Anti-Haemostatic Properties of Salivary Secretions of the Rhynchobdellid Leech Theromyzon tessulatum and the Arhynchobdellid Leech Haemopis sanguisuga: Potentially New Therapeutic Agents.

Haematophages, animals evolved to a bloodsucking lifestyle as their exclusive mode of feeding, secrete compounds capable of arresting haemostasis in the host. Ubiquitous throughout the animal kingdom, most are invertebrates of the Annelid class and insects of the Arthropod class. Since the discovery and subsequent characterisation and engineering of Hirudin, a potent thrombin inhibitor of 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. The aim of this study was to investigate the anticoagulant properties of the salivary secretions of the phylogenetically diverse haematophagous Theromyzon tessulatum and the opportunistic predatory Haemopis sanguisuga of the Rhynchobdellid and Aryhchobdellid orders of the Hirudinae respectively. Leeches of both species were collected from their natural habitat. Adult leeches of the species T. tessulatum (total weight 2.828g) and of the species H. sanguisuga (total weight 4.758g) were anaesthetised with ethanol vapour. Homogenates of the salivary gland secretion containing anterior portions of the leeches were prepared and added to commercial pooled citrated plasma (Dade Behring Ci trol 1) which was subsequently centrifuged and the supernatant aspirated. The posterior two thirds of the leeches of the same weights were treated identically to serve as control material. Standard coagulation parameters of each preparation (PT, APTT, TCT and Clauss fibrinogen) were determined along with anti-FXa activity, assays of intrinsic and extrinsic coagulation factors, multi dilutional assays (MDA) and platelet aggregation studies employing the aggregation inducers thrombin (10 units/ml), collagen (10μg/ml), ristocetin (1.5 mg/ml) and ADP (5μm/ml).

Results: Anterior portion preparations from both species caused a prolongation of the APTT. Prolongation of the TCT and the presence of anti-FXa activity were observed in T. tessulatum test preparations. Factor V was the factor most reduced following exposure of plasma to extract of T. tessulatum. A marked reduction in FV (0.24 of control value) and moderate reduction in FVIII and FIX (0.6 of control values) were seen following similar exposure to H. sanguisuga extract. MDA of all factors exhibited parallelism for both species. Platelet aggregation responses to thrombin, collagen and ADP agonists were abolished and that to ristocetin was reduced in T. tessulatum salivary gland preparations. Platelet aggregation responses to all inducers in presence of H. sanguisuga extract showed no difference with control preparations. This study confirms the presence of anti-thrombin and anti-FXa properties in the salivary secretions of T. Tessulatum. The presence of platelet aggregation inhibitors in T. tessulatum and the factor V, VIII and IX reducing properties of H. sanguisuga are novel findings. The ability of H. sanguisuga to reduce these factors may indicate general proteolytic activity directed at common domain structures in these molecules or the legacy of a haematophagous ancestry. The presence of anti-FXa activity in this species remains to be determined in order to establish whether H. sanguisuga is truly a misnomer or whether its nomenclature is taxonomically correct. These new anti-haemostatic properties in different species could have further potential therapeutic applications.