Mechanisms Responsible for the Anticoagulant Properties of Neurotoxic Dendroaspis Venoms: A Viscoelastic Analysis

Abstract

Using thrombelastography to gain mechanistic insights, recent investigations have identified enzymes and compounds in Naja and Crotalus species' neurotoxic venoms that are anticoagulant in nature. The neurotoxic venoms of the four extant species of Dendroaspis (the Black and green mambas) were noted to be anticoagulant in nature in human blood, but the mechanisms underlying these observations have never been explored. The venom proteomes of these venoms are unique, primarily composed of three finger toxins (3-FTx), Kunitz-type serine protease inhibitors (Kunitz-type SPI) and <7% metalloproteinases. The anticoagulant potency of the four mamba venoms available were determined in human plasma via thrombelastography; vulnerability to inhibition of anticoagulant activity to ethylenediaminetetraacetic acid (EDTA) was assessed, and inhibition of anticoagulant activity after exposure to a ruthenium (Ru)-based carbon monoxide releasing molecule (CORM-2) was quantified. Black mamba venom was the least potent by more than two orders of magnitude compared to the green mamba venoms tested; further, Black Mamba venom anticoagulant activity was not inhibited by either EDTA or CORM-2. In contrast, the anticoagulant activities of the green mamba venoms were all inhibited by EDTA to a greater or lesser extent, and all had anticoagulation inhibited with CORM-2. Critically, CORM-2-mediated inhibition was independent of carbon monoxide release, but was dependent on a putative Ru-based species formed from CORM-2. In conclusion, there was great species-specific variation in potency and mechanism(s) responsible for the anticoagulant activity of Dendroaspis venom, with perhaps all three protein classes-3-FTx, Kunitz-type SPI and metalloproteinases-playing a role in the venoms characterized.