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
Authors: Nakajima Yuto , Yada Koji , Nogami Keiji , Shima Midori 
Published: October 2019

Abstract
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.

Disclosures
Yada: Shire Japan Co., Ltd.: Other: Teacher at a endowed course. Nogami:Chugai Pharmaceutical Co., Ltd: Consultancy, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties: Anti-FIXa/X bispecific antibodies , Research Funding, Speakers Bureau. Shima:Chugai Pharmaceutical Co., Ltd: Consultancy, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties: Anti-FIXa/X bispecific antibodies , Research Funding, Speakers Bureau; F. Hoffmann-La Roche Ltd: Membership on an entity's Board of Directors or advisory committees.