THROMBOSIS RESEARCH Printed in the United
vol.
5, pp. 53-63,
Pergamon
States
Press,
1975 Inc.
PROTHROMBIN ACTIVATION INDUCED BY ECARIN - A PROTHROMBIN CONVERTING ENZYME FROM ECHIS CARINATUS VENOM F. Kornalik and B. Blomback Institute of Pathophysiology, Charles University, Prague, CSSR Department of Blood Coagulation Research, Karolinska Stockholm, Sweden (Received
and
InStitUtet,
in revised form 1,12,1974. 20.11.1974; Accepted by Editor A,L. Copley]
ABSTRACT Purified prothrombin was activated by a physiological activator of a thromboplastin type (AC) and by Ecarin, a procoagulant enzyme derived from Echis carinatu'svenom. Intermediate products obtained during 2 min. to 2 hours of activation were analyzed by SDS polyacrylamide gel electrophoresis and NH2-terminal analysis. Thrombin activity was measured on fibrinogen as substrate. It was found that Ecarin cleaves prothrombin at a bond, which results in release of the NH2 -terminal portion of the molecule (A-fragment; intermediate 3), A second cleavage site appears to be at the Arg-Ile bond linking the A and B chain in thrombin precursors. The latter cleavage was considered to be responsible for the appearance of the biological activity. The resulting Ecarin thrombin in constrast to thrombin produced by physiological activation appears to consist of a B-chain and an extended A-chain (A-chain + intermediate 4). Ihis is believed to be the reason for the lower specific activity of Ecarin thrombin on fibrinogen. Our results do not exclude the possibility that prothrombin is cleaved by Ecarin as well as by physiological activator at other bonds than already identified.
INTRODUCTION In the last decade,evidence has accumulated
that prothrombin can be convert-
ed to thrombin by specific enzymes present in snake venoms.
The snake ven-
oms in question are those from Echis coloratus(l), Echis carinatus (2), Notechis scutatus scutatus - Tiger snake (3) and Oxyuranus scutellatus scutellatus (4) - Taipan.
Using partially purified enzyme from Taipan venom
and human prothrombin, Lanchantin et al.
53
found that the resulting thrombin
PROTHROMBIN
j'c
AND
SNAKE
VENO>I
VOl.6,NO.l
is different from the normal thrombin produced by f.Xa or in concentrated citrate solution (5).
Some years ago we isolated a procoagulant enzyme from the venom of Sawscaled viper, Echis carinatus, which converts prothrombin directly into thrombin (6).
Its activity is independent on other cofactors of the plasma
thromboplastin system and does not require phospholipids or calcium.
In de-
scribing some of the pharmacological and biological properties of this enzyme - now called Ecarin - we have suggested that the resulting thrombin might be of different nature from the physiologically produced enzyme (7).
The present paper is a report about the probable structure of the Ecarinthrombin (ET). An attempt is made to explain the differences in the sequence of events in an activation of prothrombin with the physiological activator and Ecarin, respectively. MATERIALS AND METHODS Ecarin was prepared as described earlier (8). - Bovine prothrombin (1200 Units NIH/mg) prepared according to a modification of the method by Ingwall and Scheraga (9) was obtained from Dr. S. Iwanaga of Osaka University. Activator was prepared according to Miller and Copeland (LO). it is referred to as AC -0
In the text
Basically it is bovine brain thromboplastin with
serum coagulation factor adsorbed to the particles, which are suspended in 0.15 M calcium chloride solution before use.
%-
terminal analysis with
35 S-phenylisothiocyanate was performed as
described elsewhere (12,13,14,15). Discelectrophoresis with SDS was carried out according to previously described techniques (11).
Thrombin
activity was determined using 0.4% solution of bovine fibrinogen (lot No BO 19 from ImCo, Stockholm).
The activity (NIH-units) was calculated from
a standard curve using purified bovine thrombin with a specific activity of
VOl.6,NO.
1
PROTHRO?KBI?r' AXD
SXAKE
VESOW
207 NIH units per mg.
RESULTS
Prothrombin (prepared according to Morita et al (18)) was dissolved in 0.1 M tris-HCl buffer, pH 7.3 to a concentration of 1 mg per ml.
To 1.0 ml
aliquots of prothrombin solution were added 0.2 ml Ecarin (10 pg/ml) or 0.2 ml AC and the mixture incubated at 37'.
At time intervals of 10, 20,
40, 60 and 120 min, samples of 0.15 ml were withdrawn and added to 0.2 ml of 50% acetic acid to stop the reaction.
The samples were subsequently
freeze-dried and subjected to SDS-gel electrophoresis. Thrombin activity of the initial incubation mixture was determined during the incubation. The results are shown in Fig. 1.
In case of activation by AC, prothrombin is gradually split in two frag;'; ments, one of m.w. 29,000 - corresponding to intermediate 3 (16) (or A fraga ment according to Magnusson (17) and the other of m.w. 58,000 which corresponds to intermediate 1 (or neoprothrombin S (17). Within the first hour of incubation a faint band of m.w. around 16,000, corresponding to the intermediate 4 (S fragment (17)),was detected and at the same time a new band of m.w. 39,000 became visible.
The latter represents intermediate
2 (neoprothrombin T (17) and/or thrombin (IIa).
This interpretation is in
reasonable agreement with the findings of Morita et al. (18), except for a slower appearance of intermediate 4 in their experiments.
This discrepancy
may be explained by differences in conditions for activation.
When Ecarin was used for activation we could observe a somewhat different picture (Fig. 1).
Already after the first 10 min. the band corresponding
to prothrombin disappears completely being replaced by two main bands. One band of 25,000 m.w. corresponds to intermediate 3, the other band of 58.000 m.w. corresponds to intermediate 1.
A faint band of m.w. of
(*) throughout this paper, m-w. stands for molecular weight when non-reduced samples were analyzed.
SO1.6,?;0.1
41.000,appearing during the aciivation, night represent intermediate 2. In fact, after 2 hours incubation a very faint band (not visible in Fig. 1) corresponding to intermediate 4, could also be observed.
c_
..
A
E
E
, 13
2c)
13
511
123
.rcJiS/rnin
PC’13 I m,r
FIG. 1 a) SDS-gel electrophoresis in 7% gel, 9 mA/tube of prothrombin activated with AC (above)and Ecarin (below) in time intervals of 10,20,40 and 120 min. b) Thrombin activity of the mixtures expressed in NIH units/ml. -----, Ecarin; -, AC.
The fact that intermediate 1, a precursor
FIG. 2 a) SDS-gel electrophoresis of prothrombin activated with AC and Ecarin (E) in time intervals of 0,2,5 and 10 min. Nonreduced samples (above) run under the same conditions as in Fig. 1. Samples reduced with DTT run in 10% gel. b) Thrombin activity for AC(-----) and Ecarin (-) activated thrombin in NIH units/ml. of thrombin activity,
appeared as
a main component during activation with Ecarin suggested that it is the carrier of the thrombin activity that evolves during activation (Fig, 1).
vo1.6,50.1
However,
tion.
PROTHROMBIN
AND SNAKE VEXOM
57
intermediate 1 is known to be inactive during the biological activa.
We therefore assumed that intermediate 1 has been internally split by
Ecarin, presumably at the Arg-Ile bond, which is split by Xa during the last stage of prothrombin activation (16).
Experiments under the same conditions
were performed to support this assumption, with the exception that samples were withdrawn from the incubation mixture at 0,2,5 and 10 min. intervals. Aliquots of the samples were subjected to SDS-gel after reduction with dithiothreitol (DTT).
electrophoresis before and
The results are shown in Fig. 2.
With AC practically no biological activity was produced in the course of the experiment.
On the other hand, with Ecarin roughly 40% of the activity
obtained after complete activation (2 hours or more) was achieved.
In the
case of prothrombin activated with AC, the SDS-gels of non-reduced and reduced samples showed, in addition to a prothrombin band, a weak band corresponding to intermediate 1.
After activation with Ecarin the non-reduced
samples showed the appearance of the bands already described in the experiments with longer incubation times (Fig. 1 and 2).
In the reduced samples
three new bands appeared during activation with Ecarin. of 58,000 represents intermediate 1. to be the B-chain of thrombin.
One band with m.w.
The band with m.w. 34,000 is assumed
The band of m.w. 22,000 is likely to
represent a mixture of intermediate 3 together with a fragment of prothrombin composed of intermediate 4 (m.w. 13,OOO(16)),plus the A-chain of thrombin (m.w. 7,000).
This explanation was corroborated by NH2-terminal analysis of activation mixtures of prothrombin with Ecarin and AC, respectively. activated with Ecarin and AC for 25 min.
Prothrombin was
After this time Ecarin produced
over 50% activation in terms of the biological activity, which can be obtained by longer incubation (2 hours or more).
On the other hand AC had
only produced less than 2% of the expected activity of the prothrombin used
PROTHROMBIN
58
(1,200 NIH units/mg).
AND
SNAKE
VENOM
In the activation mixture with Ecarin, isoleucine
(and/or leucine)appeared as new NH2-terminal residues.
Only traces of this
amino acid were present in the samples activated by AC and in prothrombin before activation (Table I).
TABLE I NH2-TERMINAL AMINO ACIDS IN PROTHROMBIN ACTIVATING MIXTURES Thin-layer chromatogram of PTH-derivatives of prothrombin before activation, and of prothrombin activated with Ecarin and AC for .&5 min., respectively. About 2.5 mg of material was used for coupling with S-PIIC (20 pCi/jlmol). 4,ul was applied to the thin-layer plates. The plates were scanned for radioactivity and the radioactive material of the peaks eluted with 95% ethanol (3.5 ml). The radioactivity was determined in a Beckman (CPM-200) liquid scintillation counter, using 10 ml of Instagel (Packard). CPM OF ELUTED SPOTS IN Amino acid Alanine Isoleucine/leucine
Prothrombin 2,900 734
Prothrombin + Ecarin 7,168 15,110
Prothrombin + AC 3,455 1,245
It should be noted that the values for alanine were higher in the Ecarin activated sample than in the other two.
This might indicate that another
bond involving this amino acid has also been cleaved by Ecarin.
In a pre-
liminary experiment it was found that in both the AC and Ecarin activated prothrombin after 2 hours incubation, the NH2-terminal alanine was higher than in prothrombin before activation.
This might suggest that also in
normal activation a bond involving alanine is split.
Smaller amounts of
other NH -terminal amino acids were also detected in the prothrombin and 2 activation mixtures.
The failure to demonstrate the appearance of signifi-
cant amounts of NH2-terminal serine and threorine during the activation is most likely explained by the well known degradation of these amino acids during NH2-terminal analysis.
We have noted in our experiments that yield of thrombin activity after
Yol.d,?;o.
PROTHROK3IX
1
XiD SSAKE VEXOX
59
activation with Ecarin, as determined on fibrinogen as substrate, never reached more than about4OX
of the specific activity (NIH-unFts/mg) of the
starting prothrombin preparation used.
However, if AC was added to pro-
thrombin, which had been activated with Ecarin for two hours, substantial increase in biological activity, reaching almost the expected value for the prothrombin preparation in question, was achieved.
This increase was in
gel electrophoresis (Fig. 3) shown to be accompanied by the appearance of a band of m.w. 16,000 coresponding to the intermediate 4 and by an apparent increase in the band of m.w. 39,000 representing intermediate 2 and/or thrombin.
On addition of Ecarin to a prothrombin solution, which has been
activated for 2 hours by AC, no obvious change in band pattern occurred (Fig. 3), but the biological activity of the mixture was appreciably increased.
AC+E
AC
160
780
E
E+AC
420
990 NIH U/ml
NIH U/ml FIG. 3
SDS-gel electrophoresis of prothrombin activated 2 h. with AC or Ecarin (E). Further activation of AC-thrombin for 30 min. with Ecarin (AC+E), and of Ecarin-thrombin (E) for 30 min. with AC (E-MC).
DISCUSSION As has been shown previously venom from Echis carinatus (6,7,16) as well as the purified enzyme Ecarin (68),
has no clot promoting effect on fibrinogen
but rapidly activates prothrombin.
The experiments presented in this report show that the activation of prothrombin by Ecarin is different from that obtained in presence of an activator of the thromboplastin type.
With regard to the latter our results
confirm qualitatively those, obtained by other investigators with other "physiological" activators of prothrombin (17,18,19).
On the basis of the structure of prothrombin worked out by Magnusson (18) and on the similarity with fragments produced during physiological activation (19), we can conclude that Ecarin splits the prothrombin molecule at the Arg-Ser bond, which is split during the release of the NH2-terminal portion of the molecule (intermediate 3).
The other preferential cleavage
by Ecarin appears to be the Arg-Ile bond, which links the A-and &chain in precursors of thrombin.
This bond is present in intermediate 1 and its
cleavage is presumably responsible for the appearance of the thrombin-like activity produced by Ecarin.
This cleavage is also a "sine qua non" for
"physiological" prothrombin activation (17,20, 21).
It is puzzling to us that the amount of alanine was higher in the Ecarin activated sample than in the prothrombin before activation (compare Table I). Our preliminary results indicated that it was higher also in the AC activated samples after longer incubation.
Further experiments need to be per-
formed to show if this represents a new cleavage in the intermediate 1 or if it simply reflects differences in yield of alanine in the mixtures. It has not escaped our attention that Seegers et al (20) previously have noted an excess of NH2- terminal alanine in activation mixtures involving prethrombin
Vol.6,Xo.l
PROTHROMBIN
AND
SNAKE
VENOM
61
Since a band (approx. m.w. 39,000) of increasing intensity appears during the activation with Ecarin, we assume that also the Arg-Thr bond, linking the inter mediate 2 with intermediate 4, is cleaved.
That means that eventually a mole-
cule, chemically indistinguishable from thrombin, may be formed. It was observed that addition of Ecarin to prothrombin, which has been partially activated by AC, considerably increased the biological activity.
Since the
incubation mixture contained predominantly intermediate 1, the increase in biological activity is explained by cleavage of the above mentioned Arg-Ile bond in this precursor.
On the other hand, the increase in activity of Ecarin-
thrombin on addition of AC is best explained by the release of intermediate 4 from Ecarin-thrombin (intermediate 1 with the Arg-Ile bond cleaved), and thereby a molecule identical with thrombin is produced.
It has been demonstrated
here and also described earlier (7) that Ecarin-thrombin has a lower specific activity on fibrinogen as compared to thrombin.
It is therefore quite feasible
that the conversion of Ecarin-thrombin into thrombin,in the presence of the brain thromboplastin activator (AC), is responsible for the increase in activity. Our results with regard to Ecarin are essentially in agreement with those of Iwanaga and coworkers (personal cormnunication, 1974).
ACKNOWLEDGEMENTS The authors express their gratitude for skillful technical assistance to Mrss. Elvy Andersson, Birgit Hessel, Helga Messel and Sonja Soderman,
This work was supported by grants from 'IheSwedish Medical Research Council (Nos. 13X-2475-08, and The National Institutes of Health (No. 5 ROl HLO737909).
62
PROTHROMBIN
AND
SNAKE
VENOI"I
REFERENCES
l. GITTER, S., LEVI, G., KOCHWA, S., DE VRIES, A., RECHNIc,,J., Aii CA=‘% J. Studies on the venom of Echis coloratus. Am. J. Trap. Med. Hvg. 2, 391, 1960. 2. KORNA_I,IK, F. Uber den Einfluss Von Echis carinata Toxin auf die Blutgerinnung in vitro. Folia Haematol. 3, 73, 1963. 3. JOBIN, F. and ESNOUF, M.P. Coagulant activity of Tiger snake (Notechis scutatus scutatus) venom. Nature 211, 873, 1966. 4. PIRKLE, H., MCINTOSH, M., THEODOR, I. AND VERNON, S. Activation of prothrombin with Taipan snake venom. Thromb. Res. 1, 559, 1972. 5. LANCHANTIN. G.F.. FRIEDMANN. J.A. and HART, D.W. _ bin. Journ: Biol: Chem. 248; 5956,1973.
Iwo forms of human throm
6. KORNALIK, F. Fibrinolytische Proteasen aus Schlangengiften. Folia Haemato1. 95, 193, 1971. 7..SCHIECK, ._ A., - HABERMANN, E. AND KORNALIK, F. The prothrombin activating principle from Echis carinatus venom. II. Coagulation studies in vitro and in vivo. Naunvn Schmiedebergs Arch. Pharmacol. 2, 7, 1972. 8. SCHIECK, A., KORNALIK, F. and HABERNANN, E. The prothrombin activating principle from Echis carinatus venom. I. Preparation and biochemical properties. Naunm SchxniedebergsArch. Pharmacol. 272, 402, 1972. 9. LNGWALL,J.S.and SCHERAGA, H.A. Purification and properties of bovine prpthrombin. Biochemistry 8, 1860, 1969. 10. MILLER, K.D. and COPEIAND, W.H. Human thrombin: Isolation and stability. Exper. Molecular Pathol. 4, 431, 1965. 11. MCDONAGH, J., MESSEL, H., MCDONAGH, R.P., MURANO, G. JR. and BLOMBACK B. Molecular weight analysis of fibrinogen and fibrin chains by an improved sodium-dudecylsulphate gel electrophoresis method. Bioch. Biophys. Acta 257, 135, 1972. 12. EDMAN, P. In: Protein Sequence Determination (Needleman, S.B. ed.), Springer-Verlag, New York 1970, p. 21. 13. IWANAGA, S., WALLEN, P., GRONDAHL, N.J., HENSCHEN, A. and BLOMBACK, B. On the primary structure of human fibrinogen. Isolation and characterization of N-terminal fragments from plasmic digests. European J. Biochem. 8, 189, 1969. 14. BLOMBACK, B. and YAMtUHINA, I. gen and fibrin. Arkiv Kemi 2,
On the N-terminal amino acid in fibrino299, 1958.
15. IRION,E. and BLOMB&CK, B. N-terminal amino acid sequence of intact human fibrinogen. FEBS Letters 7, 143, 1970.
Vol.6,No.l
PROTHROMBIN
tiD
SNAKE
VENOSI
63
16.
COPLEY, A.L., BANERJEE, S. and DEVI, A. Studies of snake venoms on blood coagulation. I. The thromboserpentin (thrombin-like) enzyme in the venoms. -Thromb. Res. 2, 487, 1973.
17a
HELDEBRANTS, C.M., BUTKOWSKI, R.J., BAJA-J, S.P. and MANN, K.G. The Activation of Prothrombin. II. Partial reactions, physical and chemical characterization of the intermediates of activation. Chem. 248: 20, 7149, 1973. --
17b
MANN, K.G., HELDEBRAND, C.M., FASS, D.N., BAJAJ, S.P. and BUTKOWSKI, R.J. The Molecular Mechanism of Prothrombin Activation. Thromb. Diath. Haemorr. Supple.57, 179, 1974.
18.
MAGNUSSON, S. Primary structure studies on thrombin and prothrombin. Thromb. Diath. Haemorrh. Suppl. 54, 31, 1973.
19.
MORITA, T., IWANAGA, S., SUZUKI, T. AND FUJIKAWA, K. Characterization of amino terminal fragment liberated from bovine prothrombin by activated factor X. FEBS Letters 36, 313, 1973.
20.
SEEGERS, W.H., MURANO, G., MCCOY, L. and MARCINIACK, E. The coagulation of blood: Preliminary survey of thrombin and autoprothrombin zymogen structure. Life Sci. 8, 925, 1969.
21. MAGNUSSON, S. Terminal amino acid residues in bovine prothrombin and in prothrombin activation mixture. Arkiv Kemi 23, 271, 1965.
vol.
5, pp. 53-63,
Pergamon
States
Press,
1975 Inc.
PROTHROMBIN ACTIVATION INDUCED BY ECARIN - A PROTHROMBIN CONVERTING ENZYME FROM ECHIS CARINATUS VENOM F. Kornalik and B. Blomback Institute of Pathophysiology, Charles University, Prague, CSSR Department of Blood Coagulation Research, Karolinska Stockholm, Sweden (Received
and
InStitUtet,
in revised form 1,12,1974. 20.11.1974; Accepted by Editor A,L. Copley]
ABSTRACT Purified prothrombin was activated by a physiological activator of a thromboplastin type (AC) and by Ecarin, a procoagulant enzyme derived from Echis carinatu'svenom. Intermediate products obtained during 2 min. to 2 hours of activation were analyzed by SDS polyacrylamide gel electrophoresis and NH2-terminal analysis. Thrombin activity was measured on fibrinogen as substrate. It was found that Ecarin cleaves prothrombin at a bond, which results in release of the NH2 -terminal portion of the molecule (A-fragment; intermediate 3), A second cleavage site appears to be at the Arg-Ile bond linking the A and B chain in thrombin precursors. The latter cleavage was considered to be responsible for the appearance of the biological activity. The resulting Ecarin thrombin in constrast to thrombin produced by physiological activation appears to consist of a B-chain and an extended A-chain (A-chain + intermediate 4). Ihis is believed to be the reason for the lower specific activity of Ecarin thrombin on fibrinogen. Our results do not exclude the possibility that prothrombin is cleaved by Ecarin as well as by physiological activator at other bonds than already identified.
INTRODUCTION In the last decade,evidence has accumulated
that prothrombin can be convert-
ed to thrombin by specific enzymes present in snake venoms.
The snake ven-
oms in question are those from Echis coloratus(l), Echis carinatus (2), Notechis scutatus scutatus - Tiger snake (3) and Oxyuranus scutellatus scutellatus (4) - Taipan.
Using partially purified enzyme from Taipan venom
and human prothrombin, Lanchantin et al.
53
found that the resulting thrombin
PROTHROMBIN
j'c
AND
SNAKE
VENO>I
VOl.6,NO.l
is different from the normal thrombin produced by f.Xa or in concentrated citrate solution (5).
Some years ago we isolated a procoagulant enzyme from the venom of Sawscaled viper, Echis carinatus, which converts prothrombin directly into thrombin (6).
Its activity is independent on other cofactors of the plasma
thromboplastin system and does not require phospholipids or calcium.
In de-
scribing some of the pharmacological and biological properties of this enzyme - now called Ecarin - we have suggested that the resulting thrombin might be of different nature from the physiologically produced enzyme (7).
The present paper is a report about the probable structure of the Ecarinthrombin (ET). An attempt is made to explain the differences in the sequence of events in an activation of prothrombin with the physiological activator and Ecarin, respectively. MATERIALS AND METHODS Ecarin was prepared as described earlier (8). - Bovine prothrombin (1200 Units NIH/mg) prepared according to a modification of the method by Ingwall and Scheraga (9) was obtained from Dr. S. Iwanaga of Osaka University. Activator was prepared according to Miller and Copeland (LO). it is referred to as AC -0
In the text
Basically it is bovine brain thromboplastin with
serum coagulation factor adsorbed to the particles, which are suspended in 0.15 M calcium chloride solution before use.
%-
terminal analysis with
35 S-phenylisothiocyanate was performed as
described elsewhere (12,13,14,15). Discelectrophoresis with SDS was carried out according to previously described techniques (11).
Thrombin
activity was determined using 0.4% solution of bovine fibrinogen (lot No BO 19 from ImCo, Stockholm).
The activity (NIH-units) was calculated from
a standard curve using purified bovine thrombin with a specific activity of
VOl.6,NO.
1
PROTHRO?KBI?r' AXD
SXAKE
VESOW
207 NIH units per mg.
RESULTS
Prothrombin (prepared according to Morita et al (18)) was dissolved in 0.1 M tris-HCl buffer, pH 7.3 to a concentration of 1 mg per ml.
To 1.0 ml
aliquots of prothrombin solution were added 0.2 ml Ecarin (10 pg/ml) or 0.2 ml AC and the mixture incubated at 37'.
At time intervals of 10, 20,
40, 60 and 120 min, samples of 0.15 ml were withdrawn and added to 0.2 ml of 50% acetic acid to stop the reaction.
The samples were subsequently
freeze-dried and subjected to SDS-gel electrophoresis. Thrombin activity of the initial incubation mixture was determined during the incubation. The results are shown in Fig. 1.
In case of activation by AC, prothrombin is gradually split in two frag;'; ments, one of m.w. 29,000 - corresponding to intermediate 3 (16) (or A fraga ment according to Magnusson (17) and the other of m.w. 58,000 which corresponds to intermediate 1 (or neoprothrombin S (17). Within the first hour of incubation a faint band of m.w. around 16,000, corresponding to the intermediate 4 (S fragment (17)),was detected and at the same time a new band of m.w. 39,000 became visible.
The latter represents intermediate
2 (neoprothrombin T (17) and/or thrombin (IIa).
This interpretation is in
reasonable agreement with the findings of Morita et al. (18), except for a slower appearance of intermediate 4 in their experiments.
This discrepancy
may be explained by differences in conditions for activation.
When Ecarin was used for activation we could observe a somewhat different picture (Fig. 1).
Already after the first 10 min. the band corresponding
to prothrombin disappears completely being replaced by two main bands. One band of 25,000 m.w. corresponds to intermediate 3, the other band of 58.000 m.w. corresponds to intermediate 1.
A faint band of m.w. of
(*) throughout this paper, m-w. stands for molecular weight when non-reduced samples were analyzed.
SO1.6,?;0.1
41.000,appearing during the aciivation, night represent intermediate 2. In fact, after 2 hours incubation a very faint band (not visible in Fig. 1) corresponding to intermediate 4, could also be observed.
c_
..
A
E
E
, 13
2c)
13
511
123
.rcJiS/rnin
PC’13 I m,r
FIG. 1 a) SDS-gel electrophoresis in 7% gel, 9 mA/tube of prothrombin activated with AC (above)and Ecarin (below) in time intervals of 10,20,40 and 120 min. b) Thrombin activity of the mixtures expressed in NIH units/ml. -----, Ecarin; -, AC.
The fact that intermediate 1, a precursor
FIG. 2 a) SDS-gel electrophoresis of prothrombin activated with AC and Ecarin (E) in time intervals of 0,2,5 and 10 min. Nonreduced samples (above) run under the same conditions as in Fig. 1. Samples reduced with DTT run in 10% gel. b) Thrombin activity for AC(-----) and Ecarin (-) activated thrombin in NIH units/ml. of thrombin activity,
appeared as
a main component during activation with Ecarin suggested that it is the carrier of the thrombin activity that evolves during activation (Fig, 1).
vo1.6,50.1
However,
tion.
PROTHROMBIN
AND SNAKE VEXOM
57
intermediate 1 is known to be inactive during the biological activa.
We therefore assumed that intermediate 1 has been internally split by
Ecarin, presumably at the Arg-Ile bond, which is split by Xa during the last stage of prothrombin activation (16).
Experiments under the same conditions
were performed to support this assumption, with the exception that samples were withdrawn from the incubation mixture at 0,2,5 and 10 min. intervals. Aliquots of the samples were subjected to SDS-gel after reduction with dithiothreitol (DTT).
electrophoresis before and
The results are shown in Fig. 2.
With AC practically no biological activity was produced in the course of the experiment.
On the other hand, with Ecarin roughly 40% of the activity
obtained after complete activation (2 hours or more) was achieved.
In the
case of prothrombin activated with AC, the SDS-gels of non-reduced and reduced samples showed, in addition to a prothrombin band, a weak band corresponding to intermediate 1.
After activation with Ecarin the non-reduced
samples showed the appearance of the bands already described in the experiments with longer incubation times (Fig. 1 and 2).
In the reduced samples
three new bands appeared during activation with Ecarin. of 58,000 represents intermediate 1. to be the B-chain of thrombin.
One band with m.w.
The band with m.w. 34,000 is assumed
The band of m.w. 22,000 is likely to
represent a mixture of intermediate 3 together with a fragment of prothrombin composed of intermediate 4 (m.w. 13,OOO(16)),plus the A-chain of thrombin (m.w. 7,000).
This explanation was corroborated by NH2-terminal analysis of activation mixtures of prothrombin with Ecarin and AC, respectively. activated with Ecarin and AC for 25 min.
Prothrombin was
After this time Ecarin produced
over 50% activation in terms of the biological activity, which can be obtained by longer incubation (2 hours or more).
On the other hand AC had
only produced less than 2% of the expected activity of the prothrombin used
PROTHROMBIN
58
(1,200 NIH units/mg).
AND
SNAKE
VENOM
In the activation mixture with Ecarin, isoleucine
(and/or leucine)appeared as new NH2-terminal residues.
Only traces of this
amino acid were present in the samples activated by AC and in prothrombin before activation (Table I).
TABLE I NH2-TERMINAL AMINO ACIDS IN PROTHROMBIN ACTIVATING MIXTURES Thin-layer chromatogram of PTH-derivatives of prothrombin before activation, and of prothrombin activated with Ecarin and AC for .&5 min., respectively. About 2.5 mg of material was used for coupling with S-PIIC (20 pCi/jlmol). 4,ul was applied to the thin-layer plates. The plates were scanned for radioactivity and the radioactive material of the peaks eluted with 95% ethanol (3.5 ml). The radioactivity was determined in a Beckman (CPM-200) liquid scintillation counter, using 10 ml of Instagel (Packard). CPM OF ELUTED SPOTS IN Amino acid Alanine Isoleucine/leucine
Prothrombin 2,900 734
Prothrombin + Ecarin 7,168 15,110
Prothrombin + AC 3,455 1,245
It should be noted that the values for alanine were higher in the Ecarin activated sample than in the other two.
This might indicate that another
bond involving this amino acid has also been cleaved by Ecarin.
In a pre-
liminary experiment it was found that in both the AC and Ecarin activated prothrombin after 2 hours incubation, the NH2-terminal alanine was higher than in prothrombin before activation.
This might suggest that also in
normal activation a bond involving alanine is split.
Smaller amounts of
other NH -terminal amino acids were also detected in the prothrombin and 2 activation mixtures.
The failure to demonstrate the appearance of signifi-
cant amounts of NH2-terminal serine and threorine during the activation is most likely explained by the well known degradation of these amino acids during NH2-terminal analysis.
We have noted in our experiments that yield of thrombin activity after
Yol.d,?;o.
PROTHROK3IX
1
XiD SSAKE VEXOX
59
activation with Ecarin, as determined on fibrinogen as substrate, never reached more than about4OX
of the specific activity (NIH-unFts/mg) of the
starting prothrombin preparation used.
However, if AC was added to pro-
thrombin, which had been activated with Ecarin for two hours, substantial increase in biological activity, reaching almost the expected value for the prothrombin preparation in question, was achieved.
This increase was in
gel electrophoresis (Fig. 3) shown to be accompanied by the appearance of a band of m.w. 16,000 coresponding to the intermediate 4 and by an apparent increase in the band of m.w. 39,000 representing intermediate 2 and/or thrombin.
On addition of Ecarin to a prothrombin solution, which has been
activated for 2 hours by AC, no obvious change in band pattern occurred (Fig. 3), but the biological activity of the mixture was appreciably increased.
AC+E
AC
160
780
E
E+AC
420
990 NIH U/ml
NIH U/ml FIG. 3
SDS-gel electrophoresis of prothrombin activated 2 h. with AC or Ecarin (E). Further activation of AC-thrombin for 30 min. with Ecarin (AC+E), and of Ecarin-thrombin (E) for 30 min. with AC (E-MC).
DISCUSSION As has been shown previously venom from Echis carinatus (6,7,16) as well as the purified enzyme Ecarin (68),
has no clot promoting effect on fibrinogen
but rapidly activates prothrombin.
The experiments presented in this report show that the activation of prothrombin by Ecarin is different from that obtained in presence of an activator of the thromboplastin type.
With regard to the latter our results
confirm qualitatively those, obtained by other investigators with other "physiological" activators of prothrombin (17,18,19).
On the basis of the structure of prothrombin worked out by Magnusson (18) and on the similarity with fragments produced during physiological activation (19), we can conclude that Ecarin splits the prothrombin molecule at the Arg-Ser bond, which is split during the release of the NH2-terminal portion of the molecule (intermediate 3).
The other preferential cleavage
by Ecarin appears to be the Arg-Ile bond, which links the A-and &chain in precursors of thrombin.
This bond is present in intermediate 1 and its
cleavage is presumably responsible for the appearance of the thrombin-like activity produced by Ecarin.
This cleavage is also a "sine qua non" for
"physiological" prothrombin activation (17,20, 21).
It is puzzling to us that the amount of alanine was higher in the Ecarin activated sample than in the prothrombin before activation (compare Table I). Our preliminary results indicated that it was higher also in the AC activated samples after longer incubation.
Further experiments need to be per-
formed to show if this represents a new cleavage in the intermediate 1 or if it simply reflects differences in yield of alanine in the mixtures. It has not escaped our attention that Seegers et al (20) previously have noted an excess of NH2- terminal alanine in activation mixtures involving prethrombin
Vol.6,Xo.l
PROTHROMBIN
AND
SNAKE
VENOM
61
Since a band (approx. m.w. 39,000) of increasing intensity appears during the activation with Ecarin, we assume that also the Arg-Thr bond, linking the inter mediate 2 with intermediate 4, is cleaved.
That means that eventually a mole-
cule, chemically indistinguishable from thrombin, may be formed. It was observed that addition of Ecarin to prothrombin, which has been partially activated by AC, considerably increased the biological activity.
Since the
incubation mixture contained predominantly intermediate 1, the increase in biological activity is explained by cleavage of the above mentioned Arg-Ile bond in this precursor.
On the other hand, the increase in activity of Ecarin-
thrombin on addition of AC is best explained by the release of intermediate 4 from Ecarin-thrombin (intermediate 1 with the Arg-Ile bond cleaved), and thereby a molecule identical with thrombin is produced.
It has been demonstrated
here and also described earlier (7) that Ecarin-thrombin has a lower specific activity on fibrinogen as compared to thrombin.
It is therefore quite feasible
that the conversion of Ecarin-thrombin into thrombin,in the presence of the brain thromboplastin activator (AC), is responsible for the increase in activity. Our results with regard to Ecarin are essentially in agreement with those of Iwanaga and coworkers (personal cormnunication, 1974).
ACKNOWLEDGEMENTS The authors express their gratitude for skillful technical assistance to Mrss. Elvy Andersson, Birgit Hessel, Helga Messel and Sonja Soderman,
This work was supported by grants from 'IheSwedish Medical Research Council (Nos. 13X-2475-08, and The National Institutes of Health (No. 5 ROl HLO737909).
62
PROTHROMBIN
AND
SNAKE
VENOI"I
REFERENCES
l. GITTER, S., LEVI, G., KOCHWA, S., DE VRIES, A., RECHNIc,,J., Aii CA=‘% J. Studies on the venom of Echis coloratus. Am. J. Trap. Med. Hvg. 2, 391, 1960. 2. KORNA_I,IK, F. Uber den Einfluss Von Echis carinata Toxin auf die Blutgerinnung in vitro. Folia Haematol. 3, 73, 1963. 3. JOBIN, F. and ESNOUF, M.P. Coagulant activity of Tiger snake (Notechis scutatus scutatus) venom. Nature 211, 873, 1966. 4. PIRKLE, H., MCINTOSH, M., THEODOR, I. AND VERNON, S. Activation of prothrombin with Taipan snake venom. Thromb. Res. 1, 559, 1972. 5. LANCHANTIN. G.F.. FRIEDMANN. J.A. and HART, D.W. _ bin. Journ: Biol: Chem. 248; 5956,1973.
Iwo forms of human throm
6. KORNALIK, F. Fibrinolytische Proteasen aus Schlangengiften. Folia Haemato1. 95, 193, 1971. 7..SCHIECK, ._ A., - HABERMANN, E. AND KORNALIK, F. The prothrombin activating principle from Echis carinatus venom. II. Coagulation studies in vitro and in vivo. Naunvn Schmiedebergs Arch. Pharmacol. 2, 7, 1972. 8. SCHIECK, A., KORNALIK, F. and HABERNANN, E. The prothrombin activating principle from Echis carinatus venom. I. Preparation and biochemical properties. Naunm SchxniedebergsArch. Pharmacol. 272, 402, 1972. 9. LNGWALL,J.S.and SCHERAGA, H.A. Purification and properties of bovine prpthrombin. Biochemistry 8, 1860, 1969. 10. MILLER, K.D. and COPEIAND, W.H. Human thrombin: Isolation and stability. Exper. Molecular Pathol. 4, 431, 1965. 11. MCDONAGH, J., MESSEL, H., MCDONAGH, R.P., MURANO, G. JR. and BLOMBACK B. Molecular weight analysis of fibrinogen and fibrin chains by an improved sodium-dudecylsulphate gel electrophoresis method. Bioch. Biophys. Acta 257, 135, 1972. 12. EDMAN, P. In: Protein Sequence Determination (Needleman, S.B. ed.), Springer-Verlag, New York 1970, p. 21. 13. IWANAGA, S., WALLEN, P., GRONDAHL, N.J., HENSCHEN, A. and BLOMBACK, B. On the primary structure of human fibrinogen. Isolation and characterization of N-terminal fragments from plasmic digests. European J. Biochem. 8, 189, 1969. 14. BLOMBACK, B. and YAMtUHINA, I. gen and fibrin. Arkiv Kemi 2,
On the N-terminal amino acid in fibrino299, 1958.
15. IRION,E. and BLOMB&CK, B. N-terminal amino acid sequence of intact human fibrinogen. FEBS Letters 7, 143, 1970.
Vol.6,No.l
PROTHROMBIN
tiD
SNAKE
VENOSI
63
16.
COPLEY, A.L., BANERJEE, S. and DEVI, A. Studies of snake venoms on blood coagulation. I. The thromboserpentin (thrombin-like) enzyme in the venoms. -Thromb. Res. 2, 487, 1973.
17a
HELDEBRANTS, C.M., BUTKOWSKI, R.J., BAJA-J, S.P. and MANN, K.G. The Activation of Prothrombin. II. Partial reactions, physical and chemical characterization of the intermediates of activation. Chem. 248: 20, 7149, 1973. --
17b
MANN, K.G., HELDEBRAND, C.M., FASS, D.N., BAJAJ, S.P. and BUTKOWSKI, R.J. The Molecular Mechanism of Prothrombin Activation. Thromb. Diath. Haemorr. Supple.57, 179, 1974.
18.
MAGNUSSON, S. Primary structure studies on thrombin and prothrombin. Thromb. Diath. Haemorrh. Suppl. 54, 31, 1973.
19.
MORITA, T., IWANAGA, S., SUZUKI, T. AND FUJIKAWA, K. Characterization of amino terminal fragment liberated from bovine prothrombin by activated factor X. FEBS Letters 36, 313, 1973.
20.
SEEGERS, W.H., MURANO, G., MCCOY, L. and MARCINIACK, E. The coagulation of blood: Preliminary survey of thrombin and autoprothrombin zymogen structure. Life Sci. 8, 925, 1969.
21. MAGNUSSON, S. Terminal amino acid residues in bovine prothrombin and in prothrombin activation mixture. Arkiv Kemi 23, 271, 1965.
