Laboratory of Hemostasis-Inflammation-Thrombosis, Institut National de la Santé et de la Recherche Médicale, UMR_S 1176, Univ. Paris-Sud, Université Paris-Saclay, Le Kremlin-Bicêtre, France.
Inovarion, Paris, France.
J Thromb Haemost. 2019 Dec;17(12):2035-2046. doi: 10.1111/jth.14615. Epub 2019 Sep 3.
Activated factor VII (FVIIa) is pertinent to the initiation of blood coagulation. Proteolytic and amidolytic activity of FVIIa are greatly enhanced by its cofactor, tissue factor (TF).
We aimed to generate a single-domain antibody (sdAb) that recognizes free FVIIa rather than TF-bound FVIIa.
A llama-derived phage library was used to screen for anti-FVIIa sdAbs.
One sdAb, KB-FVIIa-004, bound to FVIIa, but not to its precursor FVII or to homologous proteins (prothrombin, factor X, or their activated derivatives). FVIIa amidolytic activity was inhibited by KB-FVIIa-004 (K = 28-45 nM) in a competitive manner. KB-FVIIa-004 also inhibited FVIIa-mediated FX activation (K = 26 nM). In contrast, KB-FVIIa-004 was inefficient in prolonging the clotting time of the prothrombin time-test, which was prolonged by a maximum of 10 s at high sdAb concentrations (10 μM). Furthermore, FVIIa/TF amidolytic activity or FVIIa/TF-mediated FX activation remained unaffected up to a 50-fold to 1000-fold molar excess of KB-FVIIa-004. These data suggest that KB-FVIIa-004 loses its inhibitory activity in the presence of TF. A KB-FVIIa-004/albumin fusion-protein (004-HSA) was generated for in vivo testing. By using 004-HSA, we observed that this sdAb blocked the therapeutic capacity of FVIIa to correct bleeding in FVIII-deficient mice.
This observation is compatible with the view that FVIIa functions independently of TF under these conditions. In conclusion, we have generated a sdAb that specifically blocks TF-independent activity of FVIIa. This antibody can be used to gain insight into the roles of TF-bound and TF-free FVIIa.
激活的因子 VII（FVIIa）与血液凝固的启动有关。FVIIa 的蛋白水解和氨肽酶活性通过其辅因子组织因子（TF）大大增强。
我们旨在产生一种单域抗体（sdAb），该抗体识别游离的 FVIIa，而不是与 TF 结合的 FVIIa。
使用骆驼衍生的噬菌体文库筛选抗 FVIIa 的 sdAbs。
一种 sdAb，KB-FVIIa-004，与 FVIIa 结合，但不与它的前体 FVII 或同源蛋白（凝血酶原、因子 X 或它们的激活衍生物）结合。KB-FVIIa-004 以竞争性方式抑制 FVIIa 的氨肽酶活性（K i = 28-45 nM）。KB-FVIIa-004 还抑制 FVIIa 介导的 FX 激活（K i = 26 nM）。相比之下，在高 sdAb 浓度（10 μM）下，KB-FVIIa-004 最多只能延长凝血酶原时间试验的凝血时间 10 秒。此外，FVIIa/TF 氨肽酶活性或 FVIIa/TF 介导的 FX 激活在 KB-FVIIa-004 的 50 倍至 1000 倍摩尔过量下仍不受影响。这些数据表明，KB-FVIIa-004 在 TF 存在下失去其抑制活性。生成了 KB-FVIIa-004/白蛋白融合蛋白（004-HSA）用于体内测试。通过使用 004-HSA，我们观察到该 sdAb 阻断了 FVIIa 纠正 FVIII 缺乏小鼠出血的治疗能力。
这一观察结果与以下情况下 FVIIa 在没有 TF 的情况下独立发挥作用的观点是一致的。总之，我们已经产生了一种特异性阻断 FVIIa 的 TF 非依赖性活性的 sdAb。该抗体可用于深入了解 TF 结合和 TF 游离 FVIIa 的作用。
