Effects of thrombin treatment of preparations of factor VIII and the Ca2+-dissociated small active fragment.
Effects of thrombin treatment of preparations of factor VIII and the Ca2+-dissociated small active fragment.
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凝血酶处理因子 VIII 和 Ca2-解离小活性片段制剂的效果。
DOI：
10.1172/jci108146
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发表时间：
1975
期刊：
影响因子：
--
通讯作者：
R. Wagner
中科院分区：
文献类型：
--
作者：
H. Cooper;F. Reisner;M. Hall;R. Wagner
当人、犬或牛的因子VIII制剂在离子强度为0.2的4%琼脂糖上进行色谱时，因子VIII活性在空隙体积中以单峰形式洗脱，并有轻微的尾迹。这种制剂与稀释的（0.01 U/ml）高纯度凝血酶孵育可导致因子VIII的一定活化。在与之前相同的条件下，对这种孵育混合物进行色谱分析，可以洗脱出两个因子VIII活性峰，一个在空隙体积中，另一个在空隙体积中较晚的地方，有明显的尾迹。空隙体积峰具有大部分蛋白质和部分因子VIII活性。这些空体积分数也包含所有的血管性血友病因子活性的凝血酶处理的牛制剂。较长的凝血酶治疗，或较强的凝血酶治疗，似乎将更多的促凝活性转移到较晚的洗脱峰。此外，当凝血酶处理后的空隙体积中发现因子VIII活性的峰值时，再次用稀释的凝血酶孵育，因子VIII活性增加。该培养混合物的色谱分析表明，在空隙体积中只有少量活性，而大部分活性存在于第二个峰。另一方面，凝血酶处理后，因子VIII活性从2峰开始迅速下降，而不是进一步增加。我们提出，在空隙体积中发现的残留因子VIII活性代表未反应的因子VIII，而后期洗脱峰代表凝血酶活化的物质，其表观尺寸较小。后期洗脱峰与Ca2+解离获得的小活性因子VIII片段不同，后者可以被凝血酶激活。在蔗糖密度梯度上，用牛因子VIII制剂进行了一组类似的实验。实验结果与色谱法完全一致。由人血友病血浆制备的制剂，采用纯化人因子VIII的方法，也用凝血酶孵育并进行色谱分析。冯氏血液病因子仅存在于空穴体积分数中，而在洗脱的所有分数中均未发现因子VIII活性。在其他对照实验中，活化和未活化的因子VIII组分不凝血纤维蛋白原，也不含可测定的因子IX或x。凝血酶修饰的小尺寸因子VIII被天然存在的人因子VIII抑制剂和兔抗血清中针对钙解离的小活性因子VIII片段产生的γ球蛋白组分灭活。
When human, canine, or bovine factor VIII preparations are chromatographed on 4% agarose at ionic strength 0.2, the factor VIII activity elutes as a single peak in the void volume with slight tailing. Incubation of such preparations with dilute (0.01 U/ml) highly purified thrombin results in some activation of factor VIII. Chromatography of such incubation mixtures, under the same conditions as before, results in elution of two peaks of factor VIII activity one in the void volume and one much later with marked tailing. The void volume peak has most of the protein and some factor VIII activity. These void volume fractions also contain all the von Willebrand factor activity of thrombin-treated bovine preparations. Longer treatment with thrombin, or treatment with stronger thrombin, appears to shift much more of the procoagulant activity to the later eluting peak. Also, when the peak of factor VIII activity, found in the void volume after thrombin treatment, was again incubated with dilute thrombin, an increase in factor VIII activity occurred. Chromatography of this incubation mixture demonstrated only a small amount of activity in the void volume, while the bulk of the activity was present in the second peak. On the other hand, thrombin treatment of factor VIII activity from peak 2 caused a rapid decline of activity instead of a further increase. It is proposed that the residual factor VIII activity found in the void volume represents unreacted factor VIII, while the late eluting peak represents thrombin-activated material that is of smaller apparent size. The late eluting peak differs from the small active factor VIII fragment obtained by Ca2+ dissociation, as the latter can be activated by thrombin. A similar set of experiments was performed using ultracentifugation of bovine factor VIII preparations on sucrose density gradients. Results of these experiments agreed completely with those obtained with get chromatography. Preparations made from human hemophilic plasma, by the procudure employed in the purification of human factor VIII, were also incubated with thrombin and chromatographed. von Willebrand factor was again found only in the void volume fractions, but there was no factor VIII activity in any fractions eluted. In other control experiments, activated and unactivated factor VIII fractions did not clot fibrinogen and contained no assayable factor IX or X. The thrombin-modified factor VIII of small size was inactivated by both a naturally occurring human inhibitor to factor VIII and the gamma globulin fraction of a rabbit antisera produced against the calcium-dissociated small active factor VIII fragment.