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，血管性血友病因子通过与血小板膜糖蛋白Ib-V-IX复合体(GPIBα)的N-末端结合区域介导血小板与内皮下结缔组织的黏附。GPIBα与vWF结合的详细分子机制尚不清楚。为了直接回答这个问题，我们采用电荷到丙氨酸的扫描突变来确定GPIBα的N-末端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、谷氨酸和组氨酸在内的66种带电氨基酸在人GPIBα的1-302之间被单独或以小簇形式改变为丙氨酸。重组突变体与几种构象依赖的抗GPIBα的单抗的结合，以及瑞沙西汀和肉豆蔻素诱导的1251标记的人vWF的结合。获得了40个表达GPIBαa可溶性片段的突变体，表达产物的C末端带有标志标签。128、172、175、217和218位的突变减少了瑞斯托菌素和肉豆根素诱导的VWF结合。相反，第12位和第14位突变减少了依赖瑞斯托菌素的VWF结合，而正常的肉毒菌素诱导结合。在217、218285、287和301位突变的3个重组蛋白仅抑制肉豆根素诱导的VWF结合。单抗(Mab)6d1抑制了瑞斯托西汀和肉豆根素诱导的VWF结合，Glul25处的突变特异性地将这种结合减少到6d1。相反，单抗HPL7对VWF结合没有影响，121位突变降低了结合。

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.: "Complete antithrombin deficiency in mice results in embryonic lethality"J Clin Invest. 106・7. 873-878 (2000)

K. Ishiguro、T. Matsushita 等：“小鼠完全抗凝血酶缺乏导致胚胎致死”J Clin Invest 106・7 (2000)。

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）。

Y.Yoshida, M.Hirai, et al.: "Antiarrhythmic efficacy of dipyridamole in trearing reperfusion arrhythmia"Circulation. 101・6. 624-630 (2000)

Y. Yoshida、M. Hirai 等：“双嘧达莫治疗再灌注心律失常的抗心律失常功效”循环 101・6。