The overall objective of this research is to understand the mechanisms through which occupancy of cell surface receptors leads to secondary events. The hypothesis addressed is that signal transduction may be mediated by novel sites on receptors which are expressed as a consequence of ligand binding. Such sites may mediate signal transduction by acting as binding sites for receptor-receptor interaction, they may bind other membrane molecules which then serve as second messengers, or they may bind to molecules on other cells leading to aggregation or cell-cell communication. Moreover, such sites are hypothesized to be antigenic and therefore may act as antibody binding sites. These sties are therefore termed Ligand Inducible Binding Sites or LIBS. The model system used to test the hypothesis is glycoprotein IIb-IIIa, a platelet receptor for fibrinogen (Fg), and the secondary events involved in clot retraction. A monoclonal antibody, anti-LIBS1, has been developed which preferentially recognizes ligand-occupied conformers of GPIIb-IIIa and inhibits clot retraction. In order to understand how this occurs, we will: 1) Map the LIBS1 epitope by identifying the minimal proteolytic fragments of the receptor containing the epitope and by identifying mutations and deletions which affect expression of the epitope. 2) Identify the smallest ligand regulated fragment by examining ligand upregulation of epitope expression on receptor fragments. 3) Identify the functional role of the LIBS1 epitope in clot retraction by examining the interaction of the receptor with the two other critical components; fibrin and cytoskeleton. 4) Examine the role of the LIBS1 epitope in receptor clustering by EM, and chemical cross-linking. 5) Isolate LIBS1 binding molecules by chemical cross-linking and affinity chromatography. 6) Examine the role of the LIBS1 epitope on redistribution of the vitronectin receptor (VnR) during cell adhesion, and 7) Identify other LIBS which are functionally important. This research provides a unique approach to the study of signal transduction. It is also important since the signal transduction other members of the integrin superfamily of adhesion receptors. These studies may provide a novel pathway for signal transduction which is applicable to other cell surface receptors. Finally, antibodies and peptides developed in the course of this work may useful diagnostic or therapeutic anti- thrombotic agents.
