Phospholipid regulates the activation of factor X by tissue factor/factor VIIa (TF/VIIa) via substrate and product interactions
Phospholipid regulates the activation of factor X by tissue factor/factor VIIa (TF/VIIa) via substrate and product interactions
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DOI：
10.1021/bi050338b
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发表时间：
2005-06-07
期刊：
影响因子：
2.9
通讯作者：
Nemerson, Y
中科院分区：
文献类型：
--
作者：
Hathcock, JJ;Rusinova, E;Nemerson, Y
尽管组织因子 (TF) 活性所需的磷脂已被明确确定，但表面调节酶活性的机制仍不清楚。我们将磷脂囊泡添加到已经重新脂质化的 TF (30/70 PS/PC) 中，发现添加的脂质既可以增强也可以抑制 X 因子 (F.X) 的激活速率。使用活性位点抑制的 F.Xa，我们证明 F.Xa 在较低的脂质浓度下是 TF/VIIa 的更有效的抑制剂，并且这种抑制可归因于 F.Xa 在酶附近的高表面占据。我们还发现，饱和时与脂质表面结合的 F.Xa 分子数量是 F.X 的两倍，并且 F.Xa 与脂质结合的二聚体模型可以解释实验观察到的 F.Xa（与 F.X 相比）与磷脂表面的优先结合。我们通过控制囊泡大小和每个囊泡的 TF 分子数量来控制每个 TF 分子可用的磷脂量，并发现，随着每个 TF 分子可用的磷脂的 2D 半径增加，观察到的 k(cat) 以双曲线方式增加到最大值或“真实 k(cat)”。在类似于 37 nm 的 2D 脂质半径处，观察到的 k(cat) 是“真实 k(cat)”的 50%。因此，磷脂表面充当 F.X 呈递和 F.Xa 去除的管道，并且 F.Xa 通过横向扩散或从表面解吸离开酶附近的速率，调节 F.X 激活的速率。我们认为这些发现需要重新评估现有的凝血模型。
Although the phospholipid requirement for tissue factor (TF) activity has been well-established, the mechanism by which the surface regulates enzymatic activity remains unclear. We added phospholipid vesicles to already relipidated TF (30/70 PS/PC) and found that added lipid can both enhance and inhibit the rate of factor X (F.X) activation. Using active-site-inhibited F.Xa we demonstrate that F.Xa is a more potent inhibitor of TF/VIIa at lower lipid concentrations, and that this inhibition is attributable to high surface occupancy by F.Xa near the enzyme. We also find that exactly twice as many F.Xa molecules are bound to a lipid surface at saturation as F.X, and that a dimer model of F.Xa binding to the lipid can account for the experimentally observed, preferential binding of F.Xa (compared to F.X) to phospholipid surfaces. We manipulated the amount of phospholipid available to each TF molecule by controlling vesicle size and the number of TF molecules per vesicle and found that, as the 2D radius of phospholipid available to each TF molecule was increased, the observed k(cat) increased hyperbolically toward a maximum or "true k(cat)". At a 2D lipid radius of similar to 37 nm, the observed k(cat) was 50% of the "true k(cat)". Thus, phospholipid surface serves as a conduit for F.X presentation and F.Xa removal, and the rate at which F.Xa leaves the vicinity of the enzyme, either by lateral diffusion or desorption from the surface, regulates the rate of F.X activation. We argue that these findings require reevaluation of existing models of coagulation.