Characterization of the anticoagulants from taiwan cobra (Naja naja atra) snake venom

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CHARACTERIZATION OF THE ANTICOAGULANTS FROM TAIWAN COBRA (NAJA NAJA ATRA) SNAKE VENOM KUO,

CHE-MING TENG, YIH-PYNG LIH -GEN LEE and CHAOHO OUYANG Pharmacological Institute, College of Medicine, National Taiwan University, Taipei, Taiwan, R .O .C . (Accepted for publication 19 August 198 C .-M . Tetve, Y:P . Kuo, L .-(1 . Lee and C . Ouv~t~a. Characterization of the antrcoagrrlanta ' from Taiwan cobra (Ngja ngja atra) snake venom . Toxicon 2S, 201- 210, 1987 . - Taiwan cobra (Ngja ngja atra) snake venom was separated into 19 fractions by means of CM-Sepha~r C-SO oolnmn chromatography . Antiwagulant Fractions V - VII were refractionated by gel filtration on Sephadex G-SO and the purified component possessed phaspholipase A, activity and an inhibitory effect on collagen-induced platelet aggregation . The anticoagulant action could be antagonized by phospholipid or platelet factor 3 . Anticoagulant Fraction XVII was also further refraaionated by gel filtration on Sephadex G-SO and the purified compon~t was shown to be cardiotaxin. It was a weak antirnagulant, caused direct hemolysia and potentiated collagen-induced platdet aggregation . Thromboelastographic :
INTRODUCTION

Interference with blood coagulation is one of the main causes of pathological manifestation in snakebites by the families of Crotalidae and Viperidae . The active components have been purified and their properties were studied ('TENG et al., 1984x, 1985; OUYANG et al., 1978, 1982; TEND and SEEGERS, 1981 ; OUYANG and TEND, 1972) . However, there are few papers reporting on the anticoagulant action of venoms of the elapid family. OUYANG (1957) reported that Taiwan cobra (Naja ngja atra) snake venom possessed strong anticoagulant action in whole blood in vitro even though this action was not easily observed in actual snakebite and the main cause of death with this venom was peripheral respiratory paralysis (LEE, 1971). Ouyang also proposed that the anticoagulant action of the cobra venom was due to the inactivation ofthromboplastin, Ac-globulin and prothrombin. The purpose of this study was to purify the anticoagulant principles from cobra venom and to study their properties . MAT$RIALS AND METHODS Materials Ngja ngja atra snake venom was kindly supplied by Mr M . I . Liso of the Preventive Medical Institute, Nankang, Taipei, and was stored at blow - 20°C in a deslaxtor . CM-Sephadar C-30, Sephadar G-30 and G-25 were purchased from Pharmacia, Sweden. Collagen (bovine tendon, Sigma Chemial Co ., U .S .A .) was homogenized in 25 mM acetic acid and stored at -70°C at a eonantratlon of 1 mg/ml . Platelet factor 3 and thromboplastin were obtained from General Diagnoatia, U .S .A . Bovine sen~ albumin, apyrase, t)L-aphosphatidylcholine (dipalmitoyl, synthetic), rabbit brain aphalin (phaspholipid mixture), trypsinogen, insulin, cytochrome c. ß-lactoglobulin, tetracaine and ß-mercaptoethanol were purchased fra~m Sigma Chemical Co., U .S .A . 201

202

CHE-MING TENG et al.

CM-Sephadex C-SO column chromatography

One gram of the crude venom was applied to a column (3 .2 cm x 63 .0 cm) of CM-Sephadex C-S0 . Gradient elution with ammonium acetate was carried out in two stages : 0.05 M (pH S.0)-0.4 M (pH 6.8), then 0.4 M (pH 6.8)-0.8 M (pH 8.0). The flow rate was adjusted to 24 ml/hr and an eluate of 4 ml per tube was automatically collected with an LKB Fraction Collector. The effluent was monitored rnntinuously at 278 nm and 4°C with an LKB Uvicord and a Chopper Bar Recorder . Gel Jltrotion

Sephadex G-SO was swollen in 0.01 M ammonium bicarbonate and packed in a column (1 .6 cm x 100 cm, 200 ml). The elution was carried out with the same eluent as above. The flow rate was adjusted to 36 ml/hr and eluates of 6 ml per tube were collected. Phospholipasr A, activity

Determinations were by theindirect hemolysis method of BROWN and BOWL.FS (19C~ and the pH-slat titration method of STxoNa et al. (1976) . Sodium deoxycholate of a concentration equimolarto phosphatidylcholine was used as the emulsifier and 10 mM CaCla wes added. Blood coagulation test

The one-stage method of Qutct~ (1938) was used to estimate the rabbit plasma prothrombin time. The calcium clotting time of rabbit plasma was determined as described by Btoasand MwcF~ttt.~Nt? (1962). Stypven clotting time was determined by the method of O'BatHV (1957) . Platelet aggrrgotion

Washed rabbit platelets were prepared as described by Rams et al. (1971) . Aggregation was measured by the turbidimetric method of O'BweN (1962) and Bowv and Cttoss (1963) using a Payton Lumi-Aggregometer (Model 1020). 0.05M

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Anticoagulants in Cobra Venom TABLE 1 . COMPARISON OF THE BIOLOGICAL ACTIVITIES OF THE FitACTIONS OF Nq%R SNAKE VENOM

203 naja RtrR

Fraction

PPT (sec) " Platelet aggregations Indirect hemolytis (4sß Control 10 .3 0 0 Crude 13 .7 13 I 11 .0 0 1 II 11 .5 0 0 III 10.3 0 6 IV 11 .3 30 V 18 .2 -90 VI 13 .1 --39 VII 14 .0 --59 VIII 11 .0 --90 IX 13 .7 -90 X 10 .4 90 XI 10 .5 0 1 XII 10 .7 0 10 XIII 12 .2 0 6 XIV 11 .8 0 3 XV 11 .2 0 3 XVI 16 .4 + 0 XVII 16.0 ++ 4 XVIII 14 .1 + 0 XIX 10 .4 0 1 The data are means of duplicate determinations. "Plasma prothrombies time (PPT); the final concentration of each fraction was SO pg/ml. sEffect on aggregatioes of platelet-rich plasma induced by collagen (10 Yg/ml): 0, ineffective; -,inhibition; +, potentiation . *Indirect hemolytic activity is alpreased as a percentage of hemolytis. The final concentration of each fraction was 1 pg/ml in the incubation mixture of lecithin . Thrombodartoanaphy Citrated rabbit blood (0 .3 ml containing 0.384s sodium citrate) was preincubated with test solution or saline at 37°C for 1 min, thm 0.04 ml of CaC1, (1 .29~i) was added to :taK the coagulation . The r value was calculated from the time of addition of G to the time when the tracing width reacdied 1 estm ; the k value was calculated as the time when tracing width increased from 1 mm to 20 mm, and Ma was the maximal amplitude of the tracing. Cardiotaxfn artivrty. The contracture activity of ardiotoxin was detested in chicrees biventer muscle preparation using themethod of Giessborg and Warriner (1960) . The direst hemolytic activity of cardiotoxin was measured by its incubation with 1~Fs RHC at 37°C for 3 hr. The absorbance of the supernatant was measured at S30 nm after oentrifugatioes. RESULTS

Pur~artion of the anticoagulant principles

CM-Sephadea C-SO column chromatography separated Ngja ngja atra venom into 19 fractions (Fig. 1). The anticoagulant activities, distributed in fractions V - VII and XVI-XVIII, were measured by plasma prothrombin time. Fractions V-IX also inhibited collagen-induced aggregation of platelet-rich plasma, while fractions XVI-XVIII showed weak potentiating action (Table 1). Fractions V-VII and XVII wen further purified to obtain the main components which affected blood coagulation and platelet aggregation. Fractions V - VII were refractionated by gel filtration on Sephadex G-S0. Similar patterns wen obtained, with some difference in the proportions of the three peaks shown in Fig. 2a. The anticoagulant and antiplatelet activities were concentrated in peak 2. This peak also possessed indirect hemolytic activity . After further gel filtration (2 x ) a single

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Fta . 2. RBCHAOMA7~OaaAP1tY of FRAC110N VII (Fig . 1) otv SePttwoex G-S0 . The indirect hemolytic acüvity ( " - " ) and the inhibitory activity on platelet aggregation (O--O) of the ducat were measured. The solid line ~) is optical density of protein at 278 nm . Each dot is the mean of duplicate determinations . (a) The lat rahromatograph . Aggregation of platelet-rich plasma was induced by collagen (10 pg/ml) . The saond peak was collated and used for future purification . (b) The 3rd rahromatograph . Aggregation of platelctrich plasma wag induced by arachidonic acid (100 ~.

symmetrical peak was obtained . Both the antiplatelet action and indirect hemolytic activity correlated well with the protein content (Fig. 2b). Fraction XVII was also further fractionated by gel filtration on Sephadex G-S0 . After further gel filtrations (2 x) a single symmetrical peak was obtained. Both the aggregationpotentiating action and the direct hemolytic activity correlated well with the protein content (Fig. 3). Homogeneity and biological properties Both components purified from fractions VII and XVII were homogeneous as judged by the patterns in disc polyacrylamide gel electrophoresis. They were single peptide chains as revealed by SDS-polyacrylamide gel patterns obtained in the presence or absence of 290

205

Anticoagulants is Cobra Venom

mercaptoethanol . The molecular weights were estimated to be 14,000 and 6600 for purified proteins from fractions VII and XVII, respectively (Fig. 4). The purified component from fraction VII possessed phospholipase A, activity, but was devoid of S' -nucleotidase, phosphodiesterase or alkaline phosphomonoesterase activity . Phospholipase A, activity was 311111 Etmole/min/mg (n = 4), measured by pHstat titration using synthetic phosphatidylcholine as substrate. The specific activity was about 20 times that of the crude venom (16.8f2.1) ~trnole/min/mg, n = 8). The amino acid analysis of this purified protein showed a similar composition to that reported by TSAI et al. (1981) . It contains (residua/mole) : Lys(S), His(1), Arg(4), Asp(19), Thr(S), Ser(S), Glu(8), Pro(4), Gly(10), Ala(11), C~s(12), Val(4), Met(1), Ile(3), Leu(S), Tyr(8), Phe(4) . o .so

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K values were calailated from dation volume after passing through a Sephadex G-30 column udng ammonium bicarbonate (0 .01 ~ as ducat. Insulin, cytodtmme c, ß-lactoglobuUn and trypriaogea were used as standard proteins .

206 TABLE

CHE-MING TENG

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EFFECT OF VENOM PHOSPHOLIPASE A, AND CARDIOTOXIN ON CALCIUM CLOTTING TIME AND PLASMA PROTHROMBIN TIME OF RABBIT PLATELET-POOR PLASMA (PPP) OR PLATELET-RICH PLASMA (PRP)

Concentration (Ng/m1)

Phospholipase A, Plasma prothrombin time (sec) PRP 5412 1010 .1

Calcium clotting time (sec)

PPP Control 8711 0.I 94112 0.5 130112' 2 176118' 70131 83131 10 5131261 110111 50 > 600 158131 125 > 600 2681221 250 > 600 MeanslS.E . of 3-6 experiments are presented. "P < 0.02, 1P < 0.001 compared with that of the respective

1010 .1 141-0.1 1 1910 .51 2411 .21 37131 53141

Cardiotoxin Calcium clotting time (sec) PPP 8711 99-6' 116141 1491121 1941201 231191

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5. ANTIOOAGULAT7r EFFECTS OF PHOSPHOLIPASB I;y (NNAV-PLAN AIa) CARDIOTOXIN (NNAVCTX) IN TFSTS OF STYPVEN CL07TIIi0 TIME USING VARIOUS CONCEN77tATIONS OF CEPHALIN .

Platelet-poor plasma (0.1 ml) was preincubatod with 0.1 ml of cephelin at 37°C for 3 min. After 0.1 ml of test sample was added, 0.1 ml of Russell's viper venom (25 pg/ml) and 0.1 ml of Ca (25 mM) were used to trigger the clotting. MeanstS.E. (n14) are presented.

The purified component from fraction XVII was cardiotoxin, since it possessed direct hemolytic activity and also caused contracture of the chicken biventer muscle . This purified cardiotoxin was not contaminated with detectable phospholipase A, activity (less than 2%), as measured by the pH-stet titration method using phosphatidylcholine as substrate. Anticoagulant actions of venom phaspholipase A~ and cardiotoxin Table 2 shows a comparison of the anticoagulant actions of phospholipase A, and cardiotoxin in various test systems of blood coagulation. For venom phospholipase A, it was clearly shown that the anticoagulant action appeared stronger when the calcium

Anticoagulants in Cobra Venom

207

clotting time was measured than when the plasma prothrombin time was determined . Venom phospholipase A, did not destroy fibrinogen, induce fibrinolysis nor interfere with the interaction between thrombin and fibrinogen. The anticoagulant action of cardiotoain was much weaker compared to that of phospholipase A, as determinated by calcium clotting time of platelet-poor plasma (Table 2). Since phospholipase A= might prolong the clotting time by inactivating the procoagulant activity of phospholipids, the neutralization of the anticoagulant activity of the venom phospholipase A1 by cephalin was studied. As shown in Fig. S, cephalin neutralized the anticoagulant action of phospholipase A,. Cardiotoxin showed only weak anticoagulant action in this test system . Anticoagulant actions on the thromboelastogram of whole bh~od Venom phospholipase A, affected the thrombcelastogram of rabbit whole blood profoundly . At a concentration of 12.5 fig/ml, the reaction time (r) was prolonged from 5.2 to 20.8 min, k from 4.0 to 10.0 min and Ma from SO to 35 mm. The anticoagulant effect could be reversed by platelet factor 3, but not by bovine serum albumin (Fig. ~. lA) Control

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Co++ Fra . 6 . THE EFFECT OF COBRA PHOSPHOL1PA38 A, (NNAV-PLAG oN 77m 77flroMBOELA.sroaRAM of RABBIT WHOM BLOOD IN 7iû PRE.4EtiCE OR ABSENCEOF BOVINE SERUM ALBUMIN OR PLA773ISr FACTOR 3 . Citrated blood (0 .3 ml) was preincubated at 37°C for 1 min with 0.035 ml of saline (a), bovine serum albumin (b) or platelet factor 3 (c) . 'I1Ien 0 .005 ml of aalirre (A) or NNAV-PLA (B) (1 mg/ ml) was added before 0 .04 ml of CaCI, (1 .2940. aA : r, 5 .2 min; k, 4.0 min; Mo, SO mm, aB : r, 20.8 min; k, 10.0 min; Ma 35 mm . bA : r, 6 .0 min; k, 3 .6 min; Ma, SO mm . bB : r, 16.4 min; k, 10.0 min; Ma, 33 mm . cA : r, 3 .2 min; k, 1 .6 min; Ma, 54 mm . cB : r, 3 .6 min; k, 6.0 min; Ma, 43 mm.

208

CHE-MING TENG et al. (A) Control -J I cm IOmin

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SYNERGISTIC EFFECT OF OOBRA PHO6PHOLIPASE A~ (NNAV-PLA,) AND CARDIOTOXIN ON THE THROI~OELASTOGRAM OF RABBIT WHOLE BLOOD.

Citrated blood (0.3 ml) was preincubated at 37°C for 1 min with 0.06 ml of saline (A), phospholipese A (NNAV-PLA) 2.3 ~/ml (B), cardiotoxin (CTX) 50 ~/ml (C), NNAV-PLA 1.23 ~/ml and CTX 2S Kg/ml (D) or NNAV-PLA, 2.5 ~/ml and CTX SO pg/ml (E) . Thrn 0.04 ml of CaCI, (1 .29Ys) was added at the arrow. A: r, 4.8 min; k, 2.0 min; Ma, SO mm . B: r, 16 .8 min; k, 10 .4 min; Ma, 33 mm . C: r, 7.2 min; k, 3.6 mitt ; Ma, 47 mm . D: r, 13 .6 min; k, 17 .2 min; Ma, 23 mm . E: r, 25 .0 min; k, 12 .8 min; Ma, IS mm.

Figure 7 presents the synergistic anticoagulant effects of phospholipase A= and cardiotoxin. Cardiotoain (SO Fig/ml) showed only weak anticoagulant action compared with that produced by venom phospholipase A, . However, combination of phospholipase A, and cardiotoxin markedly prolonged the r and k times and also decreased the amplitude of Ma values (Fig . 7D, E) . DISCUSSION Two main anticoagulants, phospholipase A~ and cardiotoxin, were isolated from the venom of Ngja ngja atra. Venom phospholipase A, showed anticoagulant action in many blood coagulation tat systems. It prolonged the calcium clotting time of platelet-poor plasma more markedly than that of platelet-rich plasma . This indicates that platelets possess some proooagulant activity opposing the anticoagulant action . Because cephalin (Fig . S) or platelet factor 3 (Fig. ~ antagonize the anticoagulant action of phospholipase A,, its effect could be due to the inactivation of the procoagulant activity of the phospholipids . These phospholipids are essential for the cascade sequence of blood coagulation, both in the intrinsic (platelet phospholipid) and extrinsic (tissue thromboplastin) pathways (VECCHIUNE Snd ZUCI~R, 1973 ; HVATUM and PRYDZ, 1969). The anticoagulant actions of other venom phospholipase A= enzymes have also been reported (BOFFA and BOFFA, 1974; CONDREA et al., 1981, 1983 ; L1N et al., 1983 ; TENG et al., 1984x, 1985 ; OUYANG et al., 1978). However, the anticoagulant action of the cobra venom phospholipase A, is much weaker than that produced by the basic phospholipases A, isolated from the venoms of i?pera russelli ijTÉNG et al., 1984x), Trimeresurus

Anticoagulants in Cobra Venom

209

mucrosquamatus (OUYANG et al., 1981), Naja nigricolis (OUYANG et al., 1981 ; CONDREA et al., 1981) and Apis mell~fera (LIN et al., 1983). This difference might be due to the nonenzymatic phospholipid-binding activity of the basic phospholipases A, (OUYANG et al., 1978 ; CONDREA et al., 1981, 1983) or due to the ability of basic phospholipases A, to penetrate into tightly packed monolayer films of phospholipid (BOFFA and BOFFA, 1976). Cardiotoavn is a basic polypeptide devoid of enzymatic activity (Lo et al., 1966). It causes muscle contracture, direct hemolysis and has cytotoxic effects (LEE and LEE, 1979). This membrane-active venom protein was also reported to potentiate the platelet aggregation induced by ADP, thrombin, collagen and venom phospholipase A, (TÉNG et al., 1984b) . In this paper we found that cardiotoxin had only weak anticoagulant action, however it markedly potentiated the anticoagulant action of cobra venom phospholipase A,. Although the mechanism is unknown, this potentiation could be partly due to its highly basic charge and partly to the increased accessibility of phospholipid brought about by the action of this membrane-active protein. Because phospholipase A, and cardiotoxin are the only anticoagulants obtained by chromatography of cobra venom, and they show a synergistic action in whole blood, we propose that the anticoagulant action of cobra venom is due to the synergistic effect of phospholipase A, and cardiotoxin. Acknowkdgemtrets - This work wan partly supported by a grant of the National Science Council of the Republic of China (NSC74-0412-H002-89). REFERENCES Annt.te, N . G., Pt3aav, D . W ., Pwctatwnt, M . A . and Musrwxn, J . F . (1971) Influence of apyrase on stability of suspensions of washed platelets. Proc. Soc . arp. Biol. Med. 136, 1021 . Btoas, R . and MwcFwat.wrre, R . G . (1962) Human Blood Cooguiatlon and its Disorders, 3rd Edn. Oxford: Blackwell Scientific . Bow, M. C . and How, G . A. (1974) Correlations between the enzymatic activities and factors active on blood coagulation and platelet aggregation from the venom of Vipers aspic. Biockim . blophys. Acts 354, 275 . Bot~w, M . C . and Hot~w, G . A. (1976) A phospholipase A, with anticoagulant activity . II . Inhibition of the phospholipid activity in coagulation. Biockim. biophys. Acts 429, 839. Hotw, ß. V. R . and Caoss, M . J . (1963) The aggregation of blood platelets . J. Pkysiol., Lond. 16g, 178 . BaowN, J . H . and BOwLFS, M. E . (19fif)6 Studies on the phoapholipase A activity of Crotakts atrox venom . Tazkan 3, 205 . Cormnew, E ., Ywxa, C . C . and RosEtvasa0, P . (1981) Lack of correlation between anticoagulant activity and phospholipid hydrolysis by snake venom pho:pholipaaes A,. Tkromb. Haarrosta:s. 4S, 82 . Coxatetw, E., Ywwa, C . C . and Ros~aetta, P . (1983) Anticoagulant activity and plasma phoaphatidylaerine hydrolysis by make venom pho:pholipaaa A,. Tkromb . Haa»ostas. 49, IS1 . GtKSaoaa, B . L . and Wwaanvea, J . (1960) The isolated chick biventer cervida nerve munde preparation. Br. J. Pharntac. 15, 410. Hvwru~s, J . and Pamz, H. (1969) Studies on tissue thromboplaatin - its splitting into two separable parts . 77trontb. Dlatk, haemorrk . 21, 217 . Lam, C . Y . (1971) Mode of action of cobra venom and its purified twdm . In : Neuropoisons: Their Fathophydological Actions, Vol . t, p . 21 (Sao'soN, L . L ., Ed .) . New York : Plenum Preaa . Lee, C. Y . and Lee, S. Y . (1979) Cardiovascular effects of snake venoma . In: Snake Venom, Hatdbook of F.tptritnattai Pkanrtarniody, Vol. 32, p. 347 (L,ee, C. Y., Ed.) . Berlin : Springer-Verlag. Lw, S . C ., Huwrta, T . F . and Otnwrta, C . (1983) Characterirstion of the purified anticoagulant prindples from Ads ntelljjera (hoary bee) venom . !. Forrrtasan reed. Ass. >t2, 629. Lo, T. B ., Cw, Y. H . sad Lee, C. Y. (1966) Chemical studies of Formosan cobra (Ngja ngja atrn) venom . Part I . Chromatographic separation of crude venom on CM-Sephadex and preliminary chsracterizatioa of its components. J . Chin . chew . Soc., Talpef 13, 23 . O'Batert, J . R . (1957) The effect of some fatty acids and phoapholiplds on blood coagulation . Br. J. exp. Pack. 12, 45 . O'Btumv, J. R . (1%2) Platelet aggregation . II . Some results from a new method of uudy . J. din. Patk . iS, 432. Ouvwrto, C . (1937) The effects of Formosan snake venoms on blood coagulation in vitro. 1. Format~an med. Ass. S6, 433 .

210

CHE-MING TENG tt al.

Ourwac, C. and TENG, C. M. (1972) Purification and properties of the anticoagulant principle of Agkistrodon acutus venom. Biochim, biophys. Acts 27E, 135. OuYwtac, C., Tewc, C. M., Curs, Y. C. and Ltta, S. C. (1978) Purification and characterization of the anticoagulant principle of Trimeresurus mucrosquamatus venom. Biochim. biophys. Acts 541, 394 . OUYANG, C., JY, W., Zwrt, Y. P. and Ttaac, C. M. (1981) Mechanism of the anticoagulant action of phospholipase A purified from Trimerrsurus mucrosquamatus (Formosan baba) snake venom. Toxirnn 19, 113. Ouv~wc, C., Tt=.wa, C. M. and HuNac, T. F. (1982) Characterization of the purified principles of Formosan snake venoms which affect blood coagulation and platelet aggregation. J. Formosan med. Ass. al, 781 . Qutcx, A. J . (1938) The nature of bleeding in jaundice . J. Am. med. Ass. 110, 1638 . S~rrtotve, P. N., Goettxe, J., Oaeec, G. and I{PLLY, R. B. (1976) o-Bungarotoxin, a presynaptic toxin with enzymatic activity . Proc. natn. Aced. Sci. U.S.A . 73, 178. TFrac, C. M. and S~eeRS, W. H. (1981) Agkistrodon acutus snake venom inhibits prothrombinase complex formation. 77rromb. Res. 23, 253. Tetvc, C. M., Ctmta, Y. H. and Ouv,~wc, C. (19840) Purification and properties of the main procoagulant and anticoagulant principles of Vlpma russellü snake venom. Biochim biophys. Acts 786, 204. Tetac, C. M., Jv, W. and OuY~rte, C. (1984b) Cardiotoxin from Ngja ngja otra snake venom: a potentiator of platelet aggregation. Toxicon 22, 463. Tene, C. M., Caress, Y. H. and OUYANG, C. (1985) Effect of Russell's viper venom phospholipase A on blood coagulation and platelet aggregation . Semin. Thromb. Hemastas. 11, 369. TS~t, I. H., Wu, S. H. and Lo, T. B. (1981) Complete amino acid sequence of a phospholipase A, from the venom of Ngja rtgja atm (Taiwan cobra) . Taxirnn 19, 141 . VECCtuorte, J. and Zucxett, M. B. (1975) Procoagulant activity of platelets in recalcified plasma. Br. J. Haemat. 31, 423.