PADGEM (Platelet Activation Dependent Granule-External Membrane protein), is a 140 k glycoprotein expressed on the surface of activated platelets. PADGEM, also known as GMP-140, is an integral membrane protein of the platelet alpha granule which is translocated to the plasma membrane upon cell activation. The cDNA for PADGEM has been cloned and has sequence homology with two adhesive proteins; ELAM-1 and MEL-14. Recent studies have demonstrated that PADGEM mediates the binding of activated platelets with monocytes and neutrophils. The goal of this project is to investigate the mechanism and consequences of this interaction at a molecular level, employing the tools of protein chemistry, cell biology, and molecular biology. The initial phase of this project will characterize the binding domain of the PADGEM molecular. This will involve identification and isolation of the minimal amino acid sequence necessary for the protein's binding function. This will be performed by a combination of purification of active peptide fragments and epitope mapping utilizing antibodies directed against the binding site. Since PADGEM contains a lectin-like domain, potential carbohydrate binding properties will be investigated. The second stage of the project will involve identification and isolation of the receptor for PADGEM on leukocytes. The initial strategy for this study will be the production of inhibitory anti-leukocyte monoclonal antibodies and subsequent immunoaffinity purification of the receptor. Several alternative approaches and subsequent immunoaffinity purification of the receptor. Several alternative approaches will also be considered including affinity purification with the ligand PADGEM, functional cloning of the receptor, and the production of anti-idiotypic antibodies. Next the binding domain of the receptor will be characterized and the potential role of carbohydrate in binding will be investigated. The third phase of the project will study the physiologic relationship between the activated platelet and the leukocyte. A combination of immunofluorescence experiments and immunoassays will address the mechanism of receptor expression, the role of leukocyte activation in platelet adherence, and the phenotypic and functional consequences of platelet binding to leukocytes. The short term goal of this research is to gain a greater understanding of the PADGEM mediated interaction between platelets and leukocytes. The results of this studies are likely to provide insights into the biological significance this interaction.
