1549

Coagulation and Fibrinolysis

Elevated plasma TFPI activity causes attenuated TF-

dependent thrombin generation in early onset
preeclampsia
Karl Egan1,2*; Hugh O’Connor3*; Barry Kevane2,3,4; Fergal Malone3,5; Aine Lennon4; Amani Al Zadjali2; Sharon
Cooley1; Cathy Monteith3,4; Patricia Maguire2,6; Paulina B. Szklanna2,6; Seamus Allen1,2; Naomi McCallion3,4;
Fionnuala Ní Áinle1,2,3,4
1
School of Medicine and Medical Sciences, University College Dublin (UCD), Ireland; 2UCD Conway SPHERE Research Group, Dublin, Ireland; 3The Rotunda Hospital, Dublin, Ireland;
4
Department of Haematology, Mater Misericordiae University Hospital, Dublin, Ireland; 5Royal College of Surgeons in Ireland, Dublin, Ireland; 6School of Biomolecular
and Biomedical Sciences, UCD, Dublin, Ireland

Summary significantly reduced in patients with EOP compared to pregnant

con- Early onset preeclampsia (EOP) is a pregnancy-specific proinflamma- trols, most significantly in cases of severe EOP. EOP patients
displayed tory disorder that is characterised by competing thrombotic and a trend towards an increased response to endogenous

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activated pro- bleeding risks. It was the aim of this study to characterise thrombin tein C and thrombomodulin relative to
pregnant controls. Plasma tis- generation, a major determinant of thrombotic and bleeding risk, in sue factor pathway inhibitor (TFPI)
activity was increased in EOP pa- order to better understand the haemostatic balance in patients with tients. Inhibition of TFPI
abolished the attenuation of thrombin gener- EOP. Patients with EOP were recruited at the Rotunda Hospital, Dublin. ation stimulated
by low-dose TF. In conclusion, patients with EOP are Twenty-six cases of EOP were recruited over a 21-month period, out of
characterised by an attenuated coagulation response characterised by 15,299 deliveries at the Rotunda. Blood samples were collected
into reduced thrombin generation stimulated by low-dose TF and elevated sodium citrate plus corn trypsin inhibitor anticoagulated
vacutainers, plasma TFPI activity. These changes in coagulation may modulate platelet-poor plasma was prepared, and calibrated
automated throm- thrombotic risk and bleeding risk in patients with EOP.
bography was used to assess thrombin generation. Results were com-
pared to age and sex-matched non-pregnant controls (n=13) and age- Keywords
and gestation-matched pregnant controls (n=20). The rate and extent Pregnancy, tissue factor, tissue factor pathway inhibitor
of thrombin generation triggered by low-dose tissue factor (TF) was

Correspondence to: Financial support:

Fionnuala Ní Áinle This work was supported by funding from Health Research Board Ireland
Department of Haematology (HRA_POR/2013/377) and Friends of the Rotunda.
Mater Misericordiae University Hospital
Dublin 7 & Rotunda Hospital Received: December 21, 2016
Dublin 1, Ireland Accepted after major revision: May 9, 2017
E-mail: fniainle@mater.ie Epub ahead of print: June 1, 2017
https://doi.org/10.1160/TH16-12-0949
* HOC and KE contributed equally to this study and should be considered Thromb Haemost 2017; 117: 1549–1557
joint
first authors.

Introduction preeclampsia as- sociated with intrauterine

growth restriction (IUGR) (3, 4). How- ever,
Preeclampsia (defined as new hypertension preeclampsia is also associated with
presenting after 20 weeks with significant significant bleeding risks, due to
proteinuria) is a serious complication of complications such as placental abruption
pregnancy with potentially life-threatening (which may complicate severe PE), HELLP
consequences for both mother and baby (1). (haemolysis, elevated liver enzymes and low
It is a major public health issue, affecting platelets) syndrome, post-partum
more than half a million pregnancies in the haemorrhage, and renal
United States each year (2).
The risk of venous thromboembolism (VTE;
comprising deep- vein thrombosis [DVT] and
pulmonary embolism [PE]) is in- creased in
preeclampsia, particularly in severe
© Schattauer Thrombosis and Haemostasis
2017 8/2017
impairment. Accordingly, there has been
significant variation in the reported VTE risk
associated with preeclampsia (4).
We hypothesised that a detailed
characterisation of coagulation in patients
with preeclampsia would improve our
understanding of the overall haemostatic
balance in this patient cohort. We fo-
cussed on patients with early onset
preeclampsia (EOP; onset <34 completed
gestational weeks), a rare form of
preeclampsia but a purer disease
phenotype. EOP is characterised by more
pro- nounced placental dysfunction (5), an
elevated anti-angiogenic state (6), and
increased maternal inflammation (7). It
confers a 20-fold increased risk of maternal
death and an increased risk of severe fetal
complications including intrauterine growth
restric- tion (IUGR), preterm birth, and fetal
death (8).
The primary aim of this study was to
characterise coagulation activation in
patients with EOP using calibrated
automated thrombography and compare
the results to age and gestation-

Thrombosis and Haemostasis © Schattauer

8/2017 2017
155 Egan, O’Connor, et al. Coagulation in early onset
0 preeclampsia
matched pregnant controls and age- and
sex-matched non-preg- nant controls. We
subsequently performed a “mechanism of
ac- tion” study to determine factors that
could underlie the observed changes in
thrombin generation in EOP. The secondary
aim of the study was to compare coagulation
activation in EOP patients with non-severe or
severe disease with pregnant controls and
non-preg- nant controls.
Methods
Patient recruitment
hypertension.
Blood collection and preparation
Voluntary, informed, written consent was
obtained. Ten ml blood samples were
collected into vacutainer tubes containing
sodium citrate (0.105 mol/l final
concentration) plus corn trypsin inhibitor (50
µg/ml CTI; Haematological Technologies Inc.,
Essex Junction, VT, USA). Platelet-poor
plasma (PPP) was prepared by centrifu-
gation at 3000xg for 20 minutes (min) at
room temperature and stored in aliquots at
-80°C until analysis.

This study was performed in the Rotunda

Hospital, Dublin, Ire- land, a tertiary referral
centre with almost 9000 deliveries per year.

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The study was performed in line with the
Declaration of Helsinki. Ethical approval was
obtained from the Hospital Research Ethics
Committee. Pregnant women with EOP were
recruited by a senior obstetric fellow with
specific expertise in phlebotomy. In accord-
ance with local international clinical
guidelines (1, 9, 10). EOP was defined as
preeclampsia (new hypertension after 20
weeks ges- tation with significant
proteinuria) developing before 34 com-
pleted gestational weeks. Participants were
classified as having non-severe or severe
disease based on the presence of multiorgan
involvement in severe cases. Patients with
severe disease were characterised by any of
the following: severe hypertension
(SBP>160 or DBP>110 mmHg) twice ≥4
hours apart, thrombocy- topenia (platelet
count <100,000/µl), impaired hepatic
function or severe unexplained right upper
quadrant abdominal pain, new- onset renal
impairment, pulmonary oedema, new-onset
cerebral or visual disturbances. Patients with
severe anaemia and patients receiving
prophylactic or therapeutic anticoagulant
therapy or with known previous thrombosis
or inherited coagulation or in- flammatory
disorders (e.g. systemic lupus
erythematosus) were excluded. Age- and
gestation-matched healthy pregnant controls
and age- and sex-matched healthy non-
pregnant controls were also recruited at the
Rotunda Hospital, Dublin, by the same obste-
trician. Within the non-pregnant control
cohort, time within the menstrual cycle was
not recorded. Within the pregnant control co-
hort, one patient subsequently developed
late onset preeclampsia (non-severe) and
one patient developed gestational
hypertension. No pregnant control had a
history of preeclampsia or gestational
 Egan, O’Connor, et al. Coagulation in early onset preeclampsia

estimated to be required to demonstrate a

Calibrated automated thrombography ~30 % difference in thrombin gen- eration
parameters between healthy pregnant
Thrombin generation was assessed by controls and EOP patients (with severe and
calibrated automated thrombography using non-severe disease) with 90–95 % power.
a Fluoroskan Ascent Plate Reader (Thermo- Data were analysed using GraphPad Prism
Lab System, Helsinki, Finland) in (Horsham, PA, USA). Within the text, data
combination with Thrombino- scope are expressed as mean plus or minus stan-
software (Thrombinoscope BV, Maastricht, dard deviation. The Kolmogorov-Smirnov test
the Nether- lands). PPP (80 µl) was was used to deter- mine if data sets were
incubated with 20 µl “platelet-poor plasma parametric or non-parametric. Non-para-
(PPP) reagent” containing 1 pM tissue factor metric data sets were compared using the
(TF) and 4 µM phos- pholipid vesicles (60 % Kruskal-Wallis test fol- lowed by Dunn’s
phosphatidylcholine, 20 % phosphatidylser- multiple comparison test or Mann-Whitney
ine, 20 % phosphatidylethanolamine). test. Parametric data were analysed by one-
Thrombin generation was initiated by way ANOVA followed by Bonferroni’s multiple
automatic dispensation of fluorogenic comparison test or Student t-test. Within fig-
thrombin sub- strate (Z-Gly-Gly-Arg- ures, the median is used to demonstrate
AMC.HCl) and 100 mM CaCl2 into each well which data sets are non-
(final concentrations, Z-Gly-GlyArg-AMC.HCl,
0.42 mM and CaCl2, 16.67 mM). Thrombin
generation was determined using a
thrombin calibration standard and
characterised by measurement of specific
parameters, including lag time to initiation
of thrombin generation, peak thrombin
generated, time to peak thrombin gen-
eration, the velocity index, and the area
under the thrombin gener- ation curve
(endogenous thrombin potential, ETP).
Thrombin generation was characterised in
the presence and absence of acti- vated
protein C (APC), soluble thrombomodulin
(sTM) and a sheep polyclonal anti-tissue
factor pathway inhibitor (TFPI) anti- body
(Haematological Technologies Inc.). All
samples were ana- lysed in duplicate by a
single operator. Intra- and inter-assay varia-
bility in thrombin generation assay
parameters were in line with those reported
by other laboratories (11). The operator
was blinded to the patient grouping of each
sample until the time of statistical analysis.

Protein S levels, TFPI levels, and TFPI activity

Free Protein S levels were measured using
the HemosIL® Free Pro- tein S kit on an ACL
TOP 500 analyser (Instrumentation Labora-
tory, Bedford, MA, USA). Free plasma TFPI
levels were measured using the Human TFPI
Quantikine ELISA kit (R&D Systems, Oxon,
UK). Plasma TFPI activity was measured
using the Actich- rome® TFPI activity assay
(Sekisui Diagnostics, Lexington, MA, USA).

Statistics
Based on previous studies assessing
thrombin generation parame- ters in healthy
pregnant controls, a sample size > 6 was
parametric, while the mean plus or minus
the 95 % confidence in- terval are used to
demonstrate data sets which are
parametric. Correlations between variables
were assessed using the Spear- mann Rank
correlation coefficient. P-values <0.05 were
consider- ed statistically significant. * = p <
0.05. ** = p < 0.01. *** = p < 0.001.
Results
Patients with EOP were recruited at the
Rotunda Hospital, Dublin over a 21-month
period, during which 15,299 deliveries were
rec- orded. Of the 15,299 deliveries, 334
(2.2 %) were complicated by preeclampsia.
The majority of cases (n = 294) were
classified as
late-onset preeclampsia (>34 weeks
gestation) while 40 cases were classified as
EOP (<34 weeks gestation). Recruitment was
not pos- sible in two cases due to brevity of
the interval between clinical presentation
and delivery. Therefore, recruitment was
considered in 38 patients. Of these patients,
10 were excluded due to estab- lished
exclusion criteria (severe anaemia,
anticoagulant use, prior thrombosis,
inherited coagulopathy or inflammatory
disorders). Of the remaining 28 patients, 26
provided consent for recruitment. Of the 26
patients, 14 were considered to have non-
severe disease while 12 patients had severe
disease. Thirteen age- and sex- matched
non-pregnant controls and 20 age- and
gestation- matched pregnant controls were
also recruited. Patient demo-
graphic information is provided in ▶Table 1.

n Age T Gestation Smoking Nulliparous BMI

(years) a (weeks) (%) (%) kg/m2
b
Non-pregnant controls 13 33.7 ± 6.5
l n/a 0% 46.2% 24.8 ± 3.9
Pregnant controls 20 32.5 ± 5e 30.7 ± 3 5% 65% 25.8 ± 5.4
EOP 26 35.1 ± 5 31.8 ± 2 3.4% 68% 28.5 ± 5.5
1
Non-severe EOP 14 34.6 ± 5 31.6 ± 1.8 5% 71.4% 30.1 ± 5.2
:
Severe EOP 12 35.7 ± 5.8 32.1 ± 2.3 0 66.7% 27.1 ± 5.5
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i Figure 1: Thrombin generation D) Peak thrombin generation. E)
a in early onset preeclampsia. Endogenous thrombin potential
t Thrombin generation triggered Parametric data sets are
i by low-dose TF was measured in expressed as the mean ± 95 %
o plasma from non- pregnant confidence interval. Non-para-
n controls (n = 13), pregnant metric data sets are expressed
) controls (n = 20) and patients as the median. * A p < 0.05 was
. with early onset preeclampsia (n considered statistically
= 26). A) Lagtime to thrombin significant.
generation. B) Time to peak
thrombin generation. C) Velocity
index of thrombin generation.

© Schattauer Thrombosis and Haemostasis

2017 8/2017
Figure 2: Thrombin generation in non-severe and severe early = 12). A) Velocity index of thrombin generation. B) Peak Thrombin

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onset preeclampsia. Thrombin generation triggered by low-dose TF gener- ation. Parametric data sets are expressed as the mean ± 95 %
was com- pared in pregnant controls (n = 20), patients with non- confidence in- terval. Non-parametric data sets are expressed as the
severe early onset preeclampsia (n = 14), and patients with severe median. A p < 0.05 was considered statistically significant.
early onset preeclampsia (n

duced in non-pregnant controls (119.3 ± 34.5

Thrombin generation triggered by low-dose TF in EOP nM, n = 13, p < 0.05) and patients with EOP
(150 ± 80 nM, n = 26, p < 0.05)
Thrombin generation triggered by low-dose (▶Figure 1D).
TF (1 pM TF) was measured in PPP. Plasma Because thrombin generation triggered by
anticoagulated with sodium citrate plus CTI low-dose TF was at-
was used to exclude the effect of artefactual tenuated in patients with EOP, we
contact activation that occurs during blood investigated if disease severity influenced
collection. Compared to non-pregnant thrombin generation. When the rate of
controls and pregnant controls, EOP patients thrombin gen- eration and peak thrombin
were characterised by a prolongation of the generation in pregnant controls were
lagtime and time to peak thrombin gener- compared to patients with either non-severe
ation (▶Figure 1A, B). The lagtime to or severe EOP, thrombin generation was only
significantly attenuated in patients
initiation of thrombin gen-
eration was 6.8 ± 0.6 min in non-pregnant with severe EOP (▶Figure 2A, B).The rate of
controls (n = 13), 7.0 ± thrombin gener-
1.2 min in pregnant controls (n = 20) but ation was similar in pregnant controls (41 ± 27
significantly prolonged at 8.5 ± 2.0 min in nM IIa/min, n =
patients with EOP (n= 26, p < 0.05) (▶Figure 20) and patients with non-severe EOP (25 ±13
1A). In a further demonstration of nM IIa/min, n =
attenuated thrombin gener-
ation in EOP, the rate of thrombin generation
(velocity index) and peak thrombin
generated were significantly reduced in
patients
with EOP compared to pregnant controls
(▶Figure 1C, D). The
velocity index was 41 ± 27 nM/min in
pregnant controls (n = 20)
but significantly reduced in both non-pregnant
controls (19.2 ± 8.0 nM/min, n = 13, p<0.05)
and patients with EOP (25 ± 17 nM/min,
n = 26, p<0.05) (▶Figure 1C). Similarly, peak
thrombin gener-
ation was 210 ± 70 nM in pregnant controls
but significantly re-
14) but significantly reduced in patients with
severe EOP (22 ± 22 nM IIa/min, n = 12)
compared to pregnant controls ( ▶Figure 2A).
Similarly, peak thrombin generation was
similar in pregnant
controls (206 ± 70 nM, n = 20) and patients
with moderate EOP (162 ± 57 nM IIa, n =
14) but significantly reduced in patients
with severe EOP (136 ± 102 nM IIa, n = 12)
compared to pregnant con-
trols (▶Figure 2B).

TFPI and Protein S in early onset preeclampsia

Under the CAT assay conditions used in this
study, thrombin gen- eration is heavily
dependent on the levels of anticoagulant
factors, particularly TFPI and protein S (12).
As such, we investigated pos- sible changes
in protein S and TFPI levels in EOP, as a
possible mechanism underlying the
attenuated thrombin generation re- sponse
in EOP. Plasma volume constraints,
particularly in the case of EOP patients, was
responsible for the small variations in the
number of mothers contributing samples to
each assay.
Free TFPI levels were increased in patients
with EOP (27.1 ±
7.8 ng/ml, n = 21) relative to pregnant
controls (21.7 ± 10.9 ng/ml, n = 17, p < 0.05,
▶Figure 3A). The increase in free TFPI levels
in EOP was most apparent in cases of severe
disease (32.6 ± 7.4 ng/
ml, n = 10). Patients with severe disease
displayed significantly in- creased free TFPI
levels compared to both non-pregnant (22.3
±
7.8 ng/ml, n = 13, p < 0.05) and pregnant
controls (21.7 ± 10.9 ng/ ml, n = 17, p <
0.05, ▶Table 2). There was no significant
differ- ence in free TFPI levels in non-
pregnant and pregnant controls.
Within our study population, free TFPI levels
significantly corre- lated with the lagtime (r =
0.5, p < 0.05), the time to peak (r = 0.5, p <
0.05), the velocity index (r = – 0.41, p <
0.05), peak thrombin generation (r = – 0.4, p
< 0.05), and the ETP (r = – 0.3, p < 0.05) of
thrombin generation triggered by low-dose TF.

Figure 3: TFPI and protein S in early onset preeclampsia. A) Free
TFPI levels. B) Plasma TFPI activity. C) Thrombin generation triggered by
low-dose TF in the absence (white bar) and presence (black bar) of an
inhibitory poly- clonal anti-TFPI antibody (100 µg/ml). Comparison of
its effect on thrombin generation in pregnant controls (n = 11) and
patients with severe early onset preeclampsia (n = 8). D) The
percentage increase in peak thrombin gener-
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ation following inhibition of TFPI in pregnant controls (white bar, n =
11) and patients with severe early onset preeclampsia (n = 8). E) Free
protein S levels (& activity). Parametric data sets are expressed as
the mean + 95 % confi- dence interval. Non-parametric data sets are
expressed as the median.* A p
< 0.05 was considered statistically significant.
Table 2: Parameters of coagulation (mean ± standard
Non pregnant deviation).
Pregnant Early onset Non-severe Severe
controls controls preeclampsia EOP EOP
Lagtime (min) 6.8 ± 0.6 7.0 ± 1.2 8.4 ± 2.0 8.1 ± 1.3 8.8 ± 2.6
Time to Peak (min) 13.4 ± 1.2 12.7 ± 1.8 15.8 ± 3.8 14.8 ± 2.1 16.9 ± 5.1
Velocity Index (nM IIa/min) 19.2 ± 8 40.8 ± 27.0 24.7 ± 17.7 26.4 ± 13.1 22.8 ± 22.4
Peak IIa (nM IIa) 119.3 ± 34.5 205 ± 70 150 ± 80 162 ± 60 136 ± 100
ETP (nM□min) 1508 ± 186 2300 ± 400 2030 ± 800 2250 ± 560 1780 ± 970
APC sensitivity ratio– 2.5 nM 0.36 ± 0.2 0.78 ± 0.25 0.67 ± 0.18 0.71 ± 0.16 0.63 ± 0.2
APC sensitivity ratio – 5 nM 0.16 ± 0.1 0.56 ± 0.25 0.38 ± 0.23 0.45 ± 0.15 0.3 ± 0.28
APC sensitivity ratio – 10 0.1 ± 0.1 0.31 ± 0.22 0.18 ± 0.12 0.21 ± 0.11 0.16 ± 0.19
nM
sTM sensitivity ratio – 0.5 0.47 ± 0.22 0.57 ± 0.16 0.5 ± 0.22 0.57 ± 0.14 0.41 ± 0.19
nM
sTM sensitivity ratio – 1 nM 0.21 ± 0.12 0.38 ± 0.14 0.27 ± 0.19 0.36 ± 0.2 0.17 ± 0.13
sTM sensitivity ratio – 2.5 0.02 ± 0.04 0.14 ± 0.1 0.06 ± 0.12 0.11 ± 0.15 0.008 ± 0.01

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nM
TFPI levels (ng/ml) 22.3 ± 7.8 21.7 ± 10.9 27.1 ± 7.8 22 ± 3.8 32.6 ± 7.4
TFPI activity (IU/ml) 1.48 ± 0.2 1.6 ± 0.3 1.9 ± 0.8 1.5 ± 0.3 2.4 ± 1.1

TFPI activity was increased in EOP patients (1.9 between pregnant

± 0.8 IU/ml, n controls (224 ± 117 nM IIa, n=11) and
= 18) compared to non-pregnant controls patients with severe EOP patients (102 ± 57
(1.48 ± 0.2 IU/ml, n = nM IIa, n = 8, p < 0.05). After inhibition of
12) but not compared to pregnant controls TFPI, there was no significant difference in
(1.6 ± 0.3 IU/ml, n = peak thrombin gener- ation between
pregnant controls (266 ± 98 nM IIa, n = 11)
16) (▶Figure 3). TFPI activity was more
significantly increased in cases of severe EOP and pa- tients with severe EOP (198 ± 50 nM
(2.4 ± 0.4 IU/ml, n = 9) compared to non- IIa, n = 8).
pregnant controls, and also significantly Free protein S levels (% activity) were
increased relative to non- significantly decreased in both pregnant
severe EOP cases (1.5 ± 0.3 IU/ml, n = 9) controls (54.9 ± 21.4 %, n = 13, p < 0.05) and
EOP patients (57.6 ± 15.4 %, n = 17, p < 0.05)
(▶Table 2). Within our
relative to non-pregnant
study population, TFPI activity correlated
controls (95.8 ± 12.3 %, n = 13, p < 0.05,
with the lagtime (r =
0.3, p < 0.05) and time to peak (r = 0.3, p ▶Figure 3E and ▶Table
<0.05) of thrombin gen- eration triggered by 2). There was a trend towards an increase in
low-dose TF. protein S activity in
To investigate if increased TFPI activity
was partially respon- sible for the
attenuation of thrombin generation triggered
by low- dose TF, we measured thrombin
generation in the presence of an inhibitory
anti-TFPI antibody (100 µg/ml). In pregnant
controls, inhibition of TFPI increased peak
thrombin generation modestly
from 224 ± 117 nM IIa to 266 ± 98 nM IIa (n =
11; ▶Figure 3C),
corresponding to a 25 ± 20 % increase in
thrombin generation (▶Figure 4D). In patients
with severe EOP, inhibition of TFPI in- creased
peak thrombin generation from 94 ± 55 nM IIa
to 198 ± 50 nM IIa (n = 8, ▶Figure 3C),
corresponding to a 160 ± 115 % in- crease
(▶Figure 3D). Prior to TFPI inhibition, there
was a signifi-
cant difference in peak thrombin generation
cases of severe EOP (61.6 ± 17.5 %, n = 7)
relative to pregnant con- trols and non-
severe EOP patients (54.8 ± 15.4 %, n =
10). Similar to TFPI levels and TFPI activity,
there was a significant correlation between
protein S activity and thrombin generation
triggered by low-dose TF. Protein S activity
significantly correlated with the velocity
index (r = –0.45, p < 0.05), peak thrombin
generation (r =
– 0.53, p < 0.05), and the ETP (r = –0.58, p <
0.05) of thrombin generation triggered by
low-dose TF.

Protein C pathway in early onset preeclampsia

Thrombin generation triggered by low-dose
TF was measured in the presence of sTM
(0–2.5 nM) or APC (0–10 nM). Results are
expressed as APC and sTM sensitivity ratios,
calculated as the ETP in the presence of
APC or sTM / ETP in the absence of APC or
sTM (▶Figure 4A-B).
Consistent with published literature,
pregnant controls ex-
hibited a significantly reduced response to the
anticoagulant activ- ity of APC compared to
non-pregnant controls, indicated by a higher
sensitivity ratio value in response to 2.5 nM
APC (pregnant controls; 0.78 ± 0.15, n = 20
vs non-pregnant controls; 0.36 ± 0.2, n
= 13, p < 0.05), 5 nM APC (pregnant controls;
0.56 ± 0.25, n = 20
vs non-pregnant controls; 0.16 ± 0.1, n = 13,
p < 0.05), and 10 nM APC (pregnant controls;
0.31 ± 0.22, n = 20 vs non-pregnant con-
trols; 0.1 ± 0.1, n = 13, p < 0.05) (▶Figure
4A, ▶Table 2). Patients
with EOP also exhibited a significantly
reduced response to APC
compared to non-pregnant controls, as
evidenced by a higher sen- sitivity ratio value
in response to 2.5 nM APC (EOP; 0.67 ± 0.15,
n
= 25) and 5 nM APC (EOP; 0.38 ± 0.23, n =
25). There was no sig- nificant difference in
sensitivity to APC between pregnant
controls and EOP patients. However, when
EOP patients were segregated based on
disease severity, pregnant controls
exhibited a reduced
Figure 4: Protein C pathway in early onset preeclampsia.

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Thrombin generation stimulated by low-dose TF was measured in the
presence of in- creasing concentrations of activated protein C (APC) or
soluble thrombo- modulin (sTM) in non-pregnant controls (black bar),
pregnant controls (gray bar), and patients with early onset
preeclampsia (white bar). The APC or sTM sensitivity ratio was
calculated as the endogenous thrombin potential in the
presence of APC or sTM / the endogenous thrombin potential in the
absence of APC or sTM. A) APC sensitivity ratio. B) sTM sensitivity ratio.
Parametric data sets are expressed as the mean + 95 % confidence
interval. Non-para- metric data sets are expressed as the median. A p
< 0.05 was considered statistically significant.

0.12, n = 22, p < 0.05) (▶Figure 4B, ▶Table

sensitivity to APC compared to severe EOP 2) This increase in sensitivity to sTM in EOP
patients in response to 5 nM APC (severe EOP; patients was most pronounced in severe
cases who ex-
0.3 ± 0.28, n = 12, p < 0.05). In contrast there hibited an increased sensitivity to sTM
was no difference between pregnant controls compared to pregnant con- trols in response
and non-severe EOP patients in response to 5 to 1nM sTM (severe EOP; 0.17 ± 0.13, p <
nM APC (non-severe EOP; 0.45 ± 0.15, n = 13). 0.05) and 2.5 nM sTM (severe EOP; 0.008 ±
As expected, sensitivity to APC within our 0.01, n = 10, p < 0.05)
study co- hort showed a very significant
inverse correlation with protein S levels at all (▶Table 2).
APC concentrations tested (2.5 nM APC; r = –
0.6, 5 nM APC; r = –0.65, 10 nM APC; r = –
0.435, p < 0.05) and a mod-
est but significant correlation with TFPI
levels at high APC con- centrations (10 nM
APC, r = –0.32, p < 0.05).
Pregnant controls also displayed a
significantly reduced sensi- tivity to sTM
compared to non-pregnant controls, indicated
by a higher sTM sensitivity ratio in response to
1 nM sTM (non-preg- nant controls; 0.21 ±
0.12, n = 13 vs pregnant controls; 0.38 ± 0.14,
n = 20) and 2.5 nM sTM (non-pregnant
controls; 0.02 ± 0.04, n =
13, vs pregnant controls; 0.14 ± 0.1, n = 20, p
< 0.05) (▶Figure 4B,
▶Table 2). There was no significant
difference in sensitivity to sTM when EOP
patients were compared to non-pregnant
controls (▶Figure 4B). However, there was a
modest but significant in- crease in
sensitivity in EOP patients compared to
pregnant con-
trols in response to 2.5 nM sTM (EOP; 0.06 ±
Discussion major difference in our study is that blood
was collected into CTI. In contrast, we
In this study, we have demonstrated that demonstrated reduced thrombin generation
patients with EOP exhibit attenuated thrombin in EOP, a much rarer clinical phenotype. A
generation triggered by low-dose TF com- major difference in our study is that blood
pared to healthy pregnant women. This was was collected blood into CTI. Artefactual
accompanied by elev- ated plasma TFPI contact activation can occur upon blood col-
activity and a modest increase in response to lection into tubes not pre-incubated with
the anticoagulant activity to APC and sTM. CTI. This critical pre- analytical variable
The attenuated coagu- lation response seen artificially increases thrombin generation.
in EOP was most pronounced in patients with Therefore, inhibition with CTI is
severe EOP. recommended at the point of blood
Thrombin generation has been clearly collection (15). Our data suggests that when
shown to increase through pregnancy and contact acti- vation is inhibited, patients with
is highest in the third trimester (13). EOP display significantly attenu- ated
Studies have further demonstrated that thrombin generation triggered by low-dose
patients with late onset preeclampsia TF compared to non-pregnant controls.
display a further enhanced thrombin TFPI is an important regulator of thrombin
generation re- sponse compared to pregnant generation trig-

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controls (14). In contrast, we dem- gered by low-dose TF (16). Low levels of TFPI
onstrated reduced thrombin generation in are linked to an in- creased risk of venous
EOP, a much rarer clinical phenotype. A thrombosis (17). Late onset preeclampsia is
pregnant controls, possibly owing to
What is known about this topic? differences in time of blood sampling (second
vs third trimester) (18).
• Early onset preeclampsia is a pregnancy-specific pro-inflamma-
Other factors that could potentially
tory disorder characterised by competing thrombotic and
contribute to attenuated
bleed- ing risks, which makes decision making on thrombin generation triggered by low-dose TF
thrombophylaxis ex- tremely challenging. in EOP are the re- ported reduced production
of clotting factors due to preeclampsia-
What does this paper add? induced liver dysfunction (23–25). A
• In this study, we demonstrate that patients with early onset coagulopathy due to coagu- lation activation
pre- associated with consumption of clotting
eclampsia display an attenuated TF-dependent thrombin gener- factors could represent a further mechanism
ation response compared to healthy pregnant women. underlying attenuated thrombin generation
• Elevated plasma TFPI activity causes this attenuation of triggered by low-dose TF. However, we ob-
thrombin generation. served no significant difference in D-dimer or
thrombin-anti- thrombin levels between
• These alterations in the haemostatic balance may
pregnant controls and patients with EOP in
modulate our study (unpublished data), suggesting an
thrombotic risk in early onset preeclampsia and exacerbate alternative mech- anism.
bleed- ing risk in patients whose clinical course is complicated Patients with EOP were also characterised by
a modest but sig- nificant increase in sensitivity
to the anticoagulant activity of APC
characterised by elevated TFPI levels (18,
19) owing to endothelia cell activation (20)
(the major cellular source of TFP) and
elevated levels of TFPI-expressing
microparticles derived from synctiotro-
phoblasts (21, 22). Here, we also show
significantly enhanced TFPI levels and TFPI
activity in patients with EOP. The elevation
in plasma TFPI activity in EOP partially
mediated the attenuation of TF-dependent
thrombin generation in EOP as following in-
hibition of TFPI, there was no significant
difference in the rate or extent of thrombin
generation in pregnant controls and patients
with EOP. In contrast to previous studies, we
did not show a sig- nificant difference in free
TFPI levels between pregnant and non-
and sTM, most notably in cases of severe
EOP. Numerous factors, including a reduced
baseline thrombin generation response,
elev- ated levels of TFPI and protein S
(important cofactors for APC)
contribute to this response. While it is clear
that preeclamp- sia is characterised by
changes in coagulation, including changes
in TFPI and protein C pathway activity, it is
unclear if these changes are a by-product of
disease progression (e. g. liver damage) or
our actively involved in the pathogenesis of
preeclampsia.
Our data revealing attenuated thrombin
generation in EOP may have potential
clinical relevance. Elevated and reduced
thrombin generation have been reported to
be associated with thrombosis
bleeding, respectively (26–28). Severe
preeclamp- sia is a major health issue in
Ireland and worldwide. During 2011, 254
discharges with a diagnosis of severe
preeclampsia were re- ported to the Irish
Hospital Inpatient Enquiry (HIPE) National
File, and 45 deliveries were recorded in the
Rotunda Maternity Hospital, Dublin, alone.
Of these 45 women, 71 % underwent
emergency Caesarean Section,
confers an increased risk of venous
thrombosis, bleeding and infection. The
data reported here advances
understanding of overall haemostatic
balance in pre- eclampsia. Further clinical
Abbreviations
studies will be required to more pre- cisely
elucidate the overall VTE risk in a complex,
high-risk
CTI – corn trypsinindi-
inhibitor,vidual
EOP – Early with several
Onset Preeclampsia,
interplaying risk factors. A limitation of this
ETP – endogenous thrombin potential, IUGR – Intrauterine growth
study is that we did not include patients
restric- tion, PPP- Platelet poor plasma, TF – Tissue Factor, TFPI –
with late-onset pre- eclampsia
Tissue Fac- tor Pathway
comparison. While Inhibitor.
previous studies have
assessed thrombin generation in late-onset
Conflicts of interest
None declared.
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Acknowledgements
We are deeply grateful to the patients and
medical/midwifery staff of Rotunda Hospital,
Dublin, Ireland. We are very grateful to Cia- ran
Mooney for his assistance with the organization
of sample pro- cessing and sincerely
which acknowledge the invaluable assistance of the
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KE performed experiments, analysed data,
drafted/edited the manuscript. HOC
recruited patients and drafted/edited the
manu- script. BK, AAZ, and SA performed
experiments. FM, SC, PM, PBZ, CM, and NM
for drafted/edited the manuscript. FNA designed
the study, analysed data, and drafted/edited
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