IDENTIFICATION OF AMINO-ACID-RESIDUES ESSENTIAL FOR VON-WILLEBRAND-FACTOR BINDING TO PLATELET GLYCOPROTEIN IB - CHARGED-TO-ALANINE SCANNINGMUTAGENESIS OF THE A1 DOMAIN OF HUMAN VON-WILLEBRAND-FACTOR

At sites of vascular injury, von Willebrand factor (VWF) mediates plat elet 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. Rec ombinant variants of VWF were assayed for binding to conformation-depe ndent monoclonal antibody NMC-4, for ristocetin-induced and botrocetin -induced binding to platelets, and for direct binding to botrocetin. S ubstitutions at 32 amino acids had no effect on VWF function. The epit ope of NMC-4 depended on charged residues between Asp-514 and Arg-632 and not on segments previously implicated by peptide inhibition studie s, Cys-474-Pro-488 and Leu-694-Pro-708. Substitutions at Glu-626 and i n 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 f or binding of VWF to GPIb. Mutations at Glu-596 and Lys-599 decreased binding of VWF to GPIb without affecting its binding to botrocetin, su ggesting that this segment interacts directly with GPIb. Alanine subst itutions at Arg-545 and in the segments Glu-497-Arg-511 and Arg-687-Gl u-689 caused increased binding of VWF to GPIb. These results, and the locations of von Willebrand disease type 2B mutations, suggest that tw o 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 modu lators ristocetin and botrocetin, or by binding to subendothelial conn ective tissue.