ToxGaon, Vol. 18, pp . 87-96.©Pan;smon Pres Ltd. 1980 . Printed in Great Britain .

(Acceptedfor publication 121une 1979)

INTRODUCTION

87

oo4t-olollsorolol-0oa7sos.wro

IN VITRO STUDIES ON COMPLEMENT INACTIVATION BYSNAKE VENOMS

GÖsTA EGGERTSEN,' .TAN FOHLMANt and JOHN S7ÖQu1sT*

'Department of Medical and Physiological Chemistry, Box 575, 751 23 Uppsala, Sweden.tDepartment of Cell Research, Box 562, 751 22 Uppsala, Sweden.

G. Eaa>:aTSeN, J. FOllia~t~v and J. SröQulsr . In vitro studies on complement inactivation bysnake venons . Toxicon 18, 87-96, 1980.--Snake venons of the families Elapidae, Viperidaeand Crotalidae were investigated concerning their effects on the human complement system.Twenty-two of 26 venons inhibited complement activity in normal human serum . Mostefficient were the venons from Naja nala, Agkistrodon rhodostoma, A. halys, Yipera lebetirtaand 7}lmeresurus okinawensis. Conslunption of the complement factors C2, C3 and C4 wasobserved with most venons . Factor B and C3 were transformed to physiological conversionproducts . Purified C3 was converted by host venons, suggesting a direct effect on this factor.Conversion was inhibited by EDTA, but could in some cases be restored by the additionofMg'+ or Ca'+ . We conclude that viperid and crotalid venons inhibit the complement systemthrough proteolytic action on several of its components, in contrast to active elapid venons ofthe genusNaja, which activate the system owing to 'cobra venom factor' . lâ no othercase coulda clear specific attack on a single pathway be demonstrated . However, some distinctive featuresof the multipoint attack made possible a grouping of the venons into three categories whichmight be valuable in future studies of the complex factors involved in complement activation .

SNAKE venoms are among the most complex of animal secretions, containing a vast numberof substances with different pharmacological and biochemical activities . The presence ofprinciples affecting complement was observed nearly a century ago (Ew1Na, 1894 ; FLExN~tand NOGUCHI, 1903) . The action of the Indian cobra (Naja naja) venom on serum comple-ment has been studied in some detail . The active venom factor (Cobra venom factor) is aglycoprotein with a mol. wt of 144,000 (MÜLLER-EBERHARD and FJELLSTRÖM, 1971), whichcombines with the serum factor B in the presence of Mgß+ (MÜLLER-EBERHARD, 1967) .Factor B, a glycoprotein with a mol . wt of 93,000, is then split proteolytically by the serumfactor D into two fragments, a large basic one (Bb) and a smaller acidic one (Ba) which isreleased into the solution (GÖTZE and MÜLLER-EBERHARD, 1971 ; GÖTZE, 1975) . Thecomplex CVF-Bb is a highly active C3- and CS-splitting enzyme whose action leads to anactivation of the terminal complement sequence and reactive lysis of erythrocytes(PICKERING et al., 1969 ; MnrAMA et al., 1975 ; LYNEN et al., 1976) . CVF has been a veryvaluable tool in the studies of the structure and function ofthe complement system . More-over, it also has pronounced effects in vivo on the activity of complement (BIRDSEY et al.,1971 ; HOOPES and MCCALL, 1977 ; McCALL et al., 1974) . Many other snake venoms alsoexhibit complement-inhibiting effects (BIRDSEY et al., 1971), but very little is known aboutthe mechanisms . Snake venoms probably contain many potentially useful tools for investi-gation of the complement cascade. In the present investigation we have screened severalsnake venoms for complement-inhibiting activity and have tried to further characterizeunderlying reactions .

88

GÖSTAEGGERTSEN, JAN FOHLMAN and JOHN SJCyQUIST

MATERIALSAND METHODSSnake vertomsSnake venons (listed in Table 1) were obtained from the following sources : Miami Serpentarium Labora-

tories, Miami, Florida, U.S.A . ; Sigma Chem. Co ., St . Louis, Missouri, U.S.A . ; Koch-Light Labs.,Coolbrook, England; Mr. C. Tanner, Cooktown, Australia; Dr. P. A. Christensen, The Southern AfricanInstitute for Medical Research, Johannesburg ; Pasteur Institute, Paris, France . All venons were lyophilizedat -20°C, weighed and dissolved immediately before use or stored in solution at -70°C.

Complement factorsPartially purfied Cl Was prepared aocordmg to the method OfCoorEtt Snd Mt7LLkR-RaauHwan (196$) and

CZ according to that of Po "r .RV and Mtha-ER-EsBxxnßn (1968). Purified C3 was prepared as described byLvxnwwt.t, et al. (1978) .

Whole blood and serumFreshly drawn sheep blood in Alsevers' solution was obtained from the National Bacteriological Labora-

tory, Stockholm, Sweden. Frozen human serum was obtained from the Blood Center, University Hospital,Uppsala. Antihuman-C3 was raised in rabbits by intradermal igjection of 1 ~0 mg ofpurified C3 emulsified inFreund's complete adjuvant . Booster ißjections were given subcutanously in Freund's incomplete adjuvant 4weeks later. Rabbit anti-human factor B was kindly donated by Dr . B. Curman at the Department of CellResearch, Uppsala University . Zymosan treatment of normal human serum was performed with zymosanfrom Sigma Chem. Co ., U.S.A., according to MAYER (1967) .

Hemolytic titration of whole complement (CHso) and complement component activitiesCrude snake venons weredissolved in gelatin-veronal-buffer, pH7~5, to a concentration of 1~0 mg/ml, and

incubated with an equal volume ofnormal human serum at ß-37°Cfor 60 min. The material was then storedat -70°C and subsequently tested. Serum incubated with gelatin-veronal-buffer without dissolved snakevenons was used as a control . CHso test was performed according to the procedure of Mi+YEx (1967) . Efficientmolecular titration ofCl was performed according to the method of Boasos and RnrP (1963), of C2 accord-ing to that of Coorax et al. (1970), of C3 according to that of Mtlra sx-EBExx~xn et al. (19C~ and of C4according to the procedure described by COOPER and Mih.t,ex-Eeetetrutn (1968).

Immunoelectrophoretic analysisCrude snake venons were dissolved in serum to a concentration of 0~5 mg/ml and the specimens were

incubated at 37°C for 60 min. The material was immediately processed with or stored at -70°Cfor sub-sequent analysis. To study the metal ion dependence, human EDTA-plasma was used instead of serum. Theeffect of snake venom on purified C3 was investigated by mixing 50 ~1 of purified C3 in gelatin-veronal-buffer (3~0 mg/ml) with 50 ul of a snake venom solution (1~0 mg/ml) and incubated as above. Immuno-electrophoresis was performed according to the method of SctrEm~aEte (1955) in 005 M Tris-acetatebuffer containing 0-01 M EDTA, pH 8~0, for 2 hr . Anti-human C3 or anti-human factor B was applied in thecentral troughs . The slides were stained with 0~1 ~ Amidoschwartz .

RESULTS

Titration ofwhole complement activity (CHbo-test)Of the 26 snake venoms tested, 22 possessed complement inhibiting activity (Table 1) .

Most potent were the venoms from Agkistrodon rhodostoma, Agkistrodon piscivorus andTrimeresurus okinawensis. Very potent were also the venoms from Naja n . siamensis andVipers lebetina. The complement inhibiting activity of 18 of the most active venoms werethen further investigated as described lxlow.

Quantitative titration ofC1, C2, C3 and C4The venoms consumed the complement factors differently (Table 1) . However, character-

istic patterns emerged which allowed classification of some of the venoms (Table 2) . Ageneral impression was that C1 is more resistant to snake venom action than all the otherfactors . There is a good correlation with the zoological classification of snakes, since all ingroup A are elapids and B and C are viperids and crotalids. The consumption patterns ofthe cobra venoms in group A are very similar, except that Naja nigricollis venom showed ahigher consumption of C4 than the others .

Naja n . siamenstsNaja hgjeNaja rrtveaNaja nigricollis

In vitro Studies on Complement Inactivation by Snake Veaoma

TAEI.E 1 . HEMOLYTIC TITRATION OF WHOIE COMPLHMENT ACTIVITY (CHso) Arm

QUANTITAIIVB CONIPIF.I~NT FACTUR TITRATION IN NORMAL HUMAN SAUM AFTER SNAâE

VENOM TREATMENT

Equal parts of serum and snake venom in gelatin-veronal-buffer (1~0 mg/ml) weroincubated at ß-37°Cfor 60 min and subsequently tested. The activity is expressed as

ofthe activity in serum incubated without venom.

TAHLB 2. GROUPII~O OF THE TES'11~ VENOMS WTTH REGARD TO THE CONSUMPTION PATTB1tN

Group A: a high consumption of C3 compared to the other complement factors tested.

Group B: a high consumption of C2, C4 and C3 with less consumption of Cl .Agkistrodon rhodostornaAgkistrodon halys blomlw,~Agkistrodon piscivores leukostoma7Ytnltresurus jlavovlridis7Ylrnertsurus okinawensisGroupC: a high consumption of C2 compared to the consumption of the other factors .Vipers lebetinaEchts carlnatusCrotalur h . horrldus

89

DitBculties arise with venonscausing an even consumption. 'Individualistic patterns' were given by Crotalesdurissus terrifrcus and Vipers bores, which mainly inhibited C2 and C4 (see Table 1).

Immunoelectrophoretic analysisEffects on C3 in semen (See Fig . lA) . Complete conversion of C3 occurred with all the

cobra venoms tested except Naja nigricollis, which only partially converted C3 (Table 3).Complete conversion was also effected by the venoms from Trimeresurtts okittawensis andall venoms from the genus Agkistrodon . The rest of the venoms converted C3 only partially.

Snake venom CHsoRemaining activityCl C2 C4 C3

Ngja n . slamensis <20 60-80 40-60 20-40 <5Naja h~e 20~0 8l}-100 40fi0 80-100 <5Naja nivea 20~0 40-60 60~0 20~0 <5Naja nigricollis 40-60 80-100 60-80 5-20 10-20Bungarus melticlnctus 20~0 60-80 40-80 80-100 10-20Bungarus coerulees 40~0 60-80 40-80 60-80 60-80Vlpera aspls 40~0 40fi0 20-40 60-80 40~0Vipera lebetina <20 60-80 5-10 20-0fl 20-40Vipera berus 40-60 60-80 20~40 40fi0 5-20Bitis arletans 20~0 60-80 20~0 40-60 20-40Echis carinatus 20-40 60--80 5-10 10-20 20-40Crotales d. terrlfrces 20-40 40~0 10-20 5-20 20-40Crotales h . horridut 40-60 40~0 5-20 20~0 40~0Agkistrodon rhodostoma <20 80-100 <5 <5 <5Agkistrodon halys bl. <20 20-40 <5 <5 20~0Agkistrodon piscivores 1. 20~0 80-100 <5 <5 5-10Tümeresurus jlavoviridis 40fi0 40fi0 10-20 N.D . 20~07}ünertsurus okinawensls <20 20~10 <5 <5 <5Ophlophagus hannah 60-80 N.D . N.D . N.D . N.D .Bltis gabonica 60-80 N.D . N.D . N.D . N.D .Causas rhontbeatus 60-80 N.D . N.D . N.D . N.D .V~pera ammodytes 60-80 N.D. N.D . N.D . N.D .Oxyuranus scutellatus <80 N.D . N.D . N.D . N.D .Notechis scutatus <80 N.D . N.D . N.D . N.D .Bungarusfasciatus <80 N.D . N.D . N.D . N.D .Dtndroaspls jamesoni <80 N.D . N.D . N.D . N.D .

90

GÖSTA EGGERTSEN, JAN FOHLMAN and JOHN SJ(SQUIST

The electrophoretic mobility of the conversion product was identical to that obtained withserum after incubation under the same conditions with EA-cells or zymosan. No otherconversion product appeared with any of the venoms tested . In controls no conversion ofC3 or factor B was observed .

TAHLE 3 . THe C3-CONVERTINO BFFECT OF DIFFERBNT SNARF VENOMS ASEVALUATED HY IMMUNOELECTROPHORESIS

Degree of conversion of :Snake venom

C3 in serum

Purified C3

Crude snake venoms were dissolved either in serum or in a solution ofpurified C3 in gelatin-veronal-buffer to a wncentration of 03 mg/mland this was incubated at 37°C for 60 min. After immuncelectro-phoresis, the slides were allowed to diffuse and the amount ofconvertedC3 was estimated visually. -}-++ only converted C3 present ; ~-+more than 50~ conversion ; -}- less than 50% conversion .

E,~ect on purified C3 (See Fig. 1B). Conversion was obtained with all of the venomsexcept three ofthe cobras and the two Bungarus species (Table 3) . In most cases there wasagood correlation with the conversion of C3 in serum. The venom from Bitis arietansconverted purified C3 completely, but had little effect in serum. In contrast, the venom fromAgkistrodon halys blomho~I was less active on purified C3.E�~`ects on factor B (See Fig. 1 C) . Conversion of factor B in serum occurred with all the

venoms investigated . The conversion product shows Y-mobility . With the venoms fromNaja n. siamensis, Naja haje and Naja nivea an acidic component also appeared . The same,but more faint, precipitate was also seen with Bungartts multicinctus and Bungarus coeruleus

FIa . I . IMMUNOELECTROPHORECIC ANALYSIS OF THE AGTION3 OF SNAKE VENOMS ON COMPLEMENTCOMPONENTS .

(A) THB BFFECI'3 OF CRUDE SNARE VENOMS ON C3 IN SERUM (FOR DETAR3 SEB TEXT) .Central troughs : rabbit-anti-human C3 . Note that Naja nigricollis only converts C3 partially,

while Naja haje and Naja raja siamensis venoms convert C3 completely.(B) THB BFFECI'S oN PURIF~D C3 .

Central troughs : rabbit-anti-human C3 . C3 is partially converted by the venom from Yiperaaspic, but is unaffected by Naja raja stamensts venom.

(C) THE EPFECT3 ON FACTOR B .Central troughs : rabbit-anti-human-factor B . Both the split products are visible with thesecobra venoms, but only the basic fragment appears with the Trtmeresurus okinawensis venom.With the latter venom anotherprecipitate ofunknowncharacter is visible close to that of native

factor B.

Naja n. slamensis -f-++ -Naja haje +++ -Naja nivea +++ -Naja nigricollts + +(~-)Bungarus multicinctus + -Bungarus coeruleus + -Vipera aspis + +Vipera lebetina + +Vipera berus +Bitis arietans ~- +-I--FEchis carinates ++-I- ++Crotales durissus terrificus -~ +(-I-)Crotales h. horridus + -f-~-Agkistrodon rhodostoma +++ +~-+Agklstrodon halys blomho,~r -I-~--~ -f-(+)Agkistrodon piscivores leucostoma -i- ~--}-7Hmeresurus jlavovirldis + -I-Trimeresurus okinawensis +++ ++-~

91

5

FIC~IRE 1 A

NaJA N . SIAhENSIS

NAJA NIGRICALLIS

IWJA IiAJE

CONTPIIL

FIGURE 1 B

NAJA N. SIAhENS1S

VIPERA ASPIS

FIGURE 1 C

IWJR N. SIAhENSIS

NAJA FIAJE

CCNTRDL

TRIhERESURUS OKINAIVENSIS

In vitro Studies on Complement Inactivation by Snake Venoms

93

venoms . Both precipitates occurred in serum treated with rymosan, which is a well-knownactivator of the alternative pathway. Another precipitate was produced with a few of theother venoms (Agkistrodon rhodostoma, A. halys blomho�~t, A . piscivores, Trimeresttrusflavoviridis, T. okinawensis, Echis carinatus), which had nearly the same mobility as nativefactor B.

Effects of EDTA on the C3-converting activity . When incubation was performed withhuman EDTA-plasma instead of serum, no conversion of C3 was observed . With somesnake venoms attempts were made to restore the activity by subsequent addition of CaE+ orMg'+ . The most active cobra venoms were completely reactivated either by Ca'+ or Mg'+,and a few of the other venoms regained some activity after addition of Mga+ (Table 4) .EDTA-plasma without snake venoms was used as a control . When EDTA was presentpurified C3 was unaffected by the venoms from Vipers berus, Bitis arietans, Echis carinatus,Agkistrodon halys blomho~t and Trimeresurus flavoviridis. The venoms from Agkistrodonpiscivores, A . rhodostoma and Trimeresurus okittawensis showed decreased C3-convertingactivity, while the venom from Vipers lebetina retained nearly full activity . Attempts torestore the activity by addition of Cag+ or Mg$+ were not successful . The addition of CaB+changed the electrophoretic mobility of purified C3 .

TABLE 4. EFFECTS OF RECALCIFiCATiON AND REMAONESÛ'ICATION ON i'FIEC3-CONVERTIIdO ABILr[Y OF SNAKE vENOM3

After incubation of crude snake venoms dissolved in EDTA-plasma(0 " S mg/ml) (in which the C3~onverting effect of all snake venoms was in-hibited), 20pl ofa 0~1 M CaCI,-solution or 20 pl of004 M MgCI,-solutionwas added to 100 ~1 of specimen . After incubation, immuncelectrophoresiswas performed. The degree of C3-conversion in each sample was estimatedvisually and compared to the corresponding effect of the venom in serum .

DISCUSSION

Most ofthe 26 snake venoms inactivated complement in the CHsu-test . Virtually all of thetested complement factors (C1, C2, C3, C4 and factor B) were affected by 18 venoms . Ofthese, viperid and crotalid venoms converted purified C3 as judged by immunoelectro-phoresis, indicating a direct proteolytic action . Snake venoms of the families Crotalidae andViperidae are known to contain different proteolytic activities, while most elapid venomslack proteases (Mass, 1970) . Caseinolytic, esterolytic, thrombin-like and kininogenaseactivities have been identified, often in the same venom (GEIGER and KORTMANN, 1977) .Several professes from crotalids have been characterized physico-chemically (IWANAGAet al., 1976 ; SPIEKERMAN et al., 1973 ; TAKAHASHI and OHSARA, 1970) . These enrymes arethermolabile metalloendopeptidases which preferably cleave peptide bonds in which the

Snake venomDegree OfCa'+

I'P.aCtivatlOII with :Iylg~+

Naja n. siamensis complete completeNgja haje complete completeNaja mues complete completeNaja nigricollis partial partialBurRgarus multicinctus none partialBungarus coeruleus none partialVipers aspic none noneVipers berus weak weakAgkistrodon rhodostoma none weakAgkistrodon piscivores leucostoma none completeTYlmeresurus flavoviridls none none

94

GÖSTAEGGERTSEN, JANFOHLMAN and JOHN SJÖQUIST

amino group is contributed by hydrophobic amino acid residues (PRESCOrr et al., 1976).They are irreversibly inactivated by treatment with EDTA, but are unaffected by DFP.MINTA ¢t al. (1977) and MAN and MINTA (1977) purified four proteolytic enzymes with

anti-complementary activity from Crotalus atrox venom. These enzymes split severalcomplement factors, but differ slightly from each other in specificity . Two of them possessesterase activity . All were sensitive to heating to 56°C and three of them were inhibited byEDTA. Our own results show that the activity of the viperid andcrotalid venoms toward C3is completely inhibited in EDTA. However, purified C3 wasconverted by afewvenoms evenin the presence of EDTA, an effect which may not appear in serum or plasma since manydifferent protease inhibitors are present (VOGEL et al., 1968). Some C3~onverting activitycould be regained with a few venoms upon addition of Mgz+ . Whether this is due to Mgz+per se or some other metal ion released by excess Mgz+ on EDTA-metal complexes isuncertain.The immunoelectrophoretic analysis of C3 after incubation with the snake venoms

showed that the conversion products looked identical to C3 converted by EA-cells orzymosan. Concerning factor B, the larger fragment (Bb) could be identified with all thesnake venoms, while a precipitate corresponding to the smaller one (Ba) appeared only withsome of the elapid venoms . 1t might have undergone further digestion by proteaes in theother venoms . The unidentified precipitate observed with some venoms (see Results) mightrepresent another split product or possibly a complex with some common component in thevenoms . Probably certain regions of many complement factor molecules are more sensitiveto proteolytic attack (regardless of the specificity of the enzyme), as is the case with, forexample, immunoglobulins and transplantation antigens .No elapid venoms (except Naja nigricollis) converted purified C3 . The cobra venom

factor has been identified in the venom of Naja naja and Naja haje (Mt}LL~t-EBERHARD andFJELLSTRÔM, 1971 ; Sxtx et al., 1969). The consumption pattern of complement factorswould suggest that the venom of Naja nivea also contains such a principle. The report of ahigh mol. wt fraction acting on CI in the Naja naja venom (SALLOW arid COCHRANE, 1971 ;EGGERTSEN, unpublished results) would explain the slight affect on the early components .The presence of such an activity in the Naja haje and the Naja niv¢a venom cannot be ruledout. The venom of Naja nigricollis was much less efficient than the other three, and inaddition to a fairly high C3-activation, it also consumed C4, and even purified C3 to someextent. It is clear, that this venomcontains components which interact with the complementsystem in other ways than cobra venom factor.

Earlier investigations of the effects of snake venoms on the complement system alsosuggest that some cobra venoms might be unique. BIRDSEY et QI. (1971) found that thevenoms of several cobras and one crotalid (Lachesis muta) were capable of lysing un-sensitized red blood cells with guinea-pig complement, possibly owing to reactive lysis . Onesource of difficulty in working with crude snake venoms is the presence of phospholipase Aactivity (MEas, 1970), which might mimic complement-induced lysis by enzymatic catalysisof erythrocytic membrane phospholipads. Even in purified cobravenom factor preparationsphospholipase activity has been detected (LneHHtnx et al., 1976 ; 5xAw et al., 1978 ;Ec3GEßTSSx et al., to be published) . In crotalid and viperid venoms, the complement-inhibiting activity was ascribed to different enzymic effects (BIRDSSY et al., 1971). A fewvenoms (Crotalus durissus terrificus, Vipera ammodytes and Bungarus coeruleus) reported tobe non-anti-complementary by the latter authors did inactivate complement in our hands.

Little is known about the effects of snake venoms on complement in vivo. BIRDSEY et al.(1971) found adecrease in complement activity in guinea pig serum after i.v. administration

In vitro Studies on Complement Inactivation by Snake Yenoms

95

of partially purified Naja raja venom, while several other complement-affecting venomsfailed to show this effect . Purified cobra venom factor administered i.v. is able to depleteserum of C3 (CocHxAr1E et al., 1970). C~IUGH et al. (1977) reported significant depletion ofC3 in Rhesus monkeys following administration of crude venom from Vipers russelli andEchis carinatus. Depletion of C3 and factor Bwas reported in serum from humans bitten byNaja nigricollis (WARREL et al., 1976), and of C3 after a bite by Dispholidus typos(Nlcorsox et al., 1974) . It cannot be excluded that a proteolytic action on C3 with pro-duction of Cab might lead to an activation of the factors CS-C9 (THaoFiLOrouLOS et al .,1974). Some split-products from several complement factors possess anaphylatoxicproperties (Mi)L1.Ett-EHERHARD, 1976). At present, no definite statement can be made aboutthe clinical implications of complement-inhibition following snakebites .

Acknowledgements-We are in great debt to Drs. Dnvtn EAKER and LBNNART ANDER3.40N for supply ofsnake venoms. We also thank Dr. E.vcEß for valuable discussion. This work was supported by the SwedishMedical Research Council (Proj . No. 13X-02518) .

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