The effect of carbohydrate depletion on procoagulant activity and in vivo survival of highly purified human factor VIII.
The effect of carbohydrate depletion on procoagulant activity and in vivo survival of highly purified human factor VIII.
复制标题
碳水化合物消耗对高纯度人因子 VIII 的促凝血活性和体内存活的影响。
DOI：
10.1016/0304-4165(84)90054-0
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发表时间：
1984
期刊：
影响因子：
--
通讯作者：
Marder,VJ
中科院分区：
文献类型：
--
作者：
Fay,PJ;Chavin,SI;Malone,JE;Schroeder,D;Young,FE;Marder,VJ
使用我们先前描述的方法的修改来纯化人因子VIII促凝血蛋白（因子VIII），其中Sephacryl S-400洗脱，而不是QAE-纤维素层析，作为最终纯化步骤。SDS-聚丙烯酰胺凝胶电泳分析表明，该蛋白的比活力大于2500 U/mg，为单一多肽（Mr 100 000）。通过电泳后用过碘酸-希夫试剂染色，证明因子VIII是一种糖蛋白。治疗因子VIII的混合物的外切和内切glycoproteinases引起的减少约50%的高碘酸-希夫染色的强度，通过扫描光密度测定，和电泳迁移率的增加（相当于一个newMr 95 000）。去除这部分总碳水化合物对凝血因子VIII凝血活性或凝血酶增强凝血活性没有显著影响。天然和糖耗尽125 I标记的因子VIII的体内存活曲线均显示出相似的模式，初始快速衰减到60和40%的活动，分别由一个半衰减时间为4小时。这些结果表明，人因子VII的碳水化合物部分并没有显着的贡献，无论是在体外凝血功能或在体内的生物周转。
Human factor VIII procoagulant protein (factor VIII) was purified using a modification of our previously described method, in which Sephacryl S-400 elution, rather than QAE-cellulose chromatography, served as the final purification step. The protein had a specific activity of more than 2500 U/mg and consisted of a single polypeptide (Mr100 000) when analyzed by SDS-polyacrylamide gel electrophoresis. Factor VIII was shown to be a glycoprotein by staining with periodic acid-Schiff's reagent following electrophoresis. Treatment of factor VIII with a mixture of exo- and endoglycosidases caused a reduction by about 50% in the intensity of periodic acid-Schiff staining, as determined by scanning densitometry, and an increase in electrophoretic mobility (equivalent to a newMr95 000). Removal of this portion of the total carbohydrate had no significant effect on factor VIII clotting activity or on thrombin potentiation of clotting activity. The in vivo survival curves of native and sugar-depleted125I-labeled factor VIII both showed similar patterns of initial rapid decay to 60 and 40% activity, respectively, followed by a one-half decay time of 4 h for both. These results suggest that the carbohydrate portion of human factor VII does not contribute significantly to either clotting function in vitro or to biological turnover in vivo.