The objectives of the proposed work are to utilize modern rheological techniques combined with biochemical analyses to understand the biophysical basis of the pathophysiology of sickle cell anemia. This knowledge will then provide a basis for the rational development of new clinical treatments. Specifically, two 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; and (2) effect of shear stress on red cell membrane permeability and the sickle hemoglobin polymerization rate and final gel material properties. Structive interactions with endothelial cells leading to lack of tissue oxygenation and vaso- occlusion due to gel formation are two possible problems that could contribute to a clinical crisis. The techniques proposed will examine these areas. The investigations require very specialized equipment--some of which is only available in our laboratories. Employing videomicroscopy of red cells under controlled flow conditions, we can discriminate whether cells adhere to the wall of a flow chamber or each other at different shear rates. We will determine the role vessel wall composition plays in adherence by coating the chamber surface with various cytoadhesive proteins, by utilizing cultured endothelial cells from various parts of the circulation and by using monoclonal antibodies to membrane proteins and synthetic peptides which block certain membrane receptors. A specific novel hypothesis to be investigated is that the large molecular weight multimers of vWF protein, generated locally by activated or damaged endothelium, play a crucial role in modulating sickle RBC/endothelial cell adhesion. RBCs will be density separated SS fractions and all RBC from AS and normal controls. Variables to study will include the effect of pH, osmolarity, shear stress exposure, wall shear rate, calcium and potassium content and drug modified hemoglobin or altered RBC membranes. 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. The models as well as normal and sickle cells will be studied rheologically. Our technique will involve shearing the cells in viscometers (Rice Viscometer or Rheometrics Fluids Rheometer). Their response to shear will be assessed using biophysical and biochemical analyses.
