Targeting platelet EPCR for better therapeutic factor VIIa activity

Management of patients with inhibitory antibodies to factor VIII or factor IX remains one of the most challenging aspects of modern hemophilia care. Control of bleeding in patients with high titer inhibitors often relies on the use of bypassing agents such as activated prothrombin complex concentrates or recombinant factor VIIa (rFVIIa). The mechanism of action of pharmacologically dosed rFVIIa has been contentious [1]. Unlike endogenous FVIIa, which binds to tissue factor (TF) at sites of vascular injury to promote the activation of factors X and IX, high dose rFVIIa appears to function via TF-independent activation of factor X on the surface of activated platelets [2, 3]. The TF-independent mechanism is supported by clinical findings with the rFVIIa analog FVIIa-DVQ (also known as vatreptacog alfa). Compared with rFVIIa, FVIIa-DVQ has enhanced TF-independent activity [4], improved hemostatic effects in hemophilia mouse models [5], and superior efficacy in controlling bleeding episodes in hemophilia patients with inhibitors [6]. Unfortunately, the clinical development of vatreptacog alfa was discontinued due to the development of anti-drug antibodies [7].

The nature of the rFVIIa binding site on activated platelets is obscure but presumed to comprise anionic phospholipids because the γ-carboxyglutamic acid (Gla) domain of rFVIIa is required [8]. The FVIIa Gla domain has a relatively low affinity for phospholipids, however, and platelet surface proteins have been proposed to support the binding and therapeutic activity of rFVIIa [9]. Interestingly, FVIIa-DVQ binds to more sites per platelet than rFVIIa despite a similar extent of phospholipid binding, which raises the possibility that its higher therapeutic activity may be partly related to increased binding to an alternative site(s) on the platelet surface [8]. In this issue of the JTH, Fager et al. [10] provide compelling evidence that one such site may be the endothelial protein C receptor (EPCR).

EPCR is a multifunctional cell surface protein that was initially discovered by Esmon and colleagues as a receptor for protein C and activated protein C (PCa) on endothelial cells [11]. It serves as a cofactor for protein C activation by the thrombin/thrombomodulin complex and also as a co-receptor for protease-activated receptor signaling [12]. EPCR is structurally homologous with class 1 major histocompatibility complex molecules. It contains a tightly bound phospholipid in its “antigen presenting groove” that interacts directly with the Gla domain of protein C [13]. FVIIa binds to EPCR with a similar affinity as protein C, likely also via its Gla domain [14].

Despite its name, EPCR is not found just on the endothelium. It is known to be expressed at low levels on leukocytes, smooth muscle cells, keratinocytes, cardiomyocytes, and neurons [14, 15]. EPCR has not previously been demonstrated on platelets, although its mRNA has been detected by RNA sequencing [16]. Fager et al. demonstrated that human platelets indeed contain EPCR, that EPCR translocates to the cell surface upon platelet activation, and that the binding of rFVIIa or FVIIa-DVQ to EPCR on activated platelets enhances the rate of activation of factor X [10]. These findings suggest a model in which the Gla domain of rFVIIa interacts with the surface of activated platelets through two mechanisms: 1) direct binding to anionic phospholipids such as phosphatidylserine, and 2) binding to the phospholipid-containing antigen presenting groove of EPCR, which leads to more effective activation of factor X (Figure 1, Panel A). A limitation of the study by Fager et al. is that the relative contribution of EPCR to platelet-dependent generation of factor Xa and thrombin was quite modest, accounting for only 20–30% of the total activity. It has been demonstrated previously that rFVIIa, in high concentrations, can compete with protein C for binding to EPCR on endothelial cells, resulting in inhibition of protein C activation [17, 18] (Figure 1, Panel B). This mechanism also likely contributes to the therapeutic activity of rFVIIa by dampening the protein C anticoagulant system. Administration of anti-EPCR blocking antibodies augments the hemostatic effects of rFVIIa in hemophilia treatment [17, 18], but it remains to be determined whether this is due to blockade of ECPR on platelets, endothelial cells, or both.

Figure 1. Effects of EPCR on the hemostatic activity of recombinant factor VIIa.

A) Enhanced activation of factor X on activated platelets. Recombinant factor VIIa (rVIIa) is recruited to the surface of activated platelets through interaction of its Gla domain with anionic phosphatidylserine on the exoplasmic face of the plasma membrane where it can bind and directly activate factor X. Binding of the rVIIa Gla domain to EPCR enhances the rate of proteolytic activation of factor X. B) Decreased protein C activation on endothelial cells. On the surface of the endothelium, EPCR classically functions to enhance the rate of protein C (PC) activation by the thrombin/thrombomodulin (TM/IIa) complex. Recombinant factor VIIa competes with and displaces protein C from EPCR, preventing formation of activated protein C (PCa) and contributing to its overall hemostatic activity.

The discovery of functional EPCR on platelets by Fager et al. has implications for the design of more effective rFVIIa therapeutics, perhaps by bioengineering variants with enhanced affinity for EPCR. This approach must be undertaken with caution, however, given the clinical experience with the rFVIIa variants vatreptacog alfa and BAY 86–6150, which proved to be highly immunogenic in human subjects [7, 19]. We are entering an exciting era in hemophilia care, with a plethora of therapeutic options on the horizon for the prevention and treatment of bleeding. Emerging therapies include extended half-life factor VIII and factor IX products, gene therapy for hemophilia A and B, substitutive therapies such as the bispecific antibody emicizumab, and rebalancing therapeutics that target natural anticoagulants such as antithrombin, tissue factor pathway inhibitor, or PCa. [20]. With this remarkable progress, what will be the future role of bypassing agents such as rFVIIa? Until the problem of inhibitor development in hemophilia is solved, the need for better bypassing agents is unlikely to disappear any time soon.