Anti-Platelet Properties of Salivary Secretions of the Avian Leech.

Introduction

Haematophages, animals evolved to a bloodsucking lifestyle as their exclusive mode of feeding secrete compounds capable of arresting haemostasis in the host. It is clear that exploitation of host haemostasis is an absolute requirement for the survival of these species. Since the discovery and with the subsequent characterisation and engineering of Hirudin a potent thrombin inhibitor from the European medicinal leech Hirudo medicinalis, attention has been focused on the potential anticoagulant and platelet aggregation inhibitors derived from an array of different species of leech from both the Rhynchobdellid and Arhynchobdellid orders. Haematophagous leeches of the genus Theromyzon sp. of the Rhynchobdellid order, also termed duck leeches, feed directly on the nasal passages, trachea and nictating membranes of migratory birds. We present the novel observation of inhibition of aggregation of human platelets by the salivary secretion extracts of the avian leech Theromyzon tessulatum.

Methods

Twelve adult leeches of the species T. tessulatum (total weight 2.828g) were anaesthetised with ethanol vapour. The leeches were severed at the anterior end and a homogenate produced containing salivary gland secretions. The posterior two thirds of the leeches were treated identically to serve as control material. Platelet rich plasma (PRP) was prepared from blood from a normal individual (free from known platelet modifying medicines) mixed 9:1 (v:v) with 0.105 M trisodium citrate in siliconised glass vacutainers. Platelet numbers were adjusted with autologous platelet poor plasma (PPP) to obtain concentrations of approximately 300 × 10⁹/L. Leech extracts (anterior or posterior control) were added to PRP at a ratio of 1:4. Aggregation studies were performed using thrombin (10units/ml), collagen 10μg/ml, Ristocetin (1.5mg/ml) and ADP (5μm/ml).

Results

Data from this study shows that platelet aggregation was completely inhibited when stimulated by thrombin, collagen and ADP and partially inhibited (40%) on the addition of ristocetin.

Conclusion

Our observations contradict the belief that the anti-thrombocyte properties of this species of haematophagous leech are restricted to duck thrombocytes. We suggest the presence of one or more inhibitory molecules acting by various mechanisms including inhibition of vWF and platelet integrin mediated collagen interactions, inhibition of ristocetin mediated vWF and platelet GPIb receptor binding and salivary secretion derived apyrase inhibition of arachidonic acid mediated platelet aggregation.

These findings provide conclusive evidence that this blood sucking bird leech has the ability to overcome thrombocyte function in higher vertebrates.