Identification of the primary collagen-binding surface on human glycoprotein VI by site-directed mutagenesis and by a blocking phage antibody

• Published in: Blood Vol. 103; no. 3; pp. 903 - 911
• Main Authors: Smethurst, Peter A., Joutsi-Korhonen, Lotta, O’Connor, Marie N., Wilson, Erica, Jennings, Nicola S., Garner, Stephen F., Zhang, Yanjun, Knight, C. Graham, Dafforn, Timothy R., Buckle, Ashley, IJsseldijk, Martin J.W., de Groot, Philip G., Watkins, Nicholas A., Farndale, Richard W., Ouwehand, Willem H.
• Format: Journal Article
• Language: English
• Published: Washington, DC Elsevier Inc 01.02.2004; The Americain Society of Hematology
• Subjects: Amino Acid Sequence; Animals; Antibodies, Blocking; Base Sequence; Binding Sites - genetics; Biological and medical sciences; Blood coagulation. Blood cells; Carrier Proteins - metabolism; Collagen - metabolism; DNA, Complementary - genetics; Fundamental and applied biological sciences. Psychology; HLA Antigens - metabolism; Humans; In Vitro Techniques; Ligands; Lysine - chemistry; Mice; Models, Molecular; Molecular and cellular biology; Molecular Sequence Data; Mutagenesis, Site-Directed; Peptides; Platelet; Platelet Membrane Glycoproteins - chemistry; Platelet Membrane Glycoproteins - genetics; Platelet Membrane Glycoproteins - immunology; Platelet Membrane Glycoproteins - metabolism; Protein Structure, Tertiary; Receptors, Immunologic - metabolism; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Sequence Homology, Amino Acid; Species Specificity; Platelet; Molecular structure; Hemostasis; Animal; Collagen; Glycoprotein VI; Binding site
• ISSN: 0006-4971; 1528-0020
• DOI: 10.1182/blood-2003-01-0308

Glycoprotein (GP) VI is the major receptor responsible for platelet activation by collagen, but the collagen-binding surface of GPVI is unknown. To address this issue we expressed, from insect cells, the immunoglobulin (Ig)–like ectodomains (residues 1-185) of human and murine GPVI, called hD1D2 and mD1D2, respectively. Both proteins bound specifically to collagen-related peptide (CRP), a GPVI-specific ligand, but hD1D2 bound CRP more strongly than did mD1D2. Molecular modeling and sequence comparison identified key differences between hD1D2 and mD1D2. Ten mutant hD1D2s were expressed, of which 4 had human residues replaced by their murine counterpart, and 6 had replacements by alanine. CRP binding studies with these mutants demonstrated that the exchange of lysine at position 59 for the corresponding murine glutamate substantially reduced binding to CRP. The position of lysine59 on the apical surface of GPVI suggests a mode of CRP binding analogous to that used by the related killer cell Ig-like receptors to bind HLA. This surface was confirmed as critical for collagen binding by epitope mapping of an inhibitory phage antibody against GPVI. This anti-GPVI, clone 10B12, gave dose-dependent inhibition of the hD1D2-collagen interaction. Clone 10B12 inhibited activation of platelets by CRP and collagen in aggregometry and thrombus formation by the latter in whole blood perfusion. Antibody 10B12 showed significantly reduced binding to the hD1D2-E59, and, on that basis, the GPVI:10B12 interface was modeled.