The physiological role of the interaction of VWF-GPIb : Amino acid residues of the platelet GPIb to bind VWF and the generation of knock-in mice mutated at Lys599 to A1a.

VWF-GPIb相互作用的生理作用：血小板GPIb的氨基酸残基结合VWF并产生Lys599突变为A1a的敲入小鼠。

• 批准号: 14570974
• 负责人: MATSUSHITA Tadashi
• 金额: $ 2.24万
• 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: 2002
• 资助国家: 日本
• 起止时间: 2002 至 2003
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14570974/
• 关键词: von Willebrand factor; GPIb; Polymerase chain reaction; platelet; thrombosis; Knock out; Platelet; alanie scanning mutagenesis; homologous recombination

At the site of vascular injury, von Willebrand factor (VWF) mediates platelet adhesion to subendothelial connective tissue through binding to N-terminal domain of the a chain of platelet glycoprotein Ib (GPIba). We have found the loss-of-function mutations generated in soluble fragment containing the N-terminal 287 amino acids of GPIbα. 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-GPIba complex indicated that Glu128 and Asp175 form VWF binding sites and … More 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. Using targeted gene disruption and Cre-loxP gene excision technique, we investigated the role of VWF-GPIb in normal hemostasis. Oligonucleotide primer were used to obtain a partial cDNA of mouse VWF in from mRNA of C57BL/6J mice using RT-PCR. The resulting PCR product was used as a probe to isolate a genomic clone containing a segment of mouse VWF gene from a 129SVJ lambda FIX II genomic library. A targeting vector was constructed for homologous recombination in embryonic stem cells. It was based on a vector pBS/MC1DTpA+loxpGKneoW and the VWF gene fragments. The generation of targeted D3 embryonic stem (ES) cells and blastocyst injection were prepared to perform the knock-in strategies. Obtained findings indicates the novel binding sites required for VWF binding of human GPIbα Less

在血管损伤部位，血管性血友病因子（vonWillebrand factor，VWF）通过与血小板糖蛋白Ib（GPiba）α链的N端结构域结合，介导血小板粘附于内皮下结缔组织。我们发现GPIbα N端287个氨基酸的可溶性片段发生了功能缺失突变。Glu 128，Glu 172和Asp 175的突变特异性降低了瑞斯托菌素和肉毒素诱导的VWF结合，表明这些位点对血小板GPIb的VWF结合很重要。单克隆抗体6D 1抑制瑞斯托菌素和肉毒素诱导的VWF结合，Glu 125突变特异性降低了与6D 1的结合。相反，抗体HPL 7对VWF结合没有影响，突变体E121 A降低HPL 7结合。His 12和Glu 14突变降低了瑞斯托菌素诱导的VWF结合，而肉毒素诱导的结合正常。VWF-GPIba复合物的晶体学模型表明Glu 128和Asp 175形成VWF结合位点， ...更多信息 6D 1与Glu 125的结合中断了Glu 128与VWF的结合，但HPL 7与Glu 121的结合对VWF的结合没有影响。此外，His 12和Glu 14与VWF A1结构域的Glu 613和Arg 571接触，其突变表现出相似的表型。采用靶向基因破坏和Cre-loxP基因切除技术，我们研究了VWF-GPIb在正常止血中的作用。以C57 BL/6 J小鼠为材料，利用RT-PCR技术，从mRNA中扩增出小鼠VWF的部分cDNA。将所得PCR产物用作探针以从129 SVJ λ FIX II基因组文库中分离含有小鼠VWF基因片段的基因组克隆。构建了用于在胚胎干细胞中同源重组的靶向载体。它基于载体pBS/MC 1DTpA +loxpGKneoW和VWF基因片段。制备靶向D3胚胎干（ES）细胞和囊胚注射以执行敲入策略。所获得的结果表明人GPIbα Less与VWF结合所需的新结合位点

项目成果

Kunishima S: "Immunofluorescence analisis of neutrophil nonmuscle myosin heavy chain-A in MYH 9 disorders : association of subcellular localization with MYH9 mutations"Lab Invest. 83. 115-122 (2003)

Kunishima S：“MYH 9 疾病中中性粒细胞非肌肉肌球蛋白重链 A 的免疫荧光分析：亚细胞定位与 MYH9 突变的关联”Lab Invest。

Kunishima S: "Novel nonsense mutation in the platelet glycoprotein Ibbeta gene associated with Bernard-Soulier syndrome"Am J Hematol. 71. 279-284 (2002)

Kunishima S：“与 Bernard-Soulier 综合征相关的血小板糖蛋白 Ibbeta 基因中的新型无义突变”Am J Hematol。

Yamada T: "Enzyme immunoassay for measurement of murine plasminogen activator inhibitor-1, employing a specific antibody produced by the DNA vaccine method"Thromb Res. 111. 285-291 (2003)

Yamada T：“用于测量鼠纤溶酶原激活剂抑制剂-1的酶免疫测定法，采用由DNA疫苗方法产生的特异性抗体”Thromb Res。

Kunishima S: "Immunofluorescence analysis of neutrophil nonmuscle myosin heavy chain-A in MYH9 disorders : association of subcellular localization with MYH9 mutations"Lab Invest. 83. 115-122 (2003)

Kunishima S：“MYH9 疾病中中性粒细胞非肌肉肌球蛋白重链 A 的免疫荧光分析：亚细胞定位与 MYH9 突变的关联”Lab Invest。

Yamamoto K: "Plasminogen activator inhibitor-1 is a major stress-regulated gene : implications for stress-induced thrombosis in aged individuals"Proc Natl Acad Sci USA. 99. 890-895 (2002)

Yamamoto K：“纤溶酶原激活剂抑制剂-1 是一种主要的应激调节基因：对老年人应激诱导的血栓形成的影响”Proc Natl Acad Sci USA。