Improved hemostasis in hemophilia mice by means of an engineered factor Va mutant

Abstract

Background: Factor (F)VIIa-based bypassing not always provides sufficient hemostasis in hemophilia.

Objectives: To investigate the potential of engineered activated factor V (FVa) variants as bypassing agents in hemophilia A.

Methods: Activity of FVa variants was studied in vitro using prothrombinase assays with purified components and in FV- and FVIII-deficient plasma using clotting and thrombin generation assays. In vivo bleed reduction after the tail clip was studied in hemophilia A mice.

Results and conclusions: FVa mutations included a disulfide bond connecting the A2 and A3 domains and ones that rendered FVa resistant to inactivation by activated protein C (APC). '(super) FVa,' a combination of the A2-A3 disulfide (A2-SS-A3) to stabilize FVa and of APC-cleavage site mutations (Arg506/306/679Gln), had enhanced specific activity and complete APC resistance compared with wild-type FVa, FVL eiden (Arg506Gln), or FVaL eiden (A2-SS-A3). Furthermore, (super) FVa potently increased thrombin generation in vitro in FVIII-deficient plasma. In vivo, (super) FVa reduced bleeding in FVIII-deficient mice more effectively than wild-type FVa. Low-dose (super) FVa, but not wild-type FVa, decreased early blood loss during the first 10 min by more than two-fold compared with saline and provided bleed protection for the majority of mice, similar to treatments with FVIII. During the second 10 min after tail cut, (super) FVa at high dose, but not wild-type FVa, effectively reduced bleeding. These findings suggest that (super) FVa enhances not only clot formation but also clot stabilization. Thus, (super) FVa efficiently improved hemostasis in hemophilia in vitro and in vivo and may have potential therapeutic benefits as a novel bypassing agent in hemophilia.

Keywords: animal experimentation; bleeding; factor V; hemophilia; hemostasis.

Fig. 1

Specific activity and cofactor activity of wild-type activated factor V (FVa) and FVa mutants. Concentrations of FVa-mutants were determined by ELISA, and cofactor activities were determined either in purified prothrombinase assays or in FV-deficient plasma. (A) Specific activity of FVa mutants (28 pmol L⁻¹) determined in purified prothrombinase assay and expressed as IIase/nmol L⁻¹ FVa species (n = 5–9). (B) Specific activity of FVa mutants (0.5 nmol L⁻¹) determined in thrombin generation assays in FV-deficient plasma. A representative example is shown. (C) Endogenous thrombin potential of FVa mutants (0.5 nmol L⁻¹) in FV-deficient plasma was calculated as area under the curve (AUC) and expressed as fold-increase of endogenous thrombin potential (ETP) over baseline (ETP FVa = 1) (n = 4). Error bars represent SEM (*all P ≤ 0.02).

Fig. 2

Correction of clotting times of factor V (FV)-deficient plasma by ^superFVa. (A) Activated partial thromboplastin time (APTT) and (B) prothrombin time (PT) clotting times were determined in FV-deficient plasma after reconstitution with FVa or ^superFVa (0.5 nmol L⁻¹). Normal plasma served as control (n = 4). Error bars represent SEM (*all P < 0.0001).

Fig. 3

Activated protein C (APC) resistance of ^super FVa. Western blot analysis with an anti-FV heavy chain polyclonal antibody of (A) FV and (B) ^superFV (both 4 nmol L⁻¹) in the absence and presence of thrombin and/or APC (2 nmol L⁻¹). (C) Time course of FVa cofactor activity in purified prothrombinase assays of A2-SS-A3 disulfide-linked FVa-mutants (4 nmol L⁻¹) in the presence of APC (2 nmol L⁻¹). Conditions for the prothrombinase assay were FVa (28 pmol L⁻¹), FXa (1.42 nmol L⁻¹), and prothrombin (0.42 µmol L⁻¹) (n = 4–5). Error bars represent SEM (all P ≤ 0.05). APC resistance of ^superFVa in (D) FV-deficient plasma and (E) FVIII-deficient plasma. Thrombin generation was determined in FV-deficient plasma reconstituted with FVa or ^superFVa both at 1 nmol L⁻¹, and in FVIII-deficient plasma at 10 nmol L⁻¹, respectively, in the presence of increasing concentrations of APC. Endogenous thrombin potential (ETP) was expressed relative to the absence of APC (n = 3). Error bars represent SEM.

Fig. 4

Improved thrombin generation in factor VIII (FVIII)-deficient plasma by ^superFVa. Thrombin generation in normal plasma (NP) and FVIII-deficient plasma supplemented with FVa, ^superFVa (both 30 nmol L⁻¹), or FVIII (1 U mL⁻¹). (A) Representative example of thrombin generation curves of FVIII-deficient plasma supplemented with FVa or ^superFVa compared with FVIII-deficient plasma reconstituted with FVIII and NP. (B) Relative fold-change of thrombin generation in FVIII-deficient plasma, expressed as endogenous thrombin potential (ETP FVa = 1), in the presence of FVa mutants and compared with reconstitution with FVIII and NP (n = 3–4). Error bars represent SEM (*P ≤ 0.03).

Fig. 5

Correction of initial bleeding by ^superFVa. Factor VIII (FVIII)-deficient mice were injected intravenously with FVIII, saline, or either FVa or ^superFVa dosed at equal activity as determined in prothrombinase assays (1 unit = activity of 20 nmol L⁻¹ wild-type FVa). For wild-type FVa, 2 units = 0.8 mg kg⁻¹ and 5 units = 2 mg kg⁻¹. Based on increased specific activity of ^superFVa, 2 units = 0.27 mg kg⁻¹ and 5 units = 0.67 mg kg⁻¹. Bleeding during the first 10 min after the tail clip is expressed as blood loss in microliter blood per gram mouse. Error bars represent SEM.

Fig. 6

Correction of late rebleeding by ^superFVa but not wild-type FVa. Wild-type (wt) mice were injected intravenously with saline, and FVIII-deficient mice with FVIII, saline, or either FVa or ^super-FVa dosed at equal activity (5 units), respectively (1 unit = activity of 20 nmol L⁻¹ wild-type FVa in the prothrombinase assay). For wild-type FVa, 5 units = 2 mg kg⁻¹. Based on increased specific activity of ^superFVa, 5 units = 0.67 mg kg⁻¹. Bleeding during the (A) second 10 min (10–20 min after the tail clip) or (B) during the combined first and second 10 min (0–20 min after the tail clip) is expressed as blood loss in microliter blood per gram mouse. Error bars represent SEM.