The role of proteolytic cleavage at Arg³³⁶ and Arg³⁷² of the A1 domain in factor VIIa/tissue factor-catalyzed reactions of B domain-deleted factor VIII

Background: We previously demonstrated that factor (F)VIII was rapidly activated through proteolytic cleavage at Arg(372) and Arg(740) by activated FVII (FVIIa)/tissue factor (TF) in very early coagulation phase, followed by inactivation by cleavage at Arg(336). The influence of the absence of FVIII B domain in this series of FVIIa/TF-catalyzed reaction remains unclear, however.Aim: To examine the FVIIa/TF-catalyzed reaction of B domain-deleted (BDD-)FVIII.Methods and results: The FVIII activity (FVIII:C) of commercial full-length (FL-)FVIII and BDD-FVIII increased by similar to 1.7-fold within 0.5 min after addition of FVIIa/TF (1 nM/0.1 nM).Fviii: C decreased to initial levels with inactivation rate constant (k; similar to 0.035) within 15 min of FL-FVIII activation, but decreased gradually to initial levels (k; similar to 0.017) within 30 min of BDD-FVIII activation. SDS-PAGE analyses demonstrated that the FVIIa/TF-catalyzed cleavage of BDD-FVIII occurred at Arg(336) within 0.5 min in parallel with elevation of FVIII:C, but cleavage at Arg(372) was not evident. FVIIa/TF-catalyzed activation of both recombinant BDD-FVIII R336A and R372A mutants that were prepared, were similar to that of wild-type (WT) BDD-FVIII. However, FVIII:C returned to initial levels (k; similar to 0.046) within 30 min of R336A mutant activation, but little reduction of FVIII:C was observed with R372A mutant (k; similar to 0.0046). SDS-PAGE analysis indicated that FVIIa/TF-catalyzed cleavage of WT and R372A mutant was predominant at Arg(336), whereas that of R336A mutant was observed at Arg(372).Conclusions: FVIIa/TF-catalyzed activation of BDD-FVIII was initiated by cleavage at Arg(336), and the FVIII B domain appeared to control FVIIa/TF-catalyzed reactions by altering pattern of cleavage at Arg(336) and Arg(372).