Western Diamondback Rattlesnake (Crotalus atrox) venoms show increased snake venom metalloproteinase abundance and activity at their northeastern distributional limits.

To evaluate effectiveness of a commercially available toxoid manufactured from western diamondback (WD) rattlesnake (Crotalus atrox) venom against envenomation of mice with WD, northern Pacific (NP) rattlesnake (Crotalus oreganus oreganus), and southern Pacific (SP) rattlesnake (Crotalus oreganus helleri) venom. 90 specific pathogen-free female mice. Mice were allocated into 3 cohorts (30 mice/cohort). Mice received SC injections of C atrox toxoid (CAT) vaccine (n = 15/group) or adjuvant (15/group) at day 0 and again at 4 weeks. At 8 weeks, mice were challenge-exposed with 1 of 3 venoms. Survival until 48 hours was evaluated by use of log-rank analysis of survival curves and the z test for proportions. 6 of 15 WD-challenged CAT-vaccinated mice, 3 of 15 NP-challenged CAT-vaccinated mice, and 0 of 15 SP-challenged CAT-vaccinated mice survived until 48 hours. All adjuvant-only vaccinates survived ≤ 21 hours. Mean survival time of CAT vaccinates was longer than that of adjuvant-only vaccinates for all venoms (1,311 vs 368 minutes for WD, 842 vs 284 minutes for NP, and 697 vs 585 minutes for SP). Results of the z test indicated a significantly increased survival rate for vaccinates exposed to WD rattlesnake venom but not for vaccinates exposed to NP or SP rattlesnake venom. Log-rank analysis revealed a significant difference between survival curves of vaccinated versus unvaccinated mice exposed to NP but not WD or SP venom. CAT vaccination improved survival rate and survival time after challenge exposure with WD rattlesnake venom and may offer limited protection against NP rattlesnake venom but did not provide significant cross-protection against SP rattlesnake venom.

Characterization of two hemorrhagic zinc proteinases, toxin c and toxin d, from western diamondback rattlesnake ( Crotalus atrox) venom

Antigenic relationships of fractionated western diamondback rattlesnake ( Crotalus atrox) hemorrhagic toxins and other rattlesnake venoms as indicated by monoclonal antibodies

The resistance of three species of warm-blooded animals to western diamondback rattlesnake ( Crotalus atrox) venom

Preliminary observations on the effects of hyperbaric oxygen therapy on western diamondback rattlesnake (Crotalus atrox) venom poisoning in the rabbit model.

Five previously unknown hemorrhagic proteins, designated hemorrhagic toxins a,b,c,d, and e, were isolated from the venom of the western diamondback rattlesnake (Crotalus atrox). Molecular weights of hemorrhagic toxins a-e were determined to be 68 000, 24 000, 24 000, 24 000, and 25 700, respectively, by sodium dodecyl sulfate-phosphate gel electrophoresis using various polyacrylamide gel concentrations. Amino acid composition showed a total of 636, 200, 213, 214, and 219 amino acids for hemorrhagic toxins a-e, respectively. All the hemorrhagic toxins were found to lose their hemorrhagic activities with the metal chelators ethylenediaminetetraacetic acid and 1, 10-phenanthroline. All the hemorrhagic toxins were found to contain approximately 1 mol of zinc/mol of toxin, and they were all demonstrated to be proteolytic when dimethylcasein and dimethylhemoglobin were used as substrates. When zinc was removed from hemorrhagic toxin e with 1,10-phenanthroline, both both the proteolytic and hemorrhagic activities were equally inhibited. When the apohemorrhagic toxin e thus produced was incubated with zinc, the hemorrhagic and proteolytic activities were regenerated to the same extent. CD, UV, and Raman spectroscopy were used to study the structure of native hemorrhagin toxin e as well as the structural changes caused by zinc removal. From CD spectroscopy the native toxin was estimated to consist of 23% alpha helix, 6% beta structure, and 71% random-coil conformation. When over 90% of the zinc was removed, the alpha-helix content dropped from 23 to 7%.

Unique degradation of human fibrinogen by proteases from western diamondback rattlesnake ( Crotalus atrox) venom

Production of a monoclonal antibody against hemorrhagic activity of Crotalus atrox (western diamondback rattlesnake) venom

Crotalus atrox venom contains agents that render human fibrinogen and plasma incoagulable by thrombin. To elucidate the mechanism of alteration of fibrinogen clotting function by the venom, four immunochemically different proteases, I, II, III, and IV, were purified from the venom by anion-exchange chromatography and column gel filtration. All four proteases had anticoagulant activity rendering purified fibrinogen incoagulable. Proteases I and IV do not affect fibrinogen in plasma but in purified fibrinogen cleave the A alpha chain first and then the B beta and gamma chains. Both enzymes are metalloproteases containing a single polypeptide chain with 1 mol of zinc, are inhibited by (ethylenedinitrilo)tetraacetate and human alpha 2-macroglobulin, and have an optimal temperature of 37 degrees C and an optimal pH of 7. Protease I has a molecular weight (Mr) of 20 000 and is the most cationic. Protease IV has an Mr of 46 000 and is the most anionic glycoprotein with one free sulfhydryl group. Proteases II and III degrade both purified fibrinogen and fibrinogen in plasma, cleaving only the B beta chain and leaving the A alpha and gamma chains intact. Both enzymes are alkaline serine proteases, cleave chromogenic substrates at the COOH terminal of arginine or lysine, are inhibited by diisopropyl fluorophosphate and phenylmethanesulfonyl fluoride, and have an optimal temperature of 50-65 degrees C. Protease II is a single polypeptide chain glycoprotein with an Mr of 31 000. Protease III is a two polypeptide chain protein with an Mr of 24 000, each of the two chains having an Mr of 13 000; its activity is not affected by major protease inhibitors of human plasma. Proteases II and III are enzymes with unique and limited substrate specificity by cleaving only the B beta chain, releasing a peptide of Mr 5000 and generating a fibrinogen derivative of Mr 325 000, with intact A alpha and gamma chains and poor coagulability. Since the two enzymes are active in human plasma and serum, it is postulated that proteases II and III can mediate anticoagulant effects in vivo after envenomation.

A comparative electron microscopic study of myonecrosis induced by Crotalus atrox (Western diamondback rattlesnake) in gray woodrats and mice

Integrin β 4 is a target of rattlesnake venom during inducing apoptosis of vascular endothelial cells

One of the significant changes induced by the injection of rattlesnake (Crotalidae) venom is hemorrhage. Since crotaline antivenin does not prevent such local tissue damage, a more effective treatment of snakebite is needed. To aid in the development of such a treatment the pathogenesis of venom-induced hemorrhae was investigated.Swiss-Webster white mice were injected intramuscularly with Western diamondback rattlesnake (Crotalus atrox) venom. Two minutes after the injection, muscle tissue was obtained by bioosy from the thigh and fixed in 6% glutaraldehyde in Milloniq's phosphate buffer (DH 7.4, 2 hrs., 4°C). After post-fixation in 2% osmium tetroxide in Milloniq's phosphate buffer (pH 7.4, 1hr., 4°C) the tissue was dehydrated routinely in ethanol and embedded in Epon 812. The thin sections were stained with uranyl acetate in methanol and lead citrate then observed with either a Zeiss EM 9A or an Hitachi HS-8 electron microscope.

The proteolytic specificity of hemorrhagic toxin a from the venom of Crotalus atrox (western diamondback rattlesnake) has been investigated by using the oxidized B chain of bovine insulin and other peptides as substrates. The toxin appears highly specific for X--Leu bonds (cleaving the His10--Leu11, Ala14--Leu15, and Tyr16--Leu17 bonds), with no detectable activity against the Gly--Phe, Phe--Phe, Phe--Tyr, and Leu--Tyr bonds also present in the insulin B chain. The X--Leu bond of the peptides Tyr-Gly-Gly-Phe-Leu, Phe-Ala-Leu, and Ala-Leu was also cleaved. The toxin seems to be a strict endopeptidase, in that the cleavage of the two most susceptible bonds, Ala14--Leu15 and Tyr16--Leu17, are mutually exclusive; i.e., cleavage of either bond results in the other being too close to either the amino- or carboxyl-terminal of its respective fragment for the enzyme to be effective against it. The X--Met bond of Tyr-Gly-Gly-Phe-Met was cleaved, although a dipeptide Gly-Met was not hydrolyzed after 16 h of incubation. The substrates not hydrolyzed are furylacryloylglycyl-L-leucinamide, carbobenzoxy-L-glutamylglycine, carbobenzoxyglycyl-L-glutamic acid, benzoyl-L-arginine-p-nitroanilide, L-lysine-p-nitroanilide, (L-Ala)3-p-nitroanilide, Gly-Met, Gly-Phe-Phe, Gly-Gly-Ala, TAME, and ATEE. The absence of hydrolytic activity against the last two substrates indicates that hemorrhagic toxin a does not possess trypsin- or chymotrypsin-like activity.

Viperidae snakes belonging to the genus Crotalus, subfamily Crotalinae, include the species durissus terrificus, durissus durissus, adamanteus, atrox, cerastes, horridus, molossus, scutulatus, viridis and others. All of them, except for the first 2, are found in North America. Crotalus atrox, or diamondback rattlesnakes, live in the southwest United States and in Mexico. This paper describes the fractionation of C. atrox venom in order to isolate and identify its hypotensive agents.

Effects of constriction bands on rattlesnake venom absorption: A pharmacokinetic study