Factor VIIa (FVIIa), complexed with tissue factor (TF), is a trigger of blood coagulation through activation of factor X in the initiation phase. FVIIa can catalyze intrinsic clotting factors such as not only factor IX, but also factor VIII (FVIII). However the role and the mechanisms of the FVIIa-catalyzed FVIII are poorly understood. We first examined FVIIa-catalyzed FVIII activation in the presence of phospholipid (PL) using a one-stage clotting assay. The levels of FVIII activity elevated rapidly by ~4-fold within 30 sec after the addition of FVIIa (1 nM)-TF (1 nM)complex, and subsequently decreased to the initial level within 20 min. This time-dependent reaction was enhanced by the presence of TF and PL in dose-dependent manners, but was moderately inhibited (~50%) in the presence of von Willebrand factor at physiological concentration of 10 μg/mL. FVIII cleavage was evaluated using western blotting immediately after the addition of FVIIa-TF complex. The heavy chain of FVIII was proteolyzed more rapidly (at 15 sec) by cleavages at Arg740 (A2-B junction) and Arg372 (A1-A2 junction) by FVIIa-TF complex, whilst the cleavage at Arg336 in the A1 domain was appeared at ~2.5 min. However little cleavage of the light chain of FVIII was observed, supporting that cleavages at Arg740/Arg372 and Arg336 by FVIIa-TF complex contribute to the up- and down-regulation of FVIII(a) activity, respectively. Of interest, no proteolysis of isolated intact heavy chain was observed, indicating that the proteolysis of the heavy chain was governed by the presence of the light chain. Compared to FVIII activation by thrombin (0.1–1 nM), the activation by FVIIa (0.1–1 nM)-TF (1 nM) complex was observed more rapidly. The activation rate observed by the addition of FVIIa-TF complex was ~50-fold greater than that by thrombin. Kinetics by the chromogenic Xa generation assay showed the catalytic efficiency (kcat/Km; 8.9 min−1/32.8 nM) on FVIIa-TF complex-catalyzed FVIII activation showed ~4-fold greater than that on thrombin-catalyzed activation (kcat/Km; 7.5 min−1/86.4 nM). Furthermore, the catalytic efficiencies on cleavages at Arg740 and Arg372 of FVIII by FVIIa-TF complex were ~3- and ~20-fold greater compared to those by thrombin, respectively. These findings suggested that FVIIa-TF complex was a greater FVIII activator than thrombin in very early phase. In order to localize the binding region for FVIIa, we evaluated the interactions between FVIII subunit and Glu-Gly-Arg-active site modified FVIIa, lacking enzymatic activity, in a surface plasmon resonance-based assay. The heavy chain of FVIII bound to EGR-FVIIa with higher affinity than the light chain (Kd; 2.1 and 45 nM, respectively). Binding was particularly evident with the A2, A3, and C2 domains (Kd; 34, 37, and 44 nM, respectively), whilst the A1 domain failed to bind. In conclusion, we demonstrated that FVIIa-TF complex rapidly activated FVIII by proteolysis of the heavy chain and the activation was governed by the presence of the light chain. Furthermore, present results suggested the role of TF-dependent FVIII activation by FVIIa which is responsible for the initiation phase of blood coagulation.
Disclosures: Soeda: Chugai pharmaceutical Co., LTD.: Employment.