Studies on causative gene for thrombotic thrombocytopenic purpura, ADAMTS13

血栓性血小板减少性紫癜致病基因ADAMTS13的研究

• 批准号: 17390285
• 负责人: MIYATA Toshiyuki
• 金额: $ 10.51万
• 依托单位: National Cardiovascular Center Research Institute
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2005
• 资助国家: 日本
• 起止时间: 2005 至 2007
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-17390285/
• 关键词: Platelet; Thrombocytopenia; von Willbrand factor; genetic engineered mouse; ADAMTS13; endothelial cells; thrombosis; genetic polymorphism; 遺伝子解析; 血栓性血小板減少性紫斑病; 性差; 一般住民; 臨床検査法; メタロプロテアーゼ; 微小血管; 腎傷害; 血管障害

1. Phenotypic analysis of mouse lacking ADAMTS13 gene We generated and characterized Adamts13-knockout mice(KO) and Adamts13-congenic mice lacking the C-terminal domains of ADAMTS13(CG) . Both KO and CG were viable and fertile. In KO, unusually large von Willbrand factor(VWF) multimers were observed in the plasma. Thrombogenesis on immobilized collagen under flow and thrombocytopenia induced by a collagen plus epinephrine infusion were significantly promoted in KO than in wild-type mice(WT). However, hematological and histological analyses failed to detect any signs of TTP in KO. CG maintained the ADAMTS13 activity and normal VWF-multimer distribution in the plasma, and did not show an enhanced thrombogenesis under flow. Thrombocytopenia induced by a collagen plus epinephrine infusion was significantly more in CG than in WT. These results suggest that ADAMTS13 deficiency alone is not sufficient to cause TTP and that the mouse lacking the C-terminal domains is prone to thromosis.2. Studies on P475S mutation of ADAMTS13 We previously identified P475S mutation in ADAMTS13 gene with the allele frequency of 0.05 in the Japanese general population. Here, we expressed the recombinant wild type ADAMTS13 protein and P475S mutant and compared their activity toward the natural substrate VWF and the synthetic substrate FRETS-VWF73. We found that mutant ADAMTS13 showed about 10% activity of wild-type using natural substrate but about 70% activity using the synthetic substrate. The difference of the activity was attributable to urea in the reaction buffer for the natural substrate. The mutant protein tended to lose its activity in the presence of 1.5 M urea.

1. ADAMTS 13基因缺失小鼠的表型分析我们制备并鉴定了ADAMTS 13基因敲除小鼠（KO）和ADAMTS 13 C-末端结构域缺失的Adamts 13同源小鼠（CG）。KO和CG都是可行且可繁殖的。在KO中，在血浆中观察到异常大的von Willbrand因子（VWF）多聚体。固定胶原蛋白流动下的血栓形成和血小板减少诱导的胶原蛋白加肾上腺素输注显着促进KO比野生型小鼠（WT）。然而，血液学和组织学分析未能检测到任何迹象的TTP在KO。CG维持ADAMTS 13活性和正常的VWF-多聚体在血浆中的分布，并且在流动下没有显示出增强的血栓形成。胶原蛋白加肾上腺素输注诱导的血小板减少症在CG中显著多于WT。这些结果表明，ADAMTS 13缺陷本身不足以引起TTP，缺乏C端结构域的小鼠更容易发生血栓形成. ADAMTS 13基因P475 S突变的研究我们以前在日本普通人群中发现了ADAMTS 13基因P475 S突变，等位基因频率为0.05。在这里，我们表达了重组野生型ADAMTS 13蛋白和P475 S突变体，并比较了它们对天然底物VWF和合成底物FRETS-VWF 73的活性。我们发现突变体ADAMTS 13在使用天然底物时显示出野生型的约10%的活性，而在使用合成底物时显示出约70%的活性。活性的差异归因于天然底物的反应缓冲液中的尿素。突变体蛋白在1.5 M尿素存在下趋于失去其活性。

项目成果

「肺血栓塞栓症」THE LUNG perspectives

“肺血栓栓塞症”THE LUNG 观点

• 发表时间: 2006
• 作者: Fumiaki Banno;Koichi Kokame;Tomohiko Okuda;Shigenori Honda;Shigeki Miyata;Hisashi Kato;Yoshiaki Tomiyama;Toshiyuki Miyata;Toshiyuki Miyata;Toshiyuki Miyata;奥田智彦;宮田敏行
• 通讯作者: 宮田敏行

Inherited and novo mutations of ADAMTS13 in a patient with Upshaw-Schulman syndrome.

Upshaw-Schulman 综合征患者 ADAMTS13 的遗传性和新突变。

• 发表时间: 2008
• 期刊: J Thromb Haemost. 6
• 作者: Ohnaka K;et al.;K.Kokame;T.Okuda;K. Kokame
• 通讯作者: K. Kokame

Inherited and de novo mutations of ADAMTS13 in a patient with Upshaw-Schulman syndrome

Upshaw-Schulman 综合征患者 ADAMTS13 的遗传性和新生突变

• 发表时间: 2008
• 期刊: J. Thromb. Haemost. 6
• 作者: K. Kokame;Y. Aoyama;M. Matsumoto;Y. Fujimura;and T. Miyata
• 通讯作者: and T. Miyata

A large deletion of the PROS1 gene in a deep vein thrombosis patient with protein S deficiency

• DOI: 10.1160/th07-03-0211
• 发表时间: 2007-10
• 期刊: Thrombosis and Haemostasis
• 影响因子: 6.7
• 作者: T. Yin;S. Takeshita;Yukiko Sato;T. Sakata;Yongchol Shin;S. Honda;T. Kawasaki;H. Tsuji;T. Kojima;S. Madoiwa;Y. Sakata;M. Murata;Y. Ikeda;T. Miyata

「遺伝子診断とゲノムタイピング法」図説 分子病態学、一瀬白帝・鈴木宏治編著

《基因诊断与基因组分型方法》图解分子病理学，一之濑博亭、铃木浩二主编

• 发表时间: 2008
• 作者: Fumiaki Banno;Koichi Kokame;Tomohiko Okuda;Shigenori Honda;Shigeki Miyata;Hisashi Kato;Yoshiaki Tomiyama;Toshiyuki Miyata;Toshiyuki Miyata;Toshiyuki Miyata;奥田智彦
• 通讯作者: 奥田智彦