A Novel Mechanism of Factor VIIa/Tissue Factor (TF)-Catalyzed Activation and Inactivation of B-Domain-Deleted Factor VIII in the Early Initiation Phases of Coagulation

We have reported that factor (F)VIII was rapidly activated by FVIIa/tissue factor (TF) in vitro by limited proteolysis of the heavy chain (HCh) at Arg³⁷² and Arg⁷⁴⁰ in the very early-timed coagulation phase and inactivated by proteolysis at Arg³³⁶ (JTH 2010). Furthermore, the activation could be observed even in the presence of anti-FVIII inhibitors irrespective of their type of kinetics and the epitope recognized, whilst the inactivation was moderated by anti-C2 inhibitor with type 1 kinetics (Thromb Haemost 2011). A role of FVIII B-domain on FVIIa/TF-catalyzed activation and inactivation remain unknown, however. In this study, focusing on the roles of the B-domain of FVIII, we investigated the mechanism(s) of FVIIa/TF-catalyzed FVIIIa activation and inactivation by utilizing B-domain deleted (BDD)-FVIII as well as full-length (FL)-FVIII. We firstly examined FVIIa/TF-catalyzed activation and inactivation of FL- or BDD-FVIII(a) by a one-stage clotting assay. The FVIII activity (FVIII:C) of FL-FVIII (10nM) rapidly increased by ~1.7-fold within 0.5 min after addition of FVIIa (1nM)/TF (0.1nM), and subsequently decreased to the initial levels within 15 min (k = ~0.03). Interestingly, FVIII:C of BDD-FVIII (10nM), which increased up to ~1.7-fold of the initial level within 0.5 min after addition of FVIIa (1nM)/TF (0.1nM) similar to that of FL-FVIII, demonstrated a slower reduction to the initial level within 30 min (k = ~0.015) than that of FL-FVIII. In order to explore these inhibitory mechanisms of FVIIa/TF-catalyzed inactivation of BDD-FVIIIa, we investigated FVIIa/TF-catalyzed proteolytic cleavage of both BDD-FVIII and FL-FVIII by using SDS-PAGE. A rapid proteolysis in the heavy chain (Hch) of FL-FVIII within 0.5 min after addition of FVIIa/TF was observed by the cleavage at Arg⁷⁴⁰, followed by the cleavage at Arg³⁷² and the subsequent cleavage at Arg³³⁶, consistent with our previous study. In contrast, it was of surprise that the proteolysis in the Hch of BDD-FVIII by cleavage at Arg³⁷² was little observed after addition of FVIIa/TF, whilst that by the cleavage at Arg³³⁶ was observed within 0.5 min preceding the elevation of FVIII:C. The initial velocity of Arg³³⁶ cleavage at 0.5 min for BDD-FVIII (4.4/min) was ~3.3-times higher than that for FL-FVIII (1.4/min) by a densitometry. To the next, we examined the spontaneous dissociation of A2-domain from FVIIa/TF-catalyzed FL- or BDD-FVIIIa by a one stage clotting assay. In the presence of excess amount of A2-subunit (400nM), more than 50% of FVIIa/TF-catalyzed FVIIIa inactivation was inhibited compared to that in its absence, but no significant difference was observed between FL- and BDD-FVIII, suggesting that the spontaneous dissociation of A2-domain little affected the inhibition of the FVIIa/TF-catalyzed inactivation of BDD-FVIIIa. To further clarify the mechanism of FVIIa/TF-catalyzed BDD-FVIII activation/inactivation, we prepared and stably expressed recombinant BDD-FVIII mutants, R336A and R372A. FVIIa(1nM)/TF(0.1nM)-catalyzed activation and inactivation of R336A and R372A (10nM) was examined by a one stage clotting assay. FVIII:C of R336A and R372A rapidly increased by ~2.0-fold of the initial level within 0.5 min after addition of FVIIa/TF, similarly to that of wild type BDD-FVIII, and that of R336A subsequently decreased to the initial level within 30min (k = ~0.04), whilst little reduction of FVIII:C was observed for R372A (k = ~0.004). Evaluated by SDS-PAGE, FVIIa/TF-catalyzed proteolytic cleavage at Arg³³⁶ was predominantly observed for R372A within 0.5min after addition of FVIIa/TF, whilst cleavage at Arg³⁷² was conversely observed for R336A. Taken together, FVIIa/TF-catalyzed activation of BDD-FVIII could be predominantly initiated by the cleavage at Arg³³⁶ or secondarily at Arg³⁷² and resistance to the cleavage at Arg³⁷² would hamper the subsequent inactivation. In conclusion, the B-domain of FVIII would regulate the FVIIa/TF-catalyzed activation and inactivation of FVIII by controlling the order of proteolytic cleavage at Arg³³⁶ and Arg³⁷². We believe that our findings should also contribute to the development of more effective combination therapy of FVIIa and BDD-FVIII for hemophilia A with inhibitor.