Characterization of a protein C activator from Agkistrodon contortrix contortrix venom

The protease from Southern Copperhead venom that activates protein C was purified to homogeneity by sulfopropyl (SP)-Sephadex C-50 ion-exchange chromatography, Sephadex G-150 gel filtration, and Mono-S fast protein liquid chromatography. The purified enzyme is a glycoprotein containing 16% carbohydrate, and migrated as a single band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent molecular mass of 40,000 kDa. The enzyme is composed of a single polypeptide chain possessing an NH2-terminal sequence of Val-Ile-Gly-Gly-Asp-Glu-Cys-Asn-Ile-Asn-Glu-His. The purified venom protein C activator hydrolyzed several tripeptide p-nitroanilides. The amidolytic and proteolytic activities of the enzyme were readily inhibited by phenylmethanesulfonyl fluoride, p-amidinophenylmethanesulfonyl fluoride, chloromethyl ketones, and human antithrombin III. Covalent binding of diisopropyl fluorophosphate to the enzyme was confirmed using a tritium-labeled preparation of the inhibitor. The venom protease readily activated human and bovine protein C at 1:1000 enzyme:substrate weight ratio. The protease also cleaved human prothrombin, factor X, factor IX, factor VII, and fibrinogen. Prothrombin coagulant activity decreased upon incubation with the venom protease, and the rate of this reaction was reduced in the presence of calcium. Factor X and factor IX coagulant activity increased upon incubation with the venom protease in the presence of calcium, and decreased in the absence of calcium. Human factor VII clotting activity decreased slightly upon incubation with the venom protease. Although the venom protease did not clot human fibrinogen, it nonetheless cleaved the A alpha chain of fibrinogen, and this cleavage appeared to be associated with a measurable increase in the clottability of the protease-treated fibrinogen by thrombin. These data demonstrate that the protein C activator from Southern Copperhead venom is a typical serine protease with a relatively broad specificity.     

Characterization of a fibrinogenase from northern copperhead (Agkistrodon contortrix mokasen) venom

One of the fractions obtained by the carboxymethylcellulose ion-exchange chromatography of northern copperhead (Agkistrodon contortrix mokasen) venom prevented the thrombin-induced clotting of fibrinogen by proteolytically degrading the fibrinogen. The active component has been further purified to apparent electrophoretic homogeneity by molecular sieve chromatography. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis indicated a molecular weight of 22 900 +/- 600 for the purified enzyme. In addition to its fibrinogenase activity, it catalyzed the hydrolysis of hide power azure and had an intraperitoneal LD50 value in mice of less than 5.1 microgram/g body weight. The enzyme rapidly destroyed fibrinogen's ability to form clots. Electrophoresis of fibrinogen which had been incubated only a few minutes with the fibrinogenase revealed the rapid disappearance of the alpha-chain and the appearance of lower molecular weight fragments. The neutral pH optimum and ethylenediamine-tetraacetic acid (EDTA) and dithiothreitol sensitivity indicated that this enzyme belonged to the class metalloproteinases. Atomic absorption studies have revealed one zinc atom per molecule of protein. The apoenzyme's activity was restored by incubation with ZnCl2.     

Characterization of a protein C activator from the venom of Agkistrodon contortrix contortrix

An enzyme capable of activating protein C has been purified 60-fold from the venom of the Southern copperhead snake (Agkistrodon contortrix) by ion-exchange and gel filtration chromatography. The purified enzyme consists of a single polypeptide with an apparent molecular weight of 37,000. The isoelectric point of the protein C activator was determined to be 6.3 when measured by chromatofocusing. The enzyme was inhibited by p-nitrophenyl p-guanidinobenzoate, phenylmethanesulfonyl fluoride, and D-Phe-Pro-Arg-CH2Cl but was not affected by cysteine-directed reagents or by metal chelators. These results suggest that the enzyme is a serine protease. Protein C activator was capable of hydrolyzing the thrombin substrate tosyl-Gly-Pro-Arg-p-nitroanilide (TGPRpNA), and steady-state kinetic studies determined that the Km for amidolysis of this substrate was 1.1 mM while the Vmax was 66 s-1. The activator demonstrated considerable substrate specificity since the amidolysis of D-Phe-Pip-Arg-pNA, D-Ile-Pro-Arg-pNA, Bz-Ile-Glu-Gly-Arg-pNA, D-Val-Leu-Arg-pNA, and pyrGlu-Pro-Arg-pNA was less than 10% of that of TGPRpNA when measured under identical conditions using 1.0 mM substrate concentrations. The enzyme appears to be thrombin-like in its preference for arginyl as compared to lysyl chloromethyl ketones as well as by its inhibition by benzamidine and p-aminobenzamidine. However, the substrate specificity of the activator is distinguished from alpha-thrombin in that it does not clot fibrinogen and does not react with antithrombin III or hirudin.(ABSTRACT TRUNCATED AT 250 WORDS)     

The role of extracellular ions in the pathogenesis of myonecrosis induced by a myotoxin isolated from Broad-Banded Copperhead (Agkistrodon contortrix laticinctus) venom

Pathalogical changes in murine skeletal muscle cells induced by ACL (Agkistrodon contortix laticinctus, Broad-Banded Copperhead) myotoxin in vivo were compared to pathological changes induced by an influx of Ca²⁺ and other ions into cut skeletal muscle cells in vitro in the absence of myotoxin. In vivo, ACL myotoxin induced a rapid myonecrosis characterized by densely clumped myofibrils in the cytoplasm. In vitro, this pathological change was not produced by incubating skeletal muscle cells in Ca²⁺ concentrations as high as 200 mM, whereas skeletal muscle cells incubated in concentrations of 150 mM and 300 mM NaCl contained densely clumped myofibrils similar in morphology to muscle cells damaged by ACL myotoxin in vivo. Treatments of 300 mM KCl did not produce densely clumped myofibrils in muscle cells. These results suggest that an influx of Na⁺, possibly through disrupted regions of sarcolemma, be may primarily responsible for the pathological changes, including clumped myofibrils, induced by ACL myotoxin in vivo. However, an influx of extracellular Ca²⁺ which has been proposed to produce densely clumped myofibrils in muscle cells damaged by other snake venom myotoxins, may not be responsible for this pathological change since extracellular Ca²⁺ concentrations much higher than physiological levels did not produce this change in skeletal muscle cells in vitro.     

Primary structure of a protein C activator from Agkistrodon contortrix contortrix venom

The amino acid sequence of a protease, protein C activator, from Agkistrodon contortrix contortrix venom was determined. Peptide fragments obtained by chemical or enzymatic cleavage of the S-carboxymethylated protein were purified by gel filtration and reverse-phase high-performance liquid chromatography. The present study demonstrates that protein C activator from A. contortrix contortrix venom is a trypsin-type serine protease that is composed of 231 residues with a molecular weight of 25,095 for the polypeptide portion of the molecule. By analogy to the mammalian serine proteases, the catalytic triad in venom protein C activator consists of His-40, Asp-85, and Ser-177. The protein also contains three N-linked glycosylation sites at Asn-21, Asn-78, and Asn-129. The amino acid sequence of protein C activator exhibits a high degree of sequence identity with other snake venom proteases: 73% with batroxobin, 68% with flavoxobin, and 55% with Russell's viper venom factor V activator.     

HPLC-based two-step purification of fibrinolytic enzymes from the venom of Agkistrodon contortrix contortrix and Agkistrodon piscivorus conanti

In investigations aimed at characterizing snake venom blood clot-dissolving enzymes, we have developed a rapid two-step high-performance chromatography method for the isolation of these fibrinolytic enzymes from the venoms of Agkistrodon contortrix contortrix and Agkistrodon piscivorus conanti. The first step consisted of hydrophobic interaction chromatography on a propyl-aspartamide column. Fractions containing the fibrinolytic activity were then concentrated and applied to a hydroxylapatite column. The resulting preparation, assessed for purity by reverse-phase chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was homogeneous. The molecular weight of both venom fibrinolytic enzymes was approximately 23,000 and amino acid analysis, immunological cross-reaction, cyanogen bromide, and tryptic digestion indicate a significant degree of structural similarity. However, the general proteolytic activity of the A. p. conanti venom enzyme was significantly lower than the corresponding activity of the A. c. contortrix venom, whereas their fibrinolytic activities were quite similar.     

Purification and characterization of a fibrinolytic enzyme from venom of the southern copperhead snake (Agkistrodon contortrix contortrix)

A fibrinolytic enzyme present in Agkistrodon contortrix contortrix (southern copperhead) venom has been purified by combination of CM-cellulose chromatography, molecular sieve chromatography on Sephadex G-100, p-aminobenzamidine-agarose affinity chromatography, and DEAE-cellulose chromatography. The enzyme, fibrolase, has a molecular weight of 23,000-24,000 and an isoelectric point of pH 6.8. It is composed of approximately 200 amino acids, possesses a blocked NH2-terminus and contains little or no carbohydrate. The enzyme shows no activity against a series of chromogenic p-nitroanilide substrates and is not inhibited by diisopropylfluorophosphate, soybean trypsin inhibitor, Trasylol, or p-chloromercuribenzoate. However, the enzyme is a metalloproteinase since it is inhibited by EDTA, o-phenanthroline and tetraethylenepentamine (a specific zinc chelator). Metal analysis revealed 1 mol of zinc/mol of protein. Study of cleavage site preference of the fibrinolytic enzyme using the oxidized B chain of insulin revealed that specificity is similar to other snake venom metalloproteinases with cleavage primarily directed to an X-Leu bond. Interestingly, unlike some other venom fibrinolytic metalloproteinases, fibrolase exhibits little if any hemorrhagic activity. The enzyme exhibits direct fibrinolytic activity and does not activate plasminogen. In vitro studies revealed that fibrolase dissolves clots made either from purified fibrinogen or from whole blood.     

Purification of a protein C activator from the venom of the southern copperhead snake (Agkistrodon contortrix contortrix)

A protease has been purified by ion-exchange chromatography from the venom of Agkistrodon contortrix contortrix (Southern copperhead snake) that can activate the vitamin K dependent protein, protein C. The apparent molecular weight of this protease, determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was 20,000 under nonreducing conditions. Incubation of this protease with plasma resulted in a prolongation of the clotting time and a time-dependent increase in amidolytic activity. Incubation of the protease with purified protein C resulted in an increase in both amidolytic and anticoagulant activity. The protease had no inhibitory effect on thrombin, factor V, fibrinogen, or factor X. It had slight clotting activity toward fibrinogen. The apparent Km of the protease for protein C was 0.28 microM. Calcium ions were observed to inhibit protein C activation with an apparent Ki of 0.2 mM. Ethylenediaminetetraacetic acid, diisopropyl fluorophosphate, and soybean trypsin inhibitor were observed to inhibit the venom protease. These results suggest that the venom of the Southern copperhead snake contains a protease that is a specific activator of protein C.     

Crystallization and preliminary X-ray crystallographic studies of Protac, a commercial protein C activator isolated from Agkistrodon contortrix contortrix venom

The protein C pathway plays an important role in the control and regulation of the blood coagulation cascade and prevents the propagation of the clotting process on the endothelium surface. In physiological systems, protein C activation is catalyzed by thrombin, which requires thrombomodulin as a cofactor. The protein C activator from Agkistrodon contortrix contortrix acts directly on the zymogen of protein C converting it into the active form, independently of thrombomodulin. Suitable crystals of the protein C activator from Agkistrodon contortrix contortrix were obtained from a solution containing 2 M ammonium sulfate as the precipitant and these crystals diffracted to 1.95 A resolution at a synchrotron beamline. The crystalline array belongs to the monoclinic space group C2 with unit cell dimensions a=80.4, b=63.3 and c=48.2 A, alpha=gamma=90.0 degrees and beta=90.8 degrees.     

Amino acid sequence of fibrolase, a direct-acting fibrinolytic enzyme from Agkistrodon contortrix contortrix venom.

The complete amino acid sequence of fibrolase, a fibrinolytic enzyme from southern copperhead (Agkistrodon contortrix contortrix) venom, has been determined. This is the first report of the sequence of a direct-acting, nonhemorrhagic fibrinolytic enzyme found in snake venom. The majority of the sequence was established by automated Edman degradation of overlapping peptides generated by a variety of selective cleavage procedures. The amino-terminus is blocked by a cyclized glutamine (pyroglutamic acid) residue, and the sequence of this region of the molecule was determined by mass spectrometry. Fibrolase is composed of 203 residues in a single polypeptide chain with a molecular weight of 22,891, as determined by the sequence. Its sequence is homologous to the sequence of the hemorrhagic toxin Ht-d of Crotalus atrox venom and with the sequences of two metalloproteinases from Trimeresurus flavoviridis venom. Microheterogeneity in the sequence was found at both the amino-terminus and at residues 189 and 192. All six cysteine residues in fibrolase are involved in disulfide bonds. A disulfide bond between cysteine-118 and cysteine-198 has been established and bonds between cysteines-158/165 and between cysteines-160/192 are inferred from the homology to Ht-d. Secondary structure prediction reveals a very low percentage of alpha-helix (4%), but much greater beta-structure (39.5%). Analysis of the sequence reveals the absence of asparagine-linked glycosylation sites defined by the consensus sequence: asparagine-X-serine/threonine.     

Sporocysts isolated from the southern copperhead (Agkistrodon contortrix contortrix) produce Sarcocystis montanaensis-like sarcocysts in prairie voles (Microtus ochrogaster)

Sporulated oocysts and free sporocysts of a Sarcocystis sp. were isolated from the feces of a southern copperhead (Agkistrodon contortrix contortrix) collected in Arkansas (USA). Twenty sporocysts measured 11.2 by 8.5 microns, lacked a Stieda body, and had four sporozoites and a granular sporocyst residuum. Sarcocysts similar to those of Sarcocystis montanaensis were present in the tongues of prairie voles (Microtus ochrogaster) inoculated orally with 800 sporocysts 128 days previously. Sarcocysts were thin-walled, divided into compartments by septa, and had electron dense projections (0.14 microns) on the primary cyst wall. Infection was not pathogenic for prairie voles under the conditions of this study. No infections were observed in ICR strain laboratory mice (Mus musculus) or white-footed mice (Peromyscus leucopus) following oral inoculation of 800 sporocysts.     

Practical application of the protein C activator Protac from Agkistrodon contortrix venom

The protein C activator Protac from A. contortrix venom is being investigated as a potential antithrombotic agent and as a tool for the preparation of activated protein C. Its established major application is the zymogen activation in functional protein C determinations based on either a clotting assay or a chromogenic substrate technique. The sensitivity of the activated partial thromboplastin time as an indicator reaction for Protac activated protein C depends on the contact activator component of the reagent. Protein C dose-response increased in the following order: kaolin greater than ellagic acid greater than sulfatide. This phenomenon is due to a competition of molecular affinities between Protac, plasma components and the different activating surfaces.     

Assessment of a complex mixture of interacting proteins in the coagulant active venom from Agkistrodon contortrix contortrix

The crude venom of Agkistrodon contortrix contortrix was characterized by means of 2D-PAGE (using various separation principles in the respective directions) and high performance gel filtration chromatography. It was found that the venom presents a rich and remarkably stable mixture of proteins, mostly glycoproteins, which may interact each other. High stability of the venom in spite of the presence of many proteolytic enzymes, must most likely be attributed to the sugar moieties of venom proteins. Carbohydrate composition also causes considerable heterogeneity in charge and the presence of wide range of charge isomers. The intricate complexity of the venom makes it a real difficult-to-separate mixture.     

Characterization of clotting factors from Agkistrodon acutus venom

1. Four clotting factors, Cf-1(C), Cf-2(C), Cf-1(T) and Cf-2(T) were isolated from Agkistrodon acutus (collected on mainland China and Taiwan) venom by Komori et al. (1987). It was reported that all factors possessed coagulant activity in the conversion of fibrinogen to fibrin, although they showed different chemical properties and antigenicities. 2. Their role in the clot formation system was clarified and compared with that of thrombin. Clotting factors from A. acutus venom released only fibrinopeptide A from the A alpha chain of fibrinogen, while thrombin released fibrinopeptide A and B from the A alpha and B beta chains. 3. Cf-1(C) and Cf-2(T), like thrombin, rapidly activated factor XIII in the presence of calcium ions, whereas Cf-2(C) and Cf-1(T) had little effect on factor XIII. These effects are shown by Cf-1(C) and Cf-2(T) forming a clot that remained insoluble in 8 M urea or 0.44 M monochloroacetic acid, whereas Cf-2(C) and Cf-1(T) formed a soluble clot in these agents.     

Evaluation of individual variability in the composition of Agkistrodon contortrix contortrix venom by means of HPLC and two-dimensional PAGE

Qualitative and quantitative differences of coagulant enzymes in venom samples from individuals of the Agkistrodon c.c. were studied by means of 2D-PAGE and high performance anion-exchange chromatography. A great diversity was found among individual venoms. The two most similar venoms had an identical composition in only about 90%, the least similar ones in about 45% spots. All venoms studied contained more than two fractions with fibrinolytic activity. In three out of the nine analysed venoms two different thrombic proteases were present, one venom contained only one of these enzymes, whereas in five venoms no thrombic activity was detectable.     

Characterization of Ac3-proteinase from the venom of Agkistrodon acutus (hundred-pace snake)

Ac3-Proteinase from the venom of Agkistrodon acutus was isolated in a homogeneous form by a previously published method. Ac3-Proteinase possessed lethal, hemorrhagic, caseinolytic, azocaseinolytic, dimethylcaseinolytic and hide powder azure hydrolytic activities. These activities were inhibited when Ac3-Proteinase was incubated with the metal chelators ethylenediaminetetraacetic acid (EDTA), ethyleneglycol-bis-(beta-aminoethyl ether)-N,N'-tetraacetic acid (EGTA), tetraethylenepentamine (TEP), 1,10-phenanthroline, phosphoramidon or beta-mercaptoethanol. The toxin also hydrolyzed the oxidized A and B chains of both insulin and fibrinogen. The cleavage sites in the oxidized B chain of insulin were identified as His(10)-Leu(11), Ala(14)-Leu(15), Tyr(16)-Leu(17) and Phe(24)-Phe(25). The A alpha chain of fibrinogen was digested first followed by hydrolysis of the B beta chain. Toxicological and biochemical properties of Ac3-Proteinase were investigated further and are reported in this paper.     

Characterization of Ac1-Proteinase from the venom of Agkistrodon acutus (Hundred-pace snake) from Taiwan

1. Ac1-Proteinase from the venom of Agkistrodon acutus was isolated in a homogeneous form by a previously published method. 2. Ac1-Proteinase possessed lethal, hemorrhagic, caseinolytic, azocaseinolytic, azoalbumin hydrolytic and hide powder azure hydrolytic activities. 3. The toxin also hydrolyzed the oxidized B chain of insulin and fibrinogen. The cleavage sites in the oxidized B chain of insulin were identified as Ala(14)-Leu(15) and Tyr(16)-Leu(17). The A alpha chain of fibrinogen was digested. 4. Biological properties of Ac1-Proteinase were investigated further and are reported in this paper.     

Characterization of platelets aggregation inhibitor from Agkistrodon blomhoffii ussuriensis snake venom

Platelet aggregation inhibitor--"Blomus-B" from Agkistrodon blomhoffii ussuriensis venom has been isolated by affinity and ion-exchange chromatography. The purified inhibitor is a novel non-enzymatic single-chain protein with molecular weigth of 13 kDa. "Blomus-B" causes a change of platelets shape and takes effect on ADP- and adrenalin-induced platelet aggregation.