Effect of venom sac extract of the Oriental hornet (Vespa orientalis) on coagulation factors

T°stc°°, Vol . 26, No . 12, pp. Printed in Great Britain.

1101176, 1988 .

1"

0041-0101/88 53 .00+ .00 1988 Pergamon Press p~

EFFECT OF VENOM SAC EXTRACT OF THE ORIENTAL HORNET (VESPA ORIENTALIS) ON COAGULATION FACTORS ABRAHAM KORNBERG, 1 SUZANA KAUFMAN, 1 LILI SILBI=.Rr

and JACOB

S. IsHAY 2

'Department of Hematology, Assaf Harofeh Medical Center, Zerifin, 70300 Israel, and zDepartment of Physiology and Pharmacology, Sackler Faculty of Medicine, Tel Aviv University, Ramat-Aviv, 69978 Israel (Acceptedfor publication 12 ,luly 1988) A. KORNHERG, S. KAUFMAN, L . Sn .Bex and J. S. Isruv . Effect of venom sac extract of the Oriental hornet (Vespa orientalis) on coagulation factors. Toxicon 26 1169-1176, 1988 .-Venom sac extract of the Oriental hornet significantly prolongs the prothrombin time and the activated partial thromboplastin time both in vitro in human plasma and in vivo in cats . Activity of factors VIII and TX in plasma is reduced to less than 1 °/a within 5 min even with 1 pg of venom sac extract per ml . The activity of purified factor VIII, as well as semipurified factors DC and X, in factor IX complex was also significantly reduced after incubation with the venom . The decrease of factors II, V, VII, X, XI and XII activity to 9%, 11 %, 11 %, 29%, 1 .7% and 0.7% of normal, respectively, is dose- and timedependent. Thrombin time, plasma fibrinogen and fibrin degradation products are not affected . The anticoagulant activity is not reversed by dialysis and is abolished completely by heating; it resides mainly in fractions with mol .wts above 5000. The venom has a proteolytic activity on ~~-globin which is partially inhibited by trasylol and ethylenediaminetetraacetic acid. Thus, the venom sac extract exhibits both serine and metaloprotease activities which may affect the activity of the plasma coagulation factors .

INTRODUCTION

sac extract (VSE) of Vespa orientalis (Hymenoptera, Vespinae) has a potent anticoagulant activity . JOSHUA and ISHAY (1975) and EDERY et al. (1978) have shown that VSE inactivates exogenous as well as endogenous thromboplastin. The venom has no effect on plasma fibrinogen but has a fibrinogenolytic activity on purified fibrinogen. Its prolongation of both prothrombin time (P'T) and recalcification time and its effect on the thromboplastin generation test (TGT) suggest that VSE also affects other clotting factors. The aim of the present study was to investigate the effect of VSE on specific clotting factors. THE vENOM

MATERIALS AND METHODS A venom sac extract from adult hornets was kept in lyophilized form until used (JOSHUA and ISHAY, 1975). The protein content of the extract was estimated by the method of Lowav et al . (1951) and the concentration adjusted to 5 mg/ml. A fresh solution in saline (pH 7 .0) was prepared before each experiment . The following coagulation tests were employed : one stage PT (Qulctc, 1963), activated partial thromboplastin time (APTT) (Pxocrox and RMAPORT, 1961), thrombin time (TT) and fibrinogen degradation products (FDP) (Houcxle, 1983), fibrinogen (RATNOFF and MENZIIas, 1951) and factors II, V, VII, VIII, IX, X, XI and XII . Clotting factors were assayed by the one stage method (D~ISON and BIGG3, 1976) using factor deficient human plasma . Activated thromboplastin (for PT) was purchased from Dade Diagnostics, Agnada, Puerto Rico . Rabbit brain cephalin (for APTT) and thrombin were obtained from Sigma Co ., St Louis, MO, U .S .A . Fibro-Tec (for FDP) 1169

1170

A . KORNBERG et ai .

was from Behring Co ., W . Germany. Human plasma deficient in factors V and X was purchased from Diagnostics Stage, France . Human plasma deficient in factors II, VII, VIII and IX was from Pacific Hemostasis, U .S.A . Human plasma deficient in factors XI and XII was obtained from Merz-Dade, Dudingen, Switzerland . Studies were performed in vitro with human plasma and in vivo in cats (O'RovxKe et ai ., 1982). Blood was collected in 3 .8% sodium citrate solution, centrifuged at 6400g (Sorvall RCSB, Du Pont) for l0 min, and assayed immediately or stored at -20°C . For the in vitro studies one volume of the venom sac extract at various concentrations was added to nine volumes of normal human plasma . The final concentration of extract in the mixture thus ranged from 1 to 100 pg/ml of plasma . The mixtures were incubated for 60 min at 37°C before running the aforementioned tests. For each experiment plasma to which a one-tenth volume of saline was added served as a control. In order to study the effect of the extract on plasma at different exposure times, the plasma was mixed with a one-tenth volume of extract at a final concentration of 50 ug/ml and the various tests were performed after incubation times varying from 5 to 120 min . Also studied was the in vitro effect of the venom sac extract on purified factor VIII (Hemophil M, Hyland Therapeutic, U.S .A.) and semipurified factors IX and X in factor IX complex (Proplex, Hyland Therapeutics, U .S.A.). Purified factor VIII was added to factor VIII-deficient plasma and factor IX complex to factor IXdeficient plasma . The APTT and the activity of the factors were assayed before and after incubation of the mixtures for I S min with 1 Pg/ml and 50 pg/ml of the extract . For the in vivo studies eight healthy adult cats (3~ kg) were anesthetized with sodium pentobarbital . The femoral vein of each animal was cannulated with a polyethylene catheter and 5 mg of venom sac extract (per ml, dissolved in saline) per kg body weight were administered i .v . through the catheter . Blood was collected before and at various intervals (from 5 to 120 min) after the administration of the extract . During the experiments the catheter was flushed with saline in order to prevent clot formation . For fractionation studies lyophilized venom sac extract was dissolved in saline (pH 7 .0) in a concentration of 500 pg/ml and chromatographed on Sephadex G-25 in Tris buffer, 0 .2 M, pH 7.0. Fractions of 1 ml were assayed as follows : APTT, spectrophotometric determination at 280, 340 and 420 nm (Gilford 2600) and protein quantitation (LowxY et al., 1951) . Normal human plasma without venom sac extract served as a control . Proteolytic activity of the extract was studied by measuring its ability to hydrolyze'°C-labeled globin, and by the inhibitory effect of the serine protease inhibitor trasylol (Bayer, W . Germany) and the metaloprotease inhibitor disodium ethylenediaminetetraacetic acid (Na,EDTA) on hydrolysis . The labeled globin substrate was prepared by the method of Ro~nt et al. (1971), in which hemoglobin undergoes carbamylation with K'~NO . The resulting labeled hemoglobin was dialyzed against several changes of distilled water for 48 hr and denatured by cold acid acetone to yield "C-labeled globin . The "C-labeled globin was suspended in water to a concentration of 14.5 mg/ ml (spec . act . 230 cpm/ug protein) . Twenty-five microliters of '~-globin were incubated with 0 .4 ml of the venom sac extract or buffer at 37°C for 2 hr and the reaction was terminated by the addition of 0 .2 ml of 50% trichloroacetic acid (TCA) . A 0 .4 ml aliquot of the TCA soluble fraction was counted in 4.5 ml of Instagel II (Packard, Downers Grove, IL, U .S.A .) . The effect of the inhibitors was studied by preincubation of 100 KIU trasylol with the venom sac extract in phosphate-buffered saline (PBS) and 50 iel of Na,EDTA with the extract in 0 .1 M Tris-buffer for 5 min. Statistical analysis was performed using the Student's t-test .

RESULTS

The effect of various concentrations of the venom sac extract on the coagulation process is shown in Table 1 . Both PT and APTT were significantly prolonged by 1 ~g of extract per ml. The effect was more profound on APTT. Maximal prolongation of PT was observed at 10 pg/ml and of APTT at 50 ~g/ml of extract . The activity of factors II, V, VII and X was reduced gradually from 96%, 94%, 105% and 100% without venom sac extract to 9%, 11 %, 11 % and 29%,respectively, with 100 ~g of the extract per ml. Factors VIII and IX decreased to less than 1 % even with 1 ~g/ml of the extract. The effect of the venom on factors XI and XII was studied only in one experiment. With 1, 10 and 50 fig of the venom sac extract per ml the activity of factor XI decreased from 96% to 58%, 5.6% and 1 .7% and that of factor XII from 90% to 58%, 14% and 0.7%,respectively . Thrombin time, plasma fibrinogen and the level of FDP were not affected . The effect on the coagulation process of varying the incubation time with a constant concentration of VSE (50 fig/ml) is shown in Table 2. APTT as well as PT were markedly prolonged after 5 min . The prolongations were maximal after 30 min . Factors VII and X were reduced from 96% and 89% without the extract to 48% and 62% after 5 min, and to 15% and 34% after 30 min of

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TABLE

2.

THE in Vitro EFFECTS OF VARYING INCUBATION TIMES WITH A CONSTANT VENOM SAC EXTRACT (VSE) CONCENTRATION (SOIIg/ml) ON COAGULATION ASSAYS AND FACTORS IN NORMAL HUMAN PLASMA

Incubation time (min)

l''l' (sec

Al?TT (sec)

TT (sec)

Fibrinogen (mg%)

VII (%)

X (%)

VII (%)

0 S 1S 30 60 90 I20

l3 .Of 1 .0 14 .9f0 .4 16 .2f0 .5' 16 .8f0 .8 17 .5f0 .8' 17 .4f1 .0 17 .6f1 .2

54f 10 16Sf16" 196f22' 244f30' 298f42' 286f36 271f40

14 .2f0 .6 1S .4f0 .4 14 .6f 1 .0 1S .Of1 .2 1S .Of0 .8 14 .6f0 .S 14 .6f0 .8

244f42 252f54 230f32 240f28 230f30

96f 12 48f 8" ISf 9' l3t 6 16f 4 l3f S

89f 9 62f10' 34f12" 32f 5 3St 6

IOSf l2
IOOf 10
Abbreviations-see Table 1 . 'Significant deviation from the preceding value (P
incubation . Factors VIII and IX decreased to less than 1 % after S min. Even after 120 min of incubation TT, fibrinogen and FDP were not affected . The effect of VSE on purified factor VIII and semipurified factors IX and X is shown in Table 3. The APTT of purified factor VIII in mixture with factor VIII-deficient plasma was prolonged from 63 sec to 143 sec and 146 sec, after incubation for 15 min with 1 ~g/ml and 50 ug/ml of extract, respectively . Purified factor VIII activity was reduced from 126% to 40% and to less than 0.1 % with the same concentrations of the extract. The venom exerted similar effects on the APTT and on the activity of factors IX and X in the mixtures of factor IX complex with factor IX and with factor X-deficient plasma . The effects of the venom on the coagulation process in cats are shown in Table 4. Intravenous injection of the extract (5 mg/ml) resulted in a significant prolongation of PT TABLE

3.

EFFECT OF VENOM SAC EXTRACT (VSE) ON PURIFIED FACTOR VIII AND PARTIALLY PURIFIED FACTORS IX AND X

Incubation mixture Factor V I I I-deficient plasma Factor VIII-deficient plasma + pure factor VIII Factor V III-deficient plasma + pure factor VIII + VSE I pg/ml Factor VIII-deficient plasma + pure factor VIII + VSE SO lIg/ml Factor IX- or X-deficient plasma Factor IX- or X-deficient plasma + factor IX complex (proplex) Factor IX- or X~eficient plasma + factor IX complex (proplex) + VSE l lIg/ml Factor IX- or X-deficient plasma + factor IX complex (proplex) + VSE 50 pg/ml

APTT (sec)

VIII (%)

IX (%)

X (%)

1 SS t 9 63 f 6'


143 f 12*

40 t 12"

146 f 10

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124 f 22 64 f 13'

< 0. l' 226 f 24'

< 0 .1 192 f 16'

178 f 17 "

60 f 10'

48 f 12 "

2S2 f 28'

< 0.1 *

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A mixture of factor VIII-deficient plasma with pure factor VIII and mixtures of factors IX- and X~eficient plasmas with factor IX complex were incubated in the presence of VSE (1 pg/ml and SOIIg/ml) for 1S min . APTf and Cactors VIII, IX and X activities were then assayed . AI?TT, activated partial thromboplastin time . " Significant deviation from the preceding value (P
Hornet Venom and Coagulation Factors TABLE

4.

THE 111 YlYO EFFECTS OF S

Time (min) 0 10 30 60 90 120

PT (sec)

APTT (sec)

9.4 f 0.S 19 f 9 10.6f0 .6 63f12` 14.2f0 .8` 68t1S 17.5t1 .2` 84f20` 17.2f1 .2 127f14' 17.8f1 .8 lßSf32`

mg/kg

1 173

OF VENOM SAC EXTRACT' (VSE) IN CATS ON COAGULATION AS4AY3 AND FACTORS

TT (sec) 18 f4 19f2 20f3 20f4 21f2 20f4

FDPt Fibrinogen (kg/ml) (mg%) 2.4 1 .2 1 .2 -

230f 70 304f60 301t68 2SIfSS 2SSt50

V (%)

VII (%)

X (%)

100 12E 9' 9f11 1Sf 6 18f12 18E 8

100 13f10' llf 6 8f 6 lOf 8 -

100 3Sf16' 2St12 18t 8 1Of10` 9f 4

VII (%)

IX (%)

100 100 <1` <1`

Abbreviations - see Table 1 . `Significant deviation from the preceding value (P < 0.05). t Normal 0-S pg/ml. The results are a mean f 1 S.D . of four to six experiments .

after 30 min and of APTT . within 10 min. Factors V, VII, VIII and IX decreased to their lowest level within 10 min. The level of factor X was reduced to 35% after 10 min and to 10% after 90 min. TT, fibrinogen and FDP were unchanged even 120 min after injection of the extract. The anticoagulant activity was not prevented by dialysis for 24 hr at 4°C but was completely abolished by heating for 5 min at 80°C (Table 5). Anticoagulant activity, spectrophotometric determination at 280 nm and protein concentration were maximal in fractions 4~ of the void volume (Fig. 1). A weaker anticoagulant activity was in fractions 9-11 .

The proteolytic effect of the extract on globin is shown in Table 6. Incubation of I "Cglobin in the presence of increasing concentrations of the extract produced a significant dose-dependent increase in the degradation of ~ ° C-globin (P < 0.05). In the presence of trasylol, the degradation of I°C-globin by 100 and 500 fig of venom sac extract per ml was reduced from 10 .7 pg to 7.2 tlg (29% inhibition) and from 46.9 ~g to 33.3 ug (33% inhibition), respectively (P < 0.05). EDTA inhibited the degradation of ~ 4C-globin in the presence of 100 ~g/ml of extract by 22% and in the presence of 500 ~g/ml of the extract by 30% (P < 0.05) . TABLE S . THE EFFECT OF HEATING AND DIALYSIS ON THE ANTICOAGULANT AC'I'IVr17 OF VENOM SAC EXTRACT' (VSE)

Substances

Condition

NP NP+VSE NP+VSE

Before heating VSE After heating VSE at 80°C for 10 min Before dialysis of VSE After dialysis of VSE for 24 hr at 4°C

NP NP+ VSE NP+VSE

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APTT (sec)

TT (sec)

12.2f0.6 18.4E 1.0` 11 .4f0.6'

48~ 7 182f24` S6f 8`

16 .Sf0 .S IS .Sf 1 .2 I6 .ßf I .0

11 .Of0.4 17.0 f 0.4` 16.2f0.2

40E 2 124f 4` 130E 10

20 .Of2 .0 18 .0 f 2.0 I9 .Of3 .0

Abbreviations - see Table 1 . Coagulation assays were performed after incubation of normal plasma (NP) with SO pg/ml VSE for 1 S min. The results are the mean t 1 S.D . of four experiments . `Significant deviation from the preceding value (P<0 .05) .

1174

A. KORNBERG et al . TABLE

DEGRADATION OF ' +C-GLOBIN BY VENOM SAC EXTRACT

6.

(VSE)

VSE (leg/ml) 10 l00 500 100 500 100 500

Inhibitor of proteolysis

Degraded '~-globin (leg/120 min)

Trasylol Trasylol EDTA 0 .1 M EDTA 0 .1 M

1 .5 t 0 .5 10 .7E 1 .0 46 .9 f 5 .5 7 .2 f 2 .4 33 .3 f 6 .6 7 .8 f 1 .4 36 .6 f 4 .8

The substrate was "C-globin (14 .5 mg/ml, spec. act . 230 cpm/ leg protein) which was incubated with VSE for 120 min as described in Materials and Methods . Trasylol and EDTA were preincubated with VSE for 5 min . The released radioactivity was converted from cpm to leg of degraded '~-globin per 120 min of incubation. The results are the mean f 1 S .D . of two to three experiments . DISCUSSION

and ISHAY (1975) and EnERY et al. (1978) have shown that Vespa orientalis venom sac extract exerts multiple effects on coagulation factors. However, the question as to which factors were inactivated was not answered conclusively . Incubation of tissue thromboplastin with the extract demonstrated antithromboplastic activity, with the venom JOSHUA

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FIG . I . COMPARL90N OF APTT, SPECTROPHOTOMETRIC AND PROTEIN DETERMINATION IN FRACTIONS AFTER GEL FILTRATION OF VENOM 3AC EXTRACT (VSE; SOOIeg/ml) ON SEPHADEX G-2S COLUMN .

Blue dextran 2000 (Pharrrlacia, Uppsala, Sweden) was used to show the fractions of the void volume . NP= normal plasma . The results are shown as means f 1 S.D . of three to four experiments .

Hornet Venom and Coagulation Factors

1 l75

also inhibiting the generation of endogenous thromboplastin and thrombin . The extract had no effect on thrombin and plasma fibrinogen, but purified fibrinogen was easily lysed. The marked prolongation of the recalcification and prothrombin times suggests an effect on coagulation factors exerted either via the common or via both the instrinsic and the extrinsic pathways of coagulation. The prolonged PT of venom-treated plasma was not shortened by the addition of normal fresh BaS04absorbed plasma . The PT, however, was significantly restored towards the normal value by the addition of fresh serum. These findings argue against a significant deterioration of factor V and rather suggest separate or combined inactivation of factors VII and X. The results of the present study indicate that venom sac extract affects differently various coagulation factors. The effect on factors VIII and IX is profound and immediate, both in vitro in human plasma and in vivo in cats . Within 5 min the activity is reduced to less than 1 % even with 1 pg of extract per ml . Additional data (not shown) suggest a marked effect of the extract even after 1-2 min. The effect on factors II, V, VII and X is less pronounced, as is also apparent from the very prolonged APTT compared to PT . With a relatively high concentration of extract (100 hg/ml) the effect on factors II, V, VII and X is similar, namely, a reduction in activity to 9%, 11 %, 11 % and 29%, respectively . However, factor V is affected even by 1 hg/ml, whereas factor VII requires 10 fig/ml and factor X not less than 50 pg/ml of venom sac extract. The greater resistance of X compared to VII is further shown in the time-dependent experiments . Our results also cônfirm that the extract has neither fibrinogenolytic nor antithrombin activity. The nature of the anticoagulant activity and the mdde of action are unknown. The effect on multiple factors, and particularly on V and VIII, suggest a possible occurrence of disseminated intravascular coagulation, but the normal fibrinogen and FDP levels tend to argue against this possibility. In the fractionation studies the maximal anticoagulant activity overlapped the highest spectrophotometric reading at 280 nm and the highest protein concentration all of which were eluted in the void volume . Some of the activity, however, was detected also in the fractionation range of the gel . These findings suggest that the activity resides mainly in proteins of mol.wt > 5000, even though it occurs also in smaller proteins . Of the higher mol.wt proteins the venom sac extract of the Oriental hornet contained enzymes with varying anticoagulant activities. Phospholipases A and B activities were demonstrated by the rapid hydrolysis of egg yolk, lecithin and isolecithin (RosErrBExG et al., 1977). Phospholipases from diverse snakes and honeybee venoms are known to exert antithromboplastin activity (HECH~r and Sr..o~t-rA, 1962 ; MOHAMED and HANNA, 1973) and inhibit blood coagulation via their action on phospholipids (PRIGENT-DACHARY et al., 1980). The venom contains polysaceharidases that hydrolyse glycogen and also disaccharidases such as maltase, isomaltase, trehalase and saccharase (FisHi, et al., 1974). The poly- and disaccharidases may affect carbohydrate moieties of certain factors such as VIII (Aus~N and BmvvEr.L, 1972) and thromboplastin (Priz.icx, 1975) and reduce their coagulant activity. Protease activity was demonstrated in the venom by the digestion of gelatin in fixed X-ray film (EDERY et al., 1972). We showed that'4C-globin is degraded by the venom, thereby providing additional evidence for protease activity in the venom. The partial inhibition of degradation by EDTA and trasylol suggests that the venom has both metaloprotease and serine protease activities . It has been shown that such enzymes have a proteolytic effect (WALZ, 1974). Another example of the anticoagulant effect of the proteases is the digestion of purified fibrinogen which can be inhibited by soybean trypsin inhibitor and by normal human serum which is known to possess an a, trypsin inhibitor

1176

A. KORNBERG et al.

(JOSHUA and ISHAY, 1975). An example of a low mol.wt substance with anticoagulant

properties in the venom is the basic protein component (JOSHUA and ISHAY, 1973), which can affect coagulation by binding to the acidic groups of thromboplastin (HABERMANN, 1968).

Acknowledgements-The authors thank Dr IsewEt. GOLDBERG for helpful suggestions and Mrs BLIMErA JOSEF ând ZVEZDANA FINCI for technical assistance .

REFERENCES Ausr>:x, D. E. G. and BtuwEt.t., E. (1972) Carbohydrate structure of factor VIII . Thromb . Diath. Haemorrh. 28, 464. DExsox, K. W. E. and Btccs, R. (1976) Laboratory diagnosis, tests of clotting function and their standardization. In : Human Blood, Coagulation, Haemostasis and Thrombosis, 2nd Edn, p.325 (Btcas, R., Ed .) . Oxford : Blackwell. EnFxv, H., Istuv, J., Less, I. and GtrtEtt, S. (1972) Pharmacological activity of Oriental hornet (Vespa orientalis) venom. Toxicon 10, 13 . EnFxv, H., Ist~uv, J., GtTTEtt, S. and Josuue, H. (1978) Venoms of Vespidae . In : Handbook of Experimental Pharmacology . Arthropod Venoms, Vol. 48, p. 601 (Berrtrtt, S., Ed .) . Berlin: Springer . Ftsctn ., J., IstuY, l. and RuTFSteertc, A. (1974) Poly- and disaceharidases in Vespa orientalis . Comp . Biochem. Physiol. 488, 299. Hnetattw+xx, E. (1968) Biochemie, pharmakologie and toxikologie der inhaltsstoffe von Hymenopterengiften . Ergeb. Physiol. 60, 220. HECr-rr, E. and St .o't-r~, K. H. (1962) The chemical nature of the lipid activator of blood coagulation . Am . J. clin . Path. 37, 126. HoucHiE, C. (1983) Hemostasis studies . In Hematology, 3rd Edn, p. 1667 (WILLIAMS, W. J., BEUTt.Ett, E., Ettst,EV, A. J. and LtCHTMAN, M. A., Eds) . New York : McGraw-Hill . Josxt;n, H. and IstuY, J. (1973) The hemolytic properties of the Oriental hornet venom sac extract. Acta Pharmac. Toxicol. 33, 42 . Josxun, H. and Isew, J. (1975) The anti-coagulant properties of an extract from the venom sac of the Oriental hornet . Toxicon 13, l l . LOWRY, O. H., ROSEBROUGH, N. J., Fntut, A. L. and RANDALL, R. J. (1951) Protein measurement with the folin phenol reagent. J. hiol . Chem . 193, 265. Mox~~, A. H. and H~xxa, M. M. (1973) The in vivo anticoagulant effects of NajaJlava venom. Toxicon 12, 419. O'Roue~, A., FEt.DMAN, B. F. and Im, R. K. (1982) Coagulation, fibrinolysis, and kinin generation in adult cats . Am. J. vet. Res. 43, 478. PITLICK, F. A. (1975) Concanavalin A inhibits tissue factor coagulation activity . J. clin . Invest . 55, 175. PRIGENT-DACHARY, J., BOFFFR, M. C., Botssewu, M. R. and DuFOUac~, J. (1980) Snake venom phospholipases A2 . J. biol. Chem . 255, 7734 . PRO('.TOR, R. R. and RAPAPORT, S. I. (1981) The partial thromboplastin time with kaolin . Am. J. clip. Path . 36, 212. QutcK, A. J. (1963) Determination of prothrombin. Am . J. med. Sci. 246, 517. Rn~tnoHr, O. D. and MENZtxt:, C. (1951) A new method for determination of fibrogen in small samples of plasma . J. Lab. clin . Med. 37, 316. Rosexeenc, P., Isttnv, J. and Gti-rEx, S. (1977) Phospholipases A and B activities of the Oriental hornet (Vespa orientalis) venom and venom apparatus. Toxicon 15, 141 . RoTtt, J. A., L06TY, K, and WtEttetctct, E. (1971) Assay of proteolytic enzyme activity using a "C-labeled hemoglobin . Ann . Biochem. 42, 214. WALZ, D. A., Sgcetcs, W. H., Reu't'ettsv, J. and McCov, L. E. (1974) Proteolyticspecificity of thrombin . Thromb . Res. 4, 718.