title: Constitutive dimerization of glycoprotein VI (GPVI) in resting platelets is essential for binding to collagen and activation in flowing blood.
abstract: The platelet collagen receptor glycoprotein VI (GPVI) has been suggested to function as a dimer, with increased affinity for collagen. Dissociation constants (K(d)) obtained by measuring recombinant GPVI binding to collagenous substrates showed that GPVI dimers bind with high affinity to tandem GPO (Gly-Pro-Hyp) sequences in collagen, whereas the markedly lower affinity of the monomer for all substrates implies that it is not the collagen-binding form of GPVI. Dimer binding required a high density of immobilized triple-helical (GPO)(10)-containing peptide, suggesting that the dimer binds multiple, discrete peptide helices. Differential inhibition of dimer binding by dimer-specific antibodies, m-Fab-F and 204-11 Fab, suggests that m-Fab-F binds at the collagen-binding site of the dimer, and 204-11 Fab binds to a discrete site. Flow cytometric quantitation indicated that GPVI dimers account for ~29% of total GPVI in resting platelets, whereas activation by either collagen-related peptide or thrombin increases the number of dimers to ~39 and ~44%, respectively. m-Fab-F inhibits both GPVI-dependent static platelet adhesion to collagen and thrombus formation on collagen under low and high shear, indicating that pre-existing dimeric GPVI is required for the initial interaction with collagen because affinity of the monomer is too low to support binding and that interaction through the dimer is essential for platelet activation. These GPVI dimers in resting circulating platelets will enable them to bind injury-exposed subendothelial collagen to initiate platelet activation. The GPVI-specific agonist collagen-related peptide or thrombin further increases the number of dimers, thereby providing a feedback mechanism for reinforcing binding to collagen and platelet activation.
authors: Department of Biochemistry, University of Cambridge, Cambridge CB2 1QW, UK. smj52@cam.ac.uk
authors: Stephanie M
authors: SM
authors: Jung
authors: Masaaki
authors: M
authors: Moroi
authors: Kenji
authors: K
authors: Soejima
authors: Tomohiro
authors: T
authors: Nakagaki
authors: Yoshiki
authors: Y
authors: Miura
authors: Michael C
authors: MC
authors: Berndt
authors: Elizabeth E
authors: EE
authors: Gardiner
authors: Joanna-Marie
authors: JM
authors: Howes
authors: Nicholas
authors: N
authors: Pugh
authors: Dominique
authors: D
authors: Bihan
authors: Steve P
authors: SP
authors: Watson
authors: Richard W
authors: RW
authors: Farndale
mesh_terms: D001783:Blood Flow Velocity; D003094:Collagen / Q000494:pharmacology*; D006801:Humans; D007140:Immunoglobulin Fab Fragments; D010455:Peptides / Q000494:pharmacology*; D015539:Platelet Activation / Q000187:drug effects*; D010980:Platelet Membrane Glycoproteins / Q000819:agonists* / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects / Q000502:physiology; D055503:Protein Multimerization / Q000187:drug effects*; D017433:Protein Structure, Secondary; D013917:Thrombin / Q000494:pharmacology
chemical_list: D007140:Immunoglobulin Fab Fragments; D010455:Peptides; D010980:Platelet Membrane Glycoproteins; C098341:platelet membrane glycoprotein VI; D003094:Collagen; D013917:Thrombin