Identification of the amino acid residues of the platelet glycoprotein Ib (GPIb) essential for the von Willebrand factor binding by clustered charged-to-alanine scanning mutagenesis.
At the site of vascular injury, von Willebrand factor (VWF) mediates platelet adhesion to subendothelial connective tissue through binding to the N-terminal domain of the alpha chain of platelet glycoprotein Ib (GPIbalpha). To elucidate the molecular mechanisms of the binding, we have employed charged-to-alanine scanning mutagenesis of the soluble fragment containing the N-terminal 287 amino acids of GPIbalpha. Sixty-two charged amino acids were changed singly or in small clusters, and 38 mutant constructs were expressed in the supernatant of 293T cells. Each mutant was assayed for binding to several monoclonal antibodies for human GPIbalpha and for ristocetin-induced and botrocetin-induced binding of 125I-labeled human VWF. Mutations at Glu128, Glu172, and Asp175 specifically decreased both ristocetin- and botrocetin-induced VWF binding, suggesting that these sites are important for VWF binding of platelet GPIb. Monoclonal antibody 6D1 inhibited ristocetin- and botrocetin-induced VWF binding, and a mutation at Glu125 specifically reduced the binding to 6D1. In contrast, antibody HPL7 had no effect for VWF binding, and mutant E121A reduced the HPL7 binding. Mutations at His12 and Glu14 decreased the ristocetin-induced VWF binding with normal botrocetin-induced binding. Crystallographic modeling of the VWF-GPIbalpha complex indicated that Glu128 and Asp175 form VWF binding sites; the binding of 6D1 to Glu125 interrupts the VWF binding of Glu128, but HPL7 binding to Glu121 has no effect on VWF binding. Moreover, His12 and Glu14 contact with Glu613 and Arg571 of VWF A1 domain, whose mutations had shown similar phenotype. These findings indicated the novel binding sites required for VWF binding of human GPIbalpha.
Shimizu
Atsuya
A
First Department of Internal Medicine, Nagoya University School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan.
Matsushita
Tadashi
T
Kondo
Takahisa
T
Inden
Yasuya
Y
Kojima
Tetsuhito
T
Saito
Hidehiko
H
Hirai
Makoto
M
D000409:Alanine; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D016296:Mutagenesis; D015539:Platelet Activation; D019038:Platelet Glycoprotein GPIb-IX Complex / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D014841:von Willebrand Factor / Q000378:metabolism*
D019038:Platelet Glycoprotein GPIb-IX Complex; D014841:von Willebrand Factor; D000409:Alanine