FVIIa as used pharmacologically is not TF dependent in hemophilia B mice

Abstract

Activated factor VII is approved for treating hemophilia patients with autoantibodies to their factor IX or FVIII; however, its mechanism of action remains controversial. Some studies suggest that FVIIa requires tissue factor (TF) for function and that the reason for the high dose requirement is that it must compete with endogenous FVII for tissue factor. Others suggest that FVIIa binds platelets where it activates FX directly; the high concentration required would result from FVIIa's weak affinity for phospholipids. We address this question by infusing a chimera of mouse FIX (Gla and EGF1) with FVIIa (EGF2 and catalytic domain) into hemophilia B mice. This mutant has no TF-dependent activity because it cannot functionally bind TF at physiologically relevant concentrations. In vivo, this mutant is as effective as mouse FVIIa in controlling bleeding in hemophilia B mice. Our results suggest that the hemostatic effect of pharmacologic doses of FVIIa is TF independent.

Figure 1

Characterization of mFIX_gla-egf1FVIIa and hFIX_gla-egf1FVIIa. (A) Relative coagulant activity of mFIXgla-egf1FVIIa compared with recombinant mFVIIa in an APTT assay; 25 µL of mFVIIa or mFIXgla-egf1FVIIa (5 µg/mL) was added to 25 µL FIX -deficient human plasma and 25 µL APTT reagent, and the assay initiated with 25 µL CaCl. (B) Relative activity of mFIXgla-egf1FVIIa compared with recombinant mFVIIa in a PT assay; 25 µL of either mFVIIa or mFIXgla-egf1FVIIa (0.5 µg/mL) was added to 25 µL FVII-deficient plasma, and 100 µL Innovin was added to initiate the reaction. The Innovin concentration was determined by titration with human FVIIa. Data were derived from at least 3 experimental points. Data are shown as average ± 1 standard deviation (SD). (C) Functional binding of 5000 ng/mL mFVIIa and mFIXgla-egf1FVIIa to Innovin (human TF), measured by FXa generation and detected with an FXa chromogenic substrate. Absorbance was measured after 15 minutes. Backgrounds were subtracted. (D) Relative functional binding of 5000 ng/mL mFIXgla-egf1FVIIa compared with mFVIIa to mouse TF–positive microparticles. Substrate (S-2288, 2 mM final concentration) was added to the reaction and the absorbance at 405 nm was determined after 15 minutes. Backgrounds were subtracted. The Mann-Whitney U test was used for all statistical analysis. ND, not detectable; OD, optical density.

Figure 2

Effect of FIX_gla-egf1FVIIa chimera on saphenous vein bleeding in the hemophilia B mouse. Wild-type (control group) or hemophilia B (experimental group) mice were subjected to saphenous vein incision. After initial clot formation, the clot was disrupted repeatedly for 30 minutes and the time for each clot to form was measured; the average time to clot formation was calculated for each mouse. Ten minutes before incision, mFVIIa or mFIXgla-egf1FVIIa was injected into Hemophilia B mice. The hemophilic mice bled continuously during the entire 1800-second duration of the experiment; WT mice have an ATTH of 44 seconds. Each point represents the average of multiple bleeding times in each mouse during the 30-minute observation time. For 2 mg/kg, median ATTH was 37 seconds (n = 4) for mFVIIa-treated hemophilia B mice and 47 seconds for mFIXgla-egf1FVIIa-treated mice (n = 6). The difference in ATTH was not statistically significant (P = .24); both are significantly shorter than untreated hemophilia B mice (P < .05). For 1 mg/kg, mFVIIa-treated hemophilia B mice (n = 5) clotted in an average of 108 seconds; the ATTH was 97 seconds for mFIXgla-egf1FVIIa-treated hemophilia B mice (n = 6) (P = .75). At a dose of 0.25 mg/kg, the median ATTH for mFVIIa-treated mice (n = 5) was 86 seconds, whereas for mFIXgla-egf1FVIIa–treated mice (n = 5) it was 111 seconds (P = .83). All P values were calculated using the nonparametric Mann-Whitney U test. HB, hemophilia B.