A serine-protease inhibitor of plasma kallikrein was screened and purified from a native Korean leech species, Hirudo nipponia. The peptide, named piguamerin, potently inhibited plasma and tissue kallikreins, and trypsin. Sequence analyses by automated Edman degradation revealed 48 amino acid residues and a molecular mass for the peptide of 5090 Da. Piguamerin is similar to antistasin-type inhibitors with the same spacing of ten cysteine residues, but shows differences from hirustasin, antistasin and ghilanten at the residues surrounding Arg27, which is a common P1 reactive residue for these inhibitors. The purified inhibitor modulated plasma clotting in tests of activated partial thromboplastin time at nanomolar concentrations. The serine-protease inhibitor of this leech may be involved in leech hematophagia.Keywords : leech ; plasma kallikrein; antistasin-type inhibitor.It has been demonstrated that hematophagous animals, such demonstrated no inhibition of factor Xa or thrombin. Unlike antistasin and ghilanten, hirustasin and the guamerins revealed as leeches, vampire bats, sucking lice and ticks, contain a variety of protease inhibitors that direct the coagulation mechanism [1]. no inhibitory activity against serine-protease functions related to coagulation. In the present paper, we report a kallikrein and trypAmong these animals, the leech is the most extensively studied for anticoagulant properties and for medical applications. Vari-sin inhibitor, piguamerin, which is derived from the blood-sucking leech H. nipponia. ous anticoagulants were purified from blood-sucking leeches: hirudin NH-Np, azocasein, and activated-partial-thromboplastin-time covered with the same cysteine spacing but half the molecular (APTT) kit (Cat.# A1801/A1926) were purchased from Sigma size of antistasin or ghilanten. Even though the primary strucChemical Co. Acetonitrile (HPLC grade) was purchased from tures of these inhibitors showed close similarity, each inhibitor Merck Chemical Co. Chromozym-TH was purchased from had a different protease target. Guamerin [9] and guamerin II Boehringer Mannheim. Chromatography columns, such as [10], isolated from blood-sucking Hirudo nipponia and nonAquapore RP-300 C 8 , Vydac 208TP54 C 8 and Macrpsphere 60 blood-sucking Whitmania edentula extracts, respectively, 7U Gel-Permeation, were purchased from Pierce, Vydac and Allshowed highly potent and specific inhibitory activities against tech, respectively. Adult leeches were collected from local ponds neutrophil and pancreatic elastases. Hirustasin, isolated from and cultivated in laboratory tanks until used [12]. Hirudo medicinalis, inhibits serine proteases such as trypsin, Purification. Crude extract was prepared by the aqueous chymotrypsin, cathepsin G and tissue kallikrein, but does not acetone/trichloroacetic acid method, as described previously [9, inhibit plasma kallikrein or elastase [11]. Furthermore, hirustasin 10]. The sample was passed through a DEAE-sepharose CL-6B column (50 ml), equilibrated with 10 mM Tris/HCl pH 8.0, and trifluro...

A cysteine-rich serine protease inhibitor (Guamerin II) was isolated from the non-blood sucking leech Whitmania edentula. The new inhibitor was identified as a low molecular weight (6,012 Da) polypeptide with some sequence similarities to antistasin, hirustasin and guamerin. The inhibitor contained 56 amino acid residues with 76.8% sequence similarity to guamerin, 48.2% to hirustasin and 28.6% to the first domain of antistasin. This new inhibitor was the first completely sequenced serine protease inhibitor from a non-blood sucking leech. Analysis of the inhibitor revealed that it was active against neutrophil elastase and chymotrypsin, but had no activity against a variety of other proteases. The P1 reactive site residue was identified as methionine and the residues surrounding the P1 site were hydrophobic amino acids. The primary structure of the inhibitor showed no similarity to well-known elastase inhibitors from leeches such as eglin.