Draft genome of the European medicinal leech Hirudo medicinalis (Annelida, Clitellata, Hirudiniformes) with emphasis on anticoagulants.

The European medicinal leech has been used for medicinal purposes for millennia, and continues to be used today in modern hospital settings. Its utility is granted by the extremely potent anticoagulation factors that the leech secretes into the incision wound during feeding and, although a handful of studies have targeted certain anticoagulants, the full range of anticoagulation factors expressed by this species remains unknown. Here, we present the first draft genome of the European medicinal leech, Hirudo medicinalis, and estimate that we have sequenced between 79-94% of the full genome. Leveraging these data, we searched for anticoagulation factors across the genome of H. medicinalis. Following orthology determination through a series of BLAST searches, as well as phylogenetic analyses, we estimate that fully 15 different known anticoagulation factors are utilized by the species, and that 17 other proteins that have been linked to antihemostasis are also present in the genome. We underscore the utility of the draft genome for comparative studies of leeches and discuss our results in an evolutionary context.

Intracellular GSH and ROS levels may be related to galactose-mediated human lens epithelial cell apoptosis: role of recombinant hirudin variant III.

Oxidative processes in the lenses are the most commonly found damaging factor for the development of cataracts. Hirudin, a most potent inhibitor of thrombin as an antithrombic drug, also have potential use in cataracts. In order to investigate the mechanisms of hirudin against galactose-induced cataract at the cellular level. We used recombinant hirudin variant III (rHV3) to study the protective effect of hirudin on galactose-mediated human lens epithelial cells injury. The human lens epithelial cells (hLECs) were cultured in D/F(12)-10% FBS medium containing 125 mM D-galactose with or without rHV3. Cell viability was assessed by methylthiazol tetrazolium (MTT) assay and propidium iodide (PI) staining in situ. Cell apoptosis was elevated with comet assay (single cell gel electrophoresis, SCGE), AO/EB double staining and Annexin-V/PI double staining assay. Reactive oxygen species (ROS) were quantified with 2',7'-dichlorofluorescein (DCF), and free glutathione (GSH) levels were measured with a commercial GSH quantification kit. Decreased viability and increased apoptosis of the hLECs were observed when incubated with 125 mM galactose. These hLECs also demonstrated the increased presence of ROS, whereas GSH was reduced. rHV3 blocked the induction of cell death, apoptosis and oxidative stress in hLECs. One mechanism may be through regulating intracellular ROS and GSH levels to inhibit apoptosis of the human lens epithelial cells.

Structure-function relationships of the C-terminal functional domain of hirudin and its variants.

The C-terminal functional domain of hirudin, hirudin variant 1 (residues 55-65), binds to a non-catalytic site on thrombin. In doing so, it is capable of inhibiting the procoagulant actions of thrombin. In terms of free energy of binding, this domain, which comprises 17% of the total sequence of the protein, contributes approximately half of the binding energy of the whole protein to thrombin. This situation also appears to hold true for the known variants of hirudin, some of which differ in the functional nature of their C-terminal regions. Extensive structure-function studies on this domain yield insights into the differences and similarities in the modes of thrombin interaction of hirudin and its variants. In particular, hirudin and hirudin PA have a similar and somewhat interchangeable structure-activity relationships (SAR) profile that indicates that they interact with thrombin in a similar manner. Hirullin P18, a 62 amino acid member of the hirudin family and isolated from Hirudinaria manillensis, is substantially different in sequence and its SAR, which shows that, although it seems to utilize the same non-catalytic binding domain as hirudin, it must utilize a different mode of interaction with thrombin.