We focus on the mechanism of binding of adhesive proteins to their platelet receptors, a key process in the formation of hemostatic plugs, and in the formation of vasooclusive thrombi (coronary, cerebral, and lung microcirculatory thrombosis). The two main aspects of binding of adhesive proteins, namely, the recognition specificity of the glycoprotein IIb-IIIa complex (common platelet receptor for adhesive proteins) and regulation of this receptor from non-binding to binding mode by the platelet agonist, ADP will be studied. To investigate why the unique receptor recognition domain on the human fibrinogen gamma chain is iso-specific with the ubiquitous RGD domains in the alpha chain in regard to their interaction with GPIIb-IIIa, we will use covalent labeling with peptide probes. The GPIIb-IIIa interaction with other adhesive proteins (vWF and fibronectin) will be similarly examined. The topography of """"""""contact sites"""""""" on fibrinogen and GPIIb-IIIa decorated with specific antipeptide Fab antibody fragments in electron microscopy will be determined. 2D electron microscopic crystal- lographic analysis of GPIIb-IIIa and 2D H-1 NMR spectroscopic analysis of synthetic peptide analogs of fibrinogen and GPIIb-IIIa will help to develop a molecular model of the fibrinogen-platelet GPIIb-IIIa interaction. Regu- lation of the GPIIb-IIIa complex in platelets stimulated with ADP, will be studied with emphasis on the structure/function of the ADP receptor and protein kinase C species. We will clone cDNA for the ADP receptor and study its expression. We will identify the species of protein kinase C in platelets. We will delineate the role of diverse species of protein kinase C in phosphorylation of GPIIIa and ascertain whether this linkage is direct or involves phosphorylation of an intermediate protein. The results of these fundamental approaches to platelet adhesive receptors will provide useful information for the design of novel inhibitors of platelets.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL045994-03
Application #
3365118
Study Section
Hematology Subcommittee 2 (HEM)
Project Start
1990-05-01
Project End
1995-04-30
Budget Start
1992-05-01
Budget End
1993-04-30
Support Year
3
Fiscal Year
1992
Total Cost
Indirect Cost
Name
Vanderbilt University Medical Center
Department
Type
Schools of Medicine
DUNS #
004413456
City
Nashville
State
TN
Country
United States
Zip Code
37212
Hawiger, J (1999) Noninvasive intracellular delivery of functional peptides and proteins. Curr Opin Chem Biol 3:89-94
Ware, S; Donahue, J P; Hawiger, J et al. (1999) Structure of the fibrinogen gamma-chain integrin binding and factor XIIIa cross-linking sites obtained through carrier protein driven crystallization. Protein Sci 8:2663-71
Hawiger, J; Veach, R A; Liu, X Y et al. (1999) IkappaB kinase complex is an intracellular target for endotoxic lipopolysaccharide in human monocytic cells. Blood 94:1711-6
Zhang, L; Torgerson, T R; Liu, X Y et al. (1998) Preparation of functionally active cell-permeable peptides by single-step ligation of two peptide modules. Proc Natl Acad Sci U S A 95:9184-9
Chu, Z L; DiDonato, J A; Hawiger, J et al. (1998) The tax oncoprotein of human T-cell leukemia virus type 1 associates with and persistently activates IkappaB kinases containing IKKalpha and IKKbeta. J Biol Chem 273:15891-4
Hawiger, J (1997) Cellular import of functional peptides to block intracellular signaling. Curr Opin Immunol 9:189-94
Liu, K Y; Timmons, S; Lin, Y Z et al. (1996) Identification of a functionally important sequence in the cytoplasmic tail of integrin beta 3 by using cell-permeable peptide analogs. Proc Natl Acad Sci U S A 93:11819-24
Lin, Y Z; Yao, S Y; Hawiger, J (1996) Role of the nuclear localization sequence in fibroblast growth factor-1-stimulated mitogenic pathways. J Biol Chem 271:5305-8
Donald, R; Ballard, D W; Hawiger, J (1995) Proteolytic processing of NF-kappa B/I kappa B in human monocytes. ATP-dependent induction by pro-inflammatory mediators. J Biol Chem 270:9-12
Blumenstein, M; Matsueda, G R; Timmons, S et al. (1992) A beta-turn is present in the 392-411 segment of the human fibrinogen gamma-chain. Effects of structural changes in this segment on affinity to antibody 4A5. Biochemistry 31:10692-8

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