Macromolecular associations in dilute solutions of activated fibrinogen

THROMBOSIS RESEARCH Printed in the United

Vol. States

3,

pp.

675-689,

Pergamon

Press,

1973 Inc.

MACRCMOLEC~JIJWASSOCIATIONS IN DIIUTE SOLUTIONS OFACTIVATED FIBRINOGEN*

C. Marguerie, L. Pouit and M. Suscillon Laboratoire d'H&natologie, D.R.F. Centre d'Etudes Nucleaires de Grenoble B.P. 85 Centre de Trl, 38041 Grenoble cedex, France (Received 1.8.1973; in revised form 24.10.1973. Accepted by Editor B. Blombgck)

ABSTRACT The association of the activated form of the fibrinogen molecule has been examined in dilute solutions by means of viscosimetry and light scattering. The results are presented and discussed in terms of different models of associations. Data obtained from electron microscopic observations are compared with hydrodynamic studies In order to describe the macromolecular shape of the Intermediate polymers.

INTRODUCTION Fibrinogen Is a plasma protein which has the special interest to polymerize after its activation by the enzyme thrombin. Chemical analysis of this protein have indicated that It is a dimer which consists of two halves, containing three chains, Aa, E!@and y (for a review see 1, 2). Flbrinopeptides A and B are cleaved off from the Aa and B@ chains, respectively by the thrombin. The resulting product is the activated fibrinogen (3,

4) which Is able to

polymerize and form the fibrin flbre. A considerable amount of work has been done on fibrinown and fibri-

M The activated fibrinogen Indicates the fibrin monomer or fibrin unit (Report of Subcommittee on Nomenclature Transactions, II Congress of International Committee on Haemostasis and Thrombosis, Oslo 19713. 675

676

FIBRIN FORMATION

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noformationin order to correlatethe shape of the fibrinogenmoleculewith the morphologicalaspect of the fibrin fibre.Studyingthe fibrinogenmolecule with electronmicroscopy,Hall and Slayter (5) have suggestedthat the molecule consistsof a lineararray of three nodulesheld togetherby a very thin thread having a rod-likeaspect with a length of 475 A. This aspect has been questionedby Kappel'selectronmicroscopicexaminations(6, 7) of negatively stainedpreparationsof fibrinogen.These observationshave revealeda pentagon dodecahedricalshape for the fibrinogenmoleculewhich could be a spherical cage like particlewith a diameterof 230 8. It is of interestthat this controversyhas motivatednew physicochemical studies. Recently In a series of papers (8,

9,

10) Ledererhas demonstrated

that X-ray scatteringdata and hydrodynamicanalysisare more compatiblewith Kidppel's model. In the same way previouselectronmicroscopicstudies (11) and physicochemlcalinvestigations(12) led us to agree with this model. How can we now imaginethe transitionbetweenthe fibrinogenand the fibrin fibre?Gppel has suggestedthat the fibre is built by an association of dodecahedrical molecules.But on the basis of electronmicroscopicobservations, one of us (11) has shown that this is not really true. In fact, the examinationof differentstages of the flbre formation(11) may suggestthat the fibrinogenmoleculeundergoesa drasticstructuraltransformationduring the polymerization. In this paper we are concernedwith the foremoststage of the polymerization.We have studiedthe beginningof the polymerizationof activated fibrinogenby viscosimetryand li&t scatteringmethodsand of fibrinogenproteolysedin the presenceof small amounts of thrombinby electronmicroscopy. In this case we never obtaina fibrin fibre but only aggregatedactivated fibrinogenmoleculescommonlycalled intermediatepolymers.Studying these polymerswe expectedto find the molecularshape and size of the fibrl-

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nogen molecule before the structural change that may occur during the formation of the fibre. Viscosimetry, light scattering and electron microscopic results will then be discussed in terms of the shape and size of the intermediate polymers.

MATERIALS AND METHODS Bovine fibrinogen was purified from plasma according to the method of Keckwick (13). The purified protein was dialysed exhaustively against 0.2 M KC1 pH 6.4 at low temperature. Clarification and determination of the concentration of the solution were performed as previously described (14). Bovine activated fibrinogen was prepared from purified fibrinogen according to the method of Belitzer et al. (15). 'Iheactivated fibrinogen was dissolved in 0.05 M acetic acid, pH 3.0. The solutions were clarified by 30 minutes ultracentrifugation in a spinco model L preparative ultra centrifuge. me

concentration was determined by measuring the absorbance at 280 nm using

0.1% a specific extinction coefficient E 1 cm of 1.59 (14). The viscosity was measured with three kinds of Ubbelohde viscometers having different capillary tubes with a flow-time for water of 80, 300, 600 seconds. No differences in the results with these three viscosimeters were observed. Light scattering experiments with unpolarized li&t were performed at 25'C at angles of observation 8 ranging from 37.5' to 150' at a wavelenght > = 546 MI with a light scattering photometer (Wippler and Scheibling) manufactured by Sofica, Paris. The results were expressed by the following relation. 1 P-P 0~ (I-IO) Mw HC

-l (e) + 2Bc

In which M, is the average weight molecular wei&t,

P"

(0) is the scattering

FIBRIN FORMATION

678

v01.3,~0.6 .

factor,B is the second virial coefficient,C is the concentrationin g/ml, I and IO are the scatteredIntensityof the solutionand the constantCzis a functionof the angle 9, CY= sin e/(1 + Cos2B),Iiis a constantdependingon the apparatusand the refractionindex incrementdn/dc 2 H=

(indexb is relativeto the standardbenzeneand others parametershave the commonspecification,see reference16). The activatedfibrinogenconcentrationfor light scatteringmeasurements ranged from 0.1 to 0.5 g/l and from 1 to 5 g/l for viscosimetry. Electronmicroscopicobservationshave been made with a EM 3OC, 80 kv, Philipsmicroscope.Ionized carbon formvargrids and potassiumphosphotungstate (KFTA)as stainingmaterialwere used for these observations,as described previously(11).

RESULTS Associationat acid pH. Below pH 5.0

in 0.03 M acetatebuffersthe

activatedfibrinoepen solutionswere found to be stableand clear.Above this pH a rapid polymerizationoccurs leadingto the formationof a clot-likegel. For this reason light scatteringmeasurementshave been made at a concentration ranged from 0.1 to 0.5 g/l in the pH range between3.0 and 5.0.

In table

I we have reportedthe resultsobtainedat variouspH, Figure1 shows the correspondingcurves.At pH 4.8 the second virial coefficient,which can be evaluatedby the slope of the curve, is negativeindicatingstrong solutesolute interactions. At pH 3.0 the same radius of gyration,the same molecularweight and the same intrinsicviscosity(Fig. 2) as for the fibrinogenmolecule

(17)

were

FIBRIN

VO1.3,NO.b

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FORMATION

TABLE I Molecular weight (Mw), radius of gyration (RS) and weight average degree of polymerization (DPw) of activated fibrinogen in acetate buffer (0.05 M).

I

RG

PH

(A)

DP, x

3.00

370,~

144

1.10

4.00

480,000

207

1.45

4.50

680,000

248

4.80

1.100,ooo

510

,

2.05 3.30

* Calculated for MO = 330,000

obtained for the activated form. These results agree with the conventional idea that the fibrinogen and its activated form have the same molecular shape as far as it can be seen with these methods. In Table I it can be seen that at pH 4.5 the value of 2.05 is obtained for the weight average degree of polymerization, suggesting that an association of two activated molecules exists at this pH. This Is In good agreement with the earlier electrical blrefringence measurements of Haschmeyer (18) at this pH. Under these conditions, the Intrinsic viscosity was found to be 36.5 ml/g (Fig. 2). According to Hall and Slayter on one hand and to Kiippelon the other we have studied different models in an attempt to describe the association of two molecules. The theoretical intrinsic viscosity has been calculated for these models. The arrangement according to Ferry (lg), assuming a staggered overlapping has also been taken into consideration. The results are shown In Table II. A rod like model has been chosen for the dimer particle since it is the best average assumption in these calculations. The following relation has been taken (7 ) = z

A

(p) where N, V

680

FIBRIN

FORMATION

and M have the usual si6nifications andA

VO1.3,NO.6

is a function of the axial ratio

p, which was taken for a rod as:

l loS*p -0.8

c

o6

--1.1 c

4((1-1, -1.5

1

18=o

PH

3

/ PH4 -0

FIG. 1.

PH 4.5

- 1.0

.4

0.5

0.6

Light scattering experiment on activated fibrinogen solution: Variation of c/a (I-I ) extrapolated at %O, agains the concentration in 0.05 M acetate buffer at various PH.

0.7

FIG. 2.

PH

-30

*

3

Specific viscosity of activated fibrinogen in 0.05 M acetate buffer at pH 3.0 and 4.5 plotted against the concentration.

FIBRIN

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FORMATION

TABLE II Theoretical intrinsic viscosity calculated for various models of association of two activated fibrinogen molecules. (Experimental values in acetate buffer pH 4.5 : DPW= 2.05, (1) c 3g ml/d

Length Models

r

I

1 1

I

I

1

r-

-_ -

a) - according

a

(? 1 ml/f3

60

49.3

475 a

120

15.2

712 '

120

33.2

480 b

240

37.2

950

-1

CD

I-

1

A

I

1

Diameter

_I

to the model of Hall and Slayter

b) - according to the model of Kiippel c) - according to the Ferry arrangement

From Table II it can be seen that two models gave a theoretical intrinsic viscosity close to the experimental value. The Ferry arrangement gave a value of 8 $ lower than the experimental value while the Kiippel'smodel gave a value only 2 % higher than the obtained value. Association at basic PH. Viscosimetry and light scattering measurements have been made also at basic PH. The solutions were obtained by diluting 1 ml of a concentrated solution of activated fibrinogen (between 1 and 1.5 $ In 0.05

M acetic acid) with different amounts of 0.05 M Glycine - NaOH buffer,

PH 9.5, ~J-I 0.5 M NaCl. In Figure 3 a typical Zinnn-plotobtained in Glycine NaOH buffer, 0.5 M NaCl at pH 9.5 is shcwn. From this diagram a weight average molecular weight of 6.10~ corresponding to a weight average

degree of polymer-

682

-.lO ati-

FIBRIN

FORMATION

6

C

j’

10)

n

FIG. 3 Typical Zimm-plot obtained for activated fibrinogen solutions in 0.05 M Glycine NaOH buffer, 0.5 M NaCl, pH 9.5.

‘)sp c

sin*@/2

0.2

0.1

m/

+kC

[I‘I = 280mI / g

Ig

FIG. 4 Specific viscosity of activated fibrinogen solutions in Glycine NaOH buffer, 0.5 M NaCl pH 9.5, plotted against the concentration.

400 __/H

200 C 0.l

0.2

(L3

0.4

6.6

6.6

mg

/

ml

ization of 18 was found. Under the same conditions the intrisic viscosity of this particle was found to be 280 ml/g (Fig. 4). Cn the basis of the results obtained at acid pH, two models were considered In order to describe these hia

molecular weight aggregates in terms of

molecular shape. With the Ferry arrangement (model A), an elongated particle is obtained, which can be assimilated to a rod with a length L I (DPW + 1) Lo/2,

FIBRIN

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683

PIG. 5 Reciprocal of the theoretical particle scattering factor P(0) as a function of sin%/2 calculated for various D&,J.Model A (-), Model B (---) and experimental values (0).

2.5

DPM being the weight average degree of polymerization and Lo being the length of the fibrinogen molecule according to Hall and Slayter. The Kiippelarrangement (model B) gives a pearl necklace shape, formed by the association of sphe. rical molecules. The best approximation for this particle Is to assume that It is a rod like particle with a length L=DPN.d, d being the diameter of the fibrinogen molecule, according to Kdppel. With such an assumption the theoretical particle scattering factor P(0) was calculated for different weight average degree of polymerization. L being large, the following relation has been taken (16):

P(0) = z

-7

1

withht?

4l-r

sin@/2

The results are shown in Figure 5. Prom this figure it can be seen that the experimental values obtained at pH 9.5, fit with a theoretical particle scattering factor corresponding to a weight average degree of polymerization DPw of

684

FIBRIN FORMATION

v01.3,~0.6

13 for the model A (Ferryarrangement).The intrinsicviscosityCalCUlatedfor such a particlewas found to be 222 ml/g, a value which Is of 21 $ lower than the experimentalvalue. If the Kijppelarrangement(modelB) is taken into consideration a theoreticalDPw of 14.5 was found to be in agreementwith the experimentalvalues. The intrinsicviscosity , calculatedfor such theoreticalparticlewas 2'70ml/g, value which Is of 3.5 $ lower than the obtainedvalue (Fig.4). When taking the calculatedDP, the weight averagemolecularwelefitof 4.4.106 for the Ferry arrangementand 4.9.106 for the Kdppel arrangementwere obtained.These differenceswith the experimentalvalue of 6.106 can be explained by a side by side aggregationwhich modifiesthe molecularwei&t but influencesin a slight extent the intrinsicviscosity.This could also explain the better agreementwe found betweenthe theoreticaland experimentalvalues In the viscosimetrymeasurements. Electronmicroscopicobservations.Intermediatepolymershave been observedwith a ne&lve

stainingmethod at pH 7.0 using a 2 $ potassiumphospha-

te phosphotungstate (KFTA)solutionas stainingagent. The polymerswere prepared as follows:10 )ilof a thrombinsolution(20 NIH units/ml)was added to 1.5 ml of a fibrinogensolution (2 mg/ml)

in

0.15 M KCl. The fibrin formation

was stoppedby dilutionand by the inhibitionpropertiesof the KPTA as described previously(11).The results of these observationsare shown in Figure 6 where intermediatepolymersbuilt by the associationof sphericalparticles can be observed. These observationsare suprisinglyin contradictionwith the earlier electronmicroscopicobservationsof Krakow et al. (24). In fact it appears that differentresults can be obtainedwith electronmicroscopydependingon the procedureused. Further studiesare requiredin order to check the reliability of the resultsobtainedwith the stainingmethod. However,it Is of interest to note that the particlesobservedwith our method do not disprovethe

FIBRIN

FORMATION

685

FIG. 6 Intermediate polymers (X 126 000). The polymerization of fibrinogen (2 m&/ml) was stopped 30 set after the addition of diluted thrombin (see ref. 11). Average diameter 230 A.

assumptions made above and are in favour of the Koppel's model.

DISCUSSION Over the past few years the controversy about the molecular shape of the fibrinogen molecule has motivated many exciting works. However, one must admit that an unambiguous solution of the problem has not been given to date. Nevertheless, the careful physicochemical studies of Lederer (8, 9, 10) has shown that the model of Kdppel cannot be disproved by the physicochemical data, The purpose of this work was to obtain data concerning the shape of the fibrinogen molecule by studying high molecular aggregate forms of this protein. We have examined the aggregation of the thrombin activated fibrinogen in order to describe the shape of these intermediate polymers through the different models proposed for the fibrinogen molecule. Most of the observations on the partially polymerized fibrinogen coupled with the hydrodynamic analysis of the association of Its activated form have allowed for many authors (29, 21, 22, 23, 24, 25) to predict that the intermediate polymers are consistent with a very large particle built by stag-

686

FIBRIN

FORM&CION

VO1.3,NO.6

Current shematic representation of the Intermediate polymer.

Suggested model of aggregation of activated fibrinogen in high molecular weight lntermediate polymer.

FIG. 7

gered overlapping of thin rod units (Fig. 7). This picture appeared to agree with the well known model of Hall and Slayter. The results presented here show that viscosity and light scattering measurements can be interpreted in terms of another model of associations. Taking the model of Kijppelwe found that a theoretical particle built by the association of spherical like molecules (Fig. 7) fits more closely with the experimental values than the Ferry arrangement. Such a picture has been warranted by our electronmicroscopic observations of partially polymerized fibrinogen, using potassium phosphotungstate staining method. To our knowledge, of all the physlcochemical data, only the electric blrefringence measurements could not be explained by this model. Based on these results, Haschmeyer (18) postulated that the flbrinopeptldes A were located opposite to each other, at a distance of 23

A from the centre, on the same side of the molecule. However, it is

known from the chemical studies (2, 3) that the flbrinopeptldes A are cated

both

lo-

in the NH2-terminal part very close together. Similarly, there is evi-

dence that some of the binding sites of the activated form of the fibrinogen molecule are also located In areas different from the NH2-terminal part of the

Vo1.3,No.6

FIBRIN FORMATION

687

molecule (26, 27). On the basis of these remarks and from the previous electron microscopic observations of the different stages of the fibrin flbre formation (ll), the following suggestions can be made: - The fibrinogen molecule could be a spherical-like particle (Kappel's model). The physlcochemlcal data do not contradict such a model for this protein. - The fibrinogen molecule undergoes a slight transformation of structure as a consequence of the thrombin Induced release of fibrinopeptides A and B. This could explain the fact that the different physicochemical methods are not able to distinguish among the different shapes of the fibrinogen molecule and its activated forms. However, such a structural transformation has been indicated earlier by Haschmeyer's electrical birefringence measurements. - During the polymerization process the fibrin unit undergoes a drastic structural transition, growing into an elongated particle (11). This was shown by one of us by electron microscopic observations. This fact could explain that one cannot include the shape of the fibrinogen molecule into the fibrin fibre structure suggested by the electron microscopy findings (11, 26). We think that the development of electron microscopy as well as the use of other additional physicochemical methods may give further information with regard to the structural behaviour of fibrinogen.

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1.

BLCMBACK, B. Fibrinogen to fibrin transformation: Blood clotting enzymology. W.H. SEEGEHS (Ed). Academic Press, New York, 1967, p. 143.

2.

BLOMBACK,B. andBLOMBACK, M. The molecular structure of fibrinogen. Lecture at the conference on "the biological role of the clot stabillzing enzymes" (Transglutamlnaseand Factor XIII). 18, 19 Nov. 1971, New York, The New York Academy of Science, p.77.

3.

BLCMBACK, B., BLCMBACK, M., HESSEL, B. and IWANACA, S. Structure of N-terminal fragments of fibrinogen and specificity of thrombin. Nature, a, 1445, 1967.

688

FIBRIN

FORMATION

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IWANAGA, S., WALIEN, P., GRGNDAHL, N.J., HENSCHEN, A. andBL@&X, B. Cm the primary structure of human fibrinogen. Isolation and characterization of N-terminal fragments from plasmic digests. European J. Biochem., S, 189, 1969.

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HALL, C.E. and SIAYTER, H.S. The fibrinogen molecule: its size, shape, and mode of polymerization. J. Biophys. Blochem. Cytol., 2, 11, 1959.

6.

KijPPEL,G. Electron microscopic investigation of the shape of fibrinogen nodules: a model for certain protein. Nature, 212, 1608, 1966.

7.

KijPPEL,G. Elektronenmikroskopischeuntersuchungen zur gestalt und zum makromolekularen ban des fibrinogen moleklilsund der fibrinfasern. Z. Zellforchung, 77, 443, 1967.

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IEDERER, K. and FINKEI.STEIN, A. Hydrodynamic study of fibrinogen molecular models to test their compatibility with data from the ultracentrifuge and viscosity measurements. Biopolymer, 2, 1553, 1970.

9.

Small angle X-ray scattering measurements with dilute soLEDERER,K. lutions of bovine fibrinogen. J. Mol. Biol., 3, 315, 1972.

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LEDERER, K. and SCHURZ, J. High shear rate viscosity measurements with dilute solutions of bovine fibrinogen. Biopolymer, 11, 1989, 1972.

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PCUIT, L., MARCILLE, G., SUSCILLON, M. and HOLLARD, D. Etude en microscopie glectronique de diffbrentes &tapes de la flbrlnoformation. Thrombos. Diathes. haemorrh., 3, 559, 1972.

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MARGUERIE, G. Etude physicochimique du fibrino&ne et de la flbrinoformation. Th&se Universitg Scientifique et Mgdicale de Grenoble, France, 1972.

13.

KEKWICK, R.A., MacKAY, M.E., NANCE, M.H. and RECORD, B.R. The preparation of protein fractions from human plasma with ether. Biochem. J. 60, 671, 1955.

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BION, N., MIIR~ERIE, G., HUDRY, G. and CHAGNIEL, G. Le coefficient d'extinction du fibrinog&ne de veau d 280 mu. C.R. Acad. Sci., Paris, 112, 901, 1971. (

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BELITSER, V.A., ViRETSKAJA, T.V. and MALNEVA, G.V. Fibrinogen-fibrin interaction. Biochem. Biophys. Acta, 154, 365, 1968.

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GEIDUSCHEK, P. and HOL'IZER,A. Application of light scattering to biological systems. Adv. in biol. and med. Phys. C.A. TOBIAS, J.M. LI\WRENCE(Eds), New York, Academic Press, Inc., 1958, IV, p. 431.

17.

MARGUERIE, G. and SUSCILU)N, M. Effet de la force ionlque sur la forme et les dimensions du fibrinodne. C.R. Acad. SC. Paris, _r16, 101, 1973.

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HASCHMEYER, A.E.V. A polar intermediate in the conversion of fibrinogen to fibrin monomer. Biochemistry, 1, 851, 1963.

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BL,CMBkK, B. and LAURENT, T.C. N-terminal and light scattering studies on fibrinogen and its transformation to fibrin. Arklv Kemi, I& 137, 1958.

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STEINER, R.F. and IAKI, K. Light scattering studies on the clotting of fibrinogen. Arch. Biochem. Biophys., B, 24, 1951.

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FERRY, J.D., KATZ, S. and TINOCO, I. Some aspects of the polymerlzation of fibrinogen. J. Polym. SC., 12, 509, 1954.

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CASASSA,

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CASASSA, E.F. The conversion of fibrinogen to fibrin XVIII. Light scattering studies of the effect of hexamethyleneglycol on thermodynamic interactions in fibrinogen solutions. J. Fhys. Chem., 60, 926, 1956.

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KRAKGW, W., ENDRES, G.F., SIEGEL, B.M. and SCHERAGA, H.A. An electron microscopic investigation of the polymerization of bovine fibrin monomer. J. Mol. Biol., 71, 95, 1972.

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KUDRYK, B. andBIX)MB#CK, B. Adsorption of fibrinogen degradation products to enzyme modified fibrinogen - sepharose. IVth International Congress on Thrombosis and Haemostasis, June 1973, Vienna, Austria.

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KUDRYK, B., REUTERBY, J. and BMMBACK, B. Adsorption of plasmic fragment D to thrombin modified fibrinogen - sepharose. Thrombosis Research, 2, 297, 1973.

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KAY, D. and CUDDIGAN, B.J. Haemat., u, 341, 1967.

E.F. Light scattering from very long particles and an application to polymerized fibrinogen. J. Chem. Phys., 2_& 596, 1955.

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