Two Platelet Aggregation Inhibitors in Tsetse (Glossina) Saliva with Studies of Roles of Thrombin and Citrate in in Vitro Platelet Aggregation

Abstract

Two inhibitors of platelet aggregation have been identified in saline extracts of Glossina morsitans (tsetse) salivary glands. A protein fraction (MW > 30 000) inhibited primary and secondary aggregation to ADP, secondary I aggregation to adrenalin, and aggregation to collagen. It also caused disaggregation of platelets stimulated by ADP and adrenalin. These properties could be explained by ADP hydrolytic activity. A previously identified antithrombin fraction (MW 11 000-13 000) abolished thrombin-induced aggregation. It did not affect platelet aggregation to ADP, adrenalin or collagen, as compared to aggregation in citrate, when used as sole anticoagulant for platelet rich plasma or when added to citrated platelet rich plasma. These results fail to support hypotheses (i) that thrombin plays an important role in platelet aggregation by other agonists and (ii) that secondary platelet aggregation is an artefact induced by citrate. It is proposed that these inhibitors may be important in maintaining mouthpart and crop patency during feeding. Their discovery suggests that other arthropods may also have antiplatelet agents in their saliva which might be entomologically important, provide useful tools for platelet studies, and be of potential therapeutic interest.

The Glossina morsitans tsetse fly saliva: General characteristics and identification of novel salivary proteins

“…However, molecular information on the individual tsetse salivary proteins and their biological activity remains scanty. Previous studies on Glossina morsitans saliva have reported the presence of a 11.3 kDa inhibitor of thrombin serine protease and esterase activities (Parker and Mant, 1979) and a 430 kDa protein fraction that inhibited the ADP-induced thrombocyte aggregation (Mant and Parker, 1981). In addition, a potent 32 AA blood meal-induced tsetse thrombin inhibitor (TTI), was characterised in salivary gland extracts.…”

Insights into the Trypanosome-Host Interactions Revealed through Transcriptomic Analysis of Parasitized Tsetse Fly Salivary Glands

“…Among the known tsetse saliva components are anti-hemostatic proteins [26], [27], [28], which include a potent anticoagulant thrombin inhibitor (TTI) [29], [30], and an anti-thrombotic apyrase (5′Nuclease) with dual inhibitory action that can bind to the fibrinogen receptor (GPIIb/IIIa) and inhibit ADP-induced platelet responses [31]. In addition, two abundant proteins (Tsal1 and Tsal2) have been described with DNA/RNA non-specific nucleic acid binding [26], [32], [33].…”

Tsetse Salivary Gland Proteins 1 and 2 Are High Affinity Nucleic Acid Binding Proteins with Residual Nuclease Activity

“…The biological role of the Tsal proteins could be in the digestive tract as well as at the feeding site where saliva proteins contribute to the generation of a local blood pool [3], [8], [9]. Feeding of tsetse flies on immunized mice with high titers of anti-Tsal IgGs did not affect their feeding performance [11].…”

“…During the probing and blood feeding interaction, tsetse flies inoculate a complex mixture of salivary components from which the composition has been explored by proteome and transcriptome analyses as well as by functional genomics approaches [1], [2], [3], [4], [5], [6], [7]. It has already been established that tsetse fly saliva interferes with host hemostatic reactions that are initiated at the blood feeding site [3], [8], [9]. Anti-coagulant and anti-thrombotic compounds include respectively the highly potent tsetse thrombin inhibitor [TTI, [8]] and a recently identified apyrase with fibrinogen receptor antagonistic features [3].…”