Several leech species of the genera Hirudo, Hirudinaria, and Whitmania are widely used in traditional Chinese medicine (TCM) for the oral treatment of disorders associated with blood stasis. Among them, the non-hematophagous leech Whitmania pigra expresses a variety of components that have the potential to act on the vertebrate blood coagulation system. Whether the thrombin inhibitor hirudin, probably the most prominent leech-derived anticoagulant, is actually present in Whitmania pigra, is still a matter of debate. To answer that open question was the aim of the study. We identified several putative hirudin-encoding sequences in transcriptome data of Whitmania pigra. Upon gene synthesis and molecular cloning the respective recombinant proteins were expressed in Escherichia coli, purified, processed, and eventually functionally characterized for thrombin-inhibitory potencies in coagulation assays. We were successful in the identification and functional characterization of several putative hirudins in Whitmania pigra. Some, but not all, of these factors are indeed thrombin inhibitors. Whitmania pigra hence expresses both hirudins (factors that inhibit thrombin) and hirudin-like factors (that do not or only very weakly inhibit thrombin). Furthermore, we revealed the exon/intron structures of the corresponding genes. Coding sequences of some putative hirudins of Whitmania pigra were present also in transcriptome datasets of Hirudo nipponia, a hematophagous leech that is likewise used in TCM. Based on both structural and functional data we provide very strong evidence for the expression of hirudins in Whitmania pigra. This is the first description of hirudins in a non-hematophagous leech.

Osmotic stress in ducklings (Anas platyrhynchos) results in salt secretion and adaptive cell proliferation and differentiation in the nasal glands. We investigated whether osmotic stress in vivo or muscarinic ACh receptor activation in vitro changed the expression levels of the cellular protooncogene products Fos and Jun, which may play a role in the initiation of the adaptive processes. Using Fos- and Jun-specific polyclonal antisera in Western blot experiments, we demonstrated that Jun is constitutively expressed in nasal gland tissue, whereas Fos is not detectable in tissue from unstressed (naive) animals. Under conditions of osmotic stress imposed by replacing the drinking water of the animals with a 1% NaCl solution, Jun protein remains constant in nasal gland tissue, whereas Fos protein is transiently upregulated. Treatment of cultured nasal gland tissue with muscarinic agonists results in a transcriptionally regulated expression of Fos in an atropine-sensitive manner. Immunohistochemical experiments show that Fos accumulation occurs in the nuclei of the secretory cells. These results indicate that the activation of the c-fos gene induced by muscarinic ACh receptor-mediated signaling pathways may play an important role in the initiation of adaptive growth and differentiation processes in nasal glands of osmotically stressed ducklings.