Therostasin, a Novel Clotting Factor Xa Inhibitor from the Rhynchobdellid Leech, Theromyzon tessulatum

Abstract

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.

Inhibition of Coagulation Activation and Inflammation by a Novel Factor Xa Inhibitor Synthesized from the Earthworm Eisenia andrei

“…The advantages of such a short peptide are that it would be non-immunogenic and provide higher efficacy in the coagulation cascade. 24) Our results demonstrated that ESP would be an effective, potent FXa inhibitor that might inhibit the bidirectional pathways mediating the coagulation and inflammatory signals. In particular, ESP appeared to directly bind to the active site of FXa, thereby blocking its interaction with substrate.…”

“…All procedures were carried out at room temperature in 96-well microtiter plates following methods described previously, and the concentration for FXa inhibition (5 mg/ml) was also adopted from previously reported studies for antistasin. 20,24) The human enzyme reaction was initiated either by addition of substrate (S-2222, Chromogenix, Milano, Italy) to FXa protein (Novagen, WI, U.S.A.) or to mixtures containing pre-incubated FXa-ASP or -ESP, and the color that developed from the release of p-nitroanilide from the chromogenic substrate molecules was monitored continuously for 5 min at 405 nm on a Spectra Max Plus340 (Molecular Devices, CA, U.S.A.). The progress curves were recorded (% valueϭ[1Ϫ(DOD 405 of each sample/DOD 405 of control)]ϫ100).…”

Theromin, a Novel Leech Thrombin Inhibitor

“…However, between therostasin and theromin another region is also well conserved; i.e. a consensus sequence has been found in residues 40 - (29) are not conserved in the thrombin inhibitor theromin. Thus, the observed similarities could be the result of an evolutionary divergence from an ancestral gene, arising after gene duplication and able to generate several peptides acting toward specific substrates.…”

“…In fact, in 1994 the Merck Company deposited foreign patent applications regarding three thrombin inhibitors having masses of 3, 9, and 14 kDa. Interestingly, the N-terminal sequence of the 9-kDa inhibitor, EDDNPGPPRACPGE (47), shows homology with those of theromin (ECENTECPRACPGE), the factor Xa inhibitor therostasin (DCENTECPRACPGE) (29), and the trypsin inhibitor tessulin (MCENTECPRACPGE) (42). This 9-kDa thrombin inhibitor peptide possesses a pI of 4.9, a clotting time in a fibrinogen test of Ͼ600 s/5 l, and a specific activity at the final step of purification of 25 IU for thrombin inhibition and 0.2 IU for factor Xa inhibition.…”

“…By contrast, sequence comparisons were carried out for theromin with the four different serine protease inhibitors isolated from the leech T. tessulatum: cytin, therin, therostasin, and tessulin (5,29,(41)(42)(43). This revealed that two of the four (i.e.…”