title: Factor VIII activation by factor VIIa analog (V158D/E296V/M298Q) in tissue factor-independent mechanisms.
abstract: 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.
authors: Department of Pediatrics, Nara Medical University, Kashihara, Nara 634-8522, Japan.
authors: Kenichi
authors: K
authors: Ogiwara
authors: Keiji
authors: K
authors: Nogami
authors: Midori
authors: M
authors: Shima
mesh_terms: D001777:Blood Coagulation / Q000502:physiology; D002384:Catalysis; D020134:Catalytic Domain / Q000235:genetics; D057930:Click Chemistry; D005167:Factor VII / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005169:Factor VIII / Q000737:chemistry / Q000378:metabolism*; D015942:Factor VIIa / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006487:Hemostasis; D006801:Humans; D015202:Protein Engineering; D059748:Proteolysis; D013917:Thrombin / Q000235:genetics; D013925:Thromboplastin / Q000378:metabolism
chemical_list: C519901:NN1731; D005167:Factor VII; D005169:Factor VIII; D013925:Thromboplastin; D015942:Factor VIIa; D013917:Thrombin