title: Molecular characterization of a human monoclonal antibody that interacts with a sulfated tyrosine-containing epitope of the GPIb receptor and inhibits platelet functions.
abstract: Modification of tyrosine residues in extracellular proteins by a sulfate moity plays an important role in many ligand/receptors interactions. In the present work, we describe a unique human monoclonal antibody, termed Y1-scFv, that is specific for a sulfated epitope in the platelat receptor GPIb. The Y1-scFv single chain antibody (scFv) competes with von Willebrand factor (vWF) for binding to human platelets and thus effectively inhibits platelet aggregation. Limited proteolysis of GPIb molecule, using the endoproteases, mocarhagin and cathepsin G, revealed that a seven amino-acid epitope, Tyr-276 to Glu-282, contains the recognition site for Y1-scFv. This GPIb region contains three sulfated tyrosine residues. Binding studies of Y1-scFv to cells and to synthetic peptides in vitro indicated that of the seven residues comprising the epitope only sulfo-Tyr-276 and adjacent Asp-277 are critical for the interaction. To identify the reciprocal sequences in the antibody that recognize the sulfated epitope, we introduced mutations within the complementary-determining region of the heavy chain (CDR3H) of Y1-scFv (MRAPVI). Arginine residue in the second position was critical for the binding. Moreover, a mutant, containing two sequential arginine residues, in the second and third positions of the CDR3H (MRRPVI), showed a nine-fold increased binding to GPIb. This antibody mutant also demonstrated a significant increase in inhibition of vWF-dependent platelet aggregation and adhesion under flow. In conclusion, this unique antibody and mutants, that recognize a sulfated epitope in GP1b receptor, efficiently inhibited platelet adhesion and aggregation, making it a candidate for a new anti-thrombotic agent.
authors.affiliation: Bio-Technology General (Israel) Ltd., Research Department, Kiryat Weizmnn, Building 17, 76326 Rehovot, Israel. yochih@btgil.com
authors.forename: Yocheved
authors.initials: Y
authors.lastname: Hagay
authors.forename: Judith
authors.initials: J
authors.lastname: Lahav
authors.forename: Avigdor
authors.initials: A
authors.lastname: Levanon
authors.forename: David
authors.initials: D
authors.lastname: Varon
authors.forename: Alex
authors.initials: A
authors.lastname: Brill
authors.forename: Amos
authors.initials: A
authors.lastname: Panet
mesh_terms: D019943:Amino Acid Substitution; D000911:Antibodies, Monoclonal / Q000235:genetics / Q000276:immunology*; D000937:Antigen-Antibody Reactions; D001667:Binding, Competitive; D001792:Blood Platelets / Q000276:immunology*; D000939:Epitopes / Q000276:immunology*; D006801:Humans; D007128:Immunoglobulin Fragments / Q000235:genetics / Q000276:immunology*; D007143:Immunoglobulin Heavy Chains / Q000235:genetics / Q000276:immunology; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000276:immunology*; D008666:Metalloendopeptidases / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D010973:Platelet Adhesiveness / Q000187:drug effects; D010974:Platelet Aggregation / Q000187:drug effects; D010975:Platelet Aggregation Inhibitors / Q000276:immunology*; D019038:Platelet Glycoprotein GPIb-IX Complex / Q000276:immunology* / Q000378:metabolism; D025941:Protein Interaction Mapping; D017434:Protein Structure, Tertiary; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000737:chemistry; D014841:von Willebrand Factor / Q000378:metabolism
chemical_list: D000911:Antibodies, Monoclonal; D000939:Epitopes; D007128:Immunoglobulin Fragments; D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region; D010975:Platelet Aggregation Inhibitors; D019038:Platelet Glycoprotein GPIb-IX Complex; D014841:von Willebrand Factor; C005662:tyrosine O-sulfate; D014443:Tyrosine; D008666:Metalloendopeptidases; C099092:mocarhagin