Department of Pediatrics, Nara Medical University, Kashihara, Nara 634-8522, Japan.
Thromb Haemost. 2011 Oct;106(4):665-74. doi: 10.1160/TH11-04-0264. Epub 2011 Sep 8.
Factor (F)VIIa with tissue factor (TF) is a primary trigger of blood coagulation. The recombinant (r)FVIIa analog, NN1731 (V158D/E296V/ M298Q) containing a thrombin/FIXa-mimicking catalytic domain, is ~30-fold more effective on activated platelets without TF, but ~1.2-fold with TF, than rFVIIa for FX activation. We have recently demonstrated the FVIIa/TF-dependent FVIII activation in the early coagulation phase. We assessed the action of NN1731 on FVIII activation. NN1731/TF increased FVIII activity ~2.9-fold within 30 seconds, followed by rapid inactivation, and was slightly more active than rFVIIa/TF. NN1731-catalysed activation, however, was enhanced ~6-fold at 5 minutes (min), and its peak level persisted for ~30 min. NN1731/TF proteolysed FVIII at Arg⁷⁴⁰, Arg³⁷², and Arg³³⁶, similar to rFVIIa/TF, but cleavage by NN1731 alone was much slower at Arg³³⁶ than at Arg⁷⁴⁰ and Arg³⁷². The Km and Vmax for NN1731/TF-catalysed activation were ~1.8-fold lower and ~2.3-fold greater than rFVIIa/TF. The Km for NN1731 alone was ~1.3-fold lower than rFVIIa, whilst the Vmax was ~7.9-fold greater, indicating that the efficiency of FVIII activation by NN1731 and NN1731/TF was ~11- and ~4-fold greater, respectively, than equivalent reactions with rFVIIa. In SPR-based assays, NN1731 bound to FVIII and the heavy chain (Kd; 0.62 and 1.9 nM) with ~1.4- and ~3.1-fold higher affinity than rFVIIa, and the A2 domain contributed to this increase. Von Willebrand factor moderated NN1731-catalysed activation more significantly than NN1731/TF. In conclusion, NN1731 was a greater potential than rFVIIa in up-regulating FVIII activity, and the TF-independent FVIII activation might represent a potential extra mode of its enhanced haemostatic effect.
因子（F）VIIa 与组织因子（TF）是血液凝固的主要触发因素。重组（r）FVIIa 类似物 NN1731（V158D/E296V/M298Q）含有一个模拟凝血酶/FIXa 的催化结构域，在没有 TF 的情况下，对激活的血小板的作用比 rFVIIa 高约 30 倍，但在有 TF 的情况下，作用比 rFVIIa 高约 1.2 倍，用于 FX 激活。我们最近证明了在早期凝血阶段 FVIIa/TF 依赖性 FVIII 激活。我们评估了 NN1731 对 FVIII 激活的作用。NN1731/TF 在 30 秒内将 FVIII 活性提高约 2.9 倍，随后迅速失活，比 rFVIIa/TF 略活跃。然而，NN1731 催化的激活在 5 分钟（min）时增强了约 6 倍，其峰值水平持续约 30 分钟。NN1731/TF 在 Arg⁷⁴⁰、Arg³⁷² 和 Arg³³⁶处切割 FVIII，与 rFVIIa/TF 相似，但 NN1731 单独切割 Arg³³⁶的速度比 Arg⁷⁴⁰ 和 Arg³⁷² 慢得多。NN1731/TF 催化的激活的 Km 和 Vmax 比 rFVIIa/TF 低约 1.8 倍，高约 2.3 倍。NN1731 单独的 Km 比 rFVIIa 低约 1.3 倍，而 Vmax 高约 7.9 倍，表明 NN1731 和 NN1731/TF 对 FVIII 激活的效率分别比等效的 rFVIIa 反应高约 11 倍和 4 倍。在基于 SPR 的测定中，NN1731 与 FVIII 和重链（Kd；0.62 和 1.9 nM）的结合亲和力分别比 rFVIIa 高约 1.4 倍和 3.1 倍，A2 结构域对此增加有贡献。von Willebrand 因子对 NN1731 催化的激活的调节作用比 NN1731/TF 更为显著。总之，NN1731 比 rFVIIa 更有可能上调 FVIII 活性，TF 非依赖性的 FVIII 激活可能代表其增强止血作用的潜在额外模式。
