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.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
4R37HL034787-09
Application #
3486079
Study Section
Special Emphasis Panel (NSS)
Project Start
1984-12-01
Project End
1996-12-31
Budget Start
1993-01-01
Budget End
1993-12-31
Support Year
9
Fiscal Year
1993
Total Cost
Indirect Cost
Name
Brigham and Women's Hospital
Department
Type
DUNS #
071723621
City
Boston
State
MA
Country
United States
Zip Code
02115
Beacham, D A; Cruz, M A; Handin, R I (1995) Glycoprotein Ib can mediate endothelial cell attachment to a von Willebrand factor substratum. Thromb Haemost 73:309-17
Cruz, M A; Handin, R I; Wise, R J (1993) The interaction of the von Willebrand factor-A1 domain with platelet glycoprotein Ib/IX. The role of glycosylation and disulfide bonding in a monomeric recombinant A1 domain protein. J Biol Chem 268:21238-45
Petersen, E; Handin, R I (1992) Transient expression of recombinant glycoprotein Ib alpha polypeptides in COS cells that inhibit von Willebrand factor binding to the platelet glycoprotein Ib/IX complex. Thromb Haemost 68:203-7
Cruz, M A; Petersen, E; Turci, S M et al. (1992) Functional analysis of a recombinant glycoprotein Ib alpha polypeptide which inhibits von Willebrand factor binding to the platelet glycoprotein Ib-IX complex and to collagen. J Biol Chem 267:1303-9
Greenberg, S M; Chandrasekhar, C; Golan, D E et al. (1990) Transforming growth factor beta inhibits endomitosis in the Dami human megakaryocytic cell line. Blood 76:533-7
Ewenstein, B M; Inbal, A; Pober, J S et al. (1990) Molecular studies of von Willebrand disease: reduced von Willebrand factor biosynthesis, storage, and release in endothelial cells derived from patients with type I von Willebrand disease. Blood 75:1466-72
Michelson, A D; Adelman, B; Barnard, M R et al. (1988) Platelet storage results in a redistribution of glycoprotein Ib molecules. Evidence for a large intraplatelet pool of glycoprotein Ib. J Clin Invest 81:1734-40
Wise, R J; Pittman, D D; Handin, R I et al. (1988) The propeptide of von Willebrand factor independently mediates the assembly of von Willebrand multimers. Cell 52:229-36
Bockenstedt, P; Greenberg, J M; Handin, R I (1986) Structural basis of von Willebrand factor binding to platelet glycoprotein Ib and collagen. Effects of disulfide reduction and limited proteolysis of polymeric von Willebrand factor. J Clin Invest 77:743-9
Bockenstedt, P; McDonagh, J; Handin, R I (1986) Binding and covalent cross-linking of purified von Willebrand factor to native monomeric collagen. J Clin Invest 78:551-6

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