Recent studies established that clotting factor VIIa (FVIIa) binds endothelial cell protein C receptor (EPCR). It has been speculated that FVIIa interaction with EPCR might augment the hemostatic effect of rFVIIa in therapeutic conditions. The present study is carried out to investigate the mechanism by which FVIIa interaction with EPCR contributes to the hemostatic effect of rFVIIa in hemophilia therapy. Active-site inhibited FVIIa, which is capable of binding to EPCR but has no ability to activate factor X, reduced the concentration of rFVIIa required to correct the bleeding following the saphenous vein injury in mouse hemophilia model systems. Higher doses of rFVIIa were required to restore hemostasis in EPCR overexpressing hemophilia mice compared to hemophilia mice expressing normal levels of EPCR. Administration of FVIII antibody induced only mild hemophilic bleeding in EPCR-deficient mice, which was corrected completely with a low dose of rFVIIa. Administration of therapeutic concentrations of rFVIIa increased plasma protein C levels in EPCR overexpressing mice, indicating the displacement of protein C from EPCR by rFVIIa. EPCR levels did not significantly alter the bioavailability of rFVIIa in plasma. Overall, our data indicate that EPCR levels influence the hemostatic effect of rFVIIa in treating hemophilia. Our present findings suggest that FVIIa displacement of anticoagulant protein C from EPCR that results in down-regulation of activated protein C generation and not the direct effect of EPCR-FVIIa on FX activation is the mechanism by which FVIIa interaction with EPCR contributes to the hemostatic effect of rFVIIa in hemophilia therapy.

Summary Background Recent studies have shown that factor VIIa binds to endothelial cell protein C receptor (EPCR), a cellular receptor for protein C and activated protein C. At present, the physiologic significance of FVIIa interaction with EPCR in vivo remains unclear. Objective: To investigate whether exogenously administered FVIIa, by binding to EPCR, induces a barrier protective effect in vivo. Methods Lipopolysaccharide (LPS)-induced vascular leakage in the lung and kidney, and vascular endothelial growth factor (VEGF)-induced vascular leakage in the skin, were used to evaluate the FVIIa-induced barrier protective effect. Wild-type, EPCR-deficient, EPCR-overexpressing and hemophilia A mice were used in the studies. Results Administration of FVIIa reduced LPS-induced vascular leakage in the lung and kidney; the FVIIa-induced barrier protective effect was attenuated in EPCR-deficient mice. The extent of VEGF-induced vascular leakage in the skin was highly dependent on EPCR expression levels. Therapeutic concentrations of FVIIa attenuated VEGF-induced vascular leakage in control mice but not in EPCR-deficient mice. Blockade of FVIIa binding to EPCR with a blocking mAb completely attenuated the FVIIa-induced barrier protective effect. Similarly, administration of protease-activated receptor 1 antagonist blocked the FVIIa-induced barrier protective effect. Hemophilic mice showed increased vascular permeability, and administration of therapeutic concentrations of FVIIa improved barrier integrity in these mice. Conclusions This is the first study to demonstrate that FVIIa binding to EPCR leads to a barrier protective effect in vivo. This finding may have clinical relevance, as it indicates additional advantages of using FVIIa in treating hemophilic patients.

Summary Background Recombinant factor VIIa (rFVIIa) has been used widely for treating hemophilia patients with inhibitory autoantibodies against factor VIII or IX. Its mechanism of action is not entirely known. A majority of in vitro studies suggested that pharmacological concentrations of rFVIIa restore hemostasis in hemophilia in a phospholipid-dependent mechanism, independent of tissue factor (TF). However, a few studies suggested that a TF-dependent mechanism plays a primary role in rFVIIa correction of bleeding in hemophilia patients. Here, we investigated the potential contribution of TF in rFVIIa-induced hemostasis in hemophilia employing a model system of FVIII antibody-induced hemophilia in TF transgenic mice. Methods Mice expressing low levels of human TF (LTF mice), relatively high levels of human TF (HTF mice) or wild-type mice (WT mice) were administered with neutralizing anti-FVIII antibodies to induce hemophilia in these mice. The mice were then treated with varying concentrations of rFVIIa. rFVIIa-induced hemostasis was evaluated with the saphenous vein bleeding model. Results Administration of FVIII inhibitory antibodies induced the hemophilic bleeding phenotype in all three genotypes. rFVIIa administration rescued the bleeding phenotype in all three genotypes. No significant differences were observed in rFVIIa-induced correction in the bleeding of LTF and HTF mice administered with FVIII antibodies. Conclusions Our results provide strong evidence supporting that the hemostatic effect of pharmacological doses of rFVIIa stems from a TF-independent mechanism.

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Recent studies from our laboratory and others established that clotting factor FVII/FVIIa, which binds tissue factor and triggers the coagulation cascade, also binds endothelial cell protein C receptor (EPCR), a receptor that plays a critical role in protein C/activated protein C (APC)-mediated anticoagulation pathway. We postulated that FVIIa binding to EPCR might augment the hemostatic effect of rFVIIa in therapeutic conditions by down-regulating the EPCR-mediated anticoagulation pathway. Our recent studies showing the blockade of endogenous protein C binding to EPCR by administration of EPCR blocking antibodies augmented the hemostatic effect of rFVIIa in a mouse hemophilia model provided an indirect support to this hypothesis. However, studies from other investigators raised a possibility that FVIIa interaction with EPCR may influence the hemostatic effect of rFVIIa in vivo through EPCR-FVIIa directly activating FX or EPCR tethering FVIIa to provide an extended locale of procoagulant reactions on the endothelium. The present study is carried out to investigate the mechanism by which FVIIa interaction with EPCR contributes to the hemostatic effect of FVIIa in hemophilia therapy. The study employed wild-type, transgenic mice expressing no EPCR (EPCR-/-) or overexpressing EPCR (Tie2-EPCR). FVIII mAb (1 mg/kg) were given to mice to induce hemophilia. The saphenous vein incision bleeding model was used to evaluate the hemostatic effect of human rFVIIa. In this model, bleeding is induced by a sharp incision to the saphenous vein and the bleeding from the cut was monitored for 30 min. 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 EPCR in hemostasis by comparing the bleeding episodes in unchallenged wild-type, EPCR-/- and Tie2-EPCR mice following the saphenous vein incision. In this model, control wild-type mice had a median ATH of 55 sec (mean 63 ± 7 sec; n=9) and EPCR-/- mice had a median ATH of 67 sec (mean 74 ± 6 sec; n =12). The difference between them is not statistically significant. Surprisingly, the median ATH in EPCR overexpressing Tie2-EPCR mice was significantly lower (a median ATH of 43 sec; mean 46 ± 2 sec; n=10) compared to the ATH in wild-type or EPCR-/- mice. The reason for a shorter ATH observed in EPCR overexpressing mice is unknown at present. Analysis of blood count showed no significant differences among wild-type, EPCR-/- and Tie2-EPCR in their platelet count, RBC, and other blood cell count. Next, we induced acquired hemophilia condition in wild-type, EPCR-/- and Tie2-EPCR by injecting FVIII mAb (1 mg/kg) i.v. 2 h before inducing the bleeding. Administration of FVIII mAb markedly prolonged the bleeding time in wild-type and Tie2-EPCR mice (between 300 to 1800 sec) with a median ATH of 600 sec (mean 730 ± 277 sec) in wild-type mice and 750 sec (mean 939 ± 200 sec) in Tie2-EPCR mice. Interestingly, EPCR-/- mice were protected from the antibody-induced acquired hemophilia. Administration of FVIII mAb prolonged the bleeding time only marginally in this group of mice to a median ATH of 120 sec (mean 163 ± 38 sec). Administration of a low dose of rFVIIa (0.25 mg/kg) fully corrected the mild bleeding defect in EPCR-/- acquired hemophilia mice (median ATH of 67 sec; mean 86 ± 17 sec). Administration of 0.25 mg/kg rFVIIa is corrected the bleeding disorder only partially in wild-type acquired hemophilia mice (median ATH 164 sec; mean 303 ± 94 sec; n=5). Administration of either 1 or 4 mg/kg of rFVIIa was required to correct the bleeding in these mice (1 mg/kg dose: median ATH of 75 sec, mean 74 ± 1 sec; 4 mg/kg dose: median ATH of 37 sec; mean 40 ± 2 sec, n=5). In Tie2-EPCR hemophilia mice, administration of 1 mg/kg rFVIIa was not sufficient to fully achieve the hemostatic effect (median ATH 164 sec; mean 521 ± 237 sec; n=6). Administration of 4 mg/kg rFVIIa was required to restore the hemostasis in these mice (median ATH of 90 sec; mean 94 ± 7 sec, n= 6). In summary, the present data indicate that EPCR levels influence the hemostatic effect of rFVIIa in treating antibody-induced hemophilia. The present study rules out the possibility of direct activation of FX by FVIIa-EPCR complexes or EPCR tethering of FVIIa as probable mechanisms by which EPCR-FVIIa influences the hemostatic effect of rFVIIa. Disclosures No relevant conflicts of interest to declare.