Selective Blockade of Glycoprotein VI Clustering on Collagen Helices
2006; Elsevier BV; Volume: 281; Issue: 44 Linguagem: Inglês
10.1074/jbc.m606480200
ISSN1083-351X
AutoresMarie O’Connor, Peter A. Smethurst, Lorna W. Davies, Lotta Joutsi‐Korhonen, David J. Onley, Andrew B. Herr, Richard W. Farndale, Willem H. Ouwehand,
Tópico(s)Cell Adhesion Molecules Research
ResumoPlatelet activation by collagen relies on the interaction of the receptor glycoprotein VI (GPV121313132) 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. Platelet activation by collagen relies on the interaction of the receptor glycoprotein VI (GPV121313132) 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. Recognition of exposed subendothelial collagen by the receptor glycoprotein VI (GPVI) 2The abbreviations used are: GPVI, glycoprotein VI; Ig, immunoglobulin; CRP, collagen-related peptide; BSA, bovine serum albumin; ELISA, enzyme-linked immunosorbent assay; HRP, horseradish peroxidase. on platelets is a critical early step for platelet activation and subsequent thrombus formation. This interaction strengthens platelet adhesion through activation of integrins α2β1 and αIIbβ3 (1Kuijpers M.J. Schulte V. Bergmeier W. Lindhout T. Brakebusch C. Offermanns S. Fassler R. Heemskerk J.W. Nieswandt B. FASEB J. 2003; 17: 685-687Crossref PubMed Scopus (134) Google Scholar) and induces degranulation, aggregation, and procoagulant activity (2Heemskerk J.W. Siljander P. Vuist W.M. Breikers G. Reuteling-sperger C.P. Barnes M.J. Knight C.G. Lassila R. Farndale R.W. Thromb. Haemostasis. 1999; 81: 782-792Crossref PubMed Scopus (73) Google Scholar). Hence, elucidation of the mechanism of interaction of GPVI with collagen is valuable for developing effective platelet antagonists. We have identified the primary collagen binding surface of human GPVI on the immunoglobulin (Ig)-like domains (D1D2) of the receptor by comparing binding of human and mouse recombinant monomeric D1D2 to a synthetic collagen-related peptide (CRP) that contains ten glycine-proline-hydroxyproline (GPO) repeats in each strand of a triple helix (3O'Connor M.N. Smethurst P.A. Farndale R.W. Ouwehand W.H. J. Thromb. Haemostasis. 2006; 4: 869-873Crossref PubMed Scopus (31) Google Scholar, 4Smethurst P.A. Joutsi-Korhonen L. O'Connor M.N. Wilson E. Jennings N.S. Garner S.F. Zhang Y. Knight C.G. Dafforn T.R. Buckle A. IJsseldijk M.J. De Groot P.G. Watkins N.A. Farndale R.W. Ouwehand W.H. Blood. 2004; 103: 903-911Crossref PubMed Scopus (112) Google Scholar). Prolyl hydroxylation is necessary for recognition of collagen and such peptides by platelet GPVI (5Perret S. Eble J.A. Siljander P.R. Merle C. Farndale R.W. Theisen M. Ruggiero F. J. Biol. Chem. 2003; 278: 29873-29879Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). Both mouse and human D1D2 showed specific, dose-dependent, and saturable binding to CRP, relative to peptide GPP10, a peptide of similar structure in which hydroxyproline is replaced by proline. Using such peptides, the minimum recognition motif for GPVI has recently been identified as a tandem GPO triplet or (GPO)2. 3P. A. Smethurst, D. J. Onley, G. E. Jarvis, M. N. O'Connor, C. G. Knight, A. B. Herr, W. H. Ouwehand, and R. W. Farndale, submitted for publication. Using human recombinant D1D2 as bait, two scFvs were selected that bind to distinct epitopes on GPVI. 10B12 recognizes the primary collagen binding surface of human GPVI, and 1C3 does not (4Smethurst P.A. Joutsi-Korhonen L. O'Connor M.N. Wilson E. Jennings N.S. Garner S.F. Zhang Y. Knight C.G. Dafforn T.R. Buckle A. IJsseldijk M.J. De Groot P.G. Watkins N.A. Farndale R.W. Ouwehand W.H. Blood. 2004; 103: 903-911Crossref PubMed Scopus (112) Google Scholar). The 1C3 epitope in GPVI is as yet unknown but is conserved between human and mouse GPVI. 10B12 has been shown to inhibit collagen-induced platelet aggregation and prevent thrombus formation in vitro on a collagen-coated surface in whole blood perfusion (4Smethurst P.A. Joutsi-Korhonen L. O'Connor M.N. Wilson E. Jennings N.S. Garner S.F. Zhang Y. Knight C.G. Dafforn T.R. Buckle A. IJsseldijk M.J. De Groot P.G. Watkins N.A. Farndale R.W. Ouwehand W.H. Blood. 2004; 103: 903-911Crossref PubMed Scopus (112) Google Scholar). The effect of 1C3 on thrombus formation by human platelets under flow has also been investigated, where 1C3 was shown to alter neither the surface coverage nor the size and morphology of the thrombi formed on a collagen-coated surface. However, 1C3 halved the expression of phosphatidylserine on adherent platelets (7Siljander P.R. Munnix I.C. Smethurst P.A. Deckmyn H. Lindhout T. Ouwehand W.H. Farndale R.W. Heemskerk J.W. Blood. 2004; 103: 1333-1341Crossref PubMed Scopus (167) Google Scholar), leading us to conclude that 1C3 perturbs the function of GPVI indirectly, prompting the current investigation. In this study the scFvs 10B12 and 1C3 are shown to have an additive inhibitory effect on collagen-induced platelet aggregation. The mode of receptor blockade conferred by 1C3 was investigated by measuring its effect on the binding of D1D2 to collagen and to synthetic collagen-like peptides. Also, evidence is presented that locates the epitope of the antibody to a surface of the molecule distinct from the primary collagen-binding site, providing an explanation for the biological effects of the antibody. Materials—The anti-GPVI scFvs 10B12 and 1C3 were selected and characterized as described previously from human V gene phage display libraries (4Smethurst P.A. Joutsi-Korhonen L. O'Connor M.N. Wilson E. Jennings N.S. Garner S.F. Zhang Y. Knight C.G. Dafforn T.R. Buckle A. IJsseldijk M.J. De Groot P.G. Watkins N.A. Farndale R.W. Ouwehand W.H. Blood. 2004; 103: 903-911Crossref PubMed Scopus (112) Google Scholar). Human D1D2 (residues 1-185 of the mature protein) was cloned and expressed as a calmodulin-tagged fusion protein (4Smethurst P.A. Joutsi-Korhonen L. O'Connor M.N. Wilson E. Jennings N.S. Garner S.F. Zhang Y. Knight C.G. Dafforn T.R. Buckle A. IJsseldijk M.J. De Groot P.G. Watkins N.A. Farndale R.W. Ouwehand W.H. Blood. 2004; 103: 903-911Crossref PubMed Scopus (112) Google Scholar, 26Jennings N.S. Smethurst P.A. Knight C.G. O'Connor M.N. Joutsi-Korhonen L. Stafford P. Stephens J. Garner S.F. Harmer I.J. Farndale R.W. Watkins N.A. Ouwehand W.H. J. Immunol. Methods. 2006; (in press.)PubMed Google Scholar) using full-length human GPVI cDNA and primers designed with reference to published sequences (8Clemetson J.M. Polgar J. Magnenat E. Wells T.N. Clemetson K.J. J. Biol. Chem. 1999; 274: 29019-29024Abstract Full Text Full Text PDF PubMed Scopus (368) Google Scholar, 9Jandrot-Perrus M. Busfield S. Lagrue A.H. Xiong X. Debili N. Chickering T. Le Couedic J.P. Goodearl A. Dussault B. Fraser C. Vainchenker W. Villeval J.L. Blood. 2000; 96: 1798-1807Crossref PubMed Google Scholar). D1D2 has shown similar properties to human platelet GPVI (4Smethurst P.A. Joutsi-Korhonen L. O'Connor M.N. Wilson E. Jennings N.S. Garner S.F. Zhang Y. Knight C.G. Dafforn T.R. Buckle A. IJsseldijk M.J. De Groot P.G. Watkins N.A. Farndale R.W. Ouwehand W.H. Blood. 2004; 103: 903-911Crossref PubMed Scopus (112) Google Scholar). Horm collagen is a preparation of fibrillar type I collagen from equine tendon (Nycomed, Munich, Germany). Collagen-related peptides are triple helices, each strand composed of 37 residues, and their synthesis has been described previously (10Farndale R.W. Siljander P.R. Onley D.J. Sundaresan P. Knight C.G. Barnes M.J. Biochem. Soc. Symp. 2003; 70: 81-94Crossref PubMed Scopus (39) Google Scholar, 11Knight C.G. Morton L.F. Onley D.J. Peachey A.R. Ichinohe T. Okuma M. Farndale R.W. Barnes M.J. Cardiovasc. Res. 1999; 41: 450-457Crossref PubMed Scopus (192) Google Scholar). The primary structure of GPP10 is GCP-[GPP]10-GCPG; GPO2 is GCP-[GPP]4 [GPO]2 [GPP]4-GCPG; GPO4 is GCP-[GPP]3 [GPO]4 [GPP]3-GCPG; GPO6 is GCP-[GPP]2 [GPO]6 [GPP]2-GCPG; and monomeric (m)CRP is GCO-[GPO]10-GCOG, where O = hydroxyproline. In CRP-XL, mCRP molecules were cross-linked via terminal cysteine or lysine residues as described previously (11Knight C.G. Morton L.F. Onley D.J. Peachey A.R. Ichinohe T. Okuma M. Farndale R.W. Barnes M.J. Cardiovasc. Res. 1999; 41: 450-457Crossref PubMed Scopus (192) Google Scholar). N9A peptide (CAAARWKKAFIAVSAANRFKKIS) (12Montigiani S. Neri G. Neri P. Neri D. J. Mol. Biol. 1996; 258: 6-13Crossref PubMed Scopus (55) Google Scholar) binds calmodulin in the presence of Ca2+ ions and was conjugated to BSA by a standard method (13Bernatowicz M.S. Matsueda G.R. Anal. Biochem. 1986; 155: 95-102Crossref PubMed Scopus (94) Google Scholar) to give BSA-N9A. In this form it was used to immobilize D1D2 molecules in enzyme-linked immunosorbent assay (ELISA). To detect D1D2 binding to its ligands N9A was conjugated to peroxidase to give HRP-N9A. Protein purity was assessed by SDS-PAGE, and quantitation was performed using a BCA assay (Pierce). Platelet Aggregometry—Citrate-anticoagulated whole blood was obtained from normal blood donors (National Blood Service, Cambridge, England) homozygous for the common allele (SKTQH) of the GP6 gene (14Watkins N.A. O'Connor M.N. Rankin A. Jennings N. Wilson E. Harmer I.J. Davies L. Smethurst P.A. Dudbridge F. Farndale R.W. Ouwehand W.H. J. Thromb. Haemostasis. 2006; 4: 1197-1205Crossref PubMed Scopus (36) Google Scholar) and platelet-rich plasma (200 × 109 platelets/liter) prepared as previously described (15Joutsi-Korhonen L. Smethurst P.A. Rankin A. Gray E. Ijsseldijk M. Onley C.M. Watkins N.A. Williamson L.M. Goodall A.H. de Groot P.G. Farndale R.W. Ouwehand W.H. Blood. 2003; 101: 4372-4379Crossref PubMed Scopus (122) Google Scholar). Preparation of Monomeric and Dimeric scFvs—scFvs were expressed with c-Myc and hexahistidine affinity tags as previously described (16Watkins N.A. Brown C. Hurd C. Navarrete C. Ouwehand W.H. Tissue Antigens. 2000; 55: 219-228Crossref PubMed Scopus (19) Google Scholar) and purified using a Ni2+-charged HiTrap chelating column (Amersham Biosciences) according to the manufacturer's instructions with 0.5 m NaCl, 50 mm sodium phosphate, pH 7.6-7.9, as the base buffer, eluting with 275 mm imidazole. This eluate contained a mixture of monomeric and dimeric (≤5%) forms of each scFv. The latter species of 60 kDa likely represents a 1C3 diabody with two identical juxtaposed antigen-binding sites separated by 65 Å (17Perisic O. Webb P.A. Holliger P. Winter G. Williams R.L. Structure. 1994; 2: 1217-1226Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar). After concentration (Vivaspin 10kDa; Vivascience) the scFvs were subjected to gel filtration (Superose 12; Amersham Biosciences GE Healthcare) into 10 mm HEPES, 150 mm NaCl, pH 7.2, to desalt and separate the two forms. These were kept on ice and used without delay to minimize re-equilibration. Ligand Binding Assay for D1D2—Binding of D1D2 to collagen or collagen-related peptides was measured in a solid phase assay as previously described (4Smethurst P.A. Joutsi-Korhonen L. O'Connor M.N. Wilson E. Jennings N.S. Garner S.F. Zhang Y. Knight C.G. Dafforn T.R. Buckle A. IJsseldijk M.J. De Groot P.G. Watkins N.A. Farndale R.W. Ouwehand W.H. Blood. 2004; 103: 903-911Crossref PubMed Scopus (112) Google Scholar). Briefly, collagen or peptides were immobilized on 96-well Maxisorp™ microplates (Invitrogen). Equal coating of each peptide (GPP10, GPO2-GPO6, monomeric CRP) was ensured by measuring, in a separate experiment, the fluorescent signal obtained after derivatization of free cysteines of the peptides with Alexa 488-maleimide (Molecular Probes). 4P. A. Smethurst, personal observations. Then, after appropriate blocking and washing, the binding of D1D2 was detected with HRP-N9A using a standard peroxidase substrate on a plate reader at 450 nm. Values for nonspecific binding to blocked wells (typically less than 0.06 absorbance units) were subtracted from other readings before analysis. Data were analyzed and graphs were produced in PRISM (GraphPad, San Diego, CA). Inhibition Assay—The inhibition assay protocol is a modification of the standard ligand binding assay, with the addition of the following steps. A subsaturating concentration of D1D2 was preincubated in assay buffer either without any scFv or peptide (positive control of 100% binding) or in the presence of increasing concentrations of scFv or peptide for 2 h at room temperature before adding to the plate. Assay buffer was added to the remaining wells (negative control of 0% binding), and the plate was incubated for 20 min at room temperature. Bound D1D2 was detected as for the standard ligand binding assay. Specific binding was obtained by subtracting nonspecific binding (to the plate and/or BSA) from total binding. Site-directed Mutagenesis of D1D2—Site-specific mutations were introduced into D1D2 using the QuikChange site-directed mutagenesis kit (Stratagene), essentially following the manufacturer's instructions and as described previously (4Smethurst P.A. Joutsi-Korhonen L. O'Connor M.N. Wilson E. Jennings N.S. Garner S.F. Zhang Y. Knight C.G. Dafforn T.R. Buckle A. IJsseldijk M.J. De Groot P.G. Watkins N.A. Farndale R.W. Ouwehand W.H. Blood. 2004; 103: 903-911Crossref PubMed Scopus (112) Google Scholar). Residues Tyr-134 and Ile-148 were mutated individually to alanine using the following primer pairs: 5′-GAAGGGGACCCTGCGCCCGCCAAGAATCCCGAGAGATGG-3′ and 5′-CCATCTCTCGGGATTCTTGGCGGGCGCAGGGTCCCCTTC-3′ for Y134A, 5′-CGGGCTAGTTTCCCCATCGCCACGGTGACCGCC-3′ and 5′-GGCGGTCACCGTGGCGATGGGGAAACTAGCCCG-3′ for I148A. Capture ELISA—The effect of the single amino acid mutations on binding of D1D2 to the anti-GPVI scFvs 10B12 and 1C3 was investigated by ELISA. D1D2 mutants were captured via the calmodulin tag to BSA-N9A in the presence of Ca2+ and binding of the scFvs detected with an HRP-labeled mouse monoclonal anti-c-Myc (clone 9E10; Roche Applied Science) as described previously (4Smethurst P.A. Joutsi-Korhonen L. O'Connor M.N. Wilson E. Jennings N.S. Garner S.F. Zhang Y. Knight C.G. Dafforn T.R. Buckle A. IJsseldijk M.J. De Groot P.G. Watkins N.A. Farndale R.W. Ouwehand W.H. Blood. 2004; 103: 903-911Crossref PubMed Scopus (112) Google Scholar). Structural Analysis of GPVI—The locations of Ile-148 and Tyr-134 on the surface of GPVI were determined using the crystal structure of the collagen-binding domains of GPVI (18Horii K. Kahn M.L. Herr A.B. Blood. 2006; 108: 936-942Crossref PubMed Scopus (128) Google Scholar). Prediction of likely protein interaction interfaces on the surface of GPVI was carried out using the Sppider web server (sppider.cchmc.org), which classifies amino acid residues based on their probability of forming protein interaction sites. 5A. Porollo and J. Meller, submitted for publication. The SPPI-DER1 algorithm was used, with the threshold value corresponding to the trade-off between sensitivity and specificity set to either 0.5 (the default value) or 0.7 (higher specificity). At both settings, Sppider identified a prominent patch of residues on the side of D2 adjacent to Ile-148 as a probable interaction site. Images were generated using PyMOL (20DeLano W.L. The PyMOL User's Manual. DeLano Scientific, San Carlos, CA2002Google Scholar). scFv 1C3 Potentiates 10B12 Inhibition of Platelet Aggregation Evoked by Collagen and CRP-XL—After separating the two forms of each scFv, the monomeric forms were used in these aggregation assays. The dose-dependent inhibitory effect of 10B12 can clearly be seen, whereas preincubation with 1C3 at 20 μg/ml (a saturating dose as judged by flow cytometry, data not shown) had very little effect on the rate or extent of platelet aggregation induced by CRP-XL and fibrillar collagen (Fig. 1). However, when 1C3 was applied in combination with 10B12, the inhibitory effect was more pronounced than with 10B12 alone, particularly in response to fibrillar collagen (Fig. 1). scFv 1C3 Partially Inhibits Binding of D1D2 to Collagen and CRP-XL in a Plate Assay—The molecular basis for the above observation was investigated by measuring the effect of monomeric and dimeric 10B12 and 1C3 on binding of D1D2 to immobilized CRP-XL and fibrillar collagen by the ligand binding assay. In each experiment, a subsaturating concentration of D1D2 (3 μg/ml) was preincubated with increasing concentrations of scFv before addition to the coated plate. As expected, binding of D1D2 to CRP or collagen was completely abolished by 10B12 in monomeric or dimeric form (Fig. 2, B and D). In contrast, the effect of 1C3 was markedly different depending on its valency. The bivalent 1C3 dimer dramatically increased binding of D1D2 to both ligands (Fig. 2, A and C), whereas monomeric 1C3 partially inhibited binding (Fig. 2, B and D). The extent of inhibition was greater with CRP as the ligand (maximum effect ∼80%) than with collagen (∼50%). This partial inhibition was nevertheless dose dependent and consistent with the dose range observed previously for binding to D1D2 by ELISA (4Smethurst P.A. Joutsi-Korhonen L. O'Connor M.N. Wilson E. Jennings N.S. Garner S.F. Zhang Y. Knight C.G. Dafforn T.R. Buckle A. IJsseldijk M.J. De Groot P.G. Watkins N.A. Farndale R.W. Ouwehand W.H. Blood. 2004; 103: 903-911Crossref PubMed Scopus (112) Google Scholar). The increase in binding produced by the 1C3 dimer most likely results from the increase in avidity of the bivalent scFv, which can link two D1D2 molecules together. It demonstrates that the epitope of this antibody is remote from the primary collagen-binding site, leading to the conclusion that 1C3 inhibits an interaction of D1D2 with CRP/collagen that is separate from the primary binding interaction. scFv 1C3 Inhibits Clustering of D1D2 on Collagen-like Peptides with Increasing GPO Content—Binding of D1D2 to immobilized collagen-related peptides, GPO6, GPO4, GPO2, or the control GPP10, was measured in the ligand binding assay. Binding of D1D2 to these peptides increased relative to the GPOGPO content of each peptide as we have recently described.3 The effect of monomeric 1C3 on binding of D1D2 to peptides GPO6, GPO4, and GPO2 was then measured. A subsaturating amount of D1D2 (10 μg/ml) was preincubated with increasing concentrations of monomeric 1C3, then incubated in the peptide-coated wells, and subsequently bound D1D2 was detected. 1C3 reduced the binding of D1D2 to peptides GPO4 and GPO6 to the same level as binding to GPO2 (Fig. 3). Binding to the latter peptide was not diminished and was significantly above that to GPP10 across the range of scFv concentrations (one-way analysis of variance, p < 0.001). Hence, the direct interaction of GPVI with its minimal binding motif GPOGPO is not inhibited by scFv 1C3, whereas additional lateral clustering to adjacent motifs such as those present in peptides GPO4 and GPO6 is. Isoleucine 148 on a Side Face of GPVI Is Contained within the 1C3 Epitope—To further understand the inhibitory action of 1C3, we undertook to locate its epitope on GPVI using site-directed mutagenesis of D1D2. ScFvs 10B12 and 1C3 have been shown to bind D1D2 concurrently (4Smethurst P.A. Joutsi-Korhonen L. O'Connor M.N. Wilson E. Jennings N.S. Garner S.F. Zhang Y. Knight C.G. Dafforn T.R. Buckle A. IJsseldijk M.J. De Groot P.G. Watkins N.A. Farndale R.W. Ouwehand W.H. Blood. 2004; 103: 903-911Crossref PubMed Scopus (112) Google Scholar), indicating that 1C3 does not bind to the primary collagen binding surface recognized by 10B12, a conclusion confirmed by the data in Fig. 2. Previously published work shows that 1C3 does not bind to either Ig-like domain of GPVI when it is expressed individually, showing that both Ig-like domains are required for 1C3 binding (4Smethurst P.A. Joutsi-Korhonen L. O'Connor M.N. Wilson E. Jennings N.S. Garner S.F. Zhang Y. Knight C.G. Dafforn T.R. Buckle A. IJsseldijk M.J. De Groot P.G. Watkins N.A. Farndale R.W. Ouwehand W.H. Blood. 2004; 103: 903-911Crossref PubMed Scopus (112) Google Scholar). Also, biosensor experiments conducted in our laboratory show that the lectin derivative succinylated Concanavalin A binds to D1D2 concurrently with 1C3 and 10B12.4 Hence, the 1C3 epitope does not overlap with the N-glycosylation site on D1D2 at residue Asn-72 that occupies the surface of domain 1 distal to the interdomain apex. In addition, 1C3 binds both human and mouse GPVI (4Smethurst P.A. Joutsi-Korhonen L. O'Connor M.N. Wilson E. Jennings N.S. Garner S.F. Zhang Y. Knight C.G. Dafforn T.R. Buckle A. IJsseldijk M.J. De Groot P.G. Watkins N.A. Farndale R.W. Ouwehand W.H. Blood. 2004; 103: 903-911Crossref PubMed Scopus (112) Google Scholar); therefore residues in the epitope are likely to be conserved between species. Further information on the location of the 1C3 epitope came from a commercial peptide-based epitope-scanning screen of the Ig-like domains in which a series of overlapping 15-mer peptides covering the Ig-like domains of GPVI was synthesized and immobilized by a proprietary process (Pepscan Systems, Lelystad, Netherlands) and tested for reactivity with 1C3, 10B12, and irrelevant scFvs (acting as background controls). 10B12 reacted with peptides containing residues Lys-41 and Lys-59, consistent with previous work (3O'Connor M.N. Smethurst P.A. Farndale R.W. Ouwehand W.H. J. Thromb. Haemostasis. 2006; 4: 869-873Crossref PubMed Scopus (31) Google Scholar, 4Smethurst P.A. Joutsi-Korhonen L. O'Connor M.N. Wilson E. Jennings N.S. Garner S.F. Zhang Y. Knight C.G. Dafforn T.R. Buckle A. IJsseldijk M.J. De Groot P.G. Watkins N.A. Farndale R.W. Ouwehand W.H. Blood. 2004; 103: 903-911Crossref PubMed Scopus (112) Google Scholar). Three peptides were identified as likely to contain partial epitopes for 1C3, lying between residues Ser-144 and Ser-162 in the second Ig-like domain (data not shown). In the light of the above and information obtained from modeling, we designed and expressed two further single amino acid mutants of D1D2. Binding of 1C3 to wild-type and the two novel mutated forms of D1D2 was compared by ELISA. Each construct was immobilized via its calmodulin tag, ensuring equal loading and orientation of antigen. It was observed that, whereas binding of the scFv to Y134A and to wild-type D1D2 were of similar level, binding to I148A was completely abolished (Fig. 4A). When both mutants were tested for CRP binding activity, a slightly diminished binding was observed compared with that of wild-type D1D2 (Fig. 4B). However, binding was specific compared with the control peptide GPP10 (data not shown), indicating that the decrease in binding of 1C3 was not due to a general defect in D1D2 protein structure. Activation of platelets by collagen is an important initial step in hemostasis and its pathological expression, thrombosis. GPVI is the major activating receptor on platelets for collagen and is a promising target for therapeutic intervention in athero-thrombotic diseases (21Penz S. Reininger A.J. Brandl R. Goyal P. Rabie T. Bernlochner I. Rother E. Goetz C. Engelmann B. Smethurst P.A. Ouwehand W.H. Farndale R. Nieswandt B. Siess W. FASEB J. 2005; 19: 898-909Crossref PubMed Scopus (138) Google Scholar). To facilitate the informed development of antagonists, it is important to understand the molecular details of the GPVI-collagen interaction. As both GPVI and collagen are insoluble, a number of molecular tools have been devised to investigate the GPVI-collagen interaction: recombinant soluble Ig-like domains of GPVI, termed D1D2, recombinant single chain variable domain antibody fragments, 10B12 and 1C3, selected on D1D2, and a series of synthetic triple-helical peptides that are structurally well defined and recognized by GPVI. The primary collagen binding surface of human GPVI has been localized in this laboratory to the apical surface of the Ig-like domains, using mutagenesis and binding studies (3O'Connor M.N. Smethurst P.A. Farndale R.W. Ouwehand W.H. J. Thromb. Haemostasis. 2006; 4: 869-873Crossref PubMed Scopus (31) Google Scholar, 4Smethurst P.A. Joutsi-Korhonen L. O'Connor M.N. Wilson E. Jennings N.S. Garner S.F. Zhang Y. Knight C.G. Dafforn T.R. Buckle A. IJsseldijk M.J. De Groot P.G. Watkins N.A. Farndale R.W. Ouwehand W.H. Blood. 2004; 103: 903-911Crossref PubMed Scopus (112) Google Scholar). In an independent study, non-biased in silico docking of CRP onto a 2.4 Å crystal structure of D1D2 identified the same interaction site (18Horii K. Kahn M.L. Herr A.B. Blood. 2006; 108: 936-942Crossref PubMed Scopus (128) Google Scholar), confirming our earlier studies. This collagen binding surface is also the epitope of the anti-GPVI scFv, 10B12 (4Smethurst P.A. Joutsi-Korhonen L. O'Connor M.N. Wilson E. Jennings N.S. Garner S.F. Zhang Y. Knight C.G. Dafforn T.R. Buckle A. IJsseldijk M.J. De Groot P.G. Watkins N.A. Farndale R.W. Ouwehand W.H. Blood. 2004; 103: 903-911Crossref PubMed Scopus (112) Google Scholar). Whereas 10B12 exerts clear inhibitory effects on all platelet responses to collagen (4Smethurst P.A. Joutsi-Korhonen L. O'Connor M.N. Wilson E. Jennings N.S. Garner S.F. Zhang Y. Knight C.G. Dafforn T.R. Buckle A. IJsseldijk M.J. De Groot P.G. Watkins N.A. Farndale R.W. Ouwehand W.H. Blood. 2004; 103: 903-911Crossref PubMed Scopus (112) Google Scholar, 7Siljander P.R. Munnix I.C. Smethurst P.A. Deckmyn H. Lindhout T. Ouwehand W.H. Farndale R.W. Heemskerk J.W. Blood. 2004; 103: 1333-1341Crossref PubMed Scopus (167) Google Scholar, 21Penz S. Reininger A.J. Brandl R. Goyal P. Rabie T. Bernlochner I. Rother E. Goetz C. Engelmann B. Smethurst P.A. Ouwehand W.H. Farndale R. Nieswandt B. Siess W. FASEB J. 2005; 19: 898-909Crossref PubMed Scopus (138) Google Scholar), another anti-GPVI scFv, 1C3, selected against the same antigen, exhibited only a minor inhibitory effect on thrombus formation on immobilized collagen fibers and gave some reduction in procoagulant expression of adherent platelets, leading to the suggestion that 1C3 may exert its effect by interfering with receptor clustering on the extended collagenous ligand (4Smethurst P.A. Joutsi-Korhonen L. O'Connor M.N. Wilson E. Jennings N.S. Garner S.F. Zhang Y. Knight C.G. Dafforn T.R. Buckle A. IJsseldijk M.J. De Groot P.G. Watkins N.A. Farndale R.W. Ouwehand W.H. Blood. 2004; 103: 903-911Crossref PubMed Scopus (112) Google Scholar, 7Siljander P.R. Munnix I.C. Smethurst P.A. Deckmyn H. Lindhout T. Ouwehand W.H. Farndale R.W. Heemskerk J.W. Blood. 2004; 103: 1333-1341Crossref PubMed Scopus (167) Google Scholar). In this study, 1C3 alone did not produce a significant functional effect on collagen and CRP-XL-induced platelet aggregation, in line with previous data (4Smethurst P.A. Joutsi-Korhonen L. O'Connor M.N. Wilson E. Jennings N.S. Garner S.F. Zhang Y. Knight C.G. Dafforn T.R. Buckle A. IJsseldijk M.J. De Groot P.G. Watkins N.A. Farndale R.W. Ouwehand W.H. Blood. 2004; 103: 903-911Crossref PubMed Scopus (112) Google Scholar, 7Siljander P.R. Munnix I.C. Smethurst P.A. Deckmyn H. Lindhout T. Ouwehand W.H. Farndale R.W. Heemskerk J.W. Blood. 2004; 103: 1333-1341Crossref PubMed Scopus (167) Google Scholar). However, in combination, 1C3 potentiated the response of 10B12, revealing its own more subtle inhibitory effect (Fig. 1). Treatment of platelet suspensions with polymeric ligands of GPVI, such as CRP-XL or convulxin, triggers tyrosine phosphorylation of the Fc receptor γ chain and results in irreversible platelet aggregation (22Gibbins J. Asselin J. Farndale R. Barnes M. Law C.L. Watson S.P. J. Biol. Chem. 1996; 271: 18095-18099Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar, 23Gibbins J.M. Okuma M. Farndale R. Barnes M. Watson S.P. FEBS Lett. 1997; 413: 255-259Crossref PubMed Scopus (262) Google Scholar, 24Tsuji M. Ezumi Y. Arai M. Takayama H. J. Biol. Chem. 1997; 272: 23528-23531Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar). In contrast, monomeric forms of such agonists elicit a weak or no response (19Polgar J. Clemetson J.M. Kehrel B.E. Wiedemann M. Magnenat E.M. Wells T.N. Clemetson K.J. J. Biol. Chem. 1997; 272: 13576-13583Abstract Full Text Full Text PDF PubMed Scopus (317) Google Scholar, 25Asselin J. Knight C.G. Farndale R.W. Barnes M.J. Watson S.P. Biochem. J. 1999; 339: 413-418Crossref PubMed Scopus (60) Google Scholar), leading to the conclusion that clustering of GPVI is critical for activation of the receptor. A binding mechanism involving dimeric GPVI was first proposed by Miura et al. (6Miura Y. Takahashi T. Jung S.M. Moroi M. J. Biol. Chem. 2002; 277: 46197-46204Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar) who used a dimeric Fc-GPVI construct. Very recently, the docking of multiple triple helices onto a dimeric GPVI has been proposed based on structural modeling (18Horii K. Kahn M.L. Herr A.B. Blood. 2006; 108: 936-942Crossref PubMed Scopus (128) Google Scholar); however, the precise mode of interaction of GPVI with collagen helices has remained biochemically uncharacterized. To address this issue and to elucidate the functional behavior of scFv 1C3, we investigated the effect of the antibody on binding of D1D2 to CRP-XL, collagen, and a series of other GPO-containing peptides (Fig. 2, B and D). In sharp contrast to the partial inhibition observed for the 1C3 monomer, the 1C3 diabody greatly enhanced binding of D1D2 to both CRP-XL and collagen (Fig. 2, A and C). This may be explained by the dimeric 1C3-D1D2 complex having enhanced avidity for its ligands, which in turn demonstrates that 1C3 does not block the primary collagen-binding site. The inhibitory action of 1C3 must therefore depend upon a different feature of the GPVI-collagen interaction. Because collagen is a complex heterogeneous molecule, for further binding studies we chose to use a series of well defined, triple-helical peptides in which the number of GPO triplets varied. By using D1D2 binding and platelet activation as readouts, the minimum GPVI binding motif has been proposed to contain two GPO triplets.3 Here it was observed that, in the presence of monomeric 1C3, binding of D1D2 to the peptides GPO4 and GPO6 was reduced to the same level as binding to peptide GPO2 (Fig. 3), providing direct evidence that 1C3 interferes with the ability of GPVI to cluster closely on these peptides. This now elucidates the effect of the antibody on D1D2 binding to its polymeric ligands, CRP-XL and collagen. Binding to CRP-XL was reduced by ∼80% in the presence of monomeric 1C3 (Fig. 2B). This is consistent with the binding of several D1D2 molecules per helix to adjacent sites in the absence of the antibody and the blockade of binding to those adjacent sites (but not the first on each helix) by 1C3. Binding to collagen is reduced by a lower proportion, ∼50% (Fig. 2D), which is also consistent with the primary structure of collagen type I, which has a maximum run of two adjacent GPOGPO triplets in both α1 and α2 chains. We conclude that, in the presence of a saturating concentration of 1C3, GPVI receptors cannot cluster on adjacent binding motifs. Because platelet activation occurs even in the presence of 1C3 (4Smethurst P.A. Joutsi-Korhonen L. O'Connor M.N. Wilson E. Jennings N.S. Garner S.F. Zhang Y. Knight C.G. Dafforn T.R. Buckle A. IJsseldijk M.J. De Groot P.G. Watkins N.A. Farndale R.W. Ouwehand W.H. Blood. 2004; 103: 903-911Crossref PubMed Scopus (112) Google Scholar, 7Siljander P.R. Munnix I.C. Smethurst P.A. Deckmyn H. Lindhout T. Ouwehand W.H. Farndale R.W. Heemskerk J.W. Blood. 2004; 103: 1333-1341Crossref PubMed Scopus (167) Google Scholar), this implies that binding of GPVI to non-adjacent GPOGPO triplets on the same collagen chain, or to proximal GPOGPO triplets on other chains or collagen molecules, can induce signaling, although the magnitude of the response is reduced (7Siljander P.R. Munnix I.C. Smethurst P.A. Deckmyn H. Lindhout T. Ouwehand W.H. Farndale R.W. Heemskerk J.W. Blood. 2004; 103: 1333-1341Crossref PubMed Scopus (167) Google Scholar). To visualize the effect of 1C3 on the receptor, its epitope was mapped by site-directed mutagenesis. The mutation of D1D2 I148A abolished the ability to bind 1C3 (Fig. 4A). In contrast, another hydrophobic mutation in the same Ig-like domain, Y134A, and numerous residues in the apical surface (3O'Connor M.N. Smethurst P.A. Farndale R.W. Ouwehand W.H. J. Thromb. Haemostasis. 2006; 4: 869-873Crossref PubMed Scopus (31) Google Scholar, 4Smethurst P.A. Joutsi-Korhonen L. O'Connor M.N. Wilson E. Jennings N.S. Garner S.F. Zhang Y. Knight C.G. Dafforn T.R. Buckle A. IJsseldijk M.J. De Groot P.G. Watkins N.A. Farndale R.W. Ouwehand W.H. Blood. 2004; 103: 903-911Crossref PubMed Scopus (112) Google Scholar) did not. These new mutations do not profoundly affect the structure of the protein, as they still bind specifically to CRP in a binding assay (Fig. 4B). An inspection of the position of Ile-148 in the recent structure of D1D2 (18Horii K. Kahn M.L. Herr A.B. Blood. 2006; 108: 936-942Crossref PubMed Scopus (128) Google Scholar) shows that the residue is present on a side face of domain 2 and is close to a putative protein interaction interface distal from the primary collagen-binding site (Fig. 5). From this, one can envisage how 1C3 may occupy an epitope separate from that of 10B12, straddling both domains and thus interfering with docking of GPVI to adjacent GPOGPO triplets in its ligands. The work we present here using purified components will complement and inform other approaches that could be applied to study GPVI clustering, such as advanced microscopy. In conclusion, scFv 1C3 appears to be a valuable tool for distinguishing the effects of receptor clustering from direct ligand recognition on platelet activation by collagen through GPVI.
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