The objectives of the proposed work are to utilize modern rheological techniques combined with biochemical analysis to understand the biophysical basis of the pathophysiology of sickle cell amenia. This knowledge will then provide a basis for the rational development of new clinical treatments. Specifically, three major classes of problems are to be investigated: (1) Red cell - endothelial cell interactions under controlled flow conditions using epi-fluorescent video microscopy and image analysis techniques. (2) Flow of and oxygen transport from sickle red cells in silicone capillaries of the same sizes as found in the human microcirculation. (3) Control of the kinetics of gelation of S hemoglobin (in solution and in the whole cell) upon changes in environmental conditions. Obstructive interactions with endothelial cells, lack of tissue oxygenation and vaso occlusion due to gel formation are three possible problems that could contribute to a clinical crisis. The techniques proposed will examine each of these areas. These investigations require very specialized equipment - some of which is only available in our laboratories. By videomicroscopy of red cells under controlled flow conditions, we can discriminate whether cells adhere to the wall of a flow chamber or to each other at differernt shear rates. We will determine the role vessel wall composition plays in adherence by coating the chamber with collagen or culturing endothelial cells on it. The ability of sickle red cells to deliver 02 to tissue will be compared in the oxygen transport simulation studies. RBCs will be density separated SS fractions and also RBC from AS and normal controls. Variables to study will include the effect of 02, pH, osmolarity, shear stress exposure, calcium loading, ghost preparation and drug modification of hemoglobin or membrane proteins. By using AS and AA RBC and experimental manipulations (increased red cell calcium, dehydration, increased membrane bound hemoglobin) we plan to produce models of the sickle red cell. These models as well as normal and sickled cells will be studied rheologically. One technique will involve shearing the cells in viscometers (Rice Viscometer or the Rheometrics Fluids Rheometer). Their response to shear will be assessed biochemically. Deformability will be evaluated by the nuclepare filtration technique, and the kinetics of whole cell deformability changes will be described. The kinetics of hemoglobin gel formation in sickle cells and in the models developed will also be investigated.
