Abstract
Medicinal leeches express a broad variety of anticoagulants and other bioactive factors that are involved in the blood feeding process. For most of the anticoagulants, several genes exist that may encode isoforms of the respective proteins, including hirudins and decorsins. Decorsins negatively affect platelet aggregation, whereas hirudins are potent thrombin inhibitors. Both factors belong to the hirudin superfamily that also includes the group of hirudin-like factors, and all hematophagous leeches analyzed so far contain several gene copies that encode representatives of at least two of the three groups of these factors. Members of the hirudin superfamily may contain only one central globular domain, but others may contain two or more copies. Here we describe the molecular identification and partial functional characterization of a broad variety of putative mono-, bi and multimeric hirudins, decorsins and hirudin-like factors in two Asian medicinal leech species, namely Hirudo nipponia and H. tianjinensis. Some of the monomeric hirudins and decorsins have already been described before, but they represent only a small part of the overall diversity. For the first time, putative monomeric and one oligomeric decorsins of H. tianjinensis were expressed as recombinant proteins, functionally characterized and successfully verified as platelet aggregation inhibitors. In addition we have conducted phylogenetic analyses based on genomic and mitochondrial markers and found convincing evidence that H. nipponia and H. tianjinensis together with members of the genus Whitmania form a monophyletic clade that is clearly distinct from clades that are formed either by European members of the genus Hirudo or by members of the genus Hirudinaria.