LECTIN AND EPIDERMAL GROWTH-FACTOR DOMAINS OF P-SELECTIN AT PHYSIOLOGICAL DENSITY ARE THE RECOGNITION UNIT FOR LEUKOCYTE BINDING

P-selectin is an integral membrane glycoprotein on stimulated platelets and endothelial cells that serves as a receptor for leukocytes. To estimate the density of P-selectin in membranes necessary to support adhesion, we incorporated purified P-selectin at varying concentrations into phospholipid bilayers that encapsulated glass microspheres. Maximal binding of these lipospheres to HL60 cells, a P-selectin ligand-expressing cell line, was approached at a P-selectin density of about 100 molecules per mu m(2); half-maximal binding was observed at about 50 to 60 molecules per mu m(2). Compatible results were obtained with P-selectin expressed on Chinese hamster ovary cells. The P-selectin density on stimulated platelets was estimated to be 150 to 200 molecules/mu m(2). To identify the domains of P-selectin required for HL60 cell binding, chimeras of P-selectin and L-selectin were stably expressed in Chinese hamster ovary cells and clones that expressed the chimeras at the estimated physiologic density were selected. Chimeras containing the P-selectin lectin and epidermal growth factor (EGF) domains or the lectin, EGF, and short consensus repeats bound HL60 cells equivalently, but a chimera containing the P-selectin lectin domain alone bound HL60 cells much less well. These results indicate that at a physiologically relevant P-selectin density on membrane surfaces, the lectin, and EGF domains of P-selectin are together required for optimal leukocyte binding. (C) 1995 by The American Society of Hematology.