title: 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.
abstract: At sites of vascular injury, von Willebrand factor (VWF) mediates platelet adhesion through binding to platelet glycoprotein Ib (GPIb). The VWF-GPIb interaction was investigated by clustered charged-to-alanine scanning mutagenesis of VWF domain A1 between His-473 and Gly-716. Recombinant variants of VWF were assayed for binding to conformation-dependent monoclonal antibody NMC-4, for ristocetin-induced and botrocetin-induced binding to platelets, and for direct binding to botrocetin. Substitutions at 32 amino acids had no effect on VWF function. The epitope of NMC-4 depended on charged residues between Asp-514 and Arg-632 and not on segments previously implicated by peptide inhibition studies, Cys-474-Pro-488 and Leu-694-Pro-708. Substitutions at Glu-626 and in the segment Asp-520-Lys-534 abolished ristocetin-induced binding of VWF to GPIb but did not affect botrocetin-induced binding, suggesting that these regions are required for modulation by ristocetin but not for binding of VWF to GPIb. Mutations at Glu-596 and Lys-599 decreased binding of VWF to GPIb without affecting its binding to botrocetin, suggesting that this segment interacts directly with GPIb. Alanine substitutions at Arg-545 and in the segments Glu-497-Arg-511 and Arg-687-Glu-689 caused increased binding of VWF to GPIb. These results, and the locations of von Willebrand disease type 2B mutations, suggest that two acidic regions containing the Cys-509-Cys-695 disulfide (Glu-497-Arg-511, Arg-687-Val-698) and one predominantly basic region (Met-540-Arg-578) cooperate to inhibit a distinct GPIb binding site in the VWF A1 domain. This inhibition is relieved by specific mutations, by the modulators ristocetin and botrocetin, or by binding to subendothelial connective tissue.
authors.affiliation: Howard Hughes Medical Institute, Department of Medicine, Jewish Hospital of St. Louis, Washington University School of Medicine, Missouri 63110, USA.
authors.forename: T
authors.initials: T
authors.lastname: Matsushita
authors.forename: J E
authors.initials: JE
authors.lastname: Sadler
mesh_terms: D000409:Alanine / Q000378:metabolism; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism*; D000911:Antibodies, Monoclonal / Q000276:immunology; D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism; D002460:Cell Line; D003435:Crotalid Venoms / Q000494:pharmacology; D000939:Epitopes / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010980:Platelet Membrane Glycoproteins / Q000378:metabolism*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012310:Ristocetin / Q000494:pharmacology; D014841:von Willebrand Factor / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D000596:Amino Acids; D000911:Antibodies, Monoclonal; D003435:Crotalid Venoms; D000939:Epitopes; D010980:Platelet Membrane Glycoproteins; D011994:Recombinant Proteins; D014841:von Willebrand Factor; D012310:Ristocetin; C030961:botrocetin; D000409:Alanine