Hirudin and Decorsins of the North American Medicinal Leech Macrobdella decora: Gene Structure Reveals Homology to Hirudins and Hirudin-like Factors of Eurasian Medicinal Leeches.

Blood-sucking leeches, some of which are referred to as medicinal leeches, have caught attention not only because of their medical purposes, but also as study organisms to conduct research within fields as diverse as neurobiology, osmoregulation, ecology, and phylogeny. Of particular interest is the question whether hemophagy in leeches is of single origin or evolved independently several times. A key component in the saliva of hematophagous leeches is hirudin, a strong natural inhibitor of thrombin and hence the blood coagulation cascade. Multiple isoforms of hirudin have been described within and among several leech species and genera, often based on sequence data only. The identification of hirudin-like factors (HLFs) illustrated the necessity to underpin such predictions by functional tests. We overexpressed and purified the hirudin of the North American medicinal leech, Macrobdella decora, and proved its thrombin-inhibiting activity. In addition, analysis of the gene structure of both hirudin and some of the decorsins of M. decora clearly indicated conserved exon and intron positions when compared to genes of hirudins and HLFs of Eurasian medicinal leeches. Our data provide evidence for the incorporation of decorsins into the hirudin superfamily and support the concept of a single origin of blood feeding in jawed leeches.

[Emulsion liquid membrane extraction with D2EHPA mobile carrier Hirudinaria manillensis hirudin in experimental study].

OBJECTIVE: To study the extraction system of hirudin emulsion liquid membrane with the Poecilobdella manillensis as raw material, di-(2-ethylhexyl) phosphate (D2EHPA) as carrier, Span 80 as emulsifier, octane and D2EHPA mixed to constitute membrane solution, diluted HCl solutions as internal aqueous phase. METHOD: Using the orthogonal experiment to optimize the extraction conditions of hirudin reference substance such as membrane phase, internal aqueous phase volume ratio (MIPVR), external aqueous phase pH, internal aqueous phase pH, mobile carrier concentration and so on, and then using hirudin crude extracts to do purifying experiment, and gaining experimental samples. RESULT: The optimal conditions of hirudin extraction were as follows: MIPVR 10: 3, internal aqueous phase pH 2.6, external aqueous phase pH 3.4, the mass fraction of carrier D2EHPA 2%. In the optimal extraction conditions, when the initial concentration of hirudin was one anti-thrombin activity units (ATU) x mL(-1), ATU recovery rate of the reference substance was 83.06%. In the purifying experiment of crude extracts, ATU recovery rate was 82.99%, and the specific activity of sample was 3 289.48 the ATU x mg(-1). Discontinuous polyacrylamide gel electrophoresis and spectral scanning, the results showed that the purity and reference substance were considerable. CONCLUSION: The method of preparation hirudin was relatively simple, the purity of the experimental samples and ATU recovery were both high.

[Determination of biological activity of extract from hirudo by N-benzoyl-L-arginine ethyl ester].

As a potent anticoagulant, leech a traditional Chinese medicine, has become increasing topics. Hirudin, which is the primary effective component in leech, is a specific and efficient inhibitor of thrombin, mainly used in prevention and treatment of thrombus on the clinic practice. However, there is still no accurate and convenient method reported about the determination of it's biological activity. This paper reported a method for the determination of the biological activity the of extract from hirudo. The extra thrombin, which was not inhibited by hirudin in the extract from hirudo, reacted with N-benzoyl-L-arginine ethyl ester and was determined. The biological activity of the hirudo extract was determined, indirectly. The linear of calibration curve and accuracy were both perfect, the method was accurate and reliable.

[Inhibition of kallikrein from human plasma by salivary gland secretion and extracts of the medicinal leech Hirudo medicinalis]. / Ingibirovanie kallikreina plazmy krovi cheloveka sekretom sliunnykh zhelez i ékstraktom iz meditsinskoi piiavki Hirudo medicinalis.

It has been shown for the first time that the salivary gland secretion of the medicinal leech (Hirudo medicinalis) contains a human blood plasma kallikrein inhibitor which is capable of blocking the amidolytic activity of the enzyme in an irreversible manner (with D-Pro-Phe-Arg-pNA as substrate) and which also suppresses the kininogenase activity of kallikrein. The inhibition of the amidolytic activity of highly purified kallikrein preparations from human blood plasma obeys the pseudo-first order kinetics. The experimental results suggest that in the salivary-gland secretion of H. medicinalis the inhibitor concentration exceeds by one order of magnitude that in whole leech homogenate extracts, which indicates that the inhibitor biosynthesis may be localized in leech salivary glands.

Pharmacology of hirudin: one hundred years after the first report of the anticoagulant agent in medicinal leeches.

One hundred years after its discovery hirudin, the selective thrombin inhibitor from medicinal leeches, has been brought into the focus of interest again. Advanced methods of peptide isolation and gene technology are expected to provide the opportunity to obtain pure hirudin in sufficient yield for therapeutic uses. Therefore, the present state of knowledge of this naturally occurring anticoagulant is reported.