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)

Hirudin from Hirudo medicinalis is a bivalent alpha-Thrombin (alpha T) 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 alpha T 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 alpha T 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 alpha T 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 alpha T 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 alpha T active site (K-I = 1.9 mu M) and its potency was enhanced by 10-fold after Phe3 -> beta-Naphthylalanine exchange. Conversely to full-length haemadin, haem(45-57) displays intrinsic affinity for exosite-I (K-D = 1.6 mu M). 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 alpha T active site and exosite-I. Haemanorm can be regarded as a novel class of hirulog-like alpha T inhibitors with potential pharmacological applications.