Platelet binding and activity of a factor VIIa variant with enhanced tissue factor independent activity
Platelet binding and activity of a factor VIIa variant with enhanced tissue factor independent activity
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DOI：
10.1111/j.1538-7836.2011.04223.x
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发表时间：
2011-04-01
影响因子：
10.4
通讯作者：
Monroe, D. M.
中科院分区：
文献类型：
--
作者：
Hoffman, M.;Volovyk, Z.;Monroe, D. M.
背景和目标：血小板结合和活性在凝血因子VIIa（FVIIa）作为血友病旁路治疗剂的疗效中发挥重要作用。通过在蛋白酶结构域中引入三个氨基酸变化，产生了具有增加的组织因子（TF）非依赖性活性的FVIIa类似物NN 1731。即使在不存在TF的情况下，NN 1731的构象也与TF结合FVIIa相似。这导致更大的内在蛋白水解活性，但在TF存在下具有相似的活性。目的：我们假设这些变化不会改变与血小板或磷脂的结合，这是一种被认为定位于Gla结构域的特征。当前工作的目的是比较NN 1731和野生型FVIIa的血小板结合和活性。方法/结果：FVIIa和NN 1731与磷脂囊泡的结合相同，通过活性测定和电泳准弹性光散射技术进行评估。然而，通过流式细胞术评估，NN 1731与活化血小板上的结合位点数量多于FVIIa。去除Gla结构域消除了FVIIa和NN 1731的结合。活性位点的抑制并未将NN 1731结合降低至FVIIa水平。当校正蛋白结合量时，NN 1731在血小板表面的活性大于FVIIa。结论：虽然Gla结构域对于FVIIa与血小板结合至关重要，但NN 1731中蛋白酶结构域的变化可增强血小板结合以及蛋白水解活性。除脂质组成外，其他特征似乎有助于rFVIIa（尤其是NN 1731）与血小板的结合。
Background and objectives: Platelet binding and activity play important roles in the efficacy of factor VIIa (FVIIa) as a bypassing agent for hemophilia treatment. An analog of FVIIa with increased tissue factor (TF)-independent activity, NN1731, has been produced by introducing three amino acid changes in the protease domain. NN1731 has a conformation similar to TF-bound FVIIa, even in the absence of TF. This results in much greater intrinsic proteolytic activity, but similar activity in the presence of TF. Objectives: We hypothesized that these changes would not alter binding to platelets or phospholipid, a characteristic thought to be localized to the Gla domain. The goal of the current work was to compare platelet binding and activity of NN1731 and wild-type FVIIa. Methods/Results: FVIIa and NN1731 bound identically to phospholipid vesicles as assessed by both activity assays and electrophoretic quasielastic light scattering techniques. However, NN1731 bound to a greater number of sites on activated platelets than FVIIa, as assessed by flow cytometry. Removal of the Gla domain abolished binding of both FVIIa and NN1731. Inhibition of the active site did not reduce NN1731 binding to the level of FVIIa. When corrected for the amount of protein bound, NN1731 had greater activity than FVIIa on platelet surfaces. Conclusions: While the Gla domain is essential for FVIIa binding to platelets, changes in the protease domain in NN1731 enhanced platelet binding as well as proteolytic activity. Features in addition to lipid composition appear to contribute to binding of rFVIIa and, especially, NN1731 to platelets.