A Variant of Recombinant Factor VIIa With Enhanced Procoagulant and Antifibrinolytic Activities in an In Vitro Model of Hemophilia

Allen, Geoffrey; Persson, Egon; Campbell, Robert A.; Ezban, Mirella; Hedner, Ulla

doi:10.1161/01.atv.0000257204.82396.2b

Cited by 68 publications

(95 citation statements)

References 25 publications

(47 reference statements)

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“…NN1731 (Novo Nordisk A/S, Bagsvaerd, Denmark) is one such rFVIIa variant analog under development that has three amino acid substitutions [6,7]. The human pharmacokinetics of NN1731 has been described [8], and preliminary data have characterized its in vitro and ex vivo hemostatic effects [9][10][11]. The mechanism of action of rFVIIa and NN1731 is based upon the enhancement of thrombin generation on the activated platelets at the site of injury.…”

Section: Introductionmentioning

confidence: 99%

Factor VIIa analog has marked effects on platelet function and clot kinetics in blood from patients with hemophilia A

Brophy

Martin

Nolte

et al. 2010

Blood Coagulation &Amp; Fibrinolysis

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To evaluate the hemostatic effects of NN1731 and rFVIIa, an ex-vivo study in hemophilia patients used the Hemodyne Hemostasis Analysis System (HAS) to measure platelet contractile force (PCF), clot elastic modulus (CEM), and force onset time (FOT), and the Haemoscope Thrombelastograph (TEG) to measure reaction time (R), kinetics time (K), and maximum amplitude (MA). Blood samples from 10 healthy volunteers and 10 Factor VIII-deficient patients of varying severity (mild, moderate, severe), were spiked with rFVIIa and NN1731 (both 0.64 and 1.28 microg/ml, respectively) and analyzed to characterize platelet function and clot kinetics. There was wide variability in the rFVIIa response. NN1731 had greater and more consistent effects on PCF, CEM, FOT, R, and K relative to rFVIIa, in all hemophilia groups. The lowest NN1731 concentration (0.64 microg/ml) shortened R and FOT, and increased CEM and PCF more than rFVIIa 1.28 microg/ml. NN1731 normalized clotting parameters equivalent to values obtained in healthy volunteers. FOT and R were highly correlated (r = 0.96). No correlation was observed between CEM and MA. NN1731 produced less variable, more pronounced and predictable ex-vivo hemostatic effects on PCF, CEM, FOT, R and K than rFVIIa in all hemophilia groups. HAS and TEG assays provided similar estimates of FOT and R, however CEM appeared to be more sensitive than MA to changes in clot firmness.

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Section: Introductionmentioning

confidence: 99%

Factor VIIa analog has marked effects on platelet function and clot kinetics in blood from patients with hemophilia A

Brophy

Martin

Nolte

et al. 2010

Blood Coagulation &Amp; Fibrinolysis

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“…We have discovered several such variants through site-directed mutagenesis in the protease domain of FVIIa and have selected four FVIIa analogs with unique properties for the present study. These are FVIIa M306D , which is unresponsive to activation by TF (15), FVIIa VEAY , which has optimized activity especially toward small peptidyl substrates (16), and FVIIa DVQ and FVIIa DVQA , with dramatically improved activity toward the natural macromolecular substrate factor X (FX) (17)(18)(19).…”

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confidence: 99%

The Origins of Enhanced Activity in Factor VIIa Analogs and the Interplay between Key Allosteric Sites Revealed by Hydrogen Exchange Mass Spectrometry

Rand

Andersen

Olsen

et al. 2008

Journal of Biological Chemistry

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Factor VIIa (FVIIa) circulates in the blood in a zymogen-like state. Only upon association with membrane-bound tissue factor (TF) at the site of vascular injury does FVIIa become active and able to initiate blood coagulation. Here we used hydrogen exchange monitored by mass spectrometry to investigate the conformational effects of site-directed mutagenesis at key positions in FVIIa and the origins of enhanced intrinsic activity of FVIIa analogs. The differences in hydrogen exchange of two highly active variants, FVIIa DVQ and FVIIa VEAY , imply that enhanced catalytic efficiency was attained by two different mechanisms. Regions protected from exchange in FVIIa DVQ include the N-terminal tail and the activation pocket, which is a subset of the regions of FVIIa protected from exchange upon TF binding. FVIIa DVQ appeared to adopt an intermediate conformation between the free (zymogen-like) and TF-bound (active) form of FVIIa and to attain enhanced activity by partial mimicry of TF-induced activation. In contrast, exchange-protected regions in FVIIa VEAY were confined to the vicinity of the active site of FVIIa. Thus, the changes in FVIIa VEAY appeared to optimize the active site region rather than imitate the TF-induced effect. Hydrogen exchange analysis of the FVIIa M306D variant, which was unresponsive to stimulation by TF, correlated widespread reductions in exchange to the single mutation in the TFbinding region. These results reveal the delicate interplay between key allosteric sites necessary to achieve the transition of FVIIa into the active form.

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“…on May 12, 2018. by guest www.bloodjournal.org From such molecules reportedly increase catalytic activity on platelets, [42][43][44][45] whereas some may increase TF-dependent activity, 46 and yet others have been found to prolong the lifetime in the circulation. 47,48 Each of these changes can potentially alter the therapeutic window of TF-and phospholipid-dependent actions of rFVIIa, leading to drastically different efficacy and safety profiles.…”

Section: Discussionmentioning

confidence: 99%

Unifying the mechanism of recombinant FVIIa action: dose dependence is regulated differently by tissue factor and phospholipids

Shibeko

Woodle

Lee

et al. 2012

Blood

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Recombinant factor VIIa (rFVIIa) is used for treatment of hemophilia patients with inhibitors, as well for off-label treatment of severe bleeding in trauma and surgery. Effective bleeding control requires supraphysiological doses of rFVIIa, posing both high expense and uncertain thrombotic risk. Two major competing theories offer different explanations for the supraphysiological rFVIIa dosing requirement: (1) the need to overcome competition between FVIIa and FVII zymogen for tissue factor (TF) binding, and (2) a highdose-requiring phospholipid-related pathway of FVIIa action. In the present study, we found experimental conditions in which both mechanisms contribute simultaneously and independently to rFVIIa-driven thrombin generation in FVIIdeficient human plasma. From mathematical simulations of our model of FX activation, which were confirmed by thrombingeneration experiments, we conclude that the action of rFVIIa at pharmacologic doses is dominated by the TF-dependent pathway with a minor contribution from a phospholipid-dependent mechanism. We established a dose-response curve for rFVIIa that is useful to explain dosing strategies. In the present study, we present a pathway to reconcile the 2 major mechanisms of rFVIIa action, a necessary step to understanding future dose optimization and evaluation of new rFVIIa analogs currently under development. IntroductionThe use of a recombinant factor VIIa (rFVIIa) product, NovoSeven, which is licensed for the treatment of hemophilia patients with inhibitory Abs against factor VIII (FVIII) or factor IX (FIX), has proven to be safe and efficacious, but its dosing remains problematic. [1][2][3][4] The recommended dosing schedule is a supraphysiological dose of 90 g/kg every 2-3 hours until hemostasis is achieved, producing an approximately 250-fold increase above basal plasma concentrations of FVIIa (0.1 to 25nM). 5 Not only does such a treatment regimen incur a high cost, but ineffective drug responses and thrombotic complications have also been reported. [6][7][8][9] However, our current understanding of the mechanism of rFVIIa action is unclear, limiting our ability to optimize the safety and cost of treatment. 10,11 Off-label use of rFVIIa for nonhemophilia patients at similar high doses indicates that the high dose required for hemophilia treatment cannot be explained by deficiencies of FVIII or FIX alone. 5,12 FVIIa is a weak enzyme and its activity requires either of 2 cofactors, tissue factor (TF) or negatively charged phospholipids. 13 Disagreement over which of the 2 cofactors explains the high pharmacologic dose of the drug has led to TF-and phospholipiddependent theories of rFVIIa action. Although these mechanisms may appear to be nonexclusive, the 2 theories support opposing approaches to dose adjustment.The TF-dependent mechanism suggests that the hemostatic effect of rFVIIa is mediated by its binding to TF expressed on cell surfaces at the site of injury, 14 forming the extrinsic tenase complex, which activates factor X (FX), leading to thrombin genera...

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A Variant of Recombinant Factor VIIa With Enhanced Procoagulant and Antifibrinolytic Activities in an In Vitro Model of Hemophilia

Cited by 68 publications

References 25 publications

Factor VIIa analog has marked effects on platelet function and clot kinetics in blood from patients with hemophilia A

Factor VIIa analog has marked effects on platelet function and clot kinetics in blood from patients with hemophilia A

The Origins of Enhanced Activity in Factor VIIa Analogs and the Interplay between Key Allosteric Sites Revealed by Hydrogen Exchange Mass Spectrometry

Unifying the mechanism of recombinant FVIIa action: dose dependence is regulated differently by tissue factor and phospholipids

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