Toxlron, 1963, Vol. 1, pp . 109-112 .
Pergamon Press, Ltd. . .Printed in Grcat Britain
ACTIVATION OF THE FIBRINOLYTIC SYSTEM OF THE GUINEA PIG FOLLOWING INOCULATION OF SCHIS COLORATA VENOM* B . Monv, CH. Moxoz and A.
DE V RiFS
Rogoff Medical Research Institute, Department of Experimental Biology, Tel Aviv University and the Labor Sickfund, Beilinson Hospital, Petah Tikva, Israel Abstract-The intravascular fibrinolysis occwring following igjection of Echis colorata venom in the guinea pig is due mainly to activation of its fibrinolytic system and not to a direct fibrin-degrading action of the injected venom fibrinolysins.
Ir` A PREVIOUS Study it was shown that Echis colorata venom contains both procoagulant and fibrinolytic activity[1] . The afibrinogenemia occurring in guinea pigs following the intravenous administration of this venom was found to be due mainly to intravascular clotting induced by the venom procoagulant with subsequent fibrinolysis[2]. The question arose whether the fibrinolysis was effected by the injected venom fibrinolysin, or whether it was due to activation of the fibrinolytic system of the guinea pig . Healthy guinea pigs weighing 250-300 g were used. Echis colorata venom was obtained and chromatographic fractions were prepared as described previously[1] . Fraction I. in vitro, had strong fibrinolytic activity, while fraction II had strong procoagulant activity but only weak fibrinolytic potency . The difference in fibrinolytic potency of the two fractions was evident when tested by the plate method using purified bovine fibrinogen[3] (Fig. 1) and by the clot lysis time, both when using purified guinea pig fibrinogen prepared according to Lnxl[4j, and guinea pig plasma . Intravenous administration of whole venom or of each of its fractions I and II in the guinea pig produced a decrease in plasma fibrinogen, shortening of the serum euglobulin lysis time and increase of plasma fibrinolytic activity [Table 1]. It is noteworthy that there was no shortening of serum euglobulin lysis time when afibrinogenemia or hypofibrinogenemia were produced in guinea pig plasma by either whole venom or fractions I or II in vitro. In contrast to the comparative fibrinolytic potencies of the two fractions in vitro, larger amounts of fraction I than of fraction II were required to produce fibrinolytic activity in vivo. In fact, the property of whole venom to produce fibrinolytic activity in vivo may be ascribed wholly or mainly to the action offraction II. Of fraction I amounts with protein content of more than 60 times greater than that of fraction II were necessary to produce a similar magnitude of fibrinolytic activity in vivo . It was observed that the in vivo fibrinolytic activity produced by whole venom and fraction II became more marked with increasing intervals of time after injection and attained a maximum after about 2~ hr (the time the relationship for fraction I was not studied). "Supported by Grant E-3171 from the National Institutes of Health, Bethesda, Maryland, U.S .A . 109
c
11 0
B. MOAV, CH . MOROZ and A. DE VRIES
PERCENT ENZYME 90LUTION FIG . I . FIBRINOLYTIC ACTIVrrY OF CHROMATO('RAPHIC F'RACITONS OF ECI17S COiOf'Qlü VENOAi ON HOVINE FIBRINOGEN . PLATE METHOD [3].
TABLE I . FIBRINOLYTIC ACTIVITY OF GUINEA PIG PLASMA FOLLOWING INTRAVENOUS INIECCION COlOlQfR VENOM AND VENOM FRACCIONS
Material Venom
Fraction I I
Fraction I
Time of bleeding, Quantity min after administered injection leg protein
Plasma fibrinogen " (mg ~)
OF' EC'I1(J'
Plasma fibrinolytic activityj' (hr)
Serum cuglobulin lysis time* (min)
120 SS 75 -::1
X24 20 18 18 l4
0 30 60 90 150
280
40
0 30 90 I50
285 120 40 0
110 65 3 <1
~24 24 18 1S
2600
0 90
264 85
90 15
~24 l8
100
110 40 0
7
"Method Of RATNOFF and MENZIE[7] . *0~ 1 ml guinea pig plasma (9 vol blood I- l vol sodium oxalate, 0~ 1 M), 0'4 ml 0~2 per cent bovine fibrinogen, 10 units of thrombin in 0~1 ml saline, Observed at room temperature ; end point--complete lysis. ;0~1 ml scrum ;-I ~9 ml distilled Water, 1 per cent acetic acid added until pH 5~3. After 15 min incubation at 4''C, precipitate dissolved in borax buffer, pH 7~0 . Then added 0~4 ml 02 per cent bovine fibrinogen and ]0 units thrombin in 0~1 ml saline, observed at room temperature ; end point--complete lysis.
Snake Venom-Induced Fibrinolysis
111
The discrepancy between the comparative activities of the two fractions in vitro and vivo, as well as the time required to attain maximum fibrinolysic activity following injection of whole venom or fraction II, suggest that the circulating fibrinolysin is not identical with that in the injected material, but that it evolved in vivo . A similar suggestion was made by KoxxnLfK and PuD1 .á,K[5] concerning the mechanism of incoagulability induced in rats by administration of Echis carinata venom. In agreement with this concept is the observation that the amount of Echis colorata venom required to induce fibrinolysic activity in vivo is very small as compared to that calculated from the in vitro venom fibrinolysin titer and the plasma volume . The development of fibrinolysic activity in vivo seemed remarkable in view of the absent,°, of proactivator activity in guinea pig blood as demonstrated by LATALIA et al. [6] and confirmed by us : no plasmin activity could be obtained in normal guinea pig plasma by addition of streptokinase. Also, intravenous injection of streptokinase into the guinea pig did not induce fibrinolysic activity. However, as seen in Table 2, when streptokinase was added to plasma obtained from guinea pigs following an injection of venom or fraction II, a marked increase in fibrinolytic activity occurred, suggesting the appearance of proactivator activity in vivo . In contrast, following intravenous injection of fraction I, even in amounts large enough to cause a considerable decrease in fibrinogen, no proactivator activity could be detected in the plasma on addition of streptokinase in vitro. The mechanism by which proactivator activity appears in the circulating blood following an injection of Echis colorata venom or fraction II has not been elucidated . Appearance of in
TABLE 2. EFFECT OF STREPTOKINASE ON FIBRINOLYTIC ACCIVII'Y INDUCED BY INJECTION OF Echis COIOIRIR VENOM AND VENOM FRACFIONS
Treatment of guinea pig
Time of bleeding, min after injection
Normal controls
Streptokinase i .v ., 40,000 units
Plasma fibrinogen (mg ~)
Streptokinase (units)
Added fibrinogen
280-320 280-320 280-320
1000 1000
bovine
Clot lysis time > 24 hr > 24 hr >24 hr
60
300
>24 hr
Venom, 100 tcg protein
150 30 150 150
0 140 0 0
1000 1000 1000
bovine bovine bovine guinea pig
16 hr 22 min 6 min 11 min
Fraction li, 40 F+g protein
150 90 150 150
0 117 0 0
1000 1000 1000
bovine bovine bovine Guinea pig
IS hr 32 min 10 min 75 min
Fraction 1, 2600 Fig protein
150 150 I50
90 60 60
1000 1000
bovine bovine Guinea pig
> 24 hr > 24 hr >~24 hr
0~1 ml guinea pig plasma (9 vol bloody-1 vol sodium oxalate 0~1 M), 0~1 ml streptokinase solution, 0~4 ml 0~2 per cent fibrinogen, 0~1 ml thrombin solution containing 10 units. Observed at room temperature; end. point-complete lysis
11 2
B. MOAV, CH . MOROZ and A. DE VRIES
proactivator activity in vivo apparently was not simply a sequel of intravascular clotting produced by the venom or fraction II. Intravascular clotting and severe hypofibrinogenemia induced in the guinea pig by intravenous thrombin or thromboplastin injection was not followed by increased plasma fibrinolytic activity or shortening of serum euglobulin lysis time, nor by appearance of proactivator activity . Furthermore, it seems improbable that the evolved proactivator activity was due to an agent produced by an action of the venom on a plasmatic component, since no proactivator activity could be detected following addition of streptokinase to guinea pig plasma rendered afibrinogenemic in vitro by illcubation with venom or fraction l[. Consistent with this is the inability of venom or its fractions to produce shortening of serum euglobulin lysis time in vitro. Further investigation will be required to clarify whether or not the appearance of proactivator activity and fibrinolytic activity following the injection of venom or fraction [[ ínvolv~s release of an agent from the tissues or elimination of an inhibitor. The abovi: results indicate that the intravascular fibrinolysis occurring following injection of Echis coTa~ata venom in the guinea pig is due mainly to activation of its fibrinolytic system and not to a direct fibrin-degrading action of the injected venom fibrinolysins . REFERENCES
l-1
13l [4]
GII~FER, S., Lt~I, G., KOCHWA, S DF VRIFS, A., RECHNIC, .I . illld CASPER, .L, Ar17eP. Trop . 11-1er1. ifl;Q ., 9, 391, 1960 . RECH\IC, J., TRACHTEI~RFRG, P., CASPER, J ., MOROZ, CH . and DE VRIES, A., Blood, 20, 735, 1962 . AS~rRI~P, T. and MÜLLERT7_, M., Arch . Bioc'herrl ., 40, 346, 1952, LAKI, R., .~rc"1r . BioclrerJr., 32, 317, 1951 .
[Sl
KOKI~ALÍK, F, and P(,~nt.dk, P., L:~ner'ierrrirr, tß, 381, 1962 .
[7l
RATnOFF, O. D. and Mf~7:~lE, C., J. Lrrh . Clin. Mer1., 34, 316, 1954 .
[61
LATALLO, Z., NIF.WIARO\i'SKI, S. and COI'LFY, A. L., An:er. J. Physiol., 196, 775. 1959 .