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

描述（由申请人提供）：在剪切应力下血小板血栓的形成是由血小板受体糖蛋白（GP） Ib-IX复合物与其配体血管性血友病因子（VWF）结合引发的。这种受体-配体相互作用对于将血小板拴在受伤的血管壁上是必不可少的，这是整合素介导的firm arrest的先决条件。这种相互作用中的功能异常会导致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相互作用仅提供物理力来减缓血小板的流动。然而，最近我们开始意识到，GP Ib- IX复合物与VWF相互作用后，可以启动整合素激活的跨膜信号事件，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，通过与单个多肽的细胞质结构域相关联，介导这些事件。脂质结构域，也被称为糖鞘脂富集膜（GEMs），可以作为GP Ib-IX复合物下游信号分子组装的平台。通过膜胆固醇消耗将GP Ib-IX复合物从GEMs中解离，可消除血小板活化和对VWF的粘附。然而，关于gem关联的GP Ib-IX复合体的结构要素是什么，这种相互作用是如何调节的，以及gem关联在GP Ib-IX功能中的生理相关性是什么，这些基本问题从未得到回答。我们的初步数据表明GP Ib1与GEMs结构域的关联主要是由GP Ib2/GP IX介导的。去除GP Ib1和GP Ib2/GP IX之间的二硫键不仅可以抑制GP Ib1与GEMs结构域的结合，还可以抑制表达CHO （Chinese Hamster Ovary）复合物的GP Ib-IX细胞在高剪切下与VWF的相互作用。此外，我们发现蛋白二硫异构酶（PDI）在血小板和表达CHO细胞的GP Ib-IX复合体中都与GP Ib-IX复合体相关联，这种相互作用仅在GEMs域中可见。此外，我们证明了血小板膜脂组成的改变抑制了GP Ib-IX复合物与GEMs结构域的结合以及复合物介导的血小板与VWF的相互作用。基于这些结果和先前报道的证据，我们假设1)GP Ib2/GP IX包含介导GEMs与GP Ib-IX复合物相互作用的结构决定因素；2) GP Ib1与GP Ib2/GP IX之间形成二硫键，通过蛋白二硫异构酶的氧化还原调控使GP Ib1与GEMs结构域结合；3)特定的GEMs脂质组成对复杂关联至关重要；4) GP Ib1与GEMs结构域结合的特异性破坏可消除GP Ib-IX复合物的功能。综上所述，本研究将有助于阐明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，最终为开发新的治疗策略提供机制指导，以对抗各种复杂相关的出血性疾病和心血管疾病。

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.