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

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.