Gale A J, Radtke K-P, Cunningham M A, Chamberlain D, Pellequer J-L, Griffin J H
Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, CA 92037, USA.
J Thromb Haemost. 2006 Jun;4(6):1315-22. doi: 10.1111/j.1538-7836.2006.01951.x.
The utility of purified coagulation factor (F)VIII for treatment of hemophilia A is limited in part by its instability following activation by thrombin, which is caused by spontaneous dissociation of the A2 domain from the activated FVIII (FVIIIa) heterotrimer. To prevent this A2 domain dissociation in FVIIIa, we previously engineered a cysteine pair (C664-C1826) in recombinant FVIII that formed a disulfide bond cross-linking the A2 domain in the heavy chain to the A3 domain in the light chain. This engineered disulfide bond resulted in a more stable FVIIIa.
Here, we characterize the functional parameters of C664-C1828 FVIII and of a new disulfide bond-stabilized FVIII (C662-C1828 FVIII).
In order to assess whether these FVIII variants might be good candidates for a new therapeutic agent to treat hemophilia A, we investigated a variety of functional parameters that might affect the in vivo properties of the variants, including half-life of disulfide bond-stabilized FVIII and FVIIIa and the potency of these FVIIIa molecules in the FXase complex.
Both disulfide bond-stabilized variants had improved affinity for von Willebrand factor (VWF). In studies of FX activation by purified FIXa and FVIIIa, C662-C1828 FVIIIa had normal activity while C664-C1826 FVIIIa had reduced activity. Both C664-C1826 FVIIIa and C662-C1828 FVIIIa were inactivated by activated protein C (APC) but the rates of inactivation were different.
Overall, the specific location of the disulfide bridge between the A2 and A3 domains appears to affect functional properties of FVIIIa. In summary, introduction of engineered interdomain disulfides results in FVIIIa variants that resist spontaneous loss of activity while retaining susceptibility to APC proteolytic inactivation and maintaining VWF binding.
纯化的凝血因子VIII用于治疗甲型血友病的效用在一定程度上受到其被凝血酶激活后不稳定的限制，这是由于A2结构域从活化的FVIII（FVIIIa）异源三聚体中自发解离所致。为了防止FVIIIa中的这种A2结构域解离，我们之前在重组FVIII中设计了一对半胱氨酸（C664 - C1826），它们形成了一个二硫键，将重链中的A2结构域与轻链中的A3结构域交联起来。这种设计的二硫键使FVIIIa更稳定。
在此，我们对C664 - C1828 FVIII和一种新的二硫键稳定的FVIII（C662 - C1828 FVIII）的功能参数进行表征。
为了评估这些FVIII变体是否可能是治疗甲型血友病的新型治疗药物的良好候选者，我们研究了各种可能影响变体体内特性的功能参数，包括二硫键稳定的FVIII和FVIIIa的半衰期以及这些FVIIIa分子在FX酶复合物中的效力。
两种二硫键稳定的变体对血管性血友病因子（VWF）的亲和力均有所提高。在纯化的FIXa和FVIIIa激活FX的研究中，C662 - C1828 FVIIIa具有正常活性，而C664 - C1826 FVIIIa活性降低。C664 - C1826 FVIIIa和C（此处原文有误，应为C662 - C1828 FVIIIa）均被活化蛋白C（APC）灭活，但灭活速率不同。
总体而言，A2和A3结构域之间二硫键的特定位置似乎会影响FVIIIa的功能特性。总之，引入设计的结构域间二硫键会产生FVIIIa变体，这些变体能够抵抗活性的自发丧失，同时保留对APC蛋白水解灭活的敏感性并维持VWF结合。
