Localization of von willebrand factor-binding sites for platelet glycoprotein Ib and botrocetin by charged-to-alanine scanning mutagenesis.
At sites of vascular injury, von Willebrand factor (VWF) mediates platelet adhesion through binding to platelet glycoprotein Ib (GPIb). Previous studies identified clusters of charged residues within VWF domain A1 that were involved in binding GPIb or botrocetin. The contribution of 28 specific residues within these clusters was analyzed by mutating single amino acids to alanine. Binding to a panel of six conformation-dependent monoclonal antibodies was decreased by mutations at Asp(514), Asp(520), Arg(552), and Arg(611) (numbered from the N-terminal Ser of the mature processed VWF), suggesting that these residues are necessary for domain A1 folding. Binding of (125)I-botrocetin was decreased by mutations at Arg(629), Arg(632), Arg(636), and Lys(667). Ristocetin-induced and botrocetin-induced binding to GPIb both were decreased by mutations at Lys(599), Arg(629), and Arg(632); among this group the K599A mutant was unique because (125)I-botrocetin binding was normal, suggesting that Lys(599) interacts directly with GPIb. Ristocetin and botrocetin actions on VWF were dissociated readily by mutagenesis. Ristocetin-induced binding to GPIb was reduced selectively by substitutions at positions Lys(534), Arg(571), Lys(572), Glu(596), Glu(613), Arg(616), Glu(626), and Lys(642), whereas botrocetin-induced binding to GPIb was decreased selectively by mutations at Arg(636) and Lys(667). The binding of monoclonal antibody B724 involved Lys(660) and Arg(663), and this antibody inhibits (125)I-botrocetin binding to VWF. The crystal structure of the A1 domain suggests that the botrocetin-binding site overlaps the monoclonal antibody B724 epitope on helix 5 and spans helices 4 and 5. The binding of botrocetin also activates the nearby VWF-binding site for GPIb that involves Lys(599) on helix 3.
Matsushita
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Howard Hughes Medical Institute, Department of Medicine, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Meyer
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Sadler
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D000409:Alanine; D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000378:metabolism; D001665:Binding Sites; D003435:Crotalid Venoms / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D016296:Mutagenesis; D019038:Platelet Glycoprotein GPIb-IX Complex / Q000378:metabolism*; D013329:Structure-Activity Relationship; D014841:von Willebrand Factor / Q000737:chemistry* / Q000378:metabolism
D000911:Antibodies, Monoclonal; D003435:Crotalid Venoms; D019038:Platelet Glycoprotein GPIb-IX Complex; D014841:von Willebrand Factor; C030961:botrocetin; D000409:Alanine