Therapeutic Doses of Recombinant FVIIa in Hemophilia Generates Thrombin in Platelet-Dependent and -Independent Manner

Recombinant factor FVIIa (rFVIIa) is used successfully in the treatment of hemophilia patients with inhibitors and other bleeding disorders for more than two decades. However, its mechanism of action is still not completely understood. Most of the data in the literature indicate that platelet phospholipid-dependent and tissue factor (TF)-independent activation of factor X by pharmacological concentrations of rFVIIa is responsible to the hemostatic effect of FVIIa in hemophilia treatment. The successful use of rFVIIa in correcting bleeding associated with platelet defects suggest a platelet-independent mechanism may also contribute to the hemostatic effect of rFVIIa in clinical settings. A recent study of Ivanciu et al. (Blood 124:1705-1714, 2014) suggested that the activated endothelium plays an unexpectedly important and major role in supporting prothrombinase assembly and function at the site of damage. Our studies showed that FVIIa binds endothelial protein C receptor (EPCR) on the endothelium and downregulates EPCR-mediated protein C activation, which indirectly contributes to the hemostatic effect of rFVIIa in the treatment of hemophilia. Recently, it has been reported that platelets express EPCR, and suggested that FVIIa binding to EPCR on platelets could enhance the hemostatic activity of FVIIa (Fager et al., J Thromb Haemost 16:1817-1829, 2018). The present study is conducted to evaluate the contribution of platelets and EPCR to the hemostatic effect of rFVIIa in hemophilia treatment in a mouse model system. In this model, platelets in wild-type and hemophilic (acquired and genetic) mice were depleted using anti-murine CD42b antibodies, subjected to the saphenous vein injury, and the hemostatic effect of pharmacological concentrations of rFVIIa administered to mice was evaluated by measuring the amount of thrombin: antithrombin (TAT) complexes generated in the blood. In initial experiments, we found that TAT levels measured in the blood collected at the injury site and not from the blood collected via sub-mandibular site provide a better correlation between TAT levels and the hemostatic effect (clot formation). Therefore, in all studies, blood was collected from the injury site to measure TAT levels. Administration of anti-CD42b antibodies depleted platelets about 95% within 2 to 4 h after the administration of the antibody. Depletion of platelets in wild-type (WT) C57BL/6J mice suppressed TAT levels significantly (51 ± 18 ng/ml) (p<0.01) compared to mice that received no antibody or isotype-specific control antibodies (250 ± 41 ng/ml and 217 ± 31 ng/ml respectively). Induction of hemophilic condition in WT mice by administration of FVIII neutralizing antibodies reduced TAT levels (17 ± 4.0 ng/ml) following the injury. Platelet depletion in these mice further reduced TAT levels (4.7 ± 0.6 ng/ml). Administration of rFVIIa, 1 or 4 mg/kg, to the antibody-induced hemophilic mice, increased the generation of TAT significantly, 75 ± 13 ng/ml and 134 ± 27 ng/ml, respectively. In platelet-depleted, antibody-induced hemophilic mice, administration of the same doses of rFVIIa increased TAT levels to 25 ± 4.0 ng/ml and 58 ± 5.4 ng/ml, respectively. Although TAT levels in rFVIIa-treated platelet-depleted hemophilic mice were lower than that was observed in rFVIIa-treated hemophilic mice (administered with control IgG), still, it was significantly higher compared to TAT levels in hemophilic mice not treated with rFVIIa. Next, to assess the role of platelets and EPCR to the hemostatic effect of rFVIIa, we depleted platelets in FVIII^-/-, EPCR-deficient FVIII^-/-, and EPCR-overexpressing FVIII^-/-mice, subjected them to the saphenous vein injury and administration of rFVIIa (4 mg/kg). The TAT levels in platelet-depleted mice were - 11 ± 1.6 ng/ml in FVIII^-/-, 9 ± 0.6 ng/ml in EPCR-deficient FVIII^-/-, and 8 ± 1.0 ng/ml in EPCR-over expressing FVIII^-/- mice. Administration of rFVIIa to platelet-depleted mice increased TAT levels to 54 ± 10 ng/ml in FVIII^-/-, 53 ± 8 ng/ml in EPCR-deficient FVIII^-/-, and 42 ± 3.2 ng/ml in EPCR-overexpressing FVIII^-/-mice. The differences among them are not statistically significant. Overall, our data indicate that cellular sources other than platelets contribute to the hemostatic effect of rFVIIa. There is no evidence, at least in the murine model, platelet EPCR, if at all present, contributes to the hemostatic effect by augmenting FVIIa binding to platelets.