title: Glycoprotein (GP) VI dimer as a major collagen-binding site of native platelets: direct evidence obtained with dimeric GPVI-specific Fabs.
abstract: BACKGROUND
  The platelet collagen receptor glycoprotein (GP) VI is suggested to exist as a dimer on the platelet surface, but no direct proof of the functional importance of dimer formation has been provided.
  
  
  OBJECTIVE
  To obtain direct evidence for GPVI dimers on the platelet membrane and their functional importance, Fab antibodies were developed that bind to GPVI dimer (GPVI-Fc2) but not to GPVI monomer (GPVIex) through a phage display method.
  
  
  RESULTS
  Six Fabs were found: B-F, only reactive with GPVI-Fc2, and A, mainly reactive with GPVI-Fc2, with some reactivity towards GPVIex; each Fab (Fab-dHLX-MH) forms a bivalent dimer (b-Fab) by dimerizing the dHLX domains from two Fab molecules. Fab F was subcloned to a monovalent format by deleting its dHLX domain. All b-Fabs induced platelet aggregation, but the monomeric form of Fab F (m-Fab-F) specifically inhibited collagen-induced aggregation. All b-Fabs and m-Fab-F inhibited GPVI-Fc2 binding to fibrous collagen. Immunoblotting showed that b-Fab-F and m-Fab-F bound weakly to GPVI-Fc2. Adding the anti-GPVI monoclonal antibody 204-11 increased the B(max) of m-Fab-F binding to GPVI-Fc2, suggesting that 204-11 binds to GPVI-Fc2 molecules not already in the appropriate conformation to recognize the Fab, converting them to a conformation reactive to the Fab.
  
  
  CONCLUSIONS
  GPVI forms a specific structure by dimerization that is necessary for the binding of this receptor to collagen fibrils. The binding of m-Fab-F to platelets directly demonstrates that GPVI is present as a functionally relevant dimer on the platelet surface.
authors.affiliation: Department of Protein Biochemistry, Institute of Life Science, Kurume University, Kurume-shi, Fukuoka-ken, Japan. smj@lsi.kurume-u.ac.jp
authors.forename: S M
authors.initials: SM
authors.lastname: Jung
authors.forename: K
authors.initials: K
authors.lastname: Tsuji
authors.forename: M
authors.initials: M
authors.lastname: Moroi
mesh_terms: D001665:Binding Sites; D001792:Blood Platelets / Q000737:chemistry*; D003094:Collagen / Q000378:metabolism*; D024022:Fibrillar Collagens / Q000378:metabolism; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000378:metabolism*; D010974:Platelet Aggregation; D010980:Platelet Membrane Glycoproteins / Q000737:chemistry* / Q000276:immunology; D011485:Protein Binding; D055503:Protein Multimerization
chemical_list: D024022:Fibrillar Collagens; D007140:Immunoglobulin Fab Fragments; D010980:Platelet Membrane Glycoproteins; C098341:platelet membrane glycoprotein VI; D003094:Collagen