Characterization of the Rabbit as an In Vitro and In Vivo Model to Assess the Effects of Fibrinogenolytic Activity of Snake Venom on Coagulation

Abstract

Several in vitro investigations have demonstrated that anticoagulant effects of fibrinogenolytic snake venom metalloproteinases have been abrogated in human plasma by modifying fibrinogen with iron (Fe) and carbon monoxide (CO) to prevent catalysis or by directly inhibiting these enzymes with CO. To translate these findings, we chose to assess the rabbit as a model of envenomation with Crotalus atrox venom. It was determined with thrombelastography that 15 times the concentration of venom noted to compromise coagulation in plasma in vitro was required to cause coagulopathy in vivo, likely secondary to venom binding to blood cells and being cleared from the circulation rapidly. Unlike human plasma, rabbit plasma pre-treated with Fe/CO was not protected from fibrinogenolysis by venom. Consequently, the administration of purified human fibrinogen (with or without Fe/CO) would be required before venom administration to rabbits. Of greater interest, venom exposed to CO had complete loss of fibrinogenolytic effect in rabbit plasma and partial loss of activity in whole blood, indicative of unbinding of CO from venom and binding to haemoglobin. Thus, venom exposed to CO could remain partially or completely inhibited in whole blood long enough for clearance from the circulation, allowing rabbits to be a useful model to test the efficacy of regional CO administration to the bite site. Future investigations are planned to test these novel approaches to attenuate venom-mediated coagulopathy in the rabbit.

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

“…Exposure of CatroxMP-II to CORM-2 prior to being placed in human plasma resulted in TMRTG, MRTG and TTG values near those of plasma samples not exposed to the purified SVMP. Thus, as in the case of raw venom (Nielsen 2018; Nielsen and Bazzell 2016; Nielsen et al 2018b), the activity of CatroxMP-II was inhibited by CORM-2 (Fig. 6).…”

“…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.…”

“…Experimental conditions in the thrombelastographic experiments were represented by n = 2 replicates per condition. A greater number of replicates or conditions for the thrombelastographic experiments for statistical analysis were deemed unnecessary as CO derived from CORM-2 has been shown to completely inhibit the fibrinogenolytic effects of unfractionated C. atrox venom (Nielsen 2018; Nielsen and Bazzell 2016; Nielsen et al 2018b). Graphics were generated with commercially available programs (OrigenPro 2017, OrigenLab Corporation, Northampton, MA, USA; CorelDRAW X8, Corel Corporation, Mountain View, CA, USA).…”

“…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). …”