From haemadin to haemanorm: Synthesis and characterization of full-length haemadin from the leech Haemadipsa sylvestris and of a novel bivalent, highly potent thrombin inhibitor (haemanorm)
Basic science published in Protein Sci (2023)
Abstract
Hirudin from Hirudo medicinalis is a bivalent α-Thrombin (αT) inhibitor, targeting the enzyme active site and exosite-I, and is currently used in anticoagulant therapy along with its simplified analogue hirulog. Haemadin, a small protein (57 amino acids) isolated from the land-living leech Haemadipsa sylvestris, selectively inhibits αT with a potency identical to that of recombinant hirudin (KI = 0.2 pM), with which it shares a common disulfide topology and overall fold. At variance with hirudin, haemadin targets exosite-II and therefore (besides the free protease) it also blocks thrombomodulin-bound αT without inhibiting the active intermediate meizothrombin, thus offering potential advantages over hirudin. Here, we produced in reasonably high yields and pharmaceutical purity (>98%) wild-type haemadin and the oxidation resistant Met5 → nor-Leucine analogue, both inhibiting αT with a KI of 0.2 pM. Thereafter, we used site-directed mutagenesis, spectroscopic, ligand-displacement, and Hydrogen/Deuterium Exchange-Mass Spectrometry techniques to map the αT regions relevant for the interaction with full-length haemadin and with the synthetic N- and C-terminal peptides Haem(1-10) and Haem(45-57). Haem(1-10) competitively binds to/inhibits αT active site (KI = 1.9 μM) and its potency was enhanced by 10-fold after Phe3 → β-Naphthylalanine exchange. Conversely to full-length haemadin, haem(45-57) displays intrinsic affinity for exosite-I (KD = 1.6 μM). Hence, we synthesized a peptide in which the sequences 1-9 and 45-57 were joined together through a 3-Glycine spacer to yield haemanorm, a highly potent (KI = 0.8 nM) inhibitor targeting αT active site and exosite-I. Haemanorm can be regarded as a novel class of hirulog-like αT inhibitors with potential pharmacological applications.
Abstract sourced from PubMed (NCBI) for the cited record. See the original publication for the authoritative version.
Resumen
Synthetic haemadin from Haemadipsa sylvestris (57-aa) inhibits alpha-thrombin (Ki 0.2 pM) at active site + exosite-II, plus engineered haemanorm hybrid peptide (Ki 0.8 nM) targets active site + exosite-I.
Por qué esto importa para la hirudoterapia
Este estudio reporta la síntesis y caracterización de la haemadina, un inhibidor de trombina de 57 aminoácidos aislado de forma natural de la sanguijuela terrestre Haemadipsa sylvestris, y el diseño racional de un nuevo inhibidor bivalente denominado haemanorm. La haemadina inhibe potentemente la α-trombina (Ki = 0,2 pM), igualando a la hirudina recombinante, pero se dirige de forma única al exositio II en lugar del exositio I, lo que le permite bloquear la trombina unida a la trombomodulina sin inhibir la meizotrombina, una propiedad que los autores sugieren puede ofrecer ventajas farmacológicas sobre la hirudina. Los autores además mapearon los dominios funcionales e ingenierizaron el haemanorm (Ki = 0,8 nM), uniendo secuencias N- y C-terminales de haemadina mediante un espaciador de glicina para crear una molécula tipo hirulog dirigida tanto al sitio activo como al exositio I. Este trabajo es directamente relevante para el dominio de la ASH, ya que caracteriza una proteína anticoagulante derivada de la sanguijuela y deriva un potencial nuevo fármaco candidato a partir de su estructura. Sin embargo, estos son hallazgos bioquímicos e in vitro; no se presentan datos animales ni clínicos, y las afirmaciones de aplicación farmacológica siguen siendo preliminares.
Citación
From haemadin to haemanorm: Synthesis and characterization of full-length haemadin from the leech Haemadipsa sylvestris and of a novel bivalent, highly potent thrombin inhibitor (haemanorm).
Acquasaliente L et al. · Protein science, 2023
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Añadido a la biblioteca ASH: May 27, 2026 · Última actualización del sitio: 18 de junio de 2026