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)

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 ( K I = 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 K I 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 ( K I = 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 ( K D = 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 ( K I = 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.