THROMBOSIS RESEARCH 66; 169-177,1992 0049-3848/92 $5.00 + .OOPrinted in the USA. Copyright (c) 1992 Pergamon Press Ltd. All rights reserved.
ACT I VATI ON OF Lonomla
HUMAN PRCTHRCMf3 I N BY THE VENOM OF achelous (Craner) CATERPILLARS
Guerrero, LaboratOriO Ctentiflcas
de Ftslopatologia, (IVIC), Apartado
B.,
Arocha-Pltiango,
C.L.
lnstttuto Venezolano de lnvestlgaclones 21827, Caracas 1020, Caracas-VENEZUELA
(Received 7.8.1991; accepted in revised form 30.1.1992 by Editor J. Soria)
ABSTRACT In thts paper, we demonstrated that the procoagulant action of LOnCzmla achelous (CrWr) IS due In part to a component that activates prothrcmbln. The actlvatlon by crude venom and Fractions obtained by ftltratlon on Sephadex G-75 IS not dependent of phosphol Ipld, gel I IS Ca++ or Factor V. The actlvatlon of prothrcmbln by Fraction greatly stimulated by Factor V In the presence of phosphol~p~d and Ca++; In presence of SBTI, we found that the Fraction I and Factor Xa act In a slmllar manner. These results suggest that the Fraction I IS a Factor Xa - I Ike prothrombtn activator.
INTR0DUCTICN The convertlon of prothranbln to thrombln IS one of the key reactions of Under physlologlcal condltlons this process IS the coagulation cascade. catalysed by Factor Xa, potentlated by Factor V, Ca++ and phospholipids from the so called the “prothrcfnbInase Complex”, the platelets membranes, formlng (l-5). Prothrombln activators have been Isolated from vencms of a wide variety however there are large differences between their meChanIsm of snakes (6-8); others require the of act Ion: Some activate prothrcmbin directly (g-12), al I the and the maJOrltY needs of phospho I I p I d and Ca++ (13,14) presence complex to fully develop their activity, (8, components of the prothrombinase 15‘16).
Key words:
Prothrombln, Factor Xa.
Prothrombln
activators,
169
I Onoinla
achelous
venom,
170
ACTIVATION OF HUMAN PROTHROMBIN
Vol. 66, Nos. 213
A bleeding syndrome produced by Contact with the lOncmi8 achelous caterptllars was descrtbed by one of us In 1967 (17). Later, several cases have being studied and cosmon findtngs are: normal platelets, low fibrinogen, degradation Factor V, Factor XIII and plasminogen; with Increased flbrlnogen In the very severe cases a Products (FDP’s) and I ys I s areas on f I br I n pl ate. fall In prothrcmbln and accelerated thrcmbin generation has also been observed (18-20) . Urokinase, “In vitro” It has been demonstrated the presence of suggesting that the clinical Kallikreln and Factor Xa-IIKe activities (21), picture should be due to a combination of the effect of those enzymes. In a previous papers we reported the effect on purlfled fibrinogen In this paper we studied the activation of purlfled prothrombin by the hemolymph (00 and scme semipurified chrcmatographic fractions.
MATERIALS &Q
(22); crude
METHODS
Reagents: Chromogenic Substrates and Purlfled Human Flbrlnogen, (Grade L) were purchased frcfn A 6 Kabl DiagnostIca; Soybean Trypsin InhIbItor Bovtne Serum AlbtNnln (BSA) and other reagents were Purchased from (SBTI), Slgna Chemical Co; R.V.V. (Vipera russell~ venom) was from Wellcome Reagents Ltd. Prothrombln, Factor X and Factor Coaaulatlon Factors: from human fresh plasma as deSCrIbeed earlier (23,24). Factor from Factor X after actkvation by RVV-X (25).
V were Xa was
purified prepared
Lonomia achelous: Crude hemolymph was collected by aspiration with a needle and syringe from the body of the caterpillars. The hemolyrnph was purlfled by gel filtration chromatography on Sephadex G-75 as previously descr I bed (26) , The Fractions were pooled according to the protein peaks and tested for z#nidoIytic activities with Chromogenic Substrates S-2444, s-2222, S-2238 and S-2251 (Substrates of UK, Xa, Ila and plasmln respectively) and fibrinolytic activity on fibrin plates (26-27). Activation experiments: Prothrcnnbln activation by crude hemolymph or Fractions were carried out at 37GC in Plastic tubes. Prothrombin was prelncubated for 3 mln at 37% in buffer containing 50 N&I TrIs/HCL, 150 &I NaCl 0.5 mg/mI BSA (prelncubatlon mixture). When cofactors (PH 7.4)s Factor V or Cacl2) were Present, they were included in the (phospholipids, Prelncubation mixture. Prothrcmbtn activation was started with the addition of the activator; after different time Intervals samples were drawn for clotting and chromogenic assays. The exact Cunposition of the InCUbatiOn mixture IS given in the description of the lndivldual experiments. Clott Ing assay: 200 ul of 0.3% purified H-n Fibrinogen In buffer COntalnlng 50 mM Trls/HCI, 150 mM NaCI, (pH 7,4) were PreWarfMd to 37%. At the Indicated incubation time, 50 ul of tncubatlon mixture was added and the clotting time was recorded.
150
ChromoQenlc substrate m NaCl PH 7.4 buffer
assays: To a mixture (containing 0.5 mg/mI
of 230 ul of 50 rm Tris/HCl, BSA) and 10 UI S-2238 (5.33
ACTIVATION OF HUMAN PROTHROMBIN
Vol. 66, Nos. 213
r’r+l) prewarmed lncubatlon Increase
at
37%,
time. was >0.2
10 ul
curves Ca I I brat I on Olagnostlc Reagents Ltd. National lnstltute for actlvltles were expressed each
callbratlon
:
mtxture
was
added
at
the
tndlcated
recorded at 405 nm; volume was reduced.
tf
Were prepared with purlflea Factor Xd Plasmin and Thrombln Standards and Uroklnase, Blologlcal Standards and Control (N I BSC) . tn U/ml calculated by plotting the results
by SBTI: Adltlon 10n of= Ug/ml was
COm3itrat
Ion
Incubation
the
from from Al 1 against
curve.
lnhlbltlon f Inai Fract
of
The Increase In absorbance was absorbancy unlts/mln, the sample
171
of SBTI to the lncubatlon used to study its effect
mixture on Factor
at 2(a
a ana
I.
RESULTS Tt-ie crude Hemolymph or Sephadex fraCtlOnS UIU not Present actlvrty as no direct hydrolysis of chrcmogenlc substrate S-2238 Crude Hefnolymph, Fractions II and III presented flbrrnolytic flbrln plates and hyCtrOlYSed chromogenic sustrates S-2222, S-2444 however Fraction I Only hydrolysed S-2222 (Table I). TABLE CHARACTERISTICS
actlvlty
I
OF THE HEMOLYMPH AND SEPHADEX
Flbrlnolytlc on Fibrin
Plate
s-2222
Actlvatlon hemolymph or or amldolytic metnods. The prothrombln
Actlvltles S-2251
(U/ml
i3,o 0.0 10.2 7.2
I II III
FRACTIONS
Pmidolytlc S-2444
(U/ml )
Hemolymph Fraction Fraction Fractton
thrcmbln-I Ike was observed. aCtlVlty on and S-2251;
4.10 1.05 1 .30 0.84
S-2238
1
10.25 0.0 3.60 3.30
3.4 0.0 2.4 1.0
0 0 0 0
The velocity of thrcinbln formatjon by experiments: Fractions was very Slmllar when measured either by the method, therefore the results shown are the expresion
crude hemolymph and Fractions In the absence of phospholipids,
I,
II Ca++
and and
Ill Factor
the
readily V,
Figure
crude
coagulant of both
activated 1.
tne Vencin catalyzed protnrombln activation was proportional to In the A slow decay concentration of the activator as seen In Figure 2. thrombln activity was observed with higher concentration of CrUde hemolymph and Ca++ did not affect the aCt!vatlOn and Fraction II. Phospholiplds material but wnen Factor V was added In the produced by al 1 caterpillar presence of phOSphOl~p~Cis plus Ca++ to the incubation mixture of Fraction I, the VelOCltY of the reaction was Ur~tICally Increased showing an aCtiVltY slmllar
to
that
of
Factor
Xa,
(Table
II
and
Figure
3)
Vol. 66, Nos. 213
ACTIVATION OF HUMAN PROTHROMBIN
172
8
6
12
8
A&N
INCUB
TIME (HOURS 1
Figure GeneratIon of thrombln during hemolymph and fractions: The final of: Factor Xa (XI; and 0.1 u/ml Fraction I I (Cl); and Fraction II
t
Crude
activation by Factor Xa, of PrOthrombtn was 200 ( A 1; Fraction hemolymph
crude
prothrombln concentration
ug/ml
I
(0) ;
I (A) *
[E
FB
4
8
4
12
INCUBATION
Figure
TIME
(HOURS
8
12
I
&
GeneratIon of thrombin during prothrombln cofactors) by different concentration of crude COnCentrat ion Of ACt i VatOr was (X) 0.1 f lnal U/ml; and of Protrtrcfnbln was 200 ug/ml .
activation nemolymph, U/ml ; (0)
(in the absence and Fractions. 0.25 U/ml; (A)
of The 0.5
Vol. 66, Nos. 213
ACTIVATION
OF HUMAN PROTHROMBIN
173
TABLE I I -EFFECT OF Pl-0SPHCLIPIDS (PL), CA++ AND FACTOR V (FV) PROTHRUlBlN ACTIVATION RELATIVE CRUDE FACTOR Xa HEMCLYMPH
Act
VatOr
CN THE
VELOCITY FRACTIONS
*
I
II
III
1.00
1.00
1 *cm
1.03
1 .ocJ
7.52
1.03
1.17
1.69
1 .30
Act t vator, Ca++,PL,FV
9,290.OO
1.09
2.08
1 .50
*
/min/U
I
Activator, Ca++, PL
U Thrombln
2,370.O
Activator.
:
CRUDE
3
HEMOLYMPH (U/ml 1
z20-
FACTOR
Xa
(U/ml
1
2 FRACTION
INCUBATION
TIME FlRure
(HOURS
I (U/ml
)
1
&
GeneratIon of thrombin Uurlng prothrombin acttvatron by Factor hemol ymph and Fraction I In the absence (0) or presence (X) of 20 rr+l Cal Ct7lOrlUe and 80 ugjml pnospnol~p~d or’ the same concentration of calcium phosphollpld plus 3 ug/ml of Factor V. ( A ) The flnal concentration prothrcmbIn was 500 ug/ml. The concentration of Factor Xa ana venom represented In the figures.
Xa, c I Wn
anU of are
ACTIVATION OF HUMAN PROTHROMBIN
174
Effect of SBTI on the prOthrombln activation & Fraction the catalytic activity of Fraction I SBTI inhibited pattern of that of Factor Xa, (Figure 4) .
FRACTION
Vol. 66, Nos. 213
L. with
a
similar
I
30 t
::Ly 0
!?i
20
40
INCUBATION TIME Figure
](
60
(min )
&
Effect of SBTI on prothrombln activation In the presence of phospholipid, calciun and Factor V. The final concentration of each Component in the 0.02 U/ml; Fraction I: 0.01 mixture were: ProthrOmbin 500 ug/ml; Factor xa: The concentration of cofactors were the same as U/ml and SBTI: 50 &t/ml. presence of descrtbed in the previous exper lment. (0) Abscence of SBTI ; (X) SBTI.
DISaJSSIoN
In severe cases of caterplllar polsonlng It has been observed low values of Factor II as well as an acceleration of thrombln generation. In moderate cases the transfusion of whole blood or plasma agravates the cltnlcal situation appearing thrcfnbocytopenra; it has been suggested that two processes coexist: a very severe frbrlnolytlc SYnUrCme which maSKed a very mild DIG. The latter being manIfeSted when all coagulation factors are supplied witn plasma, as It IS not seen when the patients are treated with only hUnan purlfieci
fibrinogen
and aprotinin
(19).
Our results conftrm the hypothesis of caterpillar venom that is very similar to the I, which is free of fibrlnolytic activity.
a procoagulant activity action of Factor Xa in
In the Fraction
Vol. 66, Nos. 2L3
The
ACTIVATION OF HUMAN PROTHROMBIN
prothrombtn
acttvator
from
the
crude
hemolymph
175
or
Fracttons
IV from the vencm of C. achelous strongly resembles the activators the L carlnatus (10,28,29), 0. typus (9, 30) and S, atrox (11) direct prothrombin activators.
II
and
present which
In are
On the other hand, the activator found In the Fraction I has been found to act SlmilarlY to the PhYSlOlOglCal aCtlVatOr (Factor Xa) (5) and activators from N_ SCUtatUS (15,31,32) and K ater nlger (16) whose actlvlty IS greatly enhanced by the Presence of pho~liplds, Ca++ plus Factor V. The I IS other lnhlbitory effect produced by SBTI on the activation by Fraction lndlcatfve Therefore It appears that at of a Factor Xa-llke activity. I east two classes of prothranbin activators are present in L achelous. The decay In thrombin generatlon during the process, betng more evident for high concentrations of Fraction I, seems to be due to the Presence of one I or to a Proteolytlc InhIbItor of thrcmbln wlch coeluate with Fraction agent against thrcmbln of retarded actton. In the hemolymph of the L.achelous CaterpIllar It has been In conclusion: ldentlfled the crude
two different prothrcfnbln activators: direct, wlch hemolymph, Fractions II and III and a Factor Xa-I Ike
More different
work are activators
the
We acknowledge: ConIcIt Grant S-1352, Maraven Petroleos de Venezuela Glaxo LaboratorIeS U.S.A. for the flnanclal help, to Banco de Sangre del Federa 1 for supplylng the plasma; to Dr. N. Bosch and Dr. R. Apltz for secretarial valuable and useful CCmf?entS and to L. Rybak and A. GII for blbllographrcal work.
and Dto.
In order
to
and
In I.
CharaCterIZeed
In process Identlfled.
Isolate
IS Present In Fraction
thelr and
REFERENCES 1.
The converslbn of prothrombln Esmon, C.T. and JaCkSOn, C.M. J. III, The Factor Xa-catalyzed actlvatlon of prothrcmbln. 249: 7782-7790, 1974.
2.
and Jackson, C.M. 49: 765-811, 1980.
3.
Roslng, J., Tans, H.C. The Role complex. J. BIoI.
4.
5.
Nemerson,
Y.
Blood
coagulation
J.W.P., G., Govers-Rlemslag, of phosphollplds and factor Chem. 255: 274-283, 1980.
The klnetlcs M.E. and Mann, K.G. Netshelm, the two cleavages involved In prothrombfn 258: 5386-5391, 1983.
Zwaal, In
Va
3299,
1987.
Rev.
Ann.
Blochem.
R.F.A. and Herr)
ACT I VATI ON OF Lonomla
HUMAN PRCTHRCMf3 I N BY THE VENOM OF achelous (Craner) CATERPILLARS
Guerrero, LaboratOriO Ctentiflcas
de Ftslopatologia, (IVIC), Apartado
B.,
Arocha-Pltiango,
C.L.
lnstttuto Venezolano de lnvestlgaclones 21827, Caracas 1020, Caracas-VENEZUELA
(Received 7.8.1991; accepted in revised form 30.1.1992 by Editor J. Soria)
ABSTRACT In thts paper, we demonstrated that the procoagulant action of LOnCzmla achelous (CrWr) IS due In part to a component that activates prothrcmbln. The actlvatlon by crude venom and Fractions obtained by ftltratlon on Sephadex G-75 IS not dependent of phosphol Ipld, gel I IS Ca++ or Factor V. The actlvatlon of prothrcmbln by Fraction greatly stimulated by Factor V In the presence of phosphol~p~d and Ca++; In presence of SBTI, we found that the Fraction I and Factor Xa act In a slmllar manner. These results suggest that the Fraction I IS a Factor Xa - I Ike prothrombtn activator.
INTR0DUCTICN The convertlon of prothranbln to thrombln IS one of the key reactions of Under physlologlcal condltlons this process IS the coagulation cascade. catalysed by Factor Xa, potentlated by Factor V, Ca++ and phospholipids from the so called the “prothrcfnbInase Complex”, the platelets membranes, formlng (l-5). Prothrombln activators have been Isolated from vencms of a wide variety however there are large differences between their meChanIsm of snakes (6-8); others require the of act Ion: Some activate prothrcmbin directly (g-12), al I the and the maJOrltY needs of phospho I I p I d and Ca++ (13,14) presence complex to fully develop their activity, (8, components of the prothrombinase 15‘16).
Key words:
Prothrombln, Factor Xa.
Prothrombln
activators,
169
I Onoinla
achelous
venom,
170
ACTIVATION OF HUMAN PROTHROMBIN
Vol. 66, Nos. 213
A bleeding syndrome produced by Contact with the lOncmi8 achelous caterptllars was descrtbed by one of us In 1967 (17). Later, several cases have being studied and cosmon findtngs are: normal platelets, low fibrinogen, degradation Factor V, Factor XIII and plasminogen; with Increased flbrlnogen In the very severe cases a Products (FDP’s) and I ys I s areas on f I br I n pl ate. fall In prothrcmbln and accelerated thrcmbin generation has also been observed (18-20) . Urokinase, “In vitro” It has been demonstrated the presence of suggesting that the clinical Kallikreln and Factor Xa-IIKe activities (21), picture should be due to a combination of the effect of those enzymes. In a previous papers we reported the effect on purlfled fibrinogen In this paper we studied the activation of purlfled prothrombin by the hemolymph (00 and scme semipurified chrcmatographic fractions.
MATERIALS &Q
(22); crude
METHODS
Reagents: Chromogenic Substrates and Purlfled Human Flbrlnogen, (Grade L) were purchased frcfn A 6 Kabl DiagnostIca; Soybean Trypsin InhIbItor Bovtne Serum AlbtNnln (BSA) and other reagents were Purchased from (SBTI), Slgna Chemical Co; R.V.V. (Vipera russell~ venom) was from Wellcome Reagents Ltd. Prothrombln, Factor X and Factor Coaaulatlon Factors: from human fresh plasma as deSCrIbeed earlier (23,24). Factor from Factor X after actkvation by RVV-X (25).
V were Xa was
purified prepared
Lonomia achelous: Crude hemolymph was collected by aspiration with a needle and syringe from the body of the caterpillars. The hemolyrnph was purlfled by gel filtration chromatography on Sephadex G-75 as previously descr I bed (26) , The Fractions were pooled according to the protein peaks and tested for z#nidoIytic activities with Chromogenic Substrates S-2444, s-2222, S-2238 and S-2251 (Substrates of UK, Xa, Ila and plasmln respectively) and fibrinolytic activity on fibrin plates (26-27). Activation experiments: Prothrcnnbln activation by crude hemolymph or Fractions were carried out at 37GC in Plastic tubes. Prothrombin was prelncubated for 3 mln at 37% in buffer containing 50 N&I TrIs/HCL, 150 &I NaCl 0.5 mg/mI BSA (prelncubatlon mixture). When cofactors (PH 7.4)s Factor V or Cacl2) were Present, they were included in the (phospholipids, Prelncubation mixture. Prothrcmbtn activation was started with the addition of the activator; after different time Intervals samples were drawn for clotting and chromogenic assays. The exact Cunposition of the InCUbatiOn mixture IS given in the description of the lndivldual experiments. Clott Ing assay: 200 ul of 0.3% purified H-n Fibrinogen In buffer COntalnlng 50 mM Trls/HCI, 150 mM NaCI, (pH 7,4) were PreWarfMd to 37%. At the Indicated incubation time, 50 ul of tncubatlon mixture was added and the clotting time was recorded.
150
ChromoQenlc substrate m NaCl PH 7.4 buffer
assays: To a mixture (containing 0.5 mg/mI
of 230 ul of 50 rm Tris/HCl, BSA) and 10 UI S-2238 (5.33
ACTIVATION OF HUMAN PROTHROMBIN
Vol. 66, Nos. 213
r’r+l) prewarmed lncubatlon Increase
at
37%,
time. was >0.2
10 ul
curves Ca I I brat I on Olagnostlc Reagents Ltd. National lnstltute for actlvltles were expressed each
callbratlon
:
mtxture
was
added
at
the
tndlcated
recorded at 405 nm; volume was reduced.
tf
Were prepared with purlflea Factor Xd Plasmin and Thrombln Standards and Uroklnase, Blologlcal Standards and Control (N I BSC) . tn U/ml calculated by plotting the results
by SBTI: Adltlon 10n of= Ug/ml was
COm3itrat
Ion
Incubation
the
from from Al 1 against
curve.
lnhlbltlon f Inai Fract
of
The Increase In absorbance was absorbancy unlts/mln, the sample
171
of SBTI to the lncubatlon used to study its effect
mixture on Factor
at 2(a
a ana
I.
RESULTS Tt-ie crude Hemolymph or Sephadex fraCtlOnS UIU not Present actlvrty as no direct hydrolysis of chrcmogenlc substrate S-2238 Crude Hefnolymph, Fractions II and III presented flbrrnolytic flbrln plates and hyCtrOlYSed chromogenic sustrates S-2222, S-2444 however Fraction I Only hydrolysed S-2222 (Table I). TABLE CHARACTERISTICS
actlvlty
I
OF THE HEMOLYMPH AND SEPHADEX
Flbrlnolytlc on Fibrin
Plate
s-2222
Actlvatlon hemolymph or or amldolytic metnods. The prothrombln
Actlvltles S-2251
(U/ml
i3,o 0.0 10.2 7.2
I II III
FRACTIONS
Pmidolytlc S-2444
(U/ml )
Hemolymph Fraction Fraction Fractton
thrcmbln-I Ike was observed. aCtlVlty on and S-2251;
4.10 1.05 1 .30 0.84
S-2238
1
10.25 0.0 3.60 3.30
3.4 0.0 2.4 1.0
0 0 0 0
The velocity of thrcinbln formatjon by experiments: Fractions was very Slmllar when measured either by the method, therefore the results shown are the expresion
crude hemolymph and Fractions In the absence of phospholipids,
I,
II Ca++
and and
Ill Factor
the
readily V,
Figure
crude
coagulant of both
activated 1.
tne Vencin catalyzed protnrombln activation was proportional to In the A slow decay concentration of the activator as seen In Figure 2. thrombln activity was observed with higher concentration of CrUde hemolymph and Ca++ did not affect the aCt!vatlOn and Fraction II. Phospholiplds material but wnen Factor V was added In the produced by al 1 caterpillar presence of phOSphOl~p~Cis plus Ca++ to the incubation mixture of Fraction I, the VelOCltY of the reaction was Ur~tICally Increased showing an aCtiVltY slmllar
to
that
of
Factor
Xa,
(Table
II
and
Figure
3)
Vol. 66, Nos. 213
ACTIVATION OF HUMAN PROTHROMBIN
172
8
6
12
8
A&N
INCUB
TIME (HOURS 1
Figure GeneratIon of thrombln during hemolymph and fractions: The final of: Factor Xa (XI; and 0.1 u/ml Fraction I I (Cl); and Fraction II
t
Crude
activation by Factor Xa, of PrOthrombtn was 200 ( A 1; Fraction hemolymph
crude
prothrombln concentration
ug/ml
I
(0) ;
I (A) *
[E
FB
4
8
4
12
INCUBATION
Figure
TIME
(HOURS
8
12
I
&
GeneratIon of thrombin during prothrombln cofactors) by different concentration of crude COnCentrat ion Of ACt i VatOr was (X) 0.1 f lnal U/ml; and of Protrtrcfnbln was 200 ug/ml .
activation nemolymph, U/ml ; (0)
(in the absence and Fractions. 0.25 U/ml; (A)
of The 0.5
Vol. 66, Nos. 213
ACTIVATION
OF HUMAN PROTHROMBIN
173
TABLE I I -EFFECT OF Pl-0SPHCLIPIDS (PL), CA++ AND FACTOR V (FV) PROTHRUlBlN ACTIVATION RELATIVE CRUDE FACTOR Xa HEMCLYMPH
Act
VatOr
CN THE
VELOCITY FRACTIONS
*
I
II
III
1.00
1.00
1 *cm
1.03
1 .ocJ
7.52
1.03
1.17
1.69
1 .30
Act t vator, Ca++,PL,FV
9,290.OO
1.09
2.08
1 .50
*
/min/U
I
Activator, Ca++, PL
U Thrombln
2,370.O
Activator.
:
CRUDE
3
HEMOLYMPH (U/ml 1
z20-
FACTOR
Xa
(U/ml
1
2 FRACTION
INCUBATION
TIME FlRure
(HOURS
I (U/ml
)
1
&
GeneratIon of thrombin Uurlng prothrombin acttvatron by Factor hemol ymph and Fraction I In the absence (0) or presence (X) of 20 rr+l Cal Ct7lOrlUe and 80 ugjml pnospnol~p~d or’ the same concentration of calcium phosphollpld plus 3 ug/ml of Factor V. ( A ) The flnal concentration prothrcmbIn was 500 ug/ml. The concentration of Factor Xa ana venom represented In the figures.
Xa, c I Wn
anU of are
ACTIVATION OF HUMAN PROTHROMBIN
174
Effect of SBTI on the prOthrombln activation & Fraction the catalytic activity of Fraction I SBTI inhibited pattern of that of Factor Xa, (Figure 4) .
FRACTION
Vol. 66, Nos. 213
L. with
a
similar
I
30 t
::Ly 0
!?i
20
40
INCUBATION TIME Figure
](
60
(min )
&
Effect of SBTI on prothrombln activation In the presence of phospholipid, calciun and Factor V. The final concentration of each Component in the 0.02 U/ml; Fraction I: 0.01 mixture were: ProthrOmbin 500 ug/ml; Factor xa: The concentration of cofactors were the same as U/ml and SBTI: 50 &t/ml. presence of descrtbed in the previous exper lment. (0) Abscence of SBTI ; (X) SBTI.
DISaJSSIoN
In severe cases of caterplllar polsonlng It has been observed low values of Factor II as well as an acceleration of thrombln generation. In moderate cases the transfusion of whole blood or plasma agravates the cltnlcal situation appearing thrcfnbocytopenra; it has been suggested that two processes coexist: a very severe frbrlnolytlc SYnUrCme which maSKed a very mild DIG. The latter being manIfeSted when all coagulation factors are supplied witn plasma, as It IS not seen when the patients are treated with only hUnan purlfieci
fibrinogen
and aprotinin
(19).
Our results conftrm the hypothesis of caterpillar venom that is very similar to the I, which is free of fibrlnolytic activity.
a procoagulant activity action of Factor Xa in
In the Fraction
Vol. 66, Nos. 2L3
The
ACTIVATION OF HUMAN PROTHROMBIN
prothrombtn
acttvator
from
the
crude
hemolymph
175
or
Fracttons
IV from the vencm of C. achelous strongly resembles the activators the L carlnatus (10,28,29), 0. typus (9, 30) and S, atrox (11) direct prothrombin activators.
II
and
present which
In are
On the other hand, the activator found In the Fraction I has been found to act SlmilarlY to the PhYSlOlOglCal aCtlVatOr (Factor Xa) (5) and activators from N_ SCUtatUS (15,31,32) and K ater nlger (16) whose actlvlty IS greatly enhanced by the Presence of pho~liplds, Ca++ plus Factor V. The I IS other lnhlbitory effect produced by SBTI on the activation by Fraction lndlcatfve Therefore It appears that at of a Factor Xa-llke activity. I east two classes of prothranbin activators are present in L achelous. The decay In thrombin generatlon during the process, betng more evident for high concentrations of Fraction I, seems to be due to the Presence of one I or to a Proteolytlc InhIbItor of thrcmbln wlch coeluate with Fraction agent against thrcmbln of retarded actton. In the hemolymph of the L.achelous CaterpIllar It has been In conclusion: ldentlfled the crude
two different prothrcfnbln activators: direct, wlch hemolymph, Fractions II and III and a Factor Xa-I Ike
More different
work are activators
the
We acknowledge: ConIcIt Grant S-1352, Maraven Petroleos de Venezuela Glaxo LaboratorIeS U.S.A. for the flnanclal help, to Banco de Sangre del Federa 1 for supplylng the plasma; to Dr. N. Bosch and Dr. R. Apltz for secretarial valuable and useful CCmf?entS and to L. Rybak and A. GII for blbllographrcal work.
and Dto.
In order
to
and
In I.
CharaCterIZeed
In process Identlfled.
Isolate
IS Present In Fraction
thelr and
REFERENCES 1.
The converslbn of prothrombln Esmon, C.T. and JaCkSOn, C.M. J. III, The Factor Xa-catalyzed actlvatlon of prothrcmbln. 249: 7782-7790, 1974.
2.
and Jackson, C.M. 49: 765-811, 1980.
3.
Roslng, J., Tans, H.C. The Role complex. J. BIoI.
4.
5.
Nemerson,
Y.
Blood
coagulation
J.W.P., G., Govers-Rlemslag, of phosphollplds and factor Chem. 255: 274-283, 1980.
The klnetlcs M.E. and Mann, K.G. Netshelm, the two cleavages involved In prothrombfn 258: 5386-5391, 1983.
Zwaal, In
Va
3299,
1987.
Rev.
Ann.
Blochem.
R.F.A. and Herr)
