Recombinant human factor VIIa (rFVIIa) in hemophilia: mode of action and evidence to date

Abstract

Recombinant activated factor VII (rFVIIa) is a bypassing agent widely used both in the treatment and prevention of hemorrhagic complications due to hemophilia with inhibitor. In such cases, antihemophilic factors cannot be used. The normal physiology of factor VII/ factor VIIa (FVII/FVIIa) in the hemostatic process requires the presence of tissue factor (TF) that links to FVII leading to a FVIIa-TF complex which activates both factor X and factor IX. The therapeutic use of rFVIIa requires high amount of FVIIa. Some studies demonstrate that FVIIa at high doses still requires tissue factor for function, whereas others suggest that FVIIa activates FX directly on the platelet surface, in a TF-independent manner. In the present article, we discuss the arguments supporting both TF-dependent and TF-independent modes of action. Finally, the coexistence of both TF-dependent and TF-independent mechanisms cannot be excluded.

Introduction

The development of inhibitors to factor VIII (FVIII) or factor IX (FIX) is actually the most challenging complication in patients with hemophilia A (HA) or hemophilia B (HB). Alloantibodies that neutralize antihemophilic factor develop in 20–30% of HA and in 1–5% of HB. Although inhibitor eradication using immune tolerance induction is the best long term option, bypassing agents are necessary to manage bleeding complications in patients with inhibitors. There are two bypassing agents available: activated prothrombin complex concentrate (FEIBA^TM), infused at 50–100 U/kg twice a day and recombinant activated factor VII (rFVIIa: Novoseven^TM), infused at 90 µg/kg every 2–3 h or as a single shot of 270 µg/kg once a day. While under physiological conditions, the blood-coagulation cascade is initiated when tissue factor (TF) binds plasma factor VII (FVII) leading to a TF–FVIIa complex that activates FIX and factor X (FX). The high level of FVIIa obtained during treatment by rFVIIa raises the possibility that FVIIa at high concentrations has significant activity in the absence of cofactor. Recombinant FVIIa may not bind only to TF, but also phospholipids exposed on thrombin-activated platelet surfaces.¹ Other mechanisms of action have been proposed for rFVIIa in hemophilia with inhibitors. In this article, we shall review the evidence for the various modes of action of rFVIIa.

Structure and functions of FVII and TF

Initiation pathway overview

Under physiological conditions, the blood-coagulation cascade is initiated when TF is expressed at the cell surface and binds to zymogen factor VII or enzymatically active FVIIa. The resulting TF–FVIIa complex activates the serine protease zymogens, FIX and FX. Both FXa and FIXa enzymes assemble on the thrombin-activated platelet surface with their own cofactors FVa and FVIIIa respectively, leading to a large burst of thrombin and ultimately to conversion of soluble fibrinogen into an insoluble tight fibrin plug.²

FVII and TF: structure

FVII is a single-chain zymogen, composed of an amino-terminal Gla domain, two epidermal growth factor (EGF)-like domains homologous to the epidermal growth factor, and a short region where the proteolytic activation cleavage occurs, followed by a serine protease domain. FVII can be activated after binding to TF. FVII can also be activated by FVIIa itself [TF-dependent (auto) activation] or by FXa or FIXa (feedback activation).³

Activated FVII (FVIIa) is a two-chain enzyme resulting from a single peptide bond cleavage in FVII at Arg212. The light chain, bearing the Gla and EGF-like domains, is covalently linked to the heavy chain protease domain by a single disulfide bridge. The light chain anchors FVIIa to the phospholipid cell surface and TF whereas the heavy chain protease domain provides substrate binding specificity and catalytic activity (Figure 1).^4,5

Figure 1.

Schematic structure of the three forms of FVII. (a) FVII zymogen, (b) Free FVIIa, (c) TF–FVIIa complex. The FVII domains are in light grey, the TF domains are in dark blue.

EGF, epidermal growth factor-like domain; FVII, plasma factor VII; FVIIa, activated form of FVII; SP, serine protease; TF, tissue factor

Approximately 99% of FVII circulates as inactive zymogen whereas 1% circulates as free FVIIa. Free FVIIa is not susceptible to most plasma protease inhibitors⁶ but it has very low intrinsic catalytic activity remaining in a so-called zymogen-like functional status.^7,8

TF is an integral membrane protein and does not require proteolysis for its function. It belongs to the class 2 cytokine receptor family, characterized by an extracellular domain of homologous units of approximately 200 residues. TF is composed of 263 amino acids and has a molecular weight of 47 kDa. The highly elongated extracellular domain extends from residues Ser1 to Glu219. It is composed of two fibronectin-III domains, connected end to end at, an angle of approximately 120°.⁹ Reduction of TF abolishes its function. The transmembrane region of 23 amino acids extends from Ile 220 to Leu242. The cytoplasmic portion of the molecule is located at the carboxyl end of the molecule (His243 to Ser263) (Figure 1). This portion can be deleted with no apparent loss of function.⁹

TF–FVIIa binary complex

Being the physiologic initiator of coagulation, TF reversibly binds to FVII or FVIIa in the presence of Ca²⁺. TF must be inserted into phospholipid vesicles to function efficiently, but the role of the lipid membrane is unclear. The binding of FVII/FVIIa is independent of the membrane in which TF is embedded. However, the ability of the TF–FVIIa complex to initiate coagulation is strongly limited by the availability of phosphatidylserine, required for association with the substrate. The TF–FVIIa binary complex can be formed through capture either of free circulating FVIIa or FVII zymogen followed by conversion to FVIIa. Both FVII and FVIIa bind the extracellular domain of TF with 1:1 stoichiometry.¹⁰ The Kd estimates reported for FVIIa binding to TF vary considerably from 0.1 to 13 nm. The sites of contact with FVII and FVIIa on TF are in both fibronectin type-III domains and the interfacial region between them. The domain of contact of FVIIa and TF extends from the bottom of the carboxyterminal part of extracellular TF up to the top of the amino-terminal domain. Upon binding to TF, the catalytic rate of FVIIa increases by at least five orders of magnitude.¹¹ This increase may be the consequence of interactions with specific side chains in TF that stabilize the conformation of the corresponding structure located in the protease domain of FVIIa. The allosteric mechanism induced by TF and the structural changes observed in FVIIa are not fully understood due to the absence of well-resolved crystal structure of the FVII zymogen of and full-length TF–FVIIa complex.¹² A recent work from Prasad and colleagues using molecular dynamics simulations, confirmed the critical role of EGF2 domain by maintaining optimal distances among catalytic triad residues and providing extra stability to the protease domain.¹³

Localization of TF

Cell-associated TF

Circulating blood is not in contact with tissue factor under normal conditions: TF is constitutively expressed in fibroblasts of the adventitia and is variably present on the outer layer of medial smooth muscle cells,^10,14 ensuring rapid initiation of coagulation after vascular injury. This distribution represents a type of hemostatic envelope that is ready to activate coagulation upon disruption of vascular integrity. TF is also expressed in a cell-specific manner in vital organs such as brain, lung, heart, and placenta. Quiescent cells, such as endothelial cells and non-activated monocytes, in contact with circulating blood do not express TF.¹⁴ In vitro, monocytes express TF upon stimulation by various agents: endotoxin (bacterial lipopolysaccharide: LPS), complement fractions (C5a), tumor necrosis factor alpha (TNFα), interleukin 1, or vascular permeability factor. TF expression can be induced in endothelial cells by interleukin 1, TNFα, thrombin, LPS, or immune complexes. Apoptotic endothelial cells may also express TF. After synthesis, TF is expressed at the cell surface where it is fully functional.

TF in circulating blood: microparticle-associated TF and soluble TF

TF is also present in microparticles which are submicron-sized vesicles shed from leukocytes, endothelial cells, platelets, and vascular smooth muscle cells.¹⁵ Although circulating TF is undetectable in healthy individuals, levels of circulating levels of microparticle-associated TF can be increased in prothrombotic states such as hyperlipidemia.¹⁶ Moreover atherosclerotic plaques have been shown to contain high concentration of TF-positive microparticules.¹⁷ The exact role of this circulating form of TF is unknown, but it may play a role in the diffusion of coagulation activation.

An alternatively spliced form of TF circulates in a soluble form.¹⁸ It is released from endothelial cells and other cell types in response to inflammatory cytokines. Whether this soluble form is physiologically active remains a matter of debate, but infused rFVIIa comes into contact with these forms of circulating TF.

TF-dependent and TF-independent mechanism: two concepts for rFVIIa mode of action

The recombinant rFVIIa has been used to treat hemophilia patients with inhibitors since 1988.¹⁹ Although the basal plasma concentration of FVIIa is 0.1–25 nm,²⁰ the recommended dose schedule is 90 µg/kg every 2–3 h. This is a supraphysiological dose, producing an approximately 250-fold increase above the basal plasma concentration, raising questions about the mode of action of rFVIIa at this pharmacological dose.

Whether the pharmacologic effect of rFVIIa primarily results from its binding to TF by removing zymogen FVII (TF-dependent mechanism) or from FX activation on the platelet activated surface (TF-independent mechanism) was a matter of debate in the early 2010s.

Indeed, physiological FVII zymogen is present in large excess over FVIIa and has been shown to compete with FVIIa for TF binding. Therefore, a high dose of rFVIIa as the one used to control bleeding in hemophilia would be needed to efficiently compete with the 10 nm circulating FVII zymogen. On the other hand, a sufficient rate of FX activation could be generated on the thrombin-activated platelet surface with high doses of rFVIIa through a TF-independent pathway. These two opposite or complementary modes of action are further discussed.

TF-dependent action of rFVIIa

The rationale for using high doses of rFVIIa in patients with HA or HB who developed an inhibitor is that FVIIa can drive activation of FX by FVIIa–TF in the absence of the FIXa/FVIIIa complex, thus bypassing the need for FIX or FVIII.

In 1996, Rao and Rappaport reported that FVIIa can compete with zymogen FVII for binding to TF and increase procoagulant activity by forming active FVIIa–TF, rather than FVII–TF complexes.²¹ This competition could explain why very high levels of FVIIa are needed. The same team hypothesized that the therapeutic mechanism of rFVIIa treatment of HA was, at least in part, the result of overcoming the inhibition of TF activity by the zymogen FVII:²² they first tested the effect of FVII on FVIIa–TF activity in a thrombin generation reaction and evaluated the effects of zymogen FVII and the enzyme FVIIa in a reconstituted hemophilia model. They found that the zymogen FVII inhibits thrombin generation initiated by physiological trace amounts of active FVIIa and a low concentration of TF. In the presence of the stoichiometric coagulation inhibitors, tissue factor pathway inhibitor and antithrombin (AT), addition of FVII resulted in highly delayed thrombin generation, supporting the role of FVII as a physiological down-regulator of thrombin generation. FVIIa could overcome the inhibitory effect of FVII at a concentration of 2 nmol/l, which corresponds to the blood concentration obtained by high-dose rFVIIa treatment. The role of TF in this model was confirmed in another study.²³ The authors used HA plasma and so-called ‘acquired’ HB blood, prepared by adding anti-FIX antibody to normal plasma, as models. They found that in HA or ‘acquired’ HB blood pharmacological concentrations of FVIIa (i.e. 10–50 nm) corrected the clotting time at all TF concentrations tested, but could not restore normal thrombin generation.²³ In hemophilia, plasma TF alone had a more pronounced effect on thrombin generation: an increase in TF from 0 to 100 pM increased the maximum thrombin level in hemophilia from 120 to 400 nm. They concluded that the efficacy of FVIIa (10–50 nm) in hemophilia was dependent on TF.

In a recent work on thrombin generation measurements in HA plasma a counteractive effect of FVII (auto) activation on the regulative impact of FVII zymogen competition with FVIIa for TF was shown. The authors demonstrated that (auto) activation of FVII, either by rFVIIa in a TF-dependent manner or by FXa or FIXa, neutralized the inhibitory effect of the endogenous FVII zymogen. They concluded that although competition between FVII zymogen and rFVIIa is an inevitable phenomenon, it has limited effect at physiological or clinically relevant concentration of FVIIa (>5 nm) and at physiological concentration of FVII.²⁴

TF-independent theories have emerged concerning the mode of action of high doses of rFVIIa in hemophilia, but they cannot completely exclude a role for TF. In all situations, FVIIa binds to TF and converts FX into FXa, even if it also binds to lipid surfaces allowing the same activation. The effect of lipid-surface-bound FVIIa is much slower than the reaction of TF-bound FVIIa.²⁵ Thus, the question is not the existence or not of TF-dependent action in high-dose rFVIIa, which is likely, but the degree to which this action is relevant to the physiology of high-dose FVIIa as a therapy.

TF-independent action of rFVIIa

A TF-independent action of high-dose rFVIIa was suspected from the beginning of treatment by high-dose rFVIIa.^1,26 Bom and Bertina demonstrated the activation of FX by FVIIa on a negatively charged phospholipid surface, independent of TF. In the model used, TF contributed to the reaction rate mostly by increasing the Vmax, with the formation of a ternary complex (FVIIa–TF–phospholipid) resulting in a 15-million-fold increase in the catalytic efficiency of FX activation. Thus, Rao and Rapaport²⁷ suggested that this might underlie the hemostatic activity of FVII in hemophilia. They used a purified system and found that FVIIa can slowly activate FX in a reaction mixture containing Ca²⁺ and phospholipid, but no source of TF. The rate of activation was sufficient to account for the observed shortening of partial thromboplastin time (APTT). Another observation, coming from clinical trials, provided arguments against TF-dependent activity of rFVIIa in the absence of other factors: clinical studies comparing doses of 35 µg/kg with 70, 90, and 120 µg/kg suggested that increasing doses were generally associated with increasing efficacy.^25,28 TF is almost certainly limiting in vivo. Thus, the reason why higher doses of rFVIIa led to greater efficacy was unclear.²⁵

These observations led to the hypothesis that high-dose rFVIIa could act via phospholipids in a non-TF-dependent pathway. Monroe and colleagues demonstrated that FVIIa binds to activated platelets independently of TF and partially restores thrombin generation in an in vitro model of hemophilia.^29,30 In a cell-based model, they used inactivated platelets and TF-bearing cells that were mixed with plasma levels of zymogen and inhibitors and found that FVIIa could directly activate FX on activated platelets even though platelets do not contain TF. Platelet surface FXa could complex with FVa and generate nearly normal levels of thrombin. Moreover, thrombin generation could be increased to several times normal levels when all coagulation factors were present: fewer platelets were required to reach a given level of thrombin generation. They proposed a platelet surface mechanism in which FVIIa binds to phosphatidylserine on the membrane of activated platelets with a low affinity, explaining the localization of FVIIa activity to the site of injury. Thus, platelet-bound rFVIIa could substantially enhance thrombin generation in a hemophilia model. However, as shown by Butenas and colleagues,²³ TF-independent thrombin generation does not occur in the presence of pharmacological concentrations of rFVIIa, even when a high concentration of pre-activated platelets are added. Evidence of the TF-independent mode of action of high-dose rFVIIa has come from in vitro models using FVIIa mutants in which residues believed to function as determinants for zymogenicity were replaced.³¹ This resulted in a marked improvement of the TF-independent rate of FX activation: up to 100-fold faster than that obtained with the wildtype enzyme. Nonetheless, the mutants retained the substrate specificity of the enzyme and could be further stimulated by TF. A recent study has confirmed the critical role of FVIIa platelet binding by titrating rFVIIa into platelet-rich hemophilia A plasma and triggering coagulation cascade with either TF or direct platelet agonists.²⁴ The authors demonstrated that concentrations of rFVIIa below clinically relevant levels (<6 nm) had hardly any effect on thrombin generation when triggered with TF or no effect when triggered with platelets activators. In contrast, increasing rFVIIa concentrations above 6 nm resulted in higher thrombin peaks whatever the trigger. They concluded that the TF-independent mechanisms, most likely on platelets, is predominant at pharmacological concentrations (>6 nm) of rFVIIa where the FVII zymogen competition effect has been counteracted.²⁴ This conclusion was in agreement with another original study in HB mice which also supports the phospholipid-binding model. The authors demonstrated that a mutant murine FVIIa with no TF-dependent activity has a similar efficacy in preventing bleeding than the wildtype murine FVIIa.³²

TF-dependent and TF-independent action of rFVIIa: can they coexist?

It is obvious that the overall coagulation process still requires initiation by TF expressed on the cell surface at the site of injury.³³ At physiological doses, the effect of FVIIa is TF-dependent. It was 20 years ago that Zur and colleagues described the existence of competition between the low activity zymogen FVII and FVIIa for TF.³⁴ This dual role of FVII could constitute a mechanism of regulation of the proteolytic system. Following this model, high doses of rFVIIa are needed to shift the competition between rFVIIa and FVII for TF. The platelet phospholipid-dependent theory considers that the coagulation cascade is activated by low affinity binding of rFVIIa to phospholipids exposed on the surface of activated platelets, with no role of TF. Such low binding could explain the need of high doses of rFVIIa. These two theories have different consequences: in the TF-dependent model, increasing doses to pharmacological levels would be of little interest, whereas it would lead to the saturation of all available TF molecules. In the TF-independent model, platelet-phospholipid hemostasis is achieved by direct activation of FX by rFVIIa bound to phospholipids exposed on the surface of activated platelets. Thus, it could never be saturated, suggesting a benefit of increasing dose.

Shibeko and colleagues set up an original model using fluorogenic substrate-based thrombin generation and clotting in human plasma.³⁵ They found conditions for the presence or absence of zymogen inhibition by testing a wide range of FVII and rFVIIa concentrations. Under conditions of FVII inhibition, they showed the simultaneous existence of TF-dependent and phospholipid-dependent mechanisms. TF noteworthy plays a critical role in generating the initial small amounts of thrombin needed for platelet activation resulting in full FXa activation essential for the TF-independent mode of action of rFVIIa. Thus, both mechanisms independently contribute to rFVIIa induced activation of coagulation. Moreover, they confirmed their results using mathematical simulations, leading to a model in which rFVIIa may act under various dosing strategies, depending on the level of TF exposed and rFVIIa infused. However, these results were only obtained in vitro and would require in vivo confirmation, even if this study is in accord with previous work and provides evidence to support the two opposing theories and the action of pharmacological doses of rFVIIa.

rFVIIa in patients with hemophilia: can we draw conclusions about the mode of action?

Since the first use of rFVIIa in people with hemophilia,¹⁹ it has been widely used to treat hemophilia disease with inhibitors and other hemorrhagic diseases:³⁶ Glanzmann thrombocytopenia, thrombocytopenia, life-threatening hemorrhage in obstetrical conditions, and more recently, hemorrhage in patients treated by direct oral anticoagulant treatment.

In people with hemophilia, rFVIIa has been used to treat hemorrhage in nonsurgical conditions in hemophilia with inhibitors at a dose of 90 µg/kg every 2–3 h.^37,38 Major and minor surgical procedures have been successfully performed on inhibitor patients treated with rFVIIa.^19,39 In a prospective randomized trial, two doses of rFVIIa were compared in hemophilia patients with inhibitors undergoing surgery: 35 µg/kg and 90 µ/kg every 2 h. The conclusion was that the highest dose was an effective first-line option for these patients. All the assays and clinical reports supported the safety of this drug.⁴⁰ In a retrospective review, the Hemophilia Thrombosis Research Society noted that doses >200 µg/kg showed significantly higher efficacy than lower dose groups. These very high doses of rFVIIa were well tolerated. These observations led to the use of higher doses to both enhance hemostasis and facilitate care by allowing home treatment. A prospective randomized assay was initially published.⁴¹ A single bolus of 270 µg/kg dose of rFVIIa was found to be at least as effective and well tolerated as standard 90 µg/kg × 3 dosing. Several studies confirmed this result.⁴² Another mode of utilization was then considered: the possibility of prophylaxis with rFVIIa in patients with inhibitors. Overall, two dosages were tested in secondary prophylaxis:⁴³ 90 µg/kg three times a day versus a single 270 µg/kg/day dose. Clinically relevant reductions in bleeding frequency during prophylaxis were achieved, without raising safety concerns.

Apart from the interest for patient care (a once-a-day infusion allows home treatment) the efficacy of a 270 µg/kg once-a-day infusion may provide clues concerning the mode of action of high-dose rFVIIa. Considering the arguments previously developed concerning the TF-dependent versus TF-independent effect of high-dose rFVIIa, the fact that the highest dose increases efficacy favors a TF-independent mechanism, whereas the 90 µg/kg dose is probably sufficient to saturate all available TF molecules. The efficacy of a 270 µg/kg once-a-day infusion also raises the question of how a daily infusion can be efficient, considering that the drug has an intravascular half-life of approximately 2.5 h. The explanation proposed by Hedner⁴⁴ relates to the extravascular concentrations of rFVIIa obtained with daily dosing. This extravascular concentration at the bleeding site may be high enough to support complex formation with TF, thereby providing enough thrombin to form fibrin plugs to stop minor bleeds. This explanation opens the way to extravascular hemostasis, and suggests a TF-dependent mode of action for very high doses of rFVIIa, confirming the coexistence of both a TF-dependent and TF-independent mode of action of high-dose rFVIIa.