British Journal of Haemafology, 1972,22, 681.

Metabolism and Distribution of Fibrinogen

I. FIBRINOGEN TURNOVER IN PHYSIOLOGlCAL CONDITIONS IN HUMANS

D. COLLEN, H. CLAEYS
G. N. TYTGAT, AND R. PIESSENS

Laboratory of Blood Coagulation, Department of Medicine,

University o f Leuven, Belgium

(Received 20 March 1971 ; acceptedfor publication 28 June 1971)

SUMMARY. The metabolism of human fibrinogen, labelled with radioactive iodine,

was studied in 35 healthy subjects over a 5 yr period using eight fibrinogen pre-
parations in 23 labelling procedures. The labelled fibrinogen was highly clottable
and homogeneous on agarose gel filtration, immunoelectrophoresis and auto-
radiography. Results in the control subjects were: plasma volume 42+ 7 ml/kg;
plasma fibrinogen concentration 284+ 71 mg/ 100ml; total plasma fibrinogen pool
119+ 40 mg/kg, representing 0.72+ 0.07 of the total body pool; fibrinogen half-life
4.14+ 0.56 days; fractional catabolic rate 0.24+ 0.04 of the plasma pool/day;
fractional transcapillary efflux rate o.6of 0.26 of the plasma poollday. Comparable
results were obtained for all labelled fibrinogen batches. Anticoagulation with
heparin in five control subjects had no influenceon the fibrinogen half-life. Inhibition
of the fibrinolytic system with tranexamic acid in five control subjects had no in-
fluence on the fibrinogen half-life in four of them but resulted in a prolongation
in one subject.
A mathematical compartmental model for the metabolism of fibrinogen is
proposed, consisting of one extravascular compartment and three catabolic
pathways (basic protein turnover, intravascular fibrin formation, and intravascular
fibrino(gen0)lysis). In physiological conditions intravascular fibrin formation or
fibrino(geno)lysisare not essential in the fibrinogen turnover.

Several studies dealing with the metabolism and distribution of iodine-labelled fibrinogen
in humans have appeared (Christensen, 1958; Hammond & Verel, 1959; Adelson et al, 1961;
Amris & Amris, 1964; McFarlane et al, 1964; Hart, 1966; Blomback et al, 1966; Takeda,
1966; Izak et al, 1967). In none of them were enough data compiled to permit extensive
evaluation of the different aspects of its kinetics. For studies of the metabolism of fibrinogen
in health and disease, a quantitative determination of the rate of catabolism in the different
pathways, of the exchange rate between the plasma and extravascular spaces and of the
relative magnitudes of the pool sizes is essential. This requires an adequate metabolic tracer
and a mathematical procedure that will allow accurate determination of the metabolic
parameters. Moreover, because fibrinogen is substrate for both the coagulation and the
fibrinolytic system, experimental conditions have to be studied in which consumption in
these pathways can be differentiated from basic protein turnover. The purpose of this study
Correspondence: Dr D. Collen, Laboratory of Blood Coagulation, A.Z. St. Rafael, 3000 Leuven, Belgium.

68 I
682 D. Collen et al
was to document the validity and the reproducibility of iodine-labelled fibrinogen as a
metabolic tracer, and to evaluate the relative importance of fibrinogen catabolism by in vivo
coagulation, in vivo fibrino(geno)lysis,and basic protein turnover in healthy subjects.

Free iodine Incorporation ratio Mols Iodine per Recovery of label

Batch Isotope (%I label infibrinogen mol fibrinogen infibrin clot
m
r (%I
26-01-65 1311 0.7 20.9 0.150 93
23-02-65
01-07-65
1311
1311
- - - -
0.S 15.2 0.095 91
1311 0.8 0.148
09-08-65 23.1 94
1311 0-4
17-11-65 28.1 0.150 93
25-04-66 1311 0.7 11.1 0.121 91
08-06-66 1311 0.5 24.7 0.154 90
27-06-66 1311 0.6 25.8 0.156 94
27-07-66 1251
0.8 13.6 0.179 -
27-07-66 1311 c 1.0 21.8 0.272 -
26-11-66 1251 c 1.0 24.5 0.300 94
26-11-66 1311 c 1.0 23.7 0.295 92
18-03-67 1251 c 1.0 27.0 0.336 93
18-03-67 1311 0.5 25.8 0.321 -
17-6-67 1251 - 26.6 0.331 -
1251
04-12-67 0.2 22.5 0.280 94
30-12-67 1251 < 1.0 25.2 0-314 95
04-05-68 1251 0.6 32.6 0.406 94
zoog-68
1311
0.5 27.0 0.331 -
06-11-68 1251 - 20.7 0.263 -
06-02-69 1251 0.6 21.5 0.278 -
03-04-69 1251
0.4 19.0 0.240 -
27-11-69 1311 - 14.8 - -

Fibrinogen was labelled with Na13'I or with Na1251according to McFarlane (1958) with
slight modifications. IC1 was generated from a mixture of SKI+KIO, + 6HCl e 3 IC1+
3KI+ 3KCl+ 3H20. Non-protein bound iodide was removed by passage through an
Amberlite IRA 401column. The labelled protein solution was sterilized through a Jena GsM
filter, subdivided, and stored at - 20°C. In total eight different homologous fibrinogen
preparations in 23 labelling procedures were used over a period of 5 yr. Tracer data on the
used batches of iodinated fibrinogen are summarized in Table I. The content of free iodide
in the final solution was determined in the supernatant after precipitation of labelled fibrino-
gen with an equal volume of 10% trichloracetic acid (TCA). The incorporation ratio of
 Normal Fibrinogen Turnover 683
label in fibrinogen was calculated from the ratio of the protein-bound radioactivity to the
total amount of radioactivity added to the fibrinogen solution. The ratio of mols of iodine
per mol labelled fibrinogen was calculated by multiplying the ratio of the concentrations of
both substances in the labelling solution with the incorporation ratio. The mean substitution
level in the used batches of tagged fibrinogen varied between 0.- and 0.40 (mean 0.235)
mols iodine per mol fibrinogen (mol wt= 330 000).This low substitution level was selected
to minimize damage by overiodination (McFarlane, 1963). The recovery of label in the fibrin
clot, prepared according to Blombkk et a1 (1966)~ was always 90% or more.

Effluent volume (ml)

FIG I. Agarose AsM gel filtration of 3 ml human pIasma and 0.1 ml i2sI-labelled fibrinogen,
prepared according to Blombick & Blombick (1956).Column height I m, elution with hydrostatic
pressure of 400 mm, flow rate 3 0 ml/hr, phosphate buffer p=o.z, pH=7.4, fractions of 6 ml.

Gel filtration on Agarose (Biogel AsM) (separating globular proteins with a molecular
weight between 10ooo and 5 ooo 000) of a mixture of labelled fibrinogen and native plasma,
demonstrated an identical elution position for radioactivity and for fibrinogen (Fig I).
Immunoelectrophoresis (Scheidegger, 1955) did not reveal any change in fibrinogen by
iodination (Fig 2). Autoradiography showed a concentration of label in the precipitation line.

Metabolic Studies
At least I day before injection of the labelled protein and during the whole procedure, the
thyroid was saturated by daily administration of 500 mg of KI. Exactly 10min after intra-
venous injection of 2-40 p Ci of the labelled protein, the first blood sample was taken from
684 D. Collen et a1
the opposite arm. The amount ofradioactive solution administered was determined by weigh-
ing. Subsequent blood samples were taken at different time intervals, in general for 10-20
days. Blood was collected on 0.2 mlEDTA 10% ando.1 ml Aprotinin (Trayslol, 5000 KIU/ml)
per 10ml. In several control subjects daily quantitative urine collections were made.

FIG2. Immunoelectrophoresisof human fibrinogen in agar (Scheidegger, 1955). (A) 5 pl unlabelled

fibrinogen 2% versus 0.1ml rabbit anti-human fibrinogen antiserum. (B) 5 pl unlabelled fibrinogen
2% versus 0.1 ml rabbit anti-human serum antiserum. (C) 5 labelled fibrinogen versus 0.1 ml
rabbit anti-human fibrinogen antiserum. (C') Autoradiography of C.

Anticoagulation was performed starting 7-9 days after injecting the labelled fibrinogen
and continued for 4 days either by continuous i.v. infusion of heparin (four subjects) or by
S.C. injection of long-acting heparin every 12 hr (one subject). The daily amount of heparin
was adjusted individually to prolong the clotting time at least two to four times. All possible
precautions were taken with respect to selection and clinical surveillance of the control
 Normal Fibrinogen Turnover 68 S
subjects in whom anticoagulation was performed. No adverse effects were observed in any
of the subjects.
Inhibition of the fibrinolytic system was produced by peroral administration of tranexamic
acid (cyclocapron) I g every 8 hr for 4 days, starting 7-9 days after injection of the labelled

ri
8
iY
I
,001 I 1 I
0 5 10 15
Time [days)

FIG 3. Fibrinogen metabolism in a control subject (T.J., male, 22 yr). x(t): plasma radioactivity.
Fibrin: clottable radioactivity. U t :fractional daily urinary excretion of label. I-ZU,:injected dose
minus cumulativeurinary excretion of label. z(t):non TCA-precipitableradioactivityin the body. R(t) :
remaining total body radioactivity, determined by whole body counting. Graphical curve peeling in
two exponential fmctions x(t) = CIe-ol'x CZcaZf. The straight linear terminal portion of the plasma
radioactivity x(t) is extrapolatedto the ordinateto obtain the intercept C1. The slope of this line is -aI.
By subtracting the extrapolated line from the original curve [x(t) - C1e-"1'] a new line is obtained
f ] which the slope -a2 and intercept value C2are determined.
[ C z ~ a *for

fibrinogen. This dosage, recommended to obtain adequate inhibition of the fibrinolytic sys-
tem (Andersson et al, 1968),resulted in a marked interference with the in vitro plasminogen
assay. Only an average of 40-60% of the plasminogen activity could be measured during
cyclocapron treatment using a caseinolytic plasminogen assay (De Vreker, 1965).
At the end of the observation period, duplicate samples of plasma, urine, supernatants of
TCA precipitated plasma, and of fibrin, solubilized in alkaline urea (Blombfck & Blombfck,
D
686 D. Collen et a1
1956) were measured in a well-type scintillation counter (gammalguard; Autowell Counting
System), with a sensitivity of approximately 625 ooo cpm/pCi, above a background of 3 0
cpm.
Analysis of Tracer Data
The tracer data were analysed using a two compartmental mathematical model according
to Atencio et al (1965). Therefore the plasma radioactivity data versus time were fitted with
a function x(t)= Cle-"'+ C2e-a ', by graphical analysis on semilogarithmic paper, as
illustrated in Fig 3. This method provides estimations of the radioactivity distribution ratio
EV/IV between the extravascular (EV) and intravascular (IV) pools, the fractional trans-
capillary efflux rate constant (k,2 ) , the fractional transcapillary reflux rate constant (k2,) and
the fractional catabolic rate constant (k,o , p ) (Matthews, 1957), using following fomulae :
EV/IV= [C2(a2- a,)]/[alc2 + azC,]
k12 = [Cl Cz(a2- a1)2]/[(C1 + C2)(C,a2+ C 2 4 l
k2 1 = [a,c*+ a2 C,]/[Cl+ c21
k10?p=[ala2(Cl + Cz)]/[a,Cz+a2C1]
In six subjects in whom quantitative urine collections were obtained, the fractional catabolic
rate constant (k,,,") was also determined from the ratio of the daily urinary radioactivity to
the corresponding mean plasma radioactivity (Berson et al, 1953; Campbell et al, 1956),
corrected for a delay in urinary excretion of iodide (Reeve & Roberts, 1959). The plasma
volume was obtained from the total injected radioactivity divided by the plasma radioactivity
in the first sample. The intravascular fibrinogen pool was determined by dividing the total
injected radioactivity by the plasma specific activity in the first sample. The absolute cata-
bolic rate was obtained by multiplying the fractional catabolic rate with the plasma fibrinogen
pool.
Laboratory assays
One-stage prothrombin times were performed according to Quick (193s), thrombin times
according to Vermylen & Verstraete (1960), fibrinogen with the FPT-test (Vermylen et al,
1963) and blood platelet count using the technique of Brecher & Cronkite (1950). Analysis
of the degree of purification of the fibrinogen preparations and assessment of clottable
radioactivity in the labelled fibrinogen solution and in serial plasma samples were performed
according to Blomback et af (1966).

RESULTS
The control group consisted of 35 healthy volunteers (30 males and five females), performing
their usual activities during the study. Clinical and laboratory data are summarized in Table
11. Daily fluctuations of the plasma fibrinogen concentration were frequently observed.
Coagulation parameters, liver function tests, and plasma protein electrophoresis, were
normal in all subjects.
Tracer Data
The plasma radioactivity data, expressed as a fraction of the first sample, were fitted with
a sum of two exponential terms x(t)=Cle-"1'+Cze-"2' by graphical curve peeling on
 TABLE
If. Metabolism and distribution of fibrinogen in healthy subjects

Plasma Fg Fractional rafe consfants Dufributwn

Inifiab Date of Sex menfrafiot r+(days) rafw
injecfion Irg/Ioo ml: Cl ‘11 CZ 02 for a t EVIIV
- - - .__ -
E.J. 06.01.65 M 41 0.66 0.18 0.34 2.31 3.8 0.26 0.90 1.59 0.45 26
S.J. 13.01.65 M 51 0.67 0.16 3.46 4.4 0.23 1.25 2.37 0.45 27
M.L. 25.01.65 M 42 0.70 0.14 2.31 4.8 0.19 0.79 1.66 0.39 23
0.19 0.50 1.07 0.28
23.02.65
2;:0.26 1.38
C.P. M 42 0.74 3.7 0.24 33
W.A. 02.07.65 M 60 0.76 0.15 0.24 4.5 0.19 0.48 1.09 0.27 35
W.V. 02.07.65 M 42 0.60 0.14 0.40 ;:g 5.0 0.21 0.51 0.70 0.53 25
O.V. 10.06.66 M 35 0.64 0.15 0.36 2.31 4.6 0.23 0.93 1.53 0.50 23
R.M. 12.06.66 F 39 0.56 0.21 0.44 2.31 3.3 0.35 1.13 1.39 0.66 41
M.R. 27.07.66 F 54 0.71 0.14 0.29 0.99 5.0 0.19 0.39 0.74 0.33 31
W.J. 09.08.66 M 30 0.50 0.19 0.50 1.98 3.7 0.35 1.09 I .09 0.82 42
D.M. 13.09.66 F 48 0.62 0.21 0.38 1.26 3.3 0.31 0.60 0.86 0.46 37
D.M. 22.09.66 F 42 0.68 0.17 0.32 1-49 4.I 0.24 0.56 I .m 0.39
W.A. 22.og.66 M 0.52 0.13 0.48 0.87 5.2 0.22 0 49 0.51 0.68
R.G. 19.01.67 M 0.57 0.19 0.43 2.77 0.32 1.30 I.66 0.66
;; 3.6
E35
R.L. 19.01.67 M 32 0.62 0.22 0.38 1.39 3.’ 0.32 0.66 0.95 0.47 33
F.P. 29.03.67 M 35 0.83 0.17 0.17 1.26 4.0 0.w 0.36 1.07 0.17 31
D.F. 04.07.61 F 48 0.79 0.17 0.21 0.77 4.1 0.20 0.30 0.64 0.19 25
V.M. 12.07.67 M 58 0.75 0.16 0.25 0.87 4.3 0.w 0.34
K.M. 14.03.68 M 32 0.60 0.18 0.40 0.99 3.9 0.27 0.50 % :3 ;:
0.70 0.30 0.27 0.60 21
V.M. 25.09.68 M 40 0.w 1.54 3.4
:1.14 ;0.35
24.09.68 M 41 0.76 0.17 0.24 1.53 4.1 0.21 0.50 1.w 0.27 21
f:? 25.09.68 M 42 0.68 0.19 0.32 1.39 3.6 0.26 0.57 1.01 0.38 32
B.M. 15.01.69 M 30 0.73 0.18 0.27 1.38 3.8 0.24 0.50 1.06 0.30 25
C.D. 21.04.69 M 35 0.67 0.19 0.33 0.87 3.8 0.26 0.41 0.65 0.34 19
W.P. 21.04.69 M 40 0.60 0.16 0.40 0.99 4.4 0.24 0.49 0.66 0.50 22
T.M. 21.04.69 M 42 0.67 0.12 0.33 0.77 5.6 0.17 0.56 0.38 21
A.A. 21.04.69 M 39 0.69 0.15 0.31 0.82 4.5 0.20 ::;i 0.61 0.33 21
V.J. 21.04.69 M 0.58 0.15 0.42 1.03 4.7 0.23 0.51 0.64 0.55
L.T. 21.04.69 M :; 0.64 0.15 0.36 1.39 4.5 0.22 0.60 0.94 0.47 3
C.H. 21.04.69 M 32 0.65 0.18 0.35 1.06 3.9 0.25 0.49 0.75 0.40 19
V.R. 21.04.69 M 34 0.71 0.19 0.29 1.38 3.7 0.25 0.54 1.03 0.33 W
R.J. 21.04.69 M 39 0.61 0.17 0.39 1.38 4.1 0.26 0.65 0.91 0.52 29
O.R. 10.12.69 M so 0.72 0.17 0.28 1.53 4.1 0.23 0.55 ax
0.s 10.12.69 M 48 0.85 0.16 0.15 1.25 4.4 0.18 0.32 ;:g 3; 23
T.J. 10.12.69 M 47 0.77 0.17 0.23 r.06 4.0 0.21 0.37 0.86 0.23 23
- - -
Mean 42 0.67 0.1% 1.44 4.14 0.24 0.60 1.02 0.41 28
SD 7 0.08 0.023 0.60 0.56 0.04 0.26 0.39 0.15 9
- - -- - -
688 D. Collen et a1
semilogarithmic paper. The data are summarized in Table 11. The mean plasma radioactivity
half-life for 3 0 men and five women was 4.17 and 3.96 days respectively. The overall mean
was 4.14k0.56 days. Radioactivity decay rates of the same range were obtained for the
various batches of labelled fibrinogen.
Daily measurements of clottable radioactivity in plasma were performed in 10 subjects.
Approximately 85 (8o-g0)% of the plasma radioactivity was constantly recovered in the
fibrin clot. In all subjects the decay of clottable radioactivity was parallel to the plasma
radioactivity, up to 2 weeks after injection of the labelled substrate. The amount of circulating
radioactive breakdown products, determined as non-TCA precipitable radioactivity,
increased initially to a peak value of less than 2% and paralleled subsequently the plasma

TABLE
111. Comparison of fractional catabolic rates, calculated by
cornpartmental analyses (kl0,,) and by metabolic clearance (klo,,,)

Initials kl0.P
I tl0.,,=( .;u -.;>
kl,,-al t

C.D. 0.26 0.206 0.I 87

V.R. 0.2s 0.24 0.220
W.P. 0.24 0.233 0.217
T.J. 0.21 0.218 0.203
O.R. 0.23 0.235 0.219
W.A. 0.19 0.171 0.160

Mean 0.218 0.201

SD 0.026 0.026 0.023
I

* Mean of nine determinations.

t Correctionfor error introducedby the delay in urinary excretion
of tracer, based on the ratio between the final slope (al) and the
urinary excretion rate for iodide (kl,,).

radioactivity. The total fraction of radioactive breakdown products in the body was cal-
culated by multiplying the intravascular fraction by the distribution volume. The radioactive
breakdown products mainly consist of free iodide (McFarlane, 1963) and are distributed in a
volume approximately eight times the plasma volume (Myant et al, 1950; Takeda & Reeve,
1962).
Quantitative urine collections were performed in six subjects. The daily fractional urinary
excretion of label was approximately a constant fraction of the mean corresponding plasma
radioactivity after the first day. During the first day a small fraction was excreted, probably
because of dilution of label in the extravascular iodide pool. The curve of the remaining total
body radioactivity, calculated by subtracting the cumulative urinary excretion of label from
the injected dose, usually diverged from the plasma radioactive curve. Because this divergence
could be due either to inadvertent incomplete urine collections, to excretion of label via
other ways than urine, or to accumulation of label elsewhere in the body, total body radio-
activity was measured by whole body counting in three subjects, immediately and 2, 5 , g and
13 days after injection of the labelled fibrinogen. The total body radioactivity disappearance
 Normal Fibrinogen Turnover 689
curve was parallel to the plasma and clottable radioactivity curves, ruling out any significant
accumulation of tracer in extravascular compartments. A representative metabolic study is
summarized in Fig 3. Thyroid radioactivity uptake was measured in three subjects. In two
of them, the evolution of thyroid radioactivity was parallel to the plasma radioactivity,
indicating absence of significant uptake of label. In one subject a transient increase in thyroid
radioactivity was observed which could be ascribed to irregular daily intake of stable iodide.
In all subjects the thyroid radioactivity never exceeded the maximal permissible dose of 1.1
pCi (I.C.R.P., 1959).

Time ( d a y s )

FIG4. Influence of anticoagulation on the metabolism of fibrinogen in a control subject (W.A., male,
17 yr). x ( t ) : plasmaradioactivity. U,:fractional daily urinary excretion of label. I -XUi: injected dose
minus cumulative urinary excretion of label. y(t) = I -XU,-x(t) : extravascular radioactivity

Calculation of Metabolic Parameters

The fractional catabolic rate constant of fibrinogen, calculated in all subjects from the
plasma radioactivity disappearance curve by two compartmental analytis (k, was o.24f
0.04of the plasma pool per day (Table 11). In six healthy subjects in whom presumably com-
plete urine collections were obtained, the mean fractional catabolic rate constant calculated
by two compartmental analysis (k,o,,) was 0.23 f0.026 and calculated by the metabolic
690 D. Collen et a1
clearance method 0.20+0.02 of the plasma poollday (Table 111). Thls discrepancy was
probably mainly due to incomplete urinary excretion of released label. The mean value of the
absolute catabolic rate, determined by multiplying the mean plasma fibrinogen pool (in
mg/kg) (Table 11) by the fractional catabolic rate, was 28 k 9 mg/kg/day. The fractional efflux
,
rate constant k, was 0.60rf: 0.26 of the plasma poollday and the fractional reflux rate con-
stant k,, was 1.0240.3g/day. The distribution ratio of label between the extra- and intra-
vascular fibrinogen pool (EV/IV) was 0.41k 0.15. The corresponding intravascular fraction
was 0.72+ 0.07 of the total body pool.

0.05

0.025
t I
Cyclocapron
0 5 10
Time (doys)

FIG 5. Influence o f inhibition of the fibrinolytic system on the metabolism of fibrinogen in a control
subject. (R.G., male, 21 yr). x ( t ) : plasma radioactivity.

Inzuence of Inhibition of Coagulation and Fibrinolysis on the Metabolism of Fibrinogen

The relative importance of fibrinogen consumption by in vivo coagulation or fibrino-
(geno)lysis in the overall fibrinogen catabolism was evaluated by analysis of the effect of
pharmacological inhibition of coagulation (heparin) or fibrino(gen0)lysis(tranexamic acid)
on the metabolism of fibrinogen. The influence of heparin was studied in five normal
subjects. No change in the slope of the plasma radioactivity disappearancecurve was observed
as illustrated for one of them in Fig 4.The influence of tranexamic acid was studied in five
normal subjects. No change in the slope was observed in four of these subjects while in one
subject (R.G.) there was a prolongation of the half-life from 3.6 days before to 4.3 days during
inhibition, as shown in Fig 5.
 Normal Fibrinogen Turnover 691

DISCUSSION
The purity of the labelled fibrinogen preparations used in this study is evident from their
high clottability before and after labelling and their homogeneous behaviour on agarose gel
filtration and immunoelectrophoresis. The following in uiuo frndings suggest the absence of
detectable amounts of denatured or contaminating material : (a) maximal TCA-soluble
radioactivity in plasma of less than 2%, (b) intravascular fraction at equilibrium of more than
0.70, (c) parallel evolution of plasma and clottable radioactivity, (d) absence of high initial
urinary excretion of radioactivity, and (e) straight semi-exponential decay of radioactivity in
plasma after equilibrium with the extravascular fluid.
The reproducibility of the tracer preparation and the turnover studies is demonstrated
over a 5 yr period by using 23 labelled batches and eight fibrinogen preparations in 3 5 healthy
subjects. The metabolic parameters obtained for each batch of labelled fibrinogen are very
similar. The plasma fibrinogenconcentrationis 2841 71 mg/Ioo ml and the plasma fibrinogen
pool is 1 1 9 140 mg/kg. The fractional catabolic rate is 0.24+ 0.04 of the plasma poollday and
the daily absolute catabolic rate 28f 9 mg/kg. The intravascular fibrinogen pool contains
0.72 of the body pool. The half-life of labelled fibrinogen is 4.14 5 0.56 days. The fractional
transcapillary efflux rate is 0.601 0.26 of the plasma pool per day.
No exact knowledge is available concerning the mechanism of normal catabolism of
fibrinogen in man. Astrup (1956) has suggested that in physiological conditions there is a
dynamic equilibrium between coagulation, constantly laying down fibrin to seal any defects
of the endothelial wall, and fibrinolysis, removing such deposits after they served their
haemostatic function. This muchdiscussed hypothesis is based on the fact that coagulation
factors have a rapid turnover, compared with other plasma proteins, and that disturbances in
the haemostatic balance, either by increased coagulability or by hyperfibrinolysis, result in
accelerated fibrinogen catabolism. W e have attempted to validate this concept in man by
experimentally tipping the postulated balance between coagulant and fibrinolytic forces by
means of anticoagulation with heparin and inhibition of fibrinolysis by tranexamic acid. No
change in half-life of labelled fibrinogen is observed during heparin infusion in five healthy
subjects. These results are in agreement with the data of Lewis et al(1961), who did not find
significant differences in fibrinogen survival in three dogs in spite of markedly impaired
coagulation by heparin. No prolongation of plasma radioactivity decay is observed in four
of five control persons during inhibition of the fibrinolytic system. In one subject a pro-
longation occurred which may be explained by the occasional activation of the fibrinolytic
system in physiological conditions, by mechanisms such as severe muscular exercise (Biggs
et al, 1947; Iatridis & Ferguson, 1963) or mental stress and anxiety (MacFarlane & Biggs,
1946; Ogston et al, 1962; Cash & Allen, 1967). These results are in agreement with the
studies of Hart (1966)who also noted a prolongation of the survivaltime of labelled fibrinogen
in some of his control persons during EACA administration. However, Lewis (1963) and
Gajewski & Alexander (1963) have found that high doses of EACA do not influence the half-
life of fibrinogen in dogs and rabbits.
From these experiments we may conclude that continuous in vivo coagulation and fibrin-
olysis are not operative in the formation and removal of labelled fibrin, at least not to an
extent sufficient to affect noticeably the fibrinogen turnover rate. The validity of the concept
692 D. Collen et a1
of an equilibrium between coagulation and lysis is therefore questioned. Definite conclusions,
however, cannot be drawn because the possibility remains that fibrin formation, occurring
in relation to endothelial cells or other tissues and potentially brought about by platelet
coagulation or tissue thromboplastin, is out of range of anticoagulant control. Moreover,
local plasmin formation, which has escaped the action of endogenous and exogenous inhibi-
tors, may result from the selective adsorption of plasminogen and plasminogen activator on
the fibrin evolving in vivo. Therefore unequivocal evidence as to whether fibrinogen is
broken down directly or after fibrin formation is not provided by these data, although
it is unlikely that the normal catabolism in man proceeds by steady conversion of fibrinogen
to fibrin with subsequent fibrinolysis to a substantial extent.
The correlation between the daily excretion of label and the mean corresponding plasma
radioactivity (McFarlane, 1963; Takeda, 1966) suggests that breakdown takes place either
intravascularly or at sites in rapid exchange with the plasma compartment and also that
fibrinogen is either catabolized directly with insignificant fibrin formation, or that fibrin, if
formed in significant amounts, is broken down very rapidly. The absence of significant
amounts of intermediate breakdown products suggests that normal fibrinogen catabolism is
probably an intracellular process (Regoeczi, 1967). In conjunction with the above kinetic
considerations this behaviour seems to support the view that fibrinogen catabolism may take
place in the vascular endothelium during the passage of fibrinogen into the extravascular
space.
The plasma fibrinogen concentration occasionally fluctuates significantly from day to day.
Because the catabolism takes place at a constant fractional rate, this implies that the synthesis
occurs in waves as was already shown by Atencio et al(1969).
From these experiments we may conclude that in vim intravascular coagulation and/or
fibrino(gen0)lysis are not essential pathways in the fibrinogen catabolism in physiological
conditions in humans.

APPENDIX
MATHEMATICAL MODEL FOR THE METABOLISM OF LABELLED FIBRINOGEN IN HUMANS
Mathematical Analysis of Tracer Data
The results of a turnover study of fibrinogen consist of a series of measurements of plasma
radioactivity x i , extending over a time period ti after administration of the labelled substance.
In the compartmental model the fitting of a set of experimental data with a sum of exponen-
tial terms
n

yields a number of coefficients and exponents from which the metabolic parameters have to
be calculated (Berman & Schoenfeld, 1956).
In physiological conditions, protein molecules are continuously synthesized, are released
into the blood stream in which they are rapidly mixed, and pass into the extravascular fluid
or into the breakdown sites. Extravascular protein molecules traverse the tissue fluids,
enter lymphatics and blood capillaries and return to the circulation after different time inter-
 Normal Fibrinogen Turnover 693
vals. Even if one groups all molecules with the same in- and out-flow characteristics into one
compartment, then the physiological model will consist of an innumerable number of
compartments, all with different pool masses and kinetic constants (Reeve & Bailey, 1962).
Therefore any compartmental model will only be an approximation of the physiological
system. Theoretically, the approximation will be better when the number of compartments
increases, but at the same time non-linear curve fitting becomes increasingly difficult.
Furthermore, the fitting of a sum of exponential terms to experimental data is limited by the
fact that, in biological tracer studies, only a small number of data-points of moderate accuracy
can be obtained. The fitting with a reduced number of exponentials introduces a systematic
error in the analysis. The actual number of compartments which will give optimal resolution
will be determined by the relative magnitudes of the systematic error and the experimental
data error. When the systematic error becomes small compared with the data error, the
estimated parameters will be increasingly and unacceptably influenced by data error when
increasing the number of compartments.
The influence of systematic and data error on the estimation of the fractional catabolic
rate constant, of the exchange rates between intra- and extravascular compartments and of
the fibrinogen pool sizes has been investigated mathematically in order to answer the fol-
lowing questions:
(I) Is there a practical criterion for determining the number of terms which will give an
optimal estimate of the model parameters?
( 2 ) For which number of terms is the systematic error negligible in comparison with data
error?
(3) How are the estimated parameters influenced by data error when the systematic error is
eliminated?
(4) Are the estimated parameters still meaningful when the number of compartments is
underestimated?
(5) Is computer fitting superior to graphical curve peeling in the estimation of the metabolic
parameters?
Simulation ofexperimental data. Tracer data were simulated on a high speed digital computer
(IBM 360/44). The coefficients and exponents of the data generating function
n
x(t)= CC,e -' akt
k=l
in which ZC, was normalized to I, were given values, according to literature data and our
own experimental findings concerning fibrinogen metabolism. For two compartmental
analysis, data were generated from the function x(t)= 0 . 7 e - ~ - ' ~ ' o.3e-
+ For three com-
partmental analysis, data were generated from the function x(t)= o.7e- "+ o.ze- 0.90' +
o . ~ e - ~ . Each
~ ' . simulated decay curve contained either 10 or 15 measurements, the final
point averaging 7% of the initial value. More data points are difficult to obtain in biological
studies and much lower radioactivities are less accurately counted. More samples were taken
initially when the curve was more rapidly changing. For 10 data points, t= O.OI,O.S, 1.0,1.5,
2.0, 3.0, 5.0, 7.0, 10.0,15.oandfor 15 data points t = o . o ~ 0.5,
, 1.0, 1.5, 2.0, 2.5, 3.0, 4.0, 5.0,
6.0, 7.0, 9.0, 11.0, 13.0, 15.0. Normally distributed random error, calculated according to
Glass & de Garreta (1967) was superimposed on the simulated tracer data. The value of the
 TABLB
IV. Analysis of simulated tracer data
A. Data generating function: x(t) =0.7e-O" 5'+o.3e-1. sf
Fitting function: x(t)= Cle-g1r+C2e-'2r

Sum squares No. of

deviation x 105 EVIIV I ki2 I k1o convergence

Theoretical value 0.369 0.350 0.2055 30

No. ofdata Data error

points (%I Mean SD Mean SD Mean SD Mean SD
~~

0.369 0.018 0.355 0.030 0.2056 0.0020 30

0.371 0.045 0.371 0.075 0.2059 0.00~0 30
0.369 0.019 0.343 0.029 0.2050 0.0020 30
b
0.369 0.048 0.339 0.070 0.2044 O.OO$O 30
2-
3.0r
B. Data generating function: x(t) =o.7e-0~15r+o.2e-0~90~+o.~e-
I. Fitting function: x(t)= C,e-"i* + C , e - ' ~ ~ + C ~ e - ' 3 ~
I I I I
Sum squares No. of
deviations x 105 EV/IV kl eflux convergence

Theoretical value 0.354 0.382 0.2032 30

No. ofdata Data error

points (%) Mean SD Mean SD Mean SD Mean SD

I0 2 7.8 5.2 0.476 0.350 0.442 0.128 0.2026 0.0022 18

5 s4.5 43.1 0.460 0,090 0.476 0.172 0.2018 0.0048 I3
1s 2 11.5 7.1 0.395 0.055 0.394 0.114 0.2027 0.0020 I8
5 85.2 49.5 0.578 0.240 0.401 0.134 0.2019 0.0064 I2
Normal Fibrinogen Turnover

w m m u o o
8 - 8 2 8-8s
N.N.N. N.N.N.
0 0 0 0 0 0

N C n O H e m
- 0 - N N c 1
? ? ? ? ? ?
0 0 0 0 0 0

s w
u
696 D. Collen et a1
error was either 2% or 5% which corresponds to the error range in biological tracer studies.
Curve fitting procedure. Sums of exponential terms were fitted to the simulated tracer data,
using the Fortran IV program (in double precision version) written by Spzth (1968),based on a
damped Gauss-Newton method. The variability of the estimates of the function parameters
(ck,uk) and of the metabolic parameters (k,,, kI2, EV/IV) was studied for each data
generating function by analysing 30 simulations differing only with respect to random
numbers, used to calculate the error in the data points. The parameter values (Ck,a k ) of the
data generating function were also used as starting values in the Fortran IV computer pro-
gram. When fitting data generated from a sum of three exponentials with a sum of two, the
starting value of the second exponent in the fitting function was the mean of the second and
third exponent in the generating function.
The iterative curve fitting procedure was terminated when a stationary point was obtained
(reduction of less than 10- th of the sum of squares of residuals), after 50 iterations if conver-
gence was too slow or when the matrix of the normal equations became singular. The
solution was not accepted when one of the fitted exponents or coefficients was negative or
differed in order of magnitude from the starting values (convergence to a local minimum of
sum of squares of residuals).

Results
The means and standard deviations of the sum of squares of differences between simulated
and fitted data (residuals) were calculated for each set of 30 simulations, to determine the
validity of this criterion in estimating the number of exponential terms from which the data
were generated. As shown in Table IV,the sum of squares of residuals was mainly determined
by the magnitude of the data error and less by the systematic error, introduced by under-
estimation of the number of terms in the fitting function. Atencio et al(1965) and Takeda
(1966) have used the variance of the fit (Moore & Zeigler, 1962) as a criterion to justify their
two compartmental approach to the metabolism of fibrinogen. It is obvious that this criterion,
based on the sum of squares of residuals, does not allow one to determine, in a single experi-
mental situation, the number of terms from which the data are generated.
When fitting data generated from a sum of two exponential terms with a sum of two
exponentials, the fractional catabolic rate constant (k,o ) and the distribution ratio (EV/1V)
were accurately estimated as well for 2% as for 5% data error. The fractional efflux rate
constant (12, 2) was less accurately estimated, with a standard deviation of up to 20%.
When fitting data generated from a 3 u m of three exponentials with a sum of three, the
fractional catabolic rate constant (k,), was accurately estimated, but the distribution ratio
(EV/IV) and fractional efflux rate constant (k,), were subject to large error. Furthermore
convergence was only obtained in a half to two-thirds of the simulations, whereas conver-
gence was always obtained when approximating these data with a sum of two exponentials.
The systematic error, introduced by fitting data, generated from a sum of three expo-
nentials, with a sum of two exponentials (Table IV, error 0%) resulted in an underestimation
of the distribution ratio of radioactivity (EV/IV) and of the fractional efflux rate constant
(ki2) but had no significant influence on the estimation of the fractional catabolic rate
constant (ki0).
The combination of systematicand data error in fitting data, generated from a sum of three
 Normal Fibrinogen Turnover 697
exponentials with a sum of two exponentials, did not influence the estimation of the fractional
catabolic rate constant (klo). The radioactivity distribution ratio (EV/IV) and the fractional
efflux rate constant (k12) were always underestimated but the variability due to data error was
smallerthan when fittingwitha sumofthree exponentialsandconvergencewasalwaysobtained.
This study of the influence of systematic error and data error on the estimation of the
metabolic parameters has revealed that in the simulated experimental conditions, only
Plasma Extrovascular

PI
, ,
- F
l kl2

,421

Fibrin . Catabolic sites Fibrinogenolysis

Breakdown products

Excretion
FIG6. Compartmental model for the metabolism oflabelled fibrinogen in man. Radioactive fibrinogen
is injected in plasma in which rapid mixing occurs. The total plasma radioactivity at time t is repre-
sented by x(t). Radioactivity passes to the interstitium at a rate k,,x(t) per day and is catabolized at
a rate kl03t(t)=(klJ+Kl6+kl7)X(t). Interstitial radioactivity returns to the circulation at a rate kzl y(t),
y(f) representing the total radioactivity at time tin the interstitium. Radioactive non-TCA precipitable
breakdown products in the body z(t)are generated by basic protein turnover at a rate k34 v(t), by break-
down of fibrin at a rate ks4 w(t), and by breakdown of fibrinolytic degradation products at a rate k74
r(t). w(t) represents the total amount of fibrin, r(t) the total amount of TCA precipitable fibrinolytic
degradation products and u(t) the total amount ofradioactivitypresent in the catabolicsitesin the body.
Radioactive non-TCA precipitable degradation products in the body z(t) are excreted, mainly in the
urine, at a rate kq5 z(t).

meaningful results can be obtained for the fractional catabolic rate constant and, to a lesser
extent, for the radioactivity distribution ratio. These parameters are the most accurately
estimated when approximating the data with a sum of two exponentials regardless of the
number of terms in the generating function.
In view of the poor resolution obtained by non-linear exponential curve fitting with a
damped Gauss-Newton method, it has been investigated whether the computer analysis gives
a better estimation of the model parameters than graphical curve peeling on semilogarithmic
paper. Therefore 20 sets of experimental data have been generated from a sum of two
exponential terms x(t) = o.7e- '*I + o.3e- O r and 20 sets from sums of three exponential
terms x(t)=0.7e -'.I5: + 0.x-Oh4' +o. ~e-~.",with a normally distributed error of 5% and
698 D. Collen et a1
with 10 or 15 data points. These sets have been approximated with a sum of two exponential
terms by the computer and by visual graphical curve peeling. The estimations of the main
metabolic parameters by both methods are equivalent.
McFarlane et a1 (1964) used the extrapolation method of Sterling (1951)to calculate the
fractional catabolic rate constant (k,,= In 2/(T&). However, this method introduces a
systematic error, which amounts respectively to 4.3% and 16.3% in the estimation of the
fractional catabolic rate constant and the radioactivity distribution ratio, for plasma radio-
activity data derived from the function x ( t ) = 0 . 7 e - ~ . ' ~ ~0.3e-I.'~.
+

Mathematical Model f o r the Metabolism of Fibrinogen

The approximation of the plasma radioactivity data with a s u m of two exponential terms
corresponds to a two compartmental model with an intravascular and a single interstitial
compartment. However, such a model is oversimplified and inaccurate to represent the
metabolism and distribution kinetics of fibrinogen for two reasons: (a) daily fluctuations in
plasma fibrinogen concentration are frequently observed; (b) fibrinogen may be consumed
by coagulation or fibrino(geno)lysis in pathological and possibly physiological conditions.
Such catabolism has to be superimposed on the general plasma protein turnover. The modi-
fied diagram of labelled fibrinogen is shown in Fig 6. Such description of the metabolism
and distribution of labelled fibrinogen requires seven differential equations with seven time
varying coefftcients. These seven differential equations cannot be solved unless all rate co-
efficients are defined from the experimental data. After equilibration, x@),z(t)and I -Xui can
be described by a single exponential term with practically the same exponents, despite the
fluctuations in plasma fibrinogen concentration. This strongly implies, according to Atencio
et al (1965)~that all the rate coefficients can be considered as constants. Therefore, the flow
diagram of fibrinogen can be described in physiological conditions, by following set of linear
differential equations with constant coefficients.
(I) dx/dt= -k x(t) + k2,y(t)
(2) dy/dt=k,,x(t)- kz, y(t)
(3) W d t = k13x(t)- k34 v(t)
(4)dw/dt=kl -
k64
&(l) w(t)
(5) &dt= k 7 4 ) - k 7 4 4
+
(6) d~/dt=k,,v(t) k s q ~ ( t ) + k74r(t)- k45~(t)
(7) d@=k45 .(t)
+
with k = krz k,, + k16+ k,, and k,, = k, +kI6+ k,,
and with initial and final conditions
x(0) = I
y(o)= u(ol= w(o)=~ ( o=)r(o)= o
x(co)=y(co)=~(co)=W(oo)=f(co)=z(~)=o
u (co)=I

In vivo inhibition of coagulation by heparin (kl6--+o) and of fibrinolysk by tranexamic

acid (kI7+0) have revealed that in physiological conditions, consumption of fibrinogen by
either of these systems is not essential. However, these metabolic pathways have to be
 Normal Fibrinogen Turnover 699
included in the metabolic model, because the shortening of the fibrinogen half-life in patho-
logical conditions can be due to an activation of both systems.

ACKNOWLEDGMENTS

We thank Dr Sci. R. De Vreker, for his valuable cooperation, Mr F. De Cock and Miss A.
Verhagen for their skilful technical assistance and Dr M. De Roo and Dr M. Goris for their
advice and hospitality at the Department of Nuclear Medicine.
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