Venom variability and envenoming severity outcomes of the Crotalus scutulatus scutulatus (Mojave rattlesnake) from Southern Arizona

Twenty-one Mojave rattlesnakes, Crotalus scutulatus scutulatus (C. s. scutulatus), were collected from Arizona and New Mexico U.S.A. Venom proteome of each specimen was analyzed using reverse-phase HPLC and SDS-PAGE. The toxicity of venoms was analyzed using lethal dose 50 (LD(50)). Health severity outcomes between two Arizona counties U.S.A., Pima and Cochise, were determined by retrospective chart review of the Arizona Poison and Drug Information Center (APDIC) database between the years of 2002 and 2009. Six phenotypes (A-F) were identified based on three venom protein families; Mojave toxin, snake venom metalloproteinases PI and PIII (SVMP), and myotoxin-A. Venom changed geographically from SVMP-rich to Mojave toxin-rich phenotypes as you move from south central to southeastern Arizona. Phenotypes containing myotoxin-A were only found in the transitional zone between the SVMP and Mojave toxin phenotypes. Venom samples containing the largest amounts of SVMP or Mojave toxin had the highest and lowest LD(50s), respectively. There was a significant difference when comparing the presence of neurotoxic effects between Pima and Cochise counties (p=0.001). No significant difference was found when comparing severity (p=0.32), number of antivenom vials administered (p=0.17), days spent in a health care facility (p=0.23) or envenomation per 100,000 population (p=0.06). Although not part of the original data to be collected, death and intubations, were also noted. There is a 10× increased risk of death and a 50× increased risk of intubations if envenomated in Cochise County.     

Studies of an acidic cardiotoxin isolated from the venom of Mojave rattlesnake (Crotalus scutulatus).

A major lethal protein was isolated from the venom of Mojave rattlesnake (Crotalus scutulatus) by successive purification in DEAE column chromatography and isoelectric focusing. This homogeneous and monomeric form of toxin is designated as "Mojave toxin". Unlike basic neurotoxins or cytotoxins isolated from venoms of cobras, kraits and sea snakes, the Mojave toxin is an acidic protein with an isoelectric point of 4.7. The toxin is also different from crotoxin (from Crotalus durissus terrificus) which consists of both acidic and basic components. The molecular weight determined by Sephadex G-75 column chromatography resulted in a value of about 22 000. A singel protein band with a molecular weight of about 12 000, was observed after sodium dodecyl sulfate gel electrophoresis of the reduced Mojave toxin. Isoelectric focusing gel in the presence of 8 M urea also showed a single protein band, suggesting that the toxin is composed of subunits. Unlike the neurotoxic nature of the basic proteins from the venoms of Elapidae and sea snakes (Hydrophiidae) and crotoxin, Mojave toxin is cardiotoxic rather than neurotoxic. It is very likely that venoms of all rattlesnakes from North and Central America contain Mojave toxin as the common toxin.     

Hemorrhagic and mojave toxins in the venoms of the offspring of two mojave rattlesnakes (Crotalus scutulatus scutulatus)

• 1.1. The venoms of two Mojave rattlesnakes and those of their offsprings were analyzed for Mojave toxin and hemorrhagic toxin.
• 2.2. The venom of one female, collected in Pima County, Arizona, and the venoms of her six offspring contained hemorrhagic toxin but not Mojave toxin (venom B).
• 3.3. The venom of the second female, captured in El Paso County, Texas, contained both toxins (A + B venom). Of her 10 offspring, five contained venom with both toxins, two had hemorrhagic toxin only, and three contained neither toxin.
• 4.4. Venoms that caused hemorrhage also inactivated complement. A pool of the venoms of the venom B offspring was less toxic than adult pooled venom A.

     

Snake venomics of Crotalus tigris: the minimalist toxin arsenal of the deadliest Neartic rattlesnake venom. Evolutionary Clues for generating a pan-specific antivenom against crotalid type II venoms

We report the proteomic and antivenomic characterization of Crotalus tigris venom. This venom exhibits the highest lethality for mice among rattlesnakes and the simplest toxin proteome reported to date. The venom proteome of C. tigris comprises 7-8 gene products from 6 toxin families; the presynaptic β-neurotoxic heterodimeric PLA(2), Mojave toxin, and two serine proteinases comprise, respectively, 66 and 27% of the C. tigris toxin arsenal, whereas a VEGF-like protein, a CRISP molecule, a medium-sized disintegrin, and 1-2 PIII-SVMPs each represent 0.1-5% of the total venom proteome. This toxin profile really explains the systemic neuro- and myotoxic effects observed in envenomated animals. In addition, we found that venom lethality of C. tigris and other North American rattlesnake type II venoms correlates with the concentration of Mojave toxin A-subunit, supporting the view that the neurotoxic venom phenotype of crotalid type II venoms may be described as a single-allele adaptation. Our data suggest that the evolutionary trend toward neurotoxicity, which has been also reported for the South American rattlesnakes, may have resulted by pedomorphism. The ability of an experimental antivenom to effectively immunodeplete proteins from the type II venoms of C. tigris, Crotalus horridus , Crotalus oreganus helleri, Crotalus scutulatus scutulatus, and Sistrurus catenatus catenatus indicated the feasibility of generating a pan-American anti-Crotalus type II antivenom, suggested by the identification of shared evolutionary trends among South and North American Crotalus species.     

Isolation of two phospholipases A2 from Mojave rattlesnake (Crotalus scutulatus scutulatus) venom and variation of immunologically related venom proteins in different populations

Two phospholipases A2 of mol. wt 14,500 (P1) and 14,400 (P2) and pI 9.2 and 7.4 respectively were isolated from Crotalus scutulatus scutulatus venom. The two isoenzymes cross-reacted immunologically with phospholipase A2 from C. adamanteus and C, atrox, but not with Mojave toxin, excluding them as the basic subunit of the Mojave toxin complex. C. s. scutulatus venoms from Arizona had two common bands recognized by anti-P2 which were absent in most C. s. scutulatus venoms from Texas, suggesting two genetically different populations east and west of the Continental Divide.     

Molecular evolution of PIII-SVMP and RGD disintegrin genes from the genus Crotalus

Several types of disintegrins have been isolated from Crotalus spp rattlesnakes, including RGD disintegrins, and PIII-SVMPs. We isolated six cDNAs from snake venom glands using RT-PCR. Three RGD disintegrins (atroxatin, mojastin, and viridistatin) and three PIII-SVMPs (catroriarin, scutiarin, and viristiarin) cDNAs were isolated from the rattlesnakes Crotalus atrox, Crotalus scutulatus scutulatus, and Crotalus viridis viridis, respectively. Atroxatin and Viridistatin shared 90% amino acid identity to each other, and 87% identity to Mojastin. Scutiarin and Viristiarin were identical. All PIII-SVMPs isolated in this study shared the highest amino acid identity with Catrocollastatin. cDNA and protein sequences for RGD disintegrins, one MVD disintegrin, and PIII-SVMPs of the genus Crotalus (present in the NCBI database), were used in phylogenetic analysis. Neighbor-joining analysis of PIII-SVMP and RGD/MVD disintegrin-coding DNA sequences showed that these groups of genes separate into separate clades. A Phi(ST) pairwise comparison and Analysis of Molecular Variance (AMOVA) between PIII-SVMPs and RGD/MVD disintegrins showed significant genetic differences. Mutations observed in ten of the cDNAs analyzed did not affect Cys-coding sequences. Our K(A)/K(S) data suggest that rapid evolution occurred between the genes coding for PIII-SVMPs resulting, in the production of RGD disintegrin-coding genes. However, once these genes diverged, mutations in the PIII-SVMP-coding genes were accumulated less frequently.     

Comparative enzymatic study of HPLC-fractionated Crotalus venoms

1. Ten venoms of the genus Crotalus (Crotalus adamanteus, Crotalus atrox, Crotalus durissus durissus, Crotalus horridus horridus, Crotalus lepidus, Crotalus polystictus, Crotalus molossus molossus, Crotalus pusillus, Crotalus scutulatus scutulatus, venom B, and Crotalus viridis lutosus) were fractionated using HPLC anion and cation exchange chromatography. 2. HPLC venom fractions were tested for hemorrhagic, hemolytic, and proteolytic activities. 3. Crude Virginia opossum (Didelphis virginiana) serum neutralized the hemorrhagic activity of HPLC fractions.     

Evolution of snake venom disintegrins by positive Darwinian selection

PII-disintegrins, cysteine-rich polypeptides broadly distributed in the venoms of geographically diverse species of vipers and rattlesnakes, antagonize the adhesive functions of beta(1) and beta(3) integrin receptors. PII-disintegrins evolved in Viperidae by neofunctionalization of disintegrin-like domains of duplicated PIII-snake venom hemorrhagic metalloproteinase (SVMP) genes recruited into the venom proteome before the radiation of the advanced snakes. Minimization of the gene (loss of introns and coding regions) and the protein structures (successive loss of disulfide bonds) underpins the postduplication divergence of disintegrins. However, little is known about the underlying genetic mechanisms that have generated the structural and functional diversity among disintegrins. Phylogenetic inference and maximum likelihood-based codon substitution approaches were used to analyze the evolution of the disintegrin family. The topology of the phylogenetic tree does not parallel that of the species tree. This incongruence is consistent with that expected for a multigene family undergoing a birth-and-death process in which the appearance and disappearance of loci are being driven by selection. Cysteine and buried residues appear to be under strong purifying selection due to their role in maintaining the active conformation of disintegrins. Divergence of disintegrins is strongly influenced by positive Darwinian selection causing accelerated rate of substitution in a substantial proportion of surface-exposed disintegrin residues. Global and lineage-specific sites evolving under diversifying selection were identified. Several sites are located within the integrin-binding loop and the C-terminal tail, two regions that form a conformational functional epitope. Arginine-glycine-aspartic acid (RGD) was inferred to represent the ancestral integrin-recognition motif, which emerged from the subgroup of PIII-SVMPs bearing the RDECD sequence. The most parsimonious nucleotide substitution model required for the emergence of all known disintegrin's integrin inhibitory motifs from an ancestral RGD sequence involves a minimum of three mutations. The adaptive advantage of the emergence of motifs targeting beta(1) integrins and the role of positively selected sites located within nonfunctional disintegrin regions appear to be difficult to rationalize in the context of a predator-prey arms race. Perhaps, this represents a consequence of the neofunctionalization potential of the disintegrin domain, a feature that may underlie its recruitment into the venom proteome followed by its successful transformation into a toxin.     

Presence of zinc in proteolytic hemorrhagic toxin isolated from Agkistrodon acutus venom

1. Hemorrhagic toxin (Ac1-proteinase) was isolated from the venom of Agkistrodon acutus (Formosa) and the zinc content was determined (1.15 mol/mol protein). The results we extensively compared with hemorrhagic toxin e of Crotalus atrox venom (U.S.A.). Comparable results were obtained for zinc content, hemorrhagic and proteolytic activities for native hemorrhagic toxins from two different venoms. It is of interest that the zinc content of hemorrhagic toxins is identical even though the venoms are obtained from snakes inhabiting totally different continents. 2. Zinc content, hemorrhagic and proteolytic activities were compared before and after the removal of zinc. It was found that both hemorrhagic and proteolytic activities disappeared upon removal of the zinc. 3. Zinc content of all hemorrhagic toxins with proteolytic activity reported so far were also compared and it is concluded that regardless of their geographic origin, zinc is present in venom hemorrhagic toxins on a unimolar basis. 4. When zinc is removed there is a drastic change in the low-frequency region of the Raman spectrum suggesting the presence of a zinc ligand co-ordination. The decrease of alpha-helical content and increase of random coil are reflected in the amide I and III bands of Raman spectroscopy after the removal of zinc. Increase of random coil and the loss of zinc are probably responsible for the loss of hemorrhagic and proteolytic activities.     

Purification and characterization of mojave (Crotalus scutulatus scutulatus) toxin and its subunits

An acidic lethal protein, Mojave toxin, has been isolated from the venom of Crotalus scutulatus scutulatus. The purified toxin had an i.v. LD₅₀ of 0.056 μg/g in white mice. Disc polycrylamide gel electrophoresis at pH values of 9.6 and 3.8 and isoelectric focusing in polyacrylamide gels with a pH 3.5–10 Ampholyte gradient were used to establish the presence of one major protein band. The pI of the most abundant form of the toxin was determined to be 5.5 by polyacrylamide gel isoelectric focusing experiments. The molecular weight was established to be 24,310 daltons from amino acid composition data. Mojave toxin was shown to consist of two subunits, one acidic and one basic with isoelectric point (pI) values of 3.6 and 9.6, respectively. Amino acid analyses established molecular weights of 9593 for the acidic component and 14,673 for the basic component. The acidic subunit consisted of three peptide chains intermolecularly linked by cystine residues. The basic subunit was a single polypeptide chain with six intramolecular disulfide bonds. The basic subunit was lethal to test animals with an intravenous LD₅₀ of 0.58 μg/g. Following recombination of the subunits a recombinant toxin was isolated which was identical to the native toxin by comparisons of electrophoretic mobility and toxicities. Comparisons of circular dichroism spectra also indicated reassociation to the native toxin structure. Phospholytic activity was associated with Mojave toxin and the basic subunit was responsible for this enzymic activity. Phospholipase activity of the basic subunit was inhibited by addition of the acidic subunit.     

Structural properties of mojave toxin of crotalus scutulatus (Mojave rattlesnake) determined by laser Raman spectroscopy.

Laser Raman Spectra were obtained on aqueous and solid samples of Mojave toxin isolated from the venom of the Mojave rattlesnake ($\text{Crotalus}\text{scutulatus}$). The Raman spectra reveal that the Mojave toxin, an acidic protein of molecular weight about 22,000, contains a predominantly α-helical secondary structure and that the tyrosyl residues, on the basis of the Raman frequencies and intensities, are exposed to the solvent. These features of the Mojave toxin distinguish it structurally from the neurotoxins of sea snake venoms. However, like the sea snake venom toxins, Mojave toxin contains four disulfide bridges and is not greatly altered in structure by removal of the aqueous solvent.     

Regional variation in the presence of canebrake toxin in Crotalus horridus venom

Reverse-phase HPLC was used to isolate the PLA complex neurotoxin “canebrake toxin” from the venom of Crotalus horridus from northern Florida. Individual venoms from 107 specimens of C. horridus throughout its range were investigated for the presence of the toxin. The distribution of canebrake toxin was limited to two separate regions, including a region of Louisiana, Arkansas and Oklahama, and a separate region from southeastern South Carolina through eastern Georgia to northern Florida. Four distinct venom types were found and designated Venoms A (neurotoxic), B (hemorrhagic), A + B (neurotoxic and hemorrhagic) and C (lacking in both neurotoxic and hemorrhagic activities).     

Molecular models of the Mojave rattlesnake (Crotalus scutulatus scutulatus) venom metalloproteinases reveal a structural basis for differences in hemorrhagic activities

Rattlesnake venom can differ in composition and in metalloproteinase-associated activities. The molecular basis for this intra-species variation in Crotalus scutulatus scutulatus (Mojave rattlesnake) remains an enigma. To understand the molecular basis for intra-species variation of metalloproteinase-associated activities, we modeled the three-dimensional structures of four metalloproteinases based on the amino acid sequence of four variations of the proteinase domain of the C. s. scutulatus metalloproteinase gene (GP1, GP2, GP3, and GP4). For comparative purposes, we modeled the atrolysin metalloproteinases of C. atrox as well. All molecular models shared the same topology. While the atrolysin metalloproteinase molecular models contained highly conserved substrate binding sites, the Mojave rattlesnake metalloproteinases showed higher structural divergence when superimposed onto each other. The highest structural divergence among the four C. s. scutulatus molecular models was located at the northern cleft wall and the S’₁-pocket of the substrate binding site, molecular regions that modulate substrate selectivity. Molecular dynamics and field potential maps for each C. s. scutulatus metalloproteinase model demonstrated that the non-hemorrhagic metalloproteinases (GP2 and GP3) contain highly basic molecular and field potential surfaces while the hemorrhagic metalloproteinases GP1 and atrolysin C showed extensive acidic field potential maps and shallow but less dynamic active site pockets. Hence, differences in the spatial arrangement of the northern cleft wall, the S’₁-pocket, and the physico-chemical environment surrounding the catalytic site contribute to differences in metalloproteinase activities in the Mojave rattlesnake. Our results provide a structural basis for variation of metalloproteinase-associated activities in the rattlesnake venom of the Mojave rattlesnake.     

A new gene structure of the disintegrin family: a subunit of dimeric disintegrin has a short coding region

Disintegrin is a potent platelet aggregation inhibitor isolated from various snake venoms. The cDNA of the snake venom disintegrin family precursor is well-known to encode pre-peptide, metalloprotease, spacer, and disintegrin domains. Recently, new types of disintegrins, dimeric disintegrins, have been isolated, and their amino acid sequences were determined to be approximately 65 amino acid residues in each subunit. We isolated a novel heterodimeric disintegrin, acostatin, from the venom of Agkistrodon contortrix contortrix, which consisted of 63 and 64 amino acid residues in the alpha chain and beta chain, and both chains had the Arg-Gly-Asp (RGD) sequence for binding platelet GPIIb/IIIa. The cDNA lengths of the alpha chain and the beta chain of acostatin were 902 bp and 2031 bp, respectively. The acostatin alpha chain precursor, surprisingly, has the only disintegrin domain alone and lacked almost all of the pre-peptide and metalloprotease domains. The precursor of the acostatin beta chain belongs to a well-known motif of disintegrin precursors. Furthermore, both precursors of alpha and beta chains of another heterodimeric disintegrin, piscivostatin, also have the same domain structures as those of acostatin subunits. These results indicate that the cDNAs of heterodimeric disintegrin subunits have quite a different length of coding region and their precursors have a novel domain structure of disintegrin-family proteins.     

The complete sequence of the acidic subunit from Mojave toxin determined by Edman degradation and mass spectrometry

Mojave toxin, a heterodimeric, neurotoxic phospholipase complex from Crotalus scutulatus scutulatus, is one of a group of closely related rattlesnake toxins for which much structural information is still lacking. The complete amino-acid sequence of the acidic subunit from Mojave toxin was determined. The three individual peptide chains, derived from the acidic subunit by reductive alkylation, were separated by high-performance liquid chromatography. Fragmentations of the A and B chains were done using specific proteinases and the resulting peptide mixtures were fractionated by reverse-phase high-performance liquid chromatography. Sequence analyses on the intact chains and the fragments from digests were done by automated Edman degradation, carboxypeptidase Y degradation and triple-quadrupole and tandem-quadrupole Fourier-transform mass spectrometry. The sequence for each acidic subunit chain is very similar to the corresponding chain from the related neurotoxin complex, crotoxin, and overall the sequence is similar to the sequences of group I and II phospholipases A2. The N-terminus of the B chain is blocked by pyroglutamic acid. The existence of two distinct and closely related C chains was established. It is unlikely that the small sequence difference can account for the isoforms that are present in purified Mojave toxin and in unfractionated venom.     

Snake venomics of Central American pitvipers: clues for rationalizing the distinct envenomation profiles of Atropoides nummifer and Atropoides picadoi

We report the proteomic characterization of the Central American pitvipers Atropoides nummifer and Atropoides picadoi. The crude venoms were fractionated by reverse-phase high-performance liquid chromatography (HPLC), followed by analysis of each chromatographic fraction by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), N-terminal sequencing, matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass fingerprinting, and collision-induced dissociation-tandem mass spectrometry (CID-MS/MS) of tryptic peptides. Each venom contained a number of bradykinin-potentiating peptides and around 25-27 proteins of molecular masses in the range of 7-112 kDa, belonging to only nine different toxin families (disintegrin, DC fragment, snake venom vascular endothelial growth factor, phospholipases A2, serine protease, cysteine-rich secretory proteins, C-type lectins, L-amino acid oxidase, and Zn2+-dependent metalloproteases), albeit distinctly distributed among the two Atropoides species. In addition, A. nummifer expresses low amounts of a three-finger toxin not detected in the venom of A. picadoi. The major toxins of A. nummifer belong to the PLA2 (relative abundance, 36.5%) and the serine proteinase (22%) families, whereas the most abundant A. picadoi toxins are Zn2+-dependent metalloproteinases (66.4%). We estimate that the similarity of venom proteins between the two Atropoides taxa may be around 14-16%. The high degree of differentiation in the venom proteome among congeneric taxa emphasizes unique aspects of venom composition of related species of Atropoides snakes and points to a strong role for adaptive diversification via natural selection as a cause of this distinctiveness. On the other hand, their distinct venom toxin compositions provide clues for rationalizing the low hemorrhagic, coagulant, and defibrinating activities and the high myotoxic and proteolytic effects evoked by A. nummifer snakebite in comparison to other crotaline snake venoms and the high hemorrhagic activity of A. picadoi.     

Purification and characterization of a lethal toxin from the venom of Heloderma horridum horridum

A lethal toxin was isolated from the venom of Heloderma h. horridum by gel filtration and ion-exchange chromatography. Molecular weight of the purified toxin was determined to be 28 kDa under reducing and nonreducing conditions. Biological activity, assayed by i.v. routes of injection, shows an LD50 for this preparation of 0.135 micrograms/g. Additionally, the toxin possesses an inhibitory effect on direct electrical stimulation of the isolated mouse hemi-diaphragm. However, neither hemorrhagic nor hemolytic activities were detected. Phospholipase A2 activity, proteolytic activity and arginine esterolytic activity were absent. The amino acid composition of the lethal toxin and the NH2-terminal sequence up to residue number 33 were determined. Neither show similarities to other components from H. h. horridum venom.     

Hemorrhagic, coagulant and fibrino(geno)lytic activities of crude venom and fractions from mapanare (Bothrops colombiensis) snakes

Bothrops colombiensis venom from two similar geographical locations were tested for their hemostatic functions and characterized by gel-filtration chromatography and SDS-PAGE electrophoresis. The snakes were from Caucagua and El Guapo towns of the Venezuelan state of Miranda. Fibrino(geno)lytic, procoagulant, hemorrhagic, lethal activities, gel-filtration chromatography and SDS-PAGE profiles were analyzed and compared for both venoms. The highest hemorrhagic activity of 5.3 mug was seen in El Guapo venom while Caucagua venom had the lowest LD(50) of 5.8 mg/kg. Both venoms presented similar thrombin-like activity. El Guapo showed a factor Xa-like activity two times higher than Caucagua. Differences were observed in kallikrein-like and t-PA activities, being highest in El Guapo. Caucagua venom showed the maximum fibrin lysis. Both crude venom runs on Sephadex G-100 chromatography gave fraction SII with the high fibrinolytic activity. Proteases presented in SII fractions and eluted from Benzamidine-Sepharose (not bound to the column) provoked a fast degradation of fibrinogen alpha chains and a slower degradation of beta chains, which could possibly be due to a higher content of alpha fibrinogenases in these venoms. The fibrinogenolytic activity was decreased by metalloprotease inhibitors. The results suggested that metalloproteases in SII fractions were responsible for the fibrinolytic activity. The analysis of samples for fibrin-zymography of SII fractions showed an active band with a molecular mass of approximately 30 kDa. These results reiterate the importance of using pools of venoms for antivenom immunization, to facilitate the neutralization of the maximum potential number of toxins.     

Analysis of Vipera russelli venom using polyclonal antibodies prepared against its purified toxic phospholipase A2 VRV PL-V.

Vipera russelli venom induces predominantly neurotoxic, myotoxic necrotic and hemorrhagic symptoms in experimental animals and has several hydrolytic enzyme activities. In this study, V. russelli venom is characterized both as a PLA2 and as a toxin. Anti PL-V Ig (antibodies to a toxic phospholipase A2 VRV PL-V of V. russelli venom) nullifies the toxicity of whole V. russelli venom to a great extent. The neurotoxic symptoms vanish completely in the presence of anti PL-V Ig. The cross reacting components of whole V. russelli venom were removed by precipitating them from whole venom by the addition of anti PL-V Ig. The non-cross reacting components present in the supernatant were checked for toxicity. There was a significant reduction in toxicity. The LD50 value of the supernatant had increased from 4.1 mg/kg body weight to 11.7 mg/kg body weight and it showed about 34% of the total venom phospholipase A2 activity. It had edema forming, hemorrhagic and hemolytic activity but failed to induce neurotoxic, anticoagulant and myotoxic effects.     

乌梢蛇血清的抗出血因子:一个有前途的抗蛇毒药物原料

用柱层析和聚丙烯酰胺凝胶盘状电泳法,从乌梢蛇血清中分离纯化了一个抗出血因子。用SDS-聚丙烯酰胺凝胶电泳法测得其分子量大约为65 kD;测定了五种蝮亚科蛇毒(尖吻蝮、竹叶青蛇、原矛头蝮、哈扑和短尾蝮)的最小出血剂量和乌梢蛇血清中抗出血因子对这五种蛇毒的抗出血活性;还测定了七种蛇毒(除上述五种毒蛇外,还包括圆斑蝰和银环蛇)的半数致死量,以及抗出血因子对中毒小鼠的治疗作用。结果显示:从乌梢蛇血清中提纯的抗出血因子的抗蛇毒活性,不仅可以抵抗它的捕食者尖吻蝮的蛇毒,而且还可以抵抗具出血活性的其它蛇毒;但它对不具出血活性的银环蛇毒的致死抑制作用不明显。该抗出血因子不仅在体外实验表现出强的中和出血毒素的活性,而且在体内实验中亦表现出对中毒小鼠良好的治疗作用,因而可能成为新的抗蛇毒药物的有前途的原料。乌梢蛇血清对血循毒的中和能力的获得,可能归因于尖吻蝮与乌梢蛇之间捕食与被捕食相互作用的关系。