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Prepublished online as a Blood First Edition Paper on September 22, 2003; DOI 10.1182/blood-2003-01-0308. This Article Full Text (PDF) All Versions of this Article: 2003-01-0308v1 103/3/903    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Rights and Permissions Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Smethurst, P. A Articles by Ouwehand, W. H Search for Related Content PubMed PubMed Citation Articles by Smethurst, P. A Articles by Ouwehand, W. H Social Bookmarking                   What's this? Submitted January 31, 2003 Accepted August 22, 2003 Identification of the primary collagen binding surface on human glycoprotein VI by site-directed mutagenesis and by a blocking phage antibody Peter A Smethurst*, Lotta Joutsi-Korhonen, Marie N O'Connor, Erica Wilson, Nicola S Jennings, Stephen F Garner, Yanjun Zhang, C Graham Knight, Timothy R Dafforn, Ashley Buckle, Martin J W Ijsseldijk, Philip G de Groot, Nicholas A Watkins, Richard W Farndale, and Willem H Ouwehand Department of Haematology, University of Cambridge and National Blood Service, Cambridge, United Kingdom; Department of Biochemistry, University of Cambridge, Cambridge, United Kingdom Medical Research Council, Centre for Protein Engineering, Cambridge, United Kingdom Department of Haematology, University Medical Center, Utrecht, The Netherlands * Corresponding author; email: pas28{at}cam.ac.uk. 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 modelling and sequence comparison identified key differences between hD1D2 and mD1D2. Ten mutant hD1D2 were expressed, of which four had human residues replaced by their murine counterpart and six 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 modelled. CiteULike    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this? This article has been cited by other articles: B. Dumont, D. Lasne, C. Rothschild, M. Bouabdelli, V. Ollivier, C. Oudin, N. Ajzenberg, B. Grandchamp, and M. 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