Factor VIIa Variants Research Articles

Site-directed mutagenesis of tissue factor pathway inhibitor–binding exosite D60A on factor VII results in a new factor VII variant with lower coagulant activity

BackgroundRecombinant factor (F)VIIa (rFVIIa) has been approved by the US Food and Drug Administration for the treatment of hemophilia A and B with inhibitors and congenital FVII deficiency. Moreover, the investigational uses of rFVIIa are becoming of interest since it can be used to treat various clinical bleeding conditions. However, there is evidence showing that rFVIIa is a potent procoagulant agent that potentially leads to an increased risk of thrombotic complications. ObjectivesTo design a new rFVII with lower coagulant activity that could potentially be used as an alternative hemostatic agent aiming to minimize the risk of thrombogenicity. MethodsD60A was introduced into the F7 sequence by polymerase chain reaction–based mutagenesis. Wild type (WT) and D60A were generated in human embryonic kidney 293T cells by stable transfection. FVII coagulant activities were determined by amidolytic cleavage of the FVIIa-specific substrate, 2-step FXa generation, thrombin generation (TG), and clot-based assays. ResultsWT and D60A demonstrated similar FVIIa amidolytic activity. However, D60A showed approximately 50% activity on FX activation and significantly longer lag time in the TG assay than that shown by WT. The clotting time produced by D60A spiked in FVII-deficient plasma was significantly prolonged than that of WT. Additionally, the ex vivo plasma half-lives of WT and D60A were comparable. ConclusionD60A demonstrated lower coagulant activities, most likely due to the weakening of FX binding, leading to impaired FX activation and delayed TG and fibrin formation. Considering that a plasma FVII level of 15% to 25% is adequate for normal hemostasis, D60A is a molecule of interest for future development of an rFVII with a lesser extent of thrombogenicity.

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.

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

BackgroundBlood 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. MethodsSurface 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. ResultsThe 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. ConclusionThe 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.

Molecular Basis of Enhanced Activity in Factor VIIa-Trypsin Variants Conveys Insights into Tissue Factor-mediated Allosteric Regulation of Factor VIIa Activity

The complex of coagulation factor VIIa (FVIIa), a trypsin-like serine protease, and membrane-bound tissue factor (TF) initiates blood coagulation upon vascular injury. Binding of TF to FVIIa promotes allosteric conformational changes in the FVIIa protease domain and improves its catalytic properties. Extensive studies have revealed two putative pathways for this allosteric communication. Here we provide further details of this allosteric communication by investigating FVIIa loop swap variants containing the 170 loop of trypsin that display TF-independent enhanced activity. Using x-ray crystallography, we show that the introduced 170 loop from trypsin directly interacts with the FVIIa active site, stabilizing segment 215-217 and activation loop 3, leading to enhanced activity. Molecular dynamics simulations and novel fluorescence quenching studies support that segment 215-217 conformation is pivotal to the enhanced activity of the FVIIa variants. We speculate that the allosteric regulation of FVIIa activity by TF binding follows a similar path in conjunction with protease domain N terminus insertion, suggesting a more complete molecular basis of TF-mediated allosteric enhancement of FVIIa activity.

Modulating the Antithrombin-Mediated in Vivo Clearance of Coagulation Factor VIIa

The short half-life of coagulation factor VIIa (FVIIa) in circulation is the result of elimination through multiple pathways of which inactivation by the plasma inhibitor antithrombin (AT) accounts for as much as 65% of the total clearance in humans. Remarkably, the rate of inhibition in vivo is about 30 times greater than the uncatalyzed rate of inhibition in vitro suggesting the presence of rate enhancing components in vivo (Agersø et al. (2011) J Thromb Haemost, 9:333-338). Prime candidates include endogenous heparin-like glycosaminoglycans (GAGs) potentiating the reactivity of antithrombin, or tissue factor (TF) which upon binding to FVIIa increases its susceptibility to inhibition.In the present study site-directed mutagenesis of FVIIa was undertaken to identify variants with altered AT reactivity in order to explore the relationship between the reactivity of FVIIa with AT in vitro and in vivo as well as the nature of endogenous rate enhancing components. The pharmacokinetic properties of FVIIa variants were determined in Sprague Dawley rats as this model recapitulates the aspects of AT-mediated FVIIa clearance observed in humans and allows for interaction of human FVIIa with endogenous rat TF. Similar to the human situation, inactivation of wild-type FVIIa in rat is evident as an accumulation of circulating FVIIa-AT complexes and a progressive divergence of the pharmacokinetic profiles representing FVIIa clot activity and total FVIIa antigen.Initially, the ability to modulate the in vivo complex formation with AT was investigated using two FVIIa variants exhibiting enhanced (>200%) or reduced (<10%) in vitro reactivity with AT, respectively, regardless of the type of cofactor present. Reflecting the in vitro reactivity, clot activity and antigen PK profiles in rats were found to coincide for the AT resistant variant along with essentially no detectable AT complex formation, whereas exacerbated AT complex formation and clot activity:antigen discrepancy was observed for the variant exhibiting enhanced in vitro reactivity.Interestingly, among the generated FVIIa variants with altered AT reactivity, two subsets were identified that displayed differential in vitro reactivity with AT depending on the type cofactor present. Accordingly, one group exhibited a greater susceptibility to inhibition relative to wild-type FVIIa in the presence of heparin but not in the presence of TF, while the other group demonstrated the opposite behavior. Endowed with the ability to report on the cofactor identity from the rate of inhibition relative to wild-type FVIIa, variants from each group were tested for their tendency to accumulate as complexes with AT following intravenous administration to rats. Supporting a contribution from endogenous GAGs to the in vivo inactivation of FVIIa, the measured in vivo peak levels of accumulated FVIIa-AT complexes were found to directly correlate with the in vitro rate constants determined for the variants in the presence of heparin, but not when the cofactor was TF or the combination of TF and heparin.Altogether, these results 1) demonstrate a direct relationship between the in vitro reactivity of FVIIa with AT in the presence of heparin and the clearance of FVIIa through this pathway in vivo, and 2) identify heparin-like GAGs as the likely rate enhancing component of FVIIa inhibition in vivo. DisclosuresØstergaard:Novo Nordisk A/S: Employment. Hansen:Novo Nordisk A/S: Employment. Pelzer:Novo Nordisk A/S: Employment. Agersø:Novo Nordisk A/S: Employment. Pedersen:Novo Nordisk A/S: Employment. Glue:Novo Nordisk A/S: Employment. Johnsen:Novo Nordisk A/S: Employment. Andersen:Novo Nordisk A/S: Employment. Bjelke:Novo Nordisk A/S: Employment. Breinholt:Novo Nordisk A/S: Employment. Stennicke:Novo Nordisk A/S: Employment. Gandhi:Novo Nordisk A/S: Employment. Olsen:Novo Nordisk A/S: Employment. Hermit:Novo Nordisk A/S: Employment.

The length of the linker between the epidermal growth factor-like domains in factor VIIa is critical for a productive interaction with tissue factor.

Formation of the factor VIIa (FVIIa)-tissue factor (TF) complex triggers the blood coagulation cascade. Using a structure-based rationale, we investigated how the length of the linker region between the two epidermal growth factor (EGF)-like domains in FVIIa influences TF binding and the allosteric activity enhancement, as well as the interplay between the γ-carboxyglutamic acid (Gla)-containing and protease domains. Removal of two residues from the native linker was compatible with normal cofactor binding and accompanying stimulation of the enzymatic activity, as was extension by two (Gly-Ser) residues. In sharp contrast, truncation by three or four residues abolished the TF-mediated stabilization of the active conformation of FVIIa and abrogated TF-induced activity enhancement. In addition, FVIIa variants with short linkers associated 80-fold slower with soluble TF (sTF) as compared with wild-type FVIIa, resulting in a corresponding increase in the equilibrium dissociation constant. Molecular modeling suggested that the shortest FVIIa variants would have to be forced into a tense and energetically unfavorable conformation in order to be able to interact productively with TF, explaining our experimental observations. We also found a correlation between linker length and the residual intrinsic enzymatic activity of Ca(2+)-free FVIIa; stepwise truncation resulting in gradually higher activity with des(83-86)-FVIIa reaching the level of Gla-domainless FVIIa. The linker appears to determine the average distance between the negatively charged Gla domain and a structural element in the protease domain, presumably of opposite charge, and proximity has a negative impact on apo-FVIIa activity.

Predicting dosing advantages of factor VIIa variants with altered tissue factor‐dependent and lipid‐dependent activities

Recombinant factor VIIa (rFVIIa) is an FX-cleaving coagulation enzyme licensed for the treatment of bleeding episodes in hemophiliacs with inhibitory antibodies. Even though the optimal dosing and comparative dose efficacy of rFVIIa remain poorly understood, genetic or chemical modifications of rFVIIa have been proposed, with the goal of achieving faster and longer hemostatic action. No ongoing trial is currently comparing rFVIIa variants with each other. We used mathematical modeling to compare the pharmacokinetics, dose-response (pharmacodynamics) and dose-effect duration (pharmacokinetics/pharmacodynamics) of rFVIIa variants to predict their optimal doses. The pharmacodynamic (PD) model of FXa generation by FVIIa in complexes with tissue factor (TF) and procoagulant lipids (PLs) was validated against published ex vivo and in vitro thrombin generation (TG) experiments. To compare variants' safety profiles, the highest non-thrombogenic doses were estimated from the clinical evidence reported for the licensed rFVIIa product. The PD model correctly described the biphasic TF-dependent and PL-dependent dose response observed in TG experiments in vitro. The pharmacokinetic/PD simulations agreed with published ex vivo TG data for rFVIIa and the BAY 86-6150 variant, and explained the similar efficacies of a single dose of 270 μg kg(-1) (as reported in the literature) and repeated doses of 90 μg kg(-1) of unmodified rFVIIa. The duration of the simulated hemostatic effect after a single optimal dose was prolonged for rFVIIa variants with increased TF affinity or extended half-lives, but not for those with modulated PL activity. Some modifications of the rFVIIa molecule may not translate into a prolonged hemostatic effect.

Antenatal identification of factor VII Padua during a healthy pregnancy: implications for labor and delivery

Abstract We present a case in which a 28-year-old pregnant woman (para 1) was suspected to have inherited factor VII (FVII) deficiency following investigation for recurrent mild easy bruising. However, this was ruled out by identification of a rare FVII variant not associated with an increased bleeding risk. This allowed usual obstetric care to proceed without any restrictions, facilitating an uncomplicated spontaneous vaginal delivery at term.

Engineering FVIIa Variants with Enhanced Activity

The formation of a complex between factor VIIa (FVIIa) and tissue factor (TF) triggers the blood coagulation cascade in response to a vascular injury. TF has the dual biological function of localizing FVIIa to the site of injury and converting the latent enzyme into an efficient catalyst of factor VII, IX, and X activation. The hemostatic effect of recombinant FVIIa in hemophilia patients, where FVIIa is present at supraphysiological concentrations, is however largely mediated by factor X activation on the surface of activated platelets to which FVIIa binds with relatively low affinity. In this setting, it would be pharmacodynamically advantageous to dispose of FVIIa variants with enhanced intrinsic activity. Moreover, such variants might shed light on how TF induces maturation of the active conformation of FVIIa. Intuitively, more active variants of FVIIa could be designed by exploiting structural differences between FVIIa before and after binding to TF. To get the most out of this approach, the structures of free, uninhibited FVIIa and TF-bound FVIIa should be available for comparison. This is because an active-site inhibitor shifts FVIIa into an active conformation and locks it there, and conformational transitions in the same direction normally induced by TF binding may already have occurred upon inhibitor incorporation or be obscured or even prevented. Unfortunately, crystallization attempts with FVIIa without an active-site inhibitor have not been fruitful, neither with nor without TF. Nevertheless, a few structural clues have been identified, one of which was an attempted mimick by mutagenesis that led to the discovery of the modestly superactive L305V-FVIIa (1). The design and generation of more active FVIIa variants by exploiting sequence differences between FVIIa and homologous enzymes, whose active sites expedite catalytic events at a higher rate, has been more successful. The most striking outcome of this particular endeavour is V158D/E296V/M298Q-FVIIa (FVIIaDVQ) (2, 3).In FVIIaDVQ, three amino acid residues in FVIIa have been replaced by those occupying the corresponding positions in thrombin and factor IXa. Interestingly, the conformational changes induced by the three replacements have been shown to be a subset of those brought about by TF. It is fair to state that FVIIaDVQ is a conformational intermediate between free and TF-bound FVIIa and that the molecule strives to imitate the latter. FVIIaDVQ is dramatically more efficient in cleaving substrates in the absence of TF compared with FVIIa, whereas the specific activities of the two forms of FVIIa bound to TF are comparable. This has also been demonstrated on cellular representations of the two conditions, namely platelets and LPS-stimulated monocytes, respectively. The high activity of FVIIaDVQ has also manifested itself as an increased potency and more rapid effect in murine bleeding models compared with FVIIa and in vitro normalization of human hemophilic blood clotting. An indication of an improved efficacy rate of FVIIaDVQ compared with the efficient drug recombinant FVIIa was observed in a clinical trial (4).1. Persson E, Bak H., Olsen O.H. Substitution of valine for leucine 305 in factor VIIa increases the intrinsic enzymatic activity. J Biol Chem.2001;276(31):29195-29199.2. Persson E, Kjalke M., Olsen O.H. Rational design of coagulation factor VIIa variants with substantially increased intrinsic activity. Proc Natl Acad Sci USA.2001;98(24):13583-13588.3. Persson E., Olsen O.H., Bjørn S.E., Ezban M. Vatreptacog alfa from conception to clinical proof of concept. Semin Thromb Hemost. 2012;38(3):274-281.4. De Paula E.V., Kavakli K., Mahlangu J., et al. Recombinant factor VIIa analog (vatreptacog alfa [activated]) for treatment of joint bleeds in hemophilia patients with inhibitors: a randomized controlled trial. J Thromb Haemost. 2012;10(1):81-89. Disclosures:No relevant conflicts of interest to declare.

Is the coexistence of thromboembolic events and Factor VII deficiency fortuitous?

Over 30 thromboembolic events have been reported in factor VII (FVII) deficiency either associated with previously asymptomatic forms or bleeding diathesis. Whether this coexistence is fortuitous or not is still a mater of debate. Nevertheless, it is well admitted that (i) thrombotic events occurring in FVII-deficient patients with any apparent triggering factors are very rare, (ii) surgical procedures, replacement therapy (especially containing activated factors) but also the presence of an antiphospholipid syndrome are frequently associated with these particular thrombotic events, (iii) in the same way, R304Q and A294V FVII variants appear to be more prevalent than other FVII equally frequent mutations and finally (iv) low FVII coagulant activity levels do not protect against thrombosis. Therefore, peri-operative thrombotic prophylaxis should be relevant for these particular FVII-deficient patients. However, safety, treatment modalities and specific indications of such an antithrombotic prophylaxis remain to be established.

Advances in Hemophilia Care: Report of Two Symposia at the Hemophilia 2010 World Congress

The World Federation of Hemophilia (WFH) 2010 World Congress held in Buenos Aires, Argentina, in July 2010, attracted more than 4,300 participants from 106 countries. This report summarizes two symposia held during the congress. The first, titled "Emerging Co-Morbidities in the Aging Hemophilia Population: Healthcare Challenges and Treatment Opportunities," chaired by Gerry Dolan, MD, and Jussara Almeida Cruz, MD, examined the co-morbidities experienced by the aging hemophilic patient population, such as cardiovascular disease, cancer, arthritis, osteoporosis, hypertension, and obesity. In addition, Bayer's products in preclinical and clinical development were reviewed, including a novel factor VIIa variant and a long-acting factor VIII molecule, i.e., one that has undergone site-specific PEGylation (attachment of polyethylene glycol [PEG] polymer chains to another molecule). The other symposium, titled "Practical Steps to Making Better Care for Hemophilia Patients a Reality," chaired by Carmen Altisent, MD, and Cesar Guerrero, RN, reviewed the steps that hemophilia caregivers can take to improve the care of their patients. Issues such as the treatment of hemarthroses, the role of the research nurse, and the management of pediatric patients transitioning to adulthood were discussed.

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.

A Novel FVIIa Variant with Increased Potency and Duration of Effect Compared to Wildtype FVIIa. A Study in a Dog Model of Hemophilia A

Abstract 2252Hemophilia patients are treated by replacement therapy, receiving the plasma-derived or recombinant clotting factor in which they are deficient. A significant number of hemophilia patients, however, develop inhibitory antibodies against the factor they receive, becoming refractory to replacement therapy. In these inhibitor patients, hemostasis can be achieved using bypass agents, activated plasma-derived prothombin complex concentrates (aPCC) or recombinant activated Factor VIIa (FVIIa). However, these treatments do not match the efficacy seen in traditional replacement therapy. To overcome this limitation, 813, a modified recombinant human Factor VIIa with enhanced biological properties was developed using a rational protein design approach. 813 was selected from a series of FVIIa variants and is characterized by increased catalytic activity and prolonged duration of effect in vivo. Compared to recombinant wild-type FVIIa, 813 has 7-fold increased catalytic activity, measured by the rate of Factor Xa generation in vitro, both in the presence and absence of tissue factor (TF).813 was studied in Factor VIII (FVIII)-deficient dogs with NovoSeven RT (wt-FVIIa) as comparator. The FVIII-deficient dogs exhibit a severe hemophilia A phenotype, with less than 1% normal coagulant activity. Dogs were dosed intravenously with 813 or wt-FVIIa and blood and plasma were collected at various time points post dosing. Plasma FVIIa levels were measured using a FVIIa-specific clotting assay. Starting from initial peak plasma levels, a time-dependent decrease in FVIIa plasma concentration was observed for wt-FVIIa and variant. Compared to wt-FVIIa, 813 exhibited a smaller volume of distribution (35–48 mL/kg vs. 108–134 ml/kg), a lower rate of clearance (10–18 mL/hr/kg vs. 45 mL/kg/hr) and consequently an increased dose adjusted exposure. Hemophilia A dogs have a prolonged activated partial thromboplastin time (aPTT); treatment with wt-FVIIa or 813 led to a dose-dependent decrease in aPTT. At equivalent doses the peak effect of 813 on decreasing aPTT was approximately two-fold compared to wt-FVIIa. Similar dose-dependent effects were observed when studying whole blood clotting profiles by thromboelastography (TEG). Treatment effects were seen for all TEG parameters monitored (time to clotting onset, rate and strength of clot formation). However, compared to wt-FVIIa, 813 given at equivalent doses caused significantly stronger effects on all TEG parameters; acute peak effects on TEG parameters measured for 813 at 10 ug/kg, were similar to those seen with 50 ug/kg wt-FVIIa. This difference became even more pronounced when monitoring treatment effect over time. All dogs used in this study were monitored throughout the study for possible treatment-related changes; both 813 and wt-FVIIa were well tolerated. No overt adverse events, no significant changes in plasma clinical chemistry or cellular blood counts were observed.In conclusion, 813 is a novel FVIIa protein with increased potency, prolonged duration of effect and a preclinical safety profile comparable to wt-FVIIa. 813 is currently in early clinical development for acute and prophylactic treatment of hemophilia patients with inhibitors. Disclosures:Pittman:Pfizer: Employment. Weston:Pfizer: Employment. Shields:Pfizer: Employment. Parng:Pfizer: Employment. Arkin:Pfizer: Employment. Madison:Catalyst Biosciences: Employment. Nichols:Pfizer: Research Funding. Fruebis:Pfizer: Employment.

Platelet binding and activity of a factor VIIa variant with enhanced tissue factor independent activity

Platelet binding and activity play important roles in the efficacy of factor VIIa (FVIIa) as a bypassing agent for hemophilia treatment. An analog of FVIIa with increased tissue factor (TF)-independent activity, NN1731, has been produced by introducing three amino acid changes in the protease domain. NN1731 has a conformation similar to TF-bound FVIIa, even in the absence of TF. This results in much greater intrinsic proteolytic activity, but similar activity in the presence of TF. We hypothesized that these changes would not alter binding to platelets or phospholipid, a characteristic thought to be localized to the Gla domain. The goal of the current work was to compare platelet binding and activity of NN1731 and wild-type FVIIa. FVIIa and NN1731 bound identically to phospholipid vesicles as assessed by both activity assays and electrophoretic quasielastic light scattering techniques. However, NN1731 bound to a greater number of sites on activated platelets than FVIIa, as assessed by flow cytometry. Removal of the Gla domain abolished binding of both FVIIa and NN1731. Inhibition of the active site did not reduce NN1731 binding to the level of FVIIa. When corrected for the amount of protein bound, NN1731 had greater activity than FVIIa on platelet surfaces. While the Gla domain is essential for FVIIa binding to platelets, changes in the protease domain in NN1731 enhanced platelet binding as well as proteolytic activity. Features in addition to lipid composition appear to contribute to binding of rFVIIa and, especially, NN1731 to platelets.

Increased Platelet Binding of NN1731, a Factor VIIa Variant with Enhanced Tissue Factor-Independent Activity.

AbstractAbstract 1133 Introduction:Platelet surface activity plays an important role in the efficacy of FVIIa as a bypassing agent in hemophilia. An analog of FVIIa with increased tissue factor (TF) independent activity, NN1731, has been produced by introducing three amino acid changes in the protease domain: V158D, E296V, and M298Q. Modeling suggests that Gln in position 298 and Asp in position 158 form a hydrogen bond network with a water molecule that stabilizes the proteolytically active conformation of the molecule. The Val introduced at position 296 avoids electrostatic repulsion between a native Glu residue and the introduced Asp at 158. These changes result in a FVIIa variant that partially mimics the conformation of TF-bound FVIIa. However, in the presence of TF the activity of the wild type FVIIa and NN1731 are indistinguishable. Previous studies suggested that the increased intrinsic activity of NN1731 was not directly reflected in the level of activity on the platelet surface. The goal of the current work was to compare the platelet binding and platelet surface activity of NN1731 and wild type FVIIa. Methods:Mutant FVIIa molecules were prepared as described previously (PNAS 98:13583-8, 2001). We assessed binding to purified human platelets by flow cytometry and activity by measuring FXa generation. Results:FVIIa and NN1731 had identical binding to phospholipid vesicles designed to have lipid composition similar to platelets. However, the two exhibited different binding to platelets. At all concentrations, more NN1731 bound to thrombin or thrombin-plus-convulxin activated platelets. This was primarily because NN1731 bound to a greater number of sites per platelet. It appears that NN1731 and FVIIa bind similarly to a higher affinity site, but NN1731 binds to an additional low-affinity site. Removal of the Gla domain abolished binding. Thus, the protease domain of NN1731 was insufficient to mediate binding to the low affinity site. Two additional FVIIa mutants were tested: the single M298Q (“Q”) variant, and the double E296V and M298Q (“VQ”) variant. These variants bound to platelets identically to FVIIa. Conclusion:While the Gla domain is essential for FVIIa binding to platelets, the three amino acid changes in the protease domain in NN1731 enhance platelet binding as well as proteolytic activity. Since NN1731 and FVIIa bind similarly to phospholipid vesicles, characteristics in addition to lipid composition likely contribute to platelet binding. The platelet surface components that contribute to NN1731 binding remain to be defined. Disclosures:Hoffman:Novo Nordisk: Research Funding. Persson:Novo Nordisk: Employment. Ezban:Novo Nordisk: Employment. Monroe:Novo Nordisk: Research Funding.

The influence of different glycosylation patterns on factor VII biological activity

In this study the bioactivity of three differently glycosylated blood coagulation factor VII (FVII) variants (human plasma FVII, recombinant human FVII produced in CHO and BHK cell cultures) were analyzed and compared. Surface plasmon resonance studies of FVII interaction with soluble and full length TF together with FVII autoactivation assays revealed that BHK-derived FVII has the highest bioactivity, while human plasma and CHO-derived FVII showed very similar bioactivity. The affinity of FVII variants to TF correlates with FVII autoactivation rates – the higher the affinity, the faster the autoactivation rate.

Effect of recombinant factor VIIa variant (NN1731) on platelet function, clot structure and force onset time in whole blood from healthy volunteers and haemophilia patients

NN1731 is a novel variant of recombinant factor VIIa (rFVIIa) that binds to activated platelets, but has greater enzymatic activity than rFVIIa in generating FXa and thrombin. The effect of NN1731 on clot structure and platelet function was characterized ex vivo in whole blood from healthy volunteers and haemophilic patients. Blood samples from six healthy volunteers, nine haemophilia A patients with and without inhibitors and one acquired haemophilia A patient, were spiked with increasing concentrations (0.32, 0.64 and 1.28 microg mL(-1)) of rFVIIa and NN1731. Platelet contractile force (PCF) or platelet function, clot elastic modulus (CEM) or clot structure, and force onset time (FOT) or the thrombin generation time (TGT) were determined using the Hemodyne Hemostasis Analysis System (HAS). Baseline PCF, CEM and FOT values in patients were abnormal compared to healthy volunteers' baseline values. Overall, haemophilia blood samples with or without inhibitors spiked with NN1731 had significantly greater PCF, CEM and shorter FOT values relative to samples spiked with corresponding doses of rFVIIa. The variability in response to treatment between patients was greater with rFVIIa compared to NN1731. At 1.28 microg mL(-1) (90 microg kg(-1)), NN1731 normalized PCF, CEM and FOT in nine of 10 patients, while rFVIIa normalized these parameters in four of 10 patients. Increasing in vitro concentrations of NN1731 normalized platelet function, clot structure and thrombin generation consistently in haemophilia blood with or without inhibitors. NN1731 may be a promising haemostatic agent for patients with bleeding disorders. These results should be confirmed in an in vivo study.

Activity and Regulation of Factor VIIa Analogs with Increased Potency at the Endothelial Cell Surface.

High doses of recombinant factor VIIa (FVIIa) have been found to bypass factor IX or factor VIII deficiency and ameliorate the bleeding problems associated with hemophilia patients with inhibitors. Recent studies show that FVIIa also acts as an effective hemostatic agent in other categories of patients, and thus has become a promising candidate for prevention and treatment of excessive bleeding associated with many other diseases/injuries. Although recombinant FVIIa has proven to be a very effective and safe drug in the treatment of bleeding episodes in hemophilia patients with inhibitors and other indications, a small fraction of patients may be refractory to FVIIa treatment. The reason for this is unclear at present, but it is possible that administration of very high pharmacological doses of FVIIa or use of genetically modified FVIIa molecules with increased potencies may circumvent the problem. The most dramatic effect on the activity (a 40-fold increase in proteolytic activity) of FVIIa was obtained by occupying the corresponding positions in thrombin/factor IXa for those positions 158, 296 and 298 of FVIIa (FVIIaDVQ). A FVIIa mutant in which the hydrophobic residue Met 298 was replaced with Gln (FVIIaQ) has 7-fold higher proteolytic activity. In the present study, we investigated the interactions of FVIIaQ and FVIIaDVQ with plasma inhibitors, tissue factor pathway inhibitor (TFPI) and antithrombin (AT) in solution and at the vascular endothelium. Both TFPI and AT/heparin inhibited the FVIIa variants more rapidly than the wild-type FVIIa in the absence of TF. In the presence of TF, TFPI, TFPI-Xa and AT/heparin inhibited FVIIa and FVIIa variants at similar rates. Although the wild-type FVIIa failed to generate significant amounts of factor Xa on unperturbed endothelial cells, FVIIa variants, particularly FVIIaDVQ, generated a substantial amount of factor Xa on unperturbed endothelium (1 nM of factor VIIa generated 0.3 ± 0.15 nM factor Xa/h whereas FVIIaQ and FVIIaDVQ generated 1.26 ± 0.1 nM/h and 9.48 ± 1.32 nM/h, respectively). Annexin V fully attenuated the FVIIa-mediated activation of factor X on unperturbed endothelial cells whereas anti-TF IgG had no effect. On stimulated HUVEC, FVIIa and FVIIa variants activated factor X at similar rates (30–40 nM/h). AT/heparin and TFPI-Xa inhibited the activity of FVIIa and FVIIa variants bound to endothelial cell TF in a similar fashion. AT inhibition of FVIIa bound to stimulated endothelial cells requires exogenous heparin. Interestingly, TFPI-Xa was found to inhibit the activities of both FVIIa and FVIIa analogs bound to unperturbed endothelial cells. Despite significant differences observed in factor Xa generation on native endothelium exposed to FVIIa and FVIIa analogs, no differences were found in thrombin generation when cells were exposed to FVIIa or FVIIa analogs under plasma mimicking conditions, probably due to limited availability of anionic phospholipids and/or putative factor Xa and Va binding sites on their cell surface. Over all, our present data suggest that although FVIIa variants may generate factor Xa on native endothelium, the resultant factor Xa does not lead to enhanced thrombin generation on native endothelium compared to FVIIa. These data should reduce potential concerns about whether the use of FVIIa variants triggers unwanted coagulation on native endothelium, and may facilitate the development of FVIIa analogs as effective therapeutic agents in near future for treatment of patients with bleeding disorders.

Of four mutations in the factor VII gene in Tunisian patients, one novel mutation (Ser339Phe) in three unrelated families abrogates factor X activation

Hereditary factor VII (FVII) deficiency is a rare bleeding disorder. Dysfunctional FVII variants characterized by normal or reduced levels of FVII antigen and discordantly low FVII activity have been described. In this study, seven unrelated Tunisian patients with FVII deficiency were examined. Molecular analysis revealed that three probands harbored a novel Ser339Phe mutation, one proband was inferred to have a novel splice site mutation in intron 2, c.226-2 A>G and three probands had two previously described mutations, Arg304Gln and Cys310Phe. Expression of Ser339Phe in baby hamster kidney cells yielded secretion of FVII antigen at a concentration of 225+/-50 ng/ml, compared with 181+/-47 ng/ml in cells transfected with wild-type FVII but with no demonstrable FVII activity. FVII Ser339Phe bound to tissue factor similarly to the binding of commercial recombinant activated FVII or recombinant wild-type FVII and was normally activated by activated factor X. The major defect of FVII Ser339Phe was its inability to activate factor X in the presence of tissue factor. Modeling predicted that the substitution of Ser339 by Phe abrogated substrate docking.

Disulfide locked variants of factor VIIa with a restricted β‐strand conformation have enhanced enzymatic activity

Proteolytic processing of zymogen Factor VII to Factor VIIa (FVIIa) is necessary but not sufficient for maximal proteolytic activity, which requires an additional allosteric influence induced upon binding to its cofactor tissue factor (TF). A key conformational change affecting the zymogenicity of FVIIa involves a unique three-residue shift in the position of beta-strand B2 in their zymogen and protease forms. By selectively introducing new disulfide bonds, we locked the conformation of these strands into an active TF*FVIIa-like state. FVIIa mutants designated 136:160, 137:159, 138:160, and 139:157, reflecting the position of the new disulfide bond (chymotypsinogen numbering), were expressed and purified by TF affinity chromatography. Mass spectrometric analysis of tryptic peptides from the FVIIa mutants confirmed the new disulfide bond formation. Kinetic analysis of amidolytic activity revealed that all FVIIa variants alone had increased specific activity compared to wild type, the largest being for variants 136:160 and 138:160 with substrate S-2765, having 670- and 330-fold increases, respectively. Notably, FVIIa disulfide-locked variants no longer required TF as a cofactor for maximal activity in amidolytic assays. In the presence of soluble TF, activity was enhanced 20- and 12-fold for variants 136:160 and 138:160, respectively, compared to wild type. With relipidated TF, mutants 136:160 and 137:159 also had an approximate threefold increase in their V(max)/K(m) values for FX activation but no significant improvement in TF-dependent clotting assays. Thus, while large rate enhancements were obtained for amidolytic substrates binding at the active site, macro-molecular substrates that bind to FVIIa exosites entail more complex catalytic requirements.