American Society of Hirudotherapy

Functional phage display of leech-derived tryptase inhibitor (LDTI): construction of a library and selection of thrombin inhibitors

Research article published in FEBS letters (1999)

Last Updated: June 18, 2026Reviewed by: ASH Editorial Board
Research article — evidence reviewArticle reference
Evidence: Research reportDrug DevelopmentSalivary PharmacologyTanaka AS et al. · FEBS letters, 1999

Abstract

The recombinant phage antibody system pCANTAB 5E has been used to display functionally active leech-derived tryptase inhibitor (LDTI) on the tip of the filamentous M13 phage. A limited combinatorial library of 5.2 x 10(4) mutants was created with a synthetic LDTI gene, using a degenerated oligonucleotide and the pCANTAB 5E phagemid. The mutations were restricted to the P1-P4' positions of the reactive site. Fusion phages and appropriate host strains containing the phagemids were selected after binding to thrombin and DNA sequencing. The variants LDTI-2T (K8R, I9V, S10, K11W, P12A), LDTI-5T (K8R, I9V, S10, K11S, P12L) and LDTI-10T (K8R, I9L, S10, K11D, P12I) were produced with a Saccharomyces cerevisiae expression system. The new inhibitors, LDTI-2T and -5T, prolong the blood clotting time, inhibit thrombin (Ki 302 nM and 28 nM) and trypsin (Ki 6.4 nM and 2.1 nM) but not factor Xa, plasma kallikrein or neutrophil elastase. The variant LDTI-10T binds to thrombin but does not inhibit it. The relevant reactive site sequences of the thrombin inhibiting variants showed a strong preference for arginine in position P1 (K8R) and for valine in P1' (I9V). The data indicate further that LDTI-5T might be a model candidate for generation of active-site directed thrombin inhibitors and that LDTI in general may be useful to generate specific inhibitors suitable for a better understanding of enzyme-inhibitor interactions.

Abstract sourced from PubMed (NCBI) for the cited record. See the original publication for the authoritative version.

Publication typeJournal ArticleResearch Support, Non-U.S. Gov't
Indexed MeSH termsAmino Acid SequenceBacteriophagesBase SequenceChymasesCloning, MolecularDose-Response Relationship, DrugHumansKineticsModels, GeneticMolecular Sequence DataMutagenesis, InsertionalPeptide Library

Summary

The recombinant phage antibody system pCANTAB 5E has been used to display functionally active leech-derived tryptase inhibitor (LDTI) on the tip of the filamentous M13 phage.

Why This Matters for Hirudotherapy

This study used functional phage display to present the leech-derived tryptase inhibitor (LDTI) on M13 phage surfaces and created a combinatorial library of approximately 52,000 mutants varying at the P1–P4' reactive-site positions. After selection against thrombin, three variants were produced in yeast: LDTI-2T and LDTI-5T inhibited thrombin (Ki 302 nM and 28 nM, respectively) and prolonged blood clotting time, while LDTI-10T bound but did not inhibit thrombin. The results revealed a strong preference for arginine at P1 and valine at P1' for thrombin inhibition, with LDTI-5T identified as a model candidate for generating active-site-directed thrombin inhibitors. This is relevant to ASH's domain as it directly engineers a leech-derived inhibitor scaffold for new thrombin-inhibitory activity. The caveat is that this is an in vitro molecular engineering study with no animal or clinical data reported in the abstract.

Citation

Functional phage display of leech-derived tryptase inhibitor (LDTI): construction of a library and selection of thrombin inhibitors

Tanaka AS et al. · FEBS letters, 1999

DOI

Added to ASH library: May 27, 2026 · Site last updated: June 18, 2026

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