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The influence of calcium ions on fibrinogen conformation
Biochimica et Biophysica Acta, 995 (1989) 70-74 Elsevier
70
BBA 33328
The influence of calcium ions on fibrinogen conformation Angela Apap-Bologna, Ailsa Webster, Fiona Raitt and Graham Kemp Department of Biochemistry and Microbiology, Universityof St. Andrews, St. Andrews ( U.K.)
(Received 30 September 1988)
Key words: Fibdnogen; Calcium binding; [A]a chain; Surface labeling; Crosslinking
The conformation of fibdnogen has been examined using the techniques of dye.photosensitised surface labelling and cress-linking. The results obtained suggest that fibrinogen is a flexible molecule and its conformation is influenced by the coneentration of calcium ions. These effects are mediated through binding to the low-affinity calcium binding sites of fibflnogen. In particular the C.termlnal regions of the [Ala chain are more exposed at higher calcium concentrations creating a molecule which is more liable to form inter.molecular interactions.
introduction The conformation of fi~tinogen has been investigated by a number of workers using a variety of techniques [1-7]. The lack of general agreement on the native conformation of the molecule has led Mueller and Burchar¢~ [8] to propose that there is a flexibility to the structure. ~ J s being the case, the particular conformation adopted could be influenced by the method of preparation or the conditions imposed by the technique used, especially if the molecule is not in true solution. One factor which could be of particular importance is calCium ion concentration. Fibrinogen is a calcium binding protein, with binding sites of both high and low affinity [9] and there are several reports that calcium ioas influence both function and conformation [10-12]. There is therefore a need for further studies of fib~'inogen conformation in solution with particular refereL~ce to the influence of calcium ions. The technique of photosensitised labelfing of the solvent exposed parts of proteins, described by Hemmendorf et al. [13] is particularly suited to this type of study. In this report we de~cribe the use of this method, and of the crosslinking method derived from it [14] to investigate the eff~t, of calcium ions on the conformation of fibrinogen. Preliminary results from these studies have been reported [14,15].
Abbreviation: SDS-PAGE, sodi~:m dodecyl sulphate-polyacrylamide gel electrophoresis. Correspondence: G.D. Kemp, Department of Biochemistry and Microbiology, University of St. Andrews, North Street, St. Andrews KYI6 9AL, U.K.
Materials and Methods
Fibrinogen preparation Outdated freshly frozen plasma was obtained from the Blood Transfusion Centre, Ninewells Hospital, Dundee. Fibrinogen was prepared by ammonium sulphate precipitation followed by DEAE-cellulose chromatography as described by Lawrie et al. [16]. The fraction used has been shown to be free from factor XIII activity [16].
Photosensitised labelling of the solvent exposed regions of fibrinogen The method used was based on that of Hemmendorf et al. [13]. 0.9 ml fibrinogen at a concentration of 0.6 mg/ml in the required buffer was mixed with 0.5 ml of 3 mM fluorescein in 0.01 M NaOH and 0.1 ml 0.05 mM [3H]tryptophan (L-[G-3 H]tryptophan from Amersham International, diluted with unlabelled tryptophan to give a specific activity of 3.37/tCi/nmol). The mixture was stirred in an ice bath and irradiated for 20 s at a distance of 13.5 cm with a 1000 W lamp (Philips Watastar). Most of the excess tryptophan and fluorescein were removed by dialysis. To ensure complete removal of non-covalently bound tryptophan, labelled samples (in triplicate) were subjected to SDS-PAGE and stained with Coomassie blue. Stained bands were excised, solubilised in hydrogen peroxide (0.5 ml per 1 mm slice of a 5 mm diameter gel heated at 75 °C for 8 h) and the amount of radioactivity determined by scintillation counting.
Cross-li~king This was carried out at a protein concentration of 0.6 mg/ml using the method described by Apap-Bologna et al. [14].
01674838/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
71
Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate (SDS-PAGE) Non-reduced samples were separated on 3% polyacrylamide gels as described previously [17]. Reduced samples were separated according to the method of Laemmli [I 81. Plasmin digestion of fibrinogen Plasminogen was prepared from human plasma by affinity chromatography on lysine-agarose by the method of Deutsch and Mertz [19]. Plasminogen was activated by incubation at 37O C for 15 min with 1 Kallikrein Inhibitor Unit of streptokinase per 10 CTA (Committee on Thrombolytic Agents) Units [20] of plasminogen. Digestion to fragment X was achieved at 37OC with 0.004 CTA units of plasmin per mg of fibrinogen.
Cont.
Ca”
(mM
1
esults
Fibrinogen was irradiated with visible light in the presence of tritiated tryptophan and concentrations of calcium chloride up to 13.3 mM. The amount of label incorporated into the fibrinogen increased as the calcium concentration was increased (Fig. la), suggesting that calcium ions have a,n influence on the conformation of fibrinogen. The most striking difference was between 0.05 mM and 2 mM calcium chloride where there was a 2-fold increase in the amount of label incorporated. This difference was statistically significant (P < 0.001) as judged by the Student’s t-test for unrelated samples. As the calcium concentration was further increased, the amount of label incorporated, and hence the area of solvent accessible surface, decreased and then rose again above calcium concentrations of 3 mM. A similar set of experiments using magnesium chloride showed that the conformation of the molecule was also influenced by magnesium ions (Fig. lb). In this case there was also an increase in the proportion of the molecule which was accessible to solvent, although in contrast to calcium there was no discernible maximum in the region of 2 mM magnesium ions. Fig. lb also shows that over an identical range of ionic strengths, sodium ions had no effect on the conformation, as judged by the incorporation of surface label. The nature of the conformational change in fibrinogen which accompanied the increase in calcium concentration was investigated by determining the amount of label incorporated into the individual chains at 0.05 mM and 2 mM CaCl,. As well as a statistically significant increase in the amount of label taken up by all three chains in 2 mM calcium, there was a change in the proportion of the surface occupied by the [Ala and y-chains (Table I). The proportion of the surface attributable to the [Ala chain rose from 40% at 0.05 mM to 48% at 2 mM calcium, while the contribution from the
0
I
I
7.5 Ionic
I
15 strength
22 5
(mM1
Fig. 1. (a) The effect of calcium concentration on the incorporation of surface label into fibrinogen (fgn). The vertical bars represent the S.D. from the mean of two separate experiments. Within each experiment points were determined in triplicate. (b) The effect of ionic strength on the incorporation of surface label into fibrinogen (comparison of ) and NaCl (A)).The vertical bars represent the S.D. from the mean of two separate experiments. Within each experiment points were determined in triplicate.
y-chain dropped from 35% at 0.05 mM to 27% at 2 mM calcium. Both of these changes are statistically significant (P < 0.01). The [B]/3 chain comprised a quarter of the surface at both calcium concentrations. The apparent increase in surface area is accompanied by an increased tendancy to form intermolecular ag-
TABLE I Effect of calcium on the composition of the solvent-accessiblearea of fibrinogen
Fibrinogen samples which had been surface labelled at 0.05 mM and 2 mM calcium were reduced with mercaptoethanol and the chains separated by SDS-PAGE. The chains were located by Coomassie blue staining and appropriate gel slices dissolved and the amount of label incorporated into each chain determined by scintillation counting. The figures given are the means (+ 1 S.D.) of six separate experiments. Concentration of calcium (mM) 0.05
2
Incorporation (dpm/pmol protein) [Ala 24.0 f 1.9 62.4 + 5.0
1WP
Y
15.0f 1.2 31.2f2.5
21.0f 1.7 35.1 f 2.1
72 gregates. Fibrinogen, at a concentration of 0.38 m g / m l was cross-linked by the dye photosensitised procedure at a number of different calcium concentrations. Fig. 2 shows that the amount of polymer increased with increasing calcium concentratior,. The polymeric fibrinogen was mainly in the form of dimers, but at higher calciumconcentrations trimeric and tetrameric forms were apparent. Following separation by SDS-PAGE, the Coomassie blue stained gels were scanned on a densitometer. Comparison of the peak areas showed that at 1 mM CaCI 2 27~ of the fibrinogen had been cross-linked intermolecularly rising to 35~ at 5 mM and 45~ at 13 mM calcium (Fig. 2). A similar, but less pronounced, effect was observed when fibrinogen was cross-linked in the presence of magnesium chloride over the same range of concentrations (not shown). Sodium
polymer
1
50t
~
40't!m!
taj
.m,
i. Q. u
o ,tO
0
i Conc. Co=. (raM) Fig. Z The effect of calcium concentration on the amount of intermolecular cross-h'_n_ki_n8and the involvement of the [A]a chain. Nonreduced and reduced samples of fibrinogen, cross-linked at a range of calcium concentrations, were analysed by SDS-PAGE and scanning densitometry. The percentage of fibrinogen cross-linked intermoleculady (11) and the percentage of residual [A]a chain (z) were calculated for each calcium concentration. The vertical bars represent the S.D. from the mean of three separate experiments. The acrylamide concentration was 3% for non-reduced samples and 7.5% for reduced samples. The direction of migration was from the top of the figure.
ymer
~n.
a
b
c
d
e
f
Fig. 3. SDS-PAGE of non-reduced fibrinogen (lanes a-c) before cross.linking and (lanes d-f) after cross-linking. Lanes a and d, no plasmin digestion; lanes b and e, digested for 15 rain; and lanes c and f, digested for 30 rain. Acrylamide gel concentration was 3~. Direction of migration was from the top of the figure.
chloride up to a concentration of 20 mM had no effect on the extent of intermolecular cross-linking observed using the dye-photosensitised procedure. The fibrinogen samples cross-linked at different calcium concentrations were reduced with mercaptoethanol and separated by SDS-PAGE. The results (Fig. 2) show that as the calcium concentration was increased and there was more intermolecular cross-linking, the amount of monomeric [A]a chain decreased. At 1 mM CaCI 2 29~ of the [A]a chain was cross-linked rising to 38~ at 5 mM and 58~; at 13 mM. We were not able to resolve the [B]/3 and 3'-chains by scanning, but between 1 and 13 mM CaCI 2, the combined [B]fl and 3' peak areas did not decrease. Hence it would appear that the intermolecular cross-linking arises primarily through contacts between a-chains, which must be made more available as a consequence of increased calcium ion concentration. In addition to intermolecular cross-linking, intramolecular cross-links are formed involving all three chains. The conclusion that the intermolecular cross-linking occurs via the [A]a chains was supported and extended by cross-linking studies on partially proteolysed fibrinogen. Fibrinogen was progressively digested by plasmin to fragment X, and the degraded fibrinogen cross-linked by the dye-photosensitised procedure. The results shown in Fig. 3 indicate that as the fibrinogen was digested the amount of intermolecular cross-linking decreased. When the digestion had proceeded as far as fragments X there was no detectable polymer.
Discussion The results presented here show that calcium ions have a significant effect on the conformation of fibrinogen, particularly on the C-terminal regions of the [A]a chains. In fibrinogen there are thought to be three high-affinity sites with a dissociation constant in the micromolar
73 range and a larger number of sites with a dissociation constant in the millimolar range [9]. Two of the high-affinity sites have been located with one in each of the D domains close to the C-terminus of the ~,-chain [17]. The location of the third site is still in doubt. Marguerie and Ardaillou [21] maintain that this site involves the C-terminal regions of the [A]a chain, thus suggesting that these regions are in contact with one another. In contrast, Nieuwenhuizen et al. [22] suggest that the site is located in the centre of the molecule close to the E domain. The locations of the low-affinity ~i*.¢~ are not known. In this work, fibrinogen was prepared in the presence of calcium ions and dialysed against buffers containing 0.05 M calcium chloride. Under these conditions the high-affinity sites would be saturated. Over the range of calcium chloride concentrations used, the degree of occupancy of the low-affinity sites would vary. Hence the conformational changes which follow changes in calcium concentration are most probably a result of calcium ions binding to the low-affinity sites. The observation that magnesium ions have a similar, although less pronounced effect is in accord with this conclusion, as Marguerie et al. [9] reported that magnesium ions were capable of binding to these sites. Okada and Blomback [23] have reported that calcium (but not magnesium) ions binding to the low affinity sites of fibrinogen influence the structure of the fibrin clot. Results from both the labelling and cross-linking methods show that the main effect of calcium ions is on the position of the [A]a chains. Our results show that these chains constitute some 40% of the solvent accessible region of the molecule at low calcium concentration, but this rises to nearly 5070 in the presence of 2 mM calcium chloride. The absence of cross-linking with fragment X also implicates the C-terminal regions of the [A]a chains. However, this experiment on its own does not rule out the possible iuvolvement of the N-terminal region of the [B]fl chain, as SDS-PAGE of digests after reduction (not shown) indicate that the fragments X also lack this region. However, the results show that the surface contribution of the [B]fl chain is not affected by calcium concentration. In addition, the [A]a chains become more invo~,ved in intermolecular cross-linking at higher calcium c~mcentrations (Fig. 2) leading us to conclude that the cross-linking method is stabilising interactions between the C-terminal regions of the [A]a chains. In this context it is interesting to note that from electron microscopy, Mosesson et al. [5] suggested that the C-term;,ni of the [A]a chains were closely associated with the central, or E, domain of the molecule. Calorimetric studies led Medved' et al. [24] to suggest that there was a fourth domain in fibrinogen formed by interactions between the C-terminal regions of the [A]a chains while Erikson and Fowler [25] noted, on electron micrographs, the existence of a fourth nodule close to
the central domain. These studies suggesting the existence of a fouzth domain were all carried out in the absence of added calcium. The results presented here would extend this static model of fibrinogen into the concept of a flexible molecule the conformation of which is influenced by calcium concentration. Specifically we would suggest that an increase in calcium concentration causes an increase in the surface area of the molecule which is mainly due to the C-terminal regions of the [A]a chains moving from an inward orientation to a more solvent exposed position. This conformational change leads to a molecule which is more prone to form intermolecular interactions due to the consequent exposure of important amino acid sequences. Acknowledgement We ate grateful to Paul Talbot for excellent technical support. References 1 Hall, C.E. and Slayter, H.S. (1959) Biophys. Biochem. Cytol. 5, 11-15. 2 Hudry-Clergeon, G., Marguerie, G., PouR, L. and Suscillon, M. (1975) Thromb. Res. 6, 533-541. 3 Marguerie G. and Stuhrmann, H.B. (1976) J. Mol. Biol. 102, 143-156. 4 Lederer, K. (1979) Thromb. Haemost. 41,641-647. 5 Mosesson, M.W., Hainfield, J., Wall, J. and Haschmeyer, R.H. (1981) J. Mol. Biol. 153, 695-718. 6 Mihalyi E. and Donovan, J.W. (1985) Biochem. 24, 3443-3448. 7 Larrson, U., Blombac!-, B. and Rigler, R. (1987) Biochim. Biophys. Acta 915, 172-179. 8 Mueller, M. and Burchard, W. (1978) Biochim. Biophys. Acta 537, 208-225. 9 Marguerie, G., Chagniei, G. and Suscillon, M. (1977) Biochim. Biophys. Acta 490, 94-103. 10 Haverkate, F. and Timan G. (1977) Thromb. Res. 10, 803-812. 11 Furlan, M., Rupp, C. and Beck, E.A. (1983) Biochim. Biophys. Acta 742, 25-32. 12 Ly, B. and Godal, H.C. (1973) Haemostasis 1, 204-209. 13 Hemmendorf, B., Brandt, J. and Andersson, L.O. (1981) Biochim. Biophys. Acta 667, 15-22. 14 Apap-Bologna A., Raitt, F. Webster, A. and Kemp, G. (1987) in Fibrinogen 2. Biochemistry, Physiology and Clinical Relevance (Lowe, G.D.O. et al., eds.), pp. 31-34, Elsevier Science Publishers, Amsterdam. 15 Apap-Bologna A. and Kemp, G. (1987) in Fibrinogen 2. Biochemistry, Physiology and Cfinical Relevance (I owe, G.D.O. et al., eds.), pp. 35-38, Elsevier Science Publishers, Amsterdam. 16 Lawrie J.S., Ross, J. and Kemp, G.D. (1979) Biochem. Soc. Trans. 7, 693-694. 17 Lawrie, J.S. and Kemp, G.D. (1979) Biochim. Biophys. Acta 577, 415-423. 18 Laemmli, U.K. (1970) Nature, 227, 680-695. 19 Deutsch, D.G. and Mertz, E.T. (1970) Science, 1095-1096. 20 Johnson, A.J., Kline, D.L. and Alkjaersig, N. (1969) Thromb. Diathes. Haemorrh. 21,259-272. 21 Marguerie, G. and Ardaillou, N. (1982) Bioctfim. Biophys. Acta 701,410-412.
74 22 Nieuwenhuizen, W., Voskuilen, M. and Hennans, J. (1982) Biochim. Biophys. Acta 708, 313-316. 23 Okada, M. and Blomback, B. (1983) Ann. NY Acad. Sci. 408, 233-253.
24 Medved', L.V., Oorkun, O.V. and Privalov, P.L. (1983) FEBS Lett. 160, 291-295. 25 Erikson~ H.P. and Fowler, W.E. (1983) Ann. NY Acad. Sci. 408, 146-163.
70
BBA 33328
The influence of calcium ions on fibrinogen conformation Angela Apap-Bologna, Ailsa Webster, Fiona Raitt and Graham Kemp Department of Biochemistry and Microbiology, Universityof St. Andrews, St. Andrews ( U.K.)
(Received 30 September 1988)
Key words: Fibdnogen; Calcium binding; [A]a chain; Surface labeling; Crosslinking
The conformation of fibdnogen has been examined using the techniques of dye.photosensitised surface labelling and cress-linking. The results obtained suggest that fibrinogen is a flexible molecule and its conformation is influenced by the coneentration of calcium ions. These effects are mediated through binding to the low-affinity calcium binding sites of fibflnogen. In particular the C.termlnal regions of the [Ala chain are more exposed at higher calcium concentrations creating a molecule which is more liable to form inter.molecular interactions.
introduction The conformation of fi~tinogen has been investigated by a number of workers using a variety of techniques [1-7]. The lack of general agreement on the native conformation of the molecule has led Mueller and Burchar¢~ [8] to propose that there is a flexibility to the structure. ~ J s being the case, the particular conformation adopted could be influenced by the method of preparation or the conditions imposed by the technique used, especially if the molecule is not in true solution. One factor which could be of particular importance is calCium ion concentration. Fibrinogen is a calcium binding protein, with binding sites of both high and low affinity [9] and there are several reports that calcium ioas influence both function and conformation [10-12]. There is therefore a need for further studies of fib~'inogen conformation in solution with particular refereL~ce to the influence of calcium ions. The technique of photosensitised labelfing of the solvent exposed parts of proteins, described by Hemmendorf et al. [13] is particularly suited to this type of study. In this report we de~cribe the use of this method, and of the crosslinking method derived from it [14] to investigate the eff~t, of calcium ions on the conformation of fibrinogen. Preliminary results from these studies have been reported [14,15].
Abbreviation: SDS-PAGE, sodi~:m dodecyl sulphate-polyacrylamide gel electrophoresis. Correspondence: G.D. Kemp, Department of Biochemistry and Microbiology, University of St. Andrews, North Street, St. Andrews KYI6 9AL, U.K.
Materials and Methods
Fibrinogen preparation Outdated freshly frozen plasma was obtained from the Blood Transfusion Centre, Ninewells Hospital, Dundee. Fibrinogen was prepared by ammonium sulphate precipitation followed by DEAE-cellulose chromatography as described by Lawrie et al. [16]. The fraction used has been shown to be free from factor XIII activity [16].
Photosensitised labelling of the solvent exposed regions of fibrinogen The method used was based on that of Hemmendorf et al. [13]. 0.9 ml fibrinogen at a concentration of 0.6 mg/ml in the required buffer was mixed with 0.5 ml of 3 mM fluorescein in 0.01 M NaOH and 0.1 ml 0.05 mM [3H]tryptophan (L-[G-3 H]tryptophan from Amersham International, diluted with unlabelled tryptophan to give a specific activity of 3.37/tCi/nmol). The mixture was stirred in an ice bath and irradiated for 20 s at a distance of 13.5 cm with a 1000 W lamp (Philips Watastar). Most of the excess tryptophan and fluorescein were removed by dialysis. To ensure complete removal of non-covalently bound tryptophan, labelled samples (in triplicate) were subjected to SDS-PAGE and stained with Coomassie blue. Stained bands were excised, solubilised in hydrogen peroxide (0.5 ml per 1 mm slice of a 5 mm diameter gel heated at 75 °C for 8 h) and the amount of radioactivity determined by scintillation counting.
Cross-li~king This was carried out at a protein concentration of 0.6 mg/ml using the method described by Apap-Bologna et al. [14].
01674838/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
71
Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate (SDS-PAGE) Non-reduced samples were separated on 3% polyacrylamide gels as described previously [17]. Reduced samples were separated according to the method of Laemmli [I 81. Plasmin digestion of fibrinogen Plasminogen was prepared from human plasma by affinity chromatography on lysine-agarose by the method of Deutsch and Mertz [19]. Plasminogen was activated by incubation at 37O C for 15 min with 1 Kallikrein Inhibitor Unit of streptokinase per 10 CTA (Committee on Thrombolytic Agents) Units [20] of plasminogen. Digestion to fragment X was achieved at 37OC with 0.004 CTA units of plasmin per mg of fibrinogen.
Cont.
Ca”
(mM
1
esults
Fibrinogen was irradiated with visible light in the presence of tritiated tryptophan and concentrations of calcium chloride up to 13.3 mM. The amount of label incorporated into the fibrinogen increased as the calcium concentration was increased (Fig. la), suggesting that calcium ions have a,n influence on the conformation of fibrinogen. The most striking difference was between 0.05 mM and 2 mM calcium chloride where there was a 2-fold increase in the amount of label incorporated. This difference was statistically significant (P < 0.001) as judged by the Student’s t-test for unrelated samples. As the calcium concentration was further increased, the amount of label incorporated, and hence the area of solvent accessible surface, decreased and then rose again above calcium concentrations of 3 mM. A similar set of experiments using magnesium chloride showed that the conformation of the molecule was also influenced by magnesium ions (Fig. lb). In this case there was also an increase in the proportion of the molecule which was accessible to solvent, although in contrast to calcium there was no discernible maximum in the region of 2 mM magnesium ions. Fig. lb also shows that over an identical range of ionic strengths, sodium ions had no effect on the conformation, as judged by the incorporation of surface label. The nature of the conformational change in fibrinogen which accompanied the increase in calcium concentration was investigated by determining the amount of label incorporated into the individual chains at 0.05 mM and 2 mM CaCl,. As well as a statistically significant increase in the amount of label taken up by all three chains in 2 mM calcium, there was a change in the proportion of the surface occupied by the [Ala and y-chains (Table I). The proportion of the surface attributable to the [Ala chain rose from 40% at 0.05 mM to 48% at 2 mM calcium, while the contribution from the
0
I
I
7.5 Ionic
I
15 strength
22 5
(mM1
Fig. 1. (a) The effect of calcium concentration on the incorporation of surface label into fibrinogen (fgn). The vertical bars represent the S.D. from the mean of two separate experiments. Within each experiment points were determined in triplicate. (b) The effect of ionic strength on the incorporation of surface label into fibrinogen (comparison of ) and NaCl (A)).The vertical bars represent the S.D. from the mean of two separate experiments. Within each experiment points were determined in triplicate.
y-chain dropped from 35% at 0.05 mM to 27% at 2 mM calcium. Both of these changes are statistically significant (P < 0.01). The [B]/3 chain comprised a quarter of the surface at both calcium concentrations. The apparent increase in surface area is accompanied by an increased tendancy to form intermolecular ag-
TABLE I Effect of calcium on the composition of the solvent-accessiblearea of fibrinogen
Fibrinogen samples which had been surface labelled at 0.05 mM and 2 mM calcium were reduced with mercaptoethanol and the chains separated by SDS-PAGE. The chains were located by Coomassie blue staining and appropriate gel slices dissolved and the amount of label incorporated into each chain determined by scintillation counting. The figures given are the means (+ 1 S.D.) of six separate experiments. Concentration of calcium (mM) 0.05
2
Incorporation (dpm/pmol protein) [Ala 24.0 f 1.9 62.4 + 5.0
1WP
Y
15.0f 1.2 31.2f2.5
21.0f 1.7 35.1 f 2.1
72 gregates. Fibrinogen, at a concentration of 0.38 m g / m l was cross-linked by the dye photosensitised procedure at a number of different calcium concentrations. Fig. 2 shows that the amount of polymer increased with increasing calcium concentratior,. The polymeric fibrinogen was mainly in the form of dimers, but at higher calciumconcentrations trimeric and tetrameric forms were apparent. Following separation by SDS-PAGE, the Coomassie blue stained gels were scanned on a densitometer. Comparison of the peak areas showed that at 1 mM CaCI 2 27~ of the fibrinogen had been cross-linked intermolecularly rising to 35~ at 5 mM and 45~ at 13 mM calcium (Fig. 2). A similar, but less pronounced, effect was observed when fibrinogen was cross-linked in the presence of magnesium chloride over the same range of concentrations (not shown). Sodium
polymer
1
50t
~
40't!m!
taj
.m,
i. Q. u
o ,tO
0
i Conc. Co=. (raM) Fig. Z The effect of calcium concentration on the amount of intermolecular cross-h'_n_ki_n8and the involvement of the [A]a chain. Nonreduced and reduced samples of fibrinogen, cross-linked at a range of calcium concentrations, were analysed by SDS-PAGE and scanning densitometry. The percentage of fibrinogen cross-linked intermoleculady (11) and the percentage of residual [A]a chain (z) were calculated for each calcium concentration. The vertical bars represent the S.D. from the mean of three separate experiments. The acrylamide concentration was 3% for non-reduced samples and 7.5% for reduced samples. The direction of migration was from the top of the figure.
ymer
~n.
a
b
c
d
e
f
Fig. 3. SDS-PAGE of non-reduced fibrinogen (lanes a-c) before cross.linking and (lanes d-f) after cross-linking. Lanes a and d, no plasmin digestion; lanes b and e, digested for 15 rain; and lanes c and f, digested for 30 rain. Acrylamide gel concentration was 3~. Direction of migration was from the top of the figure.
chloride up to a concentration of 20 mM had no effect on the extent of intermolecular cross-linking observed using the dye-photosensitised procedure. The fibrinogen samples cross-linked at different calcium concentrations were reduced with mercaptoethanol and separated by SDS-PAGE. The results (Fig. 2) show that as the calcium concentration was increased and there was more intermolecular cross-linking, the amount of monomeric [A]a chain decreased. At 1 mM CaCI 2 29~ of the [A]a chain was cross-linked rising to 38~ at 5 mM and 58~; at 13 mM. We were not able to resolve the [B]/3 and 3'-chains by scanning, but between 1 and 13 mM CaCI 2, the combined [B]fl and 3' peak areas did not decrease. Hence it would appear that the intermolecular cross-linking arises primarily through contacts between a-chains, which must be made more available as a consequence of increased calcium ion concentration. In addition to intermolecular cross-linking, intramolecular cross-links are formed involving all three chains. The conclusion that the intermolecular cross-linking occurs via the [A]a chains was supported and extended by cross-linking studies on partially proteolysed fibrinogen. Fibrinogen was progressively digested by plasmin to fragment X, and the degraded fibrinogen cross-linked by the dye-photosensitised procedure. The results shown in Fig. 3 indicate that as the fibrinogen was digested the amount of intermolecular cross-linking decreased. When the digestion had proceeded as far as fragments X there was no detectable polymer.
Discussion The results presented here show that calcium ions have a significant effect on the conformation of fibrinogen, particularly on the C-terminal regions of the [A]a chains. In fibrinogen there are thought to be three high-affinity sites with a dissociation constant in the micromolar
73 range and a larger number of sites with a dissociation constant in the millimolar range [9]. Two of the high-affinity sites have been located with one in each of the D domains close to the C-terminus of the ~,-chain [17]. The location of the third site is still in doubt. Marguerie and Ardaillou [21] maintain that this site involves the C-terminal regions of the [A]a chain, thus suggesting that these regions are in contact with one another. In contrast, Nieuwenhuizen et al. [22] suggest that the site is located in the centre of the molecule close to the E domain. The locations of the low-affinity ~i*.¢~ are not known. In this work, fibrinogen was prepared in the presence of calcium ions and dialysed against buffers containing 0.05 M calcium chloride. Under these conditions the high-affinity sites would be saturated. Over the range of calcium chloride concentrations used, the degree of occupancy of the low-affinity sites would vary. Hence the conformational changes which follow changes in calcium concentration are most probably a result of calcium ions binding to the low-affinity sites. The observation that magnesium ions have a similar, although less pronounced effect is in accord with this conclusion, as Marguerie et al. [9] reported that magnesium ions were capable of binding to these sites. Okada and Blomback [23] have reported that calcium (but not magnesium) ions binding to the low affinity sites of fibrinogen influence the structure of the fibrin clot. Results from both the labelling and cross-linking methods show that the main effect of calcium ions is on the position of the [A]a chains. Our results show that these chains constitute some 40% of the solvent accessible region of the molecule at low calcium concentration, but this rises to nearly 5070 in the presence of 2 mM calcium chloride. The absence of cross-linking with fragment X also implicates the C-terminal regions of the [A]a chains. However, this experiment on its own does not rule out the possible iuvolvement of the N-terminal region of the [B]fl chain, as SDS-PAGE of digests after reduction (not shown) indicate that the fragments X also lack this region. However, the results show that the surface contribution of the [B]fl chain is not affected by calcium concentration. In addition, the [A]a chains become more invo~,ved in intermolecular cross-linking at higher calcium c~mcentrations (Fig. 2) leading us to conclude that the cross-linking method is stabilising interactions between the C-terminal regions of the [A]a chains. In this context it is interesting to note that from electron microscopy, Mosesson et al. [5] suggested that the C-term;,ni of the [A]a chains were closely associated with the central, or E, domain of the molecule. Calorimetric studies led Medved' et al. [24] to suggest that there was a fourth domain in fibrinogen formed by interactions between the C-terminal regions of the [A]a chains while Erikson and Fowler [25] noted, on electron micrographs, the existence of a fourth nodule close to
the central domain. These studies suggesting the existence of a fouzth domain were all carried out in the absence of added calcium. The results presented here would extend this static model of fibrinogen into the concept of a flexible molecule the conformation of which is influenced by calcium concentration. Specifically we would suggest that an increase in calcium concentration causes an increase in the surface area of the molecule which is mainly due to the C-terminal regions of the [A]a chains moving from an inward orientation to a more solvent exposed position. This conformational change leads to a molecule which is more prone to form intermolecular interactions due to the consequent exposure of important amino acid sequences. Acknowledgement We ate grateful to Paul Talbot for excellent technical support. References 1 Hall, C.E. and Slayter, H.S. (1959) Biophys. Biochem. Cytol. 5, 11-15. 2 Hudry-Clergeon, G., Marguerie, G., PouR, L. and Suscillon, M. (1975) Thromb. Res. 6, 533-541. 3 Marguerie G. and Stuhrmann, H.B. (1976) J. Mol. Biol. 102, 143-156. 4 Lederer, K. (1979) Thromb. Haemost. 41,641-647. 5 Mosesson, M.W., Hainfield, J., Wall, J. and Haschmeyer, R.H. (1981) J. Mol. Biol. 153, 695-718. 6 Mihalyi E. and Donovan, J.W. (1985) Biochem. 24, 3443-3448. 7 Larrson, U., Blombac!-, B. and Rigler, R. (1987) Biochim. Biophys. Acta 915, 172-179. 8 Mueller, M. and Burchard, W. (1978) Biochim. Biophys. Acta 537, 208-225. 9 Marguerie, G., Chagniei, G. and Suscillon, M. (1977) Biochim. Biophys. Acta 490, 94-103. 10 Haverkate, F. and Timan G. (1977) Thromb. Res. 10, 803-812. 11 Furlan, M., Rupp, C. and Beck, E.A. (1983) Biochim. Biophys. Acta 742, 25-32. 12 Ly, B. and Godal, H.C. (1973) Haemostasis 1, 204-209. 13 Hemmendorf, B., Brandt, J. and Andersson, L.O. (1981) Biochim. Biophys. Acta 667, 15-22. 14 Apap-Bologna A., Raitt, F. Webster, A. and Kemp, G. (1987) in Fibrinogen 2. Biochemistry, Physiology and Clinical Relevance (Lowe, G.D.O. et al., eds.), pp. 31-34, Elsevier Science Publishers, Amsterdam. 15 Apap-Bologna A. and Kemp, G. (1987) in Fibrinogen 2. Biochemistry, Physiology and Cfinical Relevance (I owe, G.D.O. et al., eds.), pp. 35-38, Elsevier Science Publishers, Amsterdam. 16 Lawrie J.S., Ross, J. and Kemp, G.D. (1979) Biochem. Soc. Trans. 7, 693-694. 17 Lawrie, J.S. and Kemp, G.D. (1979) Biochim. Biophys. Acta 577, 415-423. 18 Laemmli, U.K. (1970) Nature, 227, 680-695. 19 Deutsch, D.G. and Mertz, E.T. (1970) Science, 1095-1096. 20 Johnson, A.J., Kline, D.L. and Alkjaersig, N. (1969) Thromb. Diathes. Haemorrh. 21,259-272. 21 Marguerie, G. and Ardaillou, N. (1982) Bioctfim. Biophys. Acta 701,410-412.
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