STRUCTURE AND FUNCTION OF THE GP IB-IX COMPLEX

GP IB-IX 复合体的结构和功能

• 批准号: 2222910
• 负责人: Jose Aron Lopez
• 金额: $ 9.41万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 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/2222910
• 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.

拟议研究的目的是进一步我们的理解