Factor VIIa (FVIIa), complexed with tissue factor (TF), is a trigger of blood coagulation. Analog of recombinant FVIIa (rFVIIa), NN1731 (V158D/E296V/M298Q) possesses a greater hemostatic effect than rFVIIa and has been expected in clinical application. Factor X activation rate of NN1731 compared to rFVIIa was 1.2-fold in the presence of TF (TF(+)), and was 30-fold on activated platelets in its absence (TF(−))(

Arterioscler Thromb Vasc Biol.
). This TF-independent mechanism likely attributes to excellent effects by NN1731. More recently, we reported the physiological role of FVIIa/TF-dependent FVIII activation in the early phase of blood coagulation. Therefore, we were tempted to investigate the action of NN1731 in FVIII activation. Time-dependent change in FVIII activity after the addition of rFVIIa/NN1731 was examined by one-stage clotting assay under the presence of phospholipids (PS:PC:PE=1:6:3), CaCl2 and TF(+)/TF(−). NN1731 raised FVIII activity up to peak level rapidly within 30 sec (TF(+)), following by inactivation. Peak level of FVIII activity by NN1731 in TF(−) reached to the same peak level of that in TF(+) within 5 min, and this peak level persisted for ~30 min. Whilst, peak FVIII level by rFVIIa in TF(−) showed only ~35% of that in TF(+) even at 30 min. FVIII activating rate of NN1731 was observed to be 1.2-fold (TF(+)) and 3.8-fold (TF(−)) of rFVIIa-catalyzed activation. Kinetics by the Xa generation assay showed the Km values of NN1731 in FVIII activation were ~1.5-fold lower than those of rFVIIa (NN1731/rFVIIa; TF(+) 27.3/49.2 nM and TF(−) 50.5/68.1 nM). Vmax values of NN1731 in FVIII activation, however, showed the obvious difference between TF(+) (2.3-fold; NN1731/rFVIIa 70.0/30.4 nM•min−1) and TF(−) (7.9-fold; 92.5/11.7 nM•min−1), compared to rFVIIa. Inactivation of FVIIIa by NN1731 was somewhat faster than that by rFVIIa. FVIII cleavages by NN1731 were analyzed using SDS-PAGE/Western blotting. The heavy chain of FVIII was proteolyzed at Arg740 (A2-B junction), Arg372 (A1-A2 junction) and Arg336 (within the A1), faster by NN1731 than by rFVIIa. These predominant cleavages by NN1731 were more evident in TF(−). However, little cleavage of the light chain of FVIII was observed by both proteases. FVIII cleavages were correlated with the observations of FVIII activation and/or inactivation. To further localize the binding region for NN1731, we evaluated the interactions between FVIII and Glu-Gly-Arg-active site modified (EGR-) NN1731, lacking enzymatic activity, in a surface plasmon resonance-based assay. The Kd value of EGR-NN1731 with FVIII was similar to that of EGR-rFVIIa (6.3 and 7.8 nM, respectively). Binding was particularly evident with the A2, A3, and C2 domains, whilst the A1 domain failed to bind, similar to the results obtained by rFVIIa. We demonstrated that NN1731 possesses higher potential as an activator for up-regulation of FVIII activity than rFVIIa. Furthermore, catalytic activity of NN1731 in TF(−), rather than binding affinity, likely attributes to this potential of its analog. We concluded that the analog has another novel mechanism in its potent hemostatic effect through FVIII activation in TF-independent manner.

Disclosures: Soeda:Chugai Pharmaceutical CO., LTD.: Employment. Off Label Use: NN1731 which is recombinant factor VIIa analog, used for hemostasis.

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