THROMBOSIS RESEARCH 14; 651-663 QPergamon Press Ltd.1979. Printed in Great Britain OO49-3848/79/O415-06j1 $OZ.OO/O

SEPARATION OF HUMAN DES-AB FIBRIN AND FIBRINOGEN BY SEPHAROSE-PLASMA CHROMATOGRAPHY AT 20°C AND 37'C I. Mahn, W. Krell and G. Miiller-Berghaus Department of Medicine and Central Division of the Department of Nuclear Research Justus-Liebig-Universitat, Giessen, Germany (Received 8.12.1978; in revised form 20.12.1978. Accepted by Editor N. Goosens)

ABSTRACT Purified human 1251-des-AB fibrin was prepared from 1251-fibrinogen, solubilized in buffered 3 M urea and mixed with plasma containing 1311-fibrinogen. These fibrin-fibrinogen mixtures in plasma were gel-filtered through sepharose-CL 6B columns equilibrated with buffered plasma (sepharose-plasma columns) a5 20°C and at 37OC. Using buffered plasma for elution 1 IjI-fibrinwas separated from 13'I-fibrinogen at 200C as well as 37OC. At 2OoC, a mean of 5.8% 1311-fibrinogen was eluted together with 7251-fibrin, whereas at 37oC, only minute amounts (1.2%) of 1311-fibrinogen were recovered with 1251-fibrin. When in another series of experiments monomeric 1251-des-AB fibrin and 1311-fibrinogen obtained by previous gel chromatography in buffered 3 M urea were mixed with plasma, dialyzed against plasma and chromatographed on sepharose-plasma columns at 200C as well as at 37OC, fibrin could be separated from fibrinogen. At 20°C, some 131I-fibrinogen was eluted together with 1251-fibrin, whereas at2~:OSi~~ib31:Ef~ba~i~~g',~n~~~d'led. covered together with that high-molecular weight fibrin aggregates formed from previously monomeric fibrin. The experiments presented do not indicate the existence of fibrin-fibrinogen complexes in plasma at 37OC. INTRODUCTION In 1937, Apitz (1) described fibrinogen-fibrin intermediates Correspondence to: Dr. G. Miiller-Berghaus, Department of Medicine, Klinikstr. 36, 6300 Giessen, Germany 651

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HUMAN-DES-AB FIBRIN SEPARATION

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generated in vitro which he named profibrin. According to Apitz, these intermediates were soluble in plasma and could be converted to a fibrin gel in the absence of thranbin solely by physico-chemica1 means. In 1954, Thomas et al (2) observed similar fibrin intermediates in the blood of animals treated with endotoxin. According to the procedure used for demonstrating these fibrin intermediates in plasma, the investigators named them cold-precipitable fibrinogen (cryoprofibrin). Shainoff and Page (3,4) showed that cryoprofibrin was composed of fibrin and fibrinogen forming reversible complexes. These complexes can be made up of des-A as well as des-AB fibrin with fibrinogen (4,5). Des-A fibrin originates after removal of the fibrinopeptides A from the fibrinogen molecule whereas des-AB fibrin originates after splitting offfibrinopeptides A and B. Obviously, the fibrinopeptides B are removed by thrombin after a lag phase during which fibrin aggregation has already begun (6). Similar to the cited in vitro and in vivo experiments, soluble fibrin intermediates have been found in the plasma of patients suffering from local or generalized intravascular coagulation (7-16). According to gel filtration studies, it has been suspected that some of the soluble fibrin intermediates are composed of fibrin and fibrinogen (17-25). From these studies, it has been concluded that fibrin-fibrinogen complexes exist in the circulating blood although this concept has never been verified. In a previous study from our laboratory, it has been shown that gel filtration of mixtures of labeled fibrin monomer and fibrinogen on a buffer-equilibrated column is not a suitable method for quantification of fibrin in plasma as fibrin precipitated on the column when separated from its solvent plasma (26). In the present study, we used plasma as an equilibrium and elution medium for gel filtration. We intended to find out whether labeled fibrin can be separated from labeled fibrinogen when plasma is present during gel filtration to keep fibrin in solution.

MATERIALS AND METHODS Reagents. Imidazole, trisodiuxn citrate, urea, EDTA (disodium ethylenediamine tetracetate), tris (tris hydroxymethyl aminome-

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653

thane) and sodium azide were obtained from Merck, Darmstadt, Germany; EACA from Fluka, Buchs, Switzerland; aprotinin (Trasylol) from Bayer, Leverkusen, Germany; bovine thranbin (Thrombinum purum, 750 NIH units/mg protein) and 125I (NaI) from Behringwerke, Marburg, Germany; 131I (NaI) from Amersham Buchler, Braunschweig, Germany; hirudin (1000 AT units/mg protein) from Pentapharm, Basel, Switzerland). Human fibrinogen was prepared from freshly drawn ACD blood by glycine and ethanol precipitation (27). Minor modifications of the method used have been published elsewhere (28). The fibrinogen preparation used (No. 110978)

had a clottability of more than

95%. Agarose gel filtration revealed one single peak only. Disc electrophoresis showed one band only, and after reduction with B-mercaptoethanol, sodium dcdecyl sulphate-polyacrylamide gel electrophoresis revealed three main bands. Labeling of fibrinogen. Human fibrinogen was labeled with 125

I and 13'1 by the iodine monochloride method (29) with minor modifications (28). Labeling did not decrease the clottability, and agarose gel filtration as well as polyacrylamide gel electrophoresis did not show any changes or deteriorations of the labeled fibrinogen in comparison to unlabeled fibrinogen. Preparation of labeled urea-soluble des-AB fibrin. Labeled fibrinogen was clotted with thrombin in the presence of aprotinin and EDTA to prevent proteolysis by plasmin and factor-XIII action, respectively. Thereafter, the clot was isolated, dissolved in hue fered urea and the solubilized fibrin added to plasma containing differently labeled fibrinogen. In detail, the following reaction mixture was prepared: 0.2 ml '251-fibrinogen (approx. 0.4 mg dissolved in 0.055 M sodium citrate, 0.025 M EACA, 0.0025 M EDTA, 100 kiu/ml aprotinin, pH 7.0) ; 0.6 ml buffer (0.005 M imidazole, 0.005 M EDTA, 0.1 M NaCl, 100 kiu/ml aprotinin, pH 6.4); 0.2 ml bovine thrombin (5 NIH units/ml). The reaction mixture was kept for 3 h at room temperature. Thereafter, the formed clot was collected on a glass rod, dried on filter paper and dissolved in 150 pl buffered urea solution (3 M urea, 0.05 M tris, 0.005 M EDTA, 100 kiu/ml aprotinin, pH 7.4) for 1 h at room temperature on a rotating table.

654

RUMAN-DES-AB FIBRIN SEPARATION

Preparation of purified

125

I-des-AB fibrin-

vo1.14,Nos.4/5

131 I-fibrinogen

mixtures in Plasma. Method A: 12'1-fibrin in buffered urea was added to the same buffered and diluted human plasma as used for equilibration of sepharose-plasma columns. In addition, the buffered plasma contained 131I-fibrinogen. In detail, the mixture was made up of: 1 ml diluted and buffered plasma was mixed with 50 pl hirudin (10 AT units/ml) and 150 pl (approx. 0.3 mg; 3-5 pCi) 13'I-fibrinogen. This mixture was prepared in double, one kept at ~O'C, the other at 37'C. At a time, 30 pl 12'1-fibrin (approx. 0.06 mg; 2 pCi) in buffered urea were added to the above mixtures. The ratio of fibrin to fibrinogen was about 1:20. The reaction mixtures were allowed to stand for 30 min at 20°C and 37OC, respectively. Thereafter, 20 pl were removed for obtaining analytical data before gel filtration. The remainder (1.21 ml) was applied to prewarmed sepharose-plasma columns. 125I-des-AB fibrin and 131 Method B: Monomeric I-fibrinogen in buffered urea obtained from sepharose-urea column chromatography (see below) weremixed with buffered plasma and the mixture dialyzed against buffered plasma for removing the urea. In detail, the 2 ml of the elution volume containing the peak of monomeric 131I-fibrinogen and monomeric 125I-des-AB fibrin of the sepharose-urea column chromatography were mixed with 2 ml diluted and buffered plasma. The diluted and buffered plasma is the same as used for the equilibration of the sepharose-plasma columns. The mixture was dialyzed against 300 ml diluted and buffered plasma for 2.5 h at room temperature. Thereafter, the dialyzed reaction mixture was divided into two parts and the portions warmed up to 20°C and 37OC, respectively, for 0.5 h. Subsequently, 10 1.11were removed for obtaining analytical data before gel filtration. Identical amounts (1.79ml) were applied to sepharose-plasma columns prewarmed to 20°C and 37OC, respectively. Agarose-gel

filtration. Gel filtration was performed on 1.6 x 70 cm columns with thermostat jacket and flow adaptor (Pharmacia Fine Chemicals, Uppsala, Sweden). The columns were packed with sepharose-CL 6B and equilibrated either with urea (in the following called sepharose-urea column) or plasma (in the following called sepharose-plasma column). Sepharose-urea columns

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655

were equilibrated with buffered urea (3 M urea, 0.05 M tris, 0.005 M EDTA, 100 kiu/ml aprotinin, pH 7.4). The sepharose-plasma columns were equilibrated with diluted and buffered plasma. One part of pooled human ACD plasma was diluted with 1 part of a buffer containing 0.05 M tris, 0.04 M Na2HP04, 0.01 M KH2P04, 0.15 M NaCl, 0.004 M EDTA, 0.01 M EACA, 100 kiu/ml aprotinin, 10 AT units/ml hirudin, 0.04 mg/ml sodium azide, pH 7.4. Buffered urea was used as an elution medium with the sepharose-urea columns, and buffered and diluted plasma correspondingly with the sepharose-plasma columns. Two sepharose-plasma columns were run in parallel under identical conditions, but one column kept at 20°C, and the other at 37'C. The void volume (Vo) of the columns was determined by the leading peak of dextran blue. The flow rate of the sepharose-plasma as well as urea columns was lo-15 ml/h; the eluates were collected in fractions of 1 ml. Experimental design. Three series of experiments were performed. Experiment A: Mixtures of des-AB fibrin and fibrinogen in plasma were applied to sepharose-plasma columns and eluted with the same diluted and buffered plasma as used for equilibration. Five chranatographic runs were performed in parallel at 20°C and 37Oc. Experiment B: 125I-fibrinogen dissolved and stored in buffered 3 M urea for 1 h was added to buffered plasma containing J31I_ fibrinogen. These mixtures were applied to sepharose-plasma columns and eluted under identical conditions at 20°c and 37Oc like the fibrin-fibrinogen mixtures in Experiment A. Experiment C: 125I-des-AB fibrin solubilized with buffered urea was mixed with 131I-fibrinogen solubilized under identical conditions in buffered urea and applied to sepharose-urea columns. The samples were eluted with buffered urea at 20°c. Experiment D: Two ml of the elution volume containing the peak of monomeric 131I-fibrinogen and monomeric 125I-des-AB fibrin of the sepharose-urea column chromatography (see Fig. 3) were mixed with buffered and diluted plasma, dialyzed against buffered and diluted plasma (see Method B) and gel-filtered on sepharoseplasma columns at 20°C and 37OC.

HUMAN-DES-AB FIBRIN SEPARATION

656

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RESULTS Experiment A: Human

125

I-des-AB fibrin solubilized in urea

and mixed with human plasma remained in solution at 20°C as well 125 as at 37OC. When I-des-AB fibrin solubilized in plasma contai131 ning I-fibrinogen was applied to sepharose-plasma columns, it 131 was separated from I-fibrinogen (Fig. 1). Two distinct peaks were eluted when running sepharose-plasma columns at 20°C as well 12

I

1

A I”‘I-F,brro

42-

-

LLO

50

60 Elutron

70 volume

60

90

lmtl

FIG. 1 The elution of 1251-desAB fibrin and 1311-fibrinogen mixtures in plasma from sepharose-plasma columns at 200C and at 37OC. The fractions were eluted with diluted and buffered plasma. The radioactivity is given in % of the total radioactivity applied to the columns. V. = void volume.

TABLE I 125I-Des-AB Fibrin and 131I-Fibrinogen Recoveries (x 2 s) of Mixed with Plasma, Applied to Sepharose-CL 6B Columns and Eluted with Plasma at 20°C and 37OC *

Recovery (%) 125I-Fibrin 131I-Fibrinogen

Temperature

No. of experiments

2o"c

5

99 + 8

37Oc

5

99 + 6

.II: Per

972

5

102 + 14

cent of the radioactivities applied to the columns.

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657

SEPARATION

10 -

-

“sI-

8-

Fibrinogen

‘3’I-Fibrinogen

6-

goi,,,__+( B 2

,c

reof,2ffIG.2 Gel filt ation of a mixtuI-fibrinogen stcr red in buffered 3 M urea for 1 h and plasma containing 1311-fibrinogen on sepharose-plasma columns at 200C and 37OC. The samples were eluted with buffered plasma. The radioactivity is given in % of the total radioactivity applied to the columns. = void volume.

v,

&6 4 2 0i LO

50

60

Elution

70 volume

80

90

“0

(ml)

as at 37OC. The first peak was eluted with the void volume and contained mainly the 125 I-radioactivity corresponding to fibrin. 131 The second peak contained mainly the I-radioactivity corres125 ponding to fibrinogen. The recovery rates of I-fibrin as well as 13' I-fibrinogen

applied

to the columns

were

about

100%

(Table I). When

comparing

the runs at 20°C and 37OC, a mean of 5.8% of I-fibrinogen was eluted together with 125 I-fibrin in the void volume at 20°C and a mean of 1.2% of 131 I-fibrinogen at 37OC. 131

Experiment

B: This experiment serves as control of Experiment A. Mixtures of 125 I-fibrinogen stored in buffered 3 M urea and 13' I-fibrinogen columns Neither

aggregate

elution

pattern

red in buffered mixing

in plasma were eluted

as single peaks

at 20°C as well

from sepharose-plasma

as at 37OC

(Fig. 2).

formation was observed nor a difference in the 125 131 I-fibrinogen and I-fibrinogen sto-

between

urea and citrate

with plasma.

buffer,

respectively,

before

658

HUMAN-DES-AB FIBRIN SEPARATION

-

“‘I- Fibrinogen

-

lz51Fibrin

A

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FIG. 3 filtration of a mix1251-des-AB fibrin Ei”l sf I-fibrinogen in buffered urea on a sepharose-urea column. The column was equilibrated with buffered 3 M urea, pH 7.4. The radioactivity is given in % of the total radioactivity applied to the column. v, = void volume. The 2 ml of the elution volume containing the peak of monomeric 1311-fibrinogen and monomeric 1*51fibrin (fractions 55 and 56) were rechromatographed on sepharose-plasma columns (see Fig. 4).

Gel

Experiment C: Human 125I-des-AB fibrin solubilized in 3 M urea and chromatographed together with 131I-fibrinogen on sepharose-urea columns was eluted as two peaks (Fig. 3). The main portion was eluted together with the monomeric 131I-fibrinogen. About 20% of the '25I-fibrin applied to the columns were eluted with the void volume together with trace amounts of 131I-fibrinogen. Experiment D: If the elution fractions containing monomeric 125I-des-AB fibrin and 131 I-fibrinogen of Experiment C were mixed with plasma, dialyzed against plasma, and gel-filtered on sepharose-plasma columns, two peaks were observed at 20°C as well as at 37OC (Fig. 4). The first peak contained the total 125I-fibrin. The second peak corresponding to monomeric fibrinogen contained the entire 131I-fibrinogen when performing chromatography at 37'C. At 20°C, however, some of the 131I-fibrinogen was eluted 125 together with I-fibrin with the void volume. DISCUSSION In a previous study we intended to separate on a quantitative basis fibrin and fibrinogen contained in plasma. By using gel filtration with buffer-equilibrated agarose columns only lo-60% of the fibrin applied to the columns could be eluted (26).

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HUMAN-DES-AB

FIBRIN

659

SEPARATION

‘z51- Fibrinogen

8

FIG. 4 Sepharose-plasma gel filtration of mixtures of mo1251-des-AB fibrin ;zeW I-fibrinogen obtained from sepharose-urea column chromatography (see Fig. 3). Monomeric fibrinfibrinogen mixtures in urea were mixed with plasma and consecutively gelfiltered at 200C and 37OC. The radioactivity is given in % of the total radioactivity applied to the columns. V. = void volume.

L 2 0 10

50

60

Elution

Most

likely,

70

volume

90

fibrin precipitated

from its solvent

plasma

fore, in the present elution medium beled des-AB

80 (ml]

on the column when

in the course

fibrin

fibrin was separated

and labeled

whereas

corresponding

some fibrinogen formation

was recovered

of high-molecular complexes.

weight

aggregates,

fibrinogen experiments

fibrinogen

fibrinogen

with

fibrin

comp-

weight

ma-

eluted

At 20°C, indicating

aggregates

the

or of

of high-molecular

seems

aggregates

la-

des-AB

was mainly

fibrinogen

however,

and

at 20°C as well

fibrinogen.

The formation

could

Furthermore,

as high-molecular

together

weight

in plasma

fibrinogen

to monomeric

fibrin-fibrinogen

control

There-

this new technique,

columns.

from the labeled

in the void volume,

with a volume

Using

fibrinogen

from sepharose

as at 37OC. The fibrin was eluted terial

of gel filtration.

study plasma was used as equilibration

for gel filtration.

letely be recovered

separated

to be unlikely

were

neither

as in

observed

when 13' I-fibrinogen was stored inbuffered 3 M urea as it was 125 done with When in parallel runs gel filtraI-des-AB fibrin.

660

HUMAN-DES-AEI FIBRIN

SEPAMTION

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tion on sepharose-plasma columns was performed at 37'C, fibrin was eluted as high-molecular weight material as in the experiments at 20°C. At 37'C about 99% of the 131I-fibrinogen was eluted as a single peak corresponding to monaneric material whereas only a mean of 1.2% of the labeled fibrinogen applied to the columns was recovered with des-AD fibrin. Thus, gel filtration on sepharose-plasma columns at 37OC did not show fibrin-fibrinogen interaction which would indicate the existence of fibrinfibrinogen ccznplexes in plasma at 37'C. When des-AB fibrin in buffered 3 M urea was gel-filtered through sepharose-urea columns, the main portion of fibrin was eluted with a volume corresponding to that of monomeric fibrinogen. Sane of the fibrin was eluted in front of the monomer peak. This indicates that the fibrin partially had formed high-molecular weight aggregates which did not dissociate in buffered 3 M urea within 1 h. In previous studies, we had performed similar experiments with rabbit fibrin also solubilized in 3 M urea. Under the identical conditions as used for human fibrin, rabbit fibrin was eluted as a single monaner peak from sepharose-urea columns (26). Obviously, under identical conditions, human fibrin still forms some high-molecular weight aggregates when rabbit fibrin already dissociates canpletely. In order to study the behaviour of human fibrin monomer in plasma, the peak eluted from the sepharose-urea columns, which contained monomeric fibrin, was reLnromatographed on sepharoseplasma columns. For this purpose, monomeric fibrin was added to plasma and the .urea removed from the mixture by dialysis. In the plasma milieu, fibrin was eluted in the void volume indicating the formation of high-molecular weight fibrin aggregates from previously monomeric fibrin. During the formation of high-molecular weight aggregates, some fibrinogen was included into the aggregates at 20°C, but not at 37OC. Thus, in this last experiment, the generation of fibrin aggregates could be observed in the presence of fibrinogen in the plasma milieu. Under these conditions, fibrin-fibrinogen complex formation, however, did not occur at 37OC. The elution of 125 I-fibrin in the void volume might be explained differently as a conformational change of the fibrin mo-

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lecule imposing an enlargement of the molecule. The experiments reported in this study question the concept of the existence of fibrin-fibrinogen complexes in vivo as fibrin-fibrinogen complexes could not be demonstrated in vitro in a plasma milieu at 37OC. These conclusions are in accordance with studies demonstrating the in vivo dissociation of soluble fibrinfibrinogen complexes prepared in vitro (30). ACKNOWLEDGEMENTS The study was supported by grants (Mu 279) of the Deutsche Forschungsgemeinschaft, Bonn-Bad Godesberg. The authors thank Prof. Dr. E.L. Sattler for his advice and hospitality at the Zentrale Abteilung des Strahlenzentrums, Prof. Dr. S.F. Grebe, Abteilung Nuklearmedizin des Zentrums fiirRadiologie, for supplying radioactive iodine, and Prof. Dr. C. Mueller-Eckhardt, Abteilung fiirKlinische Immunologie und Bluttransfusion, JustusLiebig-Universitat, Giessen, for supplying fresh frozen human plasma. REFERENCES 1.

APITZ, K. Uber Profibrin. I. Die Entstehung und Bedeutung des Profibrins im Gerinnungsverlauf. Zschr. Ges. Exp. Med. 101, 552, 1937.

2. THOMAS, L., SMITH, R.T. and von KORFF, R. Cold-precipitation by heparin of a protein in rabbit and human plasma. Proc. Sot. Exp. Biol. Med. 86, 813, 1954. 3. SHAINOFF, J.R. and PAGE, I.H. Cofibrins and fibrin-intermediates as indicators of thrcznbin activity in vivo. Circul.Res. 8, 1013, 1960. 4. SHAINOFF, J.R. and PAGE, I.H. Significance of cryoprofibrin in fibrinogen-fibrin conversion. J. Exp. Med. 116, 687, 1962. 5. COPLEY, A.L. and LUCHINI, B.W. The binding of human fibrinogen to native and fraction fibrins and the inhibition of polymerization of a new human fibrin monomer by fibrinogen. Life Sciences 3, 1293, 1964. 6. BLOMBXCK, B.,HESSEL, B., HCGG, D. and THERKILDSEN, L. A twostep fibrinogen-fibrin transition in blood coagulation. Nature 275, 501, 1978. 7. KORST, D.R. and KRATOCHVIL, C.H. "Cryofibrinogen" in a case of

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lung neoplasma associated with thrombophlebitis migrans. Blood 10, 945, 1955. 8.

KALBFLEISCH, J.M. and BIRD, R.M. Cryofibrinogenemia. New Engl. J. Med. 263, 881, 1960.

9.

GODAL, H.C. and ABILDGAARD, U. Gelation of soluble fibrin in plasma by ethanol. Stand. J. Haematol. 3, 342, 1966.

10.

LIPINSKI, B. and WOROWSKI, K. Detection of soluble monomer complexes in blood by means of protamine sulphate test. Thrombos. Diathes. Haemorrh. 20, 44, 1968.

11.

FLETCHER, A.P., ALKJAERSIG, N., O*BRIEN, J. and TULEVSKI, V. G. Blood hypercoagulability and thrombosis. Transact. Assoc. Amer. Physicians 83, 159, 1970.

12.

VERMYLEN, J.,DONATI, M.B. and VERSTRAETE, M. The identification of fibrinogen derivatives in plasma and serum by agarose gel filtration. Stand. J. Haematol. Suppl. 13, 219, 1971.

13.

GRAEFF, H. and von HUGO, R. Identification of fibrinogen derivatives in plasma samples. Thromb. Diath. Haemorrh. 27, 610, 1972.

14.

HEENE, D.L. and MATTHIAS, F.R. Adsorption of fibrinogen derivatives on insolubilized fibrinogen and fibrinmonomer. Thromb. Res. 2, 137, 1973.

15.

LARGO, R.,HELLER, V. and STRAUB, P.W. Detection of soluble intermediates of the fibrinogen-fibrin conversion using erythrocytes coated with fibrin monomers. Blood 47, 991, 1976.

16.

MATTHIAS, F.R., REINICKE, R. and HEENE, D.L. Affinity chromatography and quantitation of soluble fibrin from plasma. Thromb. Res. 10, 365, 1977.

17.

SASAKI, T. PAGE, 1-H. and SHAINOFF, J.R. Stable fibrinogen and fibrin. Science 152, 1069, 1966.

18.

FLETCHER, A.P. and ALKJAERSIG, N. Laboratory diagnosis of intravascular coagulation. In: Poller, L.: Recent Advances in Thrombosis. Churchill-Livingstone,London, 1973, pp. 87111.

19.

JAKOBSEN, E., LY, B. and KIERULF, P. Incorporation of fibrinogen into soluble fibrin complexes. Thromb. Res. 4, 499, 1974.

20.

BLfiTTLER,W., STRAUB, P.W.,and PEYER, A. Effect of in vivo produced fibrinogen-fibrin intermediates on viscosity of human blood. Thranb. Res. 4, 787, 1974.

21.

BANG, N.U., CHANG, M.L. Soluble fibrin complexes. Sem. Thrombos. Hemostas. 1, 91, 1974.

CORIpleX

of

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22. von HUGO, R., HAFTER, R., STEMBERGER, A. and GRAEFF, H. Soluble fibrin monomer complexes demonstrated by agarose gel filtration and by adsorption on insolubilized fibrinogen. Thromb. Diath. Haemorrh. 34, 216, 1975. 23. SHERMAN, L.A., HARWIG, S. and LEE, J. In vitro formation and in vivo clearance of fibrinogen: fibrin complexes. J. Lab. Clin. Med. 86, 100, 1975. 24. DONATI, M.B., VERHAEGHE, R., CULASSO, D.E. and VERMYLEN, J. Molecular size distribution of fibrinogen derivatives formed in vitro and in vivo: a chromatographic study. Thrombos. Haemostas. 36, 14, 1976. 25. EDGAR, W.,MCKILLOP, C., HOWIE, ?.W. and PRENTICE, C.R.M. Composition of soluble fibrin complexes in pre-eclampsia. Thromb. Res. 10, 567, 1977. 26. KRELL, W., MAHN, I. and MULLER-BEPGHAUS, G. Gel filtration of 1251-fibrin and 13'1-fibrinogen at 20°C and 37OC. Thromb. Res. in press. 27. BLCMBACK, B. and BLCXBACK, M. Purification of human and bovine fibrinogen. Ark. Kemi 10, 415, 1956. 28. M N, I. and MULLER-BERGHAUS, G. Studies on catabolism of 1% I-labelled fibrinogen in normal rabbits and in rabbits with indwelling intravenous catheters: Methodologic aspects. Haemostasis 4, 40, 1975. 29. MCFARLANE, A.S. Efficient trace-labelling of proteins with iodine. Nature 182, 53, 1958. 30. MAHN, I., SCHUNBACH, F. and MULLER-BERGHAUS, G. Formation and dissociation of soluble fibrin in vivo. Thromb. Res.11, 67, 1977.