title: Design and reshaping of an scFv directed against human platelet glycoprotein VI with diagnostic potential.
abstract: Blood platelets play a key role in physiological hemostasis and in thrombosis. As a consequence, platelet functional analysis is widely used in the diagnosis of hemorrhagic disorders as well as in the evaluation of thrombosis risks and of the efficacy of antithrombotics. Glycoprotein (GP) VI is a platelet-specific collagen-signaling receptor. Clinical studies suggest that increased GPVI expression is associated with a risk of arterial thrombosis. Conversely, GPVI deficiencies have been identified in patients with defective platelet responses to collagen. Currently, there is no standard test available for measuring GPVI expression, essentially because antibodies usually cross-link GPVI upon binding, leading to platelet activation and consecutive changes in GPVI expression. Here, we designed a recombinant monovalent antibody fragment (scFv) derived from an anti-GPVI monoclonal IgG, 3J24, with the characteristics required to analyze GPVI expression. Guided by in silico modeling and V-KAPPA chain analysis, a Protein L (PpL) recognition pattern was engineered in the scFv, making possible its purification and detection using PpL conjugates. The PpL affinity-purified scFv is functional. It retains GPVI-binding specificity and allows detection of platelet surface-expressed GPVI without inducing platelet activation. In conclusion, the reshaped scFv may be very useful in the development of diagnostic approaches.
authors.affiliation: Université Paris-Sud 11, IFR 141, Faculté de Pharmacie, 92260 Châtenay-Malabry, France.
authors.forename: Muhammad
authors.initials: M
authors.lastname: Zahid
authors.forename: Stéphane
authors.initials: S
authors.lastname: Loyau
authors.forename: Maxime
authors.initials: M
authors.lastname: Bouabdelli
authors.forename: Nicolas
authors.initials: N
authors.lastname: Aubrey
authors.forename: Martine
authors.initials: M
authors.lastname: Jandrot-Perrus
authors.forename: Philippe
authors.initials: P
authors.lastname: Billiald
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001792:Blood Platelets / Q000276:immunology; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D010980:Platelet Membrane Glycoproteins / Q000032:analysis* / Q000276:immunology; D057127:Single-Chain Antibodies / Q000737:chemistry* / Q000302:isolation & purification; D013927:Thrombosis / Q000175:diagnosis*
chemical_list: D007074:Immunoglobulin G; D010980:Platelet Membrane Glycoproteins; D057127:Single-Chain Antibodies; C098341:platelet membrane glycoprotein VI