- Email: [email protected]
On the quantification of prothrombin from different species using echis carinatus as activator
THROMBOSIS RESEARCH 42; 737-747, 1986 0049-3848/86 $3.00 t .OO Printed in the USA. Copyright (c) 1986 Pergamon Journals Ltd. All rights reserved.
ON THE QUM?l!IFICATION OF PRCYI'BRWINFRCM DIFFEREXW! SPECIES USING ECHIS CARINATUSAS ACTIVATOR Ole KristianTollersrudand Liv Helgeland Departmentof Biochemistry,Universityof Oslo, P.Q. Box 1041,Blindern,0316 Oslo 3, Norway (Sutntitted to Editor S. Magnusson6.3.1985;Acceptedby Editors-in-Chief B. Bl&ck and A.L. Copley 2.4.1986)"
ABSTRACT
The generation of thrombin-like activity from rat, human, bovine and mouse prothrombin by Echis carinatus venom (ECV) treatment was compared using a partially purified system (i.e. whole ECV and A rapid increase in coagulant isolated prothrombin). activity was obtained within 0.5 to 2 min., being constant upon further incubation for 60 min. A large variation in coagulant activity of the ECV generated thrombin from the four species was found, whereas no differences were found for the amidolytic activities. The coagulant activities of the ECV generated thrombin was also low compared with the corresponding thrombin activities obtained by physiological activation. Coagulant activity of the ECV generated thrombin levelled off at increasing concentration of prothrombin in the sample as measured by the onestage coagulation assay. By measuring amidolytic activity a linear relationship to the concentration These findings of prothrombin was found, however. indicate that ECV converts prothrombin from the four different species to a thrombin-like protein with The lack of properties distinct from a-thrombin. linearity in the ECV generated clot activity with increasing concentration of prothrombin could be explained by assuming a dimerization of the thrombinlike protein molecules making them less accessible The significance of these observato fibrinogen. tions for the quantification of prothrombin from different species is discussed.
Key %ords; Prothronbinquantification- Echis carinatus- various species * The acceptanceof this cm icationby the Editors-in-Chief is exceptional and caused by the utter negligenceof the Editor to whcm it was originally stittd
737
738
QUANTIFICATION OF PROTHROMBIN
Vol. 42, No. 6
INTRODUCTION In contrast to factor X,, Echis carinatus venom (ECV) converts prothrombin to an active enzyme in the absence of factor V,, calcium and phospholipid (1). ECV thus activates acarboxyprothrombin as well as partially carboxylated prothrombin and congenital disorders in which prothrombin has low clotting activity in physiological systems (2,3). This has made ECV an important tool to trace and to quantify the amount of these various forms of prothrombin (2,4). A marked difference between the activation of prothrombin by ECV and the physiological activation of prothrombin has, however, been revealed. ECV cleaves prothrombin initially at one site, an Arg-Ile bond linking the A and B chains in thrombin (5,6). The intermediate thus formed, meizothrombin, is further converted to meizothrombin-des Fl (also called meizothrombin l), by splitting off This conversion has been suggested to be fragment 1 (7-10). autocatalytic by some authors (7,9) whereas others consider ECV to be responsible for the formation of meizothrombin-des Fl The coagulant activity of the two thrombin-like inter(8). mediates is low compared to that of a-thrombin whereas the The esterase and amidolytic activity is high (6,9-13). subsequent conversion of meizothrombin-des Fl to a -thrombin is autocatalytic (5,7). The formation of a-thrombin depends apparently on a highly purified system, i.e. purified prothrombin and purified ECV (Ecarin), and prolonged incubation times (5,7,12,). When prothrombin is assayed by the ECV method the whole venom is employed and prothrombin in the sample is not purified. Moreover, a-thrombin has frequently been used as standard reagent to convert clot time into thrombin units (14-16). In the present paper we have shown that the ECV generated thrombin activity is not related to the clotting time as found for a-thrombin (T=k/t). A modified method for the quantification of prothrombin from different species is described. MATERIALS
AND METHODS
Animals. Male albino rats, 200-250 g of the Wistar strain, and female black mice NMRI/BOM from Mollegaard, Havrup, Denmark, were used. Chemicals. Acacia (gum arabic) and Echis carinatus venom were obtained from Sigma Chemical Co., USA. Human fibrinogen L Grade (90% clottable) was purchased from Kabi, Stockholm, Sweden, bovine thrombin (Topostasine) from Hoffman-LaRoche Co., Basel, Switzerland, and Russel's viper venom (Stypven) from Burroughs Wellcome Co., U.K. The chromogenic substrate Th-1 (D-cyclohexylglycyl-L-alanyl-L-arginine-p-nitroanilide dihydroacetate) was a gift from Nyegaard & Co. A/S., Oslo, Norway. Preparation of plasma. Blood was obtained by cardiac or venipuncture and collected in 3.8% sodium citrate (10% v/v). Platelet-free plasma was obtained after centrifugation at 5OOOxg (20 min. 4OC) and stored at -2OOC for up to 2 weeks.
Vol. 42, No. 6
QUANTIFICATION OF PROTHROMBIN
739
Standard human plasma was pooled from 20 healthy volunteers. The procedure is the same as Purification of prothrombin. Plasma was described before with minor modifications (17). diluted 1:l with 5.14 mM veronal*HCl buffer (pH 7.4) containing To 1.0 ml of the 0.12 M NaCl and 6.06 mM sodiumcitrate. After diluted plasma was added 2.8 mg sodium citratea2H20. The suspension dissolution 35 ul of 1M BaC12 was added slowly. was stirred for 20 min and centrifuged at 6000xg for 10 min. The precipitate was solubilized in 1 ml of 5.14 mM veronal*HCl buffer (pH 7.4) containing 0.12 M NaCl and 6.06 mM sodium The citrate and the adsorption procedure was repeated. precipitate was suspended in 0.15 ml of a mixture of 0.04 M Dissolution was achieved by sodium citrate and 0.31 M NaCl. addition of 0.25 ml of 0.2 M EDTA (adjusted to pH 7.4 with 10 M NaOH). The solution was passed through a Sephadex-G 50 fine column equilibrated 'with 5.14 mM veronal.HCl buffer pH 7.4 The entire containing 0.12 M NaCl and 6.06 mM sodium citrate. Bovine prothrombin was procedure was carried out at 4oC. purified to homogeneity as described previously (18). Factor II assay. Prothrombin was measured using the one-stage The test was made specific for method of Hjort et al. (19). prothrombin by adding factor X (Stuart) to the test solution One unit of prothrombin was arbitrarily chosen as the (20). amount of prothrombin present in 1 ml of standard human plasma diluted 1:lOOO. Echis carinatus methods. Prothrombin was activated by incubating 0.1 ml isolated prothrombin solution and 0.025 ml Echis carinatus venom (1.0 mg/ml) at 37OC. The appropriate incubation time was tested out. Both the coagulant and amidolytic activity of Echis-generated thrombin were measured. Coagulant activity. Thrombin activity was measured with fibrinogen as substrate as described previously (14). The clotting times were converted to thrombin units by using a standard curve prepared by dilution of purified bovine standard a-thrombin. Amidolytic activity. A mixture of 0.125 ml of the activated prothrombin solution, 0.05 ml Th-1(2AcOH*H-D-CHG-Ala-Arg-pNA) (2 Vmol/ml) and 0.625 ml 5.14 mM veronal*HC1 buffer pH 8.2 containing 6.06 mM sodium citrate and 0.12 M NaCl was incubated for 15 min at 37OC. Subsequently 0.4 ml 40% HAc was added to stop the reaction. The formation of p-nitroaniline was measured at 405 nm. The amidolytic activity of the Echis generated thrombin is linearly related to AOD405/min. A blank omitting the activated prothrombin solution was included since Echis carinatus venom relased a small, but significant amount of free p-nitroaniline from Th-1. Microsomal sonicates were prepared as described previously (151. RESULTS Prothrombin from four species, human, bovine, rat and mouse was isolated from citrate plasma by adsorption on barium citrate as described in Materials and Methods. This isolation resulted in about 300 fold purification of prothrombin. The isolated prothrombin was diluted to obtain a concentration of 50 U/ml as measured by a one stage coagulation assay (19). In a system as
QUANTIFICATION OF PROTHROMBIN
Vol. 42, No. 6
described in fig. 1 the incubation time necessary for maximum The incubation ECV generated coagulant activity was estimated. time varied to a certain degree between the species; whereas 0.5-l min. was necessary for rat and mouse prothrombin, human and bovine prothrombin required 3-5 min. to obtain maximum This in agreement with another report thrombin activity. concerning rat prothrombin (2) but Carlisle et al. (21) found that even after incubation of mouse prothrombin for 3 hours The investigation maximum thrombin activity was not reached. was carried out on microsomal prothrombin, however, and it is possible that factors in the microsomal preparation might have When prothrombin from the four species was affected the assay. incubated with ECV (Fig. 1) the generated thrombin-like activity remained constant from 2 to 60 min. time of incubation. For further measurements of Echis-generated thrombinFig. 1 like activity an incubation time of 15 min. was chosen. also shows that coagulant activity obtained from rat prothrombin was about 3 times higher than from human and bovine prothrombin and 5 times higher than from mouse prothrombin. This may explain why ECV is more lethal to rat than to mouse
r
I
I
2
I
I
4
6
8'%i+60
TIME
(MIN.)
INCUBATION
I
I
FIG. 1 Figure 1. Time course of the generation of coagulant activity from prothrombin by ECV. A system consisting of ECV (0,2 mg/ml) and isolated plasma prothrombin (50 U/ml) was incubated at 370~ and coagulant activity was measured at various time intervals. Bovine a-thrombin was used as standard.
Vol. 42, No. 6
QUANTIFICATION OF PROTHROMBIN
741
between As seen in fig. 2 there is no linear relationship prothrombin concentration and Echis generated thrombin activity. The specific coagulant activity decreases upon increasing The same tendency was found concentrations of prothrombin. with prothrombin from all of the investigated species, and also with homogeneous bovine prothrombin (results not shown).
20 PROTHROMHIN
40 CONCENTRATION
60 (U/ml)
FIG. 2 Figure 2. A comparison of Echis-generated coagulant and prothrombin concentration. A system consisting of plasma prothrombin and ECV was incubated for 15 min. prior to measuring coagulant activity as described in Prothrombin concentration was measured in a one stage tion assay using prothrombin deficient plasma.
activity isolated at 37OC Methods. coagula-
When a rat liver microsomal sonicate was investigated (fig. 3) a similar nonlinear relation between prothrombin concentration and ECV-generated thrombin was obtained. If prothrombin was converted completely to a-thrombin one would expect a linear relation between clotting activity and prothrombin concentraAs fig. 4 shows there is linearity between prothrombin tion. concentration and amidolytic activity of ECV-generated thromThis is in agreement with other investigations bin. (4,221. Furthermore the amidolytic activity of Echis generated thrombin correlated when prothrombin from different species (human and rat) was compared (Fig. 4).
742
QUANTIFICATION OF PROTHROMBIN
Figure 3. A comparison of microsomal sonicate dilutions and ECV-generated thrombin activity. Rats were injected 10 mg warfarinlkg 20 hours before killing. A system consisting of microsomal sonicate (from 10 g of liver) and ECV (0.2 mg/ml) was incubated for 15 min. at 37OC prior to measuring thrombin activity. Bovine a-thrombin was used as standard.
Vol. 42, No. 6
---7__-
--
r-l
s H
4.
c
3-
2
2.
E &
9
jI
l I
E
I
1
1/a
I
l/4
DILUTION
l/2
OF MICROSOMAL
-I
l/l SONICATE
FIG. 3
I
PROTHROMBIN
CONCENTRATION
I
(U/ml)
FIG. 4 Fiaure 4. A comparison of Echis-generated amidolytic activity and prothrombin concentration. A system consisting of isolated plasma prothrombin and ECV was incubated for 15 min. at 370C prior to measuring amidolytic activity. Amidolytic activity was measured as the AOD405/min. Prothrombin concentration was measured in a one stage coagulation assay using prothrombin deficient plasma.
Vol. 42, No. 6
QUANTIFICATION OF PROTHROMBIN
743
DISCUSSION A complete conversion of prothrombin to a-thrombin upon prolonged incubation with a purified ECV system i.e. purified prothrombin and Echis carinatus procoagulant (Ecarin) has been If a short incubation time or a partially reported (5,111. purified system is used it has been suggested that prothrombin is only converted to the thrombin precursor, meizothrombin Our results show that using a partially purified (5,121. purified prothrombin and system i.e. whole ECV, partially protein with a low incubation for 15 min., a thrombin-like coagulant activity, probably meizothrombin, was generated. Since the ECV generated clotting activity did not change during an incubation period from 5 to 60 min (fig. 1) it seems unlikely that there is any generation of the highly active a-thrombin. It could be that factors in the whole ECV prevents The results also conversion of meizothrombin to a-thrombin. indicate that no degradation of the ECV generated clotting This is in agreement with results obtained by activity occurs. Carlisle et al. (21) on Echis generated clot activity from rat liver microsomes, remaining constant for 180 min of incubation with ECV. They found, however, a significant degradation in similar preparations from rabbit, guinea pig and chick (21). Our results show that the specific clotting activity of This meizothrombin decreases when the concentration increases. property of meizothrombin cannot be explained by the presence of inhibitors in the partially purified system employed as we obtained similar results with bovine prothrombin purified to homogeneity. The results can be explained if we assume an interaction between the meizothrombin molecules with the formation of dimers exhibiting lower coagulant activity than the monomers. Such interactions are likely to occur since it has been shown that meizothrombin is capable of autolysis The high amidolytic activity of meizothrombin and (5,7,9,10). its linear relationship to the prothrombin concentration may be due to a lack of spatial restriction upon the interaction of the small chromogenic substrate and the enzyme whether monomer or dimer. Meizothrombins have earlier been shown to exhibit a low clotting activity being thrombin-like because fibrinopeptides A and B are released from fibrinogen (12). The clot activity is considered to be due to the ECV cleavage of the Arg-Ile bond linking the A and B chains in thrombin. It has furthermore been suggested that the lower specific activity of meizothrombin on fibrinogen compared to a-thrombin is caused by It is possible that a meizothrombin the extended A-chain (6). dimer is even less accessible to the substrate fibrinogen. The model is visualized in the Appendix. The model presented explains the property of meizothrombin described in this investigation. To prove the model, however, a study of the cleavage products of prothrombin after activation by ECV in unpurified systems has to be carried out. The determination of prothrombin by ECV as activator is performed in unpurified systems with short incubation times. As pointed out these conditions lead to generation of meizothrombin rather than a-thrombin should be avoided as Accordingly a-thrombin. standard reagent when the coagulant activity of ECV-generated
744
QUANTIFICATION OF PROTHROMBIN
Vol. 42, No. 6
thrombin is measured. Moreover, we have found that the clot activity of meizothrombin from rat, human and mouse varies Therefore, isolated plasma prothrombin from the extensively. should be used to construct species under investigation Isolation can easily be carried out on a standard curves. Studying the amidolytic activity of the Echis barium salt. activity these precautions seem generated thrombin-like unnecessary as the amidolytic activity of meizothrombin is linearly related to prothrombin concentration and does not vary between species.
APPENDIX The following approach shows that if prothrombin is quantitatively converted to meizothrombin and meizothrombin forms dimers, the relation between prothrombin concentration [PI and coagulant activity of meizothrombin is nonlinear as seen in Fig. 5. The reaction between meizothrombin (M) and fibrinogen the formation of fibrin (Fi) is described as follows:
(I)
M
+
F
+
F
kM
>
M
+
Fi
M2
+
Fi
(F) with
+
M KA 11 M2
The association (2)
KA
kM2
constant KA is expressed =
as follows:
1 M*l
[Ml2
When prothrombin is quantitatively converted without generation of CL-thrombin we have:
(3)
=
1 PI
2[M21 +
to meizothrombin
[M]
As fibrinogen is present in large excess, the velocity of generation of fibrin (V,) is only dependent of [Ml: (4)
vm
=
k$M]
+
kM' 1 M21 2
Combining eqn. 2-4 the velocity, function of initial [PI:
V,, can be expressed
as a
Vol. 42, No. 6
(5)
745
QUANTIFICATIONOF PROTHROMBIN
vm
=
kM -+P] 2
2kM - kM 2
+
C!l + BK&Pj
- 1)
8KA
Equation 5 is derivated with regard to [PI curvature (Vm as a function of [PI): k M2 kMkM dVnl 2 2 -=+ (6) , dP 2 JI + 8KA[P]
to investigate
When [P) approaches zero then VMW kM'[P]. This is the situation when all meizothrombin is in the monomer form. [P] increases VM is approaching kM *[PI/2 Then all meizothrombin is in the d 1mer form. The curvature shown in Fig. 5 is obtained:
the
When
>
PROTHROMBIN
CONCENTRATION
(P)
FIG. 5 Figure
5.
Curvature
based on equation
(5).
REFERENCES 1.
KORNALIK, F. Uber den Einfluss von Echis-carinata-Toxin Folia Haematol. 80, auf die Blutgerinnung in vitro. 73-78, 1963.
2.
SUTTIE, J.W. Mechanism of action of vitamin K: DemonstraScience 179, tion of a liver precursor of prothrombin. 192-194, 1973.
746
QUANTIFICATION OF PROTHROMBIN
Vol. 42, No. 6
3.
BEZEAUD, A., DROUET, L., SORIA, C. and GUILLIN, M.-C. Prothrombin Salakta: An abnormal prothrombin characterized Thromb.Res. by a defect in the active site of thrombin. 34, 507-518, 1984.
4.
BERTINA, R.M., NIEUWKOOP, W.M. and LOELIGER, E.A. Spectrophotometric assays of prothrombin in plasma of patients using oral anticoagulants. Thromb.Haemost. 42, 1296-1304, 1979.
5.
MORITA, T., IWANAGA, S. and SUZUKI, T. The mechanism of activation of bovine prothrombin by an activator isolated from Echis carinatus venom and characterization of the new J.Biochem. 2, 1089-1108, 1976. active intermediates.
6.
KORNALIK, F. and BLOMB#CK, B. Prothrombin activation induced by Ecarin-A prothrombin converting enzyme from Echis carinatus venom. Thromb.Res. 6_, 53-63, 1975.
7.
RHEE, M., MORRIS, S. and KOSOW, D.P. Role of meizothrombin and meizothrombin-(des Fl) in the conversion of prothrombin to thrombin by the Echis carinatus venom Biochemistry 21, 3437-3443, 1982. coagulant.
8.
BRIET, E., NOYES, C.M., ROBERTS, H.R. and GRIFFITH, M.J. Cleavage and activation of human prothrombin by Echis carinatus venom. Thromb.Res, 27, 591-600, 1982.
9.
MORITA, T. and IWANAGA. S. Prothrombin activator from Echis carinatus venom. Methods of Enzym., 80 - part C, 303-311, 1981.
10.
NOVOA, E. and SEEGERS, W.H. Mechanisms of a-thrombin and 8-thrombin-E formation: Use of Ecarin for isolation of meizothrombin 1. Thromb.Res. 2, 657-668, 1980.
11.
FRANZA, Jr., B.R., ARONSON, D.L. and FINLAYSON, J.S. Activation of human prothrombin by a procoagulant fraction Identification of a from the venom of Echis carinatus. high molecular weight intermediate with thrombin activity. J.Biol.Chem. 250, 7057-7068, 1975.
12.
DYR, J.E., BLOMB#CK, B. and KORNALIK, F. The action of Thromb. prothrombin activated by ecarin on fibrinogen. Rz 30, 225-234, 1983.
13.
SCHIECK, A., HABERMAN, E. and KORNALIK, F. The prothrombin activating principle from Echis carinatus venom. II Coagulation studies in vitro and in vivo. Naunyn-Schmiederberg's Arch.Pharmacol. 274, 7-17, 1972.
14.
SHAH, D.V., SUTTIE, J.W. and GRANT, G.A. A rat liver Properties and protein with potential thrombin activity: partial purification. Arch.Biochem.Biophys. 159, 483-491, 1973.
Vol. 42, No. 6 15.
747
.QUANTIFICATIONOF PROTHROMBIN
HELGELAND, L. The submicrosomal site for the conversion of prothrombin precursor to biologically active prothrombin-in rat liver. Biochim.Biophys.Acta, 499, 181-l 93, 1977.
16.
Immunological CARLISLE, T.L. and SUTTIE, J.W. terization of rat liver prothrombin precursors. Res. 18, 405-416, 1980.
17.
HELGELAND, L. On the presence of a heat-stable macromolecular inhibitor of the thrombin-fibrinogen reaction in rat liver microsomes and its separation from prothrombin. Biochem.Biophys.Acta 386, 203-208, 1975.
18.
HENRIKSEN, AA. CHRISTENSEN, T.B. and HELGELAND, L. On the significance of the carbohydrate moieties of bovine Biochim.Biophys.Acta prothrombin for clotting activity. 421, 348-352, 1976.
19.
HJORT, P., RAPAPORT, S.J. and OWREN, P.A. A simple, specific one-stage prothrombin assay using Russel's viper venom in cephalin suspension. J.Lab.Clin.Med. 46, 89-97, 1955.
20.
HASSELBACK, R. and HJORT, P. Effect of heparin on in vivo turnover of clotting factors. J.Appl.Phys. 11, 945-948, 1960.
21.
CARLISLE, T.L., SHAH. D.V., SCHLEGEL, R. and SUTTIE, J.W. Plasma abnormal prothrombin and microsomal prothrombin precursor in various species. Proc.Soc.Exp.Biol.Med. 148, 140-144, 1975.
22.
BAUGHMAN, D.J. and LYTWYN, A. A novel chromogenic assay equivalent to one stage prothrombin assay. Thromb. Haemostas. 42, 291, 1979.
characThromb.
ON THE QUM?l!IFICATION OF PRCYI'BRWINFRCM DIFFEREXW! SPECIES USING ECHIS CARINATUSAS ACTIVATOR Ole KristianTollersrudand Liv Helgeland Departmentof Biochemistry,Universityof Oslo, P.Q. Box 1041,Blindern,0316 Oslo 3, Norway (Sutntitted to Editor S. Magnusson6.3.1985;Acceptedby Editors-in-Chief B. Bl&ck and A.L. Copley 2.4.1986)"
ABSTRACT
The generation of thrombin-like activity from rat, human, bovine and mouse prothrombin by Echis carinatus venom (ECV) treatment was compared using a partially purified system (i.e. whole ECV and A rapid increase in coagulant isolated prothrombin). activity was obtained within 0.5 to 2 min., being constant upon further incubation for 60 min. A large variation in coagulant activity of the ECV generated thrombin from the four species was found, whereas no differences were found for the amidolytic activities. The coagulant activities of the ECV generated thrombin was also low compared with the corresponding thrombin activities obtained by physiological activation. Coagulant activity of the ECV generated thrombin levelled off at increasing concentration of prothrombin in the sample as measured by the onestage coagulation assay. By measuring amidolytic activity a linear relationship to the concentration These findings of prothrombin was found, however. indicate that ECV converts prothrombin from the four different species to a thrombin-like protein with The lack of properties distinct from a-thrombin. linearity in the ECV generated clot activity with increasing concentration of prothrombin could be explained by assuming a dimerization of the thrombinlike protein molecules making them less accessible The significance of these observato fibrinogen. tions for the quantification of prothrombin from different species is discussed.
Key %ords; Prothronbinquantification- Echis carinatus- various species * The acceptanceof this cm icationby the Editors-in-Chief is exceptional and caused by the utter negligenceof the Editor to whcm it was originally stittd
737
738
QUANTIFICATION OF PROTHROMBIN
Vol. 42, No. 6
INTRODUCTION In contrast to factor X,, Echis carinatus venom (ECV) converts prothrombin to an active enzyme in the absence of factor V,, calcium and phospholipid (1). ECV thus activates acarboxyprothrombin as well as partially carboxylated prothrombin and congenital disorders in which prothrombin has low clotting activity in physiological systems (2,3). This has made ECV an important tool to trace and to quantify the amount of these various forms of prothrombin (2,4). A marked difference between the activation of prothrombin by ECV and the physiological activation of prothrombin has, however, been revealed. ECV cleaves prothrombin initially at one site, an Arg-Ile bond linking the A and B chains in thrombin (5,6). The intermediate thus formed, meizothrombin, is further converted to meizothrombin-des Fl (also called meizothrombin l), by splitting off This conversion has been suggested to be fragment 1 (7-10). autocatalytic by some authors (7,9) whereas others consider ECV to be responsible for the formation of meizothrombin-des Fl The coagulant activity of the two thrombin-like inter(8). mediates is low compared to that of a-thrombin whereas the The esterase and amidolytic activity is high (6,9-13). subsequent conversion of meizothrombin-des Fl to a -thrombin is autocatalytic (5,7). The formation of a-thrombin depends apparently on a highly purified system, i.e. purified prothrombin and purified ECV (Ecarin), and prolonged incubation times (5,7,12,). When prothrombin is assayed by the ECV method the whole venom is employed and prothrombin in the sample is not purified. Moreover, a-thrombin has frequently been used as standard reagent to convert clot time into thrombin units (14-16). In the present paper we have shown that the ECV generated thrombin activity is not related to the clotting time as found for a-thrombin (T=k/t). A modified method for the quantification of prothrombin from different species is described. MATERIALS
AND METHODS
Animals. Male albino rats, 200-250 g of the Wistar strain, and female black mice NMRI/BOM from Mollegaard, Havrup, Denmark, were used. Chemicals. Acacia (gum arabic) and Echis carinatus venom were obtained from Sigma Chemical Co., USA. Human fibrinogen L Grade (90% clottable) was purchased from Kabi, Stockholm, Sweden, bovine thrombin (Topostasine) from Hoffman-LaRoche Co., Basel, Switzerland, and Russel's viper venom (Stypven) from Burroughs Wellcome Co., U.K. The chromogenic substrate Th-1 (D-cyclohexylglycyl-L-alanyl-L-arginine-p-nitroanilide dihydroacetate) was a gift from Nyegaard & Co. A/S., Oslo, Norway. Preparation of plasma. Blood was obtained by cardiac or venipuncture and collected in 3.8% sodium citrate (10% v/v). Platelet-free plasma was obtained after centrifugation at 5OOOxg (20 min. 4OC) and stored at -2OOC for up to 2 weeks.
Vol. 42, No. 6
QUANTIFICATION OF PROTHROMBIN
739
Standard human plasma was pooled from 20 healthy volunteers. The procedure is the same as Purification of prothrombin. Plasma was described before with minor modifications (17). diluted 1:l with 5.14 mM veronal*HCl buffer (pH 7.4) containing To 1.0 ml of the 0.12 M NaCl and 6.06 mM sodiumcitrate. After diluted plasma was added 2.8 mg sodium citratea2H20. The suspension dissolution 35 ul of 1M BaC12 was added slowly. was stirred for 20 min and centrifuged at 6000xg for 10 min. The precipitate was solubilized in 1 ml of 5.14 mM veronal*HCl buffer (pH 7.4) containing 0.12 M NaCl and 6.06 mM sodium The citrate and the adsorption procedure was repeated. precipitate was suspended in 0.15 ml of a mixture of 0.04 M Dissolution was achieved by sodium citrate and 0.31 M NaCl. addition of 0.25 ml of 0.2 M EDTA (adjusted to pH 7.4 with 10 M NaOH). The solution was passed through a Sephadex-G 50 fine column equilibrated 'with 5.14 mM veronal.HCl buffer pH 7.4 The entire containing 0.12 M NaCl and 6.06 mM sodium citrate. Bovine prothrombin was procedure was carried out at 4oC. purified to homogeneity as described previously (18). Factor II assay. Prothrombin was measured using the one-stage The test was made specific for method of Hjort et al. (19). prothrombin by adding factor X (Stuart) to the test solution One unit of prothrombin was arbitrarily chosen as the (20). amount of prothrombin present in 1 ml of standard human plasma diluted 1:lOOO. Echis carinatus methods. Prothrombin was activated by incubating 0.1 ml isolated prothrombin solution and 0.025 ml Echis carinatus venom (1.0 mg/ml) at 37OC. The appropriate incubation time was tested out. Both the coagulant and amidolytic activity of Echis-generated thrombin were measured. Coagulant activity. Thrombin activity was measured with fibrinogen as substrate as described previously (14). The clotting times were converted to thrombin units by using a standard curve prepared by dilution of purified bovine standard a-thrombin. Amidolytic activity. A mixture of 0.125 ml of the activated prothrombin solution, 0.05 ml Th-1(2AcOH*H-D-CHG-Ala-Arg-pNA) (2 Vmol/ml) and 0.625 ml 5.14 mM veronal*HC1 buffer pH 8.2 containing 6.06 mM sodium citrate and 0.12 M NaCl was incubated for 15 min at 37OC. Subsequently 0.4 ml 40% HAc was added to stop the reaction. The formation of p-nitroaniline was measured at 405 nm. The amidolytic activity of the Echis generated thrombin is linearly related to AOD405/min. A blank omitting the activated prothrombin solution was included since Echis carinatus venom relased a small, but significant amount of free p-nitroaniline from Th-1. Microsomal sonicates were prepared as described previously (151. RESULTS Prothrombin from four species, human, bovine, rat and mouse was isolated from citrate plasma by adsorption on barium citrate as described in Materials and Methods. This isolation resulted in about 300 fold purification of prothrombin. The isolated prothrombin was diluted to obtain a concentration of 50 U/ml as measured by a one stage coagulation assay (19). In a system as
QUANTIFICATION OF PROTHROMBIN
Vol. 42, No. 6
described in fig. 1 the incubation time necessary for maximum The incubation ECV generated coagulant activity was estimated. time varied to a certain degree between the species; whereas 0.5-l min. was necessary for rat and mouse prothrombin, human and bovine prothrombin required 3-5 min. to obtain maximum This in agreement with another report thrombin activity. concerning rat prothrombin (2) but Carlisle et al. (21) found that even after incubation of mouse prothrombin for 3 hours The investigation maximum thrombin activity was not reached. was carried out on microsomal prothrombin, however, and it is possible that factors in the microsomal preparation might have When prothrombin from the four species was affected the assay. incubated with ECV (Fig. 1) the generated thrombin-like activity remained constant from 2 to 60 min. time of incubation. For further measurements of Echis-generated thrombinFig. 1 like activity an incubation time of 15 min. was chosen. also shows that coagulant activity obtained from rat prothrombin was about 3 times higher than from human and bovine prothrombin and 5 times higher than from mouse prothrombin. This may explain why ECV is more lethal to rat than to mouse
r
I
I
2
I
I
4
6
8'%i+60
TIME
(MIN.)
INCUBATION
I
I
FIG. 1 Figure 1. Time course of the generation of coagulant activity from prothrombin by ECV. A system consisting of ECV (0,2 mg/ml) and isolated plasma prothrombin (50 U/ml) was incubated at 370~ and coagulant activity was measured at various time intervals. Bovine a-thrombin was used as standard.
Vol. 42, No. 6
QUANTIFICATION OF PROTHROMBIN
741
between As seen in fig. 2 there is no linear relationship prothrombin concentration and Echis generated thrombin activity. The specific coagulant activity decreases upon increasing The same tendency was found concentrations of prothrombin. with prothrombin from all of the investigated species, and also with homogeneous bovine prothrombin (results not shown).
20 PROTHROMHIN
40 CONCENTRATION
60 (U/ml)
FIG. 2 Figure 2. A comparison of Echis-generated coagulant and prothrombin concentration. A system consisting of plasma prothrombin and ECV was incubated for 15 min. prior to measuring coagulant activity as described in Prothrombin concentration was measured in a one stage tion assay using prothrombin deficient plasma.
activity isolated at 37OC Methods. coagula-
When a rat liver microsomal sonicate was investigated (fig. 3) a similar nonlinear relation between prothrombin concentration and ECV-generated thrombin was obtained. If prothrombin was converted completely to a-thrombin one would expect a linear relation between clotting activity and prothrombin concentraAs fig. 4 shows there is linearity between prothrombin tion. concentration and amidolytic activity of ECV-generated thromThis is in agreement with other investigations bin. (4,221. Furthermore the amidolytic activity of Echis generated thrombin correlated when prothrombin from different species (human and rat) was compared (Fig. 4).
742
QUANTIFICATION OF PROTHROMBIN
Figure 3. A comparison of microsomal sonicate dilutions and ECV-generated thrombin activity. Rats were injected 10 mg warfarinlkg 20 hours before killing. A system consisting of microsomal sonicate (from 10 g of liver) and ECV (0.2 mg/ml) was incubated for 15 min. at 37OC prior to measuring thrombin activity. Bovine a-thrombin was used as standard.
Vol. 42, No. 6
---7__-
--
r-l
s H
4.
c
3-
2
2.
E &
9
jI
l I
E
I
1
1/a
I
l/4
DILUTION
l/2
OF MICROSOMAL
-I
l/l SONICATE
FIG. 3
I
PROTHROMBIN
CONCENTRATION
I
(U/ml)
FIG. 4 Fiaure 4. A comparison of Echis-generated amidolytic activity and prothrombin concentration. A system consisting of isolated plasma prothrombin and ECV was incubated for 15 min. at 370C prior to measuring amidolytic activity. Amidolytic activity was measured as the AOD405/min. Prothrombin concentration was measured in a one stage coagulation assay using prothrombin deficient plasma.
Vol. 42, No. 6
QUANTIFICATION OF PROTHROMBIN
743
DISCUSSION A complete conversion of prothrombin to a-thrombin upon prolonged incubation with a purified ECV system i.e. purified prothrombin and Echis carinatus procoagulant (Ecarin) has been If a short incubation time or a partially reported (5,111. purified system is used it has been suggested that prothrombin is only converted to the thrombin precursor, meizothrombin Our results show that using a partially purified (5,121. purified prothrombin and system i.e. whole ECV, partially protein with a low incubation for 15 min., a thrombin-like coagulant activity, probably meizothrombin, was generated. Since the ECV generated clotting activity did not change during an incubation period from 5 to 60 min (fig. 1) it seems unlikely that there is any generation of the highly active a-thrombin. It could be that factors in the whole ECV prevents The results also conversion of meizothrombin to a-thrombin. indicate that no degradation of the ECV generated clotting This is in agreement with results obtained by activity occurs. Carlisle et al. (21) on Echis generated clot activity from rat liver microsomes, remaining constant for 180 min of incubation with ECV. They found, however, a significant degradation in similar preparations from rabbit, guinea pig and chick (21). Our results show that the specific clotting activity of This meizothrombin decreases when the concentration increases. property of meizothrombin cannot be explained by the presence of inhibitors in the partially purified system employed as we obtained similar results with bovine prothrombin purified to homogeneity. The results can be explained if we assume an interaction between the meizothrombin molecules with the formation of dimers exhibiting lower coagulant activity than the monomers. Such interactions are likely to occur since it has been shown that meizothrombin is capable of autolysis The high amidolytic activity of meizothrombin and (5,7,9,10). its linear relationship to the prothrombin concentration may be due to a lack of spatial restriction upon the interaction of the small chromogenic substrate and the enzyme whether monomer or dimer. Meizothrombins have earlier been shown to exhibit a low clotting activity being thrombin-like because fibrinopeptides A and B are released from fibrinogen (12). The clot activity is considered to be due to the ECV cleavage of the Arg-Ile bond linking the A and B chains in thrombin. It has furthermore been suggested that the lower specific activity of meizothrombin on fibrinogen compared to a-thrombin is caused by It is possible that a meizothrombin the extended A-chain (6). dimer is even less accessible to the substrate fibrinogen. The model is visualized in the Appendix. The model presented explains the property of meizothrombin described in this investigation. To prove the model, however, a study of the cleavage products of prothrombin after activation by ECV in unpurified systems has to be carried out. The determination of prothrombin by ECV as activator is performed in unpurified systems with short incubation times. As pointed out these conditions lead to generation of meizothrombin rather than a-thrombin should be avoided as Accordingly a-thrombin. standard reagent when the coagulant activity of ECV-generated
744
QUANTIFICATION OF PROTHROMBIN
Vol. 42, No. 6
thrombin is measured. Moreover, we have found that the clot activity of meizothrombin from rat, human and mouse varies Therefore, isolated plasma prothrombin from the extensively. should be used to construct species under investigation Isolation can easily be carried out on a standard curves. Studying the amidolytic activity of the Echis barium salt. activity these precautions seem generated thrombin-like unnecessary as the amidolytic activity of meizothrombin is linearly related to prothrombin concentration and does not vary between species.
APPENDIX The following approach shows that if prothrombin is quantitatively converted to meizothrombin and meizothrombin forms dimers, the relation between prothrombin concentration [PI and coagulant activity of meizothrombin is nonlinear as seen in Fig. 5. The reaction between meizothrombin (M) and fibrinogen the formation of fibrin (Fi) is described as follows:
(I)
M
+
F
+
F
kM
>
M
+
Fi
M2
+
Fi
(F) with
+
M KA 11 M2
The association (2)
KA
kM2
constant KA is expressed =
as follows:
1 M*l
[Ml2
When prothrombin is quantitatively converted without generation of CL-thrombin we have:
(3)
=
1 PI
2[M21 +
to meizothrombin
[M]
As fibrinogen is present in large excess, the velocity of generation of fibrin (V,) is only dependent of [Ml: (4)
vm
=
k$M]
+
kM' 1 M21 2
Combining eqn. 2-4 the velocity, function of initial [PI:
V,, can be expressed
as a
Vol. 42, No. 6
(5)
745
QUANTIFICATIONOF PROTHROMBIN
vm
=
kM -+P] 2
2kM - kM 2
+
C!l + BK&Pj
- 1)
8KA
Equation 5 is derivated with regard to [PI curvature (Vm as a function of [PI): k M2 kMkM dVnl 2 2 -=+ (6) , dP 2 JI + 8KA[P]
to investigate
When [P) approaches zero then VMW kM'[P]. This is the situation when all meizothrombin is in the monomer form. [P] increases VM is approaching kM *[PI/2 Then all meizothrombin is in the d 1mer form. The curvature shown in Fig. 5 is obtained:
the
When
>
PROTHROMBIN
CONCENTRATION
(P)
FIG. 5 Figure
5.
Curvature
based on equation
(5).
REFERENCES 1.
KORNALIK, F. Uber den Einfluss von Echis-carinata-Toxin Folia Haematol. 80, auf die Blutgerinnung in vitro. 73-78, 1963.
2.
SUTTIE, J.W. Mechanism of action of vitamin K: DemonstraScience 179, tion of a liver precursor of prothrombin. 192-194, 1973.
746
QUANTIFICATION OF PROTHROMBIN
Vol. 42, No. 6
3.
BEZEAUD, A., DROUET, L., SORIA, C. and GUILLIN, M.-C. Prothrombin Salakta: An abnormal prothrombin characterized Thromb.Res. by a defect in the active site of thrombin. 34, 507-518, 1984.
4.
BERTINA, R.M., NIEUWKOOP, W.M. and LOELIGER, E.A. Spectrophotometric assays of prothrombin in plasma of patients using oral anticoagulants. Thromb.Haemost. 42, 1296-1304, 1979.
5.
MORITA, T., IWANAGA, S. and SUZUKI, T. The mechanism of activation of bovine prothrombin by an activator isolated from Echis carinatus venom and characterization of the new J.Biochem. 2, 1089-1108, 1976. active intermediates.
6.
KORNALIK, F. and BLOMB#CK, B. Prothrombin activation induced by Ecarin-A prothrombin converting enzyme from Echis carinatus venom. Thromb.Res. 6_, 53-63, 1975.
7.
RHEE, M., MORRIS, S. and KOSOW, D.P. Role of meizothrombin and meizothrombin-(des Fl) in the conversion of prothrombin to thrombin by the Echis carinatus venom Biochemistry 21, 3437-3443, 1982. coagulant.
8.
BRIET, E., NOYES, C.M., ROBERTS, H.R. and GRIFFITH, M.J. Cleavage and activation of human prothrombin by Echis carinatus venom. Thromb.Res, 27, 591-600, 1982.
9.
MORITA, T. and IWANAGA. S. Prothrombin activator from Echis carinatus venom. Methods of Enzym., 80 - part C, 303-311, 1981.
10.
NOVOA, E. and SEEGERS, W.H. Mechanisms of a-thrombin and 8-thrombin-E formation: Use of Ecarin for isolation of meizothrombin 1. Thromb.Res. 2, 657-668, 1980.
11.
FRANZA, Jr., B.R., ARONSON, D.L. and FINLAYSON, J.S. Activation of human prothrombin by a procoagulant fraction Identification of a from the venom of Echis carinatus. high molecular weight intermediate with thrombin activity. J.Biol.Chem. 250, 7057-7068, 1975.
12.
DYR, J.E., BLOMB#CK, B. and KORNALIK, F. The action of Thromb. prothrombin activated by ecarin on fibrinogen. Rz 30, 225-234, 1983.
13.
SCHIECK, A., HABERMAN, E. and KORNALIK, F. The prothrombin activating principle from Echis carinatus venom. II Coagulation studies in vitro and in vivo. Naunyn-Schmiederberg's Arch.Pharmacol. 274, 7-17, 1972.
14.
SHAH, D.V., SUTTIE, J.W. and GRANT, G.A. A rat liver Properties and protein with potential thrombin activity: partial purification. Arch.Biochem.Biophys. 159, 483-491, 1973.
Vol. 42, No. 6 15.
747
.QUANTIFICATIONOF PROTHROMBIN
HELGELAND, L. The submicrosomal site for the conversion of prothrombin precursor to biologically active prothrombin-in rat liver. Biochim.Biophys.Acta, 499, 181-l 93, 1977.
16.
Immunological CARLISLE, T.L. and SUTTIE, J.W. terization of rat liver prothrombin precursors. Res. 18, 405-416, 1980.
17.
HELGELAND, L. On the presence of a heat-stable macromolecular inhibitor of the thrombin-fibrinogen reaction in rat liver microsomes and its separation from prothrombin. Biochem.Biophys.Acta 386, 203-208, 1975.
18.
HENRIKSEN, AA. CHRISTENSEN, T.B. and HELGELAND, L. On the significance of the carbohydrate moieties of bovine Biochim.Biophys.Acta prothrombin for clotting activity. 421, 348-352, 1976.
19.
HJORT, P., RAPAPORT, S.J. and OWREN, P.A. A simple, specific one-stage prothrombin assay using Russel's viper venom in cephalin suspension. J.Lab.Clin.Med. 46, 89-97, 1955.
20.
HASSELBACK, R. and HJORT, P. Effect of heparin on in vivo turnover of clotting factors. J.Appl.Phys. 11, 945-948, 1960.
21.
CARLISLE, T.L., SHAH. D.V., SCHLEGEL, R. and SUTTIE, J.W. Plasma abnormal prothrombin and microsomal prothrombin precursor in various species. Proc.Soc.Exp.Biol.Med. 148, 140-144, 1975.
22.
BAUGHMAN, D.J. and LYTWYN, A. A novel chromogenic assay equivalent to one stage prothrombin assay. Thromb. Haemostas. 42, 291, 1979.
characThromb.