Abstract
Thromboembolic disease is a common cardio-cerebral vascular disease that threatens human life and health. Thrombin not only affects the exogenous coagulation pathway, but also the endogenous pathway. Thus, it becomes one of the most important targets of anticoagulant drugs. RGD-hirudin is an anticoagulant drug targeting thrombin, but it can only be administered intravenously. We designed a low molecular weight peptide based on RGD-hirudin that could prevent blood clots. We first used NMR to identify the key amino acid residues of RGD-hirudin that interacted with thrombin. Then, we designed a novel direct thrombin inhibitor peptide (DTIP) based on the structure and function of RGD-hirudin using homology modeling. Molecular docking showed that the targeting and binding of DTIP with thrombin were similar to those of RGD-hirudin, suggesting DTIP interacted directly with thrombin. The active amino acids of DTIP were identified by alanine scanning, and mutants were successfully constructed. In blood clotting time tests in vitro, we found that aPTT, PT, and TT in the rat plasma added with DTIP were greatly prolonged than in that added with the mutants. Subcutaneous injection of DTIP in rats also could significantly prolong the clotting time. Thrombelastography analysis revealed that DTIP significantly delayed blood coagulation. Bio-layer interferometry study showed that there were no significant differences between DTIP and the mutants in thrombin affinity constants, suggesting that it might bind to other sites of thrombin rather than to its active center. Our results demonstrate that DTIP with low molecular weight can prevent thrombosis via subcutaneous injection.
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References
Timmis A, Townsend N, Gale C, Grobbee R, Maniadakis N, Flather M, et al. European society of cardiology: cardiovascular disease statistics 2017. Eur Heart J. 2018;39:508–79.
Mackman N. Triggers, targets and treatments for thrombosis. Nature. 2008;451:914–8.
Franchini M, Mengoli C, Cruciani M, Bonfanti C, Mannucci PM. Effects on bleeding complications of pharmacogenetic testing for initial dosing of vitamin K antagonists: a systematic review and meta-analysis. J Thromb Haemost. 2014;12:1480–7.
Lane MA, Zeringue A, McDonald JR. Serious bleeding events due to warfarin and antibiotic co-prescription in a cohort of veterans. Am J Med. 2014;127:657–63.
Shen AY, Yao JF, Brar SS, Jorgensen MB, Chen W. Racial/ethnic differences in the risk of intracranial hemorrhage among patients with atrial fibrillation. J Am Coll Cardiol. 2007;50:309–15.
Vanassche T, Vandenbriele C, Peerlinck K, Verhamme P. Pharmacotherapy with oral Xa inhibitors for venous thromboembolism. Expert Opin Pharmacother. 2015;16:645–58.
Krenzlin H, Lorenz V, Danckwardt S, Kempski O, Alessandri B. The importance of thrombin in cerebral injury and disease. Int J Mol Sci. 2016;17:84.
Strukova SM. Role of platelets and serine proteinases in coupling of blood coagulation and inflammation. Biochem (Mosc). 2004;69:1067–81.
Crawley JT, Zanardelli S, Chion CK, Lane DA. The central role of thrombin in hemostasis. J Thromb Haemost. 2007;5(Suppl 1):95–101.
Lee CJ, Ansell JE. Direct thrombin inhibitors. Br J Clin Pharmacol. 2011;72:581–92.
Huntington JA. Molecular recognition mechanisms of thrombin. J Thromb Haemost. 2005;3:1861–72.
Lechtenberg BC, Freund SM, Huntington JA. GpIbα interacts exclusively with exosite II of thrombin. J Mol Biol. 2014;426:881–93.
Coppens M, Eikelboom JW, Gustafsson D, Weitz JI, Hirsh J. Translational success stories: development of direct thrombin inhibitors. Circ Res. 2012;111:920–9.
Huang Y, Zhang Y, Zhao B, Xu Q, Zhou X, Song H, et al. Structural basis of RGD-hirudin binding to thrombin: Tyr3 and five C-terminal residues are crucial for inhibiting thrombin activity. BMC Struct Biol. 2014;14:26.
Lu WF, Mo W, Liu Z, Fu WG, Guo DQ, Wang YQ, et al. The antithrombotic effect of a novel hirudin derivative after reconstruction of carotid artery in rabbits. Thromb Res. 2010;126:e339–43.
Zhao B, Zhang Y, Huang Y, Yu J, Li Y, Wang Q, et al. A novel hirudin derivative inhibiting thrombin without bleeding for subcutaneous injection. Thromb Haemost. 2017;117:44–56.
NIGMS. 50 Years of Protein Structure Determination Timeline-HTML Version-National Institute of General Medical Sciences. ed.: https://publications.nigms.nih.gov/psi/timeline_text.html 2011.
Berman H, Henrick K, Nakamura H. Announcing the worldwide protein data bank. Nat Struct Biol. 2003;10:980.
Cavasotto CN, Phatak SS. Homology modeling in drug discovery: current trends and applications. Drug Discov Today. 2009;14:676–83.
Muhammed MT, Aki-Yalcin E. Homology modeling in drug discovery: overview, current applications, and future perspectives. Chem Biol Drug Des. 2019;93:12–20.
Werner T, Morris MB, Dastmalchi S, Church WB. Structural modelling and dynamics of proteins for insights into drug interactions. Adv Drug Deliv Rev. 2012;64:323–43.
Marti-Renom MA, Stuart AC, Fiser A, Sanchez R, Melo F, Sali A. Comparative protein structure modeling of genes and genomes. Annu Rev Biophys Biomol Struct. 2000;29:291–325.
Huang Y, Zhang Y, Wu Y, Wang J, Liu X, Dai L, et al. Expression, purification, and mass spectrometric analysis of 15N, 13C-labeled RGD-hirudin, expressed in Pichia pastoris, for NMR studies. PLoS One. 2012;7:e42207.
Kuwahara K, Hikosaka M, Kaneko T, Takamatsu A, Nakajima Y, Ogawa R, et al. Analysis of cranial morphology of healthy infants using homologous modeling. J Craniofac Surg. 2019;30:33–8.
Osmani SA, Bak S, Moller BL. Substrate specificity of plant UDP-dependent glycosyltransferases predicted from crystal structures and homology modeling. Phytochemistry. 2009;70:325–47.
Rydel TJ, Ravichandran KG, Tulinsky A, Bode W, Huber R, Roitsch C, et al. The structure of a complex of recombinant hirudin and human alpha-thrombin. Science. 1990;249:277–80.
Liu CC, Brustad E, Liu W, Schultz PG. Crystal structure of a biosynthetic sulfo-hirudin complexed to thrombin. J Am Chem Soc. 2007;129:10648–9.
Song X, Mo W, Liu X, Zhu L, Yan X, Song H, et al. The NMR solution structure of recombinant RGD-hirudin. Biochem. Biophys. Res. Commun. 2007;360:103–8.
Carter NJ, McCormack PL, Plosker GL. Enoxaparin: a review of its use in ST-segment elevation myocardial infarction. Drugs. 2008;68:691–710.
WHO. About cardiovascular diseases. ed.: http://www.who.int/cardiovascular_diseases/about_cvd/en/.
Posma JJ, Posthuma JJ, Spronk HM. Coagulation and non-coagulation effects of thrombin. J Thromb Haemost. 2016;14:1908–16.
Zavyalova E, Ustinov N, Golovin A, Pavlova G, Kopylov A. G-Quadruplex aptamers to human thrombin versus other direct thrombin inhibitors: the focus on mechanism of action and drug efficiency as anticoagulants. Curr Med Chem. 2016;23:2230–44.
Kong Y, Chen H, Wang YQ, Meng L, Wei JF. Direct thrombin inhibitors: patents 2002-2012 (Review). Mol Med Rep. 2014;9:1506–14.
Fareed J, Jeske WP. Small-molecule direct antithrombins: argatroban. Best Pract Res Clin Haematol. 2004;17:127–38.
Fugate JE, Rabinstein AA, McBane RD, Lanzino G. Dabigatran: a primer for neurosurgeons. World Neurosurg. 2013;79:154–8.
Mo W, Zhang YL, Chen HS, Wang LS, Song HY. A novel hirudin derivative characterized with anti-platelet aggregations and thrombin inhibition. J Thromb Thrombolysis. 2009;28:230–7.
Acknowledgements
This study was supported by the National Natural Science Foundation of China (NSFC 81673498) and the Science and Technology Commission of Shanghai Municipality (STCSM 16431904600).
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YRL, MFW, and YLZ performed all of the experiments; BZ, YNH, MFW, TYL, and DC participated in the research; YRL and WM designed experiments, analyzed data, and wrote the paper; MY is the supervisor of YRL. All authors read and approved the final manuscript.
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Li, Yr., Huang, Yn., Zhao, B. et al. RGD-hirudin-based low molecular weight peptide prevents blood coagulation via subcutaneous injection. Acta Pharmacol Sin 41, 753–762 (2020). https://doi.org/10.1038/s41401-019-0347-0
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DOI: https://doi.org/10.1038/s41401-019-0347-0
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