title: Selective blockade of glycoprotein VI clustering on collagen helices.
abstract: Platelet activation by collagen relies on the interaction of the receptor glycoprotein VI (GPVI) with collagen helices. We have previously generated two recombinant single chain human antibodies (scFvs) to human GPVI. The first, 10B12, binds to the collagen-binding site on the apical surface between the two immunoglobulin-like domains (D1D2) of the receptor and so directly inhibits GPVI function. The second, 1C3, binds D1D2 independently of 10B12 and has been shown to have a more subtle effect on platelet responses to collagen. Here we have shown that 1C3 potentiates the effect of 10B12 on platelet aggregation induced by collagen and cross-linked collagen-related peptide (CRP-XL). We investigated this by measuring the effect of both scFvs on the binding of D1D2 to immobilized collagen and CRP. As expected, 10B12 completely inhibited binding of GPVI to each ligand in a dose-dependent manner. However, 1C3 inhibited only a proportion of GPVI binding to its ligands, implying that it interferes with another aspect of ligand recognition by GPVI. To further understand the mode of inhibition, we used a unique set of CRPs in which the content of critical glycine-proline-hydroxyproline (GPO) triplets was varied in relation to an "inert" scaffold sequence of GPP motifs. We observed that a stepwise increase in D1D2 binding with (GPO)(2) content was blocked by 1C3. Together these results indicate that 1C3 inhibits clustering of the immunoglobulin-like domains of GPVI on collagen/CRPs, a conclusion that is supported by mapping the 1C3 epitope to the region including isoleucine 148 in D2.
authors.affiliation: Department of Haematology, University of Cambridge, Cambridge CB2 2PT, UK.
authors.forename: Marie N
authors.initials: MN
authors.lastname: O'Connor
authors.forename: Peter A
authors.initials: PA
authors.lastname: Smethurst
authors.forename: Lorna W
authors.initials: LW
authors.lastname: Davies
authors.forename: Lotta
authors.initials: L
authors.lastname: Joutsi-Korhonen
authors.forename: David J
authors.initials: DJ
authors.lastname: Onley
authors.forename: Andrew B
authors.initials: AB
authors.lastname: Herr
authors.forename: Richard W
authors.initials: RW
authors.lastname: Farndale
authors.forename: Willem H
authors.initials: WH
authors.lastname: Ouwehand
mesh_terms: D000906:Antibodies / Q000276:immunology; D003094:Collagen / Q000378:metabolism*; D000939:Epitopes / Q000276:immunology; D006801:Humans; D007532:Isoleucine / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010974:Platelet Aggregation; D010980:Platelet Membrane Glycoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary
chemical_list: D000906:Antibodies; D000939:Epitopes; D010455:Peptides; D010980:Platelet Membrane Glycoproteins; C098341:platelet membrane glycoprotein VI; D007532:Isoleucine; D003094:Collagen