Proton bridging in the interactions of thrombin with hirudin and its mimics

Thrombin is the pivotal serine protease enzyme in the blood cascade system and thus a target of drug design for control of its activity. The most efficient nonphysiologic inhibitor of thrombin is hirudin, a naturally occurring small protein. Hirudin and its synthetic mimics employ a range of hydrogen bonding, salt bridging, and hydrophobic interactions with thrombin to achieve tight binding with K(i) values in the nano- to femtomolar range. The one-dimensional (1)H nuclear magnetic resonance spectrum recorded at 600 MHz reveals a resonance 15.33 ppm downfield from silanes in complexes between human α-thrombin and r-hirudin in pH 5.6-8.8 buffers and between 5 and 35 °C. There is also a resonance between 15.17 and 15.54 ppm seen in complexes of human α-thrombin with hirunorm IV, hirunorm V, an Nα(Me)Arg peptide, RGD-hirudin, and Nα-2-naphthylsulfonyl-glycyl-DL-4-amidinophenylalanyl-piperidide acetate salt (NAPAP), while there is no such low-field resonance observed in a complex of porcine trypsin and NAPAP. The chemical shifts suggest that these resonances represent H-bonded environments. H-Donor-acceptor distances in the corresponding H-bonds are estimated to be <2.7 Å. Addition of Phe-Pro-Arg-chloromethylketone (PPACK) to a complex of human α-thrombin with r-hirudin results in an additional signal at 18.03 ppm, which is 0.10 ppm upfield from the observed signal [Kovach, I. M., et al. (2009) Biochemistry 48, 7296-7304] for thrombin covalently modified with PPACK. In contrast, the peak at 15.33 ppm remains unchanged. The fractionation factors for the thrombin-hirudin complexes are near 1.0 within 20% error. The most likely site of the short H-bond in complexes of thrombin with the hirudin family of inhibitors is in the hydrophobic patch of the C-terminus of hirudin where Glu(57') and Glu(58') are embedded and interact with Arg(75) and Arg(77) and their solvate water (on thrombin). Glu(57') and Glu(58') present in the hirudin family of inhibitors make up a key binding epitope of fibrinogen, thrombin's prime substrate, which lends substantial interest to the short hydrogen bond as a binding element at the fibrinogen recognition site.

Abstract sourced from PubMed (NCBI) for the cited record. See the original publication for the authoritative version.

1H NMR studies at 600 MHz reveal a short hydrogen-bond network at the C-terminal hirudin-thrombin interface (Glu57', Glu58' to Arg75, Arg77), providing structural insight into hirudin's extremely high affinity for thrombin.

Leech-derived molecules this paper discusses, each with a full profile in the ASH compound registry:

Erfahren Sie, wie diese Forschung mit der klinischen Praxis verknüpft ist

More peer-reviewed studies in Speichel-Pharmakologie.

• Novel Leech Antimicrobial Peptides — Hirunipins — Kumar et al. (2025)
• 434 Salivary Proteins — Integrated Proteomics of Hirudo medicinalis — Liu et al. (2019)
• Medicinal Leech Genome Assembly — Anticoagulant Gene Catalog — Kvist et al. (2020)
• Effects of hirudotherapy on liver functions, lipid profile, and insulin sensitivity in rats with metabolic syndrome — Bilden A et al. (2026)
• Efficacy and Safety of Hirudotherapy for Improving Sperm Quality Parameters in Male Infertility: A Randomized Controlled Trial — Abtahi-Forooshani M et al. (2026)
• [Medicinal leeches and hirudotherapy] — Gödekmerdan A et al. (2011)
• PEROXIDATION OF SALIVA LIPIDS IN PATIENTS WITH POSTCOVID SYNDROME DURING HIRUDOTHERAPY — Serikbayeva S et al. (2024)
• Medicinal leech therapy in pain syndromes: a narrative review — Koeppen D, Aurich M, Rampp T (2014)
• Leech therapy in the treatment of a penile haematoma in a stallion — Nowicki A, Jaworska J, Baranski W (2021)
• Leech therapy (Hirudo medicinalis) attenuates testicular damages induced by testicular ischemia/reperfusion in an animal model — Davoodi F et al. (2021)

Browse all ASH-analysed research →