The long term objective of this proposal is to develop a detailed understanding of the rheological and adherence properties of blood cells from patients with sickle cell disease, as they relate to flow disruption and stasis in the microcirculation. The principal objective of this renewal application is to develop a detailed molecular understanding of the material behavior and adhesive properties of sickle blood cells. The hypotheses we want to test are: 1) Perturbations in normal organization of membrane proteins is responsible for abnormal membrane rheology of sickle cells; 2) Reticulocyte dehydration and sickling-unsickling induced dehydration of mature red cells contribute to generation of dense, rheologically compromised sickle cells; and 3) Integrins and their ligands are involved in mediating sickle cell adherence to endothelial cells. In order to accomplish our stated objective, a series of studies are designed with the following specific aims: 1) Define the contribution of skeletal protein components and sickle hemoglobin to membrane rheology of sickle cells; 2) Define the contribution of fetal hemoglobin to the rheological properties of sickle red cells in the oxygenated and deoxygenated state; 3) Define the contributions of reticulocyte dehydration and sickling-unsickling induced dehydration of mature red cells to generation of dense sickle cells; 4) Define the functional involvement of integrin receptors and their ligands in mediating sickle cell adherence to vascular endothelial cells; and 5) Define the potential contributions of surface changes to increased interaction of sickle cells with cellular components of the reticuloendothelial system. Micromechanical experiments on individual cells will be used to quantitate membrane and cellular properties of blood cells, as well as adhesive cell interactions. A microfluorometric system will be used to study surface diffusion and/or distribution of fluorescently-labelled molecules in membranes of single cells. We anticipate that successful accomplishment of these proposed aims will provided detailed molecular understanding of the rheological and adherence properties of sickle cells. This in turn, should enable the development and critical testing of hypotheses concerning the contributions of various cellular abnormalities to the pathophysiology of sickle cell disease.
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