STRUCTURE AND FUNCTION OF THE GP IB-IX COMPLEX

GP IB-IX 复合体的结构和功能

• 批准号: 3473513
• 负责人: Jose Aron Lopez
• 金额: $ 11.81万
• 依托单位: J. DAVID GLADSTONE INSTITUTES
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-03-15 至 1997-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3473513
• 关键词: blood coagulation; carbohydrates; cell membrane; clone cells; complementary DNA; disulfide bond; fatty acylation; flow cytometry; gene mutation; glycoprotein biosynthesis; glycoproteins; glycosylation; immunofluorescence technique; laboratory rabbit; membrane proteins; monoclonal antibody; platelet aggregation; platelets; posttranslational modifications; protein purification; protein sequence; protein structure function; thrombasthenia; thrombin; transfection /expression vector; tunicamycin; von Willebrand factor

The aim of the proposed research is to further our understanding of the structure and function of the glycoprotein (GP) Ib-IX complex of human blood platelets. The GP Ib-IX complex is a trimeric complex of three polypeptides, GP Ib-alpha, GP Ib-beta, and GP IX, that mediates the attachment of platelets to von Willebrand factor (vWf) at a site of blood vessel injury, a reaction that is a crucial initial step in forming a hemostatic platelet plug. The interaction between GP Ib-IX and vWf is also an essential step in platelet aggregation induced at high shear rates and thus may mediate aggregation in areas of the vasculature such as stenotic coronary arteries. Additionally, GP Ib-IX provides a high-affinity binding site for thrombin on the platelet surface that ap- pears necessary for the activation of platelets at low thrombin concentration. Hereditary deficiency of the GP Ib-IX complex results in a potentially fatal bleeding disorder, the Bernard-Soulier syndrome. In this application, we propose to study three aspects of the structure and function of this vital membrane complex: 1) to determine whether all three subunits of the GP Ib-IX complex are required for the formation of a functional membrane complex; 2) to identify the features of the individual subunits of the GP Ib-IX complex that are important in the formation of a membrane complex; and 3) to identify the structural features of the GP Ib-IX complex that are necessary for the binding of vWf. Cell lines expressing the GP Ib-IX complex on the cell surface have been established by transfection with expression vectors containing cDNAs for GP Ib-alpha, GP Ib-beta, and GP IX. Experiments will now be performed to determine whether the individual subunits are expressed on the cell membrane of different cell types when less than the full complement of expression constructs is transfected. Defined mutations will be made in the cDNAs encoding the individual subunits of the complex and the effect of these mutations on complex assembly, its membrane expression, and vWf binding will be evaluated by comparison to cell lines expressing wildtype complex. The effect of O- and N-glycosylation on the formation, stability, and function of the complex will also be investigated by expressing the complex in a cell line defective in O-glycosylation and by the use of tunicamycin, an inhibitor of N-glycosylation. It is expected that these studies will further elucidate the structure and biosynthesis of the GP Ib-IX complex, provide insight into the nature of disorders of the GP Ib-IX complex (e.g., Bernard-Soulier syndrome), and answer some basic questions about the role of the complex in platelet physiology and thrombotic disorders.

这项研究的目的是为了进一步了解 人糖蛋白（GP）Ib-IX复合物的结构和功能 血小板 GP Ib-IX复合物是三个GP Ib-IX的三聚体复合物。 多肽，GP Ib-α，GP Ib-β和GP IX，其介导 血小板与血管性血友病因子（vWf）在血液部位的附着 血管损伤，这是一个反应，是一个关键的初始步骤，形成一个 止血血小板塞。 GP Ib-IX和vWf之间的相互作用是 这也是在高剪切下诱导的血小板聚集的必要步骤 并因此可介导脉管系统区域中的聚集， 冠状动脉狭窄 此外，GP Ib-IX提供了一个 血小板表面上凝血酶的高亲和力结合位点， 梨必要的血小板活化在低凝血酶 浓度. GP Ib-IX复合物的遗传性缺陷导致 一种可能致命的出血性疾病，伯纳德-苏利尔综合征。 在 本申请中，我们建议研究结构的三个方面， 这个重要的膜复合物的功能：1）确定是否所有 GP Ib-IX复合物的三个亚基是形成 功能性膜复合物; 2）识别膜的特征， GP Ib-IX复合物的单个亚基，这些亚基在 膜复合物的形成;和3）鉴定结构 GP Ib-IX复合物的结合所必需的特征 vWf. 在细胞表面上表达GP Ib-IX复合物的细胞系已经被发现， 通过用含有cDNA的表达载体转染来建立， GP Ib-α、GP Ib-β和GP IX。 现在将进行实验， 确定单个亚基是否在细胞上表达 不同细胞类型的膜，当小于完整的补充， 转染表达构建体。 定义的突变将在 编码复合物的各个亚基的cDNA和 这些突变对复合物组装、膜表达和vWf的影响 结合将通过与表达野生型的细胞系进行比较来评估 复杂. O-和N-糖基化对形成的影响， 稳定性和功能的复杂性也将研究， 在O-糖基化缺陷的细胞系中表达所述复合物， 使用衣霉素，一种N-糖基化的抑制剂。 预计 这些研究将进一步阐明其结构和生物合成 GP Ib-IX复合物，提供对疾病性质的洞察， GP Ib-IX复合物（例如，Bernard-Soulier综合征），并回答一些 关于复合物在血小板生理学中的作用的基本问题， 血栓性疾病