Engineering of a membrane-triggered activity switch in coagulation factor VIIa

Abstract

Recombinant factor VIIa (FVIIa) variants with increased activity offer the promise to improve the treatment of bleeding episodes in patients with inhibitor-complicated hemophilia. Here, an approach was adopted to enhance the activity of FVIIa by selectively optimizing substrate turnover at the membrane surface. Under physiological conditions, endogenous FVIIa engages its cell-localized cofactor tissue factor (TF), which stimulates activity through membrane-dependent substrate recognition and allosteric effects. To exploit these properties of TF, a covalent complex between FVIIa and the soluble ectodomain of TF (sTF) was engineered by introduction of a nonperturbing cystine bridge (FVIIa Q64C-sTF G109C) in the interface. Upon coexpression, FVIIa Q64C and sTF G109C spontaneously assembled into a covalent complex with functional properties similar to the noncovalent wild-type complex. Additional introduction of a FVIIa-M306D mutation to uncouple the sTF-mediated allosteric stimulation of FVIIa provided a final complex with FVIIa-like activity in solution, while exhibiting a two to three orders-of-magnitude increase in activity relative to FVIIa upon exposure to a procoagulant membrane. In a mouse model of hemophilia A, the complex normalized hemostasis upon vascular injury at a dose of 0.3 nmol/kg compared with 300 nmol/kg for FVIIa.

Keywords: coagulation; disulfide engineering; factor VIIa; serine proteases; tissue factor.

Fig. 1.

Design and expression of disulfide-linked FVIIa–sTF complexes. (A) Crystal structure of the FVIIa+sTF complex (3) with the position of the five residue pairs targeted for cysteine replacement marked by spheres and interconnecting dashed lines. Labels refer to the mutation introduced in FVIIa and sTF, respectively. Calcium ions (yellow) and the active site inhibitor (light blue) are shown. (B) Each variant pair was transiently coexpressed in CHO cells and the culture supernatant analyzed 48 h posttransfection. Immediately after harvest, the culture supernatants were treated with 10 mM EDTA and 5 mM N-ethylmaleimide to dissociate any noncovalent complexes and block free thiols. Disulfide-linked complexes were detected by IP of the culture supernatant with anti-TF antibody (αTF-5G9) (51) followed by nonreducing SDS/PAGE and immunodetection of FVII with a polyclonal antibody (αFVII). (C) Immunostaining for FVII and sTF in the culture supernatant of a BHK cell line stably expressing 7TF-64 at 48 h after medium exchange. (D) Detection of complex formation in culture supernatants (S) and cell extracts (C) of 7TF-64–expressing BHK cells cultured for 24 h in the presence of vitamin K₃ or warfarin (25 μM) to promote or prevent γ-carboxylation, respectively. (Upper) Detection of FVII with αFVII after IP with αTF-5G9. (Lower) Detection with monoclonal antibody against the Ca²⁺-complexed Gla-domain [αFVII-Gla (30)] after acetone precipitation. The amount loaded in lanes 3–5 were four times that of lane 2. (E) Reducing and nonreducing SDS/PAGE analysis of purified 7TF-40, 7TF-64, and 7TF-275 complexes followed by Coomassie staining. Where indicated, FVIIa or HEK293F-derived sTF were included for comparison.

Fig. 2.

Activation state of FVIIa in covalent complex with sTF. (A) Residual amidolytic activity of FVIIa and FVIIa–sTF complexes following exposure to 0.2 M KOCN for 2 h to assess the extent of I153 burial. (B) Residual amidolytic activity relative to antibody-free sample of FVIIa (○), FVIIa+sTF (⋄), 7TF-40 (■), 7TF-64 (●), 7TF-275 (□), or 7TF-64z (∆) following incubation with increasing concentrations of the zymogen conformation-specific inhibitory antibody F3-3.2a (10). (C) Catalytic constants (k_cat/K_M) for the activation of des-Gla FX (1.3 mM calcium) and FX (10 mM calcium) in solution. Results are shown as mean ± SD (n = 3).

Fig. 3.

Potencies of FVIIa–sTF complexes in human and murine hemophilia A whole blood. (A) Effect of of FVIIa (○), 7TF-64 (●), or 7TF-64z (∆) on clotting times (mean ± SD, n = 3) in recalcified human whole blood depleted for FVIII by addition of a neutralizing antibody. From the concentration-response profiles, EC₅₀-values of 0.40 ± 0.33 nM, 0.10 ± 0.03 pM, and 4.4 ± 1.3 pM, respectively, were estimated. (B) Effect of 5 µM FVII Gla-domain–specific antibody (αFVII-Gla) (34) and an isotype control antibody (αCtrl) on clotting times in normal or FVIII-deficient whole blood from one representative donor. (C) The effect of 5 µM αFVII-Gla or αCtrl antibody on EC₅₀-values based on clotting time for 7TF-64 in FVIII deficient whole blood (mean ± SD, n = 3). (D) Effect of FVIIa (○), 7TF-64 (●), or 7TF-64z (∆) on clotting times in recalcified murine hemophilia A whole blood (mean ± SD, n = 4). EC₅₀-values of 5.4 ± 2.6 nM, 5.9 ± 2.3 pM, and 32 ± 13 pM, respectively, were estimated from the concentration-response profiles.

Fig. 4.

Effect of FVIIa and FVIIa–sTF complexes on tail bleeding in hemophilia A mice. Fully anesthetized hemophilia A mice were administered the indicated amounts of FVIIa, 7TF-64, or 7TF-64z at 5 min before tail-tip transection. Blood loss was determined from the amount of hemoglobin (µmol) collected in the following 30 min. Vehicle-treated hemophilia A and normal mice (wild-type) were included for comparison. Results are shown as mean ± SEM (n = 5 or greater). Asterisks indicate samples for which the blood loss was significantly different (P < 0.05) from vehicle treated hemophilia A controls as determined by one-way ANOVA followed by a Bonferroni test for significance.