Factor VIII mutated with Lys1813Ala within the factor IXa-binding region enhances intrinsic coagulation potential

Abstract

Factor VIII (FVIII) functions as a cofactor of FIXa for FX activation in the intrinsic tenase complex. The 1811-1818 region in the FVIII A3 domain was observed to contribute to FIXa binding, and the K1813A/K1818A mutant increased the binding affinity for FIXa. The current study aims to identify mutated FVIII protein(s) that increase FVIIIa cofactor activity in the 1811-1818 region. FVIII mutants with K1813A, K1818A, and K1813A/K1818A were expressed in baby hamster kidney cells and were followed by assessments using purified and global coagulation assays for mouse models with hemophilia A (HA). A surface plasmon resonance-based assay revealed that the Kd value of FVIII-K1813A for FIXa interaction was lower than that of the wild-type (WT) (3.9±0.7/6.3±0.3 nM). However, the Km value of FVIII-K1813A for FIXa on tenase activity was comparable with that of the WT, whereas the kcat of this mutant was significantly greater than that of the WT. Thrombin-catalyzed FVIII-K1813A activation was ∼1.3-fold more enhanced than that of the WT, and the spontaneous decay of activated FVIII-K1813A was ∼2.5-fold slower than that of WT. The heat stability assay revealed that the decay rate of FVIII-K1813A was ∼2.5-fold slower than that of WT. Thrombin generation assay and rotational thromboelastometry using blood samples from patients with HA demonstrated that the addition of FVIII-K1813A (0.5 nM) exhibited a coagulation potential compatible with that of WT (1 nM). In the tail clip assay of HA mice, FVIII-K1813A showed a two- to fourfold higher hemostatic potential than that of the WT. FVIII-K1813A, with higher a FIXa binding affinity, enhances the global coagulation potential because of the stability of FVIII/FVIIIa molecules.

Graphical abstract

Figure 1.

Effectsof FVIII K1813A, K1818A, and K1813A/K1818A mutants on the kinetics of FIXa-catalyzed FX activation. (A) FIXa association. FVIII WT or mutants (1 nM) were activated by thrombin (30 nM) for 30 seconds in the presence of PL vesicles (20 μM). FXa generation was initiated by the addition of FX (300 nM) and various concentrations of FIXa (0-40 nM). (B) FX association. The WT or mutant FVIII (1 nM) was activated by thrombin (30 nM) for 30 seconds in the presence of PL vesicles (20 μM). FXa generation was initiated by the addition of FIXa (40 nM) and various concentrations of FX (0-400 nM). WT, open circles; K1813A, closed circles; K1818A, open squares; and K1813A/K1818A, closed squares. Experiments were performed 3 separate times, and the average and standard deviation values are shown. The initial rates of FXa generation were plotted as a function of FIXa or FX concentration and fitted to Equation 1 using nonlinear least-squares regression.

Figure 2.

Thrombin-catalyzed activation of FVIII mutants of K1813A, K1818A, and K1813A/K1818A. FVIII WT or mutants (10 nM) were incubated with thrombin (0.4 nM) before measuring FVIII activity at the indicated times in a 1-stage clotting assay, as described in the Methods section. The symbols used are defined in Figure 1. The initial FVIII activities of WT, K1813A, K1818A, and K1813A/K1818A mutants were 7.8, 16.1, 14.3, and 15.1 IU/mL at t=0, respectively. FVIII activity was expressed as a fold of initial (t=0) and was plotted as a function of incubation time. Experiments were performed 3 separate times, and the average and standard deviation values are shown.

Figure 3.

Comparison with FVIIIa stability of WT and K1813A mutant. FVIII WT or mutant (1 nM) were incubated with thrombin (30 nM) before measuring FVIII activity at the indicated times in a 1-stage clotting assay. The symbols used are defined in Figure 1. FVIIIa activity was expressed as the fold change of the initial value and was plotted as a function of incubation time. The experiments were performed 3 times, and the average and standard deviation calculations are shown. The data were fitted to the formula given in Equation 2.

Figure 4.

FVIII stability of K1813A, K1818A, and K1813A/K1818A mutants. FVIII WT or mutant (0.5 nM) were incubated at 55°C before measuring FVIII activity at the indicated times in a 1-stage clotting assay. The symbols used are defined in Figure 1. FVIII activity was expressed as the fold change of the initial value and was plotted as a function of incubation time. The experiments were performed 3 times, and the average and standard deviation calculations are shown. The data were fitted to the formula given in Equation 2.

Figure 5.

Global coagulation function potentials on FVIII K1813A, K1818A, and K1813A/K1818A mutants. (A). FVIII WT (1 nM) or mutants (K1813A; 0.5 and 1 nM; K1818A, 1 nM; K1813A/K1818A, 1 nM) were added to commercial FVIII-deficient plasma. These samples were incubated with TF (1 pM) and PL vesicles (4 μM) before the addition of fluorogenic substrate and CaCl₂ at the start of the assay. Experiments were performed 3 separate times, and the mean values are shown thrombin generation curves are shown (black; WT 1 nM, red; K1813A 1 nM, tangerine; K1813A 0.5 nM, blue; K1818A 1 nM, green; K1813A/K1818A 1 nM, gray; buffer). (B) ROTEM. CaCl₂ was added to citrated whole blood sample (residual FVIII activity of 2.3 and <1 IU/dL) in 2 patients with severe HA, together with various concentrations of FVIII mutants (WT and K1813A), followed by a ROTEM assay. The raw blood data in 2 patients and the representative curves in 1 patient are shown. The average parameters (CT and CT+CFT) in 2 patients are shown below each figure. The CT and CT+CFT obtained from normal controls (n=20) were 938±128 seconds and 1273±217 seconds, respectively. CT, clot time; CFT, clot formation time.

Figure 6.

In vivo hemostatic effect of FVIII K1813A in HA mice by a tail clip assay. HA mice were infused with PBS, 1 and 2 μg/kg FVIII K1813A, 2 μg/kg K1818A, 2 μg/kg K1813A/K1818A, or 2 and 4 μg/kg WT. Five minutes after infusion, the terminal 5 mm of the tail was amputated, and shed blood was collected for 40 minutes. Each point represents a mouse. The short horizontal lines in each graph indicate the average values. A one-way ANOVA with Dunnett multiple comparison test was used to determine significance relative to HA mice PBS controls. A Student t test was used to compare between each group. ANOVA, analysis of variance; NS, not significant; PBS, phosphate buffered saline.