Regulation of Platelet Glycoprotein (GP) Ib-IX Complex Function by Membrane Lipid

描述（由申请方提供）：血小板受体（糖蛋白（GP）Ib-IX复合物）与其配体（血管性血友病因子（VWF））结合，在剪切应力下引发血小板血栓形成。这种受体-配体相互作用是将血小板束缚在受损血管壁上的必要条件，是整合素介导的牢固停滞的先决条件，这种相互作用的功能障碍可引起Bernard-Soulier综合征（BSS）或血小板型血管性血友病（VWD）。长期以来，人们一直认为GP Ib-IX复合物/VWF相互作用仅提供使流动的血小板减速的物理力。然而，最近我们开始认识到，在与VWF相互作用时，GP Ib- IX复合物可以启动跨膜信号传导事件，从而激活整联蛋白，导致血小板牢固粘附和聚集.信号分子，如Src家族激酶、14-3-3>和PI-3-激酶，通过与单个多肽的胞质结构域结合，介导这些事件。脂质结构域，也称为鞘糖脂富集膜（GEM），可以作为GP Ib-IX复合物下游信号分子组装的平台。GP Ib-IX复合物通过膜胆固醇耗竭从GEM解离，消除了血小板活化和与VWF的粘附。然而，基本的查询，什么样的结构元素的GP Ib-IX复杂的GEM协会，这种相互作用是如何调节，以及什么样的生理相关性的GEM协会在GP Ib-IX功能，从来没有得到回答。我们的初步数据表明，GP Ib 1与GEM结构域的关联主要是由GP Ib 2/GP IX介导的。去除GP Ib 1和GP Ib 2/GP IX之间的二硫键不仅抑制了GP Ib 1与GEM结构域的结合，而且抑制了表达GP Ib-IX复合物的CHO细胞在高剪切下与VWF的相互作用。此外，我们发现蛋白质二硫键异构酶（PDI）与GP Ib-IX复合物在血小板和表达GP Ib-IX复合物的CHO细胞中结合，仅在GEM结构域中观察到相互作用。此外，我们证明了血小板膜脂质组成的改变抑制GP Ib-IX复合物与GEM结构域的结合以及复合物介导的血小板与VWF的相互作用。基于这些结果和已有的证据，我们推测：1）GP Ib 2/GP IX含有介导GEM与GP Ib-IX复合物相互作用的结构决定簇：2）二硫键异构酶的氧化还原调节在GP Ib 1与GP Ib 2/GP IX之间形成二硫键以使GP Ib 1与GEM结合; 3）特异性GEM脂质组成对于复合物缔合是关键的;和4）GP Ib 1与GEM结构域缔合的特异性破坏消除了GP Ib-IX复合物功能。总之，这项研究将有助于阐明GP Ib-IX复合物在血小板表面正确定位的结构基础，探索GP Ib-IX复合物功能调节的新机制，并最终为开发新的治疗策略提供机制指导，以对抗各种复杂的出血性疾病和心血管疾病。 公共卫生相关性：血小板膜脂质结构域对血栓形成和止血中的血小板功能很重要。血小板糖蛋白GP Ib-IX复合物是受膜脂结构域调控的蛋白质之一。在本项目中，我们着眼于研究这种调节机制。

DESCRIPTION (provided by applicant): The formation of platelet thrombi at shear stress is initiated by the binding of the platelet receptor, glycoprotein (GP) Ib-IX complex, to its ligand, von Willebrand factor (VWF). This receptor-ligand interaction is essential for tethering platelets to the injured vessel wall as a prerequisite for integrin-mediated firm arrest.Amalfunction in this interaction causes either Bernard-Soulier Syndrome (BSS) or platelet-type von Willebrand disease (VWD). It has long been thought that the GP Ib-IX complex/VWF interaction only provided the physical force to decelerate the flowing platelets. Recently, however, we have begun to realize that upon interacting with VWF, the GP Ib- IX complex can initiate transmembrane signaling events for integrin activation, leading to platelet firm adhesion and aggregation. Signaling molecules, such as Src family kinase, 14-3-3> and PI-3-Kinase, through association with the cytoplasmic domains of individual polypeptides, mediate these events. Lipid domains, also known as glycosphingolipid-enriched membranes (GEMs), can act as a platform for the assembly of downstream signaling molecules of the GP Ib-IX complex. Dissociation of the GP Ib-IX complex from the GEMs by membrane cholesterol depletion abolishes platelet activation and adhesion to VWF. Nevertheless, basic inquiries as to what the structural elements of the GP Ib-IX complex for GEMs association are, how such interaction is regulated, and what the physiological relevance of GEMs association in the GP Ib-IX function is, have never been answered. Our preliminary data demonstrate that GP Ib1 association with the GEMs domain is primarily mediated by GP Ib2/GP IX. Removal of disulfide linkage between GP Ib1 and GP Ib2/GP IX not only inhibits GP Ib1 association with GEMs domain, but also inhibits GP Ib-IX complex-expressing CHO (Chinese Hamster Ovary) cells interaction with VWF under high shear. In addition, we found that protein disulfide isomerase (PDI) associates with the GP Ib-IX complex in both platelets and the GP Ib-IX complex-expressing CHO cells, an interaction only being seen in GEMs domain. Furthermore, we demonstrated that alteration of platelet membrane lipid composition inhibits both GP Ib-IX complex association with GEMs domain and complex- mediated platelet interaction with VWF. Based on these results and previously reported evidence, we hypothesize that 1) GP Ib2/GP IX contains the structural determinants to mediate the GEMs interaction with the GP Ib-IX complex; 2) redox regulation by protein disulfide isomerase plays a role in the formation of disulfide linkage between GP Ib1 and GP Ib2/GP IX for GP Ib1 association with GEMs domain; 3) specific GEMs lipid composition is critical for complex association; and 4) specific disruption of GP Ib1 association with GEMs domain abolishes GP Ib-IX complex function. Overall, this proposed study will help to elucidate the structural basis for the proper localization of the GP Ib-IX complex on platelet surface, explore a novel mechanism for GP Ib-IX complex function regulation, and finally, to provide a mechanistic guide for developing novel therapeutic strategies to combat various complex related bleeding disorders and cardiovascular disease. PUBLIC HEALTH RELEVANCE: Platelet membrane lipid domain is important for platelet function in thrombosis and haemostasis. The platelet glycoprotein GP Ib-IX complex is one of the proteins regulated by membrane lipid domain. In this project, we sight to investigate this regulatory mechanism.