Geographic isolation of venomous snakes within the same genus is associated with evolution-driven diversity of the enzymatic composition of their venom, which is also affected by diet [1
]. A prime example of this sort of diversity are the numerous Ovophis
species of Asian pit viper that are scattered across the Indian subcontinent, Southeast Asia, Japanese islands, and islands throughout and just beyond the South China Sea [4
]. Based on the diversity of mitochondrial deoxyribonucleic acid, many of these pit vipers, including the ones displayed in Table 1
, are posited to be in different genera than they are presently assigned [4
]. Proteomic investigation of the effects of geography-driven diversity of venom composition is important, as the toxic enzymes/compounds of any particular venom may or may not be neutralized by antivenom prepared with venom obtained from related but physically separated and genetically distinct species [1
]. Critically, even if the various classes of enzyme (e.g., phospholipase, serine protease, metalloproteinase) within a venom are quantified, proteomic analyses cannot predict which of the enzymes will have the most predominant effect on target molecules (e.g., prothrombin activator, fibrinogenolytic enzyme). Given that coagulopathy following envenomation by snakes with hemotoxic agents continues to be a major cause of international morbidity and mortality [5
], identification of the major effects of any specific venom on coagulation provides insight into potential therapies [6
]. Using thrombelastography and human plasma, it has been possible to identify the contribution of diverse venom enzymatic activities on coagulation kinetics between species or within the same venom. Thrombelastographic methods also permit comparisons of the potency of different venoms to achieve the same hemostatic derangement. Further, this method facilitates assessment of the vulnerability of specific venom activities to heme modulation in isolation with carbon monoxide (CO) or O
-phenylhydroxylamine (PHA), an agent that forms metheme [6
]. Numerous proteins have been found to be heme-bound, and their functions are controlled by the ligand (e.g., oxygen, nitric oxide, CO) that binds to the iron center of the heme group as recently reviewed [12
]. Heme modulation has potential therapeutic applications in the setting of envenomation, and we have recently described a heme group bound to a fibrinogenolytic enzyme obtained from Crotalus atrox
venom, and this enzyme was inhibited by CO [13
]. Taken as a whole, the analyses of changes in coagulation kinetics in human plasma mediated by hemotoxic venom can complement the elegant proteomic analyses of these venoms, potentially providing insight into the relative impact of major enzyme types (e.g., snake venom metalloproteinases (SVMP), snake venom serine proteases (SVSP)) on human coagulopathy after envenomation.
Given the preceding, the goals of this study were to: (1) use human plasma to thrombelastographically characterize the effects on coagulation kinetics of hemotoxic venoms obtained from geographically separated and genetically diverse species within the same genus (Table 1
); (2) assess any CO-inhibition of venom activities; (3) assess if PHA inhibits venom effects; (4) in the case of prothrombotic venoms, to utilize heparin–antithrombin anticoagulation to separate venom mediated thrombin generation (e.g., prothrombin-activating properties) from thrombin-like activity that is antithrombin resistant. Prior to this investigation, of the species displayed in Table 1
, Ovophis okinavensis
has been noted to have serine proteases and metalloproteinases in its venom, with one enzyme found to be fibrinogenolytic [14
]; and Trimeresurus purpureomaculatus
venom was noted to have both thrombin-like and fibrinogenolytic activity [16
This investigation achieved its stated goals of characterizing the potency, anticoagulant/procoagulant nature, and heme-modulated behavior of these diverse Asian pit viper venoms. Beginning with the physically separated, genetically distinct [4
species, the two venoms were similar in potency (µg/ml venom to achieve the same coagulation kinetic derangement) with a predominant fibrinogenolytic-like activity exerting the most influence on coagulation. With regard to inhibition by CO, the fibrinogenolytic-like activity of O. monticola
was resistant to the smaller concentration of CO than that of O. okinavensis
. The addition of PHA enhanced the fibrinogenolytic-like activity of both species’ venoms. An antithrombin-resistant, CO-inhibitable thrombin-like activity was detectable in O. monticola
venom, whereas an iRM-enhanced thrombin-generating activity was identified in O. okinavensis
venom. Critically, the thrombin-generating activity is essentially silent without iRM, with fibrinogenolytic-like activity the major player kinetically in the case of O. okinavensis
venom. In sum, the venom of the two Ovophis
species had similar but different characteristics that could be determined thrombelastographically.
With regard to the three genetically distinct [4
species on landmasses separated by the South China Sea, there was a ten-fold difference in potency between the venoms to achieve the same degree of coagulation derangement, which were all fibrinogenolytic-like in nature. However, there were different degrees of susceptibility to CO inhibition of this anticoagulant activity between the venoms. Further, depending on the venom, a metheme state enhanced fibrinogenolytic-like activity, diminished activity, or did nothing to activity. In this genus, iRM remained essentially silent, not interacting with fibrinogenolytic-like activity or enhancing otherwise silent procoagulant activities. Taken as a whole, while the venoms of Trimeresurus
species primarily had only one predominant anticoagulant activity, the potency and response to heme modulation were remarkably different between vipers.
The use of inactivated CORMs was originally designed to implicate CO-mediated heme modulation, with the concept that the iRM would remain biochemically silent while the CORM would produce an effect in the system tested. Instead, there is a number of works demonstrating CO-independent effects of iRM [7
] wherein various enzymes are modulated via interaction of the iRM without an intermediary attached heme. We have specifically seen northern copperhead fibrinogenolytic-like venom activity enhanced via thrombelastography with iRM [7
]. While this may seem confounding, the use of iRM can provide further confirmation of a particular venom activity by enhancing it [7
], or as was the case with the present study, decreasing activity (Table 2
). Further, as in the case of O. okinavensis
venom, a previously kinetically silent activity was activated by iRM (Figure 3
, a thrombin-generating activity), providing insight into the complexity of this particular venom. In sum, the use of CORMs, iRMs, and other heme modulators may provide direct, heme-mediated insights or heme-independent information as biochemical probes that query the characteristics of any particular venom.
An important issue that was raised in review was the ability of our methodology to discern if the coagulation kinetic profile derived from each venom could provide insight into its enzymatic composition (e.g., SVMP, SVSP). This is unfortunately not possible, as SVMP and SVSP are both capable of fibrinogenolytic, thrombin-like, factor X-activating, prothrombin-activating, and other activities. The relative percentage of any particular enzyme type (e.g., 20% SVMP with 30% SVSP) in any given venom also cannot be determined with our techniques. Thus, the contribution of any particular enzyme by biochemical type is not possible; however, the power of our methods is the revelation as to which of any particular mix of enzymes is the predominant one responsible for the observed coagulopathy in vitro, which, given the milieu (human plasma), would likely predict the clinical situation. When data derived from our methodology are combined with complimentary venomics, the specific enzyme responsible can likely be identified.
The present work focuses on some unique species related by genus in Asia and South East Asia, but in earlier works prior to systematic use of metheme formation [6
], these thrombelastographic methods have been utilized to characterize the coagulation kinetic profiles of medically important species in other parts of the world. North American Agkistrodon
species venom tested by our laboratory was uniformly anticoagulant in nature, displaying a fibrinogenolytic coagulation kinetic profile [9
]. In contrast, while most North American Crotalus
species venom seemed fibrinogenolytic by our methods [6
], there were some that were procoagulant in nature, displaying thrombin-like activity coagulation kinetic patterns in human plasma [8
]. In terms of evolution, the South American Lachesis muta muta
is considered a progenitor species for pit vipers throughout the Americas, and our methods identified both fibrinogenolytic and thrombin-like activities differentially susceptible to CO inhibition [11
]. Of interest, Naja
species venoms across continents appear to have a highly conserved anticoagulant coagulation kinetic profile that is CO inhibited but may be mediated either by phospholipases and/or SVMP or SVSP [6
]. Lastly, Oxyuranus
species in Australia and Papua New Guinea, separated by the Torres Strait, display essentially the same procoagulant, prothrombin activator coagulation kinetic profile that is CO inhibitable [11
]. In summary, continued utilization and refinement of our methodology is anticipated to provide insight into the predominant nature and vulnerability to heme modulation of diverse species venoms.
In conclusion, the present work demonstrated that human plasma-based thrombelastographic analyses utilizing heme-modulating approaches and antithrombin activation with heparin can be used to generate a unique coagulation kinetic profile of individual snake venom activities. Just as with the chromatographic and mass-spectroscopic approach with proteomic analyses documenting the presence of key enzymes and compounds to differentiate various venoms [1
], it may be of utility to similarly kinetically profile the very same venoms to provide a functional assessment of the relative importance of any particular enzyme class (e.g., prothrombin activator, thrombin-like activity, fibrinogenolytic activity) in effecting coagulopathy. These thrombelastograph-based analyses of the impact of various classes of hemotoxic venom activity could perhaps be termed “venom kinetomics”, to complement the specific venomics methods used to assess which particular enzymes of the venom are responsible for coagulopathy. Put another way, while more than one coagulation-modifying activity can be identified in a venom (e.g., fibrinogenolytic-like, thrombin-like activity) with venomics, venom kinetomics can identify which of the enzymes predominate and inflict clinical coagulopathy. This venom-kinetomic approach could also potentially determine the utility of antivenoms across genetically related (same genus) but geographically separated species of venomous snake. Future investigation using this venom kinetomic methodology will demonstrate in time its laboratory and clinical utility.