Hirudins and fenestrins of the African medicinal leech Asiaticobdella fenestrata.

African hematophagous leeches are largely underinvestigated both in terms of their biodiversity and the characterization of the repertoire of bioactive components of their salivary gland secretions. Asiaticobdella (formerly Aliolimnatis) fenestrata Moore, 1939, also referred to as the African medicinal leech, is hence a promising candidate for the identification and functional characterization of putative anticoagulants that may serve as inspirations for the development of new or the enhancement of existing drugs for medical purposes. A. fenestrata inhabits flooded plains of southern African regions and mainly feeds on mammals like black lechwe (Kobus leche Gray, 1850). Whereas several anticoagulants, including two putative hirudins of A. fenestrata, have already been identified in previous investigations, functional characterizations of these components are yet missing. We conducted an in-depth re-analysis of the salivary gland transcriptomic data set of A. fenestrata and identified two additional hirudin-like factors that exhibit a remarkable structural feature-the presence of an RGD motif at or near the N-terminal end of the molecules. The presence of such an RGD motif is a key feature of leech-derived platelet aggregation inhibitors such as ornatins and decorsins. However, both in ornatins and in decorsins, the respective RGD motif is located within the molecule between the 5th and the 6th cysteine residues, but not at or near the N-terminus. We named the two putative platelet aggregation inhibitors "fenestrins." Both the two putative hirudins and the two fenestrins of A. fenestrata were expressed as recombinant proteins, purified, and functionally characterized in either coagulation or platelet aggregation assays.