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.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
First Independent Research Support & Transition (FIRST) Awards (R29)
Project #
5R29HL046416-02
Application #
3473513
Study Section
Hematology Subcommittee 2 (HEM)
Project Start
1992-03-15
Project End
1997-02-28
Budget Start
1993-03-01
Budget End
1994-02-28
Support Year
2
Fiscal Year
1993
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 F; Berndt, M C; Schade, A et al. (2001) Ristocetin-dependent, but not botrocetin-dependent, binding of von Willebrand factor to the platelet glycoprotein Ib-IX-V complex correlates with shear-dependent interactions. Blood 97:162-8
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
Dong, J F; Gao, S; Lopez, J A (1998) Synthesis, assembly, and intracellular transport of the platelet glycoprotein Ib-IX-V complex. J Biol Chem 273:31449-54
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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