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

描述(申请人提供)：在剪切力下，血小板血栓的形成是由血小板受体糖蛋白(GP)Ib-IX复合体与其配体von Willebrand因子(VWF)结合而开始的。这种受体-配体的相互作用是将血小板与受损的血管壁捆绑在一起，作为整合素介导的牢固阻止的先决条件。这种相互作用的异常会导致Bernard-Soulier综合征(BSS)或血小板型von Willebrand病(VWD)。长期以来，人们一直认为GP Ib-IX复合体/VWF相互作用只提供了使流动的血小板减速的物理力。然而，最近我们开始意识到，当GP Ib-IX复合体与VWF相互作用时，可以启动跨膜信号事件以激活整合素，导致血小板牢固的黏附和聚集。信号分子如Src家族激酶14-3-3&GT和PI-3-Kinase，通过与单个多肽的胞质结构域结合，介导这些事件。脂类结构域，也被称为糖鞘糖脂富集膜(GEMS)，可以作为GP Ib-IX复合体下游信号分子组装的平台。膜胆固醇耗竭使GP Ib-IX复合体从宝石中解离，从而消除了血小板的活化和与VWF的黏附。然而，关于宝石结合的GP Ib-IX复合体的结构元素是什么，这种相互作用是如何调节的，以及GEMS结合在GP Ib-IX功能中的生理相关性等基本问题从未得到回答。我们的初步数据表明，GP Ib1与GEMS结构域的联系主要是由GP Ib2/GP IX介导的。去除GP Ib1和GP Ib2/GP IX之间的二硫键不仅抑制GP Ib1与GEMS结构域的结合，而且在高剪切力下也抑制表达GP Ib-IX复合体的CHO(中国仓鼠卵巢)细胞与VWF的相互作用。此外，我们还发现蛋白质二硫键异构酶(PDI)与血小板中的GP Ib-IX复合体以及表达GP Ib-IX复合体的CHO细胞中的GP Ib-IX复合体相关联，这种相互作用仅见于GEMS结构域。此外，我们还证明，血小板膜脂组成的改变既抑制了GP Ib-IX复合体与GEMS结构域的结合，也抑制了复合体介导的血小板与VWF的相互作用。根据这些结果和以前报道的证据，我们假设：1)GP Ib2/GP IX含有介导GEMS与GP Ib-IX复合体相互作用的结构决定因素；2)蛋白质二硫键异构酶在GP Ib1与GP Ib2/GP IX之间形成二硫键连接的过程中起着重要作用；3)特定的宝石脂组成是复杂结合的关键；以及4)GP Ib1与GEMS结构域的结合被特异性地阻断，取消了GP Ib-IX复合体的功能。总之，这项研究将有助于阐明GP Ib-IX复合体在血小板表面正确定位的结构基础，探索GP Ib-IX复合体功能调节的新机制，最终为开发新的治疗策略以对抗各种复杂的出血性疾病和心血管疾病提供机制指导。 公共卫生相关性：血小板膜脂域在血栓形成和止血中对血小板功能非常重要。血小板膜糖蛋白GP Ib-IX复合体是受膜脂结构域调控的蛋白质之一。在本项目中，我们着眼于研究这一调控机制。

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.