The C-terminal acidic region in the A1 domain of factor VIII facilitates thrombin-catalyzed activation and cleavage at Arg³⁷².

• Material Type: 博士論文
• Title: The C-terminal acidic region in the A1 domain of factor VIII facilitates thrombin-catalyzed activation and cleavage at Arg³⁷².
• Author/Editor: Nakajima, Yuto; Nogami, Keiji
• Author Heading: Nakajima, Yuto; Nogami, Keiji
• Publication, Distribution, etc.: Wiley
• Publication Date: 2021-03
• Publication Date (W3CDTF): 2021-03
• Alternative Title: 第VIII因子A1ドメインC末端酸性領域は、トロンビン惹起活性化とArg³⁷²開裂を制御する
• Periodical title: Journal of thrombosis and haemostasis
• No. or year of volume/issue: 19 3
• Volume: 19
• Issue: 3
• Pages: 677-688
• ISSN (Periodical Title): ISSN : 1538-7933
• Degree grantor/type: 奈良県立医科大学
• Date Granted: 2021-03-15
• Dissertation Number: 甲第788号
• Degree Type: 博士（医学）
• Text Language Code: eng
• Subject Heading: factor VIII; hirudin; mutant protein; protein-protein interaction domain; thrombin
• Target Audience: 一般
• Note (General): type:Thesis; Background: Factor VIII (FVIII) is activated by thrombin-catalyzed cleavage at three sites. Previous reports indicated that the A2 domain contained thrombin-interactive sites responsible for cleavage at Arg³⁷². We have also found that the A1 domain of FVIII bound to the anion-binding exosite I of thrombin. The present study focused, therefore, on thrombin interaction with A1 residues 337-372 containing clustered acidic and hirugen-like sequences. Aim: To identify specific thrombin-interactive site(s) within the A1 acidic region of FVIII. Methods and results: The synthetic peptide of residues 337-353 with sulfated Tyr³⁴⁶ (337-353S) significantly blocked thrombin-catalyzed FVIII activation and cleavage at Arg³⁷², while a corresponding peptide of residues 354-372 had no significant effect. Treatment with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide to cross-link thrombin and 340-350S suggested that the 344-349 clustered acidic region was involved in thrombin interaction. Alanine-substituted FVIII mutants, Y346A and D347A/D348A/D349A, depressed thrombin-catalyzed activation and cleavage at Arg³⁷², with peak activation at ~ 50% and cleavage rates of ~ 10% to 20% compared to wild type (WT). The peak level of thrombin-catalyzed activation and the cleavage rate at Arg³⁷² using FVIII mutants with 337-346 residues substituted with hirugen-sequences (MKNNEEAEDY337-346GDFEEIPEEY) were ~ 1.5- and ~ 2.5-fold of WT, respectively. Surface plasmon resonance-based analysis demonstrated that the Kd for active-site modified thrombin interactions using Y346A and D347A/D348A/D349A mutants was ~ 3- to 6-fold higher than that of WT, and that the hirugen-hybrid mutant facilitated association kinetics ~ 1.8-fold of WT. Conclusion: Residues 346-349 with sulfated Tyr provided a thrombin-interactive site responsible for activation and cleavage at Arg³⁷². A hirugen-hybrid A1 mutant showed more efficient thrombin-catalyzed cleavage at Arg³⁷².; 博士（医学）・甲第788号・令和3年3月15日; © 2020 International Society on Thrombosis and Haemostasis.; This is the peer reviewed version of the following article: https://onlinelibrary.wiley.com/doi/10.1111/jth.15201, which has been published in final form at https://doi.org/10.1111/jth.15201. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived Versions.; identifier:Journal of thrombosis and haemostasis Vol.19 No.3 p.677-688 (2021 Mar); identifier:15387933; identifier:http://ginmu.naramed-u.ac.jp/dspace/handle/10564/3912; identifier:Journal of thrombosis and haemostasis, 19(3): 677-688
• Source: fulltext; http://ginmu.naramed-u.ac.jp/dspace/bitstream/10564/3912/1/01%e7%94%b2788%e6%9c%ac%e6%96%87%e3%81%ae%e8%a6%81%e6%97%a8.pdf; http://ginmu.naramed-u.ac.jp/dspace/bitstream/10564/3912/2/02%e7%94%b2788%e5%af%a9%e6%9f%bb%e8%a6%81%e6%97%a8.pdf; http://ginmu.naramed-u.ac.jp/dspace/bitstream/10564/3912/6/03%e7%94%b2788%e6%9c%ac%e6%96%87.pdf
• Access Restrictions: インターネット公開
• Related Material (DOI): 10.1111/jth.15201
• Periodical Title (NCID): AA11811179; https://ci.nii.ac.jp/ncid/AA11811179
• Data Provider (Database): 国立情報学研究所 : 学術機関リポジトリデータベース（IRDB）（機関リポジトリ）; https://irdb.nii.ac.jp
• Original Data Provider (Database): 奈良県立医科大学 : 奈良県立医科大学機関リポジトリ GINMU