Identification of Amino Acid Residues Essential for von Willebrand Factor Binding to Platelet Glycoprotein Ib. CHARGED-TO-ALANINE SCANNING MUTAGENESIS OF THE A1 DOMAIN OF HUMAN VON WILLEBRAND FACTOR

摘要: At sites of vascular injury, von Willebrand factor (VWF) mediates platelet adhesion through binding to glycoprotein Ib (GPIb). The VWF-GPIb interaction was investigated by clustered charged-to-alanine scanning mutagenesis VWF domain A1 between His-473 and Gly-716. Recombinant variants were assayed for conformation-dependent monoclonal antibody NMC-4, ristocetin-induced botrocetin-induced platelets, direct botrocetin. Substitutions at 32 amino acids had no effect on function. epitope NMC-4 depended charged residues Asp-514 Arg-632 not segments previously implicated peptide inhibition studies, Cys-474-Pro-488 Leu-694-Pro-708. Glu-626 in the segment Asp-520-Lys-534 abolished GPIb but did affect binding, suggesting that these regions are required modulation ristocetin GPIb. Mutations Glu-596 Lys-599 decreased without affecting its botrocetin, this interacts directly with Alanine substitutions Arg-545 Glu-497-Arg-511 Arg-687-Glu-689 caused increased These results, locations disease type 2B mutations, suggest two acidic containing Cys-509-Cys-695 disulfide (Glu-497-Arg-511, Arg-687-Val-698) one predominantly basic region (Met-540-Arg-578) cooperate inhibit a distinct site domain. This is relieved specific modulators or subendothelial connective tissue.