Recombinant factor VIIa: new insights into the mechanism of action through product innovation

Abstract

Management of bleeding in persons with hemophilia and inhibitors involves treatment with bypassing agents, including recombinant activated factor VII (rFVIIa). Two rFVIIa products are commercially approved for use in the United States and the European Union. Eptacog alfa and eptacog beta share the same amino acid sequence but differ in posttranslational modifications. Although rFVIIa has been used to manage bleeding in persons with hemophilia and inhibitors for over 30 years, its mechanisms of action is still being studied. In vitro and in vivo studies have suggested that rFVIIa could promote hemostasis by (1) increasing tissue factor-dependent activation of factor (F)X (FX); (2) directly activating FX on the surface of activated platelets; and (3) downregulating protein C anticoagulant activity through binding to the endothelial protein C receptor (EPCR). Studies of rFVIIa and rFVIIa variants in murine models demonstrate that platelet-dependent activity is sufficient for hemostatic efficacy. Dosing levels required in clinical practice are most consistent with a platelet-dependent mechanism of action. However, in vivo models also suggest that pathways involving EPCR binding contribute to rFVIIa hemostatic activity. Eptacog beta displays increased platelet- and EPCR-dependent endothelial cell binding compared to eptacog alfa. Thus, the relative contribution of these mechanisms to the overall hemostatic efficacy of eptacog alfa and eptacog beta may differ. Further research is required to assess the clinical relevance of these differences. A better understanding of the mechanisms by which rFVIIa promotes hemostasis in patients will provide insights when evaluating clinical outcomes of safety and efficacy for innovative bypassing therapies.

Keywords: blood platelets; endothelial protein C receptor; factor VIIa; hemophilia A; hemophilia B; hemostasis; tissue factor.

Figure 1

The protein domain structure of recombinant activated factor VII (rFVIIa). Factor (F)VII is a vitamin-K-dependent glycoprotein characterized by the posttranslational γ-carboxylation of glutamic acid residues at conserved locations. FVII undergoes several other posttranslational modifications including N- and O-linked glycosylation and β-hydroxylation. FVII contains 4 domains: an N-terminal Gla domain, 2 epidermal growth factor (EGF1 and EGF2)-like domains, and a C-terminal serine protease domain. Activation of FVII involves a single cleavage at the Arg152-Ile153 peptide bond, resulting in the formation of the active serine protease, FVIIa. FVIIa consists of a light and a heavy chain linked by a disulfide bond between Cys135 and Cys262. The light chain contains the Gla domain that mediates phospholipid membrane binding and EGF1 and EGF2 domains that facilitate interactions between FVIIa and TF, FX, and FIX. The heavy chain contains the serine protease domain responsible for enzymatic activity. EGF, epidermal growth factor; aa, amino acids; γ, γ carboxyglutamic acids; S–disulfide bond; O, O-linked glycan; N, N-linked glycan; β, β hydroxylation.

Figure 2

O- and N-linked glycans of recombinant activated factor VII (rFVIIa). (A) rFVIIa carries similar O-linked glycans [16,49,50]. (B) Eptacog alfa contains complex-type, partially sialylated, core-fucosylated biantennary and triantennary N-linked glycans [16]. (C) Eptacog beta contains high-mannose, complex, and hybrid-type partially fucosylated biantennary N-linked glycans [49]. The N-linked glycans of eptacog alfa and eptacog beta differ based on the presence of GalNAc-containing glycans and increased core-fucosylation on eptacog alfa, and the presence of high-mannose glycans expressed on eptacog beta [16,49]. Ser, serine; Asn, asparagine; Fuc, fucose; Gal, galactose; GalNAc, N-acetylgalactosamine; Glc, glucose; Man, mannose; GlcNAc, N-acetylglucosamine; Neu5Ac, acetylneuraminic acid (sialic acid); Xyl, xylose.

Figure 3

Mechanisms of action of recombinant activated factor VII (rFVIIa). rFVIIa has been proposed to exert hemostatic efficacy through tissue factor (TF)-dependent, platelet-dependent, and endothelial cell protein C receptor (EPCR)-dependent pathways. Upon vascular injury, circulating rFVIIa binds TF expressed by subendothelial cells and activates the coagulation cascade by converting coagulation factor (F)X (FX) to FXa. FXa, along with FVa and prothrombin form the prothrombinase complex on active platelets, generating small amounts of thrombin. Thrombin amplifies the activation of coagulation by fully activating platelets that had been partially activated through exposure to collagen at the site of vessel injury, and by activating FV, FVIII, and FXI, leading to amplification of coagulation. Platelet-bound rFVIIa can directly activate FX, enhancing prothrombinase activity and leading to a burst of thrombin generation and fibrin formation. The binding of rFVIIa to EPCR can attenuate the formation of activated protein C by the thrombin-thrombomodulin complex, thereby impairing the inactivation of FVa and FVIIIa and enhancing thrombin generation. EPCR, endothelial cell protein C receptor; rFVIIa, recombinant activated factor VII; TF, tissue factor.