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

通过聚集电荷丙氨酸扫描诱变鉴定血小板 GPIbα 中冯维勒布兰德因子结合所必需的氨基酸残基

• 批准号: 12670659
• 负责人: HIRAI Makoto
• 金额: $ 2.11万
• 依托单位: Nagoya University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2000
• 资助国家: 日本
• 起止时间: 2000 至 2001
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12670659/
• 关键词: von Willebrand factor; GPIb; Polvmerase chain reaction; platelet; thrombosis; coronary heart disease; alanine scanning mutagenesis; 初期血栓形成; von Willebrand因子; 血小板糖蛋白GPIb; リストセチン; ボトロセチン

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.

在血管损伤部位，血管性血友病因子 (VWF) 通过与血小板糖蛋白 Ib-V-IX 复合物 (GPIbα) 的 a 链 N 端 VWF 结合区结合，介导血小板与内皮下结缔组织的粘附。负责 VWF 结合的 GPIbα 仍有待阐明 为了提供这个问题的直接答案，我们采用了电荷丙氨酸扫描诱变。定义了 GPIbα N 端 302 个氨基酸的功能氨基酸残基，包括精氨酸、赖氨酸、天冬氨酸、谷氨酸和组氨酸，在人重组 GPIbα 的 1 至 302 之间单独或以小簇形式改变为丙氨酸。测定了突变体与几种构象依赖性 GPIbα 单克隆抗体的结合，用于瑞斯托菌素诱导的根据 128、172、175、217 和 218 处的 FLAG 标签，产生了 40 个表达 GPIbα a 可溶性片段的 Botrocetin 诱导的结合。相反，12 和 14 处的突变降低了瑞斯托菌素和 Botrocetin 诱导的 VWF 结合。瑞斯托菌素依赖性 VWF 结合与正常 botrocetin 诱导的结合。在 217、218 285、287 和 301 处突变的三种重组蛋白仅减少 botrocetin 诱导的 VWF 结合。单克隆抗体 (Mab) 6D1 抑制瑞斯托菌素和 botrocetin 诱导的 VWF 结合和突变。 Glul25 特异性减少与 6D1 的结合。相比之下，Mab HPL7 对 VWF 结合没有影响，并且 121 处的突变减少了结合。

项目成果

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S. Kunishima, T. Matsushita, et al: "Identification of six novel MYH9 mutations and genotype-phenotype relationships in autosomal dominant macrothrombocytopenia"J Hum Genet. 46・12. 722-729 (2001)

S. Kunishima、T. Matsushita 等人：“常染色体显性巨血小板减少症中六种新的 MYH9 突变和基因型-表型关系的鉴定”J Hum Genet 46·12（2001）。

K. Ishiguro, T. Matsushita, et al.: "Syndecan-4 deficiency leads to high mortality of lipopolysaccharide-injected mice"J Biol Chem. 276・50. 47483-47488 (2001)

K. Ishiguro、T. Matsushita 等：“Syndecan-4 缺乏导致脂多糖注射小鼠的高死亡率”J Biol Chem 276・50 (2001)。