Pharmacological concentrations of recombinant factor VIIa restore hemostasis independent of tissue factor in antibody-induced hemophilia mice

Abstract

ESSENTIALS: The role of tissue factor (TF) in recombinant factor VIIa (rFVIIa) therapy in hemophilia is unclear. An acquired mouse hemophilia model with very low or normal levels of human TF was used in the study. rFVIIa is equally effective in correcting the bleeding in mice expressing low or normal levels of TF. Pharmacological doses of rFVIIa restore hemostasis in hemophilia independent of TF.

Background: Recombinant factor VIIa (rFVIIa) has been used widely for treating hemophilia patients with inhibitory autoantibodies against factor VIII or IX. Its mechanism of action is not entirely known. A majority of in vitro studies suggested that pharmacological concentrations of rFVIIa restore hemostasis in hemophilia in a phospholipid-dependent manner, independent of tissue factor (TF). However, a few studies suggested that a TF-dependent mechanism has a primary role in correction of bleeding by rFVIIa in hemophilia patients. Here, we investigated the potential contribution of TF in rFVIIa-induced hemostasis in hemophilia employing a model system of FVIII antibody-induced hemophilia in TF transgenic mice.

Methods: Mice expressing low levels of human TF (LTF mice), mice expressing relatively high levels of human TF (HTF mice) and wild-type mice (WT mice) had neutralizing anti-FVIII antibodies administered in order to induce hemophilia in these mice. The mice were then treated with varying concentrations of rFVIIa. rFVIIa-induced hemostasis was evaluated with the saphenous vein bleeding model.

Results: Administration of FVIII inhibitory antibodies induced the hemophilic bleeding phenotype in all three genotypes. rFVIIa administration rescued the bleeding phenotype in all three genotypes. No significant differences were observed in rFVIIa-induced correction of bleeding between LTF and HTF mice that had FVIII antibodies administered.

Conclusions: Our results provide strong evidence supporting the suggestion that the hemostatic effect of pharmacological doses of rFVIIa stems from a TF-independent mechanism.

Keywords: factor VIIa; hemophilia A, acquired; hemorrhage; hemostasis; thromboplastin.

Fig. 1

Antibody-induced hemophilia in mice and effect of rFVIIa in restoring hemostasis following the saphenous vein incision. Wild-type mice (C57BL) were administered with saline or FVIII mAb (1 mg/kg) intravenously via the tail vein. After 2 h, mice administered with FVIII mAb were treated with varying concentrations of rFVIIa intravenously. Five minutes after rFVIIa administration, mice were subjected to saphenous vein incision. Average time to achieve hemostasis (A) and blood loss (B) was determined as described in methods (n = 5). For comparison, data obtained with hemophilia A (FVIII^−/−) was also shown. All p values were calculated using the nonparametric Mann-Whitney test comparing the group receiving rFVIIa with the group receiving saline. In the group receiving saline, one of the mice had an 1800 sec hemostasis time and 4 mice with an average of about 450 seconds. Exclusion of 1800 sec data point from the statistical analysis did not alter the overall outcome of statistical significance analysis between the groups but the significance values are slightly altered (recalculated p values for Fig. 1A: the group receiving saline vs. the group receiving 0.25 mg/kg rFVIIa, 0.19; : the group receiving saline vs. the group receiving 1 mg/kg rFVIIa, 0.015; : the group receiving saline vs. the group receiving 4 mg/kg rFVIIa, 0.015; recalculated p values for Fig. 1B: the group receiving saline vs. the group receiving 0.25 mg/kg rFVIIa, 0.73; the group receiving saline vs. the group receiving 1 mg/kg rFVIIa, 0.015; : the group receiving saline vs. the group receiving 4 mg/kg rFVIIa, 0.015).

Fig. 2

Comparison of bleeding times and blood loss following the saphenous vein incision in wild-type, HTF, LTF, LTF heterozygote littermate controls, and LTF mice administered with TF Ab, FVIII mAb or both. Wild-type, HTF, LTF and LTF littermate controls were subjected to saphenous vein incision and an average time to achieve hemostasis (A) and blood loss (B) were measured as described in methods. In additional groups, LTF mice were injected with rabbit polyclonal antibodies against human TF, TF mAb (5G9), FVIII mAb (GMA 8015) or both TF mAb and FVIII mAb (1 mg/kg, each) intravenously 2 h before the saphenous vein incision (n = 3 to 9). *, p < 0.05; **, p <0.01; ns, not statistically significant (p > 0.05).

Fig. 3

Factor VIIa restores hemostasis in Ab-induced hemophilia independent of TF. LTF and HTF mice were administered intravenously with FVIII mAb (1 mg/kg) to induce hemophilia. LTF mice were also administered with TF mAb (1 mg/kg) along with FVIII mAb. After 2 h, 0.25, 1 or 4 mg/kg rFVIIa was given to mice intravenously and the bleeding was initiated by saphenous vein incision. Average time to achieve hemostasis (A) and blood loss (B) were determined (n = 5 to 9). ns, not statistically significant difference.