The fundamental long range goals of this grant proposal are to improve our understanding of platelet function and then use that knowledge to improve human health. Recent advances in the structural biology of the important platelet receptor aIIb?3, conducted in part under this grant, provide an opportunity to understand the mechanism(s) of aIIb?3 activation and ligand binding at an atomic scale. Such knowledge has the potential to advance our understanding of platelet-mediated hemostasis and thrombosis and deepen our insights into the allosteric mechanisms that control the ligand binding properties of aIIb?3 and other integrin receptors. Thus, we propose a multidisciplinary effort to integrate data from monoclonal antibody binding, site-directed mutagenesis, X-ray crystallography, cellular biology, and novel animal models with molecular modeling and molecular dynamics and high throughput screening of molecular libraries.
Specific Aim 1 : Provide new information on aIIb?3 structure and function. We will develop refined models of aIIb?3 structure and its mechanism(s) of activation and ligand binding using data from structural biology coupled with molecular dynamics simulations and then test those models experimentally by altering residues and regions predicted to be important in receptor activation. Specifically, we will modify aIIb?3 so as to selectively limit receptor extension and the B3 swing-out motion thought to confer high ligand binding affinity. We will utilize novel methods to assess the functional consequences of the alterations, including in vitro systems designed to simulate physiologic activation mechanisms and both hematologically reconstituted and transgenic animals. We will also prepare new monoclonal antibodies based on our molecular models and simulations to provide information about receptor dynamics.
Specific Aim 2 : Apply high throughput screening to provide new information on aIIb?3 structure and function and to identify potential candidates for future therapeutic development. We will study further the compound we identified in our initial high throughput screen (RUC-1) by using the results of molecular docking and molecular dynamics to synthesize rationally designed derivatives to define the properties of the human aIIb ligand binding pocket. Further insights will be obtained by analyzing the effects of RUC-1 and its derivatives on aIIb?3 from other species and aIIb?3 receptors containing 1IIb mutations. We plan to also identify the chemical and structural features necessary to induce conformational changes in the receptor and priming of the receptor for ligand binding. Compounds will also be tested in in vivo models to assess their effects on hemostasis and thrombosis. Finally, we will expand our high throughput screening to new chemical libraries to identify other novel compounds that affect aIIb?3 activation and/or ligand binding. The goal of this project is to understand the mechanism underlying the function of human blood platelets, which are vital in preventing bleeding, but also contribute to thrombotic disorders such as heart attacks and stroke by studying one of the platelet's key molecules, the receptor aIIb?3 receptors containing 1IIb mutations. We plan to also identify the chemical and structural features at the atomic level both functionally and mathematically, we hope to obtain insights into platelet function and the function of other receptors in the same family. Finally, using new high throughput technology, we hope to identify new compounds that may form the basis of improved therapies for heart attack and stroke.
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