Characterization of anticoagulant proteins, kallikrein- and factor Xa inhibitors from the Korean leech, Hirudo nipponia

Abstract

I. Plasma kallikrein inhibitor: A new serine proteinase inhibitor of plasma kallikrein was screened and purified from a native Korean leech species, Hirudo nipponia. The inhibitor, named piguamerin, showed potent inhibitions of plasma and tissue kallikreins as well as trypsin. Sequencing analyses by automated Edman degradation revealed 48 amino acid residues with molecular mass of 5090 Da. Piguamerin is similar to antistasin-type inhibitors with the same spacing of ten cysteines, but shows differences from hirustasin, antistasin, and ghilanten at the surrounding residues of arginine27. The purified inhibitor modulated plasma clotting in tests of activated partial thromboplastin time. The new inhibitor was the first plasma kallikrein inhibitory member of the antistasin-type inhibitors specifically found from leeches, and may be related to the regulation of blood sucking process modulating the action of kallikreins in host vessels. II. Coagulation factor Xa inhibitor: A coagulation factor Xa inhibitor was screened and purified from the body extract of the blood-sucking leech H. nipponia. The inhibitor was found to be a polypeptide of 7366 Da named as tenastasin, with extraordinary richness in proline and lysine. It showed a specific inhibitory activity against human coagulation factor Xa with 0.5 nM of Ki value and 1:1 stoichiometric ratio, but no effect on various serine proteinases such as thrombin, trypsin, chymotrypsin, and elastase. It could prolong the clotting of human plasma at nanomolar concentrations with in vitro potency comparable to those of antistasin or tick anticoagulant peptide (TAP). The partially obtained sequence did not display high similarity toward different factor Xa inhibitors as well as proteinase inhibitors. Therefore, tenastasin, as a specific factor Xa inhibitor, might be useful for the application to various thrombotic diseases.