THE MOLECULAR BASIS OF LIGAND BINDING TO ALPHAIIB BETA3

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The alphaIIb and alphav integrins have been shown to play a significant role in a variety of diseases processes. AlphaIIbbeta3 is a platelet-specific fibrinogen receptor that is critical for thrombosis and hemostasis. Determination of the basis of ligand recognition by alphaIIbbeta3 is critical for the modulation of platelet function. The objective of this proposal is to define the molecular basis of ligand binding to alphaIIbbeta3. We hypothesize that discrete regions of alphaIIb regulate the ligand recognition specificity of alphaIIbbeta3. We further hypothesize that multiple ligand binding points are required for high affinity binding to alphaIIbbeta3. To test these hypotheses, the specific aims are 1) to identify regions and specific amino acids within alphaIIb that determine ligand recognition specificity of alphaIIbbeta3 utilizing a directed approach based upon molecular modeling and 2) identify additional amino acid residues on alphaIIb and beta3 that are required for ligand binding function utilizing an unbiased genetic screen. To approach aim 1, we will replace the predicted loop regions of alphav with the corresponding region of alphaIIb and test for a positive phenotypic shift in ligand specificity. To accomplish aim 2, we will randomly mutagenize cells expressing either the alphaIIb or the beta3 subunits and test for loss of ligand binding. Once mutations are identified, we will introduce these mutations into wild type alphaIIBbeta3 and verify that ligand binding is destroyed.