2001 Fiscal Year Final Research Report Summary
Identification of the amino acid residues of the platelet GPIbα essential for the von Willebrand factor binding by the clustered charged-to-alanine scanning mutagenesis
| Project/Area Number |
12670659
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Circulatory organs internal medicine
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| Research Institution | Nagoya University |
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Principal Investigator |
HIRAI Makoto University Hospital, Nagoya University Assistant Professor, 医学部・附属病院, 講師 (90242875)
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| Co-Investigator(Kenkyū-buntansha) |
MATSUSHITA Tadashi University Hospital, Nagoya University Research Associate, 医学部・附属病院, 助手 (30314008)
KONDO Takahisa Graduate School of Medicine, Nagoya University Research Associate, 大学院・医学研究科, 助手 (00303644)
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| Project Period (FY) |
2000 – 2001
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| Keywords | von Willebrand factor / GPIb / Polvmerase chain reaction / platelet / thrombosis / coronary heart disease / alanine scanning mutagenesis |
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| Research Abstract |
At the site of vascular injury, von Willebrand factor (VWF) mediates platelet adhesion to subendothelial connective tissue through binding to N-terminal VWF binding region of the a chain of platelet glycoprotein Ib-V-IX complex (GPIbα). The detailed molecular mechanisms responsible for GPIbα binding of VWF remain to be elucidated. In order to provide a direct answer to this question, we have employed charged-to-alanine scanning mutagenesis to define functional amino acid residues of the N-terminal 302 amino acids of GPIbα. Sixty six charged amino acids including arginine, lysine, aspartate, glutamate, and histidine were changed singly or in small clusters to alanine between 1 and 302 of human GPIbα. Recombinant mutants were assayed for binding to several conformation-dependent monoclonal antibodies to GPIbα, for ristocetin-induced and botrocetin-induced binding of 1251-labeled human VWF. Forty mutant constructs expressing a soluble fragment of GPIbα a were produced according with FLAG-tag at the C-terminal end. Mutations at 128, 172, 175, 217, and 218 decreased both ristocetin- and botrocetin-induced VWF binding. In contrast, mutations at 12 and 14 decreased the ristocetin-dependent VWF binding with normal botrocetin-induced binding. Three recombinant proteins mutated at 217, 218 285, 287, and 301reduced only the botrocetin induced VWF binding. Monoclonal antibody (Mab) 6D1 inhibits ristocetin and botrocetin-induced VWF binding and a mutation at Glul25 specifically reduced the binding to 6D1. In contrast, Mab HPL7 has no effect for VWF binding and a mutation at 121 reduced the binding.
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Research Products
(12 results)
