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• W1965622650 abstract "SummaryMonoclonal antibodies to platelet membrane receptors have been used extensively for analysis of receptor structure and function. Function-blocking human antibodies are being used for the development of antiplatelet drugs. We isolated human monoclonal antibodies from a library of single-chain Fv (scFv) antibodies displayed on the surface of filamentous phage, by selection on whole platelets. Eight different platelet-binding clones were isolated, of which three bound to the platelet-membrane glycoprotein (GP) GPIb in an ELISA assay. Specific elution with a recombinant polypeptide of von Willebrand factor (VWF) spanning the GPIbα binding site, yielded the same three phage clones. Two of the three anti-GPIb clones could be purified as scFv monoclonal antibodies, and they competed with each other for binding to intact platelets, suggesting that they bind at or near the same site on GPIb. Their binding affinities differed, however, and the clone with higher affinity inhibited ristocetin-induced platelet aggregation. These data indicate that selection from a phage display library of human scFvs using whole platelets can be applied for the isolation of functional antiplatelet-GPIb antibodies useful for the development of new therapeutic and diagnostic strategies. Monoclonal antibodies to platelet membrane receptors have been used extensively for analysis of receptor structure and function. Function-blocking human antibodies are being used for the development of antiplatelet drugs. We isolated human monoclonal antibodies from a library of single-chain Fv (scFv) antibodies displayed on the surface of filamentous phage, by selection on whole platelets. Eight different platelet-binding clones were isolated, of which three bound to the platelet-membrane glycoprotein (GP) GPIb in an ELISA assay. Specific elution with a recombinant polypeptide of von Willebrand factor (VWF) spanning the GPIbα binding site, yielded the same three phage clones. Two of the three anti-GPIb clones could be purified as scFv monoclonal antibodies, and they competed with each other for binding to intact platelets, suggesting that they bind at or near the same site on GPIb. Their binding affinities differed, however, and the clone with higher affinity inhibited ristocetin-induced platelet aggregation. These data indicate that selection from a phage display library of human scFvs using whole platelets can be applied for the isolation of functional antiplatelet-GPIb antibodies useful for the development of new therapeutic and diagnostic strategies. Platelet aggregation plays a critical role in normal hemostasis and in disorders caused by inappropriate thrombogenesis. In both processes the initiating event is platelet adhesion, in which von Willebrand factor (VWF) forms a bridge between the subendothelial extracellular matrix and glycoprotein (GP)Ib on the surface of circulating platelets [1Sakariassen K.S. Fressinaud E. Girma J.P. Meyer D. Baumgartner H.R. Role of platelet membrane glycoproteins and von Willebrand factor in adhesion of platelets to subendothelium and collagen.Ann NY Acad Sci. 1987; 516: 52-65Crossref PubMed Google Scholar, 2Roth G.J. Platelets and blood vessels: the adhesion event.Immunol Today. 1992; 13: 100-5Abstract Full Text PDF PubMed Google Scholar, 3Kroll M.H. Hellums J.D. McIntire L.V. Schafer A.I. Moake J.L. Platelets and shear stress.Blood. 1996; 88: 1525-41Crossref PubMed Google Scholar, 4Hawiger J. Platelets: receptors, adhesion, secretion.Methods Enzymol. 1992; 169: 131-6Crossref Scopus (6) Google Scholar]. Adhesion of platelets to the subendothelium results in their activation and the release of secretagogues. These in turn activate additional platelets, leading to the binding of fibrinogen to GPIIb/IIIa, its receptor on the activated platelet, and resulting in thrombus formation [5De Marco L. Girolami A. Russell S. Ruggeri Z.M. Interaction of asialo von Willebrand factor with glycoprotein Ib induces fibrinogen binding to the glycoprotein IIb/IIIa complex and mediates platelet aggregation.J Clin Invest. 1985; 75: 1198-203Crossref PubMed Google Scholar, 6Ruggeri Z.M. Ware J. Von Willebrand factor.FASEB J. 1993; 7: 308-16Crossref PubMed Google Scholar]. Therefore, the development of molecules that inhibit both adhesion and aggregation of platelets might be useful in preventing thrombosis and reocclusion in coronary arteries. A chimeric monoclonal Fab was the first of several new antiplatelet drugs directed at the platelet GPIIb/IIIa and inhibiting thrombosis [7Coller B.S. Platelet GPIIb/IIIa antagonists: the first anti-integrin receptor therapeutics.J Clin Invest. 1997; 100: S57-60PubMed Google Scholar, 8Coller B.S. A new murine monoclonal antibody reports an activation-dependent change in the conformation and/or microenvironment of the platelet glycoprotein IIb/IIIa complex.J Clin Invest. 1985; 76: 101-8Crossref PubMed Google Scholar, 9Tcheng J.E. Ellis S.G. George B.S. Kereiakes D.J. Kleiman N.S. Talley J.D. Wang A.L. Weisman H.F. Califf R.M. Topol E.J. Pharmacodynamics of chimeric glycoprotein IIb/IIIa integrin antiplatelet antibody Fab 7E3 in high-risk coronary angioplasty.Circulation. 1994; 90: 1757-64Crossref PubMed Google Scholar]. A novel strategy for isolating functionally active human antibodies to cell membrane receptors is based on biopanning on whole cells of a library of human single-chain Fv (scFv) monoclonal antibodies displayed on phage coat. Display of a repertoire of antibody fragments on the surface of a filamentous bacteriophage offers a new way to produce antibodies with defined binding specificities [10Winter G. Griffiths A.D. Hawkins R.E. Hoogenboom H.R. Making antibodies by phage display technology.Annu Rev Immunol. 1994; 12: 433-55Crossref PubMed Google Scholar, 11McCafferty J. Griffiths A.D. Winter G. Chiswell D.J. Phage antibodies: filamentous phage displaying antibody variable domains.Nature. 1990; 348: 552-4Crossref PubMed Scopus (1916) Google Scholar]. Heavy- and light-chain variable domains are displayed on the viral coat protein, allowing phages with antigen-binding activities (and encoding the antibody fragments) to be selected by panning on the desired antigen [11McCafferty J. Griffiths A.D. Winter G. Chiswell D.J. Phage antibodies: filamentous phage displaying antibody variable domains.Nature. 1990; 348: 552-4Crossref PubMed Scopus (1916) Google Scholar, 12Nissim A. Hoogenboom H.R. Tomlinson I.M. Flynn G. Midgley C. Lane D. Winter G. Antibody fragments from a ‘single pot’ phage display library as immunochemical reagents.EMBO J. 1994; 13: 692-8Crossref PubMed Scopus (527) Google Scholar]. In this study we used a library in which the complementary determining region (CDR3H) of the antibody heavy chain was completely randomized by introducing random synthetic nucleotide sequences in the CDR3H, resulting in increased repertoire diversity [12Nissim A. Hoogenboom H.R. Tomlinson I.M. Flynn G. Midgley C. Lane D. Winter G. Antibody fragments from a ‘single pot’ phage display library as immunochemical reagents.EMBO J. 1994; 13: 692-8Crossref PubMed Scopus (527) Google Scholar]. When selected phages are grown in non-suppressor strains of Escherichia coli, the soluble antibody polypeptides are secreted into the bacterial periplasm [13Carter P. Bedouelle H. Winter G. Improved oligonucleotide site-directed mutagenesis using M13 vectors.Nucl Acids Res. 1985; 13: 4431-43Crossref PubMed Scopus (0) Google Scholar, 14Harrison J.L. Williams S.C. Winter G. Nissim A. Phage display libraries.Methods Enzymol. 1996; 267: 83-109Crossref PubMed Google Scholar]. The antibody library displays many different specificities against a variety of ligands, including highly conserved regions in proteins and non-immunogenic foreign and self-antigens [15Hoogenboom H.R. Winter G. By-passing immunisation. Human antibodies from synthetic repertoires of germline VH gene segments rearranged in vitro.J Mol Biol. 1992; 227: 381-8Crossref PubMed Google Scholar, 16De Kruif J. Van Der Vuurst de Vries A.R. Cilenti L. Boel E. Van Ewijk W. Logtenberg T. New perspectives on recombinant human antibodies.Immunol Today. 1996; 17: 453-5Abstract Full Text PDF PubMed Scopus (0) Google Scholar]. Because the antibodies are of human origin [15Hoogenboom H.R. Winter G. By-passing immunisation. Human antibodies from synthetic repertoires of germline VH gene segments rearranged in vitro.J Mol Biol. 1992; 227: 381-8Crossref PubMed Google Scholar], the problem of immunogenic response in human therapy is circumvented [17Vitaliti A. Wittmer M. Steiner R. Wyder L. Neri D. Klemenz R. Inhibition of tumor angiogenesis by a single-chain antibody directed against vascular endothelial growth factor.Cancer Res. 2000; 60: 4311-4PubMed Google Scholar]. Moreover, relative to conventional mAbs, they can be produced in a short period of time in large amounts, and being smaller than conventional mAbs, their penetration into tissues is more efficient. Phage selection by biopanning of the library on purified antigens coated onto plastic surfaces has been extensively reported [11McCafferty J. Griffiths A.D. Winter G. Chiswell D.J. Phage antibodies: filamentous phage displaying antibody variable domains.Nature. 1990; 348: 552-4Crossref PubMed Scopus (1916) Google Scholar, 18Parsons H.L. Earnshaw J.C. Wilton J. Johnson K.S. Schueler P.A. Mahoney W. McCafferty J. Directing phage selections towards specific epitopes.Protein Eng. 1996; 9: 1043-9Crossref PubMed Google Scholar]. However, biopanning on intact cells offers a higher probability that membrane-active antibodies will be selected. Successful use of this approach for the isolation of antibodies against human thymic stromal cells [19Van Ewijk W. De Kruif J. Germeraad W.T. Berendes P. Ropke C. Platenburg P.P. Logtenberg T. Subtractive isolation of phage-displayed single-chain antibodies to thymic stromal cells by using intact thymic fragments.Proc Natl Acad Sci USA. 1997; 94: 3903-8Crossref PubMed Scopus (0) Google Scholar], human melanoma cells [20Cai X. Garen A. Anti-melanoma antibodies from melanoma patients immunized with genetically modified autologous tumor cells: selection of specific antibodies from single-chain Fv fusion phage libraries.Proc Natl Acad Sci USA. 1995; 92: 6537-41Crossref PubMed Scopus (0) Google Scholar, 21Pereira S. Van Belle P. Elder D. Maruyama H. Jacob L. Sivanandham M. Wallack M. Siegel D. Herlyn D. Combinatorial antibodies against human malignant melanoma.Hybridoma. 1997; 16: 11-6Crossref PubMed Google Scholar, 22Noronha E.J. Wang X. Desai S.A. Kageshita T. Ferrone S. Limited diversity of human scFv fragments isolated by panning a synthetic phage-display scFv library with cultured human melanoma cells.J Immunol. 1998; 161: 2968-76PubMed Google Scholar], human blood leukocytes [23De Kruif J. Terstappen L. Boel E. Logtenberg T. Rapid selection of cell subpopulation-specific human monoclonal antibodies from a synthetic phage antibody library.Proc Natl Acad Sci USA. 1995; 92: 3938-42Crossref PubMed Google Scholar], and adipocytes [24Edwards B.M. Main S.H. Cantone K.L. Smith S.D. Warford A. Vaughan T.J. Isolation and tissue profiles of a large panel of phage antibodies binding to the human adipocyte cell surface.J Immunol Methods. 2000; 245: 67-78Crossref Scopus (30) Google Scholar] has been reported. In this report we describe the isolation and characterization of antiplatelet-membrane-receptor scFvs from a human-derived antibody library. Using biopanning on intact platelets, we developed selection procedures that preserved the native conformation of the membrane proteins and allowed selection of antiplatelet-GPIb antibodies. One of the selected scFv antibodies showed inhibition of platelet aggregation by modulating the interaction of VWF with platelet GPIb-mediated aggregation. The human antibody library used in this study was the Nissim library [11McCafferty J. Griffiths A.D. Winter G. Chiswell D.J. Phage antibodies: filamentous phage displaying antibody variable domains.Nature. 1990; 348: 552-4Crossref PubMed Scopus (1916) Google Scholar], which was constructed as a phagemid library displaying scFv of the IgG molecule. In this library, the variable region of the heavy chain (VH) and of the light chain (VL) are linked by a flexible polypeptide. The repertoire of antibody fragments was generated by polymerase chain reaction (PCR) from rearranged V genes of peripheral blood lymphocytes withdrawn from non-immunized humans. To increase the diversity of the repertoire, random nucleotide sequences 4–12 residues long were introduced into the CDR3 region of a bank of 49 cloned human VH gene segments [11McCafferty J. Griffiths A.D. Winter G. Chiswell D.J. Phage antibodies: filamentous phage displaying antibody variable domains.Nature. 1990; 348: 552-4Crossref PubMed Scopus (1916) Google Scholar]. The fused VL fragment in all the clones was derived from a single non-mutated V gene, creating a single pot library of approximately 108 different clones. Antibody polypeptides were fused to the N-terminus of the minor coat protein pIII of the phage M13 and cloned into the pHEN1vector [11McCafferty J. Griffiths A.D. Winter G. Chiswell D.J. Phage antibodies: filamentous phage displaying antibody variable domains.Nature. 1990; 348: 552-4Crossref PubMed Scopus (1916) Google Scholar, 15Hoogenboom H.R. Winter G. By-passing immunisation. Human antibodies from synthetic repertoires of germline VH gene segments rearranged in vitro.J Mol Biol. 1992; 227: 381-8Crossref PubMed Google Scholar, 25Griffiths A.D. Williams S.C. Hartley O. Tomlinson I.M. Waterhouse P. Crosby W.L. Kontermann R.E. Jones P.T. Low N.M. Allison T.J. et al.Isolation of high affinity human antibodies directly from large synthetic repertoires.EMBO J. 1994; 13: 3245-60Crossref PubMed Google Scholar]. One-day-old platelet concentrate in acid–citrate–dextrose was obtained from the Central Blood Bank of Israel. Platelets were isolated by centrifugation at 800 ×g for 10 min and resuspended in Tyrode's solution (2 mm MgCl2, 137 mm NaCl, 2.68 mm KCl, 3 mm NaH2PO4, 0.1% glucose, 5 mm HEPES and 0.35% albumin, pH 7.35) at a final concentration of 109 cells mL−1. Platelet concentrate was incubated at 37 °C for 1 h, and an equal volume of 2.0% paraformaldehyde was then added. After 18 h at 4 °C, the platelets were washed twice with cold saline and resuspended in 0.01% HEPES in saline at a final concentration of 109 cells mL−1. The biopanning procedure comprised four steps. Approximately 1011 antibody-bearing phage particles in 1 mL buffer [phosphate-buffered saline (PBS), 10 mm HEPES pH 7.3, 1% bovine serum albumin (BSA)] were mixed with 108 fixed human platelets. Binding was allowed to proceed for 1 h at room temperature, with rotation. Following binding, cells were washed five times in the above buffer by centrifugation (13000 ×g) for 10 min and resuspended in the same buffer. Two elution protocols were developed, a low pH ‘non-specific’ and a specific elution protocol. Non-specific elution protocol. Bound phages were eluted from the fixed platelets by the addition of 200 µL of 0.1 m glycine–HCl pH 2.2, followed by incubation for 10 min at room temperature. The mixture was then neutralized with 14 µL of 2 m Tris–HCl pH 11. Specific elution protocol. Bound phages were competitively eluted from the fixed platelets by incubation with a recombinant polypeptide (VCL) derived from VWF sequence (amino acids 504–728; Bio-Technology General, Rehovot, Israel). This fragment binds to the platelet membrane GPIb and inhibits the interaction of VWF with this receptor [26Gralnick H.R. Williams S. McKeown L. Kramer W. Krutzsch H. Gorecki M. Panet A. Garfinkel L.I. A monomeric von Willebrand factor fragment, Leu-504–Lys-728, inhibits von Willebrand factor interaction with glycoprotein Ib-IX.Proc Natl Acad Sci USA. 1992; 89: 7880-4Crossref PubMed Google Scholar, 27Gralnick H.R. Williams S.B. McKeown L.P. Magruder L. Hansmann K. Vail M. Parker R.I. Platelet von Willebrand factor.Mayo Clin Proc. 1991; 66: 634-40Abstract Full Text Full Text PDF PubMed Google Scholar]. Incubation was carried out at a VCL concentration of 24 µm for 15 min at room temperature. VCL displaced the phages that bind specifically to the GPIb receptor. Eluted phages obtained by either protocol were used to infect E. coli TG-1, and the infected bacteria were plated on TYE plates containing 100 µg mL−1 ampicillin and 1% glucose [14Harrison J.L. Williams S.C. Winter G. Nissim A. Phage display libraries.Methods Enzymol. 1996; 267: 83-109Crossref PubMed Google Scholar]. The bacterial colonies were counted, scraped from the plates, and pooled. An aliquot of ampicillin-resistant E. coli was grown in liquid culture until turbidity of 0.5 at A600 was reached. It was then infected with a helper phage to produce a large, amplified phage stock [14Harrison J.L. Williams S.C. Winter G. Nissim A. Phage display libraries.Methods Enzymol. 1996; 267: 83-109Crossref PubMed Google Scholar]. Phages were concentrated by polyethyleneglycol precipitation [14Harrison J.L. Williams S.C. Winter G. Nissim A. Phage display libraries.Methods Enzymol. 1996; 267: 83-109Crossref PubMed Google Scholar]. The amplified stock (approximately 1013 phages mL−1) was used for subsequent rounds of panning. Phage from individual ampicillin-resistant colonies, after the third round of panning, were grown in 2 × TY medium [14Harrison J.L. Williams S.C. Winter G. Nissim A. Phage display libraries.Methods Enzymol. 1996; 267: 83-109Crossref PubMed Google Scholar] purified by polyethyleneglycol precipitation, resuspended in PBS, and stored at 4 °C until further use. The entire DNA of all scFv fragments of selected phage clones were amplified by PCR with the commonly used phage vector primers, LMB-3 (5′ GAGGAAACAGCTATGAC) and fd-Seq (5′ GAATTTTCTGTATGAGG). Fragments were fully sequenced from both ends by automatic ABI PRISM Big Dye termination cycle sequencing kit (310 Genetic Analyzer, Foster City, CA, USA). The phagemid vector pHEN1 possesses an amber stop codon encoded at the junction of the antibody gene and the phage pIII gene, therefore it can be directly used to express soluble single-chain antibodies. This was done as previously described [14Harrison J.L. Williams S.C. Winter G. Nissim A. Phage display libraries.Methods Enzymol. 1996; 267: 83-109Crossref PubMed Google Scholar]. Briefly, soluble monoclonal scFv antibodies (mAb-scFvs) were obtained from the periplasmic fraction of E. coli non-suppressor strain HB2151 containing the phagemid vector pHEN1, induced with 1 mm isopropyl thio-β-d-galactosidase, and grown for 18 h at room temperature with shaking. Infected bacterial cultures (200 mL) were centrifuged, and the pellets were resuspended in buffer containing 20% (w/v) sucrose, 1 mm EDTA and 30 mm Tris, pH 7.0. They were then kept on ice for 15 min, centrifuged again, and the supernatants were used as the periplasmic fraction. All the scFvs of the selected clones belong to the VH3 family, allowing purification on protein A affinity beads. The periplasmic fraction of each clone was incubated with protein A Sepharose beads (Pharmacia, Little Chalfont, Bucks, UK) for 1 h at room temperature. Bound scFvs were recovered by acid elution (0.1 m glycine pH 3.0) followed by neutralization with Tris buffer pH 8.0, and the eluted protein solutions were passed over a G-25 exchange column equilibrated with PBS. The concentration of the recovered protein was determined by optical density (OD) at A280. The extracellular proteolytic fragment of GPIb (glycocalicin) was purified from fresh human platelets as described previously [28Andrews R.K. Booth W.J. Gorman J.J. Castaldi P.A. Berndt M.C. Purification of botrocetin from Bothrops jararaca venom. Analysis of the botrocetin-mediated interaction between von Willebrand factor and the human platelet membrane glycoprotein Ib–IX complex.Biochemistry. 1989; 28: 8317-26Crossref PubMed Google Scholar, 29Andrews R.K. Gorman J.J. Booth W.J. Corino G.L. Castaldi P.A. Berndt M.C. Cross-linking of a monomeric 39/34-kDa dispase fragment of von Willebrand factor (Leu-480/Val-481-Gly-718) to the N-terminal region of the alpha-chain of membrane glycoprotein Ib on intact platelets with bis (sulfosuccinimidyl) suberate.Biochemistry. 1989; 28: 8326-36Crossref PubMed Google Scholar]. Polystyrene microtiter plates (PoliSorp; Nunc, Roskilde, Denmark) were incubated overnight at 4 °C with 108 formaldehyde-fixed platelets or with 20 µg mL−1 glycocalicin in saline. The plates were washed twice with PBS containing 0.05% Tween 20 (PBS–T), incubated with 2% skim milk in PBS for 1 h. Phages (1010) were added and incubated for 1 h at room temperature. After extensive washing with PBS–T, bound phage clones were detected by incubation with rabbit anti-M13 (produced by injecting rabbits with phage M13) diluted 1 : 200 in PBS–T containing 2% skim milk for 1 h at room temperature, followed by addition of antirabbit horseradish peroxidase (HRP; Sigma Israel Chemicals, Rehovot, Israel) and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt substrate (Boehringer-Mannheim, Mannheim, Germany). Color intensity was read by an ELISA plate reader (Anthos, Salzburg, Austria) at A405. Each sample was assayed in duplicate and the average was calculated. Anti-scFc antibodies were generated by immunization of a rabbit with a mixture of scFvs. Anti-scFv was labeled with the aid of a Phycolink® R-Phycoerythrin conjugation kit (ProZyme, San Leandro, CA, USA) according to the manufacturer's instructions. Aliquots (10 µg mL−1) of scFvs were incubated with 107 fixed or washed platelets for 1 h at room temperature. The platelets were then washed in PBS containing 1% BSA and incubated with the R-phycoerythrin (PE)-labeled anti-scFv for 1 h at room temperature. Platelets were then washed, resuspended in PBS, and 104 platelets were analyzed by fluorescence-activated cell sorter scanning (FACScan flow cytometer; Becton Dickinson, Mountain View, CA, USA). Purified soluble mAb-scFv clones were conjugated to biotin via free amines using EZ-link Biotin-PEO-Amine (Pierce, Rockford, IL, USA), according to the manufacturer's instructions. Washed platelets (108 cells) were lyzed in 1 mL of lyze buffer (1% Triton, 1 mm phenylmethylsulfonyl fluoride, 5 mg mL−1 leupeptin, and 5 mm EDTA in PBS) and kept for 10 min on ice. Thereafter, 5 × 106 platelets per lane were electrophoresed under reducing or non-reducing conditions on 10% sodium dodecyl sulfate-polyacrylamide gel and transferred to a nitrocellulose membrane (Schleicher & Schuell, Dassel, Germany). The nitrocellulose membrane was blocked with 5% skim milk in PBS–T and incubated with the biotin-labeled mAb-scFvs. After another washing with PBS–T, mAb-scFv binding was detected with streptavidin–HRP (Pierce). Peroxidase activity was detected with the aid of the enhanced chemiluminescence detection system (ECL) (Amersham, Little Chalfont, Bucks, UK). Platelet-rich plasma (PRP) and washed platelets were stirred at 500 r.p.m. at 37 °C in a Lumiaggregometer (Chronolog, Havertown, PA, USA). The difference in light transmission through the platelet suspension and suspending medium was taken as 100% aggregation. The effect of mAb-scFvs on platelet aggregation was evaluated by adding increasing concentrations of the mAb-scFvs and incubating for 2 min at 37 °C before adding the agonists and recording aggregation for 4 min. With the aim of isolating single-chain Fv monoclonal antibodies (mAb-scFvs) that recognize platelet surface antigens, a phage displayed antibody library with a repertoire of approximately 108 antibody clones was panned over fixed platelets. After binding to the platelets, phages were eluted at pH 2.2 and amplified. Thirty phage clones were randomly picked after the third round of biopanning and their DNA sequences were determined. The deduced amino acid sequences of the CDR3 of the antibody variable heavy (VH) chain show that the 30 isolated clones comprised eight unique clones at different frequencies (Table 1). Three clones (Y-1, Y-17, and Y-27) were dominant (83% of total selected clones).Table 1Deduced VH-CDR3 sequences and frequencies of selected platelet-eluted phage clonesClone nameVH-CDR3 sequenceFrequency in low pH elutionY-1MRAPVI15/30 (50%)Y-2GKARRHNGPNLN1/30 (3%)Y-16TGQSIKRS1/30 (3%)Y-17LTHPYF7/30 (23%)Y-27LRPPES3/30 (10%)Y-44TSKNTSSSKRH1/30 (3%)Y-45RYYCRSSDCTVS1/30 (3%)Y-52FRRNGTVPAP1/30 (3%) Open table in a new tab To examine the platelet-binding specificity of the phage clones, we developed a platelet-ELISA test. All eight phage clones showed some binding to platelets in the ELISA test, relative to M13 the negative control phage (Fig. 1). Out of eight clones, six clones exhibited a higher level of binding and were selected for further analysis. Of the six positive clones, clone Y-27 contained a stop codon in the CDR3, and thus could be produced as a phage clone, and not as a single-chain Fv. Each of the other five positive clones was produced as a single-chain Fv antibody in E. coli HB2151 non-suppressor strain. These antibodies, now designated Y-scFv, were assessed by FACS analysis for their ability to bind platelets. All five bound to both fixed and washed platelets (data not shown). These results clearly indicate that while biopanning was done on fixed platelets, the selected mAb clones recognized and bound washed native platelets. Western blot analysis was carried out to determine the platelet-membrane antigen to which an individual mAb-scFv bound (Fig. 2A). Platelet proteins were separated by SDS–PAGE under reducing and non-reducing conditions, transferred to nitrocellulose filter, and the blot was incubated with the five biotin-labeled mAb-scFvs. We found that Y1-scFv and Y17-scFv reacted with a protein band of molecular mass of approximately 135 kDa under reducing conditions, and with a protein of approximately 160 kDa under non-reducing condition. Mouse mAb specific to human GPIbα (PM6/40; Serotec, Oxford, UK) reacted with bands of a similar molecular mass, i.e. approximately 135 kDa and approximately 160 kDa. The other three scFvs, Y16-scFv, Y45-scFv and Y52-scFv, did not react with any platelet proteins under the conditions employed. The identity of the platelet protein was revealed by the binding of both antibodies, Y1-scFv and Y17-scFv, to purified glycocalicin, a proteolytic fragment derived from GPIb (Fig. 2B). Phage clone Y-27, which could not be produced as an mAb-scFv, showed high binding capacity to purified glycocalicin when tested by ELISA, as did phage clones Y1-scFv and Y17-scFv (Fig. 2C). Thus, biopanning on fixed platelets selected for two major mAb-scFv clones, Y1 and Y17 that specifically bind to platelet GPIb. The two selected antibodies, Y1-scFv and Y17-scFv that bound specifically to platelet GPIb were next analyzed for inhibition of platelet aggregation in vitro. Aggregation experiments were performed with ristocetin, a platelet agonist which enables VWF binding to GPIb on platelets. While the Y1-scFv effectively inhibited ristocetin-induced platelet aggregation, Y17-scFv was not active (Fig. 3). The inhibitory effect of Y1-scFv on VWF-dependent aggregation was dose dependent with an IC50 of 1.2 µm in PRP. In washed platelets its inhibitory effect was more efficient with an IC50 of 0.8 µm. To determine whether the difference in inhibitory activity of Y1-scFv and Y17-scFv towards platelets derives from the site of binding in the GPIb or from their respected affinities, a binding and competition assay between Y1-scFv and Y17-scFv were performed. Both Y1-scFv and Y17-scFv bound to washed platelets in a concentration-dependent manner, reaching saturation at 15 µg mL−1 (Fig. 4). However, the level of bound Y1-scFv at saturation was higher than that of Y17-scFv. In the competition assay, binding of 1.6 µg mL−1 of biotin-labeled Y1-scFv or Y17-scFv to washed platelets was assessed by FACS analysis in the presence of 60 µg mL−1 and 200 µg mL−1 of competitor mAb-scFv, in its unlabeled form. As shown in Fig. 5, Y1-scFv inhibited binding of labeled Y17-scFv to the platelets, and reciprocally, Y17-scFv inhibited binding of labeled Y1-scFv. Thus, Y17-scFv binds at or near the binding site of Y1-scFv. It should be noted that 60 µg mL−1 of unlabeled Y1-scFv inhibited binding of Y17-scFv by 70%, whereas Y17-scFv at the same concentration inhibited Y1-scFv binding by only 30%. This difference in displacement capacity persisted at 200 µg mL−1 of mAb-scFv competitor. Three of the six platelet-binding phage clones selected by low-pH elution bound to GPIb. It was therefore of interest to examine whether a specific elution technique would yield additional clones that bind GPIb. VCL, a recombinant polypeptide derived from VWF (amino acids 504–728 of mature VWF), was used to elute competitively the phages that bound to fixed platelets [26Gralnick H.R. Williams S. McKeown L. Kramer W. Krutzsch H. Gorecki M. Panet A. Garfinkel L.I. A monomeric von Willebrand factor fragment, Leu-504–Lys-728, inhibits von Willebrand factor interaction with glycoprotein Ib-IX.Proc Natl Acad Sci USA. 1992; 89: 7880-4Crossref PubMed Google Scholar]. This recombinant polypeptide of 224 amino acids is produced in E. coli and purified as described before [26Gralnick H.R. Williams S. McKeown L. Kramer W. Krutzsch H. Gorecki M. Panet A. Garfinkel L.I. A monomeric von Willebrand factor fragment, Leu-504–Lys-728, inhibits von Willebrand factor interaction with glycoprotein Ib-IX.Proc Natl Acad Sci USA. 1992; 89: 7880-4Crossref PubMed Google Scholar]. The VCL molecule binds specifically to the platelet membrane GPIbα in the absence of ristocetin or botrocetin and competitively inhibits the interaction of VWF with GPIbα[26Gralnick H.R. Williams S. McKeown L. Kramer W. Krutzsch H. Gorecki M. Panet A. Garfinkel L.I. A monomeric von Willebrand factor fragment, Leu-5" @default.
• W1965622650 created "2016-06-24" @default.
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• W1965622650 creator A5046350976 @default.
• W1965622650 creator A5047313181 @default.
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• W1965622650 date "2003-08-01" @default.
• W1965622650 modified "2023-09-30" @default.
• W1965622650 title "Function-modulating human monoclonal antibodies against platelet-membrane receptors isolated from a phage-display library" @default.
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