Lectin and epidermal growth factor domains of P-selectin at physiologic 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 microns 2; half-maximal binding was observed at about 50 to 60 molecules per microns 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/microns 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.
Gibson
R M
RM
Center for Hemostasis and Thrombosis, New England Medical Center, Boston, MA 02111.
Kansas
G S
GS
Tedder
T F
TF
Furie
B
B
Furie
B C
BC
D000818:Animals; D001792:Blood Platelets / Q000737:chemistry; D016466:CHO Cells; D002448:Cell Adhesion; D015815:Cell Adhesion Molecules / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006224:Cricetinae; D004815:Epidermal Growth Factor / Q000737:chemistry*; D005898:Glass; D006801:Humans; D019041:L-Selectin; D037102:Lectins / Q000737:chemistry*; D015473:Leukemia, Promyelocytic, Acute / Q000378:metabolism; D007962:Leukocytes / Q000378:metabolism*; D008051:Lipid Bilayers / Q000378:metabolism; D008081:Liposomes / Q000378:metabolism; D008863:Microspheres; D019007:P-Selectin; D010980:Platelet Membrane Glycoproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013329:Structure-Activity Relationship; D014162:Transfection
D015815:Cell Adhesion Molecules; D037102:Lectins; D008051:Lipid Bilayers; D008081:Liposomes; D019007:P-Selectin; D010980:Platelet Membrane Glycoproteins; D011993:Recombinant Fusion Proteins; D019041:L-Selectin; D004815:Epidermal Growth Factor