An RNA aptamer exploits exosite-dependent allostery to achieve specific inhibition of coagulation factor IXa.

Hemostasis relies on a reaction network of serine proteases and their cofactors to form a blood clot. Coagulation factor IXa (protease) plays an essential role in hemostasis as evident from the bleeding disease associated with its absence. RNA aptamers specifically targeting individual coagulation factors have potential as anticoagulants and as probes of the relationship between structure and function. Here, we report X-ray structures of human factor IXa without a ligand bound to the active site either in the apo-form or in complex with an inhibitory aptamer specific for factor IXa. The aptamer binds to an exosite in the catalytic domain and allosterically distorts the active site. Our studies reveal a conformational ensemble of IXa states, wherein large movements of Trp215 near the active site drive functional transitions between the closed (aptamer-bound), latent (apo), and open (substrate-bound) states. The latent state of the apo-enzyme may bear on the uniquely poor catalytic activity of IXa compared to other coagulation proteases. The exosite, to which the aptamer binds, has been implicated in binding VIIIa and heparin, both of which regulate IXa function. Our findings reveal the importance of exosite-driven allosteric modulation of IXa function and new strategies to rebalance hemostasis for therapeutic gain.

Intermediate molecular weight-fucosylated chondroitin sulfate from sea cucumber Cucumaria frondosa is a promising anticoagulant targeting intrinsic factor IXa.

Thromboembolic diseases pose a serious risk to human health worldwide. Fucosylated chondroitin sulfate (FCS) is reported to have good anticoagulant activity with a low bleeding risk. Molecular weight plays a significant role in the anticoagulant activity of FCS, and FCS smaller than octasaccharide in size has no anticoagulant activity. Therefore, identifying the best candidate for developing novel anticoagulant FCS drugs is crucial. Herein, native FCS was isolated from sea cucumber Cucumaria frondosa (FCScf) and depolymerized into a series of lower molecular weights (FCScfs). A comprehensive assessment of the in vitro anticoagulant activity and in vivo bleeding risk of FCScfs with different molecule weights demonstrated that 10 kDa FCScf (FCScf-10 K) had a greater intrinsic anticoagulant activity than low molecular weight heparin (LMWH) without any bleeding risk. Using molecular modeling combined with experimental validation, we revealed that FCScf-10 K can specifically inhibit the formation of the Xase complex by binding the negatively charged sulfate group of FCScf-10 K to the positively charged side chain of arginine residues on the specific surface of factor IXa. Thus, these data demonstrate that the intermediate molecular weight FCScf-10 K is a promising candidate for the development of novel anticoagulant drugs.

Antidote-controlled DNA aptamer modulates human factor IXa activity.

Thrombosis leads to elevated mortality rates and substantial medical expenses worldwide. Human factor IXa (HFIXa) protease is pivotal in tissue factor (TF)-mediated thrombin generation, and represents a promising target for anticoagulant therapy. We herein isolated novel DNA aptamers that specifically bind to HFIXa through systematic evolution of ligands by exponential enrichment (SELEX) method. We identified two distinct aptamers, seq 5 and seq 11, which demonstrated high binding affinity to HFIXa (Kd = 74.07 ± 2.53 nM, and 4.93 ± 0.15 nM, respectively). Computer software was used for conformational simulation and kinetic analysis of DNA aptamers and HFIXa binding. These aptamers dose-dependently prolonged activated partial thromboplastin time (aPTT) in plasma. We further rationally optimized the aptamers by truncation and site-directed mutation, and generated the truncated forms (Seq 5-1t, Seq 11-1t) and truncated-mutated forms (Seq 5-2tm, Seq 11-2tm). They also showed good anticoagulant effects. The rationally and structurally designed antidotes (seq 5-2b and seq 11-2b) were competitively bound to the DNA aptamers and effectively reversed the anticoagulant effect. This strategy provides DNA aptamer drug-antidote pair with effective anticoagulation and rapid reversal, developing advanced therapies by safe, regulatable aptamer drug-antidote pair.

Binding of coagulation factor IXa to procoagulant platelets revisited: Low affinity and interactions with other factors.

Binding of activated factor IX (fIXa) to the phosphatidylserine-expressing procoagulant platelets is a critical step in blood coagulation, which is necessary for the membrane-dependent intrinsic tenase complex assembly and factor X activation. However, the nature and parameters of the fIXa binding sites on the procoagulant platelet surface remain unclear. We used flow cytometry to elucidate the quantitative details of the fluorescently labeled fIXa binding to gel-filtered activated platelets. FIXa bound to the procoagulant platelet subpopulation only, with the parameters (maximal number of binding sites at 58900 ± 3400, Kd at 1000 ± 170 nM) similar to binding observed with phospholipid vesicles. No specific high-affinity binding sites for fIXa were detected, and binding proceeded similarly for different methods of procoagulant platelet production (thrombin, thrombin receptor activation peptide, collagen-related peptide, their combinations, or calcium ionophore A23187). Factor VIII, known to form a high affinity complex with fIXa, enhanced fIXa binding to platelets. In contrast, only competition effects were observed for factor X, which binds fIXa with much lower affinity. Unexpectedly, fIXa itself, fIX, and prothrombin also dose-dependently enhance fIXa binding at concentrations below 1000 nM, suggesting the formation of membrane-bound fIXa dimers and fIXa-prothrombin complexes on platelets. These findings provide a novel perspective on the fIXa binding site on procoagulant platelets, which does not have any major differences from pure phospholipid-based model membranes, exhibits inherently low affinity (3-5 orders of magnitude below the physiologically relevant fIXa concentration) but is significantly enhanced by its cofactor VIII, and regulated by previously unknown membrane interactions.

Kinetics and regulation of coagulation factor X activation by intrinsic tenase on phospholipid membranes.

BACKGROUND: Factor X activation by the phospholipid-bound intrinsic tenase complex is a critical membrane-dependent reaction of blood coagulation. Its regulation mechanisms are unclear, and a number of questions regarding diffusional limitation, pathways of assembly and substrate delivery remain open. METHODS: We develop and analyze here a detailed mechanism-driven computer model of intrinsic tenase on phospholipid surfaces. Three-dimensional reaction-diffusion-advection and stochastic simulations were used where appropriate. RESULTS: Dynamics of the system was predominantly non-stationary under physiological conditions. In order to describe experimental data, we had to assume both membrane-dependent and solution-dependent delivery of the substrate. The former pathway dominated at low cofactor concentration, while the latter became important at low phospholipid concentration. Factor VIIIa-factor X complex formation was the major pathway of the complex assembly, and the model predicted high affinity for their lipid-dependent interaction. Although the model predicted formation of the diffusion-limited layer of substrate for some conditions, the effects of this limitation on the fXa production were small. Flow accelerated fXa production in a flow reactor model by bringing in fIXa and fVIIIa rather than fX. CONCLUSIONS: This analysis suggests a concept of intrinsic tenase that is non-stationary, employs several pathways of substrate delivery depending on the conditions, and is not particularly limited by diffusion of the substrate.

Protein S antibody as an adjunct therapy for hemophilia B.

ABSTRACT: Hemophilia B (HB) is caused by an inherited deficiency of plasma coagulation factor IX (FIX). Approximately 60% of pediatric patients with HB possess a severe form of FIX deficiency (<1% FIX activity). Treatment typically requires replacement therapy through the administration of FIX. However, exogenous FIX has a limited functional half-life, and the natural anticoagulant protein S (PS) inhibits activated FIX (FIXa). PS ultimately limits thrombin formation, which limits plasma coagulation. This regulation of FIXa activity by PS led us to test whether inhibiting PS would extend the functional half-life of FIX and thereby prolong FIX-based HB therapy. We assayed clotting times and thrombin generation to measure the efficacy of a PS antibody for increasing FIX activity in commercially obtained plasma and plasma from pediatric patients with HB. We included 11 pediatric patients who lacked additional comorbidities and coagulopathies. In vivo, we assessed thrombus formation in HB mice in the presence of the FIXa ± PS antibody. We found an accelerated rate of clotting in the presence of PS antibody. Similarly, the peak thrombin formed was significantly greater in the presence of the PS antibody, even in plasma from patients with severe HB. Furthermore, HB mice injected with PS antibody and FIX had a 4.5-fold higher accumulation of fibrin at the thrombus induction site compared with mice injected with FIX alone. Our findings imply that a PS antibody would be a valuable adjunct to increase the effectiveness of FIX replacement therapy in pediatric patients who have mild, moderate, and severe HB.

Molecular Dynamics Simulation Study of the Selective Inhibition of Coagulation Factor IXa over Factor Xa.

Thromboembolic disorders, arising from abnormal coagulation, pose a significant risk to human life in the modern world. The FDA has recently approved several anticoagulant drugs targeting factor Xa (FXa) to manage these disorders. However, these drugs have potential side effects, leading to bleeding complications in patients. To mitigate these risks, coagulation factor IXa (FIXa) has emerged as a promising target due to its selective regulation of the intrinsic pathway. Due to the high structural and functional similarities of these coagulation factors and their inhibitor binding modes, designing a selective inhibitor specifically targeting FIXa remains a challenging task. The dynamic behavior of protein-ligand interactions and their impact on selectivity were analyzed using molecular dynamics simulation, considering the availability of potent and selective compounds for both coagulation factors and the co-crystal structures of protein-ligand complexes. Throughout the simulations, we examined ligand movements in the binding site, as well as the contact frequencies and interaction fingerprints, to gain insights into selectivity. Interaction fingerprint (IFP) analysis clearly highlights the crucial role of strong H-bond formation between the ligand and D189 and A190 in the S1 subsite for FIXa selectivity, consistent with our previous study. This dynamic analysis also reveals additional FIXa-specific interactions. Additionally, the absence of polar interactions contributes to the selectivity for FXa, as observed from the dynamic profile of interactions. A contact frequency analysis of the protein-ligand complexes provides further confirmation of the selectivity criteria for FIXa and FXa, as well as criteria for binding and activity. Moreover, a ligand movement analysis reveals key interaction dynamics that highlight the tighter binding of selective ligands to the proteins compared to non-selective and inactive ligands.

Factor VIII A3 domain residues 1793-1795 represent a factor IXa-interactive site in the tenase complex.

BACKGROUND: Factor (F)VIII functions as a cofactor in the tenase complex responsible for conversion of FX to FXa by FIXa. Earlier studies indicated that one of the FIXa-binding sites is located in residues 1811-1818 (crucially F1816) of the FVIII A3 domain. A putative, three-dimensional structure model of the FVIIIa molecule suggested that residues 1790-1798 form a V-shaped loop, and juxtapose residues 1811-1818 on the extended surface of FVIIIa. AIM: To examine FIXa molecular interactions in the clustered acidic sites of FVIII including residues 1790-1798. METHODS AND RESULTS: Specific ELISA's demonstrated that the synthetic peptides, encompassing residues 1790-1798 and 1811-1818, competitively inhibited the binding of FVIII light chain to active-site-blocked Glu-Gly-Arg-FIXa (EGR-FIXa) (IC50; 19.2 and 42.9 µM, respectively), in keeping with a possible role for the 1790-1798 in FIXa interactions. Surface plasmon resonance-based analyses demonstrated that variants of FVIII, in which the clustered acidic residues (E1793/E1794/D1793) or F1816 contained substituted alanine, bound to immobilized biotin labeled-Phe-Pro-Arg-FIXa (bFPR-FIXa) with a 1.5-2.2-fold greater KD compared to wild-type FVIII (WT). Similarly, FXa generation assays indicated that E1793A/E1794A/D1795A and F1816A mutants increased the Km by 1.6-2.8-fold relative to WT. Furthermore, E1793A/E1794A/D1795A/F1816A mutant showed that the Km was increased by 3.4-fold and the Vmax was decreased by 0.75-fold, compared to WT. Molecular dynamics simulation analyses revealed the subtle changes between WT and E1793A/E1794A/D1795A mutant, supportive of the contribution of these residues for FIXa interaction. CONCLUSION: The 1790-1798 region in the A3 domain, especially clustered acidic residues E1793/E1794/D1795, contains a FIXa-interactive site.

Factor IXa variants resistant to plasma inhibitors enhance clot formation in vivo.

Factor IXa (FIXa) plays a pivotal role in coagulation by contributing to FX activation via the intrinsic pathway. Although antithrombin (AT) and other plasma inhibitors are thought to regulate FIXa procoagulant function, the impact of FIXa inhibition on thrombin generation and clot formation in vivo remains unclear. Here, we generated FIXa variants with altered reactivity to plasma inhibitors that target the FIXa active site but maintain procoagulant function when bound to its cofactor, FVIIIa. We found that selected FIXa variants (eg, FIXa-V16L) have a prolonged activity half-life in the plasma due, in part, to AT resistance. Studies using hemophilia B mice have shown that delayed FIXa inhibition has a major impact on reducing the bleeding phenotype and promoting thrombus formation following administration of FIX protein. Overall, these results demonstrate that the regulation of FIXa inhibition contributes in a major way to the spatial and temporal control of coagulation at the site of vascular injury. Our findings provide novel insights into the physiological regulation of FIXa, enhance our understanding of thrombus formation in vivo via the intrinsic pathway, and suggest that altering FIXa inhibition could have therapeutic benefits.

Factor VIII mutated with Lys1813Ala within the factor IXa-binding region enhances intrinsic coagulation potential.

Factor VIII (FVIII) functions as a cofactor of FIXa for FX activation in the intrinsic tenase complex. The 1811-1818 region in the FVIII A3 domain was observed to contribute to FIXa binding, and the K1813A/K1818A mutant increased the binding affinity for FIXa. The current study aims to identify mutated FVIII protein(s) that increase FVIIIa cofactor activity in the 1811-1818 region. FVIII mutants with K1813A, K1818A, and K1813A/K1818A were expressed in baby hamster kidney cells and were followed by assessments using purified and global coagulation assays for mouse models with hemophilia A (HA). A surface plasmon resonance-based assay revealed that the Kd value of FVIII-K1813A for FIXa interaction was lower than that of the wild-type (WT) (3.9±0.7/6.3±0.3 nM). However, the Km value of FVIII-K1813A for FIXa on tenase activity was comparable with that of the WT, whereas the kcat of this mutant was significantly greater than that of the WT. Thrombin-catalyzed FVIII-K1813A activation was ∼1.3-fold more enhanced than that of the WT, and the spontaneous decay of activated FVIII-K1813A was ∼2.5-fold slower than that of WT. The heat stability assay revealed that the decay rate of FVIII-K1813A was ∼2.5-fold slower than that of WT. Thrombin generation assay and rotational thromboelastometry using blood samples from patients with HA demonstrated that the addition of FVIII-K1813A (0.5 nM) exhibited a coagulation potential compatible with that of WT (1 nM). In the tail clip assay of HA mice, FVIII-K1813A showed a two- to fourfold higher hemostatic potential than that of the WT. FVIII-K1813A, with higher a FIXa binding affinity, enhances the global coagulation potential because of the stability of FVIII/FVIIIa molecules.

Emicizumab: a novel drug in hemophilia A prophylaxis - a narrative review.

INTRODUCTION: Hemophilia A is a genetically conditioned disease leading to hemostatic disorders due to factor VIII (FVIII) deficiency. The treatment of hemophilia has evolved throughout the past years and has significantly changed. One of the newest drugs for prophylactic treatment is the humanized bispecific IgG antibody - emicizumab, which binds with factor IXa and factor X, bridging those factors and thus mimicking the activity of factor VIII. AREAS COVERED: The literature search was done via the PubMed database, with the emphasis on clinical trials and case reports, describing the off-label emicizumab use. This review presents an extensive summary and considers the advantages and disadvantages (side-effects) of emicizumab, describing additional clinical situations, where emicizumab has been successfully used. In our review, we cover information about the mechanisms of action, indications, and efficacy and discuss some chosen case reports about off-label emicizumab use. EXPERT OPINION: Its convenient administration method (subcutaneous) and frequency of injections (from once a week to once a month) makes it a more comfortable treatment, limiting injection-site reactions, hospital stays, costs of prophylaxis, and significantly increasing patients' quality of life. Adverse effects are scarce and rarely serious - the most common ones are reactions at the injection-site and upper respiratory tract infections.

Phosphatidylserine Regulation of Coagulation Proteins Factor IXa and Factor VIIIa.

Blood coagulation is an intricate process, and it requires precise control of the activities of pro- and anticoagulant factors and sensitive signaling systems to monitor and respond to blood vessel insults. These requirements are fulfilled by phosphatidylserine, a relatively miniscule-sized lipid molecule amid the myriad of large coagulation proteins. This review limelight the role of platelet membrane phosphatidylserine (PS) in regulating a key enzymatic reaction of blood coagulation; conversion of factor X to factor Xa by the enzyme factor IXa and its cofactor factor VIIIa. PS is normally located on the inner leaflet of the resting platelet membrane but appears on the outer leaflet surface of the membrane surface after an injury happens. Human platelet activation leads to exposure of buried PS molecules on the surface of the platelet-derived membranes and the exposed PS binds to discrete and specific sites on factors IXa and VIIIa. PS binding to these sites allosterically regulates both factors IXa and VIIIa. The exposure of PS and its binding to factors IXa/VIIIa is a vital step during clotting. Insufficient exposure or a defective binding of PS to these clotting proteins is responsible for various hematologic diseases which are discussed in this review.

SAXS analysis of the intrinsic tenase complex bound to a lipid nanodisc highlights intermolecular contacts between factors VIIIa/IXa.

The intrinsic tenase (Xase) complex, formed by factors (f) VIIIa and fIXa, forms on activated platelet surfaces and catalyzes the activation of factor X to Xa, stimulating thrombin production in the blood coagulation cascade. The structural organization of the membrane-bound Xase complex remains largely unknown, hindering our understanding of the structural underpinnings that guide Xase complex assembly. Here, we aimed to characterize the Xase complex bound to a lipid nanodisc with biolayer interferometry (BLI), Michaelis-Menten kinetics, and small-angle X-ray scattering (SAXS). Using immobilized lipid nanodiscs, we measured binding rates and nanomolar affinities for fVIIIa, fIXa, and the Xase complex. Enzyme kinetic measurements demonstrated the assembly of an active enzyme complex in the presence of lipid nanodiscs. An ab initio molecular envelope of the nanodisc-bound Xase complex allowed us to computationally model fVIIIa and fIXa docked onto a flexible lipid membrane and identify protein-protein interactions. Our results highlight multiple points of contact between fVIIIa and fIXa, including a novel interaction with fIXa at the fVIIIa A1-A3 domain interface. Lastly, we identified hemophilia A/B-related mutations with varying severities at the fVIIIa/fIXa interface that may regulate Xase complex assembly. Together, our results support the use of SAXS as an emergent tool to investigate the membrane-bound Xase complex and illustrate how mutations at the fVIIIa/fIXa dimer interface may disrupt or stabilize the activated enzyme complex.

Site-specific functional roles of the Factor X activation peptide in the intrinsic tenase-mediated Factor X activation.

The conversion of zymogen Factor X (FX) to an active protease involves the removal of a 52-residue long activation peptide (AP). Through site-directed mutagenesis, we investigate the role of the AP and demonstrate that the high abundance of proline residues is important for efficient proteolysis of FX. Moreover, we identify an essential interaction site for Factor IXa (FIXa) between residues 22 and 30 (AP numbering) and find that the residues between 31 and 41 may provide an important interaction site for the intrinsic tenase complex, composed of Factor IXa (FIXa) and Factor VIIIa (FVIIIa). Finally, we suggest that the carbohydrate chain at Asn-39 restricts the activator specificity, as elimination of this glycosylation site increases the activation rate for activation by FIXa and FXa.

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.

Compensatory epistasis explored by molecular dynamics simulations.

A non-negligible proportion of human pathogenic variants are known to be present as wild type in at least some non-human mammalian species. The standard explanation for this finding is that molecular mechanisms of compensatory epistasis can alleviate the mutations' otherwise pathogenic effects. Examples of compensated variants have been described in the literature but the interacting residue(s) postulated to play a compensatory role have rarely been ascertained. In this study, the examination of five human X-chromosomally encoded proteins (FIX, GLA, HPRT1, NDP and OTC) allowed us to identify several candidate compensated variants. Strong evidence for a compensated/compensatory pair of amino acids in the coagulation FIXa protein (involving residues 270 and 271) was found in a variety of mammalian species. Both amino acid residues are located within the 60-loop, spatially close to the 39-loop that performs a key role in coagulation serine proteases. To understand the nature of the underlying interactions, molecular dynamics simulations were performed. The predicted conformational change in the 39-loop consequent to the Glu270Lys substitution (associated with hemophilia B) appears to impair the protein's interaction with its substrate but, importantly, such steric hindrance is largely mitigated in those proteins that carry the compensatory residue (Pro271) at the neighboring amino acid position.

A factor VIIIa-mimetic bispecific antibody, Mim8, ameliorates bleeding upon severe vascular challenge in hemophilia A mice.

Hemophilia A is a bleeding disorder resulting from deficient factor VIII (FVIII), which normally functions as a cofactor to activated factor IX (FIXa) that facilitates activation of factor X (FX). To mimic this property in a bispecific antibody format, a screening was conducted to identify functional pairs of anti-FIXa and anti-FX antibodies, followed by optimization of functional and biophysical properties. The resulting bispecific antibody (Mim8) assembled efficiently with FIXa and FX on membranes, and supported activation with an apparent equilibrium dissociation constant of 16 nM. Binding affinity with FIXa and FX in solution was much lower, with equilibrium dissociation constant values for FIXa and FX of 2.3 and 1.5 µM, respectively. In addition, the activity of Mim8 was dependent on stimulatory activity contributed by the anti-FIXa arm, which enhanced the proteolytic activity of FIXa by 4 orders of magnitude. In hemophilia A plasma and whole blood, Mim8 normalized thrombin generation and clot formation, with potencies 13 and 18 times higher than a sequence-identical analogue of emicizumab. A similar potency difference was observed in a tail vein transection model in hemophilia A mice, whereas reduction of bleeding in a severe tail-clip model was observed only for Mim8. Furthermore, the pharmacokinetic parameters of Mim8 were investigated and a half-life of 14 days shown in cynomolgus monkeys. In conclusion, Mim8 is an activated FVIII mimetic with a potent and efficacious hemostatic effect based on preclinical data.