Clinical Science and Molecular Medicine (1973) 45,429438.
THE PLASMA KININ-FORMING SYSTEM IN LABOUR
M . MALOFIEJEW
Department of Physiology of Smooth Muscles, Institute of Obstetrics and Gynaecology,
The Medical School, Bialystok, Poland
(Received 13 Muy 1973)
SUMMARY
1. The plasma of non-pregnant women contains practically no kinins and only
traces of kallikrein. Treatment of the plasma with kaolin produces additional amounts
of kallikrein from prekallikrein and of kinin from kininogen.
2. During the second stage of labour, plasma kallikrein is increased and the amount
of kallikrein inhibitors is decreased. The plasma kinin-forming activity is only slightly
increased due, apparently, to a fall in plasma kininogen.
3. Plasma kallikrein causes a rise in the contractile tonus of the isolated rat and
human myometrium.
Key words : kallikrein, kinins, parturition.
In recent years, attention has been drawn to the role of the plasma kinin-forming system in
labour and in the physiological adaptation of the newborn. Armstrong & Stewart (1960)
showed that the plasma of women in labour produces contractions of the isolated uterus of the
rat, that this action was intensified by previously cooling the plasma in vitro and that it was not
due to the presence of oxytocin, angiotensin or amines.
Melmon, Cline, Hughes & Nies (1968) presented a hypothesis concerning the role of plasma
kinins in the change from foetal circulation to that in the newborn. They suggested that the
sudden cooling of the umbilical cord and the increase in the number of granulocytes in the
newborn’s blood activate kininogenesis. Kinins cause contraction of the arteries and veins of
the umbilical cord and, at the same time, dilate the vessels of the pulmonary circulation, which
would facilitate the child’s ‘first breath‘.
This paper presents an account of studies on plasma of prekallikrein, kallikrein and kinins
during the second stage of normal labour. Some of this report was presented at the Symposium
of the Polish Academy of Sciences on ‘Biological and Pharmacological Properties of Peptides’,
Bialowieza, Poland, 1972, and at the Twelfth Congress of the Polish Physiological Society,
Olsztyn-Kortowo, Poland, 1972.
Correspondence:Dr M. Malofiejew, Department of Physiology of Smooth Muscles, Institute of Obstetrics
and Gynaecology, Medical School, Bialystok-8, Poland.
429
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M . MaloJiejew
METHODS
Blood (9 vol) was drawn, without stasis, from the antecubital vein, into sodium citrate solution
(3.8 g/100 ml) (1 vol) after which it was centrifuged immediately at 4000 g for 30 min. From
the moment of drawing it from the vein, the blood, and subsequently its plasma, were kept in
glass or plastic vessels coated with several layers of silicone (Silliclad, Clay Adams).
Determinations of biological activity of plasma before and after treatment were made in an
organ bath by using isolated guinea-pig intestine in Tyrode's fluid at p H 7.3 gassed with O2 and
at a temperature of 37°C. In testing, 0.1 ml of plasma was added to 9.9 ml Tyrode's fluid.
Before, during and at the end of each experiment, the reaction of the intestine to increasing
doses of synthetic bradykinin (BRS-640, Sandoz) was determined. The biological activity of
plasma and its extracts was measured in terms of the reaction to bradykinin and expressed in
,ug/ml (equiv. as BRS). Reactions with isolated pieces of rat myometrium in the oestrus phase
and of the human myometrium obtained during Caesarean sections were studied under similar
conditions.
Kinin activity was determined in plasma, before and after treatment with an equal volume
of aqueous kaolin (Fisher Scientific Co., Fairlawn, N.Y.) suspension (10 mg/ml) or of NaCl
(0.9 g/lOO ml). Kallikrein measurement was based on the amount of kinins released during
incubation of plasma (0.1 ml) with substrate (0.2 ml) and EDTA (2 g/100 ml) (0.01 ml). The
detailed procedure was as follows: after incubation for 60 s at 37"C, the whole sample was
placed in an organ bath with the guinea-pig intestine and the kinin activity assayed. The substrate was fresh human plasma heated to 61°C and then acidified to pH 2.0 and incubated at
37°C for 30 min. Plasma thus prepared does not contain prekallikrein or kallikrein, displays no
kinin activity and the kininase and kallikrein inhibitor levels are considerably decreased
(Jahrreiss & Haberman, 1971; Movat, Poon & Takeuchi, 1971; Seidel, Stucker & Vogt, 1971)
Activation of plasma kallikrein was produced either by treating plasma (1 vol) with an equal
volume of kaolin (10 mg/ml) or by incubating with acetone (0.2 vol) for 5 h at 37°C and then
evaporating the acetone. In the kallikrein determinations, 0.1 ml of acetone-treated plasma was
diluted with NaCl(O.9 g/100 ml) to give dilutions in the range of lo-' to
The kallikrein
activity was found to be reasonably stable for a few days.
A typical experiment is illustrated by Fig. 1. The untreated plasma (sample 7) did not produce a contraction of the ileum. Plasma treated with kaolin (sample 8) produced a small contraction. Plasma heated for 1 h a t 61°C and acidified, i.e. the substrate used for the kallikrein
tests (sample 9) did not produce any effect.
When plasma (0-1 ml) was shaken for 30 s with an equal volume of kaolin and then incubated
for 60 s with 0.1ml of the substrate (sample 10) it induced a marked contraction of the intestine.
Incubation with increased amount of the substrate (0.2 ml) produced greater kinin activity
(sample 11). When the amount of substrate was further increased (0.4 ml), no further rise in
kinin activity was observed and in some samples there was less activity (sample 12). This could
have been due to the presence of a small amount of kallikrein inhibitor or of kininases. In
susbsequent experiments only 0.2 ml of substrate was used. The plasma kallikrein activity was
taken to be the difference between the kinin activity of plasma samples incubated with (sample
11) and without (sample 8) substrate.
To measure 'total kinin-forming capacity' in plasma, 1 ml was incubated with 0.2 ml of
acetone and 0.1 ml of EDTA (2 g/lOO ml) for 5 h at 37°C. After evaporation of the acetone and
Plasma kinin-forming system in labour
431
termination of the enzymic reaction in the sample by heating for 10 min in boiling water, the
amount of newly formed kinins was determined by using isolated guinea-pig intestine.
In studying the effect on plasma of treatment with kaolin or acetone, material was prepared
by incubating acetone or kaolin-treated plasma (1 vol), EDTA (2 g/lOO ml) (0.1 vol) and substrate (2 vol). After incubation for 1 min at 37”C, the enzyme reaction was terminated by heating for 10 min in boiling water. The material was then centrifuged (30 min at 18 000 g) and the
supernatant dialysed for 24 h at 4°C against an equal volume of distilled water.
I
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m
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2 3 4
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5
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FIG.1. Determination of plasma kallikrein. Samples 1-6, effect of bradykinin (BRS-640) in doses
of 0.5, 1, 5, 10,50 and 100 ng respectively. Sample 7,O.l ml of untreated plasma. Sample 8,O.l ml
of plasma treated with kaolin. Sample 9,0.2 ml of substrate. Sample 10,O.l ml of plasma treated
with kaolin +0.1 ml of substrate. Sample 11, 0.1 ml of plasma treated with kaolin +0.2 ml of
substrate. Sample 12, 0.1 ml of plasma treated with kaolin +0.4 ml of substrate. The ratio of
plasma/Tyrode’sfluid was 1 :99 and the sample tested was in contact with the isolated guinea-pig
intestine for 60 s.
The pharmacological characteristics of the preparation were investigated by using isolated
guinea-pig intestine in Tyrode’s fluid to which were added successively atropine (0.1 pglrnl),
promethazine (1 pg/ml) and lysergide (10 pglml). To study the proteolytic enzymes on plasma
treated with kaolin or acetone, 1 volume was incubated with either 1 volume of a-chymotrypsin (from bovine pancreas ; Koch-Light Laboratories, Colnbrook, Bucks.) solution (final
concentration 100pg/ml) or 1 volume of trypsin (Merck) solution (final concentration 1 mg/ml)
at 37°C and at pH 7.8. In testing, 0-2volume of incubated material was used, after termination
of the enzymic reaction by heating for 10 min in boiling water.
To study the effect of plasma kininases on the plasma treated with acetone or kaolin, 1
B
432
M. Malojiejew
volume was incubated at 37°C with 1 volume of normal plasma diluted 1 in 10 with NaCl
(0-9 g/100 ml) and either I volume of NaCl(0-9 g/100 ml) or 1 volume of EDTA (2 g/l00 ml).
Synthetic bradykinin (10 ng/ 100 ml) was treated similarly.
RESULTS
Pharmacological characteristics of plasma treated with acetone or kaolin are illustrated in
Figs. 2-4. The factor responsible for the pharmacological activity appearing in the plasma after
treatment with kaolin or with acetone did not disappear during heating and passed through
the dialysis membrane.
'
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(b)
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i
4
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Inn
1
2
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O D O
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FIG.2. Reaction of isolated guinea-pig intestine to kinin-like activity of the plasma samples in
Tyrode's fluid on the successive addition of the inhibitors atropine (0.1 pg/ml, a), promethazine
(1 pg/ml, b) and lysergide (10 pg/ml, c). 1, acetylcholine (La Roche); 2, histamine (Polfa);
3, serotinin (Sandoz); 4, bradykinin (Sandoz); 5, plasma. Activities were calculated as the response
expressed as a percentage of that obtained without inhibitor.
An example of the effect of kaolin treatment on the plasma kinin and kallikrein of a normal
woman is shown in Fig. 5. In twenty normal (non-pregnant) women aged 20-35 the plasma
before treatment contained practically no kinin activity and had a mean kallikrein activity (as
BRS) of 20.85 fSEM 0.002 ng/ml. After shaking the plasma with kaolin for 30 s, it was found
to have a mean kinin concentration (as BRS) of 101.25kSEM 0.045 ng/ml and a mean
kallikrein activity (as BRS) of 3-0+0-051pg/ml. Further shaking with kaolin caused a fall in
the kinin concentration and in the kallikrein activity.
Blood was also obtained from thirty women during the second stage of labour. The fresh
plasma had no kinin activity whereas the mean kallikrein activity (as BRS) was l.O+SEM
0.063 pg/ml. After shaking the plasma with kaolin for 30 s, the plasma contained only traces of
kinin but the mean kallikrein activity (as BRS) was 70-0kSEM 0.123 pg/ml. A typical example
of this effect is shown in Fig. 5(b). Further treatment with kaolin resulted in a complete dis-
Plasma kinin-forming system in labour
433
150 -
I
-2I
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Dumtkn of incubation (h)
Fro. 3. The effect of a-chymotrypsin ( 0 ) trypsin ( 0 ) on the kinin activity of plasma treated with
acetone. The arrow indicates the addition of a-chymotrypsin to the system containing trypsin.
See text for the conditions of incubation and concentration of reactants.
A
10
20
30
Duration of Incubation (mi)
FIG.4. The effect of incubating plasma, previously treated with kaolin to activate kinins, with
fresh plasma in presence ( 0 )and absence ( 0 )of EDTA and synthetic bradykinin with fresh plasma
in the presence (m) and absence ( 0 )of EDTA. See text for conditions of incubation and concentration of reactants.
M . Malofiejew
434
appearance of kinins from the plasma and the mean kallikrein activity (as BRS) fell to 2.7 1
0.052 pg/ml; it was not influenced by shaking with kaolin for a further 60 min.
Treatment of plasma with acetone showed a similar qualitative effect of labour but the increase in kallikrein activity was considerably greater than with kaolin treatment. The mean
kallikrein activity of the acetone-treated plasma of twenty women in the second stage of labour
was 60.5 times greater than the mean kallikrein activity in twenty non-pregnant women. For
example, kallikrein with activity of 0.1 pg/ml (as BRS) occurs in the plasma dilution of
women in labour in the range
x 4-3(f0.044), and in the plasma of non-pregnant women
in the dilution in the range
x 7.1 (f0.047). This difference was statistically significant
( P <0.05) despite the fact that there was considerable within-group variation.
L
c
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0
05
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1060
Duration of treatment (min)
FIG.5. Typical effect of kaolin treatment on kinin activity (0)and kallikrein ( 0 ) in the plasma of
(a) a non-pregnant woman and (b) of a woman in the second stage of labour.
The total kinin forming capacity of plasma was measured in twenty non-pregnant women
and in twenty women in the second stage of normal labour. The total kinin-forming capacity
in the plasma during the second stage of labour was three times higher than that of women
who were not pregnant (Fig. 6). The differences in the mean values for the test and control
groups were statistically significant ( P = 0.05).
A crude kallikrein preparation obtained from plasma incubation with acetone, was dialysed
for 24 h at 4°C against distilled water to remove as many kinins as possible. This preparation
did not itself cause a contraction of the isolated guinea-pig intestine whereas, when incubated
with kininogen, it released kinins. The kallikrein preparation was found to cause an increase
in the tonus of the isolated rat uterus, determined by the isotonic technique. When the same
dose of the crude plasma-kallikrein preparation was applied repeatedly at half-hour intervals, a
gradual fall in the contractile effect was noted.
Plasma kinin-forming system in labour
435
The effect of crude plasma kallikrein on the contractile activity of an isolated pregnant myometrium was studied by means of the isometric technique on five different samples of human
uteri obtained during Caesarean sections. Under the influence of kallikrein, the contractile
activity became more rhythmical and the amplitude and force of the various contractions
increased.
T
C
L
FIG.6. Total plasma kinin-formingcapacity expressed in pg/ml as BRS.C, controls (non-pregnant
women); L, women in second stage of labour. Column heights represent mean values (n = 20),
vertical lines represent 1 SD either side of the mean.
DISCUSSION
Treating plasma with kaolin or incubating it with acetone produced material that caused
contractions in isolated guinea-pig intestine in Tyrode's fluid made up with atropine, promethazine and lysergide. This pharmacological activity was destroyed by incubation with achymotrypsin or on contact with fresh plasma but it remained if EDTA was added. On incubation of plasma treated with trypsin, there was a slight increase in activity and in the next stage
when the sample was subsequently treated with a-chymotrypsin, the activity declined very
gradually. This may have been due to the formation of additional products which potentiated
the kinin activity.
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M . Malo$ejew
These findings suggest that polypeptides with kinin-like properties are formed in plasma on
contact with kaolin or incubation with acetone, as a result of an enzymic reaction (Moskowitz,
Schwartz, Michel, Ratnoff & Astrup, 1970). When contact with a rough surface is minimized,
fresh plasma contains practically no kinins and has only traces of kallikrein activity. The contact of plasma with kaolin causes a sudden activation of the kinin-forming enzymes. This
activity disappears within 10 min. The kinin-forming activity is accompanied by a brief appearance of kinins.
As several authors have shown, the method employed in this study is aimed primarily at
determining plasma kallikrein in terms of kinin-forming activity (Jahrreiss & Haberman, 1971;
Movat et al., 1971; Seidel et al., 1971). For that reason it has been assumed, with some reservation, that the kinin-forming activity reflects mainly the plasma kallikrein activity.
The change in kallikrein activity in the plasma of non-pregnant women on treatment with
kaolin is similar to the effect noted by Sherry & Colman (1968), who determined the amount of
kallikrein by measuring the plasma esterase activity. It is not yet known whether determination
of the esterolytic activity of the plasma is equivalent to measurement of its kinin-forming
activity. Some authors (e.g. Jahrreiss & Haberman, 1971) consider the two activities to be unrelated; parallel evaluations of plasma kallikrein by means of the two methods were not possible
in the present study. It seems likely that the evaluation of kinin-forming activity of kallikrein
based on the amount of kinins released from kininogen is a less accurate but a more specific
method than that involving determination of the esterase activity.
The explanation of the increase in kinin-forming activity observed after a brief contact of
plasma with kaolin and its fall during the next few minutes of incubation, offered by Sherry &
Colman (1958) and Colman, Mason & Sherry (1969), seems likely to be correct. Harpel (1970)
has presented a similar view which suggests that the difference between the amount of kallikrein initially present in plasma and the amount of kallikrein present after contact with kaolin
is a valid measure of prekallikrein. In contrast, the speed with which kallikrein disappears is a
measure of plasma kallikrein inhibitor activity with the reservation that a small amount of
kallikrein may be absorbed on the surface of the kaolin.
Fresh systemic blood plasma drawn during the second stage of labour has a kallikrein
activity approximately 20 times greater than that of the plasma from non-pregnant women.
Brief contact of such plasma with kaolin produces a further increase in kallikrein activity
which persists for at least 1 h. The plasma prekallikrein content is less during labour than it is
normally.
On incubation with acetone at 37"C, a gradual increase in plasma kallikrein activity occurs,
reaching a plateau after 4 h. In plasma incubated with acetone alone, the endogenous kininogen
is converted by kallikrein into kinins and the kinins broken down by the kininase present.
After incubation kinins are absent; kallikrein, on the other hand, can be found only when
incubation is carried out in the presence of kininogen.
The difference between the kallikrein level in the plasma incubated with kaolin and that in
the plasma incubated with acetone is difficult to explain. It seems that in the presence of
acetone not only does a conversion of prekallikrein take place in the plasma but also that
kallikrein is released from inactive complexes with the plasma proteins such as q m a c r o globulin or IgG (Jahrreiss & Haberman, 1971).
When EDTA (sodium salt) is present, kinins released by kallikrein are not broken down by
kininase in this system. The kinin activity determined in this experiment reflects the total
Plasma kinin-forming system in labour
437
kinin-forming capacity of fresh plasma since it indicates the amount of kinins that can be
released from the endogenous kininogen by the endogenous kallikrein produced on incubation
with acetone. The amount of kallikrein in blood plasma obtained during the second stage of
labour and activated by acetone is 60-5times greater than that ofthecontrol plasma. In contrast,
the total kinin-forming activity of the plasma from women in labour is only three times higher
than the total kinin-forming activity of the control plasma.
These results reveal a complicated pattern of changes taking place in the kinin-forming
system of systemic blood during normal labour. In spite of the high level of kallikrein activity in
the plasma, the possibility of more being formed from prekallikrein and a simultaneous fall in
plasma kallikrein inhibitors, the formation of plasma kinins is minimal. This may be due to the
fall in the level of plasma kininogen during labour possibly as a result of its being used up
(Periti & Gasparri, 1966; Wiegershausen, Hennighausen, Paegelow & Klausch, 1970).
The results throw light on kininogenesis in blood only, and they cannot be regarded as a
reflection of processes which take place in the blood vessels of the uterus. Bradykinin and
plasma kinins are known to be factors in inducing contractions of the rat myometrium. Preliminary investigations have shown, however, that plasma kallikrein devoid of kinins also induces a temporary rise in initial tonus with a simultaneous increase in the spontaneous contractile activity of the uterus. These studies were carried out on the isolated rat uterus, with the
blood vessels washed several times with physiological solution.
It has also been found that the crude plasma kallikrein preparation causes a rhythmic, spontaneous, contractile activity and a rise in the amplitude of the various contractions of the isolated sections of the human myometrium. This suggests that plasma kallikrein is capable not
only of releasing kinins from the plasma kininogen but also from kininogen in the uterine tissue
or with kininogen which is pressed out of the uterine tissue during the spontaneous contractions.
From these observations, it appears that during labour the kinin-forming system in the
circulating blood is more active and this may have a significant effect on the contractile activity
of the uterus.
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J.W. (1960) Spontaneous plasma kinin formation in human plasma collected
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