The long term objective of this proposal is to determine the underlying pathophysiology that leads to remarkably divergent cellular events in alpha vs. Beta thalassemia. Accelerated apoptosis or programmed cell death (PCD) is most likely responsible for intramedullary hemolysis in both human and murine beta thalassemia. Integrally associated with PCD, is the very specific alteration in the asymmetry of the phospholipid bilayer with an outward movement of phosphatidylserine (PS). The mechanism by which PS is exposed and recognized on thalassemic red cells or red cell precursors, rendering them ~edible~ to macrophages, is not clear. The principal objectives of this proposal are to define the contribution of enhanced PCD to ineffective erythropoiesis in beta thalassemia and further our understanding of the mechanism(s) responsible for enhanced PCD. In particular, we will discover how and why PCD leads to alterations in the phospholipid bilayer, resulting in PS exposure at the surface of affected cells, and how this in turn acts either alone or in concert with other signaling systems to lead to the recognition and removal of the affected erythroid precursors. To pursue these goals, we propose the following three specific aims: 1. Investigate programmed cell death in murine thalassemia, 2. Investigate the mechanisms responsible for the movement of PS from the inner to outer monolayer and 3. Determine the factors that render a cell with PS on its outer surface recognizable and removable. The murine alpha and beta thalassemic models we have created provide us with mice with graded degrees of clinical severity necessary to test the proposed hypotheses. We will use fluorescently labeled annexin V to quantitate, isolate and study the biochemical and functional characteristics of subpopulations of cells with PS on their surface, define the PCD driven mechanism for PS exposure, and identify factors modulating macrophagic recognition. We will explore our hypothesis that the heme/hemichromes associated with the globin chains mediate oxidant attack and play a role in PCD and macrophagic attack on apoptotic erythroid precursors. The successful accomplishment of these objectives should enable us to develop a detailed mechanistic understanding of the pathophysiology of an import human disease. The insights generated are also likely to further our understanding of the loss of PS asymmetry and the role of oxidative damage in programmed cell death.
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