A platelet function inhibitor purified from Vipera russelli siamensis (Smith) snake venom

Y.-S. Li, K.-F. Liu, Q.-C. Wang, Y.-L. Rpan and G.-C. Tu. A platelet function inhibitor purified from Vipera russelli siamensis (Smith) snake venom. Toxicon23, 895–903, 1985. — By means of CM-Sephadex C-50 column chromatography and gel filtration on Sephadex G — 75, a potent platelet function inhibitor was purified from Vipera russelli siamensis venom. It appeared as a single protein band on polyacrylamide gel electrophoresis in the presence or absence of SDS, and consists of 123 amino acid residues. Its NH₃-terminal residue is serine. It showed the following characteristics: molecular weight, 13,800; isoelectric point, 10.4; ld(In50), 0.5 ± 0.12 mg/kg (i.v.). The platelet inhibitor exhibited phospholipase A₂ activity with a specific activity of 35 μmoles/min/mg. From 2 g of the venom, 70 mg of the purified inhibitor was obtained. Inhibition of human platelet aggregation induced by ADP or adrenaline was dose-dependent, with id(In50) of 1.14μg/ml or 0.37 μg/ml, respectively. The platelet aggregation induced by thrombin or collagen was also inhibited and the inhibitory activity on platelet aggregation was heat stable (at 100°C, 20 min) in an acidic medium (pH 5.8), while its phospholipase A₂ activity was relatively heat labile under the same condition. The release of ³H-serotonin in platelets stimulated by ADP was also inhibited and this was positively correlated with inhibition of platelet aegregation induced by ADP (r = 0.998, P < 0.002).     

Relaxant effect of phospholipase A2 from Vipera russelli snake venom on rat aorta

The effect of the acidic phospholipase A2 (PLA2) from Vipera russelli venom on the rat aortic ring was studied and compared with that of acetylcholine (ACh). PLA2 induced relaxation of the aortic ring precontracted with noradrenaline (NA) in a dose-dependent manner. Removal of the endothelium did not reduce the relaxant effect of PLA2. Replacement of Ca2+ by Sr2+ in the medium to inhibit the PLA2 enzyme activity reduced the relaxant effect. Atropine, a muscarinic receptor antagonist, did not affect the relaxant response. The cyclooxygenase inhibitor indomethacin, when equilibrated for 50 min, potentiated the relaxation. The lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA) partially reduced the relaxation. This relaxation was also partially reduced by the guanylate cyclase inhibitor methylene blue. In contrast, the relaxation elicited by ACh was abolished by de-endothelialization, atropine, NDGA or methylene blue. 6-keto-PGF1 alpha (degradation product of prostacyclin) and PGE2 produced by aortic rings were measured by radioimmunoassay. PLA2 (3 X 10(-6) g/ml) increased the output of 6-keto-PGF1 alpha about 10-fold. The production of PGE2 was also increased but to a lesser extent. ACh also increased the output of 6-keto-PGF1 alpha and PGE2. However, prostacyclin released by PLA2 and ACh appears not to contribute to the relaxant effect, since prostacyclin does not relax the rat aorta. It is concluded that the relaxation elicited by PLA2 in the rat aorta is endothelium-independent and partially mediated by lipoxygenase product(s) and cyclic GMP whereas the relaxation induced by ACh was endothelium-dependent, mediated by lipoxygenase product(s) and cyclic GMP, and blocked by atropine.     

Differences between the venoms of two sub-species of Russell's viper: Vipera russelli pulchella and Vipera russelli siamensis

Ion exchange chromatography was carried out using venoms obtained from two sub-species of Russell's viper; V. russelli siamensis from Burma and V. russelli pulchella from Sri Lanka. Differences were observed in the elution position of venom components having haemolytic and procoagulant activity but not those causing fibrinolysis. Only the V. russelli siamensis venom exhibited any platelet aggregating activity. The Indian (Haffkine) polyspecific and the Burmese (Burma Pharmaceutical Industries) monospecific antivenoms, when used in cross immunoelectrophoresis against the two venoms, revealed differences in the number and/or intensity of the precipitin bands present. An important functional consequence of this was that the Burmese antivenom did not neutralize the haemolytic activity of the V. russelli pulchella venom in vitro and would thus probably not be effective in treating this consequence of envenoming by Russell's viper in Sri Lanka. Differences in the composition and the clinical effects of the two venoms emphasizes the importance of using venom from the local snake for antivenom production if optimal clinical efficacy is to be achieved.     

Effect of Russell's viper (Vipera russelli siamensis) venom on renal hemodynamics in dogs

The effect on renal hemodynamics of Russell's viper (Vipera russelli siamensis) venom was studied in 8 mongrel dogs. The venom (0.10 mg/kg) was injected i.v. Measurements of general circulation and renal function were carried out over 48 hr. During the initial post-injection period, mean arterial blood pressure, pulse pressure and heart rate decreased. Total peripheral vascular resistance and renal vascular resistance showed a tendency to increase. There was no change in cardiac output. Thereafter the blood pressure and heart rate returned to the control level 2 hr after injection and remained stable throughout the experiment. The cardiac output remained unchanged, but the pulse pressure later increased. Renal blood flow, glomerular filtration rate, renal fraction and the rate of urine flow were decreased 24 hr after venom injection and rose to the control levels at 48 hr. Renal vascular resistance remained relatively increased, while peripheral resistance decreased, at 24 hr. Blood volume was unchanged throughout the 48 hr. Disseminated intravascular coagulation was not observed, although the clotting time was prolonged. Renal histological studies showed no remarkable changes; tubular necrosis was not seen. The renal hemodynamic changes may initially be due to catecholamine release and later to renin-angiotensin activation with renal vasoconstriction.     

Isolation and pharmacological properties of phospholipases A2 from Vipera russelli (Russell's viper) snake venom

By means of Sephadex G-75 column chromatography, Vipera russelli venom was separated into five fractions. The phospholipase A2 (PLA2) activity was concentrated in Frs. II and III. These two PLA2 fractions were rechromatographed on CM-Sephadex C-50. Several subfractions of Fr. II and Fr. III contained PLA2 activities. Frs. III-3, III-6 and III-10 showed single bands in SDS-polyacrylamide gel electrophoresis with molecular weights estimated to be 15,054, 15,167 and 15,029, and isoelectric points of 4.15, 8.80 and greater than 10, respectively. Fr. III-3 had the most potent neuromuscular blocking action on the chick biventer cervicis nerve-muscle preparation, causing a complete neuromuscular blockade at 3 micrograms/ml. The response of the muscle to ACh, tested after complete blockade, was not altered. Most PLA2 subfractions had hypotensive actions in rats at 0.1 mg/kg. In the guinea-pig lung, Frs. II-5, II-7, III-3, III-6 and III-10 increased the perfusion pressure, which may account for part of their hypotensive actions.     

Mechanism of action of the platelet aggregation inhibitor purified from Agkistrodon halys (mamushi) snake venom

The platelet aggregation inhibitor purified from Agkistrodon halys snake venom inhibited rabbit platelet aggregations induced by thrombin, sodium arachidonate, collagen or ionophore A-23187. The ic₅₀ was about 11 μg/ml in platelet aggregation regardless of which aggregation inducer was used. β-Mercaptoethanol abolished both the phospholipase A enzymatic and platelet aggregation inhibitory activities of this venom inhibitor. p-Bromophenacyl bromide-treated venom inhibitor lost almost completely its phosphilipase A enzymatic activity, but retained its platelet aggregation inhibitory effect. In the presence of EGTA, the venom inhibitor still showed the same inhibitory activity on thrombin-, sodium arachidonate-, collagen- or ionophore A23187-induced platelet aggregations triggered by successive addition of Ca²⁺. The activation of platelet phospholipase A and the serotonin release reaction triggered by Ca²⁺ influx were unaffected by this venom inhibitor. It also inhibited the clot retraction of platelet-rich plasma. It is concluded that the inhibitory effect of the venom inhibitor on platelet aggregation is independent of its phospholipase A enzymatic activity. Its mode of action is different from those of other known platelet inhibitory drugs. This venom inhibitor possibly acts on a common step subsequent to platelet shape change, leading to inhibition of platelet aggregation.     

Release of slow reacting substance from the guinea-pig lung by phospholipases A2 of Vipera russelli snake venom

Phospholipases A2 (PLA2) of Vipera russelli venom were isolated by column chromatography. The ability of PLA2 fractions to release slow reacting substance (SRS) was studied in the guinea-pig lung perfused with Krebs' solution. The relationship between the perfusion pressure change produced by PLA2 and SRS release was also studied. Two PLA2 fractions (II-5 and III-3; 3-100 micrograms), injected into the lung increased the perfusion pressure and released SRS. Pretreatment of the lung with indomethacin (10 micrograms) reduced the pressure response induced by the PLA2 fractions. The SRS released in the lung effluent by PLA2 was identified by bioassay as a mixture of thromboxane A2 (TXA2), prostacyclin (PGI2) and leukotrienes. TXA2 and PGI2 release was also quantitated by radioimmunoassay of the degradation products TXB2 and 6-keto-PGF1 alpha, respectively. There was a positive linear correlation between the pressure increases and the ratios of TXB2 to 6-keto-PGF1 alpha (r = 0.87). It appears that the relative amounts of TXA2 and PGI2 released determine the effects of PLA2 fractions on the guinea-pig lung. The release of arachidonic acid metabolites, prostaglandins and leukotrienes may account for part of the hypotensive action of PLA2.     

Purification and partial characterization of two proteolytic enzymes from the venom of Vipera russelli

Two proteolytic enzymes (proteinase I and proteinase II) were isolated from the venom of Vipera russelli by successive application of starch block electrophoresis, gel filtration and ion-exchange chromatography. Homogeneity was verified by acrylamide disc electrophoresis, sedimentation velocity and N-terminal amino acid determination. They differ in their sedimentation coefficients, u.v. absorption spectra and the biological action on rabbit skin. Proteinase I was found to contain acid labile non-heme iron. It was demonstrated that the proteolytic enzymes as well as phosphodiesterase, 5′-nucleotidase and l-amino acid oxidase form complexes with other snake venom components which are in a state of association-dissociation equilibrium.     

Acute effect of Russell's viper (Vipera russelli siamensis) venom on renal hemodynamics and autoregulation of blood flow in dogs

Renal hemodynamics and autoregulation of blood flow were investigated following intravenous injection of Russell's viper venom (0.1 mg/kg) in dogs anesthetized with sodium pentobarbital. After venom injection, the glomerular filtration rate fell significantly throughout the experimental period of three hr. Urine flow rate and renal blood flow also decreased and the filtered load of electrolytes declined significantly. The fractional excretion of sodium, potassium and phosphorus increased following venom administration. These data suggest that the venom may depress both glomerular and tubular functions. The renal autoregulation of blood flow was maintained during the experimental reduction of renal arterial pressure. We conclude that the ability of renal vasculature to autoregulate renal blood flow is not inhibited by Russell's viper venom, even though renal function is depressed.     

Purification and partial biochemical characterization of an edema inducing phospholipase A2 from Vipera russelli (Russell's viper) snake venom

A phospholipase A2 (VRV PL-VI) from Vipera russelli venom was purified to homogeneity in a single step on CM-Sephadex C-25 column. VRV PL-VI is a basic protein with a mol. wt of about 12,000 and showed a basic pH optimum and a high temperature maximum. It hydrolysed purified phospholipids in the order of phosphatidylethanolamine greater than phosphatidylcholine much greater than phosphatidylserine greater than phosphatidylinositol = 0. It is toxic with an LD50 value (i.p.) of 3.5 micrograms/g body weight in mice and it induced persistent edema in the mouse foot pad.     

Amount of venom injected by Russell's viper (Vipera russelli)

The total yield of venom (desiccated) in 25 adult V. russelli (mean length 111 +/- 1.8 cm (S.E.) ranges from 21-268 mg (127 +/- 13 mg, mean +/- S.E.) and that of 13 juvenile snakes (mean length 79 +/- 2.8 cm) is 8-79 mg (45 +/- 7 mg). The venom yield per milking correlated well with the length of the snake. The average amount of venom injected in the first bite of 31 adults (mean length 107 +/- 1.4 cm) is 63 +/- 7 mg (range 6-147 mg) and by 17 juvenile snakes (mean length 83 +/- 1.1 cm) is 41 +/- 8 mg (range 3-138 mg). Adults inject 45% of the glands' content in the first bite. More than 75 mg of venom (desiccated) was injected by 11 out of 31 adults and 2 out of 17 juvenile snakes. This indicates that in a substantial proportion of cases of envenomation, 40 ml of Burma Pharmaceutical Industry monovalent antivenom, which is commonly used, may not be adequate.     

Effects of phospholipases A2 from Vipera russelli snake venom on blood pressure, plasma prostacyclin level and renin activity in rats

Vipera russelli venom contains several isoenzymes of phospholipase A2 (PLA2) which were isolated by column chromatography. The effects of PLA2 fractions on blood pressure, plasma prostacyclin level and renin activity were studied in normotensive and renal hypertensive rats. PLA2 fractions II-5, II-7, III-3 and III-6 (0.1 mg/kg) injected i.v. into rats decreased the arterial blood pressure. The hypotensive action of PLA2 fractions was not affected by heat treatment (70-80 degrees C, 30 min, pH 6.8). After indomethacin (30 mg/kg, i.v.), the hypotensive response to PLA2 was markedly reduced. Plasma prostacyclin (PGI2) and thromboxane A2 (TXA2) levels were measured by radioimmunoassays of their degradation products, 6-keto-PGF1 alpha and TXB2, respectively. PLA2 fractions (0.1 mg/kg) induced an increase in plasma PGI2 and TXA2 levels. There was a positive linear correlation between the PLA2-induced hypotensive effect and the ratio of increased 6-keto-PGF1 alpha to TXB2 (r = 0.83) in normotensive rats. In renal hypertensive rats, the increase in PGI2 level was larger than in normotensive rats. Plasma renin activity was also measured by the radioimmunoassay. Plasma renin activity was reduced by PLA2 fractions in renal hypertensive rats, but not in normotensive rats. These results suggest that the hypotensive effect of PLA2 fractions in normotensive rats may be partly due to the increase in plasma prostacyclin and thromboxane A2 levels. In addition to the larger increase in plasma PGI2 level, the reduction in plasma renin activity may also contribute to the greater hypotensive effect of PLA2 fractions in renal hypertensive rats.     

Eight cases of acute renal failure from Vipera russelli formosensis venom after administration of antivenom

The six-valent antivenom from Vipera russelli formosensis in Taiwan has recently become available in Taiwan. Between June 1998 and December 2001 8 Vipera russelli formosensis snakebite victims were treated with antivenom within 24 h of snakebite. Bleeding stopped and coagulopathy improved after antivenom use, and neurotoxic symptoms were mild. Prothrombin time was prolonged on the 1st day and thrombocytopenia changed within 2 d. In the majority of cases, renal dysfunction persisted for several days after coagulation abnormalities normalized: 3 cases required hemodialysis. Although Vipera russelli formosensis antivenom is capable of preventing hemorrhage, antivenom administration may not alter the course of severe renal damage.     

Biochemical characterization of fibrinogenolytic serine proteinases from Vipera lebetina snake venom.

Two glycosylated serine fibrinogenases isolated from Vipera lebetina venom have homologous N-terminal sequences and antigenic determinants but can be clearly differentiated according to substrate specificity, glycosylation levels, molecular mass and fibrinogen degradation. alpha-Fibrinogenase has no homolog among known serine proteinases. It has N-terminal similarity with snake venom arginine esterases but does not hydrolyze the esters of arginine, lysine and tyrosine. The enzyme has strong proteolytic activity and degrades alpha-chain of fibrinogen altering its clottability by thrombin. beta-Fibrinogenase is a typical arginine esterase which hydrolyzes esters and amides of arginine and attacks the beta-chain of fibrinogen.     

Purification and partial characterizations of coagulant protein Fla from Daboia russelli siamensis （Myanmar） venom

Aim： To purify and characterize the coagulant protein FIa from Daboia russelli siamensis （Myanmar） venom. Methods： FIa was purified from Daboia russelli siamensis （Myanmar） venom by ion-exchange chromatography on CM-Sephadex C-50, and gel filtration on Sephadex G-75 and a Superdex 75 column. The hemostatic activity of FIa was determined by the method of Williams and Esnouf. The specific chromogenic substrates were used respectively to determine the activation of factor X and prothrombin. The fibrinogen-clotting activity of FIa was determined by the method of Gao et al. Normal saline was used as a negative control while factor Xa and thrombin were used as positive controls, respectively. Results： FIa, a coagulant protein, was achieved by ion-exchange chromatography and gel filtration with a molecular weight of 34 479 and an isoelectric point of 7.2. FIa was shown to have strong hemostatic activity. The hemostatic activity of 0.5 mg FIa was equal to that of 1.5625 u thrombin. FIa primarily activated factor X, however, had no influence on prothrombin, nor did it cleave or clot fibrinogen. Condusion： FIa is a factor X-activating enzyme, which could activate factor X to factor Xa, but has no effect on prothrombin and fibrinogen.     

Synergistic interaction of a protease and protease inhibitors from Russell's viper (Vipera russelli) venom

An acidic proteolytic enzyme, RVVX, was purified from Vipera russelli venom by successive chromatography on CM-Sephadex C-25, DEAE-cellulose and Sephadex G-100 columns. RVVX is a glycoprotein with a mol. wt of 79,000. It exhibited caseinolytic and factor X activating properties. Two trypsin inhibitors, TI-I and TI-II, were purified from V. russelli venom in a single step by CM-Sephadex C-25 column chromatography. The trypsin inhibitors interacted with the proteolytic enzyme RVVX. TI-I inhibited only the factor X activating property of RVVX while TI-II inhibited both, the caseinolytic and also factor X activating properties of RVVX. The edema inducing activity of RVVX increased markedly in the presence of non-edema inducing doses of TI-I and TI-II. RVVX, TI-I and TI-II were non-lethal in mice. The combination of RVVX and TI-II demonstrated enhanced toxicity.     

Structure and function of snake venom proteins affecting platelet plug formation

Many snake venom proteins have been isolated that affect platelet plug formation by interacting either with platelet integrins, membrane glycoprotein Ib (GPIb), or plasma von Willebrand factor (VWF). Among them, disintegrins purified from various snake venoms are strong inhibitors of platelet aggregation. Botrocetin and bitiscetin derived from Bothrops jararaca and Bitis arietans venom, respectively, induce VWF-dependent platelet agglutination in vitro. Several GPIb-binding proteins have also been isolated from snake venoms. In this review, we focus on the structure and function of those snake venom proteins that influence platelet plug formation. These proteins are potentially useful as reagents for the sub-diagnosis of platelet disorder or von Willebrand disease, as well as for clinical and basic research of thrombosis and hemostasis.