Persson E, Nielsen L S
Vessel Wall Biology, Health Care Discovery, Novo Nordisk A/S, Gentofte, Denmark.
Blood Coagul Fibrinolysis. 1998 Mar;9 Suppl 1:S79-81.
Activated factor VII (FVIIa) needs to be bound to tissue factor (TF) to express biological activity, and biochemical and structural data show that the first epidermal growth factor (EGF)-like domain of FVIIa contributes essential contacts with TF. To investigate the role of Ca2+ binding to this domain in FVIIa we used site-directed mutagenesis to replace Asp46 and Asp63 by Asn, which abolished Ca2+ binding, and characterized the double mutant (D46,63N-FVIIa) with respect to its TF-binding properties and the functional status of its complex with TF. D46,63N-FVIIa had a lower amidolytic activity than FVIIa at optimal Ca2+ concentrations. A slightly lower amidolytic activity was also observed in complex with soluble TF, apparently due to a lower catalytic turnover rate of D46,63N-FVIIa. However, D46,63N-FVIIa and FVIIa bound to lipidated TF were equally efficient activators of factor X. The dissociation constant for the interaction between D46,63N-FVIIa and soluble TF, derived from amidolytic activity and direct binding measurements, was approximately 20-fold higher than that for the interaction between FVIIa and soluble TF. The same difference was observed in the affinity for lipidated TF. These findings suggest that a functional Ca2+-binding site in the first EGF-like domain adds approximately 7 kJ/mol to the total binding energy of the interaction with both lipidated and soluble TF.
活化的凝血因子 VII（FVIIa）需要与组织因子（TF）结合才能表达生物学活性，生化和结构数据表明，FVIIa 的第一个表皮生长因子（EGF）样结构域与 TF 形成了关键的相互作用。为了研究 Ca2+ 与 FVIIa 中该结构域结合的作用，我们使用定点诱变将 Asp46 和 Asp63 替换为 Asn，这消除了 Ca2+ 的结合，并就其与 TF 的结合特性及其与 TF 复合物的功能状态对双突变体（D46,63N-FVIIa）进行了表征。在最佳 Ca2+ 浓度下，D46,63N-FVIIa 的酰胺水解活性低于 FVIIa。在与可溶性 TF 形成的复合物中也观察到酰胺水解活性略低，这显然是由于 D46,63N-FVIIa 的催化周转速率较低。然而，与脂化 TF 结合的 D46,63N-FVIIa 和 FVIIa 是同等有效的因子 X 激活剂。根据酰胺水解活性和直接结合测量得出的 D46,63N-FVIIa 与可溶性 TF 之间相互作用的解离常数比 FVIIa 与可溶性 TF 之间相互作用的解离常数高约 20 倍。在对脂化 TF 的亲和力方面也观察到同样的差异。这些发现表明，第一个 EGF 样结构域中的功能性 Ca2+ 结合位点使与脂化和可溶性 TF 相互作用的总结合能增加了约 7 kJ/mol。
