Hemorrhagic Toxin Research Articles

Isolation and characterization of hemorrhagic toxin from the venom of Trimeresurus elegans

1. 1. Hemorrhagic toxin from the venom of Trimeresurus elegans was purified in a homogeneous form using gel filtration and ion exchange chromatographies. 2. 2. Hemorrhagic toxin possessed hemorrhagic and TAME (tosyl- l-arginine methyl ester) hydrolytic activities. These activities were inhibited when hemorrhagic toxin was incubated with benzamidine or N-bromosuccinimide. 3. 3. Its mol. wt was 28,500 and the isoelectric point was 7.4. 4. 4. This toxin contains ca 1.5 mol Ca per mol of protein.

Antihemorrhagic factors from the blood serum of the western diamondback rattlesnake Crotalus atrox

Several antihemorrhagic factors isolated from C. atrox serum are glycoproteins with mol. wt ranging from 65,000 to 80,000. The antihemorrhagic activity of these factors was stable at a pH range of 1.3–11.5 and at temperatures up to 85°C, for 30 min. The isolated antihemorrhagins also neutralized the proteolytic activity of C. atrox venom, as tested with azocollagen and gelatin, and formed a complex with hemorrhagic toxin e isolated from the same venom. The neutralization capacity of the isolated antihemorrhagins was six times as high as the commercial polyvalent antivenom produced for clinical use.

Primary Structure of a Hemorrhagic Metalloproteinase, HT-2, Isolated from the Venom of Crotalus ruber ruber1

Crotalidae and Viperidae snake venoms contains several kinds of metalloproteinases which cause localized hemorrhage by direct action on blood vessel walls. We report here the entire amino acid sequence and the disulfide bridge locations of HT-2, one of the hemorrhagic toxins isolated from the venom of Crotalus ruber ruber (red rattlesnake). The non-reduced protein was first cleaved at methionine residues to provide a set of 8 fragments, which covered the entire sequence of HT-2. The disulfide bridge locations of HT-2 were also determined by using these primary fragments. The unambiguous sequence for the whole protein was then established by conventional methods using lysyl endopeptidase and thermolysin digests. HT-2 consisted of 202 amino acid residues with two disulfide bridges, which were assigned to Cys-117-Cys-197 and Cys-157-Cys-164. HT-2 had a typical zinc-chelating sequence His-Glu-X-X-His (residues 142-146) found in thermolysin, and its overall sequence showed, respectively, 50, 52, and 53% identities to those of HR2a, H2-proteinase, and the metalloproteinase domain of HR1B. However, the disulfide bridge locations of HT-2 were different from those in the other metalloproteinases. The primary structure of HT-2 was more closely related to that of Ht-d from Crotalus atrox recently determined (81% identity). From the structural comparison with five metalloproteinases so far elucidated, six conservative amino acid residues, which may possibly be related to the induction of the hemorrhagic activity, were suggested to be present in these toxins.

The complete amino acid sequence of the high molecular mass hemorrhagic protein HR1B isolated from the venom of Trimeresurus flavoviridis.

Hemorrhage is a common occurrence in a victim bitten by crotalid and viperid snakes, and hemorrhagic components in these various venoms have been isolated and characterized. Previously, we have shown that a low molecular weight hemorrhagic protein (HR2a, 202 amino acid residues) isolated from the venom of Trimeresurus flavoviridis is a member of a new subfamily of metalloproteinases. We now report the complete amino acid sequence of a high molecular mass hemorrhagic protein isolated from the same venom. This protein, HR1B, is a mosaic protein composed of 416 residues containing four asparagine-linked oligosaccharide chains. The amino-terminal half (residues 1-203) of HR1B contains a metalloproteinase domain, the sequence of which is 62% identical to that of HR2a and 52% identical to that of hemorrhagic toxin d isolated from Crotalus atrox venom. The most interesting finding is that the middle region (residues 204-300) of HR1B shows a striking similarity to disintegrins, Arg-Gly-Asp-containing platelet aggregation inhibitors, recently found in several viper venoms. Interestingly, however, this region of HR1B does not contain the Arg-Gly-Asp sequence which is known to be a putative binding site in the disintegrins for the platelet fibrinogen receptor, the glycoprotein IIb-IIIa complex. We also found that the carboxyl-terminal region (residues 213-336) of the middle part of HR1B shows 30% identity to residues 1543-1656 of von Willebrand factor and that the remaining region at the carboxyl-terminal end is unique and has a cysteine-rich sequence. These results suggest that the middle portion of HR1B, which shows structural similarities to the disintegrins and von Willebrand factor, may be important in synergistically stimulating hemorrhagic activity in the NH2-terminal metalloproteinase domain.

ChemInform Abstract: Reaction of 4‐Hydroxycoumarin Derivatives with Activated Dimethyl Sulfoxide.

AbstractIn the course of studies on the haemorrhagic toxins from Ferula communis L. the oxidation of isomeric ω‐hydroxyferulenols such as (I) to the corresponding aldehydes is needed.

Interaction of hemorrhagic metalloproteinases with human .alpha.2-macroglobulin

The interaction between four Crotalus atrox hemorrhagic metalloproteinases and human alpha 2-macroglobulin was investigated. The proteolytic activity of the hemorrhagic toxins Ht-c, -d, and -e against the large molecular weight protein substrates, gelatin type I and collagen type IV, was completely inhibited by alpha 2-macroglobulin. The proteolytic activity of Ht-a against the same substrates was not significantly inhibited. Each mole of alpha 2-macroglobulin bound maximally 2 mol of Ht-e and 1.1 mol of Ht-c and Ht-d. These proteinases interacted with alpha 2-macroglobulin rapidly at 22 degrees C. Rate constants based on intrinsic fluorescence measurements were 0.62 X 10(5) M-1 s-1 for interaction of alpha 2-macroglobulin with Ht-c and -d and 2.3 X 10(5) M-1 s-1 for the interaction of alpha 2-macroglobulin with Ht-e. Ht-a interacted with alpha 2-macroglobulin very slowly at 22 degrees C. Increasing the temperature to 37 degrees C and prolonging the time of interaction with alpha 2-macroglobulin resulted in the formation of Mr 90,000 fragments and high molecular weight complexes (Mr greater than 180,000), in which Ht-a is covalently bound to the carboxy-terminal fragment of alpha 2-M. The identification of the sites of specific proteolysis of alpha 2-macroglobulin shows that the cleavage sites for the four metalloproteinases are within the bait region of alpha 2-macroglobulin. Ht-c and -d cleave only at one site, the Arg696-Leu697 peptide bond, which is also the site of cleavage for plasmin, thrombin, trypsin, and thermolysin. Ht-a cleaves alpha 2-macroglobulin primarily at the same site, but a secondary cleavage site at the His694-Ala695 peptide bond was also identified.(ABSTRACT TRUNCATED AT 250 WORDS)

Pathogenesis of hemorrhage induced by bilitoxin, a hemorrhagic toxin isolated from the venom of the common cantil ( Agkistrodon bilineatus bilineatus)

The pathogenesis of hemorrhage induced by the i.m. injection of the hemorrhagic toxin, bilitoxin, was studied using light and electron microscopy. White mice were injected with sublethal doses of the toxin, and tissue samples were obtained at 5 and 30 min, and 1, 3 and 24 hr after the injection. There was a good correlation between amount of toxin injected and amount of hemorrhage observed. Microscopically, hemorrhage was visible in all parts of the connective tissue surrounding muscle cells just 5 min after injection and fibrin was present both intravascularly and extravascularly. At later time periods the hemorrhage was more extensive and there was more fibrin. Many vessels were plugged with platelets. At 30 min after the injection, muscle cells appeared to be damaged having either delta lesions or disrupted myofibrils. Electron microscopy revealed damaged capillaries with ruptured endothelial cells, disrupted basal lamina and intact intercellular junctions. Thus, this hemorrhagic toxin acts rapidly to disrupt the capillary endothelium without damaging the intercellular junctions, and it also appears to damage skeletal muscle cells.

Isolation of a hemorrhagic toxin from Mojave rattlesnake ( Crotalus scutulatus scutulatus) venom

A hemorrhagic toxin was isolated from Mojave rattlesnake venom. The isoelectric point of the toxin was 4.7 and its mol. wt was 27,000. Concentrations as low as 2 μg injected s.c. in mice caused hemorrhage greater than 5 mm in diameter. The toxin was fibrinogenolytic and hydrolyzed hide powder azure, casein and collagen. The toxin also partially inactivated complement. It had no activity against elastin, fibrin, and the chromogenic substrates S-2805, S-2302 and S-2238. Its esterolytic activity was 3% of the activity of the unfractionated venom. The enzymatic and hemorrhagic activities were inhibited by EDTA. The hemorrhagic toxin was absent or in low quantities in Mojave rattlesnake venoms containing Mojave toxin. Chromatography by HPLC easily distinguishes Mojave rattlesnake venoms into two types by the presence or absence of the hemorrhagic toxin.

Isolation of two hemorrhagic toxins from Crotalus basiliscus basiliscus (Mexican West coast rattlesnake) venom and their effect on blood clotting and complement

1. 1. Two hemorrhagic toxins of mol. wt 27,000 (Bl) and 27.500 (B2) and pi 9.8 and 5.2 respectively were isolated from Crotalus basiliscus venom. 2. 2. The two proteinases did not cross-react antigenically. 3. 3. Both toxins caused hemorrhage in mice and each was capable of hydrolyzing hide powder azure, casein, collagen and fibrin. 4. 4. Bl hydrolyzed the Aga, Bgb and gg chains of fibrinogen. B2 hydrolyzed the Aa and Bgb chains of fibrinogen, but not the gg chain. 5. 5. Both proteinases inactivated guinea pig complement.

Preparation of a monoclonal antibody to a hemorrhagic toxin from Prairie rattlesnake (Crotalus viridis viridis) venom: its characteristics and use in isolating the toxin: Charlotte L. Ownby, 1Jane Halpern, 2 and William H. Habig2 ( 1Department of Physiological Sciences, Oklahoma State University, Stillwater, OK 74078, U.S.A., and 2Center for Biologics Evaluation and Research, Laboratory of Bacterial Toxins, Division of Bacterial

Preparation of a monoclonal antibody to a hemorrhagic toxin from Prairie rattlesnake (Crotalus viridis viridis) venom: its characteristics and use in isolating the toxin: Charlotte L. Ownby, 1Jane Halpern, 2 and William H. Habig2 ( 1Department of Physiological Sciences, Oklahoma State University, Stillwater, OK 74078, U.S.A., and 2Center for Biologics Evaluation and Research, Laboratory of Bacterial Toxins, Division of Bacterial

Characterization of hemorrhagic toxins isolated from Agkistrodon contortrix laticinctus (broad banded copperhead) venom: Edward K. Johnson and Charlotte L. Ownby (Department of Physiological Sciences, Oklahoma State University, Stillwater, OK 74078, U.S.A.)

Characterization of hemorrhagic toxins isolated from Agkistrodon contortrix laticinctus (broad banded copperhead) venom: Edward K. Johnson and Charlotte L. Ownby (Department of Physiological Sciences, Oklahoma State University, Stillwater, OK 74078, U.S.A.)

Amino acid sequence of a Crotalus atrox venom metalloproteinase which cleaves type IV collagen and gelatin

The hemorrhagic toxin Ht-d from venom of the Western diamondback rattlesnake is a metalloproteinase with a molecular weight of 23,234. Peptides were obtained from enzymatic and chemical digestions, separated by reverse-phase chromatography, and sequenced in a gas-phase sequenator. The sequence showed a putative zinc binding site similar to that of thermolysin and other metalloproteinases but no overall significant similarity to the sequences of other metalloproteinases and may represent a new subfamily of metalloproteinases. Ht-d was shown to degrade type IV collagen and gelatin types I, III, and V but not interstitial collagens. The digestion of type IV collagen and other basement membrane proteins may allow this proteinase to disrupt capillary membranes causing hemorrhage in surrounding tissues.

Hemorrhagic toxin from the venom of Agkistrodon bilineatus (Common cantil)

1. 1. Hemorrhagic toxin was isolated from Agkistrodon bilineatus (Common cantil) venom using a three-step purification procedure to obtain 32.8 mg of purified hemorrhagic toxin from 700 mg of crude venom. 2. 2. The purified toxin was homogeneous by disc polyacrylamide gel electrophoresis at pH 8.3, and by isoelectric focusing. 3. 3. Hemorrhagic toxin possessed lethal, hemorrhagic and proteolytic activities. These activities of this toxin were inhibited by ethylenediaminetetraacetic acid (EDTA) and ethyleneglycol-bis-(β-aminoethylether) N, N'-tetraacetic acid (EGTA), but not by cysteine or soybean trypsin inhibitor (SBTI). 4. 4. Its molecular weight was approximately 48 kDa and the isoelectric point was 4.2. 5. 5. Purified preparation hydrolyzed the Asn(3)-Gln(4), His(10)-Leu(11), Ala(14)-Leu(15), Tyr(16)-Leu(17), Arg(22)-Gly(23) and Phe(24)-Phe(25) bonds of oxidized insulin B chain. 6. 6. The Aα chain of fibrinogen was first split and Bβ chain was cleaved later by this toxin. 7. 7. Hemorrhagic toxin contains 1 mol of zinc and 2 mol of calcium per mol of protein.

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

R. A. Martinez, S. Y. Huang and J. C. Perez. Antigenic relationships of fractionated western diamondback rattlesnake ( Crotalus atrox) hemorrhagic toxins and other rattlesnake venoms as indicated by monoclonal antibodies. Toxicon 27, 239–245, 1989.—Seven hemorrhagic factors have been isolated from Crotalus atrox venom, but their antigenic relationships have not been well studied. In this study, two different monoclonal antibodies, C. atrox peak 8 (CA-P-8) and C. atrox subclone 5 (CA-5+), were produced against two C. atrox venom hemorrhagic fractions and used in an ELISA (enzyme-linked immunosorbent assay) to determine if the hemorrhagic factors in C. atrox venom are antigenically related. The same ELISA test was used to determine cross-reactivity of seven other crude Crotalidae venoms. The two monoclonal antibodies were tested for their ability to neutralize each hemorrhagic HPLC fraction separated from C. atrox venom. C. atrox venom was fractionated into 22 fractions using HPLC analytical DEAE ion exchange. Fractions 4–17 were hemorrhagic. The CA-P-8 monoclonal antibody reacted strongly with hemorrhagic fraction 8; CA-5+ had a broader reactivity and reacted with several HPLC hemorrhagic and non-hemorrhagic fractions. Crude venoms of C. adamanteus, C. scutulatus scutulatus and C. viridis lutosus reacted with CA-P-8, while C. viridis lutosus, C. viridis oreganus, C. scutulatus scutulatus and C. horridus horridus reacted with CA-5+. C. molossus molossus and C. lepidus lepidus did not react with CA-P-8 and CA-5+. Hemorrhagic HPLC fractions 6, 7, 8, were completely neutralized by monoclonal antibody CA-P-8; fraction 9 was partially neutralized. The present study indicated that some C. atrox venom HPLC hemorrhagic fractions have both common and unique epitopes. Antigenic determinants were also found to be shared among different Crotalus species. The availability of a panel of monoclonal antibodies would be useful in elucidating the antigenic components and biological functions related to venom toxins.

Cytopathological effects of crude venom and isolated hemorrhagic toxin from the Egyptian sand viper (Cerastes cerastes): Electron microscopic studies: Tarek R. Rahmy 1, Anthony T. Tu 1, Mahmoud A. Elmbanhawy 2, Mohamed F. El-Asmar 2 and Fathalla M. Hassan3 ( 1Biochemistry Department, CSU, Fort Collins, CO 80523, U.S.A.; 2Ain Shams and 3Suez Canal Universities, Egypt)

Cytopathological effects of crude venom and isolated hemorrhagic toxin from the Egyptian sand viper (Cerastes cerastes): Electron microscopic studies: Tarek R. Rahmy 1, Anthony T. Tu 1, Mahmoud A. Elmbanhawy 2, Mohamed F. El-Asmar 2 and Fathalla M. Hassan3 ( 1Biochemistry Department, CSU, Fort Collins, CO 80523, U.S.A.; 2Ain Shams and 3Suez Canal Universities, Egypt)

Biochemical characterization of two hemorrhagic proteases from the venom of Lachesis muta (bushmaster).

Two hemorrhagic proteases, Lachesis hemorrhagic toxins a and b (LHTa and LHTb), were isolated from the venom of Lachesis muta, which is distributed in Central and South America. One protease showed strong hemorrhagic action, while the other showed weak hemorrhagic activity even though the two enzymes are very similar in their chemical properties. Neither enzyme hydrolyzed arginine esters, but both hydrolyzed casein and reduced fibrinogen. The A alpha chain of fibrinogen was hydrolyzed first, and the B beta chain was hydrolyzed later. The gamma chain of fibrinogen was resistant to hydrolysis. The molecular weights of LHTa and LHTb were very similar, 22,000 and 23,000, respectively. The amino acid composition of LHTa was also similar to that of LHTb. The secondary structure of LHTa as determined by Lippert's equation was 52% alpha helix, 17% beta sheet, and 31% random coil; that of LHTb was 47% alpha helix, 13% beta sheet, and 40% random coil.

Isolation and characterization of horridum toxin with arginine ester hydrolase activity from Heloderma horridum (beaded lizard) venom

A hemorrhagic toxin with lethal and arginine ester hydrolytic activities was isolated from Heloderma horridum (beaded lizard) venom by Sephadex G-75, DEAE-Sephacel, and Q-Sepharose column chromatography. The hemorrhagic toxin was shown to be homogeneous as demonstrated by a single band on acrylamide gel electrophoresis and immunodiffusion. Its molecular weight is approximately 31,000 with an isoelectric point of 3.9. Hemorrhagic, lethal, and benzoyl- l-arginine ethyl ester hydrolytic activities of this preparation were inhibited by diisopropyl fluorophosphate (DFP), N-bromosuccinimide, and β-mercaptoethanol, suggesting that serine, tryptophan, and disulfide bonds are involved in these activities. Also there was an increase in creatine kinase activity in mice serum which is an indicator that the toxin is involved in muscle damage. This protein was stable to heat and pH ranges between 2 and 11. The Michaelis constant ( K m), for benzoyl- l-arginine ethyl ester, and inhibition constant ( K i), for DFP, were found to be 6.9 × 10 −3 and 1.93 × 10 −4 m, respectively.

Purification and biochemical characterization of atroxase, a nonhemorrhagic fibrinolytic protease from western diamondback rattlesnake venom.

Crotalus atrox venom contains a variety of proteases which render fibrinogen incoagulable and solubilize fibrin. One of these proteases was purified by using ion-exchange and gel permeation liquid chromatography. The protease, called atroxase, consists of a single nonglycosylated polypeptide chain with a molecular weight of 23,500 and an isoelectric point of pH 9.6. Amino acid analysis indicates atroxase to contain 206 residues with no sulfhydryl groups. Metal analysis found zinc and potassium at 1 mol/mol of protein, and calcium at 0.3 mol/mol of protein. Proteolytic activity is inhibited by ethylenediaminetetraacetate and alpha 2-macroglobulin. Maximal proteolytic activity occurs at pH 9.0 and 55 degrees C. Proteolytic specificity, using oxidized insulin B chain, is similar to that of several hemorrhagic toxins found within the same venom, yet atroxase shows no hemorrhagic activity and exhibits low lethality when tested on Swiss Webster mice. Atroxase, an A alpha, B beta fibrinogenase, cleaves the A alpha chain of fibrinogen first followed by the B beta chain and shows no effect on the gamma chain. The nonspecific action of the enzyme results in the extensive hydrolysis of fibrinogen which releases a variety of fibrinopeptides. Fibrin solubilization appears to occur primarily from the hydrolysis of alpha-polymer and unpolymerized alpha and beta chains. Although crude venom induces platelet aggregation, atroxase demonstrated no ability to induce or inhibit aggregation.

Classification of myonecrosis induced by snake venoms: Venoms from the prairie rattlesnake ( Crotalus viridis viridis), Western diamondback rattlesnake ( Crotalus atrox) and the Indian cobra ( Naja naja naja)

C. L. Ownby and T. R. Colberg. Classification of myonecrosis induced by snake venoms: venoms from the prairie rattlesnake ( Crotalus viridis viridis), western diamondback rattlesnake ( Crotalus atrox) and the Indian cobra ( Naja naja naja). Toxicon 26, 459 – 474, 1988. — The pathogenesis of myonecrosis induced by three different snake venoms was studied by light microscopic examination of skeletal muscle tissue taken at time periods ranging from 0.25 hr to 4 weeks after an intramuscular injection of the venom into mice. It was possible to identify different types of myonecrosis based on the abnormal morphologic states of the damaged cells. The types of myonecrosis observed correlated with the types of components present in the venom injected. Venoms containing direct acting toxins such as myotoxin a or phospholipase A 2 induced specific types of myonecrosis. Also, venoms containing hemorrhagic toxins produced a type of myonecrosis similar to that induced by pure hemorrhagic toxins. The pathogenesis of each type of myonecrosis could be divided into the same four phases based on the pathologic states of the affected cells and the time after injection. During the ‘early phase’ (0.25 – 3 hr) affected muscle cells were in several different pathologic states reflecting the types of components present in the venom injected. During the ‘intermediate phase’ (6 – 24 hr) the pathologic state of the damaged cells had changed and depending on the venom new states might be present. By the ‘late phase’ (48 – 96 hr) all damaged cells have reached a common pathologic state of necrosis. The ‘final phase’ (1 – 4 weeks) is characterized by regeneration (partial or complete) of muscle cells. Although the number of different types of myonecrosis depended on the type of venom injected, i.e. Naja naja naja venom produced only two different types whereas Crotalus atrox venom produced at least four different types, cells of each type of myonecrosis progressed through the same four phases. In studies of the myotoxicity of snake venoms it is important to examine tissues taken during the early and intermediate phases to obtain accurate and useful information on the types of myonecrosis caused by the venom.

Hemorrhagic Toxins from Snake Venoms

AbstractOne of the more dramatic consequences of envenomation by crotalid and viperid snakes is the occurrence of hemorrage. In cases where the envenomation is less severe, the hemorrhagic is generally observed to be localized at the site of the bite. However, hemorrhage can be found disseminated through a substantial area of the involved extremity. In cases where the envenomation is severe, bleeding in organs such as heart, lungs, kidneys and brain may also occur. From the biochemical investigations on these toxins over the past 30 years, the nature of the venom toxins and their mechanism of activity are now becoming clear. Virtually all of the hemorrhagic toxins isolated and characterized thus far have been determined to be metalloproteinases. In this review we discuss the history of the isolation and characterization of these toxins in an attempt to clarify some of the confusion surrounding these toxins and their biochemical activities. We also survey the data available on the natural and synthetic inh...