並列タイトル等第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
タイトル（掲載誌）Journal of thrombosis and haemostasis
一般注記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
DOIinfo:doi/10.1111/jth.15201
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