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J Biol Chem, Vol. 275, Issue 15, 11044-11049, April 14, 2000

Localization of von Willebrand Factor-binding Sites for Platelet Glycoprotein Ib and Botrocetin by Charged-to-Alanine Scanning Mutagenesis^*

Tadashi Matsushita^§, Dominique Meyer^¶, and J. Evan Sadler

From the  Howard Hughes Medical Institute, Departments of Medicine and of Biochemistry & Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110 and ^¶ INSERM U.143, Hôpital Bicêtre, 94275 le Kremlin-Bicêtre, Cedex, France

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 Asp⁵¹⁴, Asp⁵²⁰, Arg⁵⁵², and Arg⁶¹¹ (numbered from the N-terminal Ser of the mature processed VWF), suggesting that these residues are necessary for domain A1 folding. Binding of ¹²⁵I-botrocetin was decreased by mutations at Arg⁶²⁹, Arg⁶³², Arg⁶³⁶, and Lys⁶⁶⁷. Ristocetin-induced and botrocetin-induced binding to GPIb both were decreased by mutations at Lys⁵⁹⁹, Arg⁶²⁹, and Arg⁶³²; among this group the K599A mutant was unique because ¹²⁵I-botrocetin binding was normal, suggesting that Lys⁵⁹⁹ 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 Lys⁵³⁴, Arg⁵⁷¹, Lys⁵⁷², Glu⁵⁹⁶, Glu⁶¹³, Arg⁶¹⁶, Glu⁶²⁶, and Lys⁶⁴², whereas botrocetin-induced binding to GPIb was decreased selectively by mutations at Arg⁶³⁶ and Lys⁶⁶⁷. The binding of monoclonal antibody B724 involved Lys⁶⁶⁰ and Arg⁶⁶³, and this antibody inhibits ¹²⁵I-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 Lys⁵⁹⁹ on helix 3.

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