Abstract

The hemostatic effect of bypassing agents such as recombinant (r) factor (F)VIIa and activated prothrombin complex concentrates (aPCC) for hemophilia A with inhibitors (HA-inh) is not always stable (Berntope, Haemophilia 2009). The mechanism(s) of its instability remain unclear, however. We have recently reported the HA-inh case showing the attenuated responsiveness to aPCC (Ogiwara, Int J Hematol. 2014). Some groups reported the hemostatic effects of the complex concentrates of FVIIa and FX (Shirahata, Haemophilia 2012) in HA-inh, suggesting that FX would play the key role in the hemostatic effect by FVIIa. ACE910, a humanized bispecific antibody to FIXa and FX mimicking the functions of FVIIIa, exerting FXase activities without FVIII(a) (Kitazawa, Nature Medicine 2012). In this study, we attempted to elucidate the dependency on FX of the FVIIa- and/or ACE910-driven coagulation. Firstly, the global hemostatic potentials in the whole blood samples obtained from the four HA-inh cases (Case 1, 2, 3 and 4) under perioperative hemostatic treatment with the intermittent administration of rFVIIa every 2-3hr were evaluated by Ca2+-triggered viscoelastometric assay with ROTEM. The first infusion of rFVIIa shortened CT (from 5,087 ± 1,261 to 1,157 ± 208 sec) and increased MCF (from 17 ± 8.7 to 58.8 ± 1.3 mm) in each case. Additional rFVIIa after the 7th administration in Case 1, the 13th in Case 2 and the 12th in Case 3 little affected CT and MCF as well as clinical symptom, indicative of poor responsiveness, while Case 4 showed the improvement of the parameters even after the frequent infusion of rFVIIa, identified as a responsive case. Thrombin generation (TG) triggered by TF (1pM) or TF (1pM) together with ellagic acid (0.3μM) was evaluated in the plasma from the cases with poor response. Peak thrombin (PeakTh) was little changed between pre- and post-additional infusion of rFVIIa in the cases with poor response, similar to the pattern of ROTEM. The level of FX antigen measured by an ELISA in the plasma was 90.5 ± 9.6 nM, showing 67% of normal control (~140 nM), of little difference among the four cases at the first administration of rFVIIa, while that in Case 1, 2 or 3 at the 7th, 13th or 12th administration, respectively, decreased to 39.1 ± 7.0 nM, equivalent to ~45% of that (86.8 ± 12.9 nM) kept in the responsive Case 4. Addition of FX (300nM) in the plasma of poor response to rFVIIa ex vivo increased PeakTh to ~80% of normal control, suggesting that FVIIa-driven hemostatic effect would be dependent upon FX. Furthermore, to investigate the FX-dependency of FVIIa- and ACE910-driven coagulation, TG in the reconstituted HA-inh model plasmas consisting of FX-deficient plasma in which FVIII was inactivated by an anti-FVIII polyclonal antibody (10BU/ml) with/without rFVIIa (50 and 150 nM) or ACE910 (10, 30 and 60 μg/ml) was evaluated in the presence of various concentrations of FX (f.c. 0 - 300 nM). The control experiment without rFVIIa or ACE910 showed the FX dose-dependent increase of PeakTh. In the plasmas with FX ranged from 50 to 300nM, PeakTh improved to almost normal level by rFVIIa as well as ACE910. Of note, with the lower concentration of FX (10-20 nM), PeakTh improved to almost normal level in the presence of ACE910, increased by 38 ± 2.4%, 45 ± 1.7% and 48 ± 0.8% compared to those in its absence, respectively, in an ACE910 dose-dependent manner, whilst the presence of rFVIIa little affected TG compared to those in its absence. Taken together, ACE910 could exert its hemostatic effect with the lower amount of FX than that required for the rFVIIa-driven coagulation.

Disclosures

Yada:Chugai Pharmaceutical Co., ltd: Research Funding. Nogami:Bayer, NovoNordisk, Baxalta, Chugai, Kaketsuken, Pfizer, Biogen: Honoraria; Bayer, Novo Nordisk, Baxalta. Biogen: Research Funding; Chugai: Membership on an entity's Board of Directors or advisory committees. Kitazawa:Chugai Pharmaceutical Co., Ltd: Employment, Equity Ownership, Patents & Royalties. Hattori:Chugai Pharmaceutical Co., Ltd: Employment, Equity Ownership, Patents & Royalties. Shima:Biogen: Honoraria, Research Funding; Bayer: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Chugai Pharmaceutical Co., Ltd: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Baxalta: Honoraria, Research Funding; Novo Nordisk: Honoraria, Research Funding; Kaketsuken: Honoraria.

Author notes

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Asterisk with author names denotes non-ASH members.