb-IX复合物的结构和功能仍不清楚，其在进化过程中的演变尚不明确。本课题将深入研究低等脊椎动物鸡和斑马鱼GPIb-IX复合物的组装和凝血功能，并与哺乳动物GPIb-IX复合物进行全面比较，揭示GPIb-IX复合物中各亚基参与凝血过程的进化机制。由此了解由血小板参与的哺乳动物所独有的动脉血栓性疾病发病机理，为动脉血栓性疾病的治疗提供新思路。

GPIb-IX复合物是血小板膜上主要受体蛋白，参与血小板的凝血功能。GPIb-IX复合物由三个亚基GPIbα GPIbβ和GPIX以1：2：1的比例表达在血小板膜上。我们前期在转染的CHO细胞中，对GPIb-IX复合物在膜上表达和正确组装进行深入研究，证实了各亚基通过跨膜区以及GPIbβ和GPIX亚基胞外区相互作用组装成GPIb-IX复合物。然而，在其他低等脊椎动物中是否有GPIb-IX复合物形成，GPIb-IX复合物各亚基在进化过程中从低等动物凝血细胞膜上到高等动物血小板膜上表达和组装是否发生变化并不是很清楚。在该项目中，我们证明了鸡凝血细胞中存在GPIb-IX复合物各亚基，各亚基组装和人GPIb-IX复合物各亚基组装方式不同。我们首先从鸡凝血细胞中克隆了鸡GPIbα, GPIbβ和GPIX基因。我们进一步研究显示鸡GPIb-IX复合物在膜上表达与人类似：只有三个亚基共同存在时，复合物才能在膜上高效表达。人血小板有大量GPIb（SS-连接的 GPIbβ-GPIbα-GPIbβ）和极少量non-GPIb（SS-连接的 GPIbβ-GPIbα-GPIbα-GPIbβ），只有当GPIX亚基不存在，GPIbα和GPIbβ才会形成大量的non-GPIb。GPIb的形成是人GPIb-IX复合物正确组装和行使功能的前提。然而，我们对鸡GPIb-IX复合物组装的研究发现，鸡GPIb-IX复合物中GPIbα和GPIbβ主要形成non-GPIb。我们进一步对人和鸡GPIb-IX复合物中各亚基及各亚基相互作用区域进行替换发现人GPIX亚基在膜上的表达仅依赖于人而非鸡GPIbβ跨膜区，反之亦然。我们对多个物种间GPIbβ跨膜区进行分析发现它在哺乳动物，鸟类，鱼类，爬行类和两栖类中并不保守。上述这些结果证实了鸡GPIbα, GPIbβ 和 GPIX可以形成类似人的GPIb-IX复合物，但是组装方式完全不同。GPIbβ和GPIX胞外相互作用区域在人和鸡中是保守的，但是各物种间GPIbβ跨膜区完全不同，从而决定了不同物种间GPIb-IX的组装方式不同。GPIbβ跨膜区序列差异，序列差异对整个复合物组装的影响与高等动物血小板和低等动物凝血细胞差异一致，揭示了低等动物GPIb-IX复合物组装对于低等动物凝血细胞的功能起到非常重要的作用，有助于我们更好理解这一复合物的起源和进化，加深对该复合物认识。