Inhibitors of Platelet Aggregation from Diverse Haematophagous and Predaceous Hirudinea from around the World.

Introduction

Haematophages, animals evolved to a blood sucking lifestyle as their exclusive mode of feeding secrete compounds capable of arresting haemostasis in the host. Since the discovery and subsequent characterisation and engineering of hirudin, attention is being focused on the potential anticoagulant and platelet aggregation inhibitors from an array of different species of leech from both the Rhynchobdellid and Arhynchobdellid orders. The purpose of this study was to survey the presence of inhibitors of platelet aggregation in cephalic extracts prepared from phylogenetically diverse proboscis-bearing and jawed blood-sucking leeches compared to those which are predaceous in nature.

Methods

Cephalic extracts from specimens of each species were prepared by homogenising in tris buffer the anterior one-third region of the bodies containing the salivary glands. In the case of the giant leech, Haementeria ghilianii, paired anterior salivary glands were excised through the oesophagus. The posterior musculature served as control material. Homogenates were double centrifuged and supernatants micro-filtered. Individual extracts or imidazole buffer as control were pre-incubated 1:4 with platelet rich plasma for 5 minutes prior to testing for inhibition of platelet aggregation. Inducers of aggregation were: ADP (5 and 10 μM/ml), Collagen (COL) (4μg/ml), Thrombin (THR) (0.2 U/ml), Ristocetin (RIS) (1.5 mg/ml) and Adrenaline (ADR) (1 μM). Maximal aggregation responses were measured over 10 minutes. All extracts were kept on ice and tested within 1 hour of preparation.

Results

The species of leeches examined and maximal aggregation responses (%) after 10 minutes are shown in table 1.

None of the extracts induced the spontaneous aggregation of platelets suspended in autologous plasma. Posterior musculatures did not inhibit platelet aggregation.

Conclusion

Cephalic extracts from all the haematophagous leeches examined in this study contain inhibitors of platelet aggregation. Our findings suggest that anti-haemostatic properties in Hirudinea appear to have evolved in response to biological adaptations rather than in keeping with phylogenetic affinities. Further work is required to define the precise nature of such mechanisms. These new anti-platelet properties in different species could have further potential therapeutic applications.