We purified the most potent thrombin inhibitor described to date from the rhynchobdellid leech Theromyzon tessulatum. Designated theromin, it was purified to apparent homogeneity by gel permeation and anion exchange chromatography followed by two reverse-phase steps of high performance liquid chromatography. The primary sequence of theromin (a homodimer of 67 amino acid residues including 16 cysteine residues) was determined by a combination of reduction and s-␤-pyridylethylation, Edman degradation, trypsin enzymatic digestion, and matrix-assisted laser desorption mass spectrometry measurement. Theromin exhibits no sequence homology with any other thrombin inhibitors. Furthermore, theromin significantly diminishes, in a dose-dependent manner, the level of human granulocyte and monocyte activation induced by lipopolysaccharides. In summary, this potent thrombin inhibitor promises to have high biomedical significance.

Therostasin is a potent naturally occurring tightbinding inhibitor of mammalian Factor Xa (K i , 34 pM), isolated from the rhynchobdellid leech Theromyzon tessulatum. Therostasin is a cysteine-rich protein (8991 Da) consisting of 82 amino acid residues with 16 cysteine residues. Its amino acid sequence has been determined by a combination of techniques, including Edman degradation, enzymatic cleavage, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) on the native and s-␤-pyridylethylated compound. Sequence analysis reveals that it shares no significant homology with other Factor Xa inhibitors except for the putative reactive site. Moreover, it contains a signature pattern for proteins of the endothelin family, potent vasoconstrictors isolated in mammal and snake venom. Therostasin cDNA (825 bp) codes for a polypeptide of 82 amino acid residues preceded by 19 residues, representing a signal peptide sequence. As for the other known inhibitors of Factor Xa, therostasin is expressed and stored in the cells of the leech salivary glands.

We purified a new trypsinϪchymotrypsin inhibitor, designated tessulin, from the rhynchobdellid leech Theromyzon tessulatum. This 9-kDa peptide was purified to apparent homogeneity by gel-permeation and anion-exchange chromatographies followed by reverse-phase HPLC. The structure of tessulin was determined by reduction, S-β-pyridylethylation, trypsin digestion, automated Edman degradation and matrix-assisted laser desorption mass spectrometry (m/z 8985 Da). The 81-amino-acid peptide possesses 16 cysteines and exhibits a 16% sequence similarity with antistasin-type inhibitors. Tessulin inhibits trypsin (K i 1 pM) and chymotrypsin (K i 150 pM) and exhibits no activity with thrombin, factor Xa, cathepsin G and elastase. This is the first trypsinϪchymotrypsin inhibitor isolated from leeches that does not inhibit elastase or cathepsin G, except for cytin and therin. Furthermore, tessulin, in conjunction with other serine-protease inhibitors isolated from Theromyzon (therin, theromin), significantly diminishes the level of human granulocyte and monocyte activation induced by lipopolysaccharides (10 µg). The combined level of inhibition is higher than that of aprotinin, another serine-protease inhibitor used biomedically. Thus, tessulin may be clinically significant in reducing inflammatory events.Keywords : protease inhibitors; trypsin-chymotrypsin inhibitor.In numerous pathological processes, the crucial role played by proteases is becoming more apparent. This critical role creates a need for the development of non-toxic protease inhibitors for in vivo clinical applications. Because of the specific recognition by proteases of defined amino acid sequences, it is possible to inhibit these enzymes when they are involved in pathological processes [1]. The involvement of proteolytic enzymes in a number of degenerative diseases, e.g., pancreatitis [zymogenic forms of proteolytic enzymes (trypsin 1 and 2, chymotrypsin A [2, 3] and elastase [4])], is becoming more evident as is the need for curtailing this activity in uncalled-for situations [5Ϫ7]. In this regard, aprotinin, a 6.5-kDa trypsin and kallikrein inhibitor isolated from bovine organs [8] has been used for more than three decades as an intensive care drug for acute pancreatitis [9], demonstrating the significance of this class of enzyme.In hematophageous leeches, animals that must escape hostimmune surveillance, studies on serine-protease inhibitors have yielded significant findings relating to the clinical importance of these enzyme inhibitors. Of two groups of serine-protease [19]. However, some of these inhibitors exhibit different amino acid sequences and inhibitory activities; their three-dimensional structures share the same structural motif with the leech antihemostatic protein (L.A.P.) [20]. Interestingly, their mechanisms of action and epitopes important for binding to their respective targets are distinct as well [20], suggesting that through evolution different strategies have emerged to deal with a similar problem.Among the second group of serine-protea...

We purified a trypsin inhibitor, designated therin, from the rhynchobdellid leech Theromyzon tessulatum. Therin was purified to apparent homogeneity by gel-permeation and anion-exchange chromatography followed by reverse-phase HPLC. By a combination of reduction and S-β-pyridylethylation, Edman degradation and electrospray mass spectrometry measurement, the complete sequence of therin (48 amino acid residues ; m/z, 5376.35Ϯ0.22 Da) was determined. Therin exhibits an approximately 30% sequence similarity with peptides of the antistasin-type inhibitors family, i.e. the first and second domains of antistasin, hirustasin, ghilanthen and guamerins (I, II). Therin is a tight-binding inhibitor of trypsin (K i , 45Ϯ 12 pM) and has no action towards elastase or cathepsin G. Furthermore, therin (10 Ϫ6 M) in conjunction with theromin, a Theromyzon thrombin inhibitor (10 Ϫ6 M) significantly diminish the level of human leucocytes activation induced by lipopolysaccharide (10 µg) in a manner similar to that of aprotinin. These data suggest a leech trypsin inhibitor with possible biomedical signifance.

We purified a chymotrypsin inhibitor, designated cytin, from the rhynchobdellid leech Theromyzon tessulatum. This 7.4-kDa peptide was purified to apparent homogeneity by gel-permeation and anionexchange chromatographies, followed by reverse-phase HPLC. The structure of cytin was determined by reduction, S-p-pyridilethylation, automated Edman degradation, and electrospray mass spectrometry. Cytin is formed by the association of two protein chains, which are linked by a disulfide bridge. Chain A consists of 43 and chain B of 22 amino acid residues. Chain B exhibits 40-63% sequence similarity with the N-terminal sequences of subtilisinkhymotrypsin inhibitors isolated from barley seeds. Cytin inhibited chymotrypsin (K, 600 pM) and weakly inhibited trypsin (Kl 350 nM). This chymotrypsin inhibitor, in contrast to others isolated from leeches, does not inhibit elastase or cathepsin G. Furthermore, cytin (10 pM) significantly diminishes the level of human granulocyte and monocyte activation induced by lipopolysaccharide (1 U/ml) in a manner similar to that of aprotinin. These data indicate that this chymotrypsin inhibitor may be biomedically significant.