Decreased snake venom metalloproteinase effects via inhibition of enzyme and modification of fibrinogen

Abstract

Since the introduction of antivenom administration 120 years ago to treat venomous snake bit, it has been the gold standard for saving life and limb. However, this therapeutic approach 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 by modification of fibrinogen with iron and carbon monoxide and by inhibiting these Zn(2+) dependent metalloproteinases directly with carbon monoxide exposure. Assessment of the fibrinogenolytic effects of venoms collected from Puff adder, Gaboon viper and Indian cobra snakes on plasmatic coagulation kinetics was performed with thrombelastography. Pretreatment of plasma with iron and carbon monoxide exposure markedly attenuated the effects of all three venoms, and direct pretreatment of each venom with carbon monoxide also significantly decreased the ability to compromise coagulation. These results demonstrated that the introduction of a transition metal (e.g., modulation of the α-chain of fibrinogen with iron), modulation of transition metal in heme (e.g., carbon monoxide modulation of fibrinogen-bound heme iron), and direct inhibition of transition metal containing venom enzymes (e.g., CO binding to Zn(2+) or displacing Zn(2+) from the catalytic site) significantly decreased fibrinogenolytic activity. This biometal modulation strategy to attenuate the anticoagulant effects of snake venom metalloproteinases could potentially diminish hemostatic injury in envenomed patients until antivenom can be administered.

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

“…Alteration of the human fibrinogen molecule has been shown to create resistance to venom‐induced hypocoagulability . Nielsen et al showed that pre‐incubating normal human plasma with CORM‐2 and ferric chloride (FeCl 3 ) mitigated the deleterious effects of venom from both North American and non‐North American vipers.…”

“…Rattlesnake envenomation occurs frequently in dogs throughout the United States and often causes coagulopathy. Rattlesnake venom decreases fibrinogen function by abnormally cleaving fibrinogen alpha and beta chains and rendering it poorly functional for polymerization . In general, antivenom lessens coagulopathy induced by crotalid species; however, it is expensive (especially if multiple doses are necessary), time‐sensitive and poorly available, and some formulations have a short half‐life .…”

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). …”