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

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因子活性的极低水平有很好的相关性，范围为0.2Iu/dl~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。我们对常规凝血试验诊断为重症的血友病A患者进行了凝血因子VIII活性低水平的检测。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因子活性。2.因子的激活和失活机制：因子在因激活因子IX而导致的X因子激活反应中起辅助作用。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。第VIII因子因活化蛋白C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

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