Comparative Study of Leech-Derived Tryptase Inhibitor Genes in Four Medicinal Leech Species

Leeches are aquatic annelids of significant medicinal value, known to produce a variety of antithrombotic proteins. However, the extent of interspecies variation in these bioactive components remains poorly understood. In this study, next-generation sequencing and the Pichia pastoris eukaryotic expression system were employed to systematically analyze the diversity of leech-derived tryptase inhibitor (LDTI) genes and their functional activities across four dominant medicinal leech species: Hirudinaria manillensis, Hirudo nipponia, Whitmania pigra, and Whitmania laevis. Our results revealed a slightly higher genetic variation in LDTI genes and their protein among non-hematophagous leeches (W. pigra and W. laevis) compared to hematophagous species (H. manillensis and H. nipponia). Selection analysis indicated purifying selection in H. manillensis (ω = 0.47), while the other species exhibited either neutral or weak positive selection (ω ≥ 1). LDTI expression levels were significantly higher in hematophagous leeches, with peak expression observed in H. manillensis (transcripts per million = 2942.07 ± 1593.12). In vitro chromogenic substrate-based anticoagulant assays of recombinant proteins confirmed anticoagulation activity across all species, with H. manillensis displaying the most potent inhibition. These findings demonstrate the antithrombotic potential of all four species, regardless of their feeding ecology. Moreover, H. manillensis emerges as the optimal candidate for therapeutic development, owing to its superior LDTI sequence conservation, expression levels, and specific anticoagulant activity per unit concentration.