ISOLATED P-SELECTIN GLYCOPROTEIN LIGAND-1 DYNAMIC ADHESION TO P-SELECTIN AND E-SELECTIN

Leukocyte adhesion to vascular endothelium under flow involves an adhe sion cascade consisting of multiple receptor pairs that may function i n an overlapping fashion. P-selectin glycoprotein ligand-l (PSGL-1) an d L-selectin have been implicated in neutrophil adhesion to P- and E-s electin under flow conditions. To study, in isolation, the interaction of PSGL-1 with P- and E-selectin under flow, we developed an in vitro model in which various recombinant regions of extracellular PSGL-1 we re coupled to 10-mu m-diameter microspheres. In a parallel plate chamb er with well defined flow conditions, live time video microscopy analy ses revealed that microspheres coated with PSGL-1 attached and rolled on 4-h tumor necrosis factor-alpha-activated endothelial cell monolaye rs, which express high levels of E-selectin, and CHO monolayers stably expressing E- or P-selectin. Further studies using CHO-E and -P monol ayers demonstrate that the first 19 amino acids of PSGL-1 are sufficie nt for attachment and rolling on both E- and P-selectin and suggest th at a sialyl Lewis x-containing glycan at Threonine-16 is critical for this sequence of amino acids to mediate attachment to E- and P-selecti n, The data also demonstrate that a sulfated, anionic polypeptide segm ent within the amino terminus of PSGL-1 is necessary for PSGL-1-mediat ed attachment to P- but not to E-selectin. In addition, the results su ggest that PSGL-1 has more than one binding site for E-selectin: one s ite located within the first 19 amino acids of PSGL-1 and one or more sites located between amino acids 19 through 148.