Dabigatran Attenuates the Binding of Thrombin to Platelets—A Novel Mechanism of Action

Abstract

Background

Thrombin is a multifunctional regulatory enzyme of the haemostasis and has both pro- and anticoagulant roles. It has, therefore, been a main target for drug discovery over many decades. Thrombin is a serine protease and possesses two positively charged regions called exosites, through which it is known to bind to many substrates. Dabigatran is a thrombin inhibitor and is widely used as an oral anticoagulant for the antithrombotic treatment of atrial fibrillation and venous thromboembolism. The mechanism by which dabigatran inhibits thrombin is the blockage of the active site, however, its effect on thrombin binding to its substrates has not been studied thoroughly and is thus poorly understood.

Material and Methods

The effect of dabigatran on thrombin binding to platelets was evaluated by flow cytometry using fluorescently labelled thrombin and washed platelets. Further, to confirm the results we utilized modern techniques for biomolecular binding studies, microscale thermophoresis (MST) and surface plasmon resonance (SPR), which validated the results.

Results

Dabigatran inhibited thrombin binding to platelets as analysed by flow cytometry. The inhibition was dose dependent with IC50 of 118 nM which was slightly lower than for inhibition of platelet activation and is close to the clinically relevant plasma concentration of dabigatran. MST and SPR also confirmed inhibitory effect of dabigatran on thrombin binding to platelets.

Conclusion

Apart from blocking the active site, dabigatran also inhibits thrombin binding to platelets. Since thrombin has numerous functions beyond the cardiovascular system, this finding may have important implications.

Keywords

thrombin - dabigatran - microscale thermophoresis - platelets

Authors' Contribution

A.M. and T.L. conceived the study and designed the flow cytometry and MST experiments. A.M., T.H., A.P.K., and A.A.H. performed MST experiments. A.P.K. and E.A. performed flow cytometry experiments. A.R. and K.U. designed the SPR experiments, analyzed the data, and prepared the figures. A.R. performed the SPR experiments. A.M. and T.L. wrote the manuscript. All authors have read, commented, and approved the final version of the manuscript.

^* These authors contributed equally.

Publication History

Received: 11 May 2024

Accepted: 09 October 2024

Accepted Manuscript online:
25 November 2024

Article published online:
19 December 2024

• References

• 1 Chen J, Ishii M, Wang L, Ishii K, Coughlin SR. Thrombin receptor activation. Confirmation of the intramolecular tethered liganding hypothesis and discovery of an alternative intermolecular liganding mode. J Biol Chem 1994; 269 (23) 16041-16045
• 2 Sheehan JP, Sadler JE. Molecular mapping of the heparin-binding exosite of thrombin. Proc Natl Acad Sci U S A 1994; 91 (12) 5518-5522
• 3 Verhamme IM, Olson ST, Tollefsen DM, Bock PE. Binding of exosite ligands to human thrombin. Re-evaluation of allosteric linkage between thrombin exosites I and II. J Biol Chem 2002; 277 (09) 6788-6798
• 4 Boknäs N, Faxälv L, Sanchez Centellas D. et al. Thrombin-induced platelet activation via PAR4: pivotal role for exosite II. Thromb Haemost 2014; 112 (03) 558-565
• 5 Lane DA, Philippou H, Huntington JA. Directing thrombin. Blood 2005; 106 (08) 2605-2612
• 6 Nimjee SM, Oney S, Volovyk Z. et al. Synergistic effect of aptamers that inhibit exosites 1 and 2 on thrombin. RNA 2009; 15 (12) 2105-2111
• 7 Vu TK, Wheaton VI, Hung DT, Charo I, Coughlin SR. Domains specifying thrombin-receptor interaction. Nature 1991; 353 (6345): 674-677
• 8 Jacques SL, Kuliopulos A. Protease-activated receptor-4 uses dual prolines and an anionic retention motif for thrombin recognition and cleavage. Biochem J 2003; 376 (Pt 3): 733-740
• 9 De Candia E, Hall SW, Rutella S, Landolfi R, Andrews RK, De Cristofaro R. Binding of thrombin to glycoprotein Ib accelerates the hydrolysis of Par-1 on intact platelets. J Biol Chem 2001; 276 (07) 4692-4698
• 10 Gandhi PS, Chen Z, Di Cera E. Crystal structure of thrombin bound to the uncleaved extracellular fragment of PAR1. J Biol Chem 2010; 285 (20) 15393-15398
• 11 Lund M, Macwan AS, Tunströmer K, Lindahl TL, Boknäs N. Effects of heparin and bivalirudin on thrombin-induced platelet activation: differential modulation of PAR signaling drives divergent prothrombotic responses. Front Cardiovasc Med 2021; 8: 717835
• 12 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
• 13 Stangier J. Clinical pharmacokinetics and pharmacodynamics of the oral direct thrombin inhibitor dabigatran etexilate. Clin Pharmacokinet 2008; 47 (05) 285-295
• 14 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
• 15 Wienen W, Stassen JM, Priepke H, Ries UJ, Hauel N. In-vitro profile and ex-vivo anticoagulant activity of the direct thrombin inhibitor dabigatran and its orally active prodrug, dabigatran etexilate. Thromb Haemost 2007; 98 (01) 155-162 [pii]
• 16 Yeh CH, Stafford AR, Leslie BA, Fredenburgh JC, Weitz JI. Dabigatran and argatroban diametrically modulate thrombin exosite function. PLoS One 2016; 11 (06) e0157471
• 17 Macwan AS, Boknäs N, Ntzouni MP. et al. Gradient-dependent inhibition of stimulatory signaling from platelet G protein-coupled receptors. Haematologica 2019; 104 (07) 1482-1492
• 18 Ramström S, Oberg KV, Akerström F, Enström C, Lindahl TL. Platelet PAR1 receptor density—correlation to platelet activation response and changes in exposure after platelet activation. Thromb Res 2008; 121 (05) 681-688
• 19 Li S, Tarlac V, Christanto RBI, French SL, Hamilton JR. Determination of PAR4 numbers on the surface of human platelets: no effect of the single nucleotide polymorphism rs773902. Platelets 2021; 32 (07) 988-991
• 20 Coller BS, Peerschke EI, Scudder LE, Sullivan CA. Studies with a murine monoclonal antibody that abolishes ristocetin-induced binding of von Willebrand factor to platelets: additional evidence in support of GPIb as a platelet receptor for von Willebrand factor. Blood 1983; 61 (01) 99-110
• 21 Mazet V. Background correction. . MATLAB Central File Exchange. Accessed December 7, 2024; available at: https://www.mathworks.com/matlabcentral/fileexchange/27429-background-correction
• 22 Breitsprecher D, Schlinck N, Witte D, Duhr S, Baaske P, Schubert T. Aptamer binding studies using MicroScale thermophoresis. Methods Mol Biol 2016; 1380: 99-111
• 23 Jerabek-Willemsen M, Wienken CJ, Braun D, Baaske P, Duhr S. Molecular interaction studies using microscale thermophoresis. Assay Drug Dev Technol 2011; 9 (04) 342-353
• 24 Petoral RM, Herland A, Broo K, Uvdal K. G-protein interactions with receptor-derived peptides chemisorbed on gold. Langmuir 2003; 19(24): 10304-10309
• 25 Wan S, Cui S, Jiang M. et al. Dual-target synergistic antithrombotic mechanism of a dabigatran etexilate analogue (HY023016). Clin Exp Pharmacol Physiol 2022; 49 (05) 567-576
• 26 Mo M, Kong D, Ji H. et al. Reversible photocontrol of thrombin activity by replacing loops of thrombin binding aptamer using azobenzene derivatives. Bioconjug Chem 2019; 30 (01) 231-241
• 27 Tollefsen DM, Feagler JR, Majerus PW. The binding of thrombin to the surface of human platelets. J Biol Chem 1974; 249 (08) 2646-2651
• 28 Harmon JT, Jamieson GA. Thrombin binds to a high-affinity approximately 900 000-dalton site on human platelets. Biochemistry 1985; 24 (01) 58-64
• 29 Bakhtiar R. Surface plasmon resonance spectroscopy: a versatile technique in a biochemist's toolbox. J Chem Educ 2013; 90(2): 203-209
• 30 Jerabek-Willemsen M, André T, Wanner R. et al. MicroScale thermophoresis: interaction analysis and beyond. J Mol Struct 2014; 1077: 101-113
• 31 Schmaier AH, Meloni FJ, Nawarawong W, Jiang YP. PPACK-thrombin is a noncompetitive inhibitor of alpha-thrombin binding to human platelets. Thromb Res 1992; 67 (05) 479-489
• 32 Reilly PA, Lehr T, Haertter S. et al; RE-LY Investigators. The effect of dabigatran plasma concentrations and patient characteristics on the frequency of ischemic stroke and major bleeding in atrial fibrillation patients: the RE-LY Trial (Randomized Evaluation of Long-Term Anticoagulation Therapy). J Am Coll Cardiol 2014; 63 (04) 321-328
• 33 Treuheit NA, Beach MA, Komives EA. Thermodynamic compensation upon binding to exosite 1 and the active site of thrombin. Biochemistry 2011; 50 (21) 4590-4596
• 34 Croy CH, Koeppe JR, Bergqvist S, Komives EA. Allosteric changes in solvent accessibility observed in thrombin upon active site occupation. Biochemistry 2004; 43 (18) 5246-5255
• 35 Dólleman SC, Agten SM, Spronk HMH. et al. Thrombin in complex with dabigatran can still interact with PAR-1 via exosite-I and instigate loss of vascular integrity. J Thromb Haemost 2022; 20 (04) 996-1007
• 36 Chen B, Soto AG, Coronel LJ, Goss A, van Ryn J, Trejo J. Characterization of thrombin-bound dabigatran effects on protease-activated receptor-1 expression and signaling in vitro. Mol Pharmacol 2015; 88 (01) 95-105
• 37 Wang X, Zhang Y, Yang Y, Wu X, Fan H, Qiao Y. Identification of berberine as a direct thrombin inhibitor from traditional Chinese medicine through structural, functional and binding studies. Sci Rep 2017; 7: 44040
• 38 Huntington JA. Molecular recognition mechanisms of thrombin. J Thromb Haemost 2005; 3 (08) 1861-1872
• 39 Lee-Rivera I, López E, López-Colomé AM. Diversification of PAR signaling through receptor crosstalk. Cell Mol Biol Lett 2022; 27 (01) 77
• 40 Bai M. Dimerization of G-protein-coupled receptors: roles in signal transduction. Cell Signal 2004; 16 (02) 175-186
• 41 Lechtenberg BC, Freund SM, Huntington JA. GpIbα interacts exclusively with exosite II of thrombin. J Mol Biol 2014; 426 (04) 881-893
• 42 Arachiche A, Mumaw MM, de la Fuente M, Nieman MT. Protease-activated receptor 1 (PAR1) and PAR4 heterodimers are required for PAR1-enhanced cleavage of PAR4 by α-thrombin. J Biol Chem 2013; 288 (45) 32553-32562
• 43 de la Fuente M, Noble DN, Verma S, Nieman MT. Mapping human protease-activated receptor 4 (PAR4) homodimer interface to transmembrane helix 4. J Biol Chem 2012; 287 (13) 10414-10423
• 44 Leger AJ, Jacques SL, Badar J. et al. Blocking the protease-activated receptor 1-4 heterodimer in platelet-mediated thrombosis. Circulation 2006; 113 (09) 1244-1254
• 45 Smith TH, Li JG, Dores MR, Trejo J. Protease-activated receptor-4 and purinergic receptor P2Y12 dimerize, co-internalize, and activate Akt signaling via endosomal recruitment of β-arrestin. J Biol Chem 2017; 292 (33) 13867-13878
• 46 Bode W, Greyling HJ, Huber R, Otlewski J, Wilusz T. The refined 2.0 A X-ray crystal structure of the complex formed between bovine beta-trypsin and CMTI-I, a trypsin inhibitor from squash seeds (Cucurbita maxima). Topological similarity of the squash seed inhibitors with the carboxypeptidase A inhibitor from potatoes. FEBS Lett 1989; 242 (02) 285-292
• 47 Stubbs MT, Laber B, Bode W. et al. The refined 2.4 A X-ray crystal structure of recombinant human stefin B in complex with the cysteine proteinase papain: a novel type of proteinase inhibitor interaction. EMBO J 1990; 9 (06) 1939-1947
• 48 Rezaie AR, Yang L. Deletion of the 60-loop provides new insights into the substrate and inhibitor specificity of thrombin. Thromb Haemost 2005; 93 (06) 1047-1054
• 49 Jansson M, Själander S, Sjögren V, Renlund H, Norrving B, Själander A. Direct comparisons of effectiveness and safety of treatment with apixaban, dabigatran and rivaroxaban in atrial fibrillation. Thromb Res 2020; 185: 135-141