Modifications of the Prothrombin Active Site S4 Subpocket Confer Resistance to Dabigatran

Abstract

Background

Direct anticoagulants inhibit coagulation serine proteases by reversibly engaging their active site with high affinity. By modifying the S4 active site subpocket of factor (F)Xa, we introduced inhibitor resistance while preserving catalytic activity. Given the homology between FXa and thrombin in active site architecture and direct anticoagulant binding, we have targeted the S4 subsite to introduce inhibitor resistance in (pro)thrombin.

Methods

Recombinant prothrombin variants were generated in which I174 was substituted or sequence R92-N98 was exchanged with that of human kallikrein-3.

Results

Specific prothrombin clotting activity of the variants was 6-fold (intrinsic clotting) to 10-fold (extrinsic clotting) reduced relative to wild-type prothrombin. Further analyses revealed that modification of the S4 subsite hampers fibrinogen and thrombomodulin-mediated protein C conversion by thrombin. Consistent with this, the thrombin variants displayed a reduced catalytic efficiency toward the peptidyl substrate used in thrombin generation assessments. The variants displayed a 2-fold reduced sensitivity for dabigatran relative to wild-type prothrombin, while argatroban inhibition was unaffected. Analyses using a purified component system revealed an up to 24-fold and 4-fold reduced IC₅₀ for inhibition of thrombin by dabigatran and argatroban, respectively. Molecular dynamics (MD) simulations of both dabigatran-bound and unbound (apo) modified thrombin variants indicated these to comprise a larger inhibitor binding pocket relative to wild-type thrombin and display reduced inhibitor binding. As a net effect, (pro)thrombin variants with S4 subsite modifications supported detectable fibrin formation at therapeutic dabigatran concentrations.

Conclusion

Our findings provide proof-of-concept for the engineering of thrombin variants that are resistant to direct thrombin inhibitors by modulating the S4 subsite.

Keywords

anticoagulants - direct thrombin inhibitors - anticoagulant reversal agents - serine proteases - recombinant fusion proteins

Data Availability Statement

For original data, please contact the corresponding author.

Authors' Contribution

Da.V., V.J.F.S., and M.H.A.B. designed the study. K.L.C., De.V., T.R., and B.B. performed the research. V.J.F.S., De.V., K.L.C., and M.H.A.B. analyzed and interpreted data. V.J.F.S. and De.V. drafted the manuscript. Da.V. and M.H.A.B. critically revised the manuscript. All authors revised the manuscript, agreed with its content, and approved of submission.

^* These authors contributed equally to this study.

Publication History

Received: 09 April 2024

Accepted: 11 February 2025

Accepted Manuscript online:
12 February 2025

Article published online:
24 March 2025

• References

• 1 Bode W, Mayr I, Baumann U, Huber R, Stone SR, Hofsteenge J. The refined 1.9 A crystal structure of human alpha-thrombin: interaction with D-Phe-Pro-Arg chloromethylketone and significance of the Tyr-Pro-Pro-Trp insertion segment. EMBO J 1989; 8 (11) 3467-3475
• 2 Hedstrom L. Serine protease mechanism and specificity. Chem Rev 2002; 102 (12) 4501-4524
• 3 Connolly SJ, Ezekowitz MD, Yusuf S. et al; RE-LY Steering Committee and Investigators. Dabigatran versus warfarin in patients with atrial fibrillation. N Engl J Med 2009; 361 (12) 1139-1151
• 4 Patel MR, Mahaffey KW, Garg J. et al; ROCKET AF Investigators. Rivaroxaban versus warfarin in nonvalvular atrial fibrillation. N Engl J Med 2011; 365 (10) 883-891
• 5 Connolly SJ, Eikelboom J, Joyner C. et al; AVERROES Steering Committee and Investigators. Apixaban in patients with atrial fibrillation. N Engl J Med 2011; 364 (09) 806-817
• 6 Granger CB, Alexander JH, McMurray JJ. et al; ARISTOTLE Committees and Investigators. Apixaban versus warfarin in patients with atrial fibrillation. N Engl J Med 2011; 365 (11) 981-992
• 7 Giugliano RP, Ruff CT, Braunwald E. et al; ENGAGE AF-TIMI 48 Investigators. Edoxaban versus warfarin in patients with atrial fibrillation. N Engl J Med 2013; 369 (22) 2093-2104
• 8 Cohen AT, Spiro TE, Büller HR. et al; MAGELLAN Investigators. Rivaroxaban for thromboprophylaxis in acutely ill medical patients. N Engl J Med 2013; 368 (06) 513-523
• 9 Goldhaber SZ, Leizorovicz A, Kakkar AK. et al; ADOPT Trial Investigators. Apixaban versus enoxaparin for thromboprophylaxis in medically ill patients. N Engl J Med 2011; 365 (23) 2167-2177
• 10 Schulman S, Kearon C, Kakkar AK. et al; RE-MEDY Trial Investigators, RE-SONATE Trial Investigators. Extended use of dabigatran, warfarin, or placebo in venous thromboembolism. N Engl J Med 2013; 368 (08) 709-718
• 11 Cohen AT, Harrington RA, Goldhaber SZ. et al; APEX Investigators. Extended thromboprophylaxis with betrixaban in acutely ill medical patients. N Engl J Med 2016; 375 (06) 534-544
• 12 Bauersachs R, Berkowitz SD, Brenner B. et al; EINSTEIN Investigators. Oral rivaroxaban for symptomatic venous thromboembolism. N Engl J Med 2010; 363 (26) 2499-2510
• 13 Büller HR, Prins MH, Lensin AW. et al; EINSTEIN–PE Investigators. Oral rivaroxaban for the treatment of symptomatic pulmonary embolism. N Engl J Med 2012; 366 (14) 1287-1297
• 14 Roehrig S, Straub A, Pohlmann J. et al. Discovery of the novel antithrombotic agent 5-chloro-N-((5S)-2-oxo-3- [4-(3-oxomorpholin-4-yl)phenyl]-1,3-oxazolidin-5-ylmethyl)thiophene- 2-carboxamide (BAY 59-7939): an oral, direct factor Xa inhibitor. J Med Chem 2005; 48 (19) 5900-5908
• 15 Pinto DJ, Orwat MJ, Koch S. et al. Discovery of 1-(4-methoxyphenyl)-7-oxo-6-(4-(2-oxopiperidin-1-yl)phenyl)-4,5,6,7-tetrahydro-1H-pyrazolo[3,4-c]pyridine-3-carboxamide (apixaban, BMS-562247), a highly potent, selective, efficacious, and orally bioavailable inhibitor of blood coagulation factor Xa. J Med Chem 2007; 50 (22) 5339-5356
• 16 Fischer PM. Design of small-molecule active-site inhibitors of the S1A family proteases as procoagulant and anticoagulant drugs. J Med Chem 2018; 61 (09) 3799-3822
• 17 Gómez-Outes A, Suárez-Gea ML, Lecumberri R, Terleira-Fernández AI, Vargas-Castrillón E. Direct-acting oral anticoagulants: pharmacology, indications, management, and future perspectives. Eur J Haematol 2015; 95 (05) 389-404
• 18 Schreuder M, Reitsma PH, Bos MHA. Reversal agents for the direct factor Xa inhibitors: biochemical mechanisms of current and newly emerging therapies. Semin Thromb Hemost 2020; 46 (08) 986-998
• 19 van Es N, De Caterina R, Weitz JI. Reversal agents for current and forthcoming direct oral anticoagulants. Eur Heart J 2023; 44 (20) 1795-1806
• 20 Pollack Jr CV, Reilly PA, Bernstein R. et al. Design and rationale for RE-VERSE AD: a phase 3 study of idarucizumab, a specific reversal agent for dabigatran. Thromb Haemost 2015; 114 (01) 198-205
• 21 Milling Jr TJ, Middeldorp S, Xu L. et al; ANNEXA-4 Investigators. Final study report of andexanet alfa for major bleeding with factor Xa inhibitors. Circulation 2023; 147 (13) 1026-1038
• 22 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
• 23 Schreuder M, Jourdi G, Veizaj D. et al. Minimally modified human blood coagulation factor X to bypass direct factor Xa inhibitors. J Thromb Haemost 2024; 22 (08) 2211-2226
• 24 Bos MH, Camire RM. Procoagulant adaptation of a blood coagulation prothrombinase-like enzyme complex in Australian elapid venom. Toxins (Basel) 2010; 2 (06) 1554-1567
• 25 Skala W, Utzschneider DT, Magdolen V. et al. Structure-function analyses of human kallikrein-related peptidase 2 establish the 99-loop as master regulator of activity. J Biol Chem 2014; 289 (49) 34267-34283
• 26 Borgoño CA, Diamandis EP. The emerging roles of human tissue kallikreins in cancer. Nat Rev Cancer 2004; 4 (11) 876-890
• 27 Debela M, Magdolen V, Bode W, Brandstetter H, Goettig P. Structural basis for the Zn2+ inhibition of the zymogen-like kallikrein-related peptidase 10. Biol Chem 2016; 397 (12) 1251-1264
• 28 Lechtenberg BC, Murray-Rust TA, Johnson DJ. et al. Crystal structure of the prothrombinase complex from the venom of Pseudonaja textilis. Blood 2013; 122 (16) 2777-2783
• 29 Toso R, Camire RM. Removal of B-domain sequences from factor V rather than specific proteolysis underlies the mechanism by which cofactor function is realized. J Biol Chem 2004; 279 (20) 21643-21650
• 30 Hemker HC, Giesen P, Al Dieri R. et al. Calibrated automated thrombin generation measurement in clotting plasma. Pathophysiol Haemost Thromb 2003; 33 (01) 4-15
• 31 Bloemen S, Kelchtermans H, Hemker HC. Thrombin generation in low plasma volumes. Thromb J 2018; 16: 10
• 32 Case DA, Cheatham III TE, Darden T. et al. The Amber biomolecular simulation programs. J Comput Chem 2005; 26 (16) 1668-1688
• 33 Case DA, Aktulga HM, Belfon L. et al. Amber 2022. University of California; 2022
• 34 Mirdita M, Schütze K, Moriwaki Y, Heo L, Ovchinnikov S, Steinegger M. ColabFold: making protein folding accessible to all. Nat Methods 2022; 19 (06) 679-682
• 35 Jumper J, Evans R, Pritzel A. et al. Highly accurate protein structure prediction with AlphaFold. Nature 2021; 596 (7873) 583-589
• 36 Genheden S, Ryde U. The MM/PBSA and MM/GBSA methods to estimate ligand-binding affinities. Expert Opin Drug Discov 2015; 10 (05) 449-461
• 37 Miller III BR, McGee Jr TD, Swails JM, Homeyer N, Gohlke H, Roitberg AE. MMPBSA.py: an efficient program for end-state free energy calculations. J Chem Theory Comput 2012; 8 (09) 3314-3321
• 38 Brandstetter H, Turk D, Hoeffken HW. et al. Refined 2.3 A X-ray crystal structure of bovine thrombin complexes formed with the benzamidine and arginine-based thrombin inhibitors NAPAP, 4-TAPAP and MQPA. A starting point for improving antithrombotics. J Mol Biol 1992; 226 (04) 1085-1099
• 39 Hauel NH, Nar H, Priepke H, Ries U, Stassen JM, Wienen W. Structure-based design of novel potent nonpeptide thrombin inhibitors. J Med Chem 2002; 45 (09) 1757-1766
• 40 Wienen W, Stassen JM, Priepke H, Ries UJ, Hauel N. Effects of the direct thrombin inhibitor dabigatran and its orally active prodrug, dabigatran etexilate, on thrombus formation and bleeding time in rats. Thromb Haemost 2007; 98 (02) 333-338
• 41 Fitzgerald D, Murphy N. Argatroban: a synthetic thrombin inhibitor of low relative molecular mass. Coron Artery Dis 1996; 7 (06) 455-458
• 42 Kim JM, Noh J, Park JW. et al. Dabigatran acylglucuronide, the major metabolite of dabigatran, shows a weaker anticoagulant effect than dabigatran. Pharmaceutics 2022; 14 (02) 257
• 43 Davie EW, Kulman JD. An overview of the structure and function of thrombin. Semin Thromb Hemost 2006; 32 (Suppl. 01) 3-15
• 44 Gale AJ, Griffin JH. Characterization of a thrombomodulin binding site on protein C and its comparison to an activated protein C binding site for factor Va. Proteins 2004; 54 (03) 433-441
• 45 Stojanovski BM, Pelc LA, Di Cera E. Role of the activation peptide in the mechanism of protein C activation. Sci Rep 2020; 10 (01) 11079
• 46 Ayala YM, Arosio D, Di Cera E. Mutation of W215 compromises thrombin cleavage of fibrinogen, but not of PAR1 or protein C. Ann N Y Acad Sci 2001; 936: 456-458
• 47 Peacock RB, Davis JR, Markwick PRL, Komives EA. Dynamic consequences of mutation of tryptophan 215 in thrombin. Biochemistry 2018; 57 (18) 2694-2703
• 48 Sondag D, Verhoeven S, Löwik DWPM. et al. Activity sensing of coagulation and fibrinolytic proteases. Chemistry 2023; 29 (18) e202203473
• 49 Skejić J, Hodgson WC. Population divergence in venom bioactivities of elapid snake Pseudonaja textilis: role of procoagulant proteins in rapid rodent prey incapacitation. PLoS One 2013; 8 (05) e63988
• 50 Reza MA, Minh Le TN, Swarup S, Manjunatha Kini R. Molecular evolution caught in action: gene duplication and evolution of molecular isoforms of prothrombin activators in Pseudonaja textilis (brown snake). J Thromb Haemost 2006; 4 (06) 1346-1353
• 51 Bock PE, Panizzi P, Verhamme IM. Exosites in the substrate specificity of blood coagulation reactions. J Thromb Haemost 2007; 5 (Suppl. 01) 81-94
• 52 Bauer CA, Brayer GD, Sielecki AR, James MN. Active site of alpha-lytic protease: enzyme-substrate interactions. Eur J Biochem 1981; 120 (02) 289-294
• 53 Heemskerk JW, Mattheij NJ, Cosemans JM. Platelet-based coagulation: different populations, different functions. J Thromb Haemost 2013; 11 (01) 2-16
• 54 Posma JJ, Posthuma JJ, Spronk HM. Coagulation and non-coagulation effects of thrombin. J Thromb Haemost 2016; 14 (10) 1908-1916
• 55 Bos MHA, van't Veer C, Reitsma PH. Molecular biology and biochemistry of the coagulation factors and pathways of hemostasis. In: Kaushansky K, Prchal JT, Burns LJ, Lichtman MA, Levi M, Linch DL. ed. Williams Hematology. 10th ed.. McGraw Hill; 2021: 2017-2050
• 56 Cer RZ, Mudunuri U, Stephens R, Lebeda FJ. IC50-to-Ki: a web-based tool for converting IC50 to Ki values for inhibitors of enzyme activity and ligand binding. Nucleic Acids Res 2009; 37 (Web Server issue): W441-5