In vitro study for clotting function aid activation mechanism in the gene therapy for haemophilia A

A型血友病基因治疗中凝血功能辅助激活机制的体外研究

• 批准号: 14570761
• 负责人: SHIMA Midori
• 金额: $ 2.3万
• 依托单位: Nara Medical 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 2004
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14570761/
• 关键词: Clot waveform; factor VIII; hemophilia; 血友病A; トロンビン生成; 遺伝子治療

1.Clot waveform analysis : For the purpose of monitoring the very low level of factor VIII activity expressed during the gene therapy for hemophilia A, we performed apt-based clot waveform analysis by real time-monitored transmittance of clotting process and measured the clotting time, clotting velocity, maximum clotting velocity, clotting acceleration and its maximum acceleration. We found that the clot waveform parameters such as clotting time, maximum clotting velocity and maximum clot acceleration correlates well with the very low level of factor VIII activity with range of 0.2-1.0 IU/dl. This result indicated that the presence and significance of factor VIII less than 1.0 IU/dl. Furthermore the results also indicated that the measurement of lsuch low level of factor VIII activity is possible. We measured for the presence of low level of factor VIII activity in hemophilia A patients diagnosed as severe type by conventional clotting assay. We found that severe hemophilia A patients … More with low level of factor VIII less than 1.0 IU/dl. No factor VIII activity was detected in patients with intron 22 -related inversion.2.Factor VIII activation and inactivation mechanism : Factor VIII acts as cofactor in factor X activating reaction due to activated factor IX. Factor VIII itself can be activated by thrombin and inactivated by activated protein C. We found that synthetic peptide corresponding to the factor Xa binding region of factor VIII inhibited intrinsic pathway coagulation. Therefore these results indicated that the factor Xa-catalyzed FVIII activation is essential in the clotting function. Factor VIII inactivation due to activated protein C is known to be protected by von Willebrand factor. We found that the activated protein C compete with von Willebrand factor in binding to factor VIII. These suggested that the factor VIII inactivation by activated protein C is regulated by von Willebrand factor and the binding site for activated protein C locates in close to von Willebrand binding site. Less

1.凝血波形分析：为了监测血友病A基因治疗过程中凝血因子VIII活性表达的极低水平，我们通过实时监测凝血过程的透过率，进行了基于APT的凝血波形分析，测量了凝血时间、凝血速度、最大凝血速度、凝血加速度及其最大加速度。我们发现，凝血时间、最大凝血速度和最大凝血加速度等凝血波形参数与凝血因子VIII活性的极低水平有很好的相关性，范围为0.2-1.0IU/dl。提示第VIII因子<1.0IU/dl的存在及其意义。此外，结果还表明，测量如此低水平的凝血因子VIII活性是可能的。我们对常规凝血法诊断为重症的血友病A患者进行了低水平凝血因子活性的检测。我们发现严重的血友病A患者…因子VIII水平低于1.0IU/dl的患者较多。在内含子22相关倒置的患者中未检测到第VIII因子的活性。2.第VIII因子的激活和失活机制：第VIII因子在因第IX因子被激活而激活X因子的反应中起辅助因子的作用。凝血酶能激活凝血因子VIII，活化的蛋白C能灭活凝血因子VIII。我们发现，与凝血因子Xa结合区对应的合成肽能抑制内源性凝血途径。因此，这些结果表明，因子Xa催化的FVIII激活在凝血功能中是必不可少的。因子因蛋白C的活化而失活，已知受到von Willebrand因子的保护。我们发现活化的蛋白C与von Willebrand因子竞争结合第VIII因子。这表明活化的蛋白C失活的第VIII因子受von Willebrand因子的调节，活化蛋白C的结合部位位于von Willebrand结合部位附近。较少

项目成果

ELSaenko, Ananyeva, NM, Shima M, Mausert CAE et al.: "The future of recombinant coopelation factors"Journal of Thrombosis and Thrombolysis. 1. 922-930 (2003)

ELSaenko、Ananyeva、NM、Shima M、Mausert CAE 等人：“重组协同因子的未来”血栓形成与溶栓杂志。

Understanding the hemostatic effects of recombinant factor VIIa by clot waveform, analysis

• DOI: 10.1053/j.seminhematol.2003.11.021
• 发表时间: 2004-01-01
• 期刊: SEMINARS IN HEMATOLOGY
• 影响因子: 3.6
• 作者: Shima, M

Replacement therapy with plasma-derived factor VIII concentrates induces skew in T-cell receptor usage and clonal expansion of CD8+ T-cell in HIV-seronegative hemophilia patients.

在 HIV 血清阴性血友病患者中，血浆衍生因子 VIII 浓缩物的替代治疗会导致 T 细胞受体使用和 CD8 T 细胞克隆扩增出现偏差。

• 发表时间: 2003
• 期刊: Thrombosis and Haemostasis 90
• 作者: Takaji Matsutani;Yoshihiko Sakurai;Takeshi Yoshioka;Yuji Tsuruta;Ryuji Suzuki;Midori Shima;Akira Yoshioka
• 通讯作者: Akira Yoshioka

Sakurai Y, Shima M, Matsumoto T, Takatsuka H et al.: "Anticoagulant activity of M-LAO, -amino acid oxidase purified from Agkistrodon halys blomhoffii, through selective inhibition of factor IX"Biochimics Biophysica Acta. 1649. 51-57 (2003)

Sakurai Y、Shima M、Matsumoto T、Takatsuka H 等人：“通过选择性抑制因子 IX，从 Agkistrodon halys blomhoffii 中纯化的 M-LAO，β-氨基酸氧化酶具有抗凝血活性”Biochimics Biophysicala Acta。

Inversions of the factor VIII gene in Japanese patients with severe hemophilia A

日本严重甲型血友病患者的因子 VIII 基因倒位

• 发表时间: 2004
• 期刊: Int J Hematol 79
• 作者: Kazuyoshi K;Hiroyuki Naka;Midori Shima et al.
• 通讯作者: Midori Shima et al.