Abstract

The purpose of this study was to survey the anti-haemostatic nature of cephalic extracts prepared from phylogenetically diverse rhyncobdellid (proboscis-bearing) and arhynchobdellid (jawed) leeches and to evaluate the significance in the context of their evolutionary development. 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 removed by excision 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 pooled normal citrated plasma for 5 minutes prior to testing for inhibition of coagulation or with platelet rich plasma prior to assessing their effect on platelet aggregation. All extracts were kept on ice and tested within 1 hour of preparation. Coagulation screening tests using Dade-Behring reagents on a Sysmex CA-1500 coagulometer included PT, APTT, thrombin clotting time, atroxin clotting time, euglobulin clot lysis and anti-Xa assay. Collagen (2μg/ml) and ADP (5μM) were used as inducers of platelet aggregation. The species of leeches examined and their country of origin were:

Proboscid LeechesSourceSub-Family
Haementeria ghilianii Brazil Haementeriinae 
Oosthuizobdella garoui South Africa Haementeriinae 
Placobdella parasitica Canada Glossiphoniinae 
Jawed Leeches   
Macrobdella decora N. America Macrobdellinae 
Hirudinaria manillensis China Hirudinariinae 
Hirudinaria javanica Indonesia Hirudinariinae 
Poecilobdella granulosa India Hirudinariinae 
Myxobdella africana Kenya Praodbellinae 
Proboscid LeechesSourceSub-Family
Haementeria ghilianii Brazil Haementeriinae 
Oosthuizobdella garoui South Africa Haementeriinae 
Placobdella parasitica Canada Glossiphoniinae 
Jawed Leeches   
Macrobdella decora N. America Macrobdellinae 
Hirudinaria manillensis China Hirudinariinae 
Hirudinaria javanica Indonesia Hirudinariinae 
Poecilobdella granulosa India Hirudinariinae 
Myxobdella africana Kenya Praodbellinae 

Our findings indicate that cephalic extracts from all the species examined contain anti-thrombin properties. However, only the giant leech H. ghilianii appears to possess fibrin(ogen)olytic activity. No evidence of a plasminogen activator was detected in any of the extracts. Platelet aggregation responses induced by both agonists were completely inhibited in the presence of all cephalic extracts. No such inhibition was detectable in the posterior musculature control material. Anti-Factor Xa activity was present in extracts from H. ghilianii. 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 but further work is required to define the precise nature of such mechanisms in related species The different anti-haemostatic properties in these extracts may offer new therapeutic potentials.

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