The aggregation of blood platelets, the central cellular event in thrombus formation, is essential to prevent excessive blood loss at sites of injury; but, at the same time, underlies the thrombotic diseases including myocardial infarction, unstable angina, and stroke, the leading causes of death in industrialized countries. At a molecular level, platelet aggregation depends upon the binding of adhesive ligands, such as the plasma protein fibrinogen, to the major membrane protein on the platelet surface, integrin alphalibBeta3 (GPllb-llla). alphalibBeta3 shares the same beta subunit with alphavBeta3. This integrin is more broadly distributed and is involved in both physiologic and pathophysiologic responses including bone resorption, in osteoporosis, angiogenesis and tumor metastasis. Binding of fibrinogen to these two beta3 integrins, as well as most ligands to most integrins (there are more than 20 integrins), is divalent ion dependent, and there is an intimate, but unresolved relationship between the divalent cation and ligand binding sites in the integrins.
Aim 2 of HL54924 examines the molecular basis for ligand binding to alphallbBeta3. This FIRCA seeks to expand these studies to examine the molecular basis for divalent cation binding to alphalibBeta3 and to determine why divalent cations differentially influence the functions of the two Beta3 integrins. The impetus for this FIRCA application comes from three sources. First, Drs. Plow and Cierniewski have had a longstanding and productive collaboration in examining the interaction of divalent cations with alphalibBeta3. Second, recently, their investigations have led to findings which have the potential to provide novel insights into how divalent cations regulate integrin function. Third, the crystal structure of the extracellular domain of integrin alphavBeta3 has just been published, providing a major breakthrough and an opportunity to design incisive approaches to precisely define how divalent cations influence ligand binding. The preliminary data developed in the application support the hypothesis that a small segment of the Beta3 subunit, Beta3 (109-352), contains a non-selective divalent cation binding site, which must be occupied for ligands to bind, a ligand competent (LC) site, and a calcium-specific, inhibitory (I) site which inhibits ligand binding. This hypothesis will be tested in the FIRCA by expressing recombinant fragments in which the coordination sites for these two cation bindings are localized by systematic mutagenesis in the first Specific Aim. The second Specific Aim will address why calcium exerts differential effects on the function of alphallbBeta3 and alphavBeta3 by performing mutagenesis studies on """"""""mini receptors"""""""" composed of portions of the alpha and beta subunits expressed in baculovirus systems. Taken together, these studies will provide insights into the role of divalent cations in regulating the ligand binding functions of the Beta integrins. These studies will complement the goals of the parent grant. In concert, the two applications will provide fundamental insights into the basic molecular mechanisms associated with the functions of integrins that are of biomedical importance.
