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首页> 外文期刊>Biochemistry >Substitution of asparagine for arginine 347 of recombinant factor Xa markedly reduces factor Va binding.
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Substitution of asparagine for arginine 347 of recombinant factor Xa markedly reduces factor Va binding.

机译：用天冬酰胺替代重组因子Xa的精氨酸347可显着降低因子Va的结合。

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摘要

Herein we describe a recombinant factor X (fX) with a single substitution at position 347 (fXR347N). Activated fXR347N had a reduced affinity for factor Va (fVa), although the catalytic impact of fVa binding remained intact. The mutation was selective as demonstrated by normal activation and inhibition, except in the presence of subsaturating heparin where the rate of inhibition by antithrombin III (ATIII) was 15% of normal. The reactivity of fXaR347N toward prothrombin was equivalent to wild-type fXa (fXaWT) in the absence of fVa and phospholipid. Addition (without phospholipid) of fVa dramatically increased the catalytic efficiency of fXaWT toward prothrombin but had a negligible effect on fXaR347N. On addition of phosphatidylcholine:phosphatidylserine (PC:PS, 3:1) vesicles, fXaR347Ndisplayed an increased catalytic activity in response to fVa, but the apparent affinity for fVa on the phospholipid surface was 5-20-fold lower than that of fXaWT. On an activated platelet surface, however, fXaWT and fXaR347N activated prothrombin similarly. In a competitive binding assay that measures the displacement of radiolabeled fXa from fVa on a phospholipid surface, fXaR347N was approximately 10-fold less effective than fXaWT. Substitution of fXa at position 347 selectively attenuates the interaction between fXa and fVa without affecting its catalytic activity.
机译：在本文中，我们描述了在位置347（fXR347N）具有单个取代的重组因子X（fX）。活化的fXR347N对因子Va（fVa）的亲和力降低，尽管fVa结合的催化作用仍保持不变。如正常激活和抑制所示，该突变是选择性的，除了存在亚饱和肝素的情况下，抗凝血酶III（ATIII）的抑制率为正常值的15％。在不存在fVa和磷脂的情况下，fXaR347N对凝血酶原的反应性与野生型fXa（fXaWT）相当。 fVa的添加（不含磷脂）显着提高了fXaWT对凝血酶原的催化效率，但对fXaR347N的影响可忽略不计。在添加磷脂酰胆碱：磷脂酰丝氨酸（PC：PS，3：1）囊泡时，fXaR347N表现出对fVa的催化活性增强，但对磷脂表面上fVa的表观亲和力比fXaWT低5-20倍。然而，在活化的血小板表面上，fXaWT和fXaR347N同样活化了凝血酶原。在一种竞争性结合试验中，该试验测量了放射性标记的fXa从磷脂表面上的fVa的置换中，fXaR347N的效力比fXaWT低约10倍。 fXa在位置347处的取代选择性地减弱了fXa和fVa之间的相互作用，而不影响其催化活性。

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