Generation of active coagulation factor VIII from isolated subunits.
Generation of active coagulation factor VIII from isolated subunits.
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从分离的亚基中生成活性凝血因子 VIII。
DOI：
10.1016/s0021-9258(19)57273-8
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发表时间：
1988
期刊：
影响因子：
--
通讯作者：
M. Ezban
中科院分区：
文献类型：
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作者：
O. Nordfang;M. Ezban
因子VIII-轻链（FVIII-LC）和因子VIII-重链（FVIII-HC）分别从人血浆中分离，无凝血活性。当FVIII-LC和FVIII-HC制剂混合时，在Mn 2+、Ca 2+或Co 2+存在下产生凝血活性。Mn ~（2+）最有效，Ca ~（2+）在8天后首次获得最大活性。当FVIII-LC/HC与Ca 2+孵育20小时时，牛因子X（FX）加速重组，并能够将产生的FVIII-C的量增加至10倍。当在微摩尔浓度的巯基试剂存在下进行重组时，产生的FVIII：C的总量增加至4倍，并且超过FVIII-HC时，可以将70%的FVIII-LC驱动为活性复合物。如果在巯基试剂存在下制备FVIII-HC，则有可能将75%的FVIII-HC与过量的FVIII-LC驱动成活性复合物。重组所必需的Me 2+催化二硫键形成，并且提出FVIII亚基具有游离巯基。在FVIII-HC纯化过程中和重组过程中巯基试剂的存在使单个亚基保持适合重组的构象。
Factor VIII-light chain (FVIII-LC) and Factor VIII-heavy chain (FVIII-HC) were isolated separately from human plasma and were without coagulation activity. When FVIII-LC and FVIII-HC preparations were mixed, coagulation activity was generated in the presence of Mn2+, Ca2+, or Co2+. Mn2+ was most effective and with Ca2+ maximal activity was first obtained after 8 days. Bovine Factor X (FX) accelerated recombination and was able to increase the amount of FVIII:C generated up to 10-fold when FVIII-LC/HC were incubated with Ca2+ for 20 h. When recombination was performed in the presence of micromolar concentrations of sulfhydryl reagents, the total amount of FVIII:C generated was increased up to 4-fold and in excess of FVIII-HC it was possible to drive 70% of FVIII-LC into active complex. If FVIII-HC was prepared in the presence of a sulfhydryl reagent it was possible to drive 75% of FVIII-HC into active complex with FVIII-LC in excess. Me2+, which is necessary for recombination, catalyzes disulfide formation, and it is proposed that FVIII subunits have free sulfhydryl groups. The presence of sulfhydryl reagents during purification of FVIII-HC and during recombination retains the individual subunits in a conformation suitable for recombination.