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. Maximal Responses (%) to Aggregation Inducers in Haematophagous and Predaceous Leeches Species Source ADP COL THR RIS ADR Haematophagus Haementeria ghilianii Brazil <10 <10 <10 <10 <10 Oothuizobdell garoui South Africa <10 <10 <10 20 15 Placobdella parasitica Canada 10 10 <10 15 20 Hirudinaria manillensis China <10 <10 <10 <10 <10 Macrobdella decora North America <10 <10 <10 <10 <10 Hirudinaria javanica Indonesia <10 <10 <10 <10 <10 Poecilobdella granulose India <10 <10 <10 <10 <10 Myxobdella Africana Kenya 15 20 <10 20 25 Hirudo medicinalis Turkey <10 <10 <10 <10 <10 Predaceous Haemopis sanguisuga UK 100 100 100 100 100 Helobdella stagnalis UK 90 90 95 100 100 Erpobdella octoculata UK 100 95 95 100 100 Glossiphonia complanata UK 100 100 100 100 100 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.

Anti-Haemostatic Properties of Diverse Haematophagous Hirudinea from around the World Could Have New Therapeutic Potentials.

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 Leeches Source Sub-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.