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Ktin, Wschr.

55, 175-t79 (1977)


Klinische
Wochen-
schrift
© by Springer-Verlag 1977

Evaluation of Hypercoagulability in Users of Oral Contraceptives*


H. Graeff, P. Battis, R. Hafter, and J. Zander
I. Frauenklinik der Universitfit Mfinchen (Direktor: Prof. Dr. J. Zander)

Erfassung der Hyperkoagulabilit~it bei Frauen per 100 ml plasma) amount of SFMC. Increased le-
unter oralen Kontrazeptiva vels of SFMC were observed in users of oral con-
traceptives, when compared to age, parity and socio-
Zusammenfassung. L6sliche Fibrinmonomerkom- economic background matched control groups. This
plexe (LFMK) wurden bei Frauen bestimmt, die ver- increase was more pronounced in users of Neogynon
schiedene Ovulationshemmer einnahmen. Plasmapro- (0.05 mg ethinyl estradiol, 0.25 mg norgestrel) than
ben wurden mit fl-Atanin gefS.1tt und durch Gelfiltra- in the ones using Microgynon (0.03 mg ethinyl estra-
tion der relative Gehalt an L F M K (Prozent des Ge- diol, 0.15mg norgestrel). Users of Ovulen (0.1 mg
samtfibrinogens) und der absolute Gehalt (mg pro mestranol, 1 mg ethynodiol diacetate) in addition to
100ml Plasma) bestimmt. Es wurden Frauen, die the rise of SFMC levels exhibited elevated fibrinogen
Ovulationshemmer einnahmen, mit Kontrollgruppen levels in plasma. The data presented indicate a limited
verglichen, die hinsichtlich Alters, Zahl der Schwan- state of hypercoagutability in users of oral contracep-
gerschaften und sozio6konomischen Milieus/.iberein- tives.
stimmten. Es konnte ein signifikant erh6hter Spie-
gel yon L F M K gegenfiber den Kontrollgruppen Key words: Hypercoagulability - Oral contraceptives
festgestellt werden. Dieser Anstieg war bei der - Soluble fibrin monomer complexes - Gel filtra-
tion.
Gruppe, die Neogynon (0,05mg)kthinyl6stradiol,
0,25 mg D-Norgestrel) einnahmen, gr6Ber als bei ei-
ner Gruppe die Microgynon (0,03 mg )kthinyl6stra-
diol, 0,15mg D-Norgestrel) einnahmen. Bei der
Gruppe, die Ovulen (0, t mg Mestranol, 1 mg Ethyno- Retrospective and prospective studies both show that
diotdiacetat) nahmen, konnte zusfitzlich zu dem An- the risk of developing various thromboembolic disor-
stieg der L F M K ein erh6hter Fibrinogenspiegel nach- ders is about 5 to 10 times greater among users of
gewiesen werden. Die Befunde zeigen, dab die Ein- oral contraceptives than among nonusers [1-3]. Un-
nahme yon Ovulationshemmern zu einer begrenzten fortunately no laboratory tests are yet available which
Hyperkoagulabilitfit ffihrt. will predict those individual women most at risk.
Sehliisselwiirter: Hyperkoagutabilitfit - orale Ovula- A so called state of hypercoagulability is observed
tionshemmer - 16sliche Fibrinmonomerkomplexe - in patients with predisposing conditions for throm-
Gelfiltration. boembolic disease, such as the early puerperium [4,
5], the postoperative course [6], or advanced stages
Summary. Soluble fibrin monomer complexes of cancer [7]. This hypercoagulability is reflected by
(SFMC) were determined in users of different oral the occurrence of thrombin mediated catabolic pro-
contraceptives. Quantitative gel filtration of/~-alanin ducts of fibrinogen, i.e. soluble fibrin monomer com-
precipitated plasma samples yielded the relative (per plexes (SFMC), in plasma. The physicochemical and
cent of total fibrinogen content) and absolute (rag immunologic properties of these components are in
agreement with the assumption of a dimeric structure,
which predominantly exists in plasma as a fibrinogen
* Supported by the "Deutsche Forschungsgemeinschaft, Son- fibrin monomer complex [8-10].
derforschungsbereich 51, M/inchen", grant no. B/6 Recently abnormal plasma fibrinogen chroma-
176 H. Graeff et al. : Hypercoagulability and Oral Contraceptives

0.4 ~

0.3-

0.2-

Fig. 1. Elution pattern following 4%


agarose gel filtration (Biogel A-15 m) of
a/~-alanine precipitated plasma sample.
0.1-
The percentage of SFMC (in relation to
the fibrinogen content) is determined by
comparing the cut of the eluate A with
the one of the fibrinogen peak (B)
according to the formula SFMC (%)
i. . . . =(A x 100/A+B)
2; 40 60 80 100 120 140 160 m l

ELUTION VOLUME (ml)

tographic findings [11, 12], and an increased uptake (average time of taking Neogynon: 21/2 years). The second group
of a 14C-glycine ethyl ester into soluble fibrin were consisted of females aged 18 tilt 26 years (average age 22 years),
who had taken Microgynon® (150 tag D-norgestrel and 30 p.g
observed in oral contraceptive users [13]. Both meth- ethinyl estradiol) for at least 3 months before the start of the
ods indicate the presence of SFMC in plasma. Estima- study (average time 9 months).
tion of SFMC by gel filtration of plasma samples, A third group consisted of 17 females aged 18-29 years (aver-
which were purified by fi-alanine precipitation, en- age age 22) who had never taken any oral contraceptive. Both
studies were performed under strict control such that investigator
ables a more direct and quantitative approach for making the analyses was not aware of the identity of the blood
the determination of the relative (percentage of donor in each case.
SFMC in relation to the total fibrinogen content)
and absolute (mg/100 ml plasma) amount of SFMC Collection of Blood Specimens and Plasma Preparation
in plasma [4, 5]. This method was therefore applied
for the evaluation of hypercoagulability in users of Blood was collected at the 20th or 21th day after beginning of
different oral contraceptives. menstruation in the time between 11 a.m. and 2 p.m. The blood
samples were drawn from the cubital vein by clean venepuncture
under constant mixing with the anticoagulant (1 : 10) (0.129 M triso-
dium citrate, 0.06 M N-Tris(hydroxymethyl)methyl-2-aminoethane-
Material and Methods sulfonicacid (TES), 1,000 KIU/ml aprotinine (Trasylol®, Bayer-
Leverkusen, Germany), pH 7.5). The hematocrit of the anticoagu-
Two studies were performed with healthy female volunteers of lated blood was determined with micro-hematocrit-tubes (Clay
similar socioeconomic background (hospital nurses and students Adams, Parsipanny N.J., USA). Platelet poor plasma was prepared
of medicine). The first one lasted from Dec. 1974 till Febr. 1975. by two subsequent runs in a refrigerated centrifuge (1,400 g, i5 rain,
Twenty one women between the ages of 18-30 years (average age 3,600 g, 10 min). The ethanol gelation test (EGT) was performed
23 years) took Ovulen®1 (1 mg ethynodiol diacetate and 100 gg according to Godal and Abildgaard [14] using Tris buffered
mestranol) for 2t days. They changed to Ovulen® after they had (pH 7.5) ethanol [15]. Plasma fibrinogen was assayed according
taken a different kind of oral contraceptive for at least 9 months to Blomb/ick and Blomb/ick [16] from 0.5 ml plasma samples in
(average 3.2 years). Twenty two female volunteers between the ages duplicate using an extinction coefficient of fibrin in alkali of 15.87
of I8 28 years (average age 22 years) served as control group. They (E 1%/t cm) [17]. The fibrinogen values were corrected for the
had never taken any oral contraceptive (n=14) or at least not hematocrit dependent dilution of the anticoagulant according to
in the last 9 months before the beginning of the study. Seligsohn et al. [18]. If it was necessary to store plasma samples,
The second study was performed in the period from March heparin was added (10 I.U./ml Liquemin®, Hoffmann-La Roche,
1975 till June 1975. The first group was composed of 13 females Grenzach, Germany) and the samples held at - 2 5 ° C. Plastic tubes
between the ages of 19 29 years (average 23 years). They had taken were used for handling and storage.
Neogynon®Z (250 p.g D-norgestrel and 50 gg ethinyl estradiol)
previously for at least 9 months before the beginning of the study Purification Procedure

1 Ovulen® (Boehringer, Mannheim, Germany) Fibrinogen and its derivatives were precipitated from plasma with
2 Neogynon® and Microgynon® (Schering, West-Berlin) fi-alanine. A 40 per cent aqueous solution of fi-alanine was added
H. Graeff et al. : Hypercoagulability and Oral Contraceptives I77

at 0°C under constant stirring to the plasma sample (3 mI) up Table 1. Amount of SFMC and fibrinogen in plasma of controls
to a final concentration of 2.5 M. The precipitate was spun down and of Ovulen users (mean values and standard deviation)
at 3,600 g (5 min, 4°C) and redissolved in 2 ml TES-Ct-citrate buf-
fer, pH 7.6 (0.06 M TES, 0.081 M NaC1, 0.0t29 M tri-Na-Citrate, Control Ovulen Significance
containing t00 KIU/ml Trasylol®) [19]. users

n 22 21
Agarose Gel Filtration and Quantitation of SFMC (number of subjects)
SFMC 2.9±0.5 3 . 8 _ + 0 . 7 p<0.001
4 per cent agarose get filtration was performed using Biogel A-15 M
(per cent of
spherical agarose, 100-200 mesh (Bio-Rad Laboratories). A 2 ml
fibrinogen content)
sample containing 6 mg protein was applied to a 1.5 x 95 cm col-
umn and eluted with Tris-Cl-citrate buffer, pH 7.5 (0.05 M Tris, SFMC 6.5_+1.8 9.9±2.3 p<0.001
0.116 M NaC1, 0.0129 M trisodium citrate, 0.025 M e-amino ca- (mg per I00 mI)
proicacid (EACA), 0,025% sodium azide). The flow rate was kept
Fibrinogen 224 -+45 263 ±41 p<0.005
at t3 ml/h (3 ml per fraction). The optical density was measured
(mg per 100 mt)
at 280 and 325 nm with a Hitachi 181 spectrophotometer and
the elution profile plotted on millimeter paper. The quantitation
of SFMC in per cent of the fibrinogen content was achieved by
plan±metric evaluation of the elution pattern (area of the shoulder tically significant difference. The calculated value of
between void volume and ascending part of the fibrinogen peak U was 77 and hence smaller than the critical value
versus fibrinogen peak), (see Fig. I). For further details and re- U 99 for c~0.001. The mean value and standard devia-
liability criteria of this method see Graeff et al. [5] and Hailer
et at. [4]. The absolute amount of SFMC in mg per 100 ml plasma
tion (S.D.) of the relative amount of SFMC (per cent
was calculated by relating the relative amount of SFMC to the of fibrinogen content) was 3.8+0.7. The respective
fibrinogen concentration in the respective plasma sample. values of the controls were 2.9_+0.5. (We are aware
that it is not quite correct to calculate the standard
deviation if the normal distribution of a population
Statistical methods
has not been proved. On the other hand these values
The chi square test was employed to prove the hypothesis of a are more useful than the range). The absolute amount
normal distribution in the populations of the groups. This hypothe- of SFMC (mg per 100 ml plasma) was also increased
sis had to be dropped in both studies (probably due to the small to a statistically significant difference. The calculated
number of individuals in the groups). Therefore the non parametric
test of Mann and Whitney was utilized. As a matter of principle
value of U was 44.5 and hence considerably smaller
we employed a onetailed test. Calculations were done on a com- than the critical value U 99 for 0.001. Mean value
puter Hp 98.30. and S.D. was 9,9_+2,3 for the group of Ovulen users,
and 6.5 +_ 1.8 for the control group.
The fibrinogen content of plasma was also statisti-
Results cally increased to a 0.005 level of probability. The
calculated value of U was 114.5 and accordingly smaller
The results of the two studies are summarized in Ta- than the critical value U 118 for ~ 0.005. The mean
bles 1 and 2. Additional statistical data are given in value and S.D. was 263_+41 and 224_+45 for the
the text. The first study shows that the level of SFMC control group.
had been increased under Ovuten dosage to a star±s- The second study revealed that in spite of a low

Table 2. Amount of SFMC and fibrinogen in plasma of controls and of Microgynon- and
Neogynon users

A B C Significance
Control Microgynon Neogynon I A: B
users users I1 B :C

n 17 13 13
(number of subjects)
SFMC 2.8_+0.3 3.4+0.3 3.7±0.5 I p<0.001
(per cent of lI p < 0.05
fibrinogen content)
SFMC 5.6±0.9 7.6_+ 1.7 7.7_+ 1.2 I p <0.005
(mg per t00 ml) II none
Fibrinogen 203 ±28 223 ±39 208 ±30 I none
(mg per 100 ml) II none
178 H. Graeff et al. : HypercoagulabiIityand Oral Contraceptives

dosage of 30 gg ethinylestradiol and t 50 ~tg D-norges- tion showed that the material which is eluted prior to
trel (Microgynon) a statistically significant increase the fibrinogen peak is fibrinogen related [8, 20]. A
(p<0.00t) in the level of SFMC was obtained. The main component showed an electrophoretic mobility
calculated U value was 15.5 and hence considerably in polyacrytamide (PAA)-get-electrophoresis at
smaller than the critical value U 38 for 0.001. The pH 8.6 of 0.28 x 10- 5 cm2/V x s [8]. Using affinity
mean value and S.D. was 3.4+0.3. The respective chromatography on insolubilized fibrinogen, the deri-
values of the controls were 2.8_+0.3. The absolute vative dissociated at a ratio of ahnost 1:1 into two
amount of SFMC (rag/100 ml plasma) also increased parts, one part, which was adsorbed, and into fibrino-
to values proved statistically significant (/)<0.005). gen, which was not adsorbed [10]. It might be con-
Further the second study revealed that taking a cluded that this complex, according to its elution be-
contraceptive with a normal dosage of 50 gg ethinyl haviour on agarose gel, its migration behaviour on
estradiol and 250 gg D-norgestrel (Neogynon) addi- PAA-gel, N-terminal analysis [9] and its elution pat-
tionally increased the level of SFMC to a statistically tern following affinity chromatography, is a dimeric
significant difference beyond the one induced by a drug fibrin-fibrinogen complex (at present designated:
with only 30 gg ethinyl estradiol (Microgynon). The soluble fibrin monomer complex, SFMC).
calculated value U was 50 and hence smaller than It might be assumed in correspondence with in
the critical value U 51 for c~0.05. The mean value vitro findings, that SFMC are produced in vivo by
and S.D. of per cent SFMC of Neogynon users was the action of thrombin also. The increase of the
3.7 _+0.5. There were no statistically significant differ- thrombin mediated catabolic products of fibrinogen
ences in the content of fibrinogen between users of (SFMC) thus reflects a state of hypercoagulability
Microgynon on the one hand and users of Neogynon via an activation of the coagulation system. This
on the other hand compared to the controls. means that the users of oral contraceptives in our
The ethanol gelation test was negative as defined study had a certain, though limited state of hypercoa-
by Godal and Abildgaard as well among members gulability. Interestingly this hypercoagulability was
of the control group as among all oral contraceptive more pronounced in users of Neogynon than in those
users. using Microgynon. If the different estrogen content
is considered, this observation could be related to
studies claiming a relationship between estrogen dos-
Discussion age and thromboembolism incidence [21].
The increase of fibrinogen levels in users of Ovulen
The data presented indicate that increased levels of might be discussed in view of the findings that steroid
SFMC are observed in users of oral contraceptives, agents are capable of increasing fibrinogen produc-
when compared to age, parity and socioeconomic tion by the liver. This is well in agreement with the
background matched control groups. This increase observation that mestranol increased the fibrinogen
was more pronounced in users of Neogynon (0.05 mg production by embryo hepatic parenchymal cells [22].
ethinyl estradiol, 0.25 mg norgestrel) than in the ones Yet ethinyl estradiol is also capable, though to a
using Microgynon (0.03 mg ethinyl estradiol, 0.15 mg somewhat lesser extent, of inducing an increased syn-
norgestrel). Users of Ovulen (0.1 mg mestranol, 1 mg thesis of fibrinogen in the tissue system whereas our
ethynodiot diacetate) in addition to the rise of SFMC results show that users of Neogynon and Microgynon
levels exhibited elevated fibrinogen levels in plasma. failed to exhibit increased fibrinogen levels. Therefore
SFMC levels were estimated in the present study additional regulatory or inhibitory factors have to
by planimetric evaluation of the elution pattern fol- be considered.
lowing agarose gel filtration of fractionated plasma Hypercoagutability per se might not be directly
samples. The reproducibility of the method is satisfac- related to an increased incidence of thromboembolic
tory according to the reliability criteria as recently disease, and pregnancy is often cited as an example
described [4, 5]. However, a source of error exists of hypercoagulabilitiy which is not accompanied by
since SFMC are not completely separated from fibri- an increased risk (for review see [23, 24]). Yet it could
nogen. The planimetric evaluation of the gel filtration be argued that if hypercoagulability of pregnancy is
pattern tends, according to the procedure involved, complicated by the additional risk factor of delivery
to result in probably too high estimations, if the elu- the risk of developing thromboembolic disorders is
tion area prior to the fibrinogen peak is very small. 20 times greater in the postpartum than in the ante-
Therefore, the indicated level of 2.8 per cent SFMC partum patient [23]. The SFMC level of approximate-
for normal plasma could well be overestimated. Chain ly 6 per cent of the total fibrinogen content well re-
characterization and the staphylococcal clumping flects the increased state of hypercoagulability of the
reaction in addition to the immunological identifica- early puerperium [5]. This interrelation between by-
H. Graeff et al. : Hypercoagutabitity and Oral Contraceptives t79

percoagulability and additional risk factor may also 10. von Hugo, R., Halter, R., Stemberger, A., Graeff, H. : Soluble
be of importance in the observation that the risk fibrin monomer complexes demonstrated by agarose gel filtra-
tion and by adsorption on insolubitized fibrinogen. A compara-
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oral contraceptive use and thromboembolism : A new approach
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