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.
Highlights
From the Howard Hughes Medical Institute, Departments of Medicine and Biochemistry & Molecular Biophysics, The Jewish Hospital of St
The results suggest that several discontinuous segments ofVWF domain Al interact with glycoprotein Ib (GPIb), ristocetin, and botrocetin
Construction, and Expression of Human Recombinant von Willebrand factor (VWF) Variants-The segment ofVWF that was targeted for mutagenesis consists of =254 amino acid residues between His-463 and Gly-716
Summary
Materials-Restriction enzymes were obtained from New England BioLabs (Beverly, MA). Taq DNA polymerase was from Perkin Elmer Corp. The wells were washed with PBS containing 0.1% Tween 20 and incubated for 105 min at room temperature with 15 JLI of various concentrations of wild type or mutant rVWF diluted in PBS containing 3% BSA. The wells were washed again and incubated for 105 min at room temperature with 20 ILl of peroxidase-conjugated rabbit polyclonal anti-human VWF P226 (DAKO) diluted 1:5000 in PBS containing 3% BSA. Each reaction mixture (25 ILl) contained 570 ng/ml rVWF, 0.2% BSA and various concentrations (0-20 ILg/ml) of botrocetin in TBS. The mutant rVWF proteins did not bind spontaneously to platelets, and the results of platelet binding assays are expressed as the percentage of unbound VWF antigen relative to the value obtained with platelets but without ristocetin or botrocetin. Specific binding for each mutant rVWF was calculated by subtracting nonspecific from total binding and normalized to the value obtained for wild type rVWF
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