Persson E, Ezban M
Department of Coagulation Research, Novo Nordisk A/S, Gentofte, Denmark.
Biochem Biophys Res Commun. 1994 Apr 15;200(1):233-8. doi: 10.1006/bbrc.1994.1439.
The activity of factor VIIIa was enhanced and stabilized by treatment of factor VIII with the crosslinker disuccinimidyl suberate. The activity was > 200-fold higher compared with that of native factor VIIIa and was stable for at least 15 days at 4 degrees C and pH 7.2. The crosslinked factor VIIIa was purified by immunoaffinity chromatography and gel filtration. Electrophoretic analysis revealed high-molecular-mass (approximately 150 kDa) molecules as well as the three bands characteristic of native factor VIIIa. Thus crosslinking appeared to yield molecules stabilized by intra- and/or inter-subunit crosslinks. The material was further fractionated using immobilized von Willebrand factor and the factor VIIIa activity could be ascribed to trimers containing only intra-subunit crosslinks. Moreover, reduction of crosslinked factor VIIIa produced using dithiobis(succinimidylpropionate) suggested that the molecules containing intersubunit crosslinks had not been cleaved by thrombin at arginine 372.
用交联剂辛二酸二琥珀酰亚胺酯处理因子VIII可增强并稳定因子VIIIa的活性。与天然因子VIIIa相比，其活性高出200倍以上，并且在4℃和pH 7.2条件下至少15天保持稳定。交联后的因子VIIIa通过免疫亲和层析和凝胶过滤进行纯化。电泳分析显示出高分子质量（约150 kDa）的分子以及天然因子VIIIa特有的三条条带。因此，交联似乎产生了通过亚基内和/或亚基间交联而稳定的分子。使用固定化血管性血友病因子对该材料进一步分级分离，因子VIIIa活性可归因于仅含有亚基内交联的三聚体。此外，用二硫代双（琥珀酰亚胺丙酸酯）还原交联的因子VIIIa表明，含有亚基间交联的分子未被凝血酶在精氨酸372处切割。
