FXa-α2-Macroglobulin Complex Neutralizes Direct Oral Anticoagulants Targeting FXa In Vitro and In Vivo

Georges Jourdi; Isabelle Gouin-Thibault; Virginie Siguret; Sophie Gandrille; Pascale Gaussem; Bernard Le Bonniec

doi:10.1055/s-0038-1667014

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 2018; 118(09): 1535-1544
DOI: 10.1055/s-0038-1667014

Coagulation and Fibrinolysis

Georg Thieme Verlag KG Stuttgart · New York

FXa-α₂-Macroglobulin Complex Neutralizes Direct Oral Anticoagulants Targeting FXa In Vitro and In Vivo

Georges Jourdi

¹INSERM UMR_S1140, Faculté de Pharmacie, Paris, France

²Université Paris Descartes, Sorbonne Paris Cité, Paris, France

³AP-HP, Hôpital Cochin, Paris, France

,

Isabelle Gouin-Thibault

¹INSERM UMR_S1140, Faculté de Pharmacie, Paris, France

²Université Paris Descartes, Sorbonne Paris Cité, Paris, France

⁴CHU Pontchaillou, Rennes, France

,

Virginie Siguret

¹INSERM UMR_S1140, Faculté de Pharmacie, Paris, France

²Université Paris Descartes, Sorbonne Paris Cité, Paris, France

⁵AP-HP, Hôpital Lariboisière, Paris, France

,

Sophie Gandrille

¹INSERM UMR_S1140, Faculté de Pharmacie, Paris, France

²Université Paris Descartes, Sorbonne Paris Cité, Paris, France

⁶AP-HP, Hôpital Européen Georges Pompidou, Paris, France

,

Pascale Gaussem

¹INSERM UMR_S1140, Faculté de Pharmacie, Paris, France

²Université Paris Descartes, Sorbonne Paris Cité, Paris, France

⁶AP-HP, Hôpital Européen Georges Pompidou, Paris, France

,

Bernard Le Bonniec

¹INSERM UMR_S1140, Faculté de Pharmacie, Paris, France

²Université Paris Descartes, Sorbonne Paris Cité, Paris, France

› Author Affiliations

Abstract

Increasing number of patients are treated with direct oral anticoagulants (DOAC). An antidote for dabigatran inhibiting thrombin (idarucizumab) is available but no antidote is yet approved for the factor Xa (FXa) inhibitors (xabans). We hypothesized that a complex between Gla-domainless FXa and α₂-macroglobulin (GDFXa-α₂M) may neutralize the xabans without interfering with normal blood coagulation.

Purified α₂M was incubated with GDFXa to form GDFXa-α₂M. Affinity of apixaban and rivaroxaban for GDFXa-α₂M was only slightly decreased compared to FXa. Efficacy and harmlessness of GDFXa-α₂M were tested in vitro and in vivo. Stoichiometric excess of GDFXa-α₂M neutralized rivaroxaban and apixaban as attested by clot waveform assay and rotational thromboelastometry, whereas GDFXa-α₂M alone had no effect on these assays. Efficacy and pro-thrombotic potential of GDFXa-α₂M were also assessed in vivo. Half-life of GDFXa-α₂M in C57BL6 mice was 4.9 ± 1.1 minutes, but a 0.5 mg/mouse dose resulted in uptake saturation such that 50% persistence was still observed after 170 minutes. Single administration of GDFXa-α₂M significantly decreased the rivaroxaban-induced bleeding time (p < 0.001) and blood loss (p < 0.01). GDFXa-α₂M did not increase D-dimer or thrombin–antithrombin complex formation, suggesting a lack of pro-thrombotic potential.

GDFXa-α₂M is therefore an attractive candidate for xaban neutralization neither pro- nor anticoagulant in vitro as well as in vivo.

Keywords

antidote - apixaban - rivaroxaban - oral anticoagulant

Full Text

References

References
1 Weitz JI, Harenberg J. New developments in anticoagulants: past, present and future. Thromb Haemost 2017; 117 (07) 1283-1288
2 Greinacher A, Thiele T, Selleng K. Reversal of anticoagulants: an overview of current developments. Thromb Haemost 2015; 113 (05) 931-942
3 Siegal DM, Garcia DA, Crowther MA. How I treat target-specific oral anticoagulant-associated bleeding. Blood 2014; 123 (08) 1152-1158
4 Reiffel JA, Weitz JI, Reilly P. , et al; Cardiac Safety Research Consortium presenters and participants. NOAC monitoring, reversal agents, and post-approval safety and effectiveness evaluation: a cardiac safety research consortium think tank. Am Heart J 2016; 177: 74-86
5 Raval AN, Cigarroa JE, Chung MK. , et al; American Heart Association Clinical Pharmacology Subcommittee of the Acute Cardiac Care and General Cardiology Committee of the Council on Clinical Cardiology; Council on Cardiovascular Disease in the Young; and Council on Quality of Care and Outcomes Research. Management of patients on non-vitamin K antagonist oral anticoagulants in the acute care and periprocedural setting: a scientific statement from the American Heart Association. Circulation 2017; 135 (10) e604-e633
6 Crowther M, Crowther MA. Antidotes for novel oral anticoagulants: current status and future potential. Arterioscler Thromb Vasc Biol 2015; 35 (08) 1736-1745
7 Wolzt M, Weltermann A, Nieszpaur-Los M. , et al. Studies on the neutralizing effects of protamine on unfractionated and low molecular weight heparin (Fragmin) at the site of activation of the coagulation system in man. Thromb Haemost 1995; 73 (03) 439-443
8 Ageno W, Büller HR, Falanga A. , et al. Managing reversal of direct oral anticoagulants in emergency situations. Anticoagulation Education Task Force White Paper. Thromb Haemost 2016; 116 (06) 1003-1010
9 Goldstein JN, Refaai MA, Milling Jr TJ. , et al. Four-factor prothrombin complex concentrate versus plasma for rapid vitamin K antagonist reversal in patients needing urgent surgical or invasive interventions: a phase 3b, open-label, non-inferiority, randomised trial. Lancet 2015; 385 (9982): 2077-2087
10 Schiele F, van Ryn J, Canada K. , et al. A specific antidote for dabigatran: functional and structural characterization. Blood 2013; 121 (18) 3554-3562
11 Glund S, Stangier J, Schmohl M. , et al. Safety, tolerability, and efficacy of idarucizumab for the reversal of the anticoagulant effect of dabigatran in healthy male volunteers: a randomised, placebo-controlled, double-blind phase 1 trial. Lancet 2015; 386 (9994): 680-690
12 Pollack Jr CV, Reilly PA, van Ryn J. , et al. Idarucizumab for dabigatran reversal - full cohort analysis. N Engl J Med 2017; 377 (05) 431-441
13 Majeed A, Ågren A, Holmström M. , et al. Management of rivaroxaban- or apixaban-associated major bleeding with prothrombin complex concentrates: a cohort study. Blood 2017; 130 (15) 1706-1712
14 Martin AC, Le Bonniec B, Fischer AM. , et al. Evaluation of recombinant activated factor VII, prothrombin complex concentrate, and fibrinogen concentrate to reverse apixaban in a rabbit model of bleeding and thrombosis. Int J Cardiol 2013; 168 (04) 4228-4233
15 Lu G, DeGuzman FR, Hollenbach SJ. , et al. A specific antidote for reversal of anticoagulation by direct and indirect inhibitors of coagulation factor Xa. Nat Med 2013; 19 (04) 446-451
16 Siegal DM, Curnutte JT, Connolly SJ. , et al. Andexanet alfa for the reversal of factor Xa inhibitor activity. N Engl J Med 2015; 373 (25) 2413-2424
17 Stone SR, Le Bonniec BF. Inhibitory mechanism of serpins. Identification of steps involving the active-site serine residue of the protease. J Mol Biol 1997; 265 (03) 344-362
18 Johnson DJ, Li W, Adams TE, Huntington JA. Antithrombin-S195A factor Xa-heparin structure reveals the allosteric mechanism of antithrombin activation. EMBO J 2006; 25 (09) 2029-2037
19 Connolly SJ, Milling Jr TJ, Eikelboom JW. , et al; ANNEXA-4 Investigators. Andexanet alfa for acute major bleeding associated with factor Xa inhibitors. N Engl J Med 2016; 375 (12) 1131-1141
20 Ansell JE, Bakhru SH, Laulicht BE. , et al. Use of PER977 to reverse the anticoagulant effect of edoxaban. N Engl J Med 2014; 371 (22) 2141-2142
21 Ansell JE, Bakhru SH, Laulicht BE. , et al. Single-dose ciraparantag safely and completely reverses anticoagulant effects of edoxaban. Thromb Haemost 2017; 117 (02) 238-245
22 Thalji NK, Ivanciu L, Davidson R, Gimotty PA, Krishnaswamy S, Camire RM. A rapid pro-hemostatic approach to overcome direct oral anticoagulants. Nat Med 2016; 22 (08) 924-932
23 Parsons-Rich D, Hua F, Li G, Kantaridis C, Pittman DD, Arkin S. Phase 1 dose-escalating study to evaluate the safety, pharmacokinetics, and pharmacodynamics of a recombinant factor Xa variant (FXa^I16L ). J Thromb Haemost 2017; 15 (05) 931-937
24 Verhoef D, Visscher KM, Vosmeer CR. , et al. Engineered factor Xa variants retain procoagulant activity independent of direct factor Xa inhibitors. Nat Commun 2017; 8 (01) 528
25 Sottrup-Jensen L. Alpha-macroglobulins: structure, shape, and mechanism of proteinase complex formation. J Biol Chem 1989; 264 (20) 11539-11542
26 Kolodziej SJ, Wagenknecht T, Strickland DK, Stoops JK. The three-dimensional structure of the human α 2-macroglobulin dimer reveals its structural organization in the tetrameric native and chymotrypsin α 2-macroglobulin complexes. J Biol Chem 2002; 277 (31) 28031-28037
27 Marrero A, Duquerroy S, Trapani S. , et al. The crystal structure of human α2-macroglobulin reveals a unique molecular cage. Angew Chem Int Ed Engl 2012; 51 (14) 3340-3344
28 Sottrup-Jensen L, Sand O, Kristensen L, Fey GH. The alpha-macroglobulin bait region. Sequence diversity and localization of cleavage sites for proteinases in five mammalian alpha-macroglobulins. J Biol Chem 1989; 264 (27) 15781-15789
29 Meijers JCM, Tijburg PNM, Bouma BN. Inhibition of human blood coagulation factor Xa by α 2-macroglobulin. Biochemistry 1987; 26 (18) 5932-5937
30 Heeb MJ, Gruber A, Griffin JH. Identification of divalent metal ion-dependent inhibition of activated protein C by α 2-macroglobulin and α 2-antiplasmin in blood and comparisons to inhibition of factor Xa, thrombin, and plasmin. J Biol Chem 1991; 266 (26) 17606-17612
31 Gettins PG, Boel E, Crews BC. Thiol ester role in correct folding and conformation of human alpha 2-macroglobulin. Properties of recombinant C949S variant. FEBS Lett 1994; 339 (03) 276-280
32 Qazi U, Gettins PG, Stoops JK. On the structural changes of native human alpha2-macroglobulin upon proteinase entrapment. Three-dimensional structure of the half-transformed molecule. J Biol Chem 1998; 273 (15) 8987-8993
33 Qazi U, Kolodziej SJ, Gettins PG, Stoops JK. The structure of the C949S mutant human alpha(2)-macroglobulin demonstrates the critical role of the internal thiol esters in its proteinase-entrapping structural transformation. J Struct Biol 2000; 131 (01) 19-26
34 Wyatt AR, Kumita JR, Farrawell NE, Dobson CM, Wilson MR. Alpha-2-macroglobulin is acutely sensitive to freezing and lyophilization: implications for structural and functional studies. PLoS One 2015; 10 (06) e0130036
35 Sottrup-Jensen L, Hansen HF, Pedersen HS, Kristensen L. Localization of epsilon-lysyl-gamma-glutamyl cross-links in five human alpha 2-macroglobulin-proteinase complexes. Nature of the high molecular weight cross-linked products. J Biol Chem 1990; 265 (29) 17727-17737
36 Le Bonniec BF, Guinto ER, Esmon CT. The role of calcium ions in factor X activation by thrombin E192Q. J Biol Chem 1992; 267 (10) 6970-6976
37 Arnold JN, Wallis R, Willis AC. , et al. Interaction of mannan binding lectin with alpha2 macroglobulin via exposed oligomannose glycans: a conserved feature of the thiol ester protein family?. J Biol Chem 2006; 281 (11) 6955-6963
38 French K, Yerbury JJ, Wilson MR. Protease activation of α2-macroglobulin modulates a chaperone-like action with broad specificity. Biochemistry 2008; 47 (04) 1176-1185
39 Jourdi G, Siguret V, Martin AC. , et al. Association rate constants rationalise the pharmacodynamics of apixaban and rivaroxaban. Thromb Haemost 2015; 114 (01) 78-86
40 Bianchini EP, Pike RN, Le Bonniec BF. The elusive role of the potential factor X cation-binding exosite-1 in substrate and inhibitor interactions. J Biol Chem 2004; 279 (05) 3671-3679
41 Hantgan RR, Hermans J. Assembly of fibrin. A light scattering study. J Biol Chem 1979; 254 (22) 11272-11281
42 Johansen PB, Tranholm M, Haaning J, Knudsen T. Development of a tail vein transection bleeding model in fully anaesthetized haemophilia A mice - characterization of two novel FVIII molecules. Haemophilia 2016; 22 (04) 625-631
43 Fuchs HE, Pizzo SV. Regulation of factor Xa in vitro in human and mouse plasma and in vivo in mouse. Role of the endothelium and plasma proteinase inhibitors. J Clin Invest 1983; 72 (06) 2041-2049
44 Bergsma J, Vije J, Duursma AM, Schutter WG, Bouma JM, Gruber M. The alpha-macroglobulins from rat plasma: structure, plasma clearance and endocytosis of complexes with subtilisin. Biomed Biochim Acta 1986; 45 (11-12): 1549-1556
45 Debanne MT, Bell R, Dolovich J. Uptake of proteinase-alpha-macroglobulin complexes by macrophages. Biochim Biophys Acta 1975; 411 (02) 295-304
46 Imber MJ, Pizzo SV. Clearance and binding of two electrophoretic “fast” forms of human alpha 2-macroglobulin. J Biol Chem 1981; 256 (15) 8134-8139
47 Gliemann J, Larsen TR, Sottrup-Jensen L. Cell association and degradation of alpha 2-macroglobulin-trypsin complexes in hepatocytes and adipocytes. Biochim Biophys Acta 1983; 756 (02) 230-237
48 Eddeland A, Ohlsson K. The elimination in dogs of trypsin-alpha-macroglobulin complexes inactivated by the Kazal or the Kunitz inhibitor. Hoppe Seylers Z Physiol Chem 1978; 359 (03) 379-384
49 Narita M, Rudolph AE, Miletich JP, Schwartz AL. The low-density lipoprotein receptor-related protein (LRP) mediates clearance of coagulation factor Xa in vivo. Blood 1998; 91 (02) 555-560
50 Herrmann R, Thom J, Wood A, Phillips M, Muhammad S, Baker R. Thrombin generation using the calibrated automated thrombinoscope to assess reversibility of dabigatran and rivaroxaban. Thromb Haemost 2014; 111 (05) 989-995