A Thrombin-Activatable Factor X Variant Corrects Hemostasis in a Mouse Model for Hemophilia A

Abstract

Engineered recombinant factor X (FX) variants represent a promising strategy to bypass the tenase complex and restore hemostasis in hemophilia patients. Previously, a thrombin-activatable FX variant with fibrinopeptide-A replacing the activation peptide (FX-delAP/FpA) has been described in this regard. Here we show that FX-delAP/FpA is characterized by a sixfold shorter circulatory half-life compared with wild-type FX, limiting its therapeutical applicability. We therefore designed a variant in which the FpA sequence is inserted C-terminal to the FX activation peptide (FX/FpA). FX/FpA displayed a similar survival to wt-FX in clearance experiments and could be converted into FX by thrombin and other activating agents. In in vitro assays, FX/FpA efficiently restored thrombin generation in hemophilia A and hemophilia B plasmas, even in the presence of inhibitory antibodies. Expression following hydrodynamic gene transfer of FX/FpA restored thrombus formation in FVIII-deficient mice in a laser-induced injury model as well as hemostasis in a tail-clip bleeding model. Hemostasis after tail transection in FVIII-deficient mice was also corrected at 5 and 90 minutes after injection of purified FX/FpA. Our data indicate that FX/FpA represents a potential tenase-bypassing agent for the treatment of hemophilia patients with or without inhibitors.

Keywords

hemophilia - factor X - factor VIII

Authors' Contributions

V.M., S.V., C.C., O.D.C., G.C., L.P-.D., P.G., C.D., P.J.L., and C.V.D. performed experiments and analyzed data. M.T. provided essential reagents for this study. C.V.D., O.D.C., and P.J.L. wrote the manuscript. All authors contributed to the editing of the final manuscript.

^* C.V.D. and O.D.C. contributed equally to this study.

• References

• 1 Stonebraker JS, Bolton-Maggs PH, Michael Soucie J, Walker I, Brooker M. A study of variations in the reported haemophilia B prevalence around the world. Haemophilia 2012; 18 (03) e91-e94
• 2 Stonebraker JS, Bolton-Maggs PH, Soucie JM, Walker I, Brooker M. A study of variations in the reported haemophilia A prevalence around the world. Haemophilia 2010; 16 (01) 20-32
• 3 Berntorp E, Shapiro AD. Modern haemophilia care. Lancet 2012; 379 (9824): 1447-1456
• 4 Witmer C, Young G. Factor VIII inhibitors in hemophilia A: rationale and latest evidence. Ther Adv Hematol 2013; 4 (01) 59-72
• 5 Christophe OD, Lenting PJ, Cherel G. , et al. Functional mapping of anti-factor IX inhibitors developed in patients with severe hemophilia B. Blood 2001; 98 (05) 1416-1423
• 6 Walsh CE, Jiménez-Yuste V, Auerswald G, Grancha S. The burden of inhibitors in haemophilia patients. Thromb Haemost 2016; 116 (Suppl. 01) S10-S17
• 7 Berntorp E. Differential response to bypassing agents complicates treatment in patients with haemophilia and inhibitors. Haemophilia 2009; 15 (01) 3-10
• 8 Mauser-Bunschoten EP, Koopman MM, Goede-Bolder AD. , et al; Recombinant Factor VIIa Data Collection Group. Efficacy of recombinant factor VIIa administered by continuous infusion to haemophilia patients with inhibitors. Haemophilia 2002; 8 (05) 649-656
• 9 Gringeri A, Leissinger C, Cortesi PA. , et al. Health-related quality of life in patients with haemophilia and inhibitors on prophylaxis with anti-inhibitor complex concentrate: results from the Pro-FEIBA study. Haemophilia 2013; 19 (05) 736-743
• 10 Carpenter SL, Khair K, Gringeri A, Valentino LA. Prophylactic bypassing agent use before and during immune tolerance induction in patients with haemophilia A and inhibitors to FVIII. Haemophilia 2018; 24 (04) 570-577
• 11 Young G, Auerswald G, Jimenez-Yuste V. , et al. PRO-PACT: retrospective observational study on the prophylactic use of recombinant factor VIIa in hemophilia patients with inhibitors. Thromb Res 2012; 130 (06) 864-870
• 12 Konkle BA, Ebbesen LS, Erhardtsen E. , et al. Randomized, prospective clinical trial of recombinant factor VIIa for secondary prophylaxis in hemophilia patients with inhibitors. J Thromb Haemost 2007; 5 (09) 1904-1913
• 13 Morfini M, Batorova A, Mariani G. , et al; International FVII [IF7] and Seven Treatment Evaluation Registry [STER] Study Groups. Pharmacokinetic properties of recombinant FVIIa in inherited FVII deficiency account for a large volume of distribution at steady state and a prolonged pharmacodynamic effect. Thromb Haemost 2014; 112 (02) 424-425
• 14 Nogami K, Shima M. New therapies using nonfactor products for patients with hemophilia and inhibitors. Blood 2019; 133 (05) 399-406
• 15 Oldenburg J, Mahlangu JN, Kim B. , et al. Emicizumab prophylaxis in hemophilia A with inhibitors. N Engl J Med 2017; 377 (09) 809-818
• 16 Pasi KJ, Rangarajan S, Georgiev P. , et al. Targeting of antithrombin in hemophilia A or B with RNAi therapy. N Engl J Med 2017; 377 (09) 819-828
• 17 Bunce MW, Toso R, Camire RM. Zymogen-like factor Xa variants restore thrombin generation and effectively bypass the intrinsic pathway in vitro. Blood 2011; 117 (01) 290-298
• 18 Ivanciu L, Toso R, Margaritis P. , et al. A zymogen-like factor Xa variant corrects the coagulation defect in hemophilia. Nat Biotechnol 2011; 29 (11) 1028-1033
• 19 Louvain-Quintard VB, Bianchini EP, Calmel-Tareau C, Tagzirt M, Le Bonniec BF. Thrombin-activable factor X re-establishes an intrinsic amplification in tenase-deficient plasmas. J Biol Chem 2005; 280 (50) 41352-41359
• 20 Guéguen P, Cherel G, Badirou I, Denis CV, Christophe OD. Two residues in the activation peptide domain contribute to the half-life of factor X in vivo. J Thromb Haemost 2010; 8 (07) 1651-1653
• 21 Kurdi M, Cherel G, Lenting PJ, Denis CV, Christophe OD. Coagulation factor X interaction with macrophages through its N-glycans protects it from a rapid clearance. PLoS One 2012; 7 (09) e45111
• 22 Johansson L, Karpf DM, Hansen L, Pelzer H, Persson E. Activation peptides prolong the murine plasma half-life of human factor VII. Blood 2011; 117 (12) 3445-3452
• 23 Muczynski V, Bazaa A, Loubière C. , et al. Macrophage receptor SR-AI is crucial to maintain normal plasma levels of coagulation factor X. Blood 2016; 127 (06) 778-786
• 24 Muczynski V, Aymé G, Regnault V. , et al. Complex formation with pentraxin-2 regulates factor X plasma levels and macrophage interactions. Blood 2017; 129 (17) 2443-2454
• 25 Bi L, Lawler AM, Antonarakis SE, High KA, Gearhart JD, Kazazian Jr HH. Targeted disruption of the mouse factor VIII gene produces a model of haemophilia A. Nat Genet 1995; 10 (01) 119-121
• 26 Marx I, Christophe OD, Lenting PJ. , et al. Altered thrombus formation in von Willebrand factor-deficient mice expressing von Willebrand factor variants with defective binding to collagen or GPIIbIIIa. Blood 2008; 112 (03) 603-609
• 27 Levigne S, Thiec F, Cherel G, Irving JA, Fribourg C, Christophe OD. Role of the alpha-helix 163-170 in factor Xa catalytic activity. J Biol Chem 2007; 282 (43) 31569-31579
• 28 Hemker HC, Giesen P, AlDieri R. , et al. The calibrated automated thrombogram (CAT): a universal routine test for hyper- and hypocoagulability. Pathophysiol Haemost Thromb 2002; 32 (5–6): 249-253
• 29 Girma JP, Fressinaud E, Houllier A, Laurian Y, Amiral J, Meyer D. Assay of factor VIII antigen (VIII:CAg) in 294 haemophilia A patients by a new commercial ELISA using monoclonal antibodies. Haemophilia 1998; 4 (02) 98-103
• 30 Lenting PJ, Westein E, Terraube V. , et al. An experimental model to study the in vivo survival of von Willebrand factor. Basic aspects and application to the R1205H mutation. J Biol Chem 2004; 279 (13) 12102-12109
• 31 Lind P, Larsson K, Spira J. , et al. Novel forms of B-domain-deleted recombinant factor VIII molecules. Construction and biochemical characterization. Eur J Biochem 1995; 232 (01) 19-27
• 32 Dubois C, Panicot-Dubois L, Gainor JF, Furie BC, Furie B. Thrombin-initiated platelet activation in vivo is vWF independent during thrombus formation in a laser injury model. J Clin Invest 2007; 117 (04) 953-960
• 33 Fazavana JG, Muczynski V, Proulle V. , et al. LDL receptor-related protein 1 contributes to the clearance of the activated factor VII-antithrombin complex. J Thromb Haemost 2016; 14 (12) 2458-2470
• 34 Versteeg HH, Heemskerk JW, Levi M, Reitsma PH. New fundamentals in hemostasis. Physiol Rev 2013; 93 (01) 327-358
• 35 de Visser MC, Poort SR, Vos HL, Rosendaal FR, Bertina RM. Factor X levels, polymorphisms in the promoter region of factor X, and the risk of venous thrombosis. Thromb Haemost 2001; 85 (06) 1011-1017
• 36 Rietveld IM, Lijfering WM, le Cessie S. , et al. High levels of coagulation factors and venous thrombosis risk: strongest association for factor VIII and von Willebrand factor. J Thromb Haemost 2019; 17 (01) 99-109
• 37 von Drygalski A, Bhat V, Gale AJ. , et al. An engineered factor Va prevents bleeding induced by anticoagulant wt activated protein C. PLoS One 2014; 9 (08) e104304
• 38 Bhat V, von Drygalski A, Gale AJ, Griffin JH, Mosnier LO. Improved coagulation and haemostasis in haemophilia with inhibitors by combinations of superFactor Va and Factor VIIa. Thromb Haemost 2016; 115 (03) 551-561
• 39 Parng C, Markiewicz V, Chen J. , et al. Preclinical pharmacokinetics, pharmacodynamics, tissue distribution, and interspecies scaling of recombinant human coagulation factor Xa^I16L . J Pharm Sci 2017; 106 (08) 2136-2143
• 40 Gale AJ, Bhat V, Pellequer JL, Griffin JH, Mosnier LO, Von Drygalski A. Safety, stability and pharmacokinetic properties of (super)factor Va, a novel engineered coagulation factor V for treatment of severe bleeding. Pharm Res 2016; 33 (06) 1517-1526