Molecular biological analysis of single nucleotide polymorphisms in the D'D3 domain of von Willebrand factor

• Project/Area Number: 17K10128
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Research Field: Pediatrics
• Research Institution: Nara Medical University
• Principal Investigator: Ogiwara Kenichi 奈良県立医科大学, 医学部, 講師 (50623500)
• Co-Investigator(Kenkyū-buntansha): 野上 恵嗣 奈良県立医科大学, 医学部, 教授 (50326328)
• Project Period (FY): 2017-04-01 – 2023-03-31
• Project Status: Completed (Fiscal Year 2022)
• Budget Amount: ¥4,680,000 (Direct Cost: ¥3,600,000、Indirect Cost: ¥1,080,000); Fiscal Year 2019: ¥1,430,000 (Direct Cost: ¥1,100,000、Indirect Cost: ¥330,000); Fiscal Year 2018: ¥1,430,000 (Direct Cost: ¥1,100,000、Indirect Cost: ¥330,000); Fiscal Year 2017: ¥1,820,000 (Direct Cost: ¥1,400,000、Indirect Cost: ¥420,000)
• Keywords: フォン・ウィルブランド因子 / フォン・ウィルブランド病 / 血液凝固第VIII因子 / 血友病A / 一塩基多型 / 機能増強型変異 / 第VIII因子 / 凝固第VIII因子 / 遺伝子 / 細胞・組織 / 蛋白質

Outline of Final Research Achievements

The D'D3 domain of von Willebrand factor (VWF) plays an important role in binding to coagulation factor VIII (FVIII). We searched for gene variants that could cause functional enhancement of the D' domain of VWF by in silico analysis. (1) Wild-type VWF protein was expressed in HEK293T cells by using a plasmid transfected with human VWF cDNA (pPG-CAG-huVWF-PGKpuro) and the normal functions of the expressed protein were confirmed. The type 2N VWD mutations in the D' domain and neighboring single nucleotide polymorphisms were selected by in silico analysis to identify candidate variants with enhanced function of VWF. We generated VWF variants and analyzed its FVIII binding ability, but could not identify a variant that showed higher FVIII binding ability compared to wild-type VWF.

Academic Significance and Societal Importance of the Research Achievements

FVIIIは血漿中でVWFに保護され、FVIII欠乏は血友病Aを、VWF上のFVIII結合領域であるD'D3ドメインの変異はFVIII結合能低下による2N型VWDをきたす。D'ドメインの一塩基多型をヒントにFVIII結合能増強型VWF遺伝子の存在を探ることは、血友病Aの新規治療製剤の開発や、2N型VWDへの治療応用を含むVWFの基礎的知見につながる意義があったが、野生型VWFのFVIII結合能を上回るバリアントを同定するに至らなかった。このことはVWFのFVIII結合能がすでに極めて高い親和性にあることを示し、VWFとFVIIIの結合が進化的に温存されてきたことを示す最新知見とも合致した。

Research Products

• [Journal Article] Factor VIII pharmacokinetics associates with genetic modifiers of VWF and FVIII clearance in an adult hemophilia A population (2021)
  • Author(s): Ogiwara Kenichi、Swystun Laura L.、Paine A. Simonne、Kepa Sylvia、Choi Seon Jai、Rejt? Judit、Hopman Wilma、Pabinger Ingrid、Lillicrap David
  • Journal Title: Journal of Thrombosis and Haemostasis Volume: 19 Issue: 3 Pages: 654-663
  • DOI: 10.1111/jth.15183
  • Peer Reviewed
• [Journal Article] valuation of clinical severity in patients with type 2N von Willebrand disease using microchip-based flow-chamber system. (2020)
  • Author(s): Nakajima Y, Nogami K, Yada K, Kawamura T, Ogiwara K, Furukawa S, Shimonishi N, Takeyama M, Shima M.
  • Journal Title: Int J Hematol. Volume: 111 Issue: 3 Pages: 369-377
  • DOI: 10.1007/s12185-019-02782-z
  • Peer Reviewed
• [Presentation] DDAVP-trial has a possible role for auxiliary diagnosis in type 1 and type 2 von Willebrand disease. (2023)
  • Author(s): Kenichi Ogiwara, Naruto Shimonishi, Yuto Nakajima, Shoko Furukawa, Masahiro Takeyama, Masaki Hayakawa, Masanori Matsumoto, Keiji Nogami
  • Organizer: The XXXI Congress of the International Society on Thrombosis and Haemostasis
  • Int'l Joint Research
• [Presentation] A possible role of DDAVP trial as an auxiliary diagnosis in von Willebrand disease type 1 and type 2 (2022)
  • Author(s): Ami Morimoto, Kenichi Ogiwara, Naruto Shimonishi, Yuto Nakajima, Shoko Furukawa, Masahiro Takeyama, Masaki Hayakawa, Masanori Matsumoto, Keiji Nogami
  • Organizer: 第84回日本血液学会
• [Presentation] A new global assay for detection of protein C pathway-associated thrombophilia using thrombomodulin-mediated clot waveform analysis (2020)
  • Author(s): Naoki Hashimoto, Kenichi Ogiwara, Keiji Nogami, Midori Shima
  • Organizer: International Society on Thrombosis and Hemostasis 2020
  • Int'l Joint Research
• [Book] 新臨床内科学（第10版）第8章 血液・造血器疾患 血小板減少症以外の原因による出血傾向 6.その他の先天性凝固因子欠乏症 (2020)
  • Author(s): 荻原 建一，嶋 緑倫
  • Total Pages: 1
  • Publisher: 医学書院
  • ISBN: 9784260038065