SOLUBLE MONOMERIC P-SELECTIN CONTAINING ONLY THE LECTIN AND EPIDERMALGROWTH-FACTOR DOMAINS BINDS TO P-SELECTIN GLYCOPROTEIN LIGAND-1 ON LEUKOCYTES

Under shear stress, leukocytes use P-selectin glycoprotein ligand-1 (P SGL-1) to tether to and roll on P-selectin expressed on activated plat elets or endothelial cells. P-selectin has an NH2-terminal lectin doma in, an epidermal growth factor (EGF)-like motif, nine consensus repeat s (CRs), a transmembrane domain, and a cytoplasmic tail. To determine whether the CRs are required for P-selectin to bind PSGL-1. we express ed a soluble protein (Lec-EGF) that contained only the lectin and EGF domains, plus a short C-terminal epitope tag. Electron microscopy and hydrodynamic analysis confirmed that Lec-EGF was monomeric, as previou sly shown for soluble P-selectin (sPS) that contained the lectin and E GF domains plus all nine CRs. Fluid-phase Lec-EGF or sPS inhibited bin ding of oligomeric I-125-labeled membrane-derived P-selectin (mPS) to PSGL-1 on neutrophils and binding of I-125-PSGL-1 to immobilized mPS. The IC50 for inhibiting binding of mPS to neutrophils was fivefold gre ater for Lec-EGF than for sPS, whereas the IC50, for inhibiting bindin g of mPS to purified PSGL-1 was indistinguishable for Lec-EGF and sPS. Under static or shear conditions, neutrophils used PSGL-1 to tether t o or roll on Lec-EGF that was captured by an immobilized monoclonal an tibody to the C-terminal epitope, These data show that P-selectin requ ires only the lectin and EGF domains to bind to PSGL-1. (C) 1997 by Th e American Society of Hematology.