THROMBOSIS @Pergamon
RESEARCH 14; 561-572 Press Ltd.1979.
FORMATION OF SOLUBLE FIBRIN
Printed
in Great Britain 0049/3848/79/3415 -Oj61 $02.00/O
AND DISSOCIATION
COMPLEXES
IN PLASMA
AT 20'C AND AT 37'C
G. Miiller-Berghaus, I. Mahn and W. Krell Department of Medicine and Central Division of the Department of Nuclear Research Justus-Liebig-Universitat, Giessen, Germany (Received 26.10.1978; in revised form 6.12.1978. Accepted by Editor P.W. Straub)
ABSTRACT
Small amounts of thrombin were added to human plasma containing 12SI-fibrinogen to generate fibrin. Thereafter, thrombin was quenched with hirudin and 1311-fibrinogen added for internal calibration. Gel filtration of this mixture on sepharose CL-6B columns equilibrated with plasma revealed two peaks, one (peak A) eluting with the void volume, the other (peak B) with the volume ~o:~~p~f"t;sIr~i~~~n~~~~inogen peak. At 2OoC, a small 4.5%) was eluted together with the thrombin-tr edl 151-fibrinogen in peak A, whereas at 37OC, no es! I-fibrinogen was recovered in peak A. If isolated fractions of peak A eluted at 20°C were rechromatographed at 2OoC, they were eluted again in the void volume. At 37OC, however, this material was mainly eluted in peak B corresponding to the fibrinogen peak. If isolated fractions of peak B at 37OC were rechromatographed at 37OC, they were eluted with the same volume as in the original chromatography. At 20°C, however, the material was eluted in peak A as well as in peak B. These data show that at 200C fibrin aggregates generated in a plasma medium from monomeric fibrin without incorporating fibrinogen. From these experiments, we conclude that so-called fibrin-fibrinogen complexes not stabilized by factor XIIIa generate only in vitro at 2OoC, but do not exist at 370C.
Correspondence to: Dr. G. Miiller-Berghaus, Department of Medicine, Klinikstr. 36, 6300 Giessen, Germany 561
562
SOLUBLE FIBRIN COMPLEXES
Vo1.14,Nos.4/5
INTRODUCTION Intravascular coagulation is reflected by the presence of so-called soluble fibrin complexes. These have been demonstrated in vitro in the plasma of patients suffering from local or generalized intravascular coagulation or incipient thrombosis (l-11). The complexes are preferably composed of fibrin with fibrinogen and/or fibrinolytic degradation products. Using gel chromatogra-
phy, it has been shown that these complexes elute from an agarose gel column with an elution volume smaller than that of fibrinogen indicating the formation of high-molecular weight aggregates or complexes (3,5,12). The method was applied on the assumption that fibrin complexes exist in the circulating blood, although this concept has never been verified. In previous studies, we observed a dissociation of in vitro prepared fibrin-fibrinogen complexes after injection into animals (13). Similarly, BlZttler et al. (14) concluded from their blood viscosity experiments that fibrinogen-fibrin intermediates are not present in high-molecular form at 37OC. According to in vitro studies of Edgar and Prentice (231, complexes formed by plasmin digestion of fibrin are unstable at 37OC if only the fibrinopeptides A have been removed, b;lt are stable at 37OC if the fibrinopeptides A and B have been split off from the molecule. In the present study, we examined the gel chromatographic behaviour of soluble fibrin complexes in plasma. Using plasma as equilibration and elution medium for gel filtration, we intended to find out whether canplex formation of fibrin and fibrinogen is temperature-dependent. MATERIAL AND METHODS Reagents. The following reagents were used: Urea, EDTA (disodium ethylenediamine tetracetate), sodium azide, and tris (tris hydroxymethyl aminomethane) from Merck, Darmstadt, Germany; EACA from Fluka, Buchs, Switzerland; aprotinin (Trasylol) from Bayer, Leverkusen, Germany; bovine thrombin (Thrombinum purum, 750 NIH u/mg protein) and 125 I (NaI) from Behringwerke, Marburg, Germany; hirudin (1000 AT units/mg protein) from Pentapharm, Basel, Switzerland.
Vo1.14,NOS.4/j
Human
SOLUBLE
fibrinogen
FIBRIN
was prepared
from freshly
by glycine
and ethanol
the method
used have been published
gen preparation
used
than 95%. Agarose
(No. 110978)
Disc electrophoresis duction
with
acrylamide
gel electrophoresis of fibrinogen.
elsewhere
revealed
and agarose
dodecyl
gel filtration
fibrinogen
as well
fibrinogen
in comparison
Preparation in plasma.
of mixtures
1251-fibrinogen
was labeled with
method
(20) with minor
the clottability,
as SDS-PAA
gel electrophoreof the labeled
fibrinogen.
of labeled
was added
and after re-
sulphate-poly-
or deteriorations
to unlabeled
peak only.
three main bands.
did not decrease
sis did not show any changes
of more
one single
(19) revealed
of
(16). The fibrino-
one band only,
sodium
Human
(16). Labeling
modifications
had a clottability
I and 13' I by the iodine monochloride
modifications
drawn ACID blood
(15). Minor
(17,181 showed
S-mercaptoethanol,
Labeling 125
precipitation
gel filtration
563
COMPLEXES
fibrin
and fibrinogen
to the same plasma
as used
for equilibration of the agarose column (see below). The plasma 125 containing I-fibrinogen was treated by small amounts of thrombin
not causing
visible
time of 20 min the thrombin fibrinogen
consisted
human platelet-poor ml) diluted
100
ACD plasma
1 ml buffer
pH 7.4);
(0.05 M tris,
aprotinin,
citrate,
was added slowly
room temperature.
After
fibrinogen after,
(0.08 mg) were
the mixture
time, the mixtures
were
(approx. 0.48 mg dis-
over a period
added to the reaction
of 5 min
at 37OC for 30 min, centrifuged
at
Thereone kept
respectively.
At this
at 10 000 9 for 2 min and
(1.2 ml each) was applied
chromatographic
mixture.
into two equal portions,
filtration.
Seven
(2 units/
time of 20 min, 0.1 ml 5 min, 0.1 ml 131I_
for obtaining
columns.
0.0025 M EDTA,
thrombin
0.25 ml were removed The rest
0.01
100 kiu/ml
and after another
was divided
at 20°C, and the other
2 mg/
0.04 M Na2HP04,
0.025 M EACA,
an incubation
(250 AT units/ml)
the reac-
1 ml pooled
concentration:
pH 7.0); 0.2 ml bovine
ml). The thrombin
hirudin
In detail,
0.01 M EACA,
0.6 ml 1251-fibrinogen
in 0.055 M sodium
kiu/ml
an incubation hirudin and 131I_
substances:
(fibrinogen
0.004 M EDTA,
After
with
calibration.
of the following
0.15 M NaCl,
aprotinin, solved
with
strands.
was quenched
added for internal
tion mixture
M KH2P04,
fibrin
analytical
data before
gel
to prewarmed
runs were performed
in parallel
564
SOLUBLE FIBRIN COMPLEXES
Vo1.14,Nos.4/5
125 I-fibrinogen and 13'I-fibrinogen in plasma not treated with thrombin were at 20°C and at 37OC. In the control experiments,
applied to sepharose columns to determine the elution volume of untreated fibrinogen under identical conditions. Ethanol gelation test was performed according to Godal and Abildgaard (4). 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), packed with sepharose CL6B (Pharmacia). The columns were equilibrated with pooled human ACD plasma diluted with a buffer. 1 1 pooled plasma was diluted with 1 1 buffer containing 0.05 M tris, 0.04 M Na2HP04, 0.01 M XH2PG4, 0.15 M NaCl, 0.004 M EDTA, 0.01 M EACA, 100 kiu/ml aprotinin, 10 AT units/ml hirudin, 0.4 mg/ml sodium azide, pH 7.4). Two columns were prepared in parallel using the same gel and the same buffered plasma, 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 elution was performed with the same buffered plasma as used for equilibration. The flow rate of the columnrwas lo-15 ml/h; the eluates were collected in fractions of 1 ml. Rechromatography was performed with the same columns using buffered plasma for equilibration and elution. SDS-PAA (sodium dodecyl sulphate-polyacrylamide) gel electro. phoresis was performed according to Swank and Munkres (19) using samples reduced with p-mercaptoethanol in urea. Plasma samples eluted from the chromatography column were clotted in the presence of EDTA and aprotinin, and the isolated clot dissolved in the reducing buffer. If radioactivities were determined in sections of the gels, these were cut into 8 pieces according to the stained bands (Table II), and the pieces monitored for 1 =I_ as well as 131I-activities. RESULTS Plasma containing ,125I-fibrinogen and 131I-fibrinogen not treated with thrombin was applied to sepharose CL-6B columns equilibrated with plasma. Labeled fibrinogen was eluted at a single peak with an elution volume of about 62 ml at 20°C as well
SOLUBLE
VOl.14,NOS.4/j
10
8
FIBRIN
-
"'I-Ftbrmogen
PO
"'I-Ftbrmogen
$5
COMPLEXES
FIG. 1 Gel filtration of 1251_fibrinogen and '31I-fibrinogen in human plasma not treated with thrombin. Sepharose CL-6B columns were equilibrated with buffered plasma and the samples eluted with buffered plasma at 20°C and at 370C. The radioactivity is given in % of the total radioactivity applied to the columns.
6
0
1
LO
50
as at 37OC
60 70 80 90 Elut~on volume (ml)
(Fig. 1).
Plasma
containing
was regularly
found to yield
radioclottabilities between
of labeled
positive
on sepharose
revealed CL-6B
main peak
peaks
equilibrated
the void volume
(peak B) was eluted
volume
before
with
gelation
tests.
fibrin
The
were
and fibrino-
after gel filtration
with
plasma
(Fig. 2).
of 44-48 ml, whereas
a volume
of peak B corresponded
and fibrinogen
gel filtration
of labeled
two different
columns
Peak A was eluted with
fibrin
ethanol
of these mixtures
92 and 94%. These mixtures
gen in plasma
elution
mixtures
the
of 60-64 ml. The
to that of labeled
fibri-
nogen under identical conditions (Fig. 1). At 20°C, a small 131 I-fibrinogen (? = 4.5% + 0.9; n = 7) was eluted toamount of 125I-fibrinogen in peak A, gether with the thrombin-treated 131 I-fibrinogen was recovered in peak A. whereas at 37OC, no 125 I-radioactivity W&S recovered from the All the applied columns
at 20°C as well
manner,
all the applied
columns
at 20°C as well as at 37OC.
To control
as at 37OC (Table I). In the same 131 I-fibrinogen was eluted from the
the influence
of possibly
activated
factor XIII,
566
SOLUBLE FIBRIN COMPLEXES
Vo1.14,Nos.4/5
FIG. 2 Gel filtration of 1.2 ml human plasma each containing thrombin-treated
10
8 6
$2 pA 20 :: 2 z 6 1 2 0 LO
60
60
Elutlon
70
volume
80
90
Imll
plasma were prepared at room temperature, divided into two equal portions and chromatographed at 20°C and at 370C. Sepharose CL6B columns were equilibrated with buffered plasma and the samples also eluted with buffered plasma. The radioactivity is given in % of the total radioactivity applied to the columns. The 44th and 45th ml of the elution volume of the 20°C run and the 63rd and 64th ml of the elution volume of the 370C run were rechromatographed (see Figs. 3 and 4).
VO
8
+
6
$j2 \ .t 0IA,_i = 2 $ 10 $ 4 6
;b
8 I
2ooc
6
4 2 01!:._ LO 50
60
Nut/on
70
80
volume (ml)
90
FIG. 3 Rechromatography of the isolated 44th and 45th ml of the elution volume of the 20°C run (see Fig. 2). The fractions were divided into two equal portions and rechromatographed on columns equilibrated with plasma. The radioactivity is given in % of the total radioactivity applied to the columns.
SOLUBLE
Vo1.14,Nos.4/j
FIBRIN
567
COMPLEXES
VO 1
12
-
‘*sI
-
“‘I
10
8
137”c]
1 i
g;
t
FIG. 4 Rechromatography of the isolated 63rd and 64th ml of the elution volume of the 370C run (see Fig. 2). The fractions were divided into two equal portions and rechromatographed on plasma-equilibrated columns at 20°C and at 37OC. The radioactivity is given in % of the total radioactivity applied to the columns.
A_ i
U 2 5 88 L 2 0 LO
50
60
Elutron
SDS-PAA
90
were performed.
region
at different the eluates
of reduced
gels did neither
nor in the two peaks temperatures
samples
The radioactivities
of the SDS-PAA
two isotopes,
ween
80 (ml1
gel electrophoresis
filtration f-x
70 volume
obtained
from gel
measured
in the
differ
between
of gel chromatography,
the
nor
(Table II). The radioclottabilities
of peaks A and B at 20°C as well
as 37OC varied
of bet-
07 and 94%.
TABLE I and 13' I-Radioactivities after Gel FilRecovery (x + s) of 1 q_ tration of Piasma Containing Thrombin-Treated 1251-Fibrinogen and The Gel Filtration Was Performed on Untreated 1311-Fibrinogen. Sepharose CL-6B Columns at 20°C and at 37OC Temperature
'Per
No. of experiments
Recovery
(%I* 131I
125I
2o"c
7
106.6 + 13.2
107.1 + 11.7
37Oc
7
104.0 + - 13.0
103.7 + 12.0
cent of the radioactivities
applied
to the columns.
566
SOLUBLE FIBRIN COMPLEXES
Vo1.14,Nos.4/5
If the fractions of peak A eluted at 20°C were rechromatographed, different elution patterns were observed depending on the temperature used for rechromatography (Fig. 3). At 20°C, most of the applied material (approx. 90%) was eluted with the void volume, whereas at 37'C the main peak (91% of the radioactivity applied to the column) was found with the elution volume corresponding to that of fibrinogen. 131I-radioactivity is not repre131 sented, as there was not enough I-radioactivity in peak A before rechromatography for reliable measurements. In a second rechromatography experiment, fractions of peak B eluted at 37'C (Fig. 2) were rechromatographed at 20°C and at 37'C (Fig. 4). During rechromatography at 37OC, the peak of the 125 131I-activities appeared with the same elution I-as well as volume as in the preceding experiment where the fractions derived from. At 20°C, however, 13% of the 125I-radioactivity applied to the columns was eluted with the void volume, but no '3'I_ radioactivity was recovered in peak A (Fig. 4). DISCUSSION Soluble fibrin complexes at room temperature may consist of different complexes made up of fibrin and fibrinogen or fibrin and fibrinolytic degradation products. They may be stabilized by factor XIIIa or unstabilized. The present study deals with so-called unstabilized fibrin-fibrinogen complexes only. These were prepared by addition of small amounts of thrombin to plasma containing 125I-fibrinogen. Thus, a portion of the plasma fibrinogen as well as the 125I-fibrinogen was converted to fibrin. The main portion of the plasma fibrinogen and the '25I_ fibrinogen was not affected by thrombin as not more than 20% of the plasma fibrinogen can be transferred to fibrin without gelating (II, 21, 22). To demonstrate fibrin-fibrinogen interaction, fibrinogen labeled with a different isotope was added. The ethanol gelation test was always found positive with these mixtures. If these mixtures were applied to gel filtration columns, two peaks were eluted, the peak eluted with the void volume being larger at 20°C than at 37OC. Some of the '251radioactivity corresponding to thrombin-treated 125I-fibrinogen
131 125;
131 125;
13'1
131 125;
131125;
3
4
5
6
7
8
I
131 125;
2
12SI
131 125;
1
I
Isotope
131 125;
of PAA-gel piece
No.
+ + T -
5.3
1.0 1.1
+
+ 7
T
+
+ T
+ 7
+ T
+ T
5.2
20.4 20.7
47.0
47.1
22.5 22.1
1.8 1.8
0.7 0.7
1.2 1.1
0.3 0.4
0.8
0.8
0.6 0.8
2.0
1.6
2.0 1.6
0.4 0.5
0.1 0.1
0.1 0.1
Before gel filtration (%I
1.2
4.1
20.3
45.2
22.5
3.0
1.1
2.7
2.7
0.3
0.2
1.0
I
+
+
+
\.
I
. .A .&
0.5
0.5
1.7
-I-2.3
+
+
+
+
at 20°C (%I
Peak A
0.8 0.9
3.8
4.0
20.0 20.0
44.7
47.4
23.4 22.7
1.9 2.8
1.0 1.1
1.3 2.4
r
-i
+ T
+
+
+ T
T
+
+ +
+ T
+ T
+ F I
I
___- J
0.3 0.2
0.8
0.5
1.5 1.8
3.3
1.3
1.8 3.1
0.6 1.7
0.2 0.3
0.4 1.5
at 20°C (%I
0.8 0.7
4.0
4.4
19.3 20.0
48.1
48.5
22.6 22.3
2.4 2.6
0.7 0.9
1.5 1.5 0.2 0.2
0.2 0.2
+ T -
T
+
+ + T
+
+ T
0.4 0.2
0.7
0.5
0.8 0.9
4.1
3.0
4.2 4.7
-I-0.5 T - 0.5
+ T -
+ T
at 37OC (%I
Peak B
.
,'
/
/ . /
. I-
_----.
.'
'
/'
_---
. _ -_
.
- --.
-----
__
*\.
--
gel
PAA
TABLE II Distribution of Radioactivities in SDS-PAA Gels of Reduced Samples Obtained from Peak A and Fibrinogen in Peak B of Gel Filtration at 20°C and 37OC. 125I Corresponds to Thrombin-Treated Plasma and 13'1 to Untreated Fibrinogen. The Values of the Samples before Gel Filtration and Were during Gel Filtration Are Given in Means and Standard Deviation /n=5). Radioactivities Calculated in % of the Activity of Each PAA Gel. The Measured I3 I-Activities in Peak A Eluted in Peak A at 370C Were too Low for Reasonat 20°C as well as at 37OC and the 1251-Activities able Evaluation
c: tr _c‘4 0 "1 .I-\
cn
0
570
SOLUBLE FIBRIN COMPLEXES
V01.14,8os.4/j
was eluted with the void volume indicating the formation of high-molecular weight aggregates. At 20°C, these aggregates eluting in the void volume contained some 131I-fibrinogen which had not been treated with thrombin. The elution of thrombintreated 125I-fibrinogen together with untreated 131I-fibrinogen may be caused by adsorption of fibrinogen to the high-molecular weight fibrin aggregates or the formation of so-called fibrinfibrinogen complexes. Most interestingly, these fibrin-fibrinogen interactions could not be observed at 37OC as no 131I-fibrinogen was eluted together with the fibrin aggregates at this temperature. Furthermore, the reduction of the peak eluted with the void volume at 37OC in comparison to 20°C indicates that the so-called fibrin-fibrinogen complexes dissociated at 37'C. To prove the theory that so-called fibrin-fibrinogen complexes generate at 20°C and dissociate at 37OC, rechromatography experiments were performed. Peak A eluted at 20°C was observed in the same position after rechromatography at 20°C, but changed its position after rechromatography at 37OC. Nearly all the radioactivity was found in the peak corresponding to the fibrinogen peak. Thus, so-called fibrin-fibrinogen complexes isolated at 20°C dissociated at 37OC. If peak B eluted at 37OC was rechromatographed at 20°C, the elution pattern was similar to the one seen with gel filtration of the starting material at 20°C. Thus, at 20°C fibrin formed aggregates from mixtures of monomeric fibrin in plasma. No fibrinogen was eluted together with these fibrin aggregates. From these experiments we conclude that so-called unstabilized fibrin-fibrinogen complexes generate only in vitro at an unphysiologic low temperature but do not exist at 37OC. ACKNOWLEDGEMENTS The study was supported by grants (Mu 279) of the Deutsche Forschungsgemeinschaft, Bonn-Bad Codesberg. 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
Vol.14,Nos.
4/j
SOLUBLE
supplying
radioactive
Abteilung
fiir Klinische
FIBRIN
iodine,
Justus-Liebig-Universitat,
371
CO?4PLEXES
and Prof.
Immunologie Giessen,
Dr. C. Mueller-Eckhardt,
und Bluttransfusion, for supplying
fresh
frozen
human plasma.
REFERENCES 1.
BANG, N.U. and CHANG, M.L. Soluble Thrombos. Hemostas. 1, 91-, 1974.
fibrin
complexes.
Sem.
2. EDGAR, W. McKILLOP, C., HOWIE, P.W. and PRENTICE, C.R.M. Composition of soluble fibrin complexes in pre-eclampsia. Thrombos. Res. 10, 567, 1977. 3. FLETCHER, A.P., ALKJAERSIG, N., O'BRIEN, J. and TULEVSKI, V. G. Blood hypercoagulability and thrombosis. Transact. Assoc. Am. Physicians 83, 159, 1970. 4. GODAL, plasma
H.C. and Abildgaard, U. Gelation of soluble fibrin by ethanol. Stand. J. Haematol. 3, 342, 1966.
in
5. GRAEFF, H., von HUGO, R. and HAFTER, R. Evaluation of hypercoagulability and intravascular coagulation by estimation and characterization of soluble fibrin monomer complexes (SFMCS) . Progr. Chem. Fibrinolys. Thrombolys. 3, 435, 1978. 6. KIERULF, screened
P. and GODAL, H.C. Fibrinemia in medical patients br the ethanol test. Acta Med. Stand. 190, 185,
1971.
7. 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. 8. MATTHIAS, F.R., REINICKE, R. and HEENE, D.L. Affinity chromatography and quantitation of soluble fibrin from plasma. Thrombos. Res. 10, 365, 1977. 9. NIEWIAROWSKI, S. and GUREWICH, V. Laboratory identification of intravascular coagulation. The serial dilution protamine sulfate test for the detection of fibrin monomer and fibrin degradation products. J. Lab. Clin. Med. 77, 665, 1971. 10. SHAINOFF, J.R. and PAGE, I.H. Cofibrins and fibrin-intermediates as indicators of thrombin activity in vivo. Circul. Res. 8, 1013, 1960. 11. 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. 12. DONATI, M.B., VERHAEGHE, R., CULASSO, D.E. and VERMYLEN, J. Molecular size distribution of fibrinogen derivatives formed study. Thrombos. in vitro and in vivo: a chromatographic
SOLUBLE
572
FIBRIN
COMPLEXES
Haemostas. 36, 14, 1976. 13. MAHN, I., SCHbNBACH, F., MULLER-BERGHAUS, G. Formation and dissociation of soluble fibrin in vivo. Thrombos. Res.11, 67, 1977. 14. BLATTLER, W., STRAUB, P.W. and PEYER, A. Effect of in vivo produced fibrinogen-fibrin intermediates on viscosity of human blood. Thrombos. Res. 4, 787, 1974. 15. BLCMBACK, B. and BLOMBACK, M. Purification of human and bovine fibrinogen. Ark. Kemi 10, 415, 1956. 16. MAHN, I. and MULLER-BERGHAUS, G. Studies on catabolism of 1251-labelled fibrinogen in normal rabbits and in rabbits with indwelling intravenous catheters: Methodologic aspects. Haemostasis 4, 40, 1975. 17. ORNSTEIN, L. Disc electrophoresis-I. Background and theory. Ann. New York Acad. Sci. 121, 321, 1964. 18. DAVIS, B.J. Disc electrophoresis-II. Method and application to human serum proteins. Ann. New York Acad. Sci. 121, 404, 1964. 19. SWANK, R.T. and MUNKRES, K.D. Molecular weight analysis of oligopeptides by electrophoresis in polyacrylamide gel with sodium dodecyl sulfate. Analyt. Biochem. 39, 462, 1971. 20.
A.S. Efficient trace-labelling of proteins with iodine. Nature 182, 53, 1958.
McFARLANE,
21. SASAKI, T. PAGE, I.H. and SHAINOFF, J.R. Stable complex of fibrinogen and fibrin. Science 152, 1069, 1966. 22. YUDELMAN, I., SPANONDIS, K. and NOSSEL, H.L. Comparative behaviour of 125I-fibrinogen and 1251-fibrin in solution. Thrombos. Res. 5, 495, 1974. 23. EDGAR, W. and PRENTICE, C.R.M. The effect of fibrinopeptide B release on temperature dependent fibrin association. (Abstract). Thrombos. Haemostas. 38, 104, 1977.
RESEARCH 14; 561-572 Press Ltd.1979.
FORMATION OF SOLUBLE FIBRIN
Printed
in Great Britain 0049/3848/79/3415 -Oj61 $02.00/O
AND DISSOCIATION
COMPLEXES
IN PLASMA
AT 20'C AND AT 37'C
G. Miiller-Berghaus, I. Mahn and W. Krell Department of Medicine and Central Division of the Department of Nuclear Research Justus-Liebig-Universitat, Giessen, Germany (Received 26.10.1978; in revised form 6.12.1978. Accepted by Editor P.W. Straub)
ABSTRACT
Small amounts of thrombin were added to human plasma containing 12SI-fibrinogen to generate fibrin. Thereafter, thrombin was quenched with hirudin and 1311-fibrinogen added for internal calibration. Gel filtration of this mixture on sepharose CL-6B columns equilibrated with plasma revealed two peaks, one (peak A) eluting with the void volume, the other (peak B) with the volume ~o:~~p~f"t;sIr~i~~~n~~~~inogen peak. At 2OoC, a small 4.5%) was eluted together with the thrombin-tr edl 151-fibrinogen in peak A, whereas at 37OC, no es! I-fibrinogen was recovered in peak A. If isolated fractions of peak A eluted at 20°C were rechromatographed at 2OoC, they were eluted again in the void volume. At 37OC, however, this material was mainly eluted in peak B corresponding to the fibrinogen peak. If isolated fractions of peak B at 37OC were rechromatographed at 37OC, they were eluted with the same volume as in the original chromatography. At 20°C, however, the material was eluted in peak A as well as in peak B. These data show that at 200C fibrin aggregates generated in a plasma medium from monomeric fibrin without incorporating fibrinogen. From these experiments, we conclude that so-called fibrin-fibrinogen complexes not stabilized by factor XIIIa generate only in vitro at 2OoC, but do not exist at 370C.
Correspondence to: Dr. G. Miiller-Berghaus, Department of Medicine, Klinikstr. 36, 6300 Giessen, Germany 561
562
SOLUBLE FIBRIN COMPLEXES
Vo1.14,Nos.4/5
INTRODUCTION Intravascular coagulation is reflected by the presence of so-called soluble fibrin complexes. These have been demonstrated in vitro in the plasma of patients suffering from local or generalized intravascular coagulation or incipient thrombosis (l-11). The complexes are preferably composed of fibrin with fibrinogen and/or fibrinolytic degradation products. Using gel chromatogra-
phy, it has been shown that these complexes elute from an agarose gel column with an elution volume smaller than that of fibrinogen indicating the formation of high-molecular weight aggregates or complexes (3,5,12). The method was applied on the assumption that fibrin complexes exist in the circulating blood, although this concept has never been verified. In previous studies, we observed a dissociation of in vitro prepared fibrin-fibrinogen complexes after injection into animals (13). Similarly, BlZttler et al. (14) concluded from their blood viscosity experiments that fibrinogen-fibrin intermediates are not present in high-molecular form at 37OC. According to in vitro studies of Edgar and Prentice (231, complexes formed by plasmin digestion of fibrin are unstable at 37OC if only the fibrinopeptides A have been removed, b;lt are stable at 37OC if the fibrinopeptides A and B have been split off from the molecule. In the present study, we examined the gel chromatographic behaviour of soluble fibrin complexes in plasma. Using plasma as equilibration and elution medium for gel filtration, we intended to find out whether canplex formation of fibrin and fibrinogen is temperature-dependent. MATERIAL AND METHODS Reagents. The following reagents were used: Urea, EDTA (disodium ethylenediamine tetracetate), sodium azide, and tris (tris hydroxymethyl aminomethane) from Merck, Darmstadt, Germany; EACA from Fluka, Buchs, Switzerland; aprotinin (Trasylol) from Bayer, Leverkusen, Germany; bovine thrombin (Thrombinum purum, 750 NIH u/mg protein) and 125 I (NaI) from Behringwerke, Marburg, Germany; hirudin (1000 AT units/mg protein) from Pentapharm, Basel, Switzerland.
Vo1.14,NOS.4/j
Human
SOLUBLE
fibrinogen
FIBRIN
was prepared
from freshly
by glycine
and ethanol
the method
used have been published
gen preparation
used
than 95%. Agarose
(No. 110978)
Disc electrophoresis duction
with
acrylamide
gel electrophoresis of fibrinogen.
elsewhere
revealed
and agarose
dodecyl
gel filtration
fibrinogen
as well
fibrinogen
in comparison
Preparation in plasma.
of mixtures
1251-fibrinogen
was labeled with
method
(20) with minor
the clottability,
as SDS-PAA
gel electrophoreof the labeled
fibrinogen.
of labeled
was added
and after re-
sulphate-poly-
or deteriorations
to unlabeled
peak only.
three main bands.
did not decrease
sis did not show any changes
of more
one single
(19) revealed
of
(16). The fibrino-
one band only,
sodium
Human
(16). Labeling
modifications
had a clottability
I and 13' I by the iodine monochloride
modifications
drawn ACID blood
(15). Minor
(17,181 showed
S-mercaptoethanol,
Labeling 125
precipitation
gel filtration
563
COMPLEXES
fibrin
and fibrinogen
to the same plasma
as used
for equilibration of the agarose column (see below). The plasma 125 containing I-fibrinogen was treated by small amounts of thrombin
not causing
visible
time of 20 min the thrombin fibrinogen
consisted
human platelet-poor ml) diluted
100
ACD plasma
1 ml buffer
pH 7.4);
(0.05 M tris,
aprotinin,
citrate,
was added slowly
room temperature.
After
fibrinogen after,
(0.08 mg) were
the mixture
time, the mixtures
were
(approx. 0.48 mg dis-
over a period
added to the reaction
of 5 min
at 37OC for 30 min, centrifuged
at
Thereone kept
respectively.
At this
at 10 000 9 for 2 min and
(1.2 ml each) was applied
chromatographic
mixture.
into two equal portions,
filtration.
Seven
(2 units/
time of 20 min, 0.1 ml 5 min, 0.1 ml 131I_
for obtaining
columns.
0.0025 M EDTA,
thrombin
0.25 ml were removed The rest
0.01
100 kiu/ml
and after another
was divided
at 20°C, and the other
2 mg/
0.04 M Na2HP04,
0.025 M EACA,
an incubation
(250 AT units/ml)
the reac-
1 ml pooled
concentration:
pH 7.0); 0.2 ml bovine
ml). The thrombin
hirudin
In detail,
0.01 M EACA,
0.6 ml 1251-fibrinogen
in 0.055 M sodium
kiu/ml
an incubation hirudin and 131I_
substances:
(fibrinogen
0.004 M EDTA,
After
with
calibration.
of the following
0.15 M NaCl,
aprotinin, solved
with
strands.
was quenched
added for internal
tion mixture
M KH2P04,
fibrin
analytical
data before
gel
to prewarmed
runs were performed
in parallel
564
SOLUBLE FIBRIN COMPLEXES
Vo1.14,Nos.4/5
125 I-fibrinogen and 13'I-fibrinogen in plasma not treated with thrombin were at 20°C and at 37OC. In the control experiments,
applied to sepharose columns to determine the elution volume of untreated fibrinogen under identical conditions. Ethanol gelation test was performed according to Godal and Abildgaard (4). 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), packed with sepharose CL6B (Pharmacia). The columns were equilibrated with pooled human ACD plasma diluted with a buffer. 1 1 pooled plasma was diluted with 1 1 buffer containing 0.05 M tris, 0.04 M Na2HP04, 0.01 M XH2PG4, 0.15 M NaCl, 0.004 M EDTA, 0.01 M EACA, 100 kiu/ml aprotinin, 10 AT units/ml hirudin, 0.4 mg/ml sodium azide, pH 7.4). Two columns were prepared in parallel using the same gel and the same buffered plasma, 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 elution was performed with the same buffered plasma as used for equilibration. The flow rate of the columnrwas lo-15 ml/h; the eluates were collected in fractions of 1 ml. Rechromatography was performed with the same columns using buffered plasma for equilibration and elution. SDS-PAA (sodium dodecyl sulphate-polyacrylamide) gel electro. phoresis was performed according to Swank and Munkres (19) using samples reduced with p-mercaptoethanol in urea. Plasma samples eluted from the chromatography column were clotted in the presence of EDTA and aprotinin, and the isolated clot dissolved in the reducing buffer. If radioactivities were determined in sections of the gels, these were cut into 8 pieces according to the stained bands (Table II), and the pieces monitored for 1 =I_ as well as 131I-activities. RESULTS Plasma containing ,125I-fibrinogen and 131I-fibrinogen not treated with thrombin was applied to sepharose CL-6B columns equilibrated with plasma. Labeled fibrinogen was eluted at a single peak with an elution volume of about 62 ml at 20°C as well
SOLUBLE
VOl.14,NOS.4/j
10
8
FIBRIN
-
"'I-Ftbrmogen
PO
"'I-Ftbrmogen
$5
COMPLEXES
FIG. 1 Gel filtration of 1251_fibrinogen and '31I-fibrinogen in human plasma not treated with thrombin. Sepharose CL-6B columns were equilibrated with buffered plasma and the samples eluted with buffered plasma at 20°C and at 370C. The radioactivity is given in % of the total radioactivity applied to the columns.
6
0
1
LO
50
as at 37OC
60 70 80 90 Elut~on volume (ml)
(Fig. 1).
Plasma
containing
was regularly
found to yield
radioclottabilities between
of labeled
positive
on sepharose
revealed CL-6B
main peak
peaks
equilibrated
the void volume
(peak B) was eluted
volume
before
with
gelation
tests.
fibrin
The
were
and fibrino-
after gel filtration
with
plasma
(Fig. 2).
of 44-48 ml, whereas
a volume
of peak B corresponded
and fibrinogen
gel filtration
of labeled
two different
columns
Peak A was eluted with
fibrin
ethanol
of these mixtures
92 and 94%. These mixtures
gen in plasma
elution
mixtures
the
of 60-64 ml. The
to that of labeled
fibri-
nogen under identical conditions (Fig. 1). At 20°C, a small 131 I-fibrinogen (? = 4.5% + 0.9; n = 7) was eluted toamount of 125I-fibrinogen in peak A, gether with the thrombin-treated 131 I-fibrinogen was recovered in peak A. whereas at 37OC, no 125 I-radioactivity W&S recovered from the All the applied columns
at 20°C as well
manner,
all the applied
columns
at 20°C as well as at 37OC.
To control
as at 37OC (Table I). In the same 131 I-fibrinogen was eluted from the
the influence
of possibly
activated
factor XIII,
566
SOLUBLE FIBRIN COMPLEXES
Vo1.14,Nos.4/5
FIG. 2 Gel filtration of 1.2 ml human plasma each containing thrombin-treated
10
8 6
$2 pA 20 :: 2 z 6 1 2 0 LO
60
60
Elutlon
70
volume
80
90
Imll
plasma were prepared at room temperature, divided into two equal portions and chromatographed at 20°C and at 370C. Sepharose CL6B columns were equilibrated with buffered plasma and the samples also eluted with buffered plasma. The radioactivity is given in % of the total radioactivity applied to the columns. The 44th and 45th ml of the elution volume of the 20°C run and the 63rd and 64th ml of the elution volume of the 370C run were rechromatographed (see Figs. 3 and 4).
VO
8
+
6
$j2 \ .t 0IA,_i = 2 $ 10 $ 4 6
;b
8 I
2ooc
6
4 2 01!:._ LO 50
60
Nut/on
70
80
volume (ml)
90
FIG. 3 Rechromatography of the isolated 44th and 45th ml of the elution volume of the 20°C run (see Fig. 2). The fractions were divided into two equal portions and rechromatographed on columns equilibrated with plasma. The radioactivity is given in % of the total radioactivity applied to the columns.
SOLUBLE
Vo1.14,Nos.4/j
FIBRIN
567
COMPLEXES
VO 1
12
-
‘*sI
-
“‘I
10
8
137”c]
1 i
g;
t
FIG. 4 Rechromatography of the isolated 63rd and 64th ml of the elution volume of the 370C run (see Fig. 2). The fractions were divided into two equal portions and rechromatographed on plasma-equilibrated columns at 20°C and at 37OC. The radioactivity is given in % of the total radioactivity applied to the columns.
A_ i
U 2 5 88 L 2 0 LO
50
60
Elutron
SDS-PAA
90
were performed.
region
at different the eluates
of reduced
gels did neither
nor in the two peaks temperatures
samples
The radioactivities
of the SDS-PAA
two isotopes,
ween
80 (ml1
gel electrophoresis
filtration f-x
70 volume
obtained
from gel
measured
in the
differ
between
of gel chromatography,
the
nor
(Table II). The radioclottabilities
of peaks A and B at 20°C as well
as 37OC varied
of bet-
07 and 94%.
TABLE I and 13' I-Radioactivities after Gel FilRecovery (x + s) of 1 q_ tration of Piasma Containing Thrombin-Treated 1251-Fibrinogen and The Gel Filtration Was Performed on Untreated 1311-Fibrinogen. Sepharose CL-6B Columns at 20°C and at 37OC Temperature
'Per
No. of experiments
Recovery
(%I* 131I
125I
2o"c
7
106.6 + 13.2
107.1 + 11.7
37Oc
7
104.0 + - 13.0
103.7 + 12.0
cent of the radioactivities
applied
to the columns.
566
SOLUBLE FIBRIN COMPLEXES
Vo1.14,Nos.4/5
If the fractions of peak A eluted at 20°C were rechromatographed, different elution patterns were observed depending on the temperature used for rechromatography (Fig. 3). At 20°C, most of the applied material (approx. 90%) was eluted with the void volume, whereas at 37'C the main peak (91% of the radioactivity applied to the column) was found with the elution volume corresponding to that of fibrinogen. 131I-radioactivity is not repre131 sented, as there was not enough I-radioactivity in peak A before rechromatography for reliable measurements. In a second rechromatography experiment, fractions of peak B eluted at 37'C (Fig. 2) were rechromatographed at 20°C and at 37'C (Fig. 4). During rechromatography at 37OC, the peak of the 125 131I-activities appeared with the same elution I-as well as volume as in the preceding experiment where the fractions derived from. At 20°C, however, 13% of the 125I-radioactivity applied to the columns was eluted with the void volume, but no '3'I_ radioactivity was recovered in peak A (Fig. 4). DISCUSSION Soluble fibrin complexes at room temperature may consist of different complexes made up of fibrin and fibrinogen or fibrin and fibrinolytic degradation products. They may be stabilized by factor XIIIa or unstabilized. The present study deals with so-called unstabilized fibrin-fibrinogen complexes only. These were prepared by addition of small amounts of thrombin to plasma containing 125I-fibrinogen. Thus, a portion of the plasma fibrinogen as well as the 125I-fibrinogen was converted to fibrin. The main portion of the plasma fibrinogen and the '25I_ fibrinogen was not affected by thrombin as not more than 20% of the plasma fibrinogen can be transferred to fibrin without gelating (II, 21, 22). To demonstrate fibrin-fibrinogen interaction, fibrinogen labeled with a different isotope was added. The ethanol gelation test was always found positive with these mixtures. If these mixtures were applied to gel filtration columns, two peaks were eluted, the peak eluted with the void volume being larger at 20°C than at 37OC. Some of the '251radioactivity corresponding to thrombin-treated 125I-fibrinogen
131 125;
131 125;
13'1
131 125;
131125;
3
4
5
6
7
8
I
131 125;
2
12SI
131 125;
1
I
Isotope
131 125;
of PAA-gel piece
No.
+ + T -
5.3
1.0 1.1
+
+ 7
T
+
+ T
+ 7
+ T
+ T
5.2
20.4 20.7
47.0
47.1
22.5 22.1
1.8 1.8
0.7 0.7
1.2 1.1
0.3 0.4
0.8
0.8
0.6 0.8
2.0
1.6
2.0 1.6
0.4 0.5
0.1 0.1
0.1 0.1
Before gel filtration (%I
1.2
4.1
20.3
45.2
22.5
3.0
1.1
2.7
2.7
0.3
0.2
1.0
I
+
+
+
\.
I
. .A .&
0.5
0.5
1.7
-I-2.3
+
+
+
+
at 20°C (%I
Peak A
0.8 0.9
3.8
4.0
20.0 20.0
44.7
47.4
23.4 22.7
1.9 2.8
1.0 1.1
1.3 2.4
r
-i
+ T
+
+
+ T
T
+
+ +
+ T
+ T
+ F I
I
___- J
0.3 0.2
0.8
0.5
1.5 1.8
3.3
1.3
1.8 3.1
0.6 1.7
0.2 0.3
0.4 1.5
at 20°C (%I
0.8 0.7
4.0
4.4
19.3 20.0
48.1
48.5
22.6 22.3
2.4 2.6
0.7 0.9
1.5 1.5 0.2 0.2
0.2 0.2
+ T -
T
+
+ + T
+
+ T
0.4 0.2
0.7
0.5
0.8 0.9
4.1
3.0
4.2 4.7
-I-0.5 T - 0.5
+ T -
+ T
at 37OC (%I
Peak B
.
,'
/
/ . /
. I-
_----.
.'
'
/'
_---
. _ -_
.
- --.
-----
__
*\.
--
gel
PAA
TABLE II Distribution of Radioactivities in SDS-PAA Gels of Reduced Samples Obtained from Peak A and Fibrinogen in Peak B of Gel Filtration at 20°C and 37OC. 125I Corresponds to Thrombin-Treated Plasma and 13'1 to Untreated Fibrinogen. The Values of the Samples before Gel Filtration and Were during Gel Filtration Are Given in Means and Standard Deviation /n=5). Radioactivities Calculated in % of the Activity of Each PAA Gel. The Measured I3 I-Activities in Peak A Eluted in Peak A at 370C Were too Low for Reasonat 20°C as well as at 37OC and the 1251-Activities able Evaluation
c: tr _c‘4 0 "1 .I-\
cn
0
570
SOLUBLE FIBRIN COMPLEXES
V01.14,8os.4/j
was eluted with the void volume indicating the formation of high-molecular weight aggregates. At 20°C, these aggregates eluting in the void volume contained some 131I-fibrinogen which had not been treated with thrombin. The elution of thrombintreated 125I-fibrinogen together with untreated 131I-fibrinogen may be caused by adsorption of fibrinogen to the high-molecular weight fibrin aggregates or the formation of so-called fibrinfibrinogen complexes. Most interestingly, these fibrin-fibrinogen interactions could not be observed at 37OC as no 131I-fibrinogen was eluted together with the fibrin aggregates at this temperature. Furthermore, the reduction of the peak eluted with the void volume at 37OC in comparison to 20°C indicates that the so-called fibrin-fibrinogen complexes dissociated at 37'C. To prove the theory that so-called fibrin-fibrinogen complexes generate at 20°C and dissociate at 37OC, rechromatography experiments were performed. Peak A eluted at 20°C was observed in the same position after rechromatography at 20°C, but changed its position after rechromatography at 37OC. Nearly all the radioactivity was found in the peak corresponding to the fibrinogen peak. Thus, so-called fibrin-fibrinogen complexes isolated at 20°C dissociated at 37OC. If peak B eluted at 37OC was rechromatographed at 20°C, the elution pattern was similar to the one seen with gel filtration of the starting material at 20°C. Thus, at 20°C fibrin formed aggregates from mixtures of monomeric fibrin in plasma. No fibrinogen was eluted together with these fibrin aggregates. From these experiments we conclude that so-called unstabilized fibrin-fibrinogen complexes generate only in vitro at an unphysiologic low temperature but do not exist at 37OC. ACKNOWLEDGEMENTS The study was supported by grants (Mu 279) of the Deutsche Forschungsgemeinschaft, Bonn-Bad Codesberg. 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
Vol.14,Nos.
4/j
SOLUBLE
supplying
radioactive
Abteilung
fiir Klinische
FIBRIN
iodine,
Justus-Liebig-Universitat,
371
CO?4PLEXES
and Prof.
Immunologie Giessen,
Dr. C. Mueller-Eckhardt,
und Bluttransfusion, for supplying
fresh
frozen
human plasma.
REFERENCES 1.
BANG, N.U. and CHANG, M.L. Soluble Thrombos. Hemostas. 1, 91-, 1974.
fibrin
complexes.
Sem.
2. EDGAR, W. McKILLOP, C., HOWIE, P.W. and PRENTICE, C.R.M. Composition of soluble fibrin complexes in pre-eclampsia. Thrombos. Res. 10, 567, 1977. 3. FLETCHER, A.P., ALKJAERSIG, N., O'BRIEN, J. and TULEVSKI, V. G. Blood hypercoagulability and thrombosis. Transact. Assoc. Am. Physicians 83, 159, 1970. 4. GODAL, plasma
H.C. and Abildgaard, U. Gelation of soluble fibrin by ethanol. Stand. J. Haematol. 3, 342, 1966.
in
5. GRAEFF, H., von HUGO, R. and HAFTER, R. Evaluation of hypercoagulability and intravascular coagulation by estimation and characterization of soluble fibrin monomer complexes (SFMCS) . Progr. Chem. Fibrinolys. Thrombolys. 3, 435, 1978. 6. KIERULF, screened
P. and GODAL, H.C. Fibrinemia in medical patients br the ethanol test. Acta Med. Stand. 190, 185,
1971.
7. 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. 8. MATTHIAS, F.R., REINICKE, R. and HEENE, D.L. Affinity chromatography and quantitation of soluble fibrin from plasma. Thrombos. Res. 10, 365, 1977. 9. NIEWIAROWSKI, S. and GUREWICH, V. Laboratory identification of intravascular coagulation. The serial dilution protamine sulfate test for the detection of fibrin monomer and fibrin degradation products. J. Lab. Clin. Med. 77, 665, 1971. 10. SHAINOFF, J.R. and PAGE, I.H. Cofibrins and fibrin-intermediates as indicators of thrombin activity in vivo. Circul. Res. 8, 1013, 1960. 11. 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. 12. DONATI, M.B., VERHAEGHE, R., CULASSO, D.E. and VERMYLEN, J. Molecular size distribution of fibrinogen derivatives formed study. Thrombos. in vitro and in vivo: a chromatographic
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FIBRIN
COMPLEXES
Haemostas. 36, 14, 1976. 13. MAHN, I., SCHbNBACH, F., MULLER-BERGHAUS, G. Formation and dissociation of soluble fibrin in vivo. Thrombos. Res.11, 67, 1977. 14. BLATTLER, W., STRAUB, P.W. and PEYER, A. Effect of in vivo produced fibrinogen-fibrin intermediates on viscosity of human blood. Thrombos. Res. 4, 787, 1974. 15. BLCMBACK, B. and BLOMBACK, M. Purification of human and bovine fibrinogen. Ark. Kemi 10, 415, 1956. 16. MAHN, I. and MULLER-BERGHAUS, G. Studies on catabolism of 1251-labelled fibrinogen in normal rabbits and in rabbits with indwelling intravenous catheters: Methodologic aspects. Haemostasis 4, 40, 1975. 17. ORNSTEIN, L. Disc electrophoresis-I. Background and theory. Ann. New York Acad. Sci. 121, 321, 1964. 18. DAVIS, B.J. Disc electrophoresis-II. Method and application to human serum proteins. Ann. New York Acad. Sci. 121, 404, 1964. 19. SWANK, R.T. and MUNKRES, K.D. Molecular weight analysis of oligopeptides by electrophoresis in polyacrylamide gel with sodium dodecyl sulfate. Analyt. Biochem. 39, 462, 1971. 20.
A.S. Efficient trace-labelling of proteins with iodine. Nature 182, 53, 1958.
McFARLANE,
21. SASAKI, T. PAGE, I.H. and SHAINOFF, J.R. Stable complex of fibrinogen and fibrin. Science 152, 1069, 1966. 22. YUDELMAN, I., SPANONDIS, K. and NOSSEL, H.L. Comparative behaviour of 125I-fibrinogen and 1251-fibrin in solution. Thrombos. Res. 5, 495, 1974. 23. EDGAR, W. and PRENTICE, C.R.M. The effect of fibrinopeptide B release on temperature dependent fibrin association. (Abstract). Thrombos. Haemostas. 38, 104, 1977.
