We have reported that factor (F)VIIa/tissue factor (TF) rapidly activated FVIII by proteolysis of the heavy chain (HCh), and that appeared to serve physiologically as an activator for up-regulation of FVIII activity in very early-timed coagulation phase (J Thromb Haemost. 2010;8:2494). Furthermore, the activation phase could be observed even in the presence of anti-FVIII inhibitors, independently of their types of kinetic and epitope of inhibitors, whilst the inactivation was moderated by anti-C2 with type 1 behavior (Thromb Haemost. 2011;105:989). More recently, the other group has reported that the combination of FVIII and bypassing agent, APCC, also potentiated the thrombin generation in hemophilia A plasmas with inhibitors (Klintman et al. Br J Haematol. 2010;151:381), but the mechanisms have remained unknown. In this study, we investigated the hemostatic effect of the combination therapy with APCC and FVIII. We first examined FVIII activation catalyzed by APCC in one-stage clotting assay. The activity level of FVIII (10 nM) elevated rapidly by ∼3-fold within 1 min after addition of APCC (0.05 U/mL)/TF (0.5 nM), and subsequently decreased to the initial level within 10 min. However, the addition of APCC without TF little affected FVIII activity within 10 min, but after then gradually elevated its activity. The presence of E-76, FVIIa-specific inhibitor, significantly moderated the reaction triggered by APCC/TF, but hirudin, FIIa-specific inhibitor, little affected this activation. We further evaluated the velocity of APCC-induced thrombin generation in the presence or absence of FVIII. The increase rate of thrombin production triggered by APCC/TF in the presence of FVIII was greater than that by APCC/TF in the absence of FVIII by ∼1.6-fold, whilst the increase was little observed in the absence of TF. To clarify its enhancing effects, we performed APCC-catalyzed FVIII cleavage in SDS-PAGE and Western blot. APCC contains FVII (mainly active form) and FII, FIX and FX (mainly non-active forms). In general, FVIIa/TF, FIIa, and FXa rapidly proteolyze the HCh at Arg372 (and Arg740), and FVIIa/TF and FXa proteolyze at Arg336. FVIII proteolysis by FVIIa/TF is dependent on the presence of PL, but not FIIa. Interestingly, APCC/TF proteolyzed the HCh at Arg372 and Arg740, followed by at Arg336 in the presence of PL, whilst did not proteolyze in its absence. The presence of TF accelerated the proteolysis by ∼6.6-fold compared to the absence of TF. However, the addition of E-76 significantly delayed these cleavages. These findings supported that APCC as well as rFVIIa possessed a potential to activation/inactivation of FVIII in early-timed coagulation phase, and that FVIIa in APCC appeared to play a major role in APCC-catalyzed FVIII activation/inactivation. Furthermore, to confirm this enhancing effect in the presence of anti-FVIII inhibitors, we prepared the anti-FVIII inhibitor IgGs (3 of anti-A2, 4 of anti-C2 with type 1, and 2 of anti-C2 with type 2). The presence of anti-FVIII inhibitors did not significantly affect the APCC-catalyzed FVIII activation (by ∼3-fold), independently of their epitopes, in one-stage clotting assay. Of surprise, anti-C2 with type 1 significantly moderated APCC-catalyzed FVIIIa inactivation, and the peak level of FVIIIa retained over 30 min. In contrast, the other inhibitors little affected this inactivation, similar to FVIIa/TF-catalyzed FVIII reaction. In conclusion, we demonstrated the putative mechanism of enhancing hemostatic effects in the combination therapy using FVIII and APCC. In addition, only a small amount of APCC relative to the standard dosage (1–2 U/mL) for clinical use could activate FVIII even in the presence of anti-FVIII inhibitors, and this combination therapy would provide new therapeutic strategy in congenital hemophilia A with inhibitor and/or acquired hemophilia A.
Yada:Baxter Hemophilia Scientific Research and Education Fund 2011: Research Funding. Nogami:Bayer Award 2009: Research Funding.
Asterisk with author names denotes non-ASH members.