Pharmacological Concentrations of Recombinant Factor VIIa Restores Hemostasis in Acquired Hemophilia Primarily Independent of Tissue Factor

High doses of recombinant FVIIa (rFVIIa) are used successfully to treat hemophilia patients with inhibitory antibodies against FVIII and FIX. Despite its widespread and successful use of treating bleeding disorders for many years, the understanding of the mechanism of rFVIIa action in hemophilia therapy is incomplete. A majority of the studies, mostly in vitro studies, suggested that activation of FX by rFVIIa bound to phospholipids exposed on the activated platelets is responsible for the hemostatic effect of rFVIIa in hemophilia patients. However, other in vitro studies and mathematical modeling indicated that the action of rFVIIa at therapeutic doses is dominated by the TF-dependent pathway, partly because of the need to overcome the inhibitory effect of zymogen FVII, with a minor contribution from phospholipid-dependent mechanism. In the present study, we investigated whether TF plays a role in rFVIIa-mediated hemostasis in hemophilia by employing transgenic mice expressing low levels of human TF (Low TF mice)  or normal levels of human TF (HTF mice; breeding pairs for these mice were provided by Nigel Mackman). Mice were administered with FVIII mAb to induce acquired hemophilia. The saphenous vein incision bleeding model was used to evaluate the hemostasis. In this model, saphenous vein was exposed and bleeding was induced by a sharp incision of the vein with a fine scissors. The bleeding from the cut was monitored for 30 min while blood was collected onto a filter paper by capillary action.  After each hemostasis incident, the clot was disrupted gently to reinitiate a new bleeding episode, and average time to achieve hemostasis (ATH) was calculated.

                First, we investigated the role of TF in hemostasis by comparing the bleeding episodes in low TF and HTF mice following the saphenous vein incision. In the saphenous vein bleeding model, control wild-type mice had a median ATH of 60 sec (n=6) and hemophilia A mice failed to achieve hemostasis within 30 min experimental time frame (n=6). HTF mice had essentially very similar ATH compared to that of the wild-type mice, i.e., 64 sec (n=5).  In contrast, low TF mice had a substantially longer median ATH, 90 sec (n=6), which is statistically significantly longer than that of ATH of wild-type or HTF mice. Administration of TF antibodies (1 mg/kg) to neutralize remaining traces of TF in Low TF mice further prolonged the bleeding time to a median  ATH of 309 sec (n=6). These data clearly illustrates the role of TF in hemostasis.

                Next, we created acquired hemophilic condition in low TF and HTF mice by injecting FVIII mAb (1 mg/kg).  Low TF mice also received TF antibodies. Administration of FVIII mAb markedly prolonged the bleeding time in both groups of mice (between 300 to 1800 sec),  with a median ATH of 900 sec.  Administration of rFVIIa (4 mg/kg) to HTF hemophilic mice reduced the ATH to a median of 51 sec (mean ± SD, 50 ± 5 sec, n=6).  The same concentration of rFVIIa also restored hemostasis in hemophilic low TF mice with a median ATH of 66 sec (mean ±SD, 70 ± 18 sec, n=6). This value is not statistically significantly different from that of ATH of wild-type mice.  Although rFVIIa restored the hemostasis in both HTF and low TF mice, it appears that rFVIIa is slightly, but statistically significantly, more effective in HTF mice compared to low TF mice in restoring the hemostasis (p = 0.025).  However, we did not observe any significant differences between these two groups in the volume of blood loss following rFVIIa administration.  In summary, our data strongly support the hypothesis that high concentrations of rFVIIa restore hemostasis in hemophilia primarily in TF-independent manner.  More detailed studies are needed to completely eliminate the role of TF in rFVIIa mediated hemostasis.