The factor V-activating enzyme (RVV-V) from Russell's viper venom. Identification of isoproteins RVV-V alpha, -V beta, and -V gamma and their complete amino acid sequences

The complete amino acid sequences of two isoproteins of the factor V-activating enzyme (RVV-V) isolated from Vipera russelli (Russell's viper) venom were determined by sequencing S-pyridylethylated derivatives of the proteins and their peptide fragments generated by either chemical (cyanogen bromide and 2-(2-nitrophenylsulfenyl)-3-methyl-3-bromoindolenine) or enzymatic (trypsin, alpha-chymotrypsin, and lysyl endopeptidase) cleavages. Both enzymes, designated RVV-V alpha and RVV-V gamma, consist of 236 amino acid residues and have a N-linked oligosaccharide chain at Asn229. The six amino acid substitutions between RVV-V alpha and -V gamma are: Thr22(alpha)-Ala22(gamma), Gly29(alpha)-Ala29(gamma), Gln191(alpha)-Glu191(gamma), Ile192(alpha)-Met192(gamma), Gln193(alpha)-His193(gamma), and Asn224(alpha)-Ser224(gamma). The molecular weights were calculated as 26,182 for RVV-V alpha and 26,167 for RVV-V gamma. The sequences of the RVV-V isoproteins exhibited 62% identity with that of batroxobin, a thrombin-like enzyme present in Bothrops atrox venom, and 33% identity with that of human thrombin B chain. The most interesting difference between the structures of RVV-V and other trypsin-type serine proteases is that the conservative Ser214-Trp215-Gly216 sequence (chymotrypsinogen numbering), considered as the site of antiparallel beta-sheet formation between the protein substrate and most serine proteases, has been replaced by the corresponding sequence Ala-Gly-Gly.     

Purification and characterization of a coagulant protein from the venom of Russell's viper.

The coagulant protein from the venom of Russell's viper was purified by means of successive chromatography on Sephadex G-50, DEAE-cellulose and Sephadex G-200. The purified coagulant protein was homogeneous by polyacrylamide gel electrophoresis and ultracentrifugation. The molecular weight was estimated to be about 100 000 by ultracentrifuge analysis and 130 000 by gel filtration. The coagulant protein contains 11.1% carbohydrate which includes 5.1% hexose (galactose: mannose = 1:1), 5% hexosamine (glucosamine), and 1% neuraminic acid (N-acetylneuraminic acid and N-glycolyneuraminic acid). The isoelectric point is pH 6.3. The results of both sodium dodecyl sulfate electrophoresis and gel filtration in 6 M guanidium chloride suggest that it consists of four polypeptide chains. The coagulant protein functions as an enzyme in activating blood coagulation factor X in the presence of Ca2+. N-a-p-Toluenesulfonyl-L-arginine methyl ester hydrolyzing activity in the preparation definitely decreased during purification and it suggests that the clotting activity is not associated with the esterase activity. The clotting activity is inhibited by diisopropyl phosphorofluoridate and by phenylmethylsulfonyl fluoride, suggesting that the coagulant protein is a serine protease. The optimum pH is between pH 7.0 and pH 8.0. At neutral pH the coagulant protein is stable below 50 degrees C, but is rapidly inactivated above 55 degrees C.     

Observations on the Factor V activator present in Russell's viper venom and its action on Factor V

The Factor V activator (RVV-V) of Russell's viper venom can cause a 20–26-fold increase in the Factor V activity of bovine and human plasma whereas only a 2–4-fold increase was obtained under similar conditions with bovine serum Factor V activity. On incubation with RVV-V, bovine plasma Factor V underwent a significant change in behavior on Sephadex chromatography, with an apparent change in molecular weight from a value greater than 400 000 to one near 205 000. Bovine serum Factor V, as isolated, had a molecular weight (size) of approximately 230 000 which did not change on treatment with RVV-V.     

beta-RTX. A receptor-active protein from Russell's viper (Vipera russelli russelli) venom

A single chain polypeptide, termed beta-RTX, with an apparent Mr = 9600 has been isolated from the venom of Vipera russelli russelli. It was purified by cation exchange chromatography, followed by preparative isoelectric focusing and chromatofocusing. Purity was confirmed by gel filtration, high performance liquid chromatography, gel electrophoresis, and analytical ultracentrifugation. Amino acid analysis revealed the presence of eight half-cystines, one being located at the NH2 terminus, which are linked to form four intramolecular disulfide bridges. Chromatofocusing revealed some microheterogeneity yielding three isoforms with pI values of 9.3, 9.37, and 9.48, respectively. In its native configuration, beta-RTX was not susceptible to tryptic degradation but was readily digested after reduction and alkylation. beta-RTX possesses weak phospholipase A2 activity and competes with the binding of monoamines and opiate ligands to their respective receptors. No binding to histamine, gamma-aminobutyric acid, benzodiazepine, or muscarinic receptors was observed. In vivo, whereas 100 micrograms/kg intravenous beta-RTX seemed to be without apparent effects in the rat, 10 ng/kg beta-RTX injected intracerebroventricularly caused marked sedation, with full recovery within 3 h.     

Vipoxin. A protein from Russell's viper venom with high affinity for biogenic amine receptors

We have purified a small, basic protein with high affinity and selectivity for biogenic amine receptors to apparent homogeneity from the venom of Russell's viper (Vipera russelli). This protein, which we designate "vipoxin," has Mr = 13,000, and appears to exist in solution as a single polypeptide chain. It may contain 2 atypical amino acids. Vipoxin inhibits in a dose-dependent manner the binding of 3H-ligands to biogenic amine receptors, with apparent Ki values of 3 nM at alpha 1-adrenergic receptors, 5 nM at alpha 2-adrenergic receptors, 15 nM at dopamine receptors, and 32 nM at serotonin receptors. At concentrations up to 1 microM, vipoxin is inactive at beta-adrenergic, histamine, nicotinic cholinergic, muscarinic cholinergic, adenosine, gamma-aminobutyric acid, benzodiazepine, or opiate receptor binding sites. The effect of vipoxin is essentially irreversible over 20 h at alpha 1- and alpha 2-adrenergic receptors and serotonin receptors and is only slightly reversible at dopamine receptors. Norepinephrine protects alpha-adrenergic receptors from inhibition by vipoxin, while dopamine does not. Vipoxin has no protease activity but does have phospholipase A2 activity, which cannot account for its action on receptors, since receptor binding is assayed in the presence of 1 mM CoSO4 which completely and selectively inhibits the phospholipase activity. Other phospholipases A2 in the same venom lack vipoxin's action on receptors. In physiologic experiments, vipoxin behaves as an agonist at alpha 2-adrenergic receptors in the rat vas deferens and is over an order of magnitude more potent than norepinephrine itself. At alpha 1-adrenergic receptors, it is neither a simple agonist nor an antagonist, but selectively potentiates norepinephrine. Vipoxin may be a useful tool for biogenic amine receptor characterization.     

New insights into the functions and N-glycan structures of factor X activator from Russell's viper venom

The coagulation factor X activator from Russell's viper venom (RVV-X) is a heterotrimeric glycoprotein. In this study, its three subunits were cloned and sequenced from the venom gland cDNAs of Daboia siamensis. The deduced heavy chain sequence contained a C-terminal extension with four additional residues to that published previously. Both light chains showed 77-81% identity to those of a homologous factor X activator from Vipera lebetina venom. Far-western analyses revealed that RVV-X could strongly bind protein S, in addition to factors X and IX. This might inactivate protein S and potentiate the disseminated intravascular coagulation syndrome elicited by Russell's viper envenomation. The N-glycans released from each subunit were profiled and sequenced by MALDI-MS and MS/MS analyses of the permethyl derivatives. All the glycans, one on each light chain and four on the heavy chain, showed a heterogeneous pattern, with a combination of variable terminal fucosylation and sialylation on multiantennary complex-type sugars. Amongst the notable features were the presence of terminal Lewis and sialyl-Lewis epitopes, as confirmed by western blotting analyses. As these glyco-epitopes have specific receptors in the vascular system, they possibly contribute to the rapid homing of RVV-X to the vascular system, as supported by the observation that slower and fewer fibrinogen degradation products are released by desialylated RVV-X than by native RVV-X.     

Functional characterization of a slow and tight-binding inhibitor of plasmin isolated from Russell's viper venom

Background

Snake venoms are rich in Kunitz-type protease inhibitors that may have therapeutic applications. However, apart from trypsin or chymotrypsin inhibition, the functions of most of these inhibitors have not been elucidated. A detailed functional characterization of these inhibitors may lead to valuable drug candidates.

Methods

A Kunitz-type protease inhibitor, named DrKIn-II, was tested for its ability to inhibit plasmin using various approaches such as far western blotting, kinetic analyses, fibrin plate assay and euglobulin clot lysis assay. In addition, the antifibrinolytic activity of DrKIn-II was demonstrated in vivo.

Results

DrKIn-II potently decreased the amidolytic activity of plasmin in a dose-dependent manner, with a global inhibition constant of 0.2 nM. Inhibition kinetics demonstrated that the initial binding of DrKIn-II causes the enzyme to isomerize, leading to the formation of a much tighter enzyme-inhibitor complex. DrKIn-II also demonstrated antifibrinolytic activity in fibrin plate assay and significantly prolonged the lysis of the euglobulin clot. Screening of DrKIn-II against a panel of serine proteases indicated that plasmin is the preferential target of DrKIn-II. Furthermore, DrKIn-II treatment prevented the increase of FDP in coagulation-stimulated mice and significantly reduced the bleeding time in a murine tail bleeding model.

Conclusion

DrKIn-II is a potent, slow and tight-binding plasmin inhibitor that demonstrates antifibrinolytic activity both in vitro and in vivo.

General significance

This is the first in-depth functional characterization of a plasmin inhibitor from a viperid snake. The potent antifibrinolytic activity of DrKIn-II makes it a potential candidate for the development of novel antifibrinolytic agents.     

A heat stable paraoxonase (O,O-diethyl O-p-nitrophenyl phosphate O-p-nitrophenyl hydrolase) from Russell's viper venom.

Fractionation of Russell's viper venom revealed separate phosphohydrolase activities directed against p-nitrophenyl phosphate, bis(p-nitrophenyl) phosphate, p-nitrophenylthymidylic acid, and O,O-diethyl p-nitrophenyl phosphate (paraoxon). On gel fractionation, the first two activities are eluted ahead of the latter. They could be resolved further by phosphocellulose cation exchange chromatography. The hydrolytic activities directed against p-nitrophenylthymidylic acid hydrolyzing component is heat labile, while the paraoxon hydrolyzing component manifests an unusually high degree of heat stability. Gel filtration yields 9600 for the molecular weight of the "paraoxonase". This enzyme, as all known enzymes of this type, requires the presence of a divalent cation. Maximum activity is obtained in the presence of Ca2+. In the presence of Sr2+ the reaction rate is 50% of that of Ca2+; other divalent cations show lower activities. The presence of the enzyme is species specific. Of four species tested, only Russell's viper venom showed significant paraoxonase activity. Enzyme activity is intact following incubation with iodoacetate of p-chloromercuribenzoate. Activity is partially preserved even in the presence of 8 M urea.     

Activation of decarboxyfactor X by a protein from Russell's viper venom. Purification and partial characterization of activated decarboxyfactor X.

1. Incubation of decarboxyfactor X with the factor X-activating enzyme from Russell's Viper venom revealed the generation of amidase activity towards Bz-Ile-Glu-Gly-Arg-pNA, but not of activity in blood coagulation. 2. The rate of activation of both factor X and decarboxyfactor X depends on the ability of the zymogens to bind Ca2+. The relationship between Ca2+ concentration and velocity of the activation reaction is sigmoid in the case of factor X, but hyperbolic with decarboxyfactor X. 3. Activated decarboxyfactor X was purified by powder column electrophoresis. 4. Identical changes of primary structure accompanied the activation of factor X and decarboxyfactor X. Identical molecular weight and common antigenic determinants were found in factor Xa and decarboxyfactor Xa. The amino acid composition was identical except for 12 glutamic acid residues in decarboxyfactor Xa and gamma-carboxyglutamic acid residues in factor Xa. 5. Unlike factor X, activated factor X has a very low electrophoretic mobility in the presence of Ca2+ at pH 8.6. This is probably due to self association of factor Xa under the influence of Ca2+. The electrophoretic mobility of activated decarboxyfactor X is only slightly decreased compared to decarboxyfactor X in the presence of Ca2+.     

Purification and characterization of a low molecular weight multifunctional cytotoxic phospholipase A2 from Russell's viper venom

A basic toxin from Russell's viper venom of 7.2 kDa (RVV-7) has been purified to homogeneity after partial unfolding by 4 M urea followed by filtration through Centricon-30 membrane. Its N-terminal sequence showed strong homology with snake venom cytotoxins. Cytotoxic activity of RVV-7 has been demonstrated with B16F10 melanoma cells. PLA2 activity was observed in cytotoxin (CX3) from Naja kauthia bearing sequence homology with RVV-7. Phospholipase A2 and trypsin inhibitory activities were also observed with RVV-7. Chemical modification and inhibition studies suggested independent functional sites for these activities. A qualitative assessment of tumor growth inhibition by RVV-7 has been made.     

Separation of a bradykinin-releasing enzyme from the proteolytic complex of Levantine viper venom

Two serine hydrolases have been separated from the proteolytic complex of the venom of Levantine viper, Vipera lebetina turanica. The enzyme with mol. weight of 50,000 +/- 5,000, pH-optimum of 8.5 and isoelectric point in the range of 5.6--6.6 had proteolytic activity against casein and hydrolyzed benzoyl-arginine p-nitroanilide. The other enzyme with mol. weight of 37,000 +/- 2,000, pH optimum of 9 and isoelectric point in the range of 4.1--4.5 had no effect on benzoylarginine p-nitroanilide, casein or hemoglobin, but possessed a bradykinin-releasing activity. Both enzymes were stereoselective against L-arginine, hydrolyzing tosyl-L-arginine methyl ester without having any effect on D-arginine ester. The interaction of the enzymes with a number of N(alpha)-arylsulfonylarginine methyl esters has been studied. The influence of the substitute X in the arylsulfonyl part of the substrates upon their hydrolysis by the bradykinin-releasing enzyme has been described by the Hammett equation of rho omicron = 1.14 +/- 0.33 (r = 0.974).