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. changes in the hemostatic effects of enzyme-based neurotoxins in response to inhibitor exposure. Thus, the use of thrombelastography to detect neurotoxin activity via biochemical substrate nexuses of plasmatic coagulation with neurochemistry was conceived.Although the anticoagulant properties of the neurotoxic venoms of several of Naja [2-6] and one Crotalus [7] species have been studied, another genus, Dendroaspis (the mambas), that kill their prey and humans with neurotoxic venom [8][9][10], were found to possess venom that was anticoagulant in vitro over 50 years ago [11,12]. Using the clotting-based, antiquated technology that was available in the 1960s, these investigators proposed that thrombin generation was impaired, fibrinogen was digested, fibrinolysis was impaired, and platelet aggregation decreased in blood exposed to Black Mamba (Dendroaspis polylepis), Eastern Green Mamba (Dendroaspis angusticeps) or Jameson's Green Mamba (Dendroaspis jamesoni) venom [11,12]. The fourth extant species, Hallowell's Green Mamba (Dendroaspis viridis), was not investigated [11,12]. Of interest, the venom of D. polylepis appeared to be between one and two orders of magnitude less potent as an anticoagulant compared to the other two species tested [11,12]. Cri...

Ruthenium, Not Carbon Monoxide, Inhibits the Procoagulant Activity of Atheris, Echis, and Pseudonaja Venoms

“…This laboratory became aware of this work recently, and using a similar approach, demonstrated an identical outcome wherein the anticoagulant activity of the purified phospholipase A 2 (PLA 2 ) of Apis mellifera venom was inhibited by CORM-2 in a CO-independent, albumin-inhibitable fashion [15]. Furthermore, we recently demonstrated that the anticoagulant metalloproteinases of mamba venoms are inhibited by CORM-2 in a CO-independent, albumin-inhibitable manner [16]. Taken as a whole, it was entirely possible that Ru-based interactions with venom proteins could be responsible for the inhibition noted in our previous works [13]; and critically, if Ru-based modifications were the underpinning of such inhibition rather than the interaction of CO with a heme group, then Ru-based CORMs could well serve as permeant antivenom agents.…”

“…Similarly, increased RuCl3 concentrations significantly decrease TMRTG and increase MRTG values when PBS is the vehicle. However, and critically, the coagulation kinetic differences caused by RuCl3 are significantly enhanced by the fluid it is dissolved in as indicated by the two-way ANOVA significance values in each panel of As all of the venoms investigated over the past few years have been suspended in PBS for the purposes of preserving enzymatic function within a physiological pH, storage, and experimentation [1][2][3][4][5][6][7][8][9][10][11][12][13]15,16], it seemed prudent to continue with the use of PBS in the subsequently described experimental series assessing the effects of RuCl3 on the three venoms of the present work. The caveat that should be kept in mind was that small enhancements of MRTG in human plasma could be secondary to a RuCl3/PBS interaction when determining if RuCl3 inhibited venom procoagulant activity.…”

“…Taken as a whole, it was entirely possible that Ru-based interactions with venom proteins could be responsible for the inhibition noted in our previous works [13]; and critically, if Ru-based modifications were the underpinning of such inhibition rather than the interaction of CO with a heme group, then Ru-based CORMs could well serve as permeant antivenom agents. The importance of these line of investigation involving ion channels [14], phospholipase A 2 [15], and metalloproteinases [16] is that they lay the foundation to seriously reconsider the paradigm that Ru-based CORMs affect systems as simple as enzymes to as complex as whole animal models of disease in CO-independent ways-potentially affecting the interpretation of data contained in several hundred manuscripts.…”

“…Considering the aforementioned, the present investigation had the following goals. First, determination of inhibition of the procoagulant activities of these venoms by their exposure in isolation to CORM-2 in the absence or presence of albumin was to be performed as previously described with bee venom PLA 2 [15] and mamba venom [16]. Second, to further assess if Ru-based molecules may affect venom procoagulant activity, the three venoms were exposed to equimolar concentrations of ruthenium chloride (RuCl 3 ) which contains a Ru +3 state compared to the Ru +2 state of CORM-2.…”

“…Critically, the use of RuCl 3 in this investigation was purely to provide mechanistic insight, and as there is no clinical indication to administer it to humans or any other species, I am not advocating it as a therapeutic option. As previously described [1][2][3][4][5][6][7][8][9][10][11][12][13]15,16], changes in procoagulant activity were assessed with human plasma via changes in coagulation kinetics determined with thrombelastography.…”