The overall objective of this work is to produce a molecular map of the structural basis for platelet interaction (specifically GBIIb-IIIa) with adhesive proteins. The hypothesis states that multiple ligand contact points govern high affinity binding between IIb-IIIa and its various adhesive protein ligands, and the overall goal of the current work is to identify individual amino acids in each receptor subunit that are essential for ligand binding. The first Specific Aim is to generate an """"""""entire"""""""" library of potential mutations that may play a role in ligand binding through the use of a random mutagenesis approach. A screening methodology will be employed to enrich for mutants that result in a loss of ligand binding.
The second Aim i s based on the hypothesis that ligand recognition specificity is regulated by distinct regions of IIb. The regions will be identified by the development and assessment of chimeric alpha subunits that include different combination of alpha subunit from IIb-IIIa and the vitronectin receptor. Major concerns from last review were about interpreting data from loss-of-function mutation, and the need to establish feasibility of methods that had been proposed.
