Beating tissue factor at its own game: Design and properties of a soluble tissue factor-independent coagulation factor VIIa.

Two decades of research have uncovered the mechanism by which the complex of tissue factor (TF) and the plasma serine protease factor VIIa (FVIIa) mediates the initiation of blood coagulation. Membrane-anchored TF directly interacts with substrates and induces allosteric effects in the protease domain of FVIIa. These properties are also recapitulated by the soluble ectodomain of TF (sTF). At least two interdependent allosteric activation pathways originate at the FVIIa:sTF interface are proposed to enhance FVIIa activity upon sTF binding. Here, we sought to engineer an sTF-independent FVIIa variant by stabilizing both proposed pathways, with one pathway terminating at segment 215-217 in the activation domain and the other pathway terminating at the N terminus insertion site. To stabilize segment 215-217, we replaced the flexible 170 loop of FVIIa with the more rigid 170 loop from trypsin and combined it with an L163V substitution (FVIIa-VYT). The FVIIa-VYT variant exhibited 60-fold higher amidolytic activity than FVIIa, and displayed similar FX activation and antithrombin inhibition kinetics to the FVIIa.sTF complex. The sTF-independent activity of FVIIa-VYT was partly mediated by an increase in the N terminus insertion and, as shown by X-ray crystallography, partly by Tyr-172 inserting into a cavity in the activation domain stabilizing the S1 substrate-binding pocket. The combination with L163V likely drove additional changes in a delicate hydrogen-bonding network that further stabilized S1-S3 sites. In summary, we report the first FVIIa variant that is catalytically independent of sTF and provide evidence supporting the existence of two TF-mediated allosteric activation pathways.

Allostery in Coagulation Factor VIIa Revealed by Ensemble Refinement of Crystallographic Structures.

A critical step in injury-induced initiation of blood coagulation is the formation of the complex between the trypsin-like protease coagulation factor VIIa (FVIIa) and its cofactor tissue factor (TF), which converts FVIIa from an intrinsically poor enzyme to an active protease capable of activating zymogens of downstream coagulation proteases. Unlike its constitutively active ancestor trypsin, FVIIa is allosterically activated (by TF). Here, ensemble refinement of crystallographic structures, which uses multiple copies of the entire structure as a means of representing structural flexibility, is applied to explore the impacts of inhibitor binding to trypsin and FVIIa, as well as cofactor binding to FVIIa. To assess the conformational flexibility and its role in allosteric pathways in these proteases, main-chain hydrogen bond networks are analyzed by calculating the hydrogen-bond propensity. Mapping pairwise propensity differences between relevant structures shows that binding of the inhibitor benzamidine to trypsin has a minor influence on the protease flexibility. For FVIIa, in contrast, the protease domain is "locked" into the catalytically competent trypsin-like configuration upon benzamidine binding as indicated by the stabilization of key structural features: the nonprime binding cleft and the oxyanion hole are stabilized, and the effect propagates from the active site region to the calcium-binding site and to the vicinity of the disulphide bridge connecting with the light chain. TF binding to FVIIa furthermore results in stabilization of the 170 loop, which in turn propagates an allosteric signal from the TF-binding region to the active site. Analyses of disulphide bridge energy and flexibility reflect the striking stability difference between the unregulated enzyme and the allosterically activated form after inhibitor or cofactor binding. The ensemble refinement analyses show directly, for the first time to our knowledge, whole-domain structural footprints of TF-induced allosteric networks present in x-ray crystallographic structures of FVIIa, which previously only have been hypothesized or indirectly inferred.

FIXing postinfusion monitoring: Assay experiences with N9-GP (nonacog beta pegol; Refixia® ; Rebinyn® ).

N9-GP (nonacog beta pegol; Refixia® ; Rebinyn® , Novo Nordisk A/S, Bagsvaerd, Denmark) is a glycoPEGylated extended half-life recombinant factor IX (rFIX) that exhibits efficacy and potency comparable to unmodified FIX molecules in non-clinical models. Phase 3 clinical trials have confirmed the efficacy and tolerability of N9-GP for the prevention and on-demand treatment of bleeding episodes in patients with haemophilia B. Recent studies have shown that PEGylation affects clotting times in activated partial thromboplastin time (aPTT)-based one-stage activity assays due to interaction between the FIX molecule and certain aPTT reagents. In recognition of the challenges surrounding FIX activity assessment, the identification of consistent, reproducible and accurate assays to measure FIX activity has been a priority for Novo Nordisk, running in parallel to the clinical development program for N9-GP. N9-GP activity can be reliably measured using chromogenic substrate assays and specific aPTT reagents. The conjugation of the PEG moiety to the FIX molecule may affect one-stage aPTT-based clotting assays in a reagent-dependent manner. Many aPTT reagents that use silica as the contact activator dramatically overestimate N9-GP activity due to premature activation. On the other hand, the contact activator in some other aPTT reagents negatively affects the enzymatic activity of FXIa, causing the underestimation of N9-GP activity. While N9-GP activity cannot be measured consistently with all available aPTT reagents, accurate N9-GP measurements can be achieved with certain aPTT reagents. Here, we review the studies that led to these findings and summarize the current options for accurate measurement of N9-GP in patient samples.

In vitro evidence of a tissue factor-independent mode of action of recombinant factor VIIa in hemophilia.

Successful competition of activated factor VII (FVIIa) with zymogen factor VII (FVII) for tissue factor (TF) and loading of the platelet surface with FVIIa are plausible driving forces behind the pharmacological effect of recombinant FVIIa (rFVIIa) in hemophilia patients. Thrombin generation measurements in platelet-rich hemophilia A plasma revealed competition for TF, which potentially could reduce the effective (r)FVIIa:TF complex concentration and thereby attenuate factor Xa production. However, (auto)activation of FVII apparently counteracted the negative effect of zymogen binding; a small impact was observed at endogenous concentrations of FVII and FVIIa but was virtually absent at pharmacological amounts of rFVIIa. Moreover, corrections of the propagation phase in hemophilia A required rFVIIa concentrations above the range where a physiological level of FVII was capable to downregulate thrombin generation. These data strongly suggest that rFVIIa acts independently of TF in hemophilia therapy and that FVII displacement by rFVIIa is a negligible mechanistic component.

Sites involved in intra- and interdomain allostery associated with the activation of factor VIIa pinpointed by hydrogen-deuterium exchange and electron transfer dissociation mass spectrometry.

Factor VIIa (FVIIa) is a trypsin-like protease that plays an important role in initiating blood coagulation. Very limited structural information is available for the free, inactive form of FVIIa that circulates in the blood prior to vascular injury and the molecular details of its activity enhancement remain elusive. Here we have applied hydrogen/deuterium exchange mass spectrometry coupled to electron transfer dissociation to pinpoint individual residues in the heavy chain of FVIIa whose conformation and/or local interaction pattern changes when the enzyme transitions to the active form, as induced either by its cofactor tissue factor or a covalent active site inhibitor. Identified regulatory residues are situated at key sites across one continuous surface of the protease domain spanning the TF-binding helix across the activation pocket to the calcium binding site and are embedded in elements of secondary structure and at the base of flexible loops. Thus these residues are optimally positioned to mediate crosstalk between functional sites in FVIIa, particularly the cofactor binding site and the active site. Our results unambiguously show that the conformational allosteric activation signal extends to the EGF1 domain in the light chain of FVIIa, underscoring a remarkable intra- and interdomain allosteric regulation of this trypsin-like protease.

A novel B-domain O-glycoPEGylated FVIII (N8-GP) demonstrates full efficacy and prolonged effect in hemophilic mice models.

Frequent infusions of intravenous factor VIII (FVIII) are required to prevent bleeding associated with hemophilia A. To reduce the treatment burden, recombinant FVIII with a longer half-life was developed without changing the protein structure. FVIII-polyethylene glycol (PEG) conjugates were prepared using an enzymatic process coupling PEG (ranging from 10 to 80 kDa) selectively to a unique O-linked glycan in the FVIII B-domain. Binding to von Willebrand factor (VWF) was maintained for all conjugates. Upon cleavage by thrombin, the B-domain and the associated PEG were released, generating activated FVIII (FVIIIa) with the same primary structure and specific activity as native FVIIIa. In both FVIII- and VWF-deficient mice, the half-life was found to increase with the size of PEG. In vivo potency and efficacy of FVIII conjugated with a 40-kDa PEG (N8-GP) and unmodified FVIII were not different. N8-GP had a longer duration of effect in FVIII-deficient mouse models, approximately a twofold prolonged half-life in mice, rabbits, and cynomolgus monkeys; however, the prolongation was less pronounced in rats. Binding capacity of N8-GP on human monocyte-derived dendritic cells was reduced compared with unmodified FVIII, resulting in several-fold reduced cellular uptake. In conclusion, N8-GP has the potential to offer efficacious prevention and treatment of bleeds in hemophilia A at reduced dosing frequency.

Antibody-induced enhancement of factor VIIa activity through distinct allosteric pathways.

In the absence of its cofactor tissue factor (TF), coagulation factor VIIa (FVIIa) predominantly exists in a zymogen-like, catalytically incompetent state. Here we demonstrate that conformation-specific monoclonal antibodies (mAbs) can be used to characterize structural features determining the activity of FVIIa. We isolated two classes of mAbs, which both increased the catalytic efficiency of FVIIa more than 150-fold. The effects of the antibodies were retained with a FVIIa variant, which has been shown to be inert to allosteric activation by the natural activator TF, suggesting that the antibodies and TF employ distinct mechanisms of activation. The antibodies could be classified into two groups based on their patterns of affinities for different conformations of FVIIa. Whereas one class of antibodies affected both the K(m) and k(cat), the other class mainly affected the K(m). The antibody-induced activity enhancement could be traced to maturation of the S1 substrate binding pocket and the oxyanion hole, evident by an increased affinity for p-aminobenzamidine, an increased rate of antithrombin inhibition, an increased rate of incorporation of diisopropylfluorophosphate, and an enhanced fraction of molecules with a buried N terminus of the catalytic domain in the presence of antibodies. As demonstrated by site-directed mutagenesis, the two groups of antibodies appear to have overlapping, although clearly different, epitopes in the 170-loop. Our findings suggest that binding of ligands to specific residues in the 170-loop or its spatial vicinity may stabilize the S1 pocket and the oxyanion hole, and they may have general implications for the molecular understanding of FVIIa regulatory mechanisms.

Activation peptides prolong the murine plasma half-life of human factor VII.

Coagulation factors VII (FVII), IX (FIX), X (FX), and protein C share the same domain organization but display very different plasma half-lives. It is plausible that the half-life is influenced by the activation peptide, differing in length and glycosylation and missing in FVII. To test this hypothesis, the influence of activation peptides on the plasma half-life of human FVII was studied by administering human FVII variants containing activation peptide motifs to mice. Insertion of the activation peptide from FX gave 4-fold longer terminal half-life (5.5 hours vs 1.4 hours for FVII), whereas the activation peptide from FIX and protein C resulted in half-lives of 4.3 and 1.7 hours, respectively. Using FX's activation peptide we identified the N-linked glycans as structural features important for the half-life. The peptide location within the FVII molecule appeared not to be critical because similar prolongation was obtained with the activation peptide inserted immediately before the normal site of activation and at the C-terminus. However, only the latter variant was activatable, yielding full amidolytic activity and reduced proteolytic activity with preserved long half-life. Our data support that activation peptides function as plasma retention signals and constitute a new manner to extend the half-life of FVII(a).

The endothelial protein C receptor supports tissue factor ternary coagulation initiation complex signaling through protease-activated receptors.

Protease-activated receptor (PAR) signaling is closely linked to the cellular activation of the pro- and anticoagulant pathways. The endothelial protein C receptor (EPCR) is crucial for signaling by activated protein C through PAR1, but EPCR may have additional roles by interacting with the 4-carboxyglutamic acid domains of procoagulant coagulation factors VII (FVII) and X (FX). Here we show that soluble EPCR regulates the interaction of FX with human or mouse tissue factor (TF)-FVIIa complexes. Mutagenesis of the FVIIa 4-carboxyglutamic acid domain and dose titrations with FX showed that EPCR interacted primarily with FX to attenuate FX activation in lipid-free assay systems. In human cell models of TF signaling, antibody inhibition of EPCR selectively blocked PAR activation by the ternary TF-FVIIa-FXa complex but not by the non-coagulant TF-FVIIa binary complex. Heterologous expression of EPCR promoted PAR1 and PAR2 cleavage by FXa in the ternary complex but did not alter PAR2 cleavage by TF-FVIIa. In murine smooth muscle cells that constitutively express EPCR and TF, thrombin and FVIIa/FX but not FVIIa alone induced PAR1-dependent signaling. Although thrombin signaling was unchanged, cells with genetically reduced levels of EPCR no longer showed a signaling response to the ternary complex. These results demonstrate that EPCR interacts with the ternary TF coagulation initiation complex to enable PAR signaling and suggest that EPCR may play a role in regulating the biology of TF-expressing extravascular and vessel wall cells that are exposed to limited concentrations of FVIIa and FX provided by ectopic synthesis or vascular leakage.

Vatreptacog alfa from conception to clinical proof of concept.

Vatreptacog alfa is a genetically engineered variant of recombinant factor VIIa (rFVIIa) containing three amino acid changes. Aspartic acid, valine, and glutamine residues replace valine, glutamic acid, and methionine at positions 158, 296, and 298, respectively. These substitutions result in considerable enhancement of the intrinsic (tissue factor-independent) capability to activate factor X and the downstream hemostatic events are consequently augmented. The beneficial effects of vatreptacog alfa have been demonstrated in numerous in vitro systems attempting to mimic hemophilia and corroborated in in vivo models. Vatreptacog alfa has successfully passed through phase 1 and 2 clinical trials and the molecule is currently being explored in phase 3 clinical trial for the treatment of bleedings in hemophilia patients with inhibitors. This article describes the proposed mechanism behind the increased activity and action of vatreptacog alfa and reviews available data, which suggest that vatreptacog alfa could be a valuable addition to the existing portfolio of treatment options for hemophilia patients with inhibitors.

Intravascular inhibition of factor VIIa and the analogue NN1731 by antithrombin.

NN1731 is a recombinant activated factor VII (rFVIIa) analogue with increased intrinsic activity. This also applies to its reactivity towards antithrombin (AT), the role of which was investigated in a pharmacokinetic (PK) study. NN1731 or rFVIIa was administered to normal and haemophilia A dogs and elimination was measured by FVIIa clot activity, FVIIa- and FVIIa-AT antigen. In vitro AT complex formation was studied in canine plasma spiked with NN1731 or rFVIIa. Based on FVIIa antigen concentrations, PK profiles in normal and haemophilia A dogs were similar for NN1731 and rFVIIa with antigen half lives, t(½) ≈1·8 h. In contrast, PK profiles based on activity measurements were distinctly different. NN1731 induced a strong, short lasting (t(½) ≈0·5 h) pro-coagulant response, whereas rFVIIa induced a lower, longer lasting (t(½) ≈1·1 h) response. Western Blot and FVIIa-AT antigen analysis demonstrated in vivo AT complex formation that accounted for these divergences. AT complex formation with FVIIa or NN1731 in vitro in canine plasma was considerably slower than the in vivo reaction. The results suggest that in vivo inhibition by AT contributes significantly to define drug duration in haemophilia treatment with rFVIIa and in particular with the NN1731 analogue.

Effects on the conformation of FVIIa by sTF and Ca²âº binding: studies of fluorescence resonance energy transfer and quenching.

The apparent length of FVIIa in solution was estimated by a FRET analysis. Two fluorescent probes, fluorescein (Fl-FPR) and a rhodamine derivative (TMR), were covalently attached to FVIIa. The binding site of Fl-FPR was in the protease domain whereas TMR was positioned in the Gla domain, thus allowing a length measure over virtually the whole extension of the protein. From the FRET measurements, the distances between the two probes were determined to be 61.4 for free FVIIa and 65.5Å for FVIIa bound to soluble tissue factor (sTF). These seemingly short distances, compared to those anticipated based on the complex crystal structure, require that the probes stretch towards each other. Thus, the apparent distance from the FRET analysis was shown to increase with 4Å upon formation of a complex with sTF in solution. However, considering how protein dynamics, based on recent molecular dynamics simulations of FVIIa and sTF:FVIIa (Y.Z. Ohkubo, J.H. Morrissey, E. Tajkhorshid, J. Thromb. Haemost. 8 (2010) 1044-1053), can influence the apparent fluorescence signal our calculations indicated that the global average conformation of active-site inhibited FVIIa is nearly unaltered upon ligation to sTF. It is known from amidolytic activity measurements that Ca(2+) binding leads to activation of FVIIa, but we have for the first time directly demonstrated conformational changes in the environment of the active site upon Ca(2+) binding. Interestingly, this Ca(2+)-induced conformational change can be noted even in the presence of an inhibitor. Forming a complex with sTF further stabilized this conformational change, leading to a more inaccessible active-site located probe.

Extensive small-angle X-ray scattering studies of blood coagulation factor VIIa reveal interdomain flexibility.

Blood coagulation factor VIIa (FVIIa) is used in the treatment of replacement therapy resistant hemophilia patients, and FVIIa is normally activated upon complex formation with tissue factor (TF), potentially in context with structural rearrangements. The solution behavior of uncomplexed FVIIa is important for understanding the mechanism of activation and for the stability and activity of the pharmaceutical product. However, crystal structures of FVIIa in complex with TF and of truncated free FVIIa reveal different overall conformations while previous small-angle scattering studies suggest FVIIa always to be fully extended in solution. Here, small-angle X-ray scattering analysis of multiple forms of FVIIa and TF under several experimental conditions elaborate extensively on the understanding of the solution behavior of FVIIa. We reveal significant FVIIa domain flexibility in solution, whereas TF has a well-defined conformation. Unspecific formation of dimers of FVIIa is also observed and varies with experimental conditions. In particular, active site-inhibited FVIIa displays a distinct solution behavior different from that of uninhibited FVIIa, which may reflect structural rearrangements causing resistance to activation, thereby emphasizing the connection between the distribution of different conformations of FVII and the mechanism of activation.

Whole blood coagulation in children with thrombocytopenia and the response to platelet replacement, recombinant factor VIIa, and a potent factor VIIa analogue.

The present study evaluated dynamic coagulation profiles, platelet aggregation, and thrombin generation in whole blood (WB) from eight children with thrombocytopenia during chemotherapy, and the haemostatic potential of platelets (+60 x 10(9)/l), recombinant factor VIIa (rFVIIa - NovoSeven), and a potent rFVIIa analogue (NN1731) both at 1 and 4 microg/ml. Dynamic WB coagulation profiles were recorded by thrombelastometry employing activation with tissue factor (TF - Innovin) at low concentrations. The baseline WB coagulation patterns were characterised by a prolonged clotting time (CT) and a pronounced reduction in clot propagation (MaxVel). WB platelet aggregation signal was five times lower in the study group compared with measurements in modelled thrombocytopenic WB from healthy volunteers. In vitro addition of fresh platelets reversed the coagulopathy. Addition of rFVIIa induced no significant changes in the thrombelastographic profile, whereas spiking with NN1731 shortened the CT significantly. The changes in WB thrombin generation reflected the changes in the MaxVel. In modeled thrombocytopenic WB from healthy individuals, both rFVIIa and NN1731 exhibited a pronounced haemostatic effect with NN1731 showing greater potency than rFVIIa. Compromised platelet function in the study group was assumingly responsible for the weakened haemostatic potential of rFVIIa as well as that of NN1731.

Limited factor VIIa surface localization requirement of the factor VIIa-induced overall thrombin generation in platelet-rich hemophilia A plasma.

BACKGROUND: Thrombin generation assay (TGA) and thrombelastography (TEG) are increasingly employed, global, in vitro methods for assessment of the procoagulant potential of plasma/blood and possibly ideally suited tools to monitor, for example, therapy with recombinant factor VIIa (FVIIa). It remains controversial to what extent results obtained with spiked and postinfusion samples reflect the outcome in patients. OBJECTIVE: To characterize the TGA response to FVIIa in hemophilic plasma and compare with TEG data. METHODS: Hemophilia A (HA) was induced in platelet-rich plasma (PRP) from healthy volunteers, followed by spiking with FVIIa, γ-carboxyglutamic acid (Gla)-domainless FVIIa or V158D/E296V/M298Q-FVIIa (FVIIaDVQ). Samples were triggered with tissue factor and analyzed by TGA and TEG in parallel. RESULTS: Addition of 25 nmol L-1 FVIIa to HA PRP normalized TEG parameters angle and R time, as well as TGA lag time, but had poor effects on the thrombin peak height and velocity index. All parameters (at least) returned to normal levels either upon adding a much higher concentration of FVIIa (~1500 nmol L-1) or by using the superactive variant FVIIaDVQ. Surprisingly, Gla-domainless derivatives of FVIIa and FVIIaDVQ also yielded considerable effects in HA PRP. CONCLUSIONS: The good general responses to clinically effective concentrations of FVIIa (25 and 75 nmol L-1) seen in TEG analyses, as well as for TGA lag time, were accompanied by far-from-normal thrombin peaks. A near-normal thrombin peak response required the presence of considerably higher FVIIa activity but, intriguingly, relied only marginally on a functional Gla domain (ie, on platelet surface localization).

Reagent-specific underestimation of turoctocog alfa pegol (N8-GP) clotting activity owing to decelerated activation by thrombin.

BACKGROUND: Factor VIII (FVIII) procoagulant activity is commonly assessed by measuring the activated partial thromboplastin time (APTT) to clot formation using one of the many available but differently composed reagents. The majority of APTT reagents also accurately recover the activity of the extended half-life molecule N-glycoPEGylated FVIII (N8-GP; turoctocog alfa pegol), while a few silica-based reagents give a low recovery. OBJECTIVE: To identify the cause of N8-GP activity underestimation in the presence of certain silica-based APTT reagents. METHODS: Development of FVIIIa-dependent tenase activity and appearance of FVIIIa from N8-GP and its non-PEGylated counterpart (N8) were compared using clotting assays, a factor Xa (FXa) activity assay mimic thereof, and thrombin activation time courses. RESULTS: A strong correlation was demonstrated between clotting and FXa activity assays based on similar recoveries of N8-GP activity and equal responses to an altered duration of the contact activation phase, validating the latter as a useful clotting assay mimic. Contact activation phase duration influenced, and could even eliminate, N8-GP activity underestimation. Thrombin-catalyzed conversion of N8-GP to FVIIIa was considerably slower than that of N8 despite similar extents of adsorption to silica particles in APTT-SP, suggesting different modes and/or orientations of adsorption. CONCLUSIONS: Some contact activators reduce thrombin's ability to cleave N8-GP more than native FVIII. Decelerated thrombin activation of N8-GP is reflected in delayed FVIIIa-dependent appearance of FXa activity in plasma, in turn leading to prolonged clotting time. This forms the basis for underestimation of N8-GP activity as measured by one-stage clotting assay against a FVIII standard.

Crystal structure, epitope, and functional impact of an antibody against a superactive FVIIa provide insights into allosteric mechanism.

BACKGROUND: Blood coagulation factor VIIa (FVIIa) plays its critical physiological role in the initiation of hemostasis. Even so, recombinant FVIIa is successfully used as a bypassing agent for factor VIII or IX in the treatment of bleeds in patients with severe hemophilia with inhibitors. To investigate the utility of more potent FVIIa variants with enhanced intrinsic activity, molecules such as V21D/E154V/M156Q-FVIIa (FVIIaDVQ) were designed. METHODS: Surface plasmon resonance was used to characterize the binding of mAb4F5 to FVIIaDVQ and related variants. X-ray crystallography was used to determine the structure of the Fab fragment of mAb4F5 (Fab4F5). Molecular docking and small angle X-ray scattering led to a model of FVIIaDVQ:Fab4F5 complex. RESULTS: The binding experiments, functional effects on FVIIaDVQ and structure of mAb4F5 (originally intended for quantification of FVIIaDVQ in samples containing FVII(a)) pinpointed the epitope (crucial role for residue Asp21) and shed light on the role of the N-terminus of the protease domain in FVIIa allostery. The potential antigen-combining sites are composed of 1 hydrophobic and 1 negatively charged pocket formed by 6 complementarity-determining region (CDR) loops. Structural analysis of Fab4F5 shows that the epitope interacts with the periphery of the hydrophobic pocket and provides insights into the molecular basis of mAb4F5 recognition and tight binding of FVIIaDVQ. CONCLUSION: The binary complex explains and supports the selectivity and functional consequences of Fab4F5 association with FVIIaDVQ and illustrates the potentially unique antigenicity of this FVIIa variant. This will be useful in the design of less immunogenic variants.

Protein disulfide isomerase has no stimulatory chaperone effect on factor X activation by factor VIIa-soluble tissue factor.

INTRODUCTION: It was recently reported that protein disulfide isomerase (PDI) stimulates factor X (FX) activation by factor VIIa (FVIIa) bound to soluble tissue factor (sTF) in a purified system and that PDI may be responsible for activating cellular tissue factor (TF) and switching it between its roles in blood coagulation and cellular signalling. This study further investigates the former effect of PDI. METHOD: FX activations by FVIIa-sTF(1-219) were carried out in the presence of different forms of PDI, with annexin V or detergent present in the system and using various forms of FVIIa and FX. In addition, FVIIa-lipidated TF was used as the FX activator. RESULTS: Recombinant human PDI did not influence FX activation by FVIIa-sTF(1-219), whereas PDI purified from bovine liver enhanced the activation rate in a dose-dependent manner. The inclusion of annexin V or detergent abolished the stimulatory effect. Removal of the phospholipid-interactive gamma-carboxyglutamic acid (Gla)-containing domain from either FVIIa or FX obliterated the bovine PDI-induced enhancement of FX activation, as did the introduction of F4A or L8A mutation in FVIIa. The presence of 25 nM bovine PDI lowered the apparent K(m) for FX from far above 10 microM to 1-2 microM. No PDI effect was seen when FVIIa-lipidated TF was the FX activator. CONCLUSIONS: FX activation is insensitive to PDI per se and a phospholipid contaminant in the bovine PDI preparation acts stimulatory when sTF, but not lipidated TF, is the cofactor. Strong support is provided by the lacking effect of bovine PDI after removal or modification of the Gla domain in either FVIIa or FX as well as by the effects of annexin V and detergents and the decreased K(m) value.

A variant of recombinant factor VIIa with enhanced procoagulant and antifibrinolytic activities in an in vitro model of hemophilia.

OBJECTIVE: Recombinant factor VIIa (rFVIIa, NovoSeven) has proven efficacy in treating bleeding in hemophilia patients with inhibitors. A rFVIIa analog with mutations V158D/E296V/M298Q (NN1731) exhibits increased procoagulant activity in in vitro and in vivo models. The aim of this work was to define the effects of NN1731 toward factor X activation, platelet activation, thrombin generation, and fibrin clot formation and stability. METHODS AND RESULTS: In a cell-based in vitro model of hemophilia, rFVIIa and NN1731 similarly increased factor X activation on tissue factor-bearing cells; however, NN1731 exhibited 30-fold higher factor Xa generation on platelets than similar rFVIIa concentrations. NN1731-mediated thrombin generation depended on platelet activation, but NN1731 did not directly activate platelets. NN1731 produced 4- to 10-fold higher maximal thrombin generation rates than equal rFVIIa concentrations. Both rFVIIa and NN1731 shortened clotting times in the absence of factors IX and VIII; however, NN1731 did so at 50-fold lower concentrations than were required of rFVIIa. In fibrinolytic conditions, both rFVIIa and NN1731 increased fibrin formation and stability; however, NN1731 was effective at 50-fold lower concentrations than were required of rFVIIa. CONCLUSIONS: By increasing factor Xa generation, NN1731 promotes the formation of thrombin and a stable clot to a greater degree than rFVIIa.

A combined structural dynamics approach identifies a putative switch in factor VIIa employed by tissue factor to initiate blood coagulation.

Coagulation factor VIIa (FVIIa) requires tissue factor (TF) to attain full catalytic competency and to initiate blood coagulation. In this study, the mechanism by which TF allosterically activates FVIIa is investigated by a structural dynamics approach that combines molecular dynamics (MD) simulations and hydrogen/deuterium exchange (HX) mass spectrometry on free and TF-bound FVIIa. The differences in conformational dynamics from MD simulations are shown to be confined to regions of FVIIa observed to undergo structural stabilization as judged by HX experiments, especially implicating activation loop 3 (residues 365-374{216-225}) of the so-called activation domain and the 170-loop (residues 313-322{170A-175}) succeeding the TF-binding helix. The latter finding is corroborated by experiments demonstrating rapid deglycosylation of Asn322 in free FVIIa by PNGase F but almost complete protection in the presence of TF or an active-site inhibitor. Based on MD simulations, a key switch of the TF-induced structural changes is identified as the interacting pair Leu305{163} and Phe374{225} in FVIIa, whose mutual conformations are guided by the presence of TF and observed to be closely linked to the structural stability of activation loop 3. Altogether, our findings strongly support an allosteric activation mechanism initiated by the stabilization of the Leu305{163}/Phe374{225} pair, which, in turn, stabilizes activation loop 3 and the S(1) and S(3) substrate pockets, the activation pocket, and N-terminal insertion.