The objective of this project is to correlate the structure and function of the platelet membrane glycoprotein IIb-IIIa (GPIIb-IIIa) complex. GPIIb-IIIa is a calcium-dependent heterodimer whose binding site for ligands such as fibrinogen and von Willebrand factor is exposed by platelet activation. Because ligand binding to GPIIb-IIIa is directly responsible for platelet aggregation, regulation of ligand binding is a critical step in platelet function. The mechanism by which agonists convert GPIIb-IIIa to an active conformation and the features of the folded conformation of GPIIb-IIIa that underlay its function are unresolved questions.
Specific Aim 1 will address the nature of the signaling pathways involved in GPIIb-IIIa activation. Because it has been difficult to study the mechanism by which agonists activate GPIIb-IIIa using platelets, we have expressed GPIIb-IIIa in B lymphocytes. We found that GPIIb-IIIa in these cells can be induced to interact with fibrinogen using phorbol myristate acetate (PMA) and recently, by stimulating cells expressing the formyl peptide receptor with the peptide formyl-Met-Leu- Phe. We will examine the role of the actin cytoskeleton, small GTP-binding proteins, phosphoinositide 3-kinase isoenzymes, pleckstrin, and signals initiated by G protein-coupled receptors in GPIIb-IIIa activation using the B lymphocyte system.
Specific Aim 2 will continue studies of study of structure-function relationships in the GPIIb-IIIa heterodimer. Although x-ray or solution structures for all or parts of the GPIIb-IIIa molecule are not available, important features have been determined by examining the consequences of naturally-occurring and site-directed mutations of GPIIb or GPIIIa. We will continue studies of GPIIb-IIIa folding and intracellular transport, with emphasis on the effects of naturally occurring mutations responsible for Glanzmann thrombasthenia and on the role of intracellular chaperones in the expression of the thrombasthenic phenotype. Because our previous work demonstrated the importance of the putative calcium binding region of GPIIb in overall GPIIb-IIIa folding, the influence of divalent cations on the secondary structure of this region of GPIIb will be examined using biophysical techniques such as circular dichroism and nuclear magnetic resonance spectroscopy.
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