A Structure Based Study of Selective Inhibition of Factor IXa over Factor Xa

Blood coagulation is an essential physiological process for hemostasis; however, abnormal coagulation can lead to various potentially fatal disorders, generally known as thromboembolic disorders, which are a major cause of mortality in the modern world. Recently, the FDA has approved several anticoagulant drugs for Factor Xa (FXa) which work via the common pathway of the coagulation cascade. A main side effect of these drugs is the potential risk for bleeding in patients. Coagulation Factor IXa (FIXa) has recently emerged as the strategic target to ease these risks as it selectively regulates the intrinsic pathway. These aforementioned coagulation factors are highly similar in structure, functional architecture, and inhibitor binding mode. Therefore, it remains a challenge to design a selective inhibitor which may affect only FIXa. With the availability of a number of X-ray co-crystal structures of these two coagulation factors as protein–ligand complexes, structural alignment, molecular docking, and pharmacophore modeling were employed to derive the relevant criteria for selective inhibition of FIXa over FXa. In this study, six ligands (three potent, two selective, and one inactive) were selected for FIXa inhibition and six potent ligands (four FDA approved drugs) were considered for FXa. The pharmacophore hypotheses provide the distribution patterns for the principal interactions that take place in the binding site. None of the pharmacophoric patterns of the FXa inhibitors matched with any of the patterns of FIXa inhibitors. Based on pharmacophore analysis, a selectivity of a ligand for FIXa over FXa may be defined quantitatively as a docking score of lower than −8.0 kcal/mol in the FIXa-grids and higher than −7.5 kcal/mol in the FXa-grids.

In Hemophilia Α Plasma Treated with Emicizumab, Factor IXa in Activated Prothrombin Complex Concentrates Is the Dominant Contributor to Enhanced Thrombin Generation

Background. Recently a novel bifunctional antibody (emicizumab) that binds both factor IXa and factor X has been used to treat hemophilia A. Emicizumab has proven remarkably effective as a prophylactic treatment for hemophilia A; however there are patients that still experience bleeding. An approach to treating this bleeding in hemophilia A patients with inhibitors is to give an activated prothrombin complex concentrate (APCC; FEIBA) (disfavored in NHF MASAC #255). APCC contains a number of coaguation factors including prothrombin, factor X (FX), and factor IX (FIX). APCC also contains activated factor X (FXa) and factor IX (FIXa). Previous work has shown that when APCCs are added to hemophilia A plasma containing emicizumab there is a significant increase in thrombin generation [J Thromb Haemost 2018;16:1580-1591]. The goal of this work was to study thrombin generation in hemophilia A plasma with emicizumab and to examine the role of the zymogen and activated components of an APCC in the increased thrombin generation. Methods. In hemophilia A plasma, thrombin generation assays were done using 100 µM lipid and 420 µM Z-Gly-Gly-Arg-AMC with or without emicizumab at 55 µg/mL which is the clinical steady state level. The reactions were initiated with low (1 pM) tissue factor (TF). The components of APCC were studied at concentrations that should mimic the levels seen in the plasma of a patient given a dose of 50 U/kg: prothrombin 1800 nM; FX 130 nM; FIX 90 nM; and FIXa 0.4 nM. Results. When initiated with low TF, hemophilia A plasma alone had essentially no thrombin generation. As expected, adding emicizumab enhanced thrombin generation. The addition of zymogen coagulation factors, prothrombin, FIX, and FX, separately or together gave a small increase in thrombin generation. However, addition of FIXa to emicizumab gave a large increase in peak thrombin. In hemophilia A plasma with emicizumab and FIXa, addition of prothrombin further increased thrombin generation and specifically increased the peak level of thrombin. Further addition of FX or FIX, separately or together, only minimally increased thrombin generation. Discussion. The strong contribution of factor IXa to the effects of APCCs on thrombin generation in hemophilia A plasma depends on the presence of emicizumab. In the absence of emicizumab, a study of the individual components of APCC showed that a combination of FXa and prothrombin at levels found in APCCs had the major effect on thrombin generation [Haemophilia. 2016;22:615-24]. That study found that FIXa did not increase thrombin generation. However, in the presence of emicizumab, despite the weak solution phase affinity of the bifunctional antibody for FIXa, small amounts of FIXa were the most significant contributor to thrombin generation. Disclosures Monroe: Novo Nordisk:Research Funding.Ezban:Novo Nordisk:Current Employment.Hoffman:Novo Nordisk:Research Funding.

Novel Insights and New Developments Regarding Coagulation Revealed by Studies of the Anti-Factor IXa (Activated Factor IX)/Factor X Bispecific Antibody, Emicizumab

Emicizumab is a humanized anti-FIXa/FX (factor IXa/X) bispecific monoclonal antibody that mimics FVIIIa (activated factor VIII) cofactor function. The hemostatic efficacy of emicizumab has been confirmed in clinical studies of patients with hemophilia A, irrespective of the presence of FVIII inhibitors. Emicizumab differs in some properties from FVIIIa molecule. Emicizumab requires no activation by thrombin and is not inactivated by activated protein C, but emicizumab-mediated coagulation is regulatable and maintains hemostasis. A small amount of FIXa (activated factor IX) is required to initiate emicizumab-mediated hemostasis, whereas tissue factor/FVIIa (activated factor VII)-mediated FXa (activated factor X) and thrombin activation initiates FVIIIa-mediated hemostasis. Fibrin formation, followed by fibrinolysis, appears to be similar between emicizumab- and FVIIIa-mediated hemostasis. These results suggest possible future uses of emicizumab for treating hemorrhagic diseases other than hemophilia A and reveal previously unobservable behaviors of procoagulation and anticoagulation factors in conventional hemostasis. Here, we have reviewed novel insights and new developments regarding coagulation highlighted by emicizumab.

Nonasaccharide Inhibits Intrinsic Factor Xase Complex by Binding to Factor IXa and Disrupting Factor IXa–Factor VIIIa Interactions

A nonasaccharide (FG9) derived from natural fucosylated glycosaminoglycan (FG) is identified as a selective intrinsic factor Xase complex (FIXa-FVIIIa-Ca2+-phospholipid, FXase) inhibitor that possesses potential inhibition of venous thrombus in rats and shows negligible bleeding risk. The mechanism and molecular target of the nonasaccharide for intrinsic FXase inhibition were systematically investigated and compared with low molecular weight heparin (LMWH). Our results showed that FG9 dose-dependently inhibited FX activation by intrinsic FXase complex in a noncompetitive inhibition pattern, where the apparent affinity for FG9 was approximately 1.8-fold higher than that for LMWH. FG9 displayed no inhibitory effect on the activity of FIXa/phospholipid, and did not affect the decay rate of FVIIIa activity. FG9 reduced the apparent affinity of FIXa for FVIIIa in a dose-dependent manner, and accelerated the decay of intrinsic FXase complex activity. FG9 bound to FIXa with high affinity and the FIXa binding sites of FG9 were overlapped with that of LMWH, and the ability of FG-derived oligosaccharides to bind FIXa required the minimum 9 degrees of polymerization. FG9 derivatives were prepared and their structures were confirmed by one-dimensional/two-dimensional nuclear magnetic resonance. Structure–activity relationship studies showed that carboxy reduction significantly weakened its anti-FXase activity and binding affinity to FIXa, while the effects of carboxyl ethyl esterification and deacetylation were relatively weaker. Overall, our results suggest that the nonasaccharide FG9 strongly inhibits intrinsic FXase complex activity via binding to FIXa and disrupting FIXa–FVIIIa interactions, and the free carboxyl groups of FG9 are required for its potent anti-FXase activity.

Antibodies in the Treatment of Haemophilia A—A Biochemical Perspective

Replacement therapy has been proven effective in the management of bleedings in haemophilia A. Nevertheless, this approach comes with several shortcomings, like the need for frequent intravenous infusions and the development of neutralizing antibodies in 20 to 30% of the patients with severe haemophilia A replacement. This has led to the development of novel strategies to expand the spectrum of treatment options, some of which are based on antibody technology. These include a bispecific antibody that bridges enzyme factor IXa and substrate factor X, monoclonal antibodies that block the function of tissue factor pathway inhibitor, and a factor VIII–nanobody fusion protein with strongly enhanced von Willebrand factor binding. In this review, functional and mechanistic considerations on the use of these antibody variants will be discussed.

Biochemical Characteristics of Emicizumab Activity with Factors IXa, X, and VIIa

Introduction. Emicizumab (Hemlibra) is a bivalent antibody that binds to factor IXa and factor X; this binding can promote factor IXa activation of factor X. In patients on prophylaxis with emicizumab, breakthrough bleeding has been treated successfully with factor VIIa (eptacog alfa (activated) (NovoSeven)). Objective. Our goal was to study the biochemistry of the interaction of emicizumab with factor IXa and factor X. We further wanted to study how binding of emicizumab to factor X would impact factor X activation by factor VIIa. Background. Data from surface plasmon resonance binding studies has shown that solution phase interactions between emicizumab and factors IX, IXa, X, and Xa are in the micromolar range (supplement to Kitazawa et al Nature Med 2012; 18: 1570-1574). That same publication showed that a lipid surface is required for activity. Other studies have shown that emicizumab binds to EGF1 of factor IX and EGF2 of factor X (Kitazawa et al Thromb Haemost 2017; 117: 1348-1357). Methods. Lipids were large unilamellar vesicles with 14% phosphatidylserine. Factors IX and X were purified from plasma. The basic design of an assay is to vary one element while holding other elements constant. To determine the apparent Km for factor X, factor X was varied with constant factor IXa (0.1 nM), lipid (100 µM), and emicizumab (400 nM). To determine the apparent Kd for factor IXa, factor IXa was varied with constant factor X (135 nM), lipid (100 µM), and emicizumab (3, 10, or 30 nM). The binding of factor IXa was determined in the presence and the absence of plasma concentration (80 nM) factor IX. Factor VIIa/tissue factor activation of factor X was measured with 0.25 nM VIIa/TF complex with factor X (135 nM) and either no emicizumab, 400 nM, or 50 µM. Factor VIIa activation of factor X in the absence of tissue factor was measured with varied factor VIIa (25-100 nM), fixed factor X (135 nM) and lipid (100 µM) and either no emicizumab, 400 nM, or 50 µM. Results. In the presence of lipid, the apparent binding constants for formation of the IXa-emicizumab-X complex were tighter than the solution phase reactions. Under the conditions studied, the Km,app for factor X was about 25 nM. The Kd,app for factor IXa was about 5 nM. Surprisingly, when lipid and factor X were present, factor IX did not compete with factor IXa for activation of factor X. Factor VIIa/tissue factor activation of factor X was slowed considerably by emicizumab. By contrast, factor VIIa activation of factor X in the absence of tissue factor was not slowed. Conclusions. The binding of factor X to the factor VIIa/tissue factor complex involves multiple domains in factor X. Since emicizumab reduced factor X activation by the factor VIIa/tissue factor complex, it appears that binding of emicizumab to the second EGF domain of factor X interfered with formation of the activating complex. By contrast, activation of factor X by factor VIIa alone was not reduced by emicizumab suggesting that interactions between factor VIIa and the second EGF domain of factor X are not essential for formation of that lipid bound complex. We would predict from the solution phase binding constants that high concentrations of emicizumab would be required to form the complex with factors IXa and X. This is in contrast to the observation that relatively low concentrations of emicizumab give significant shortening of an aPTT assay. The tight binding constants in the presence of lipid may explain the results seen in clotting assays. Further, this significant effect of emicizumab in clotting assays is consistent with the surprising observation that factor IX does not compete with factor IXa in formation of the lipid bound complex of IXa-emicizumab-X. So even a small amount of factor IXa can form functional complexes that activate factor X. Disclosures Monroe: Novo Nordisk A/S: Honoraria, Research Funding. Hoffman:Novo Nordisk A/S: Consultancy, Honoraria, Research Funding.