The aim of the proposed research is to further our understanding of the structure and function of the glycoprotein (GP) Ib-IX surface membrane complex of human blood platelets. The GP Ib-IX complex mediates the attachment of platelets to a damaged blood vessel wall. In addition, the GP Ib-IX complex functions as an attachment site for the platelet membrane skeleton on the plasma membrane.
The aim the proposal is threefold: 1) to study the requirements for the association of GP Ib-alpha, GP Ib-beta, and GP IX into a functional membrane complex; 2) to study structural features of the complex that modulate its interaction with von Willebrand factor (vWf); and 3) to characterize the interaction of the GP Ib-IX complex with the membrane skeleton. Plasmids containing the cDNAs for GP Ib-alpha, GP Ib-beta, and GP IX will be transfected into mammalin cells. A system will be established in which the GP Ib-IX complex is expressed on the cell surface in a functional form. By comparing the functional activities of the wild type GP Ib-IX complex with those of glycoproteins in which defined modifications have been made, we will identify the important features of the glycoproteins required for complex formation, vWf binding, and association with the cytoskeleton. If the GP Ib-IX complex cannot be expressed in a mammalian cell line, alternative approaches for addressing these questions are proposed. It is expected that these studies will 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. As a postdoctoral fellow, the applicant has been involved in work that provided the background for the proposed research, including 1) screening a number of human cell lines for synthesis of GP Ib, 2) establishing conditions for maximizing synthesis of GP Ib in the cell line that had the greatest baseline synthesis, 3) constructing a human erythroleukemia cell cDNA library, and 4) cloning and sequencing full-length cDNAs for the alpha- and beta-chains of GP Ib. The proposed research is a logical outgrowth this preliminary work.
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