Interactions between the human red blood cell (RBC) membrane and its intracellular and extracellular environment mediate both normal RBC homeostasis and abnormal RBC pathology in the circulation. Polymerization of hemoglobin S within sickle RBCs leads to a spectrum of membrane derangements, including changes in protein-protein and protein- lipid interactions, that affect the mobility and surface distribution of the major transmembrane proteins, glycophorin and band 3. Alterations in the surface distribution of glycophorin-bearing negative charges may influence sickle RBC adherence to vascular endothelial cells (ECs), which may, in turn, initiate vaso-occlusive crisis. Formation of band 3 aggregates in sickle RBC membranes may promote the binding of autologous antibody to these cells, facilitating their premature removal by monocytes and macrophages. Abnormal exposure of phosphatidylserine at the surface of sickle RBCs may also mediate adherence to monocytes and macrophages. In this proposal high-speed laser and video microscopy techniques are used to measure the lateral mobility, rotational mobility, and surface distribution of transmembrane proteins and lipids in individual sickle RBC membranes and in the membranes of sickle RBCs adherent to ECs in culture, to peripheral blood monocytes, and to immobilized plasma proteins that may be important in facilitating sickle RBC-EC adhesion. These proteins include fibrin, fibrinogen, fibronectin, thrombospondin, vitronectin, and von Willebrand factor. We aim to test the hypotheses that: 1) specific abnormalities in sickle RBC band 3, glycophorin, and phospholipid mobility and surface distribution correlate, at the level of individual sickle RBCs, with increased binding of autologous antibody and increased adherence to monocytes, macrophages, and ECs; and 2) specific plasma proteins serve as important mediators of sickle RBC-EC adhesion. Results from these studies are expected to elucidate molecular defects in the sickle RBC membrane that are directly involved in the pathophysiology of sickle RBC hemolysis and vaso- occlusive crisis.