Antithrombins from the blood-sucking leech, hirudo nipponia

Abstract

Blood-sucking leeches are known to have various anticoagulant peptides that are necessary to prevent the clotting of blood during ingestion and digestion. The Korean native leech, Hirudo nipponia also has potent antithrombins and antiplatelet agents in the saliva. The internal amino acid sequence of the thrombin inhibitor secreted by H. nipponia into saliva is unexpectedly different (55%) from that of hirudin secreted by H. medicinalis. This difference in sequence is reflected in the lack of neutralization by the polyclonal antibody to hirudin from H. medicinalis. Blockage of the N-terminal in antithrombin from H. nipponia appears to be a further real difference compared to hirudin from H. medicinalis. A new thrombin-specific inhibitor, named hirujin, was purified from H. nipponia by size-exclusion, ion-exchange, affinity and high performance liquid chromatography. The molecular mass obtained by mass spectrometry was 7020 Da, which was less than the estimate from gel electrophoresis (= 10 kDa). Hirujin is an acidic inhibitor (pI = 3.8) and it is very stable in heat, pH, and chemical denaturants. The binding of hirujin to thrombin is extremely tight with a low inhibition constant, Ki value of 148 fM. Fifty amino acid residues of the C-terminal region of hiruin were determined, and estimated 10 residues of the N-terminal sequence were not determined due to blockage at the N-terminus. The sequenced N-terminal region showed a similarity to the same region of the hirudin sequence, but no matches were found in the C-terminal regions of hirudin and hirujin. The new inhibitor has an O-glycosylated serine residue in front of three consecutive proline residues in the C-terminal domain. Hirujin and its variant form a new subfamily within hirudin family. A cysteine-rich (approximately 20%), low molecular mass (MW 6 kDa) polypeptide has been isolated from Hirudo nipponia. From its amino acid composition and N-terminal amino acid sequence analysis, the new protein is sim...