Platelets adhere to vascular subendothelium by a complex process which involves both a direct interaction between a platelet membrane receptor and collagen fibers and the binding of adhesive glycoproteins to connective tissue constituents and additional platelet membrane glycoproteins. Glycoprotein Ib is the principal receptor for the von Willebrand's factor and is essential for normal platelet adhesion. We propose to study the structure and function of GpIb by molecular cloning of GpIb cDNA and expression of the cDNA in heterologous cells. We plan to: 1) construct cDNA libraries from Human Erythroleukemia Cells, a megakaryocytic cell line and normal human megakaryocytes; 2) determine the chromosomal location, genomic organization, and tissue distribution of GpIb; 3) analyze the molecular basis for Bernard-Soulier syndrome; 4) sequence GpIb cDNA and determine the organization of the GpIb polypeptide subunits and their homology with other adhesive glycoprotein receptors; 5) transfect full-length GpIb cDNA into heterologous cells and optimize its expression; 6) analyze the effects of a series of mutations which delete or modify the intracytoplasmic, transmembrane, protease- sensitive, carbohydrate-rich extracellular and vWF binding regions of the protein; 7) analyze the ligand binding, cytoskeletal interactions, regulation of phosphorylation, and lateral mobility, and surface adhesive properties of normal, recombinant, and mutated forms of GpIb in megakaryocytes, and heterologous cells; and 8) express portions of the GpIb polypeptide in E. coli to produce domain-specific antibodies and to test the role of GpIb- derived peptides as potential antithrombotic agents. These studies should provide new information regarding the molecular basis of platelet adhesion and the role of the Glycoprotein Ib/von Willebrand's Factor interaction in initiating hemostasis. The molecular cloning of GpIb cDNA and the application of somatic cell genetics, and site-directed mutagenesis to the study of GpIb function are extremely powerful and versatile techniques that have not yet been widely used in the area of hemostasis. In addition to their importance for the current studies, the development of cDNA libraries encoding platelet proteins, as well as the creation of the heterologous and megakaryocytic cell lines which express platelet proteins should be of value to other investigators in this and related areas.
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