The thalassemias arise from unbalanced globin chain synthesis due to deletions of, or mutations to, the Beta- and alpha-hemoglobin genes. As a consequence, unpaired alpha- or Beta-hemoglobin chains (alpha- and Beta-chains) are present within the thalassemic red blood cell (RBC). These unpaired chains are thought to contribute to the ineffective erythropoiesis and shortened RBC survival noted in the thalassemias. Surprisingly, while the RBC abnormalities in thalassemic are well characterized, little is known of the mechanisms by which unpaired alpha- and Beta-chains injure the cell. Our lack of knowledge regarding the cellular pathology of the thalassemic cell is due to: 1) the onset and development of cellular pathology occur quite rapidly in vivo; and 2) the most severely affected RBC are swiftly destroyed in either the bone marrow or in the peripheral blood. Consequently, it has not been possible to directly investigate the mechanism by which unpaired alpha- and Beta-chains damage the RBC. To circumvent these problems, I have developed models of the alpha and Beta thalassemic RBC. These model cells, made by entrapment of purified alpha- and Beta-chains in normal RBC, develop functional and structural changes almost identical to those seen in thalassemic patient RBC. Using the model thalassemic RBC, in conjunction with patient samples, it is now possible to experimentally examine the mechanisms by which unpaired hemoglobin chains mediate cell damage. Importantly, the model thalassemic cells also provide a unique means to experimentally evaluate possible therapeutic agents. It is the hypothesis of this proposal that the inherent instability of the unpaired globin chains results in the generation of reactive oxygen species and the release of globin free heme and iron and that these agents underlie the pathophysiology of the thalassemic RBC. Furthermore, by determining the direct mechanisms by which unpaired globin chains injure the RBC, therapeutic interventions can be designed that prevent cell damage. Based on these hypotheses, the specific aims of the research proposal are to: A) elucidate the direct mechanisms by which heme and iron are released from unpaired alpha- and Beta-chains; B) determine the fate of the globin derived heme and iron and, subsequently, identify the specific sites, mechanisms, and pathological consequences of damage to the RBC; and C) evaluate interventions (e.g., antioxidants and chelators) that diminish or block damage by unpaired alpha- and Beta-chains. The results of these studies will provide significant new insights into the pathophysiology of the thalassemic cell and give experimental evidence for therapeutic agents that might improve in vivo erythropoiesis and RBC survival.
