Molecular characterization of an human monoclonal antibody that interacts with a sulfated tyrosine-containing epitope of the GPIb receptor and inhibits platelet functions

摘要: Modification of tyrosine residues in extracellular proteins by a sulfate moity plays an important role many ligand/receptors interactions. In the present work, we describe unique human monoclonal antibody, termed Y1-scFv, that is specific for sulfated epitope platelat receptor GPIb. The Y1-scFv single chain antibody (scFv) competes with von Willebrand factor (vWF) binding to platelets and thus effectively inhibits platelet aggregation. Limited proteolysis GPIb molecule, using endoproteases, mocarhagin cathepsin G, revealed seven amino-acid epitope, Tyr-276 Glu-282, contains recognition site Y1-scFv. This region three residues. Binding studies cells synthetic peptides vitro indicated comprising only sulfo-Tyr-276 adjacent Asp-277 are critical interaction. To identify reciprocal sequences recognize introduced mutations within complementary-determining heavy (CDR3H) (MRAPVI). Arginine residue second position was binding. Moreover, mutant, containing two sequential arginine residues, third positions CDR3H (MRRPVI), showed nine-fold increased mutant also demonstrated significant increase inhibition vWF-dependent aggregation adhesion under flow. conclusion, this mutants, GP1b receptor, efficiently inhibited aggregation, making it candidate new anti-thrombotic agent.