May Salivary Gland Secretory Proteins from Hematophagous Leeches (Hirudo verbana) Reach Pharmacologically Relevant Concentrations in the Vertebrate Host?

Sarah Lemke,Jan-Peter Hildebrandt,Christian Müller,Gabriele Uhl,Elisabeth Lipke,Ben J Mans

doi:10.1371/journal.pone.0073809

Abstract

Saliva of hematophagous leeches (Hirudo sp.) contains bioactive proteins which allow the leech proper feeding and storage of ingested blood, but may also exert effects in the host. Leech therapy is used to treat many different ailments in humans, although only a small fraction of salivary proteins are characterized yet. Moreover, we do not know whether complete transfer of salivary proteins stored in the unicellular salivary glands in a leech to the host during feeding may generate concentrations that are sufficiently high to affect physiological processes in the host. Our 3D reconstruction of a portion of internal leech tissue from histological sections revealed that one leech contains approx. 37,000 salivary gland cells. Using tissue slices from pig liver and mouse skeletal muscle for reference, we obtained data for protein densities in leech salivary gland cells. As individual salivary cells are voluminous (67,000 µm3) and the stored proteins are densely packed (approx. 500 µg/mm3), we extrapolated that a single leech may contain up to 1.2 mg of salivary proteins. Analyzing protein extracts of unfed or fed leeches by 2D electrophoresis, we calculated the relative molar amounts of individual salivary proteins in the mass range of 17–60 kDa which may be released from a single leech during feeding. Distribution of these salivary proteins in the host (assumed plasma volume of 5 l) may result in concentrations of individual compounds between 3 and 236 pmol/l. Such concentrations seem sufficiently high to exert biochemical interactions with target molecules in the host.

Highlights

As a consequence of adaptation of leeches to hematophagy, leech salivary gland cells contain a variety of bioactive proteins and peptides [1,2] which are, at least in part, transferred to the host during feeding [3]
Salivary gland cells showed round or slightly elongated cell bodies with diameters of 50– 100 mm which were entirely filled with material that stained either blue or red with Azan stain (Fig. 1 A)
We used an indirect method for determining protein concentration in the storage compartment of salivary gland cells using two types of reference tissues for which weight, volume and protein content were determined

Summary

Introduction

As a consequence of adaptation of leeches to hematophagy, leech salivary gland cells contain a variety of bioactive proteins and peptides [1,2] which are, at least in part, transferred to the host during feeding [3]. Ingredients of leech saliva may suppress inflammation, reduce the intensity of pain and inhibit blood coagulation in the host [4,5,6,7]. Of the many proteinaceous compounds in leech saliva [8,9], only a few have been identified at the molecular and functional level [1,10,11,12,13]. The only one which is well characterized in pharmacological terms is hirudin [6,14], which functions as a highly effective thrombin inhibitor and prevents clotting of vertebrate blood. Hirudin has been generated in recombinant form, and this has been successfully used for many years in human patients for thrombosis prophylaxis [15,16]

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Results

Conclusion