Effects of Russell's viper venom fractions on systemic and renal hemodynamics.

Changes in systemic and renal hemodynamics induced by Russell's viper venom are well established. The component of the venom responsible for hemodynamic alteration has not been identified. By Sephadex column chromatography five fractions of Russell's viper (Daboia russellii siamensis) venom were isolated. Each venom fraction consisted of phospholipase A2, proteolytic enzyme, phosphomonoesterase, phosphodiesterase, arginine ester hydrolase and hyaluronidase of varying activities. Hemodynamic effects of each venom fraction including mean arterial pressure, cardiac output, systemic and renal vascular resistance, renal blood flow and glomerular filtration rate were studied in five groups of dogs; each group had four dogs. Minimal hemodynamic changes were observed in dogs receiving venom fraction I. Increased renal vascular resistance with diminution of renal blood flow and glomerular filtration rate was observed in dogs receiving venom fractions II, III, IV and V. A markedly increased renal vascular resistance with maximal decrease in renal blood flow and glomerular filtration rate was caused by fraction III of the venom with highest PLA2 and proteolytic enzyme activities. However, renal hemodynamic changes appeared to correlate better with proteolytic enzyme activity than PLA2 activity. The findings suggested the proteolytic enzyme as an important determinant of hemodynamic alteration. Fractional excretion of Na was increased in dogs injected with venom fraction IV, and is presumed to be due to the inhibition of tubular reabsorption of Na by a natriuretic factor in this venom fraction.     

Mediators and renal hemodynamics in Russell's viper envenomation

The effects of Russell's viper venom on vasoactive mediators and renal hemodynamics were studied in five mongrel dogs. Intravenous administration of Russell's viper venom to the dogs caused a reduction of mean arterial pressure, renal blood flow, and glomerular filtration rate. The filtration fraction was decreased. This was accompanied by a rise in plasma norepinephrine, endothelin, 6-keto-PGF1 alpha, a stable metabolite of PGI2, and TXB2, a metabolite of TXA2. Plasma levels of epinephrine and dopamine showed no significant changes. The increase of plasma levels of both vasodilatator and vasoconstrictor were critical to systemic and renal hemodynamics. While vasodilatation predominated in the systemic circulation and resulted in hypotension, vasoconstriction played a major role in decreasing renal hemodynamics. Decreased renal blood flow and decreased glomerular filtration rate were the result of renal vasoconstriction and hypotension.     

A competitive radioimmunoassay using a monoclonal antibody to detect the factor X activator of Russell's viper venom

A radioimmunoassay (RIA) has been developed for the detection of Russell's viper venom in body fluids. This is a competitive binding technique using a monoclonal antibody directed against the factor X activator of Russell's viper venom. The sensitivity of the test in urine was 4 ng/ml, in 0.1% bovine serum albumin-phosphate buffered saline it was 20 ng/ml and in serum it was 5 micrograms/ml. This was adequate to detect venom in the serum of four patients bitten by Russell's viper. Urine from an isolated kidney preparation perfused with Russell's viper venom contained coagulant activity and was positive using the competitive RIA. Testing of sera from other envenomated patients and pure venom from seven other species of snake indigenous to Thailand revealed RIA cross reactivity between cobra venom and Russell's viper venom. In practice, the absence of coagulant activity in cobra venom clearly distinguishes between the two. Although further development is required to elucidate the serum factors interfering with this assay, this is a promising technique, which is of potential value in the diagnosis and investigation of the pathophysiology of Russell's viper envenomation.     

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.     

The hyaluronidase activities of some Southeast Asian snake venoms

The hyaluronidase activities of venoms of snakes indigenous to Southeast Asia were investigated. With the exception of the venom of the Malayan krait Bungarus candidus, the elapid venoms had either little or no hyaluronidase activities, whereas the viperid venoms possessed considerable activity. A component of Russell's viper venom with hyaluronidase activities had a mol. wt of approximately 14,000. Neither MP4, a monoclonal antibody raised against the purified Russell's viper venom hyaluronidase toxin, nor a monospecific polyclonal antivenom neutralized the hyaluronidase activities of this purified hyaluronidase component of crude Russell's viper venom. The Russell's viper venom hyaluronidase activities was labile on heating and storage. The significance of these observations to envenomation and antivenom production is discussed.     

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.     

Renal function following sea snake venom (Lapemis hardwicki) administration in dogs treated with sodium bicarbonate solution

The effects of sea snake venom (SSV) on renal function were studied in two groups of anesthetized experimental dogs pretreated with intravenous infusion of 4.2 gm% NaHCO3 solution. Animals were envenomated by intramuscular injection of SSV at a dosage of 0.34 mg/kg. Systemic hemodynamics showed no significant changes except for a tendency of decrease in cardiac output (CO). The glomerular filtration rate (GFR), the rate of urine flow (V) and effective renal plasma flow (ERPF), and effective renal blood flow (ERBF) significantly decreased, while filtration fraction (FF) significantly increased at 180 min after envenomation. Envenomated animals showed a reduction in renal fraction (RF), while renal vascular resistance (RVR) increased stepwise throughout the experimental periods. Animals pretreated with sodium bicarbonate showed no significant changes of CO, TPR MAP, HR, and packed cell volume (PCV) while receiving sea snake venom. Animals pretreated with sodium bicarbonate showed no changes in GFR, ERPF, ERBF, RF, and RVR after envenomation. The rate of urine flow markedly increased in envenomated animals which received pretreatment with bicarbonate. After envenomation alone, there were no differences in the plasma concentration of sodium (PNa) and chloride (PCl) as compared to the control value, whereas the plasma concentration of potassium (PK) increased at 180 min after envenomation. Animals pre-treated with bicarbonate showed a stepwise increase in both UNaV, FE(NA), U(Cl)V, and FE(Cl) accompanying SSV injection. Neither PNa nor PCl were affected, while PK significantly decreased in animals given SSV with bicarbonate loading. UKV and FEK increased stepwise in envenomated animals treated with bicarbonate throughout the period of study. All groups of animals given SSV, with or without NaHCO3 infusion, showed a marked elevation of the concentration of urinary myoglobin (U(Mb)), plasma lactate dehydrogenase (LDH), and plasma creatine phosphokinase (CPK) throughout experimental periods. The urinary myoglobin excretion markedly increased in animals after SSV injection accompanied by NaHCO3 infusion. It can be concluded that large amounts of myoglobin present in the renal tubules in envenomated animals can precipitate, particularly under acidic conditions, resulting in increased intratubular pressure and subsequently decreased renal hemodynamics including GFR and ERBF. An infusion of NaHCO3 to render urine more alkaline could have a protective role against depression of renal function following sea snake venom administration.     

Effect of purified Russell’s viper venom-factor X activator (RVV-X) on renal hemodynamics, renal functions, and coagulopathy in rats

Acute renal failure (ARF) is the most frequent and a serious complication in victims of Russell’s viper snakebites. Russell’s viper venom-factor X activator (RVV-X) has been identified as a main procoagulant enzyme involving coagulopathy, which might be responsible for changes in renal hemodynamics and renal functions. Here, we purified RVV-X from crude Russell’s viper venom to study renal hemodynamics, renal functions, intravascular clot, and histopathological changes in Sprague-Dawley rats. Changes in renal hemodynamics and renal functions were evaluated by measuring the mean arterial pressure, glomerular filtration rate (GFR), effective renal plasma flow (ERPF), effective renal blood flow (ERBF), renal vascular resistance (RVR), and fractional excretion of electrolytes. After 10 min, rats receiving both crude venom and purified RVV-X decreased GFR, ERPF, and ERBF and increased RVR. These changes correlated to renal lesions. Along with the determination of intravascular clot, rats injected with purified RVV-X increased the average D-dimer level and reached a peak at 10 min, declined temporarily, and then reached another peak at 30 min. The temporal association between clots and renal dysfunction was observed in rats within 10 min after the injection of purified RVV-X. These findings suggested RVV-X as a major cause of renal failure through intravascular clotting in the renal microcirculation.     

Russell's viper snakebite in Taiwan: differences from other Asian countries.

Formosan Russell's viper (Daboia russelli siamensis) is the sixth most frequent cause of snakebite in Taiwan. Its venom has been thought to have both neurotoxic and hematoxic properties. This viper's snakebite is rare and thus scarcely subjected to systemic studies. In this paper, we retrospectively analyzed and described 18 cases of viper snakebite from 1987 to 1999. Like that of the Russell's viper snakebite in other South East Asian areas, varied degrees of acute renal failure, incoagulable blood with bleeding diathesis and hemolysis were the major symptoms found in the systemic envenoming patients. Systemic thrombosis seems to be the distinguishing feature in Formosan Russell's viper snakebite. Neither symptoms nor signs of neuromuscular junction blocking effects were observed, which is another difference from symptoms observed after bites of some other Russell's viper subspecies, suggesting a significant geographic variation. These findings confirmed the clinical importance of Russell's viper snakebite in Taiwan.     

Some biochemical properties of Russell's viper (Daboia russelli) venom from Eastern India: correlation with clinico-pathological manifestation in Russell's viper bite.

In the present study, some biochemical properties and pathological effects of Daboia russelli venom from Burdwan district of West Bengal, eastern India are presented. The clinical features of Russell's viper envenomation observed in patients admitted to Burdwan Medical College & Hospital are also reported. In vitro, whole venom exerts strong trypsin inhibitory, phospholipase A2 and procoagulant activities in addition to moderate adenosine monophosphatase and adenosine triphosphatase activities. Lethality (LD50) of this venom sample is 0.7 mg kg (i.v.) of mice. Significant local tissue damaging effects including edema, hemorrhage and necrosis are observed in experimental animal models. An increase in the level of serum enzymes, such as aspartate transaminase, alkaline phosphatase, creatine phosphokinase, lactate dehydrogenase after D. russelli venom injection in albino rats is indicative of cell or tissue damage. High incidence of intravascular hemolysis in addition to hemostasis, haemoptysis and haematuria are observed as the most prominent features of RVV envenomation from this part of India. The present study reinforces the hypothesis that variation in the venom composition of RVV from eastern India with respect to venom samples of Russell's vipers from other parts of India is responsible for the differences in the clinical manifestation in patients from eastern India.     

The anticoagulant effect of Bothrops castelnaudi snake venom (Castelnaud's pit viper)

An inhibitory effect of Bothrops castelnaudi venom was observed on the following systems: prothrombin time, activated partial thromboplastin time, thrombin time, thromboplastin generation time, activation of factor X by Russell's viper venom and Russell's viper venom activated factor X (factor Xa). This effect did not require previous incubation and was prevented by the addition of Bothrops-antivenom. The prolonged activated partial thromboplastin time was not shortened by increased phospholipid concentration (0.5-10 mg/ml), suggesting that the inhibitory effect is not due to an anti-phospholipid activity. No significant fibrinogenolytic activity was detected upon incubation of human fibrinogen with the venom, since physiological levels of thrombin-clottable material were still present. Compared to Bothrops jararaca venom, the proteolytic activity on casein and on azocoll was very low. Thrombin-induced clots of human plasma and fibrinogen were not lysed by the venom within 24 hr. The results indicate that the anticoagulant effect of Bothrops castelnaudi venom is exerted at least at two levels of the blood coagulation mechanism: (1) before prothrombin activation, by inhibiting factor X-activation and factor Xa activity; (2) by direct action on thrombin.     

Novel non-enzymatic toxic peptide of Daboia russelii (Eastern region) venom renders commercial polyvalent antivenom ineffective.

The snake venoms are typically complex mixtures of enzymes and non-enzymatic peptides. Regional variation in the non-enzymatic fraction of Russell's viper venom from three regions of India studied. The eastern, western and southern regional venom upon gel permeation chromatography on sephadex-G-75 column resolved into three peaks. All the three overlapping peaks differ in their lethality and enzymatic potency. Peak III of all the regional venom found to be non-enzymatic, Western and southern regional venom has trypsin inhibitory activity with varying potencies. Interestingly, the peak III of eastern region is devoid of trypsin inhibitory activity. But it is highly lethal with a LD50 0.7 mg/kg body weight and also it exhibited post-synaptic neurotoxicity. On the other hand southern and western regional venom's non-enzymatic peak is non-lethal and did not induce neurotoxic symptoms in experimental model. The antibodies developed against the eastern regional venom cross-reacted with the peaks I and II of other regional venom, but failed to cross-react with the peak III of western and southern regional Russell's viper venom. Commercial anti-venom prepared to neutralize the toxic effects of common poisonous snakes of India, showed positive cross-reaction against peaks I, II and III of all three regional venom tested, except peak III of eastern regional venom. Commercial anti-venom neutralized the lethal toxicity of both western and southern regional Russell's viper venom, and failed to neutralize the lethal effects of eastern regional Russell's viper venom.     

Histological and ultrastructural changes of the kidney in renal failure after viper envenomation

Forty experimental animals injected with lethal doses of Russell's viper venom and seven human victims of viper snake bite were studied in order to elucidate the pathogenesis of renal tubular necrosis due to viper envenomation. Autopsy, light and electron microscopic examinations were performed on animals and on 4 patients that died; renal biopsy studies were also carried out on the 3 patients that recovered. General features of disseminated intravascular coagulation, consumption coagulopathy and paradoxic haemorrhage were seen as a result of the strong coagulant effects of the venom. The salient features in the kidney are intraglomerular deposition of fibrin, fibrin degradation products and coagulation. The obstruction of glomerular capillaries by coagulated material is the most likely cause of reduction of blood supply to the renal tubules. This tubular ischemia results in tubular necrosis and subsequent renal failure.     

Multiple thrombotic occlusions of vessels after Russell's viper envenoming

Systemic bleeding due to consumption coagulopathy and thrombocytopenia due to activation of procoagulants is the leading manifestation and cause of death in Russell's viper systemic envenoming. Thrombotic occlusion of the blood vessels is rare in cases of snakebite. In this report, two adult patients with Russell's viper systemic envenoming presented multiple cerebral infarctions, digital gangrenes and ischaemic organs in addition to typical clinical manifestations of bleeding diathesis and renal involvement. Our findings in these two special cases suggest that the venom-induced coagulopathy and endothelium damage, predisposed by toxin-induced vasoconstriction, might be the possible mechanism of multiple thrombotic vascular occlusions in systemic envenoming of Formosan Russell's viper.     

Renal hemodynamics in hemorrhagic hypotension: studies on the effects of pre- and postjunctional dopamine receptor agonists

The present study was conducted to determine if selective prejunctional dopamine receptor agonists will be useful in improving renal hemodynamics in acute hemorrhagic shock in anesthetized mongrel dogs. Both bromocriptine and N-n-propyl-N-n-butyl dopamine (PBDA) effectively increased renal blood flow, due to a decrease in renal vascular resistance in intact anesthetized dogs. However, these two agents failed to increase renal blood flow after acute hemorrhage in the innervated or denervated kidneys. Dopamine was effective in increasing renal blood flow in both intact and hemorrhaged animals. However, this action of dopamine in hemorrhaged animals was associated with a significant increase in arterial blood pressure. These data could be explained based on the observations that the reduction in renal blood flow following hemorrhage was primarily due to a decrease in blood pressure as a result of the decrease in cardiac output and was not due to an increase in renal vascular resistance. Because elevation of sympathetic tone appeared to play little role, prejunctional inhibition by bromocriptine and/or PBDA of sympathetic nerve function was ineffective in altering renal vascular resistance. Dopamine was effective in increasing renal blood flow, not because of its pre- or postjunctional actions on dopamine receptors on renal nerves or vasculature, but because of its ability to increase cardiac output and arterial pressure. Based on the experimental model employed in these studies, it is concluded that agents such as PBDA and bromocriptine, which are considered to be selective prejunctional dopamine agonists, may not be clinically useful in improving renal circulation during hemorrhagic shock.     

Studies on pharmacological effects of Russell's viper and Saw-scaled viper venom and its neutralization by chicken egg yolk antibodies

Antivenom antibodies were raised in 24-week-old white leghorn chickens against hemotoxic venoms of Russell's viper and Saw-scaled viper snakes. Booster injections of increasing concentrations of venom were given at 14days of time interval to raise the antivenom level in egg yolk. Antibodies were extracted from immunized chicken egg yolk by Polson et al. (Polson A., Von Wechmar M.B., Van Regenmortel M.H.V. Isolation of viral IgY antibodies from yolks of immunized hens. Immunological Communications 1980; 9:475-493.) and further purified by DEAE cellulose ion exchange column chromatography, which gave pure (180-200kDa) specific antibodies against venom. High titre of more than 1:10,000 antibodies were detected by ELISA at the 135th day of observation. The lethal toxicity and various pharmacological activities like hemorrhagic activity, phospholipase activity, edema and procoagulant activities of venom were carried out by both in vivo and in vitro methods. The effectiveness of antivenom in neutralizing these effects was carried out involving pre-incubation type experiments. The median effective dose (ED50) for Russell's viper venom was 0.96mg/2LD50/18g mice and for Saw-scaled viper venom it was 1.28mg/2LD50/18g mice. One millilitre of specific antivenom was effective in neutralizing 0.110mg of Russell's viper and 0.137mg of Saw-scaled viper venoms respectively (PD50). Antivenom was effective in neutralization assays in a dose dependent manner. The results indicate that antibodies raised in chicken could effectively neutralize the pharmacological effects induced by venoms and chickens therefore present an alternative and cheaper source of specific antibody generation.     

Characterization of a novel pro-coagulant metalloprotease (RVBCMP) possessing alpha-fibrinogenase and tissue haemorrhagic activity from venom of Daboia russelli russelli (Russell's viper): evidence of distinct coagulant and haemorrhagic sites in RVBCMP.

A novel, basic pro-coagulation metalloprotease (Russell's viper basic coagulant metalloprotease, RVBCMP) with an approximate molecular weight of 15kDa was purified from the venom of Daboia russelli russelli (Russell's viper) from eastern India. RVBCMP exerted dose-dependent coagulation of platelet-poor human plasma; however, RVBCMP possessed less coagulant activity as compared with the coagulant activity of crude Russell's viper venom (RVV). RVBCMP did not show oedema induction, direct haemolysis of washed erythrocytes, hydrolysis of human plasma albumin or globulin, and thrombin-like activity, but exhibited caseinolytic, alpha-fibrinogenolytic, and liver tissue haemorrhagic activities. Inhibition of coagulant and protease activities of RVBCMP by EDTA suggested a metalloprotease nature of this protein. RVBCMP showed antigenicity as was evident from the immunoblotting experiment. None of the tested plant extracts, except Leucus lavandulaefolia, inhibited the coagulant or haemorrhagic activity of RVBCMP. Interestingly, aqueous extracts of the tested plants as well as the commercial polyvalent antivenom raised against crude RVV differentially inhibited the coagulant and tissue haemorrhagic activity of RVBCMP. The current investigation provides a fairly good indication that RVBCMP possesses a distinct, perhaps overlapping, site for coagulant and tissue haemorrhagic activity.     

Practical applications of snake venom toxins in haemostasis

Snake venom toxins affecting haemostasis have facilitated extensively the routine assays of haemostatic parameters in the coagulation laboratory. Snake venom thrombin-like enzymes (SVTLE) are used for fibrinogen/fibrinogen breakdown product assay and for the detection of fibrinogen dysfunction. SVTLE are not inhibited by heparin and can thus can be used for assaying antithrombin III and other haemostatic variables in heparin-containing samples. Snake venoms are a rich source of prothrombin activators and these are utilised in prothrombin assays, for studying dysprothrombinaemias and for preparing meizothrombin and non-enzymic forms of prothrombin. Russell's viper (Daboia russelli) venom (RVV) contains toxins which have been used to assay blood clotting factors V, VII, X, platelet factor 3 and, importantly, lupus anticoagulants (LA). Other prothrombin activators (from the taipan, Australian brown snake and saw-scaled viper) have now been used to assay LA. Protein C and activated protein C resistance can be measured by means of RVV and Protac^®, a fast acting inhibitor from Southern copperhead snake venom and von Willebrand factor can be studied with botrocetin^® from Bothrops jararaca venom. The disintegrins, a large family of Arg-Gly-Asp (RGD)-containing snake venom proteins, show potential for studying platelet glycoprotein receptors, notably, GPIIb/IIIa and Ib. Snake venom toxins affecting haemostasis are also used in the therapeutic setting: Ancrod (from the Malayan pit viper, Calloselasma rhodostoma), in particular, has been used as an anticoagulant to achieve ‘therapeutic defibrination’. Other snake venom proteins show promise in the treatment of a range of haemostatic disorders.     

Action of Calloselasma rhodostoma (Malayan pit viper) venom on human blood coagulation and fibrinolysis using computerized thromboelastography (CTEG)

The effects of Malayan pit viper (Calloselasma rhodostoma) venom on human blood coagulation and fibrinolysis were studied in vitro using computerized thromboelastography. At low concentrations the venom had a coagulant effect shown by faster onset of the coagulation process (shortened SP and R), faster progress of the clot (increased angle and shortened K), and increased coagulation (TEG) index. The maximum amplitude (MA) was not affected, suggesting that the venom had no apparent effect on platelet function; and clot lysis was similar to that in the controls, suggesting that there was no primary fibrinolytic activity. At higher concentrations the venom had anticoagulant effects, SP and R were progressively shortened, but there was poor/no progress in the clot formed, evident from prolonged or absent K, diminished MA and reduced angle. These results show that C. rhodostoma venom has both coagulant and anticoagulant actions. The coagulant action may be due to Factor X activator predominance at low concentrations, while the anticoagulant action could be due to ancrod action. TEG is able to demonstrate the dual effect of this venom, previously described as a paradox, and may be a useful tool in the diagnosis and monitoring of envenomation patients.     

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.