Direct Inhibitory Effects of Carbon Monoxide on Six Venoms Containing Fibrinogenolytic Metalloproteinases

Abstract

Since the introduction of antivenom administration over a century ago to treat venomous snake bite, it has been the most effective therapy for saving life and limb. However, this treatment is not always effective and not without potential life-threatening side effects. We tested a new paradigm to abrogate the plasmatic anticoagulant effects of fibrinogenolytic snake venom metalloproteinases (SVMP) by inhibiting these Zn -dependent enzymes directly with carbon monoxide (CO) exposure. Assessment of the fibrinogenolytic effects of venoms collected from the Arizona black rattlesnake, Northern Pacific rattlesnake, Western cottonmouth, Eastern cottonmouth, Broad-banded copperhead and Southern copperhead on human plasmatic coagulation kinetics was performed with thrombelastography in vitro. Isolated exposure of all but one venom (Southern copperhead) to CO significantly decreased the ability of the venoms to compromise coagulation. These results demonstrated that direct inhibition of transition metal-containing venom enzymes by yet to be elucidated mechanisms (e.g. CO, binding to Zn or displacing Zn from the catalytic site, CO binding to histidine residues) can in many instances significantly decrease fibrinogenolytic activity. This new paradigm of CO-based inhibition of the anticoagulant effects of SVMP could potentially diminish haemostatic compromise in envenomed patients until antivenom can be administered.

Crotalus atrox Venom Exposed to Carbon Monoxide Has Decreased Fibrinogenolytic Activity In Vivo in Rabbits

“…The most effective treatment against coagulopathy in this setting still remains the administration of long-acting or multiple doses of antivenom as recently demonstrated [2]; however, coagulopathy can still occur and recur. Of interest, as a therapy complementary to antivenom, our laboratory discovered that direct, isolated exposure of snake venom metalloproteinases (SVMP) and snake venom serine proteases (SVSP) to carbon monoxide (CO) in vitro resulted in a marked decrease in associated fibrinogenolytic or thrombin-like activity in human plasma [3][4][5][6]. This observation was then extended into and confirmed in an in vitro whole-blood rabbit model wherein the SVMP fibrinogenolytic activity of Crotalus atrox venom was attenuated by pre-exposure to CO [7].…”

CatroxMP-II: a heme-modulated fibrinogenolytic metalloproteinase isolated from Crotalus atrox venom

“…With regard to the concentration of CatroxMP-II used, the onset of coagulation had to be double and/or the velocity of clot formation half of plasma without CatroxMP-II addition to be acceptable for experimentation. The following elastic modulus-based parameters previously described (Nielsen 2018; Nielsen and Bazzell 2016, 2017; Nielsen et al 2016, 2018a, b; Nielsen and Losada 2017; Nielsen and Matika 2017) were determined: time to maximum rate of thrombus generation (TMRTG): this is the time interval (minutes) observed prior to maximum speed of clot growth; maximum rate of thrombus generation (MRTG): this is the maximum velocity of clot growth observed (dynes/cm 2 /second); and total thrombus generation (TTG, dynes/cm 2 ), the final viscoelastic resistance observed after clot formation. Data were collected for 30 min.…”

“…It has been recently demonstrated that the hemotoxic venom activity of several species of snakes can be inhibited in vitro and in vivo by exposure to carbon monoxide (CO) via thrombelastography (Nielsen 2018; Nielsen and Bazzell 2016, 2017; Nielsen et al 2016, 2018a, b; Nielsen and Losada 2017; Nielsen and Matika 2017). Also of interest, an agent that causes metheme formation (Nielsen et al 2011a) used to identify fibrinogen as a heme binding protein (Nielsen et al 2011b) was demonstrated to decrease the prothrombotic activity of Oxyuranus microlepidotus venom (Nielsen et al 2018a).…”

“…Second, a trypsin digest of this purified SVMP had to be analyzed with mass spectroscopy to determine if a mass consistent with heme could be identified. Lastly, it had to be determined if exposure of this heme-bearing SVMP to CO could inhibit its fibrinogenolytic activity in a well-established thrombelastographic assay (Nielsen 2018; Nielsen and Bazzell 2016, 2017; Nielsen et al 2016, 2018a, b; Nielsen and Losada 2017; Nielsen and Matika 2017). …”

Carbon monoxide releasing molecule enhances coagulation and decreases fibrinolysis in canine plasma exposed to Crotalus viridis venom in vitro and in vivo

“…Previous findings of the clot strengthening effects of CO led us to hypothesize that CO may be able to strengthen clot formation in plasma from normal dogs exposed to Prairie rattlesnake ( Crotalus viridis) venom and in plasma from naturally envenomed dogs . To date, CORM‐2 has been shown to mitigate coagulopathy induced by several crotalid venoms in human plasma . Coagulopathy is a common sequel to rattlesnake bites in human beings and canine victims.…”