Specific inhibition of P-selectin–mediated cell adhesion by phage display–derived peptide antagonists

• Published in: Blood 2002-11, Vol.100 (10), p.3570-3577
• Main Authors: Molenaar, Tom J.M., Appeldoorn, Chantal C.M., de Haas, Sonja A.M., Michon, Ingrid N., Bonnefoy, Arnaud, Hoylaerts, Marc F., Pannekoek, Hans, van Berkel, Theo J.C., Kuiper, Johan, Biessen, Erik A.L.
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Binding Sites; Binding, Competitive; Biological and medical sciences; Bones, joints and connective tissue. Antiinflammatory agents; Calcium - pharmacology; Cell Adhesion - drug effects; Dimerization; Fundamental and applied biological sciences. Psychology; HL-60 Cells; Humans; Inflammation; Ligands; Medical sciences; Membrane Glycoproteins - drug effects; Membrane Glycoproteins - metabolism; Molecular and cellular biology; Oligopeptides - antagonists & inhibitors; Oligopeptides - chemistry; P-Selectin - drug effects; P-Selectin - metabolism; P-Selectin - pharmacology; Peptide Library; Pharmacology. Drug treatments; Protein Binding - drug effects
• ISSN: 0006-4971; 1528-0020
• DOI: 10.1182/blood-2002-02-0641

P-selectin is a leukocyte adhesion receptor expressed on activated vascular endothelium and platelets that mediates leukocyte rolling and attachment. Because P-selectin is critically involved in inflammation, we used phage display libraries to identify P-selectin–specific peptides that might interfere with its proinflammatory function. Isolated phage contained a highly conserved amino acid motif. Synthetic peptides showed calcium-dependent binding to P-selectin, with high selectivity over E-selectin and L-selectin. The peptides completely antagonized adhesion of monocyte-derived HL60 cells to P-selectin and increased their rolling velocities in flow chamber experiments. Peptide truncation and alanine-scanning studies indicated that an EWVDV (single-letter amino acid codes) consensus motif sufficed for effective inhibition. Intriguingly, the apparent avidity of the peptides was increased 200-fold when presented in a tetrameric form (2 μM versus 10 nM), which is consistent with the proposed divalent interaction of P-selectin glycoprotein ligand 1 (PSGL-1) with P-selectin. As the EWVDV peptides inhibit the binding of an established glycoside ligand for P-selectin (sulfated Lewis A), it is conceivable that EWVDV interacts with or in close proximity to the actual carbohydrate recognition domain of P-selectin, without being a direct structural mimic of sialyl Lewisx. These ligands are among the most potent antagonists of P-selectin yet designed. Their high affinity, selectivity, and accessible synthesis provide a promising entry to the development of new anti-inflammatory therapeutics and might be a powerful tool to provide important information on the binding site of P-selectin.