Verbatim): The research objective is to develop a detailed understanding of the molecular and structural basis for the abnormal rheological and adherence properties of sickle red blood cells and define the in vivo pathologic sequelae of these cellular changes. To achieve our objective we propose the following series of studies: 1) Using novel experimental strategies, determine the relative contributions of the Gardos channel and the K-Cl cotransporter to the morphologic and rheologic heterogeneity of dense sickle red cells. Using intravital microscopy, document effects of the observed rheological heterogeneity on flow behavior in vivo. Generate sickle mice in which the Gardos channel and/or K-CL cotransporter are inactivated to directly establish the relative contributions of these two transport proteins to cell dehydration and compromised blood flow. 2) To develop a mechanistic understanding of premature sickle red cell destruction, distinct populations of mouse sickle cells will be isolated based on differences in their cellular and membrane characteristics and their in vivo life span will be determined. The surface characteristics and surface area of these different cell sub-populations will be documented. Data from these in vitro and in vivo studies will be used to define the contribution of surface area loss and associated membrane changes to the decreased life span of sickle cells. 3) As it is likely that the dynamic strength of a specific adhesive complex will determine its impact on in vivo flow, we will measure the dynamic strength for various specific adhesive interactions identified in mediating sickle red cell adherence to vascular endothelial cells. These data should enable us to define the physiologically relevant adhesive interactions. We will validate the in vitro studies by performing in situ studies on sickle mice in which genes encoding specific adhesive receptors are inactivated. For the proposed studies, we will use multiple techniques in biophysics, molecular biology, cell biology, mouse genetics and circulatory physiology. The sickle mice that we developed expressing exclusively human sickle hemoglobin and exhibiting many clinical features of human disease are a key component of our experimental strategy. These mice should enable us to perform deliberate and specific perturbations for defining the determinants of adherence and flow, which would not be possible in human studies. Our application of these refined systems offers great promise for elucidating the cause of painful vaso-occlusion and identifying potential targets for future therapy.
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