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.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Clinical Investigator Award (CIA) (K08)
Project #
1K08HL002463-01
Application #
3082894
Study Section
Special Emphasis Panel (SRC (KR))
Project Start
1990-07-01
Project End
1995-06-30
Budget Start
1990-07-01
Budget End
1991-06-30
Support Year
1
Fiscal Year
1990
Total Cost
Indirect Cost
Name
J. David Gladstone Institutes
Department
Type
DUNS #
047120084
City
San Francisco
State
CA
Country
United States
Zip Code
94158
Schade, Alicia J; Arya, Maneesh; Gao, Shan et al. (2003) Cytoplasmic truncation of glycoprotein Ib alpha weakens its interaction with von Willebrand factor and impairs cell adhesion. Biochemistry 42:2245-51
Dong , J; Ye, P; Schade, A J et al. (2001) Tyrosine sulfation of glycoprotein I(b)alpha. Role of electrostatic interactions in von Willebrand factor binding. J Biol Chem 276:16690-4
Afshar-Kharghan, V; Gineys, G; Schade, A J et al. (2000) Necessity of conserved asparagine residues in the leucine-rich repeats of platelet glycoprotein Ib alpha for the proper conformation and function of the ligand-binding region. Biochemistry 39:3384-91
Dong, J; Schade, A J; Romo, G M et al. (2000) Novel gain-of-function mutations of platelet glycoprotein IBalpha by valine mutagenesis in the Cys209-Cys248 disulfide loop. Functional analysis under statis and dynamic conditions. J Biol Chem 275:27663-70
Dong, J; Li, C; Schade, A J et al. (2000) Mutation in the leucine-rich repeat of platelet glycoprotein Ib alpha results in defects in its interaction with immobilized von Willebrand factor under flow. Chin Med J (Engl) 113:693-8
Sullam, P M; Hyun, W C; Szollosi, J et al. (1998) Physical proximity and functional interplay of the glycoprotein Ib-IX-V complex and the Fc receptor FcgammaRIIA on the platelet plasma membrane. J Biol Chem 273:5331-6
Fredrickson, B J; Dong, J F; McIntire, L V et al. (1998) Shear-dependent rolling on von Willebrand factor of mammalian cells expressing the platelet glycoprotein Ib-IX-V complex. Blood 92:3684-93
Dong, J F; Li, C Q; Sae-Tung, G et al. (1997) The cytoplasmic domain of glycoprotein (GP) Ibalpha constrains the lateral diffusion of the GP Ib-IX complex and modulates von Willebrand factor binding. Biochemistry 36:12421-7
Dong, J F; Sae-Tung, G; Lopez, J A (1997) Role of glycoprotein V in the formation of the platelet high-affinity thrombin-binding site. Blood 89:4355-63
Afshar-Kharghan, V; Lopez, J A (1997) Bernard-Soulier syndrome caused by a dinucleotide deletion and reading frameshift in the region encoding the glycoprotein Ib alpha transmembrane domain. Blood 90:2634-43

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