Effect of Iron and Carbon Monoxide on Fibrinogenase‐like Degradation of Plasmatic Coagulation by Venoms of Six Agkistrodon Species

Abstract

Annually, thousands suffer poisonous snakebite, often from defibrinogenating species. It has been demonstrated that iron and carbon monoxide change the ultrastructure of plasma thrombi and improve coagulation kinetics. Thus, this investigation sought to determine whether pre-treatment of plasma with iron and carbon monoxide could attenuate venom-mediated catalysis of fibrinogen obtained from Agkistrodon species with fibrinogenase activity. Human plasma was pre-treated with ferric chloride (0-10 lM) and carbon monoxide-releasing molecule-2 (CORM-2, 0-100 lM) prior to exposure to 0.5-11 lg/ml of six different Agkistrodon species' venom. The amount of venom used for experimentation needed to decrease coagulation function of one or more kinetic parameters by at least 50% of normal values for (e.g. half the normal speed of clot formation). Coagulation kinetics were determined with thrombelastography. All six snake venoms degraded plasmatic coagulation kinetics to a significant extent, especially prolonging the onset to clot formation and diminishing the speed of clot growth. Pre-treatment of plasma with iron and carbon monoxide attenuated these venom-mediated coagulation kinetic changes in a species-specific manner, with some venom effects markedly abrogated while others were only mildly decreased. Further in vitro investigation of other pit viper venoms that possess fibrinogenolytic activity is indicated to identify species amenable to or resistant to iron and carbon monoxide-mediated attenuation of venom-mediated catalysis of fibrinogen. Lastly, future pre-clinical investigation with animal models (e.g. rabbit ear-bleed model) is planned to determine whether iron and carbon monoxide can be used therapeutically after envenomation.Thousands of individuals experience venomous snakebites annually in the United States [1][2][3][4], with cottonmouth and copperhead snakes (Agkistrodon species) second only to rattlesnakes (Crotalus species) as the most identified responsible vipers [2]. Snakebites by Agkistrodon species result in a spectrum of degrees of local and systemic injury [5,6], including hypofibrinogenaemia and coagulopathy [7]. Of interest, there is heterogeneity in therapeutic approach to envenomation by Agkistrodon species in the literature. While administration of antivenom has been recommended to prevent local and circulatory morbidity [3,8], there is debate concerning the need to serially assess patient coagulation status [9,10] or even administer antivenom at all [11]. Nevertheless, significant morbidity and occasional mortality occurs after envenomation by Agkistrodon species [5,6,12].Cottonmouth and copperhead snakes in North America generally possess venom that is fibrinogenolytic [13][14][15][16][17][18]. Intact venom or purified metalloproteinases typically cleave the A (a)-chain and the B(b)-chain of fibrinogen, resulting in hypofibrinogenaemia state and decreased coagulation [13][14][15][16][17][18]. While antivenom can bind and inactivate these enzymes, another approach to maintain circula...

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

“…Extensive in vitro investigation of novel therapeutic methods to abrogate the fibrinogenolytic/thrombin-like activity of snake venom metalloproteinases (SVMP) and snake venom serine proteases (SVSP) has been pursued by our laboratory recently [1][2][3][4][5][6][7]. The first method was to modify fibrinogen in human plasma by exposure to iron (Fe) and carbon monoxide (CO), rendering this 'stealth' fibrinogen less recognizable to SVMPs [1][2][3][4]. The second approach was direct, isolated exposure of SVMP and SVSP to CO in vitro, with subsequent loss of associated fibrinogenolytic or thrombin-like activity observed in human plasma [4][5][6][7].…”

Effect of iron and carbon monoxide on fibrinogenase-like degradation of plasmatic coagulation by venoms of four Crotalus species

“…This investigation utilized changes in coagulation kinetics of citrated human plasma to characterize the effects of snake venom and the effects of Fe/CO pretreatment on those changes to assess changes in functional fibrinogen and protection from venom-mediated changes as in very recent studies [19,20]. The reason that this is a valid approach is that the effects of various snake venoms on fibrinogen have been identified as the major plasmatic legion [6,7], and we and various colleagues have demonstrated that iron and CO enhance coagulation by exclusive modulation of fibrinogen [15,16,18,23].…”

“…Also of interest, exposure to CO resulted in the inability to recover significant portions of the g chain after endopeptidase treatment of purified fibrinogen during mass spectroscopic analysis [16], indirect evidence of molecular conformational change of fibrinogen. Finally, we have demonstrated significant, but species-specific success in attenuating venom-mediated compromise of plasmatic coagulation by Crotalus atrox [19] and various Agkistrodon species [20] with iron and CO pretreatment. Taken as a whole, these data suggested that it may be possible to change the three-dimensional configuration of fibrinogen with iron and CO such that thrombin could interact with it as an enhanced substrate, but perhaps fibrinogenolytic-like activity in similar snake venoms could not.…”

Proteomic Analyses of Agkistrodon contortrix contortrix Venom Using 2D Electrophoresis and MS Techniques

“…A. c. contortrix fibrinogenases are responsible for the fibrino- and fibrinogenolytic properties of venom and, unusually for this group of species, prefer cutting off fibrinopeptide B. They can also cut the γ chain, responsible for crosslinking [38]. Thrombin-like enzymes, despite the relatively low similarity to the sequence of thrombin (30%), are able to clot fibrinogen [39].…”

“…However, most of these enzymes are capable of releasing only fibrinopeptide A or B, rarely both at once, and therefore the strength of the clot is small and it quickly dissolves [36]. Action of both of the above groups of proteins results in blood being unable to clot, since it lacks a functional fibrinogen [38,39,40,41]. The last protein of this group identified in our experiment is a protein C activator, characteristic for the venom of snakes belonging to the genus Agkistrodon .…”