Tarlcon, Vd . 2S, No. 12, DD . 1343-1346, 19a7.

0041-0101/a7 ß.00+ .00Printed in Orea Briuln .

SHORT COMMUNICATIONS

HETEROGENEITY OF ECHIS VENOMS FROMDIFFERENT SOURCES

RICHARD C. SCHAEFFER JR

Papmoo Iout~b I1d .

Microcirculatory Laboratory, Veterans Administration Medical Center andthe Department of Physiology, University of Arizona School of Medicine, Tucson, AZ 85723, U.S.A .

(AcceptedJorpublication 13 May 1987)

R. C. Scew~eA Jle . Heterogeneity of Echis venons from different sources. Toxicon 2S,1343 -1346, 1987 . - The heterogeneity of separate Echis carlnatus and Echis rnloratus venomIota were evaluated by high performance sizetxcluaion chromatography, as well as by clotting andesterolytic activities . Each lot ofE. carinarusandE. rnlorotus venoms appeared to be unique. Thevariation in E. carinatus venom activities from snakes within the same :pries could lead tornntradictory biochemical and/or physiological test results .

Echis carinates andEchis coloratus venoms contain a protein that initiates intravascularcoagulation by direct activation of prothrombin (wElss et al., 1973 ; ICORNALIK andBLOMBACx, 1975). Meizothrombin is formed when the E. carinates venom prothrombinactivator first cleaves an Arg-Ile peptide bond of prothrombin (MORTTA et al ., 1976 ;RHEE et al., 1982). This unique unstable prothombin derivative is rapidly converted tophysiologic thrombin (BRIET et al., 1982). The activation of prothrombin is notinfluenced by non-physiologic forms of prothrombin, the absence of Cat+, factor Xa orphospholipid (SUTTIE, 1973 ; SCHIECK et al., 1972; FRANZ.A et al., 1975).E. carinates venom poisoning in man or experimental animals often leads to

hemorrhage and disseminated intravascular coagulation that is characterized byhypofibrinogenemia, thrombocytopenia and a decline in coagulation factors V andVIII :C (WEISS et al., 1973 ; SCHAEFFER et al., 1986x) . The procoagulant feature of E.carinatesvenomhas bcen used to initiate the intravenous formation of fibrin microembolias a model of pulmonary microembolism (SCHAEFFER et al., 1987).The purpose of the present study was to identify separate Echis venom lots obtainod

from various colleagues andcommercial suppliers. Clotting andesterase activities, as wellas chromatographic profiles, were used as objective quality control data to characterizeseven lots ofE. carinates and two lots of E. colorates venoms. Although two lots ofE.carinates venom sharod some similar features, none of the venoms tested werequantitatively identical.An i.v . LDsp essay (REED andMUENCH, 1938) ofE. carinatesvenom wasattempted. No

correlation, however, was noted between the survivor/total (4/10 -6/10; 24 hr end-point)as the venom dose was increased from 0.5 to 3.0 mg/kg. Bloody edemafluid that exudedfrom the naries and cut lung surfaces was the main gross necropsy finding. Lightmicroscropic observations (mixed thrombus formation in pulmonary arteries) confirmed

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The per cent dry weight (~ undissolved material in 1 mg of crude venom per ml of deionized water), protein(LOwAY ct al., 1931), plasma clotting activity and esterolydc activity (hydrolysis of the synthetic substrate N-benzoyl-t .-prolyl-t.-phenyhtlanyl-t.-arginine-p-nitroanalide HCI, previously shown to be a measure of venomfôrinogeaolytic activity ; SCHASFFEa ct al., 1986x) were determined for each venom lot . The values for twoanalyses are reported.

'The time to clot control dog plasma following addition of 100 f+g of venom was used to calculate thethrombin equivalent units from a standard thrombin clotting curve (Scx.~FFea ct ol., 1984) .iThese venom lots were purchased from Sigma Chemical Co., St . Louis, MO, U.S.A .

the view that pulmonary embolism was the cause of death in each instance . Since over thedosage range used survival was not related to the venom dose, lethality appeared to beassociated with the failure to lyse lung fibrin clots . The short mean survival time (15tSmin) of the animals that expired are consistent with this view. This test was not performedon the other venom lots .Table 1 shows the per cent dry weight, Lowry protein, plasma clotting activity

($CHAEFFER et al., 1984) and esterolytic (hydrolysis of N-benzoyl-L-prolyl-L-phenyl-alanyl-L-argininep-nitroanalide HCI, previously shown to be a measure of venomfibrinogenolytic activity ; SexAEFFEIt et al., 1986x) activity, for each venom sample . Thevariability of clotting activity between venom lots (<0.1- 4 units/ 100 ~tg) are similar to thefindings of TOHORSKA (1971) . That study showed marked differences in venomthromboplastic coagulase activity from 21 separate species ofE. c. pyramidum from thesame region of West Pakistan . Thus, KORNALIK and T0130RSKA (1985) selected twogroups of E. carinatus venom: toxin A, rapid clotting activity ; toxin C, slow clottingactivity . In the present study E. carinatus venom A (clotting activity, 2.8 units/100 Ng)contained a moderate level of esterase activity (0.1310 O.D. /min/10 ~g protein), whereasE. carinatus venom C (clotting activity, 0.1 units/100 ptg) showed a high level ofesterolytic activity (0.475 dO.D./min/10 Itg protein) .

Since these measurements appeared unique for each venom lot, these samples weresubjected to high performance, size-selective chromatography ($CHAEFFER et al., 1986b) .The common absorption peak (37.5 ml) of each chromatogram (Fig . 1) represents smallmolecular weight amino and nucleic acids, since this position coincides with the totalvolume of the chromatography columns . Although each elution profile appeared unique,the pattern ofE. carinatus venom samples 103F and A144 showed some homology. Thisfinding and the similarity of their composition in Table 1 (low clotting and esteraseactivity with a high protein assay) suggests that these two lots may have been from thesame source .

Separate elution volumes were found for the clotting and esterolytic activities inselected venom lots (data not shown). The esterolytic activity eluted with a lowermolecular mass than the clotting activity in all lots tested . Thus, theE. carinatus venom

SampleTwsLa

Dryweight(~)

1 . CortrosmoNClottingactivity(units)'

oF Echis carinotus

Lowryprotenn(Ng)

wrm Echis colorata

Esterolyticactivity (A,u)(AO.D./min/10 N8)

vetroMsSpaies

ECV A 35/33 2.5/2 .7 66/69 0.125/0 .137 E. rnrinatus pyramldumECV C 19/13 <0.1/<0.1 49/50 0.440/0 .510 E. carinatus pyrarrtidum31F 20/16 4.0/4 .0 71/75 0.010/0 .020 E. carirratust39C 27/24 4.0/4 .0 77/80 0.005/0 .011 E. carlnatust103F 18/18 <0.1/<0 .1 103/99 0.030/0.046 E. rnrinatustAI44 31/29 0.8/0 .6 111/118 O.OOi2/0.008 E. carinatus leucogaster00039 24/32 4.0/4 .0 97/100 0.620/0.700 E. rnrinatus sochurcki0008 24/23 1 .6/1 .6 76/80 0.038/0.052 E. coloratusECOV K 10/10 2.8/2 .6 81/90 0.010/0.020 E. rnlomtus

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lô' . . .~ . . ..2â

. . . .~ . . . .~ . . . .Elution Volume-ml

Fta. 1 . Htatt rEte~oasurtce, SIZ&EXCLUSION CHAOMATOaRAPtiY aF Echis vtwonts .The elution profile (absorbance at 280 nm, A�~ of each sample chromatographed on aToyo SotaManufacturing Co . Ltd (TSK, Tokyo, Japan) type SW 3000 connected in aerie: with a TSK typeSW 2000 column (300 mm and 600 mm, respectively) is shown. Fach chromatogram was developedby injection of 25 ~1 of a SO mg/ml venom solution in O.OS M ammonium acetate buffer, pH 6 .0,containing 0.029s sodium azideat 1 ml/min. Thevoid volume ofthis chromatographic syntem wax13 .6 ml, as defined by the elution of thyroglobulin (669,000 mol.wt) . The dution volumes ofbovine serum albumin (67,000 mol.wt) and ribonudeaae A (13,700 mol.wt) were 20.9 and 30.5 ml,

respectively .

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prothrombin activator does not appear to possess esterolytic activity . This finding is inagreement with previous observation that this activator is not inhibited with serine proteaseinhibitors (diisopropylphosphorofluoridate ~RANZA et al ., 1975), benzamidine (MORITAet al., 1976) or D-phenylalanyl-L-prolyl-L-arginyl-L-chloromethyl ketone (SCHAEFFE1t etal ., 1986x)]. Thus, the prothrombin activator and esterolytic (fibrinogenolytic) propertiesof E. carinatus venom appear to be associated with separate venom components.

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In conclusion, these data suggest that each Echis venom lot may be unique . Thevariation in venom activites from snakes within the same species could lead tocontradictory biochemical and/or physiological test results . Therefore, biochemicalcharacterization of separate biotoxic materials within each venom lot from the same snakespecies is needed .

Acknowledgements - The author thanks the following for their generous gifts of the venoms used in this study:Dr FRANx KORNAI.QC and Dr DlsrwcH Mess for ECV A and ECV C; Dr E . Kocxvw for ECOV K ; YvesMrrrwINe of LATOXAN for ECV 00039, 0008 and A144 . The excellent technical assistance ofDONNA MwRSx,Jlbt Rmc eIId SHAWN-MARIE CHILTON 1n the preformance of this study is greatly appreciated . This study wassupported, in part, by a Merit Review Grant from the Veterans Administration Central Office .

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FRANZA, B . R., ARONSON, D . L. and FINLAYSON, J . S . (1973) Activation of human prothrombin by aprocoagulant fraction from the venom of Echis carinates. J. biol. Chan. 230, 7037 .

KORNALUt, F . sad BLOtirawcrt, B. (1973) Prothrombin-activation induced by ecarin - a prothrombin convertingenzyme from Echis carinates veaom . T7rromb . Res. 6, 53 .

KORNAI.IY, F . and TAaoRSttA, E . (1985) Correlation between the neutralizing activity ofmonovalem antivenomsand the quality of venoms used for immunization. Toxkon 23, 384 (abstract) .

Lowav, O . H ., RoseaROUaH, N . J ., FARR, A . L . and RANDALL, R . J . (1951) Protein measurement with theFolin phenol reagent . J. biol. Char . 193, 263 .

MORITA, T ., IwwNwaw, S . and Suurxl, T . (1976) The mechanism of activation of bovine prothrombin by anactivator isolated from Echlr carinates venom and characterization of the new active intermediates. J.Biochem . 79, 1089 .

REEn, L . H . andMUENCtt, H. (1938) A simple method of estimating SO per cent endpoints .Am . J. Xyg . 27, 493 .RHEE, M.-J ., MORRIS, S . and Kosow, D . P . (1982) Role of meizothrombin and meizothrombin-(ties Fl) in the

conversion of prothrombin to thrombin by the !:this cminates vwom coagulant . Biochanistry 21, 3437.ScxweFFeR, R . C . JR, Cxu.TON, S.-M ., HwnoeN, T . J . and CAALSON, R. W. (1984) Pulmonary fibrinmicroembolism with Echis carinates venom in dogs: effects of a synthetic thrombin inhibitor . J. app!.PhysioL : Reap. arvir. Exerc. Physiol. S7, 18?A .

SctIAeI~eR, R . C . JR, BRISroN, C., CHILTON, S:M . and CARLSON, R. W. (1986x) Disseminated intravascularcoagulation following Echis carinates venom in dogs : effects of a synthetic thrombin inhibitor . J. Lab . clip.Med. 107, 488 .

SCHAFFFER, R . C . JR, RENI~WICZ, R . R ., CHIL~roN, S. -M., MARSH, D . and CAAL9C)N, R . W . (1986b)Preparation aced high-performance size-exclusion chromatographic analysis of fluorescein isothiocyanate-hydroxyethyl starch: macromolecuhtr probes of the blood-lymph barrier. Miaovasc . Rea . 32, 230.

SCHwe~ER, R. C . JR, BARNHART, M . I . and CAALSON, R. W. (1987) Pulmonary fibrin deposition and increasedpermeability to protein following microthromboembolism in dogs: a atrucnrre-function relationship .Microvasc. Res. 33, 327 .

Sctnecx, A., FIwaeRMwNN, E. and KORNALIIC, F . (1972) The prothrombin-activating principle from EchLscarinates venom II . Coagulation studies in vitro and in vivo. Naenyn-Schmledeberg'sArehsPharamc. 274, 7 .

$UTr1E, J . W. (1973) Mechanism of action of vitamin K : demonstration of a fiver precursor of prothrombin.Sciaece 179, 192 .

Twaoasu, E. (19? 1) Intraspedes variability of the venom of Echis carirwtus. Physiologic bohemaslov. 20, 307 .Welss, H. J ., Pxu.urs, L. L ., Hopewel.L, W . S., PHII.L .IPS, G ., CHRI57Y, N . P . and Nrrrt, R . F . (1973)Heparin therapy in a patieat bitten by a saw-scaled viper (Echis carinates), a snake whose venom activatesprothrombin . Am . J. Mcd . 34, 633 .