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Anticoagulant properties of Apis mellifera (honey bee) venom
Short Communications
197
Ftt~ C~ancvs eH (197 Octyl-Sepharose CL-4B, Ph~yl-Sepharose CL-4B for hydrophobic interaction chromatography. Upplands Gr~rska AB . Vissait, L . and Louve, A . I . (1977) The importance of protein structure and conformation in the preparation of phospholipase-free cardiotoxin from snake venom . Biochim. btophys . Acta 491, 349 . PHARMACIA
roxlcon, vot . t7, ~ . t97-xoz . © Pet~amon Press Ltd. 1979 . 1rioted in Greet Britain .
coat-ototl791o3ot-0t97so2ool0
ANTICOAGULANT PROPERTIES OF APIS MELLIFERA (HONEY BEE) VENOM CHAOHO OUYANG, SONG-Ci-IOW
LIN"
and CI-IH-MING TENG
Phanvacological Institute, College of Medicine, National Taiwan University, Taipei, Taiwan, Republic of China (Accepted jor publication 24 July 1978)
A vAlt>srY of animal venoms, mostly of snake origin, modify blood coagulation (HOUSSAY, 1930; L~ et al., 1955 ; OUYANG, 1957 ; BoQusr, 1964; MF,AiJME, 1966 ; ROSENFELD et al., 1968 ; JI z-PO1tRAS, 1970; LEE, 1975 ; Tu, 1977) . JOSHUA and I3HAY (1975) demonstrated that an extract from the venom sac of the oriental hornet (Vespa orientalis) had an anticoagulant effect on blood coagulation. HABERMANN (1954) reported that honey bee (Apia mellifera) venom inhibited blood coagulation through inactivation of tissue thromboplastin probably by its phospholipase A component . Since the effect of this venom on other clotting factors is not well known, it was of interest to investigate the anticoagulant action of this venom and elucidate its mode of action . Apis mellifera venom, crude cephalin (chloroform extract of acetone-dried rabbit brain) and Russell's viper venom in crude cephalin were obtained from Sigma Chem. Co. Thrombin (bovine) was purchased from Parke-Davis Co. and dissolved in 50~ glycerolsaline to give a stock solution of 1000 N.LH . p/ml (S~GGI~ts and SMrrH, 1942) . Fibrinogen (bovine) was purchased from Pentex Co. and dissolved in imidazole-saline (pH 7"4j and the stock solution of 20 mg/ml was stored at -70°C before use . Rabbit brain thromboplastin was prepared according to the method of BIGGS and MACi?A1tLAN13 (1962) . Plateletpoor plasma was obtained from citrated blood (1 :9, v/v) after centrifugation for 20 min at 3000 rpm and 4°C. Blood collected from the rabbit marginal ear vein through a 19-gauge needle was mixed with 0'1 M Na9EDTA (1 :14, v/v) as anticoagulant and centrifuged for 10 min at 800 g and at room temperature . The platelet pellet was washed with Tris-buffered saline (pH 7"4) containing NaEEDTA (3 mM), apyrase (ADPase activity, 0~5 U/ml) and bovine serum albumin (1 mg/ml) and recentrifuged (modification of the methods of MUSTARD et al., 1972 and ARDIJE et al., 1971) . This wash procedure was repeated two times and the washed platelets were suspended in Tris-buffered saline, ruptured with a stirrer and then frozen and thawed three times before use . Whole blood clotting time was determined by the method of LEe and WFnre (1913) . Calcium clotting time of plasma was carried out as described by BIGGS and MACFARLANE (1962). The one-stage method of Qulcx (1938) was used to measure the plasma prothrombin time. Partial thromboplastin time was measured by the method of NYS et al. (1962) . " Present address : Department of Pharmacology, Taipei Medical College, Taipei, Taiwan, Republic of China .
19 8
Short Communications
The Russell's viper venom clotting time was determined by the method Of O'BRIBN (1957) . For the determination of the antithrombin actions, the method of SEEGERS et al. (1952) was used. Fibrinogenolytic activity was measured as deSCilbed by OUYANG and TENG (1976). Venom interference of the interaction between thrombin and fibrinogen was performed by the method Of SEEGEltS and SMITH (1942). The indirect hemolytic activity on washed red blood cells was measured by the method of BROWN and BOWEhES (19GG) . TABLE 1. EFFECTS OF Apls 71iCII%fCra VENOM ON PLASMA PROTHROMHIN TiAlB (A), RUSSELL'S VIPER VEI`OM CLOTTING TIMB ; (B), PARTIAL THROMBOPLASITN TIbIß (C) AND RECALCIFICATION TIME (D) OF RABHTr PLATELEI'POOR PLASMA AND ON THE CLOTTING TIIME OF THE WHOLE BLOOD (E)
Test system Venom conc . (lIg/ml)
A sec 12 "7 f 0~2
B
C
D
Clotting time (means f S.E .) sec sec sec 15 "6 f 0"5 54 "6 ~ 4"0 79 "8 ~ 2~1 82 "5 ~; 4~l 91 "1 ~ 4~5 72"6 ~ 3"4 96 "4 f S~8 16"0 f 0~4 98 "2 f 9~1 140"5 ~ 7"2 18 "2 ~ 0"3 125"1 ~ il "6 l54"5 f 9~7 26 "4 ~ 0~6 > 600 > 600 48 "3 f 0"8 > 600
min 4" 2 ~ 0~3 0 0"1 0"3 1 13 "9 f 0"3 3 19 "9 f 1 "0 10 26"9 f 1"1 5"7 f 0"5 30 30 "6 f 1"5 11 "7 f 1 "6 100 39 "4 ~ 4"8 24 "S f 4"6 300 51 "1 f 2"5 1555 f 21 "4t 1000 98 "9 f 9"6* *Granulated fibrin clot . fHemolysis after clotting . Clotting system : 0"1 ml of platelet-poor plasma was warmed to 37°C, then 0~1 ml of saline or bee venom of various wncentrations was added. To this mixture, 0"1 ml of (A) tissue thromboplastin (S ~), (B) cephalin with Russell's viper venom or (C) cephalin or (D) saline was added. Finally, 0~1 ml of 25 mM CaCI, was added and the clotting time was recorded . For (E) whole blood clotting time measurement, 0"1 ml of venom or saline was mixed with 0"9 ml of blood withdrawn from rabbit marginal ear vein . Each value is based upon 3-5 determinations .
Venom induce marked prolongation of the calcium clotting time, partial thromboplastin time, plasma prothrombin time and Russell's viper venom clotting time (Table 1). Whole blood clotting time was not prolonged at a concentration of 10 ltg per ml, but was markedly prolonged at 300 pg/ml, which also caused hemolytis. There was no coagulant action of the venom in concentrations from 0" 1 to 10001tg/ml . The venom did not destroy fibrinogen even when it was incubated with fibrinogen (10 mg/ml) for 1 hr at a concentration of 1 mg/ml. No appreciable inactivation of thrombin was produced by the venom (1 mg/ml) . There was also no significant effect on the interaction between thrombin and fibrinogen at a venom concentration as high as 0"8 mg/ml. Thromboplastin had strong procoagulant activity, the clotting time of platelet-poor plasma being prolonged about 3 times even when thromboplastin was diluted 100-200 times. The venom prolonged plasma prothrombin times at low concentrations of thromboplastin and this anticoagulant action could be counteracted by an increase in the concentrations of thromboplastin (Fig. 1). The neutralization of the anticoagulant effect was similar when cephalin or ruptured platelet was used . However, the procoagulant and neutralizing effects on the venom of cephalin and platelet were much weaker than tissue thromboplastin . The neutralizing effect of these phospholipid-containing substances toward the venom was competitive . At a fixed concentration of the venom, the increase of the concentration of these substances decreased the anticoagulant action, while at a fixed concentration of these substances, the increase of the concentration of the venom increased the anticoagulant action .
Short Conununications
199
ug/ml
,_
è
_,
_,
,
i
_,
,
,
~ 8 ,6 32 U. 1~ 256 TFN'OfT1t10pldStl~
FIG . 1 . NEUTRALIZATION OF THE ANTICOAßULANT EFFECT' OF THE VENOM HY ItABHTr BRAIN THROMIiOPLA3TTN.
Clotting system : 0~1 ml platelet-poor plasma + 0"1 ml thromboplastin -~- 0" 1 ml venom (10-300 ug/ml) ~- 0" l ml C'+ (25 mM). Means of 3~ experiments are presented. Variations were within 10~ of the mean value.
The venom immediately inactivated thromboplastin and the plasma prothrombin times were prolonged further from 11"8 ~ 1 " 1 to 22"3 ~ 1 "5 sec after 30 min incubation with thromboplastin . If the pre-incubation mixture of thromboplastin plus venom was diluted 20 fold (final concentration of venom 1 "251tg/ml ; this concentration show no anticoagulant effect-Table 1), the plasma prothrombin time was also prolonged significantly (Table 2). TABLE 2. EFFECT OF THE PRE-INCUBATION OF TI3ROINHOPLASTIN WITH THE VENOM, ON PLASMA PROTHROMBIN TIME . ALL VALUES ARE PRESENTED AS M1.ANS ~ S .E .
Preincubation times (min)
Dilution
Treatment
None (n=4)
Saline Crude venom'
6" 4102 11 " 811"1
6"010"3 165112
6010" 2 17 "9118
6" 310"2 18 " 811"5
6" 110"2 19911" 7
6" 210"1 22"311"5
20 fold (n=3)
Saline Crude venomf
15 " 1 f02 15910" 1 31 " 714"4 38"912"8
16 "Of0~3 46"913" 4
15 " Sf02 48 " 513 " 7
15 " 710" 5 53"5141
16 " Of02 53 "614"1
0
5
10
15
20
30
'Venom wncentrat!on in the pI'e-incubation mixture 50 ug/ml and in the final mixture 25 u8/ml . tVenom concentration in the pre-incubation mixture SO RS/ml and in the final mixture 125 ug/ml.
The anticoagulant activity of the venom measured by plasma prothrombin time, was not affected significantly after boiling at pH 7"4 and 5"6 for 15 min . However, this anticoagulant activity declined progressively after boiling for 30 min, but was not lost completely even after boiling for 180 min . The indirect hemolytic activity of the venom was completely destroyed after boiling at pH 7"4 for 120 min . At pH 5"6, the indirect hemolytic activity of the venom was not significantly affected after boiling for 180 min . An i.v. dose of 0"5 mg of the venom, per kg of body weight, did not cause prolongation of whole blood clotting time, however, four of the eight rabbits died within 10 min after the i .v. injection of 1 mg/kg of the venom . Whole blood clotting times of the four surviving rabbits were prolonged with maximum in 15 min after injection (from 3"3 to 9" 1 min), but returned to its initial value within 1 hr.
200
Short Communications
The present study showed that bee (Apis mellifera) venom had no coagulant activity toward plasma or fibrinogen, but had an anticoagulant effect . It did not destroy fibrinogen, inactivate thrombin nor interfere with the interaction between thrombin and fibrinogen . Hence, its anticoagulant effect does not occur in the fiaal step of blood coagulation (fibrinogen to fibrin transformation or proteolytic action of thrombin). The anticoagulant action of the venom was due to the inhibition of prothrombin activation, while tissue thromboplastin, cephalin and ruptured platelets were able to counteract the anticoagulant effect of the venom. Since the procoagulant activities of platelets and tissue thromboplastin were mainly to provide phospholipds (VECCHIOIVS and ZucxER, 1975 ; HVATUM and PRYD7., 1969), it is concluded that the anticoagulant action was mainly due to the inhibition of the procoagulant activity of phospholipids. JOSHUA and ISHAY (1975) also reported that l'. orientalis (Oriental hornet) venom possessed anticoagulant effect, manifested by inactivation of formed tissue thromboplastin as well as by inhibition of the formation of endogenous thromboplastin from its plasma precursors. Perhaps, the same mechanism is involved in the anticoagulant actions of these two Hymenopteran venoms. Recently, OUYANG et al. (1978) also demonstrated a similar property of the anticoagulant principle purified from Trimeresurus mucrosquamatus snake venom. The principle possessed inactivating action on the procoagulant activity of phospholipids, mediated partly by phospholipid-binding activity of the venom and partly by its enzymatic hydrolysis of phospholipids. Concerning the immediate anticoagulant action of bee venom, it seemed that the thromboplastin-bee venom complex was firmly bound because dilution of the mixture did not restore the procoagulant activity of tissue thromboplastin. On the contrary, the anticoagulant principle of Agkistrodon acuttrs snake venom (OUYANG and TsNG, 1972) did not bind tissue thromboplastin or plasma coagulation factors firmly and dilution of the anticoagulant-thromboplastin or plasma restored completely the procoagulant activities of thromboplastin and plasma (OUYANG and TsrrG, 1973). Since the preincubation caused less further prolongation of the clotting time, it suggested that the anticoagulant effect was mainly due to the immediate antagonistic action rather than the late degradation. The relationship between phospholipase A and the anti-thromboplastin action of snake and hymenopteran venoms have been the subject of considerable dispute. The authors suggested that phospholipase A, through phospholipid destruction, is probably the main causative agent responsible for the anticoagulant action of these venoms (BoQu>~r et al., 1967 ; BRISHOIS et al., 1968 ; HECHT and SLOITA, 1962 ; HABERMANN, 1968). The anticoagulant activity of Apis mellifera venom was more heat-stable than the phospholipase A (indirect hemolytic) activity. Whether phospholipase A of the bee venom is the main anticoagulant cannot be conclusively answered at present. Perhaps, heat treatment may inactivate the enzymatic activity, but not markedly affect the phoshpholipid-binding capacity of phospholipase A (OuYnIVG et al., 1978). Acknowledgement-This work was supported in part by a grant from China Medical Board of New York, Inc. REFERENCES
Amas, N. G., PERRY, D. W., PwcxxwM, M. A. and Musrwxn, J. F. (1971) Influence of apyrase on stability of suspensions of washed rabbit platelets. Proc. Soc. exp. Bloc. Med.136, 1021 . Bets., W. R. and AL~rox, H. G. (1954) A brain extract as a substitute for platelet suspension in the thromboplastin generation test . Nature, Land. 174, 880. Btaas, R. and MwcawtuaNt:, R. G. (1962) Human Blood Coagulation and its Disorders, 3rd Edn. Oxford : Blackwell. BoQusr, P. (1964) Venins de serpents (l~rc partie) physio-pathologie de l'envenimation et proprieties biologiques des venins . Toxicon 2, 5.
201 Short Communications BoQuEr, P., Iz,uen, Y., ME~u~, J. and Jov~NNE~r, M . (1%7) Recherche biochimiques et immunologigues sur de venin des serpents . Ann. Inst. Pasteur 112, 213 . BR,ISBOL9, L., ReanvovrrcEt-Mutt, N., DELORI, P. and Gni.o, L. (1968) J. Chromas. 37, 463 . BxowN, J. H. and Bows, M. E. (19C~ Studies on the phoshpolipase A activity of Crotales atrox venom. Toxlcon 3, ~5 . H~sERrrwNN, E. (1954) Untersuchungen >Sber die Hemmung der Blutgerinnung durch Bienengift . Arch. expl Path . Pharmak. 223, 182. HECxr, E. and SLOTTA, C. H. (1%2) The chemical nature of the lipid activator of blood coagulation . Acts physiol. pharmac. nal. 10, 278. HOUSSAY, B. A. (1930) Classification des actions des venins de serpents sur l'organisme animal. C.r . Slanc. Soc. Blol. 105, 308. HvnrvM, M. and Pxvnz, H. (1%9) Studies on tissue thromboplastin-its splitting into two separable parla. ?Tirombos. Diathes. luremorrh. 21, 217. JutéNEZ-PORR,AS, J. M. (1970) Hiochemiatry of snake venom. Cltn. Toxicon 3, 389. Jost3tr~, H. and IsFUY, J. (1975) Theanticoagulant properties of an extract from the venom sac of the oriental hornet . Toxicon 13, 11 . LEE, C. Y. (1975) Pharmacological classification of toxic proteins from snake vcnoms . Jap. J. Trop. Med. Hyg. 3, 219. LEE, R. I. and Wt~rE, P. D. (1913) A clinical study of the coagulation time of blood . Am. J. Med. Sct. 145, 495. LEE, C. Y., JotflvsoN, S. A. and S~oEtes, S. H. (1955) Clotting of blood with Russell's viper venom. J. Mtch . State med. Soc. 54, 801 . MEauuufl3, J. (1966) Les venins de serpents agents modificateurs de la coagulation sanguine . Toxkon 4, 25 . Mvsr~xn, J. F., PEtmv, D. W., Annc.u?, N. G. and Pit , M. A. (1972) Preparation of suspensions of washed platelets from humans. Br. J. Haemat. Z2, 193. NvE, S. W., Ga~M, J. B. and Btsnacaous, K. M. (1962) The partial thromboplastin time as a screening test for the detection of latent bleeders . Am . J. med. Scl. 243, 279. O'Bten:N, J. R. (1957) The effect of some fatty acids and phospholipids on blood coagulation. Br . J. exp. Path. 12, 45 . OUYANü, C. (1957) The efforts of Formosan snake venoma on blood coagulation in vitro. J. Formosan Med. Assoc. 56, 435. OUYAN(i, C. and TENa, C. M. (1972) Purification and properties of theanticoagulant principle of Agkistrodon acutus venom. Biochim. blophys. Acts 278, 155. Ou~rerro, C. and TENO, C. M. (1973) The effect of the purified anticoagulant principles of Agklstrodon acutus venom on blood coagulation. Toxicon 11, 287. OtnnNO, C. and TENO, C. M. (1976) Fibrinogenolytic enzymes of 7lrimeresurus mucrosqucanatus venom. Blochim. biophys. Acts 420, 298. Ovrnxo, C., TENa, C. M., CAEN, Y. C. and Lar, S. C. (1978) Purification and properties of the anticoagulant principle of 7Ylmtresurus mucrosquamatus venom. Blochim. blophys. Acts 541, 394. Qvecx, A. J. (1938) The nature of bleeding in jaundice. J. Am. Med. Ass.110,1658. ROSENFELD, G., N.~x~s, I. and KELEN, E. M. A. (l%8) Coagulant, Proteolytlc and Hemolytic Properties of Some Snake Venoms in Venomous Vertebrates, Vol. I, p.231, (BÛct~ExL, W., BOcxELY, E. E. and DEULOFUE, V., Eds.). London : Academic Press. SEEGHRS, W. H. and Six, H. P. (1942) Factors which influence the activity of purified thrombin . Am. J. Physiol. 137, 348. $EEOERS, W. H., MILLER, K. O., ANDR~ws, E. B. and MuttFav, R. C. (1952) Fundamental interactions and the effect of stooge, ether, adsorbants and blood clotting on plasma anti-thrombin activity . Am . J. Physiol. 169, 700. Tv, A. T. (1977) Vinoms : Chemistry and Molecular Biology, p. 329. New York : John Wiley & Sons . VE~oNE, J. and ZvcxER, M . B. (1975) Procoagulant activity of platelets in recalcified plasma . Br . J. Haemat. 31, 423 .
197
Ftt~ C~ancvs eH (197 Octyl-Sepharose CL-4B, Ph~yl-Sepharose CL-4B for hydrophobic interaction chromatography. Upplands Gr~rska AB . Vissait, L . and Louve, A . I . (1977) The importance of protein structure and conformation in the preparation of phospholipase-free cardiotoxin from snake venom . Biochim. btophys . Acta 491, 349 . PHARMACIA
roxlcon, vot . t7, ~ . t97-xoz . © Pet~amon Press Ltd. 1979 . 1rioted in Greet Britain .
coat-ototl791o3ot-0t97so2ool0
ANTICOAGULANT PROPERTIES OF APIS MELLIFERA (HONEY BEE) VENOM CHAOHO OUYANG, SONG-Ci-IOW
LIN"
and CI-IH-MING TENG
Phanvacological Institute, College of Medicine, National Taiwan University, Taipei, Taiwan, Republic of China (Accepted jor publication 24 July 1978)
A vAlt>srY of animal venoms, mostly of snake origin, modify blood coagulation (HOUSSAY, 1930; L~ et al., 1955 ; OUYANG, 1957 ; BoQusr, 1964; MF,AiJME, 1966 ; ROSENFELD et al., 1968 ; JI z-PO1tRAS, 1970; LEE, 1975 ; Tu, 1977) . JOSHUA and I3HAY (1975) demonstrated that an extract from the venom sac of the oriental hornet (Vespa orientalis) had an anticoagulant effect on blood coagulation. HABERMANN (1954) reported that honey bee (Apia mellifera) venom inhibited blood coagulation through inactivation of tissue thromboplastin probably by its phospholipase A component . Since the effect of this venom on other clotting factors is not well known, it was of interest to investigate the anticoagulant action of this venom and elucidate its mode of action . Apis mellifera venom, crude cephalin (chloroform extract of acetone-dried rabbit brain) and Russell's viper venom in crude cephalin were obtained from Sigma Chem. Co. Thrombin (bovine) was purchased from Parke-Davis Co. and dissolved in 50~ glycerolsaline to give a stock solution of 1000 N.LH . p/ml (S~GGI~ts and SMrrH, 1942) . Fibrinogen (bovine) was purchased from Pentex Co. and dissolved in imidazole-saline (pH 7"4j and the stock solution of 20 mg/ml was stored at -70°C before use . Rabbit brain thromboplastin was prepared according to the method of BIGGS and MACi?A1tLAN13 (1962) . Plateletpoor plasma was obtained from citrated blood (1 :9, v/v) after centrifugation for 20 min at 3000 rpm and 4°C. Blood collected from the rabbit marginal ear vein through a 19-gauge needle was mixed with 0'1 M Na9EDTA (1 :14, v/v) as anticoagulant and centrifuged for 10 min at 800 g and at room temperature . The platelet pellet was washed with Tris-buffered saline (pH 7"4) containing NaEEDTA (3 mM), apyrase (ADPase activity, 0~5 U/ml) and bovine serum albumin (1 mg/ml) and recentrifuged (modification of the methods of MUSTARD et al., 1972 and ARDIJE et al., 1971) . This wash procedure was repeated two times and the washed platelets were suspended in Tris-buffered saline, ruptured with a stirrer and then frozen and thawed three times before use . Whole blood clotting time was determined by the method of LEe and WFnre (1913) . Calcium clotting time of plasma was carried out as described by BIGGS and MACFARLANE (1962). The one-stage method of Qulcx (1938) was used to measure the plasma prothrombin time. Partial thromboplastin time was measured by the method of NYS et al. (1962) . " Present address : Department of Pharmacology, Taipei Medical College, Taipei, Taiwan, Republic of China .
19 8
Short Communications
The Russell's viper venom clotting time was determined by the method Of O'BRIBN (1957) . For the determination of the antithrombin actions, the method of SEEGERS et al. (1952) was used. Fibrinogenolytic activity was measured as deSCilbed by OUYANG and TENG (1976). Venom interference of the interaction between thrombin and fibrinogen was performed by the method Of SEEGEltS and SMITH (1942). The indirect hemolytic activity on washed red blood cells was measured by the method of BROWN and BOWEhES (19GG) . TABLE 1. EFFECTS OF Apls 71iCII%fCra VENOM ON PLASMA PROTHROMHIN TiAlB (A), RUSSELL'S VIPER VEI`OM CLOTTING TIMB ; (B), PARTIAL THROMBOPLASITN TIbIß (C) AND RECALCIFICATION TIME (D) OF RABHTr PLATELEI'POOR PLASMA AND ON THE CLOTTING TIIME OF THE WHOLE BLOOD (E)
Test system Venom conc . (lIg/ml)
A sec 12 "7 f 0~2
B
C
D
Clotting time (means f S.E .) sec sec sec 15 "6 f 0"5 54 "6 ~ 4"0 79 "8 ~ 2~1 82 "5 ~; 4~l 91 "1 ~ 4~5 72"6 ~ 3"4 96 "4 f S~8 16"0 f 0~4 98 "2 f 9~1 140"5 ~ 7"2 18 "2 ~ 0"3 125"1 ~ il "6 l54"5 f 9~7 26 "4 ~ 0~6 > 600 > 600 48 "3 f 0"8 > 600
min 4" 2 ~ 0~3 0 0"1 0"3 1 13 "9 f 0"3 3 19 "9 f 1 "0 10 26"9 f 1"1 5"7 f 0"5 30 30 "6 f 1"5 11 "7 f 1 "6 100 39 "4 ~ 4"8 24 "S f 4"6 300 51 "1 f 2"5 1555 f 21 "4t 1000 98 "9 f 9"6* *Granulated fibrin clot . fHemolysis after clotting . Clotting system : 0"1 ml of platelet-poor plasma was warmed to 37°C, then 0~1 ml of saline or bee venom of various wncentrations was added. To this mixture, 0"1 ml of (A) tissue thromboplastin (S ~), (B) cephalin with Russell's viper venom or (C) cephalin or (D) saline was added. Finally, 0~1 ml of 25 mM CaCI, was added and the clotting time was recorded . For (E) whole blood clotting time measurement, 0"1 ml of venom or saline was mixed with 0"9 ml of blood withdrawn from rabbit marginal ear vein . Each value is based upon 3-5 determinations .
Venom induce marked prolongation of the calcium clotting time, partial thromboplastin time, plasma prothrombin time and Russell's viper venom clotting time (Table 1). Whole blood clotting time was not prolonged at a concentration of 10 ltg per ml, but was markedly prolonged at 300 pg/ml, which also caused hemolytis. There was no coagulant action of the venom in concentrations from 0" 1 to 10001tg/ml . The venom did not destroy fibrinogen even when it was incubated with fibrinogen (10 mg/ml) for 1 hr at a concentration of 1 mg/ml. No appreciable inactivation of thrombin was produced by the venom (1 mg/ml) . There was also no significant effect on the interaction between thrombin and fibrinogen at a venom concentration as high as 0"8 mg/ml. Thromboplastin had strong procoagulant activity, the clotting time of platelet-poor plasma being prolonged about 3 times even when thromboplastin was diluted 100-200 times. The venom prolonged plasma prothrombin times at low concentrations of thromboplastin and this anticoagulant action could be counteracted by an increase in the concentrations of thromboplastin (Fig. 1). The neutralization of the anticoagulant effect was similar when cephalin or ruptured platelet was used . However, the procoagulant and neutralizing effects on the venom of cephalin and platelet were much weaker than tissue thromboplastin . The neutralizing effect of these phospholipid-containing substances toward the venom was competitive . At a fixed concentration of the venom, the increase of the concentration of these substances decreased the anticoagulant action, while at a fixed concentration of these substances, the increase of the concentration of the venom increased the anticoagulant action .
Short Conununications
199
ug/ml
,_
è
_,
_,
,
i
_,
,
,
~ 8 ,6 32 U. 1~ 256 TFN'OfT1t10pldStl~
FIG . 1 . NEUTRALIZATION OF THE ANTICOAßULANT EFFECT' OF THE VENOM HY ItABHTr BRAIN THROMIiOPLA3TTN.
Clotting system : 0~1 ml platelet-poor plasma + 0"1 ml thromboplastin -~- 0" 1 ml venom (10-300 ug/ml) ~- 0" l ml C'+ (25 mM). Means of 3~ experiments are presented. Variations were within 10~ of the mean value.
The venom immediately inactivated thromboplastin and the plasma prothrombin times were prolonged further from 11"8 ~ 1 " 1 to 22"3 ~ 1 "5 sec after 30 min incubation with thromboplastin . If the pre-incubation mixture of thromboplastin plus venom was diluted 20 fold (final concentration of venom 1 "251tg/ml ; this concentration show no anticoagulant effect-Table 1), the plasma prothrombin time was also prolonged significantly (Table 2). TABLE 2. EFFECT OF THE PRE-INCUBATION OF TI3ROINHOPLASTIN WITH THE VENOM, ON PLASMA PROTHROMBIN TIME . ALL VALUES ARE PRESENTED AS M1.ANS ~ S .E .
Preincubation times (min)
Dilution
Treatment
None (n=4)
Saline Crude venom'
6" 4102 11 " 811"1
6"010"3 165112
6010" 2 17 "9118
6" 310"2 18 " 811"5
6" 110"2 19911" 7
6" 210"1 22"311"5
20 fold (n=3)
Saline Crude venomf
15 " 1 f02 15910" 1 31 " 714"4 38"912"8
16 "Of0~3 46"913" 4
15 " Sf02 48 " 513 " 7
15 " 710" 5 53"5141
16 " Of02 53 "614"1
0
5
10
15
20
30
'Venom wncentrat!on in the pI'e-incubation mixture 50 ug/ml and in the final mixture 25 u8/ml . tVenom concentration in the pre-incubation mixture SO RS/ml and in the final mixture 125 ug/ml.
The anticoagulant activity of the venom measured by plasma prothrombin time, was not affected significantly after boiling at pH 7"4 and 5"6 for 15 min . However, this anticoagulant activity declined progressively after boiling for 30 min, but was not lost completely even after boiling for 180 min . The indirect hemolytic activity of the venom was completely destroyed after boiling at pH 7"4 for 120 min . At pH 5"6, the indirect hemolytic activity of the venom was not significantly affected after boiling for 180 min . An i.v. dose of 0"5 mg of the venom, per kg of body weight, did not cause prolongation of whole blood clotting time, however, four of the eight rabbits died within 10 min after the i .v. injection of 1 mg/kg of the venom . Whole blood clotting times of the four surviving rabbits were prolonged with maximum in 15 min after injection (from 3"3 to 9" 1 min), but returned to its initial value within 1 hr.
200
Short Communications
The present study showed that bee (Apis mellifera) venom had no coagulant activity toward plasma or fibrinogen, but had an anticoagulant effect . It did not destroy fibrinogen, inactivate thrombin nor interfere with the interaction between thrombin and fibrinogen . Hence, its anticoagulant effect does not occur in the fiaal step of blood coagulation (fibrinogen to fibrin transformation or proteolytic action of thrombin). The anticoagulant action of the venom was due to the inhibition of prothrombin activation, while tissue thromboplastin, cephalin and ruptured platelets were able to counteract the anticoagulant effect of the venom. Since the procoagulant activities of platelets and tissue thromboplastin were mainly to provide phospholipds (VECCHIOIVS and ZucxER, 1975 ; HVATUM and PRYD7., 1969), it is concluded that the anticoagulant action was mainly due to the inhibition of the procoagulant activity of phospholipids. JOSHUA and ISHAY (1975) also reported that l'. orientalis (Oriental hornet) venom possessed anticoagulant effect, manifested by inactivation of formed tissue thromboplastin as well as by inhibition of the formation of endogenous thromboplastin from its plasma precursors. Perhaps, the same mechanism is involved in the anticoagulant actions of these two Hymenopteran venoms. Recently, OUYANG et al. (1978) also demonstrated a similar property of the anticoagulant principle purified from Trimeresurus mucrosquamatus snake venom. The principle possessed inactivating action on the procoagulant activity of phospholipids, mediated partly by phospholipid-binding activity of the venom and partly by its enzymatic hydrolysis of phospholipids. Concerning the immediate anticoagulant action of bee venom, it seemed that the thromboplastin-bee venom complex was firmly bound because dilution of the mixture did not restore the procoagulant activity of tissue thromboplastin. On the contrary, the anticoagulant principle of Agkistrodon acuttrs snake venom (OUYANG and TsNG, 1972) did not bind tissue thromboplastin or plasma coagulation factors firmly and dilution of the anticoagulant-thromboplastin or plasma restored completely the procoagulant activities of thromboplastin and plasma (OUYANG and TsrrG, 1973). Since the preincubation caused less further prolongation of the clotting time, it suggested that the anticoagulant effect was mainly due to the immediate antagonistic action rather than the late degradation. The relationship between phospholipase A and the anti-thromboplastin action of snake and hymenopteran venoms have been the subject of considerable dispute. The authors suggested that phospholipase A, through phospholipid destruction, is probably the main causative agent responsible for the anticoagulant action of these venoms (BoQu>~r et al., 1967 ; BRISHOIS et al., 1968 ; HECHT and SLOITA, 1962 ; HABERMANN, 1968). The anticoagulant activity of Apis mellifera venom was more heat-stable than the phospholipase A (indirect hemolytic) activity. Whether phospholipase A of the bee venom is the main anticoagulant cannot be conclusively answered at present. Perhaps, heat treatment may inactivate the enzymatic activity, but not markedly affect the phoshpholipid-binding capacity of phospholipase A (OuYnIVG et al., 1978). Acknowledgement-This work was supported in part by a grant from China Medical Board of New York, Inc. REFERENCES
Amas, N. G., PERRY, D. W., PwcxxwM, M. A. and Musrwxn, J. F. (1971) Influence of apyrase on stability of suspensions of washed rabbit platelets. Proc. Soc. exp. Bloc. Med.136, 1021 . Bets., W. R. and AL~rox, H. G. (1954) A brain extract as a substitute for platelet suspension in the thromboplastin generation test . Nature, Land. 174, 880. Btaas, R. and MwcawtuaNt:, R. G. (1962) Human Blood Coagulation and its Disorders, 3rd Edn. Oxford : Blackwell. BoQusr, P. (1964) Venins de serpents (l~rc partie) physio-pathologie de l'envenimation et proprieties biologiques des venins . Toxicon 2, 5.
201 Short Communications BoQuEr, P., Iz,uen, Y., ME~u~, J. and Jov~NNE~r, M . (1%7) Recherche biochimiques et immunologigues sur de venin des serpents . Ann. Inst. Pasteur 112, 213 . BR,ISBOL9, L., ReanvovrrcEt-Mutt, N., DELORI, P. and Gni.o, L. (1968) J. Chromas. 37, 463 . BxowN, J. H. and Bows, M. E. (19C~ Studies on the phoshpolipase A activity of Crotales atrox venom. Toxlcon 3, ~5 . H~sERrrwNN, E. (1954) Untersuchungen >Sber die Hemmung der Blutgerinnung durch Bienengift . Arch. expl Path . Pharmak. 223, 182. HECxr, E. and SLOTTA, C. H. (1%2) The chemical nature of the lipid activator of blood coagulation . Acts physiol. pharmac. nal. 10, 278. HOUSSAY, B. A. (1930) Classification des actions des venins de serpents sur l'organisme animal. C.r . Slanc. Soc. Blol. 105, 308. HvnrvM, M. and Pxvnz, H. (1%9) Studies on tissue thromboplastin-its splitting into two separable parla. ?Tirombos. Diathes. luremorrh. 21, 217. JutéNEZ-PORR,AS, J. M. (1970) Hiochemiatry of snake venom. Cltn. Toxicon 3, 389. Jost3tr~, H. and IsFUY, J. (1975) Theanticoagulant properties of an extract from the venom sac of the oriental hornet . Toxicon 13, 11 . LEE, C. Y. (1975) Pharmacological classification of toxic proteins from snake vcnoms . Jap. J. Trop. Med. Hyg. 3, 219. LEE, R. I. and Wt~rE, P. D. (1913) A clinical study of the coagulation time of blood . Am. J. Med. Sct. 145, 495. LEE, C. Y., JotflvsoN, S. A. and S~oEtes, S. H. (1955) Clotting of blood with Russell's viper venom. J. Mtch . State med. Soc. 54, 801 . MEauuufl3, J. (1966) Les venins de serpents agents modificateurs de la coagulation sanguine . Toxkon 4, 25 . Mvsr~xn, J. F., PEtmv, D. W., Annc.u?, N. G. and Pit , M. A. (1972) Preparation of suspensions of washed platelets from humans. Br. J. Haemat. Z2, 193. NvE, S. W., Ga~M, J. B. and Btsnacaous, K. M. (1962) The partial thromboplastin time as a screening test for the detection of latent bleeders . Am . J. med. Scl. 243, 279. O'Bten:N, J. R. (1957) The effect of some fatty acids and phospholipids on blood coagulation. Br . J. exp. Path. 12, 45 . OUYANü, C. (1957) The efforts of Formosan snake venoma on blood coagulation in vitro. J. Formosan Med. Assoc. 56, 435. OUYAN(i, C. and TENa, C. M. (1972) Purification and properties of theanticoagulant principle of Agkistrodon acutus venom. Biochim. blophys. Acts 278, 155. Ou~rerro, C. and TENO, C. M. (1973) The effect of the purified anticoagulant principles of Agklstrodon acutus venom on blood coagulation. Toxicon 11, 287. OtnnNO, C. and TENO, C. M. (1976) Fibrinogenolytic enzymes of 7lrimeresurus mucrosqucanatus venom. Blochim. biophys. Acts 420, 298. Ovrnxo, C., TENa, C. M., CAEN, Y. C. and Lar, S. C. (1978) Purification and properties of the anticoagulant principle of 7Ylmtresurus mucrosquamatus venom. Blochim. blophys. Acts 541, 394. Qvecx, A. J. (1938) The nature of bleeding in jaundice. J. Am. Med. Ass.110,1658. ROSENFELD, G., N.~x~s, I. and KELEN, E. M. A. (l%8) Coagulant, Proteolytlc and Hemolytic Properties of Some Snake Venoms in Venomous Vertebrates, Vol. I, p.231, (BÛct~ExL, W., BOcxELY, E. E. and DEULOFUE, V., Eds.). London : Academic Press. SEEGHRS, W. H. and Six, H. P. (1942) Factors which influence the activity of purified thrombin . Am. J. Physiol. 137, 348. $EEOERS, W. H., MILLER, K. O., ANDR~ws, E. B. and MuttFav, R. C. (1952) Fundamental interactions and the effect of stooge, ether, adsorbants and blood clotting on plasma anti-thrombin activity . Am . J. Physiol. 169, 700. Tv, A. T. (1977) Vinoms : Chemistry and Molecular Biology, p. 329. New York : John Wiley & Sons . VE~oNE, J. and ZvcxER, M . B. (1975) Procoagulant activity of platelets in recalcified plasma . Br . J. Haemat. 31, 423 .