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The binding of calcium to fibrinogen: Influence on the clotting process
134
Biochimica et Biophysica Acta, 579 (1979) 1 3 4 - - 1 4 1 © E l s e v i e r / N o r t h - H o l l a n d B i o m e d i c a l Press
BBA 38215
THE BINDING OF CALCIUM TO FIBRINOGEN: INFLUENCE ON THE CLOTTING PROCESS
G E R A R D M A R G U E R I E *, Y V E T T E B E N A B I D ** and M I C H E L S U S C I L L O N
Laboratoire d 'Hdmatologie, Institut de Recherche Fondamentale, Centre d'Etudes Nucldaires, 85 X, 38041 - Grenoble-Cedex (France) (Received December llth,
1978)
Key words: Ca2+; Fibrinogen; Fibrin monomer; Fibrinogen-fibrin complex
Summary The influence of Ca ~+ on the basic reaction between thrombin and fibrinogen was investigated. The results demonstrate that: (a) A Ca2÷-dependent dimeric intermediate is formed during the early step of the clotting process. This dimeric intermediate is shown to be formed by the association of an intact fibrinogen molecule and a fibrin m o n o m e r devoid in only the peptide A, (b) Ca 2÷ enhances the proteolytic step as illustrated by the measure of the kinetics of H ÷ release at pH 8.6 On the basis of these observations it is proposed that Ca 2+ catalyses the proteolysis of fibrinogen by thrombin through the formation of a Ca2+-dependent dimer.
Introduction
A recent finding showing t h a t fibrinogen has three Ca 2+ binding sites of high affinity [1] stimulates new investigations on the influence of this cation on the structure-function relationship of this protein. While it was shown that the binding of Ca 2÷ does n o t entail a gross overall conformational change, several Ca2+-induced protective effects were however observed. Specific structural features are believed to be related to the binding of this cation to fibrinogen. It was proposed that Ca 2÷ is inserted within the structure of this protein [2].
* Present address: Scripps Clinic and Research Foundation, La Jolla. CA 92037. U.S.A. ** To whom reprint requests should be addressed. Abbreviation: TAME. tosylarginine-O-methyl ester.
135 This view has received recent support from other investigators who confirmed the existence of three high affinity Ca 2÷ binding sites in the fibrinogen molecule. Two of these sites are located in the D domain of the molecule [3,4]. The functional implication of the existence of Ca 2÷ binding sites on fibrinogen remains to be elucidated. The role of this divalent cation in the clotting of fibrinogen has been recognized for a long time. Most of the studies have led to the suggestion that the catalytic influence of Ca 2÷ is restricted to the polymerization of the fibrin monomer. Moreover the proteolytic activation by thrombin is essentially unaffected [5,9]. In the present paper, we provide additional data that suggest a possible influence of Ca 2÷ on the reaction between thrombin and fibrinogen. Material and Methods Twice distilled water was used for all the solutions from which contaminating Ca 2÷ was removed as previously detailed [ 1]. Bovine fibrinogen was purified from fresh plasma according to the m e t h o d of Kekwick et al. [10]. After clarification of the sample and determination of protein concentration [11], the purified fibrinogen (>98% clottable protein) was stored at --30°C in 0.3 M NaC1 solutions. Ca2÷-free fibrinogen was prepared as previously described [ 1 ]. Bovine thrombin from Parke-Davis and Co. was dissolved in 0.1 M NaC1, 0.05 Tris-HC1 buffer, pH 7.5 at a final concentration of 1 NIH unit/ml. Polyacrylamide gel electrophoresis on 3.75% gels were carried o u t at pH 8.9 [ 12]. Staining was done with coomassie blue and scanning of gels for densitometric determination of relative a m o u n t of proteins was performed on a densitometer DD2 (Kipp & Zonen). SDS polyacrylamide gel electrophoresis of reduced samples were run on 10% gels [13]. Agarose gel filtrations on Biogel A-5m (200--400 mesh) from Biorad Laboratories were carried o u t as previously published [12]. Hydrogen ion titrations at constant pH were done using a highly sensitive pH shift m e t h o d [2]. In a typical experiment 4 nmol of Ca2÷-free fibrinogen (1.4 mg) were dissolved in 5 ml of Ca2÷-free 0.15 M NaC1 solution. The solution was brought to 8.6 and equilibrated at 20°C-+ 0.05°C. Carbon dioxide-free helium was continuously flushed over the solution. 25 pl of thrombin were added (0.025 NIH units) and the pH shift due to the reaction was recorded. Standardized HC1 solution was used for calibration. When the reaction was performed in the presence of Ca 2÷, 0.5 ml of 10 -2 M CaC12 solution was added to the solution. Ca 2÷ concentrations were determined after each experiment b y atomic absorption spectrophotometry. Equilibrium dialyzis experiments, using [14C]EDTA (from A m e r s h a m ) w e r e done as previously detailed [ 1]. Results
I. Influence o f Ca 2÷ on complex-formation between native fibrinogen and thrombin-treated fibrinogen When fibrinogen is incubated with a small amount of thrombin over a short
136
period, soluble polymeric aggregates can be isolated according to their size by gel filtration over a Biogel A-5m column, if they are eluted at basic pH [12]. We have investigated the influence of Ca :+ on the formation of these complexes using the same procedure. 0.01 units of thrombin were added to 15 mg of fibrinogen which was dissolved in 2 ml of 0.1 M NaC1, 0.05 M Tris-HC1 buffer, pH 7.5. After 30 min incubation at 37°C the reaction was terminated b y the addition of 1.5 units of Hirudine. The sample was then applied on a Biogel A-5m column (1.8 × 50 cm) and eluted with a 0.25 M NaC1, 0.05 M Tris-HC1 buffer, pH 8.6. At this pH the elution profile consisted of three peaks, as illustrated in Fig. 1. Polyacrylamide gel electrophoresis at pH 8.9 on 3.75% gels indicated that peak 1, eluted in the void volume corresponded to soluble material of high molecular weight whicti did not penetrate the gel. Peak 2 represents material with a molecular weight
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Elution v o l u m e ( m l ) Fig. 1. G e l f i l t r a t i o n o n Biogel A - 5 m ( 2 0 0 - - - 4 0 0 m e s h ) o f f i b r i n o g e n ( 1 5 r a g ) i n c u b a t e d f o r 3 0 m i n a t 3 7 ° C w i t h 0.01 NIH units t h r o m b i n . The r e a c t i o n was t e r m i n a t e d b y H i r u d i n (1.5 units). The b u f f e r syst e m w a s 0 . 2 5 M NaC1, 0 . 0 5 M Tris-HC1 p H 8 . 5 in t h e p r e s e n c e ( . . . . . . ) a n d a b s e n c e (. . . . . . ) o f 1 0 -3 M E D T A . P o l y a c r y l a m i d e gel e l e c t r o p h o r e s i s , 3 . 7 5 % gel a t p H 8 . 9 ( w i t h o u t S D S ) . A, f i b r i n o g e n (a) a n d t h r o m b i n t r e a t e d f i b r i n o g e n ( b ) . B, c h r o m a t o g r a p h i c p e a k s 1, 2 a n d 3 o b t a i n e d u s i n g Tris-NaC1 b u f f e r . C, chromatographic peak 1 and 3 obtained using EDTA-containing buffer.
137
twice that of fibrinogen, which has already been described as a dimeric intermediate [12]. Peak 3 was unmodified fibrinogen. To confirm further the composition of the 'dimer', the electrophoretic pattern of the reduced material was compared with that of reduced fibrinogen and fibrinogen treated with thrombin and reptilase as previously described [12]. The results are shown in Fig. 2. As can be seen in the dimeric intermediate, the B~ chain is intact, whereas the band corresponding to the As chain, slightly overlaps with the ~ chain, suggesting that both polypeptidic chains are present. This is in excellent agreement with previously published data which suggested that the dimer was formed from the association of one intact fibrinogen molecule and one fibrinogen devoid in only the fibrino-peptide A [12]. When the sample was run over the same column with the same buffer at pH 8.6 in the presence of 10 -3 M EDTA, the elution profil e was different (Fig. 1). The presence of EDTA eliminated the second peak that contained the 'dimer'. Polyacrylamide gel electrophoresis of peak 1 and peak 3 revealed that peak 1 still contained high molecular weight aggregates, while peak 3 contained both unmodified fibrinogen and the dimer formed under the electrophoretic conditions. When Ca 2÷ was restored at a concentration of 5 • 10 -3 M the dimeric intermediate was again eluted in the position of peak 2.
II. Influence o f Ca 2÷ on the proteolysis o f fibrinogen by thrombin The kinetics of proton release during the proteolytic reaction of thrombin on fibrinogen was studied at pH 8.6, using the pH shift method [15]. The reaction curves are shown in Fig. 3 and are representative of average values from 3 runs. Reproducibility was good (0.02 equiv. H ÷ per mol of fibrinogen) and was tested as described elsewhere [2]. Recordings were stopped after 10 min during which time no clot or aggregates were observed in the reaction chamber.
F D Fig. 2. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, 7.5% gel, of sample reduced by 2-mercaptoethanol: fibrinogen ( F ) d i m e r (D)thrombin-treated fibrinogen ( F . T ) a n d reptilase-t~eated fibrinogen (F.R.).
138
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Fig. 3. Proton release during the pzoteolysis of fibrinogen by t h r o m b i n at pH 8.6, in the presence of 2 • 1 0 -6 M CaC12 ( e ) ' a n d 10 -3 M CaCI 2 (o). I n s e t 1 s h o w s t h e p r o t o n release d u r i n g t h e esterolysis b y t b x o m h i n o f 1 0 -2 M T A M E s o l u t i o n u n d e r t h e s a m e c o n d i t i o n s . I n s e t 2 r e p r e s e n t s t h e v a r i a t i o n of d i m e r f o r m a t i o n d u r i n g t h e r e a c t i o n . 1 0 0 pl w e r e r e m o v e d f r o m t h e r e a c t i o n c h a m b e r at s e l e c t e d t i m e i n t e r v a l s a n d s u b m i t t e d t o 5% p o l y a c r y l a m i d e gel e l e c t r o p h o r c s i s . T h e e l e c t r o p h o r e t i c b a n d c o r r e s p o n d i n g to t h e d i m e r w a s s c a n n e d a n d c o m p a r e d w i t h t h e original f i b r i n o g e n s a m p l e .
When the c o n c e n t r a t i o n of Ca 2÷ was increased from 2 • 10 -~ M to 10 -3 M, m ore H ÷ were released per mol o f fibrinogen, indicating that Ca 2÷ had an effect on the rate o f the reaction between t h r o m b i n and fibrinogen. When analyzed in terms of first order reaction, as previously detailed by Mihalyi [15], these reaction curves give points which fall into straight lines (Fig. 4) according to the equation. log d H t / d t = log A o K e -- Klo T where d H t / d t is the rate of the reaction, Ke and K10 'is the rate constant in normal and decimal logarithms and A0 is the total n u m b e r of peptide bonds taking part in the reaction. Such plots gave estimated rate constants o f 0.22 and 0.10 at Ca 2÷ concentrations o f 10 .3 M and 2 • 10 -6 M, respectively. The esterolytic activity of t h r o m b i n was measured under the same experimental conditions at pH 8.6 on 10 -2 M TAME solutions. The results shown in Fig. 3 (inset 1) indicated that the enzymatic activity was n o t affected by the presence o f Ca 2+ at 20 ° C. The percentage of fibrinogen molecules cleaved by t h r o m b i n in the presence and absence o f Ca 2+ was estimated through ability to form the dimeric inter-
139
TIME ( m i n ) 0
A
1
2
3
4
5
6
7
!
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F i g . 5. A n a l y s i s o f t h e p r o t e o l y s i s r e a c t i o n c u r v e s i n t e r m s o f s i m u l t a n e o u s f i r s t o r d e r r e a c t i o n a c c o r d i n g t o M i h a l y i [ 1 8 ] , i n t h e p r e s e n c e o f 2 • 1 0 -5 M CaC12 ( e ) a n d 1 0 -3 M CaCI 2 (o).
mediate with non treated fibrinogen. A constant volume (100 pl) was removed from the incubation mixture at regular intervals of time, and the reaction was terminated by the addition of hirudin (0.1 units). The samples were then compared on the basis of their electrophoretic pattern on 5% polyacrylamide gels at basic pH as described above. It was possible to quantify the a m o u n t of dimer formed during the proteolytic reaction in the presence and absence of Ca 2+ by scanning the gels and expressing the data in percent of the starting fibrinogen. The results are illustrated in Fig. 3 (inset 2), where the time courses of the formation of the dimer in the presence and absence of Ca 2÷ are compared. These curves are similar to the kinetics profiles of H ÷ release during the reaction. This suggests that in the presence of Ca 2÷ more fibrinogen molecules were potentially cleaved by thrombin. Discussion
Clotting of fibrinogen is classically described as a composite of the following reactions: (1) proteolysis by thrombin, (2) formation of intermediate polymers, (3) clotting. An additional step can be added to this sequence of reactions which includes the possibility t h a t native fibrinogen forms a dimeric intermediate with a fibrin m o n o m e r devoid in only the fibrinopeptide A. The formation of this dimer has already been described [12,16,17] and our results obtained from a purified system confirm its existence and demonstrate that it is Ca ~÷ dependent. The function of this dimeric intermediate is not clear. It was recently suggested [16] that this dimer acts physiologically as a buffer system inducing a lag phase in the clotting process. On the basis of the results presented in this paper, we are able to further characterize this compound. The formation of a Ca 2÷ dependent dimeric intermediate during the clotting
140
of fibrinogen can be illustrated by the following sequence of reactions: fAB~ A
fB'T~-~ f B Ca"H"
fAB + fB ~
(1)
(2)
(fAB-- fB)
(fAB -- fs) ~
n(fB) ~-~ n(f) A
(3)
B
where fAB represents intact fibrinogen, f is the fibrin m o n o m e r and A and B the peptides which are cleaved off b y thrombin from the As and B~ chain, respectively. One of the major problems when studying the kinetics of the overall fibrinogen clotting system is to separate the proteolytic step from the polymerization step. Depending on the pH, protons can be produced or adsorbed during the overall reaction. According to Mihalyi [18] hydrogen ions are liberated at pH 7 or less by the polymerization step and between pH 7 and 8 both polymerization and proteolytic reaction contribute to the release of hydrogen ions. Above pH 8 protons can only be produced by the proteolytic reaction and are absorbed b y the polymerization step. Our experiments were performed at pH 8.6. Therefore the observed increase of proton release (Fig. 3) largely concerns the proteolytic step. This suggests that the presence of Ca2+ increases the release of H ÷ and therefore catalyses the proteolytic reaction. This is in apparent contradiction with the recent observations of Blomb~ick et al. [9] who did not find any influence of Ca 2÷ o n the kinetics of release of the peptides A and B. This discrepancy may be explained either by the very low concentration of fibrinogen used in our experiments (0.02%) or by the fact that the kinetics of H ÷ release was measured during the first 5 min of the reaction where no aggregation or visible absorbance interfered. At higher concentration the different reactions occurring might overlap the effect of Ca 2÷ thus rendering less sensitivity to the measurement. Estimation of the number of molecules of fibrinogen which have been cleaved during the first five minutes of the reaction, also indicates that the reaction is more rapid in the presence of Ca 2÷. Interesting observations have been made by Kudrick et al. [24] who showed that abnormal fibrinogen Detroit lacking the fibrinopeptide A does not associate with normal fibrinogen. Moreover, some of the abnormal fibrinogens which have been studied have a delayed thrombin time that is corrected by Ca 2÷ [19-23]. It is therefore proposed that abnormal fibrinogen might have different affinity for Ca 2÷. This would confer to the Ca2÷-dependent dimeric intermediate an important role in the kinetics of the clotting of fibrinogen. Such a model would agree with the previous observations of Hansen et al. [25] on the enhancement of blood coagulation by soluble fibrinogen-fibrin complexes. Acknowledgement The authors wish to thank Miss E. Concord for her skillful technical assistance. This work was supported by INSERM F R A 21.
141
References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
Marguerie, G., Chagniel, G. and Suscillon, M. (1977) Biochim. Biophys. Acta 490, 94--103 Marguerie, G. (1977) Biochim. Biophys. Acta 494, 172--181 Purves, L.R., Lindsey~ G.G. and Franks, J.J. (1978) South. Af. J. Sci. 74, 202--209 Van Ruijven-Vermeer, I.A., Nieuwehuizen, W. and Nooijen, W.J. (1978) FEBS Lett. 9 3 , 1 7 7 - - 1 8 0 Enias, T.P. and lyer, G.Y.N. (1967) Thromb. Diath. Haem. 18, 499--509 Boyer, M.H., Shainoff, J.R. and Ratnoff, O.D. (1972) Blood 39,382--387 Endres, G.F. and Scheraga, H.A. (1972) Arch. Biochem. Biophys. 153, 266--278 Brass, E.P., Forman, W.B., Edwards, R.V. and Lindan, O. (1978) Blood 52, 654--658 Blomb~/ck, B., Hessel, B., Hogg, D. and Therkildsen, L. (1978} Nature 275, 501--505 Kekwick, R.A., Mackay, M.E., Nance, M.M. and Record, B.H. (1955) Biochem. J. 60, 6 7 1 - 6 8 3 Marguerie, G. and Stuhrmann, H.B. (1976) J. Mol. Biol. 102, 143--156 Benabid, Y., Concord, E. and Suscillon, M. (1977) Thromb. Haemost. 37, 144--153 Weber, K. and Osborne, H. (1969) J. Biol. Chem. 244, 4406--4412 Shainoff, J.R. and Page, M.D. (1962) J. Exp. Med. 116, 687--706 Mihalyi, E. (1972) in Application of Proteolytic Enzymes to Protein Structure studies, pp. 106, 161, CRC Press, Cleveland, Ohio Brass, E.P., Forman, W.B., Edwards, R.V. and Lindan, O. (1976) Thromb. Haemost. 36, 37--48 Graeff, H.R., Von Hugo, R. and Hafter, R. (1973) Thromb. Res. 3~ 465--476 Mihalyi, E. and BiUick, I.H. (1963) Biochim. Biophys. Acta 71, 97--108 Forman, W.B., Ratnoff, O.D. and Boyer, M.H. (1968) J. Lab. Clin. Med. 72, 455--472 Krause, W.M., Heene, D.L. and Lasch, H.G. (1973) Thromb. Diath. Haemorth. 29,547--561 Lacombe, M., Sofia, J., Soria, C., D'Angelo, G., LavaUee, R. and Bonny, Y. (1973) Thromb. Diath. HaemorEh, 29, 536--546 Jaeobsen~ C.D. and Hoak, J.C. (1973) Tl~omb. Res. 2~ 261--270 AI-Mondhiry, H.A.B., Bilezikian, S.B. and Nossel, H.L. (1975) Blood 45, 607--619 Kudxyk, B., Blomb~/ck, B. and Blombb'ck, M. (1976) Thzomb. Res. 9, 25--36 Hansen, M.S., Bang, N.U., Barton, R.D. and Mattler, L.E. (1975) J. Exp. Med. 141, 944--961
Biochimica et Biophysica Acta, 579 (1979) 1 3 4 - - 1 4 1 © E l s e v i e r / N o r t h - H o l l a n d B i o m e d i c a l Press
BBA 38215
THE BINDING OF CALCIUM TO FIBRINOGEN: INFLUENCE ON THE CLOTTING PROCESS
G E R A R D M A R G U E R I E *, Y V E T T E B E N A B I D ** and M I C H E L S U S C I L L O N
Laboratoire d 'Hdmatologie, Institut de Recherche Fondamentale, Centre d'Etudes Nucldaires, 85 X, 38041 - Grenoble-Cedex (France) (Received December llth,
1978)
Key words: Ca2+; Fibrinogen; Fibrin monomer; Fibrinogen-fibrin complex
Summary The influence of Ca ~+ on the basic reaction between thrombin and fibrinogen was investigated. The results demonstrate that: (a) A Ca2÷-dependent dimeric intermediate is formed during the early step of the clotting process. This dimeric intermediate is shown to be formed by the association of an intact fibrinogen molecule and a fibrin m o n o m e r devoid in only the peptide A, (b) Ca 2÷ enhances the proteolytic step as illustrated by the measure of the kinetics of H ÷ release at pH 8.6 On the basis of these observations it is proposed that Ca 2+ catalyses the proteolysis of fibrinogen by thrombin through the formation of a Ca2+-dependent dimer.
Introduction
A recent finding showing t h a t fibrinogen has three Ca 2+ binding sites of high affinity [1] stimulates new investigations on the influence of this cation on the structure-function relationship of this protein. While it was shown that the binding of Ca 2÷ does n o t entail a gross overall conformational change, several Ca2+-induced protective effects were however observed. Specific structural features are believed to be related to the binding of this cation to fibrinogen. It was proposed that Ca 2÷ is inserted within the structure of this protein [2].
* Present address: Scripps Clinic and Research Foundation, La Jolla. CA 92037. U.S.A. ** To whom reprint requests should be addressed. Abbreviation: TAME. tosylarginine-O-methyl ester.
135 This view has received recent support from other investigators who confirmed the existence of three high affinity Ca 2÷ binding sites in the fibrinogen molecule. Two of these sites are located in the D domain of the molecule [3,4]. The functional implication of the existence of Ca 2÷ binding sites on fibrinogen remains to be elucidated. The role of this divalent cation in the clotting of fibrinogen has been recognized for a long time. Most of the studies have led to the suggestion that the catalytic influence of Ca 2÷ is restricted to the polymerization of the fibrin monomer. Moreover the proteolytic activation by thrombin is essentially unaffected [5,9]. In the present paper, we provide additional data that suggest a possible influence of Ca 2÷ on the reaction between thrombin and fibrinogen. Material and Methods Twice distilled water was used for all the solutions from which contaminating Ca 2÷ was removed as previously detailed [ 1]. Bovine fibrinogen was purified from fresh plasma according to the m e t h o d of Kekwick et al. [10]. After clarification of the sample and determination of protein concentration [11], the purified fibrinogen (>98% clottable protein) was stored at --30°C in 0.3 M NaC1 solutions. Ca2÷-free fibrinogen was prepared as previously described [ 1 ]. Bovine thrombin from Parke-Davis and Co. was dissolved in 0.1 M NaC1, 0.05 Tris-HC1 buffer, pH 7.5 at a final concentration of 1 NIH unit/ml. Polyacrylamide gel electrophoresis on 3.75% gels were carried o u t at pH 8.9 [ 12]. Staining was done with coomassie blue and scanning of gels for densitometric determination of relative a m o u n t of proteins was performed on a densitometer DD2 (Kipp & Zonen). SDS polyacrylamide gel electrophoresis of reduced samples were run on 10% gels [13]. Agarose gel filtrations on Biogel A-5m (200--400 mesh) from Biorad Laboratories were carried o u t as previously published [12]. Hydrogen ion titrations at constant pH were done using a highly sensitive pH shift m e t h o d [2]. In a typical experiment 4 nmol of Ca2÷-free fibrinogen (1.4 mg) were dissolved in 5 ml of Ca2÷-free 0.15 M NaC1 solution. The solution was brought to 8.6 and equilibrated at 20°C-+ 0.05°C. Carbon dioxide-free helium was continuously flushed over the solution. 25 pl of thrombin were added (0.025 NIH units) and the pH shift due to the reaction was recorded. Standardized HC1 solution was used for calibration. When the reaction was performed in the presence of Ca 2÷, 0.5 ml of 10 -2 M CaC12 solution was added to the solution. Ca 2÷ concentrations were determined after each experiment b y atomic absorption spectrophotometry. Equilibrium dialyzis experiments, using [14C]EDTA (from A m e r s h a m ) w e r e done as previously detailed [ 1]. Results
I. Influence o f Ca 2÷ on complex-formation between native fibrinogen and thrombin-treated fibrinogen When fibrinogen is incubated with a small amount of thrombin over a short
136
period, soluble polymeric aggregates can be isolated according to their size by gel filtration over a Biogel A-5m column, if they are eluted at basic pH [12]. We have investigated the influence of Ca :+ on the formation of these complexes using the same procedure. 0.01 units of thrombin were added to 15 mg of fibrinogen which was dissolved in 2 ml of 0.1 M NaC1, 0.05 M Tris-HC1 buffer, pH 7.5. After 30 min incubation at 37°C the reaction was terminated b y the addition of 1.5 units of Hirudine. The sample was then applied on a Biogel A-5m column (1.8 × 50 cm) and eluted with a 0.25 M NaC1, 0.05 M Tris-HC1 buffer, pH 8.6. At this pH the elution profile consisted of three peaks, as illustrated in Fig. 1. Polyacrylamide gel electrophoresis at pH 8.9 on 3.75% gels indicated that peak 1, eluted in the void volume corresponded to soluble material of high molecular weight whicti did not penetrate the gel. Peak 2 represents material with a molecular weight
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C
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3
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Elution v o l u m e ( m l ) Fig. 1. G e l f i l t r a t i o n o n Biogel A - 5 m ( 2 0 0 - - - 4 0 0 m e s h ) o f f i b r i n o g e n ( 1 5 r a g ) i n c u b a t e d f o r 3 0 m i n a t 3 7 ° C w i t h 0.01 NIH units t h r o m b i n . The r e a c t i o n was t e r m i n a t e d b y H i r u d i n (1.5 units). The b u f f e r syst e m w a s 0 . 2 5 M NaC1, 0 . 0 5 M Tris-HC1 p H 8 . 5 in t h e p r e s e n c e ( . . . . . . ) a n d a b s e n c e (. . . . . . ) o f 1 0 -3 M E D T A . P o l y a c r y l a m i d e gel e l e c t r o p h o r e s i s , 3 . 7 5 % gel a t p H 8 . 9 ( w i t h o u t S D S ) . A, f i b r i n o g e n (a) a n d t h r o m b i n t r e a t e d f i b r i n o g e n ( b ) . B, c h r o m a t o g r a p h i c p e a k s 1, 2 a n d 3 o b t a i n e d u s i n g Tris-NaC1 b u f f e r . C, chromatographic peak 1 and 3 obtained using EDTA-containing buffer.
137
twice that of fibrinogen, which has already been described as a dimeric intermediate [12]. Peak 3 was unmodified fibrinogen. To confirm further the composition of the 'dimer', the electrophoretic pattern of the reduced material was compared with that of reduced fibrinogen and fibrinogen treated with thrombin and reptilase as previously described [12]. The results are shown in Fig. 2. As can be seen in the dimeric intermediate, the B~ chain is intact, whereas the band corresponding to the As chain, slightly overlaps with the ~ chain, suggesting that both polypeptidic chains are present. This is in excellent agreement with previously published data which suggested that the dimer was formed from the association of one intact fibrinogen molecule and one fibrinogen devoid in only the fibrino-peptide A [12]. When the sample was run over the same column with the same buffer at pH 8.6 in the presence of 10 -3 M EDTA, the elution profil e was different (Fig. 1). The presence of EDTA eliminated the second peak that contained the 'dimer'. Polyacrylamide gel electrophoresis of peak 1 and peak 3 revealed that peak 1 still contained high molecular weight aggregates, while peak 3 contained both unmodified fibrinogen and the dimer formed under the electrophoretic conditions. When Ca 2÷ was restored at a concentration of 5 • 10 -3 M the dimeric intermediate was again eluted in the position of peak 2.
II. Influence o f Ca 2÷ on the proteolysis o f fibrinogen by thrombin The kinetics of proton release during the proteolytic reaction of thrombin on fibrinogen was studied at pH 8.6, using the pH shift method [15]. The reaction curves are shown in Fig. 3 and are representative of average values from 3 runs. Reproducibility was good (0.02 equiv. H ÷ per mol of fibrinogen) and was tested as described elsewhere [2]. Recordings were stopped after 10 min during which time no clot or aggregates were observed in the reaction chamber.
F D Fig. 2. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, 7.5% gel, of sample reduced by 2-mercaptoethanol: fibrinogen ( F ) d i m e r (D)thrombin-treated fibrinogen ( F . T ) a n d reptilase-t~eated fibrinogen (F.R.).
138
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i
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3
4
5
6
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TiME(minute)
Fig. 3. Proton release during the pzoteolysis of fibrinogen by t h r o m b i n at pH 8.6, in the presence of 2 • 1 0 -6 M CaC12 ( e ) ' a n d 10 -3 M CaCI 2 (o). I n s e t 1 s h o w s t h e p r o t o n release d u r i n g t h e esterolysis b y t b x o m h i n o f 1 0 -2 M T A M E s o l u t i o n u n d e r t h e s a m e c o n d i t i o n s . I n s e t 2 r e p r e s e n t s t h e v a r i a t i o n of d i m e r f o r m a t i o n d u r i n g t h e r e a c t i o n . 1 0 0 pl w e r e r e m o v e d f r o m t h e r e a c t i o n c h a m b e r at s e l e c t e d t i m e i n t e r v a l s a n d s u b m i t t e d t o 5% p o l y a c r y l a m i d e gel e l e c t r o p h o r c s i s . T h e e l e c t r o p h o r e t i c b a n d c o r r e s p o n d i n g to t h e d i m e r w a s s c a n n e d a n d c o m p a r e d w i t h t h e original f i b r i n o g e n s a m p l e .
When the c o n c e n t r a t i o n of Ca 2÷ was increased from 2 • 10 -~ M to 10 -3 M, m ore H ÷ were released per mol o f fibrinogen, indicating that Ca 2÷ had an effect on the rate o f the reaction between t h r o m b i n and fibrinogen. When analyzed in terms of first order reaction, as previously detailed by Mihalyi [15], these reaction curves give points which fall into straight lines (Fig. 4) according to the equation. log d H t / d t = log A o K e -- Klo T where d H t / d t is the rate of the reaction, Ke and K10 'is the rate constant in normal and decimal logarithms and A0 is the total n u m b e r of peptide bonds taking part in the reaction. Such plots gave estimated rate constants o f 0.22 and 0.10 at Ca 2÷ concentrations o f 10 .3 M and 2 • 10 -6 M, respectively. The esterolytic activity of t h r o m b i n was measured under the same experimental conditions at pH 8.6 on 10 -2 M TAME solutions. The results shown in Fig. 3 (inset 1) indicated that the enzymatic activity was n o t affected by the presence o f Ca 2+ at 20 ° C. The percentage of fibrinogen molecules cleaved by t h r o m b i n in the presence and absence o f Ca 2+ was estimated through ability to form the dimeric inter-
139
TIME ( m i n ) 0
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1
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3
4
5
6
7
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F i g . 5. A n a l y s i s o f t h e p r o t e o l y s i s r e a c t i o n c u r v e s i n t e r m s o f s i m u l t a n e o u s f i r s t o r d e r r e a c t i o n a c c o r d i n g t o M i h a l y i [ 1 8 ] , i n t h e p r e s e n c e o f 2 • 1 0 -5 M CaC12 ( e ) a n d 1 0 -3 M CaCI 2 (o).
mediate with non treated fibrinogen. A constant volume (100 pl) was removed from the incubation mixture at regular intervals of time, and the reaction was terminated by the addition of hirudin (0.1 units). The samples were then compared on the basis of their electrophoretic pattern on 5% polyacrylamide gels at basic pH as described above. It was possible to quantify the a m o u n t of dimer formed during the proteolytic reaction in the presence and absence of Ca 2+ by scanning the gels and expressing the data in percent of the starting fibrinogen. The results are illustrated in Fig. 3 (inset 2), where the time courses of the formation of the dimer in the presence and absence of Ca 2÷ are compared. These curves are similar to the kinetics profiles of H ÷ release during the reaction. This suggests that in the presence of Ca 2÷ more fibrinogen molecules were potentially cleaved by thrombin. Discussion
Clotting of fibrinogen is classically described as a composite of the following reactions: (1) proteolysis by thrombin, (2) formation of intermediate polymers, (3) clotting. An additional step can be added to this sequence of reactions which includes the possibility t h a t native fibrinogen forms a dimeric intermediate with a fibrin m o n o m e r devoid in only the fibrinopeptide A. The formation of this dimer has already been described [12,16,17] and our results obtained from a purified system confirm its existence and demonstrate that it is Ca ~÷ dependent. The function of this dimeric intermediate is not clear. It was recently suggested [16] that this dimer acts physiologically as a buffer system inducing a lag phase in the clotting process. On the basis of the results presented in this paper, we are able to further characterize this compound. The formation of a Ca 2÷ dependent dimeric intermediate during the clotting
140
of fibrinogen can be illustrated by the following sequence of reactions: fAB~ A
fB'T~-~ f B Ca"H"
fAB + fB ~
(1)
(2)
(fAB-- fB)
(fAB -- fs) ~
n(fB) ~-~ n(f) A
(3)
B
where fAB represents intact fibrinogen, f is the fibrin m o n o m e r and A and B the peptides which are cleaved off b y thrombin from the As and B~ chain, respectively. One of the major problems when studying the kinetics of the overall fibrinogen clotting system is to separate the proteolytic step from the polymerization step. Depending on the pH, protons can be produced or adsorbed during the overall reaction. According to Mihalyi [18] hydrogen ions are liberated at pH 7 or less by the polymerization step and between pH 7 and 8 both polymerization and proteolytic reaction contribute to the release of hydrogen ions. Above pH 8 protons can only be produced by the proteolytic reaction and are absorbed b y the polymerization step. Our experiments were performed at pH 8.6. Therefore the observed increase of proton release (Fig. 3) largely concerns the proteolytic step. This suggests that the presence of Ca2+ increases the release of H ÷ and therefore catalyses the proteolytic reaction. This is in apparent contradiction with the recent observations of Blomb~ick et al. [9] who did not find any influence of Ca 2÷ o n the kinetics of release of the peptides A and B. This discrepancy may be explained either by the very low concentration of fibrinogen used in our experiments (0.02%) or by the fact that the kinetics of H ÷ release was measured during the first 5 min of the reaction where no aggregation or visible absorbance interfered. At higher concentration the different reactions occurring might overlap the effect of Ca 2÷ thus rendering less sensitivity to the measurement. Estimation of the number of molecules of fibrinogen which have been cleaved during the first five minutes of the reaction, also indicates that the reaction is more rapid in the presence of Ca 2÷. Interesting observations have been made by Kudrick et al. [24] who showed that abnormal fibrinogen Detroit lacking the fibrinopeptide A does not associate with normal fibrinogen. Moreover, some of the abnormal fibrinogens which have been studied have a delayed thrombin time that is corrected by Ca 2÷ [19-23]. It is therefore proposed that abnormal fibrinogen might have different affinity for Ca 2÷. This would confer to the Ca2÷-dependent dimeric intermediate an important role in the kinetics of the clotting of fibrinogen. Such a model would agree with the previous observations of Hansen et al. [25] on the enhancement of blood coagulation by soluble fibrinogen-fibrin complexes. Acknowledgement The authors wish to thank Miss E. Concord for her skillful technical assistance. This work was supported by INSERM F R A 21.
141
References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
Marguerie, G., Chagniel, G. and Suscillon, M. (1977) Biochim. Biophys. Acta 490, 94--103 Marguerie, G. (1977) Biochim. Biophys. Acta 494, 172--181 Purves, L.R., Lindsey~ G.G. and Franks, J.J. (1978) South. Af. J. Sci. 74, 202--209 Van Ruijven-Vermeer, I.A., Nieuwehuizen, W. and Nooijen, W.J. (1978) FEBS Lett. 9 3 , 1 7 7 - - 1 8 0 Enias, T.P. and lyer, G.Y.N. (1967) Thromb. Diath. Haem. 18, 499--509 Boyer, M.H., Shainoff, J.R. and Ratnoff, O.D. (1972) Blood 39,382--387 Endres, G.F. and Scheraga, H.A. (1972) Arch. Biochem. Biophys. 153, 266--278 Brass, E.P., Forman, W.B., Edwards, R.V. and Lindan, O. (1978) Blood 52, 654--658 Blomb~/ck, B., Hessel, B., Hogg, D. and Therkildsen, L. (1978} Nature 275, 501--505 Kekwick, R.A., Mackay, M.E., Nance, M.M. and Record, B.H. (1955) Biochem. J. 60, 6 7 1 - 6 8 3 Marguerie, G. and Stuhrmann, H.B. (1976) J. Mol. Biol. 102, 143--156 Benabid, Y., Concord, E. and Suscillon, M. (1977) Thromb. Haemost. 37, 144--153 Weber, K. and Osborne, H. (1969) J. Biol. Chem. 244, 4406--4412 Shainoff, J.R. and Page, M.D. (1962) J. Exp. Med. 116, 687--706 Mihalyi, E. (1972) in Application of Proteolytic Enzymes to Protein Structure studies, pp. 106, 161, CRC Press, Cleveland, Ohio Brass, E.P., Forman, W.B., Edwards, R.V. and Lindan, O. (1976) Thromb. Haemost. 36, 37--48 Graeff, H.R., Von Hugo, R. and Hafter, R. (1973) Thromb. Res. 3~ 465--476 Mihalyi, E. and BiUick, I.H. (1963) Biochim. Biophys. Acta 71, 97--108 Forman, W.B., Ratnoff, O.D. and Boyer, M.H. (1968) J. Lab. Clin. Med. 72, 455--472 Krause, W.M., Heene, D.L. and Lasch, H.G. (1973) Thromb. Diath. Haemorth. 29,547--561 Lacombe, M., Sofia, J., Soria, C., D'Angelo, G., LavaUee, R. and Bonny, Y. (1973) Thromb. Diath. HaemorEh, 29, 536--546 Jaeobsen~ C.D. and Hoak, J.C. (1973) Tl~omb. Res. 2~ 261--270 AI-Mondhiry, H.A.B., Bilezikian, S.B. and Nossel, H.L. (1975) Blood 45, 607--619 Kudxyk, B., Blomb~/ck, B. and Blombb'ck, M. (1976) Thzomb. Res. 9, 25--36 Hansen, M.S., Bang, N.U., Barton, R.D. and Mattler, L.E. (1975) J. Exp. Med. 141, 944--961