Cell membranes have important functions in many steps of the blood coagulation cascade, including the activation of factor X (FX) by the factor VIIa (FVIIa)-tissue factor (TF) complex (extrinsic Xase). FVIIa shares structural similarity with factor IXa (FIXa) and FXa. FIXa and FXa are regulated by binding to phosphatidylserine (PS)-containing membranes via their γ-carboxy-glutamic acid (Gla) and epidermal growth-factor (EGF) domains. Although FVIIa also has a Gla-rich region, its affinity for PS-containing membranes is much lower compared to that of FIXa and FXa. Research suggests that a more common endothelial cell lipid, phosphatidylethanolamine (PE), might augment the contribution of PS in FVIIa membrane-binding and proteolytic activity. We used soluble forms of PS and PE (C6PS, C6PE) to test the hypothesis that the two lipids bind to FVIIa jointly to promote FVIIa membrane binding and proteolytic activity. By equilibrium dialysis and tryptophan fluorescence, we found two sites on FVIIa that bound equally to C6PE and C6PS with Kd of ~ 150 -160 μM, however, deletion of Gla domain reduced the binding affinity. Binding of lipids occurred with greater affinity (Kd ~ 70-80 μM) when monitored by FVIIa proteolytic activity. Global fitting of all data sets indicated independent binding of two molecules of each lipid. The proteolytic activity of FVIIa increased by ~50-100-fold in the presence of soluble Tissue Factor (sTF) plus C6PS/C6PE. However, the proteolytic activity of Gla-deleted FVIIa in the presence of sTF was reduced drastically, suggesting the importance of Gla domain to maintain full proteolytic activity.

中文翻译：

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磷脂酰丝氨酸和磷脂酰乙醇胺调节 FVIIa 的结构和功能及其与可溶性组织因子的相互作用。

细胞膜在凝血级联的许多步骤中具有重要功能，包括因子 VIIa (FVIIa)-组织因子 (TF) 复合物（外源性 Xase）激活因子 X (FX)。FVIIa 与因子 IXa (FIXa) 和 FXa 具有结构相似性。FIXa 和 FXa 通过其 γ-羧基-谷氨酸 (Gla) 和表皮生长因子 (EGF) 结构域与含磷脂酰丝氨酸 (PS) 的膜结合来调节。虽然 FVIIa 也有一个富含 Gla 的区域，但与 FIXa 和 FXa 相比，它对含 PS 膜的亲和力要低得多。研究表明，更常见的内皮细胞脂质，磷脂酰乙醇胺 (PE)，可能会增加 PS 在 FVIIa 膜结合和蛋白水解活性中的贡献。我们使用可溶性形式的 PS 和 PE（C6PS，C6PE) 来检验两种脂质共同结合 FVIIa 以促进 FVIIa 膜结合和蛋白水解活性的假设。通过平衡透析和色氨酸荧光，我们发现 FVIIa 上的两个位点与 C6PE 和 C6PS 同等结合，Kd 约为 150 -160 μM，然而，Gla 结构域的缺失降低了结合亲和力。当通过 FVIIa 蛋白水解活性监测时，脂质的结合以更高的亲和力 (Kd ~ 70-80 μM) 发生。所有数据集的整体拟合表明每种脂质的两个分子的独立结合。在可溶性组织因子 (sTF) 和 C6PS/C6PE 存在下，FVIIa 的蛋白水解活性增加了约 50-100 倍。然而，在 sTF 存在下，Gla 缺失的 FVIIa 的蛋白水解活性急剧降低，表明 Gla 结构域对维持完整蛋白水解活性的重要性。