Clustering of platelet surface membrane glycoproteins (GPIIb and III) by multivalent ligands such as antibodies or lectins has been shown to induce transmembrane interactions between GPIIb-III and the cell cytoskeleton. Understanding the molecular basis of these interactions is a central goal of the proposed studies. Specifically, experiments are designed to evaluate, in a quantitative manner, the relationship between the degree of GPIIb-III clusters induced by polyclonal antibodies (or dimeric clusters formed by monoclonal antibodies directed against single molecular epitopes) and the subsequent induction of association of these surface molecules with the cytoskeleton. For this purpose, the concentration of surface clusters (or dimers) will be calculated from theoretical models successfully used in other cell systems. The extent of cluster formation will then be compared to the extent of associations measured after isolation of the cell cytoskeletons.
A second aim of this work is to determine the nature of the interaction between GPIIb-III and the cytoskeleton. To wit, to determine if the interactions with cytoskeletal actin or other cytoskeletal proteins is a direct transmembrane interaction or is mediated by other pre-anchored surface glycoproteins including a preanchored fraction of GPIIb and/or III. Finally, the complex between GPIIb-III and any putative linkage proteins will be solubilized using selective actin depolymerizing agents (e.g. DNAse I) and isolated by affinity purification methods and other conventional isolation techniques. This will allow characterization and identification of the molecules involved in linking GPIIb-III to the platelet cytoskeleton. In related experiments, antibodies to any putative linkage proteins and other cytoskeletal proteins will be used to localize molecules at the ultrastructural level so that their spatial relationship to clustered surface molecules can be assessed in intact cells. Associations between membrane surface proteins and the cytoskeletal apparatus are important in numerous motile phenomena including shape changes, endocytosis and clot retraction. Understanding the molecular basis and regulation of such interactions will improve the understanding of platelet-mediated processes and, by extension, other cellular processes as well.
| Painter, R G; Dukes, R; Sullivan, J et al. (1988) Function of neutral endopeptidase on the cell membrane of human neutrophils. J Biol Chem 263:9456-61 |
| Painter, R G; Zahler-Bentz, K; Dukes, R E (1987) Regulation of the affinity state of the N-formylated peptide receptor of neutrophils: role of guanine nucleotide-binding proteins and the cytoskeleton. J Cell Biol 105:2959-71 |
| James, H L; James, P L; Painter, R G et al. (1986) Subcellular localization of platelet elastase and its retention during the release reaction. Semin Thromb Hemost 12:250-2 |
