Journal of Crohn's and Colitis (2014) 8, 495–503
Available online at www.sciencedirect.com
ScienceDirect
Increased responsiveness to thrombin
through protease-activated receptors
(PAR)-1 and -4 in active Crohn's disease
Werner Schmid a,⁎, Harald Vogelsang b , Pavol Papay b , Christian Primas b ,
Alexander Eser b , Cornelia Gratzer b , Michael Handler a ,
Gottfried Novacek b,1 , Simon Panzer c,1
a
Department of Anesthesiology, General Intensive Care and Pain Medicine, Division of Cardiothoracic and Vascular
Anesthesia and Intensive Care, Medical University of Vienna, Vienna, Austria
b
Division of Gastroenterology and Hepatology, Department of Internal Medicine III, Medical University of Vienna, Vienna, Austria
c
Department for Blood Group Serology and Transfusion Medicine, Medical University of Vienna, Vienna, Austria
Received 10 August 2013; received in revised form 3 October 2013; accepted 2 November 2013
KEYWORDS
Crohn's disease;
Platelets;
Thrombin;
Aggregometry
Abstract
Background and aims: Platelets are essential in hemostasis and inflammation, thereby linking
coagulation with inflammation. Abundant thrombin generation in association with inflammation
is considered a major reason for the increased risk for thromboembolic events. We therefore
investigated platelet responsiveness to thrombin.
Methods: In this case–control study 85 patients with Crohn's disease (active CD 42, remission 43)
and 30 sex- and age-matched controls were enrolled. Clinical disease activity (Harvey–
Bradshaw-Index) was assessed and CD-related data were determined by chart review. Platelets'
response to protease activated receptor-1 and -4 (PAR-1, -4) was assessed by whole blood
platelet aggregometry (MEA), levels of platelets adhering to monocytes (PMA), and platelet
surface P-selectin.
Results: Platelets' aggregation after activation with the specific PAR-1 agonist (SFLLRN) and
PAR-4 agonist (AYPGKF) was higher in patients with active CD compared to patients in remission
and controls (p = 0.0068 and p = 0.0023 for SFLLRN, p = 0.0019 and 0.0003 for AYPGKF).
Likewise, levels of PMA after activation with PAR-1 and PAR-4 receptor agonists were higher in
patients with active CD compared to patients in remission and controls (p = 0.0001 and
p b 0.0001 for SFLLRN, p = 0.0329 and p = 0.0125 for AYPGKF). However, P-selectin expression
on human platelets showed heterogeneous results. Only PAR-1 activation of platelets resulted in
significant differences between CD patients and controls (p = 0.0001 and p = 0.0022 for active
and inactive CD versus controls, respectively).
⁎ Corresponding author at: Department of Anesthesiology, General Intensive Care and Pain Medicine, Division of Cardiothoracic and Vascular
Anesthesia and Intensive Care, Medical University of Vienna, 1090 Vienna, Austria. Tel.: + 43 40 400 2516; fax: + 43 40 400 4109.
1
GN and SP share senior authorship.
1873-9946/$ - see front matter © 2013 European Crohn's and Colitis Organisation. Published by Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.crohns.2013.11.001
496
W. Schmid et al.
Conclusions: Our data suggest a new mechanism of platelet activation which has the potential
to increase risk for thromboembolism in patients with active CD which might be due to platelets
poised for thrombin-inducible activation.
© 2013 European Crohn's and Colitis Organisation. Published by Elsevier B.V. All rights reserved.
1. Introduction
The two major forms of inflammatory bowel disease (IBD),
Crohn's disease (CD) and ulcerative colitis (UC), result
from complex interactions between evolving environmental
changes, more than 1501 of predisposing genetic mutations, a
complex gut microbiota that may be continuously varied, the
intricacies of individual immune systems2 and non-immune
systems such as the hemostatic system.3 Among these,
platelets are believed to play a crucial role as it is evident
that, in addition to their role in primary hemostasis, platelets
also play an active role in inflammatory processes.4,5 The
concept of enhanced platelet activity in IBD is supported
byreports showing significantly increased platelet aggregation
in response to agonists like ADP, collagen, and ristocetin6–8
in both forms of IBD.9–11 While platelets are traditionally
seen as the major players under high shear conditions like
in atherosclerosis, there is growing evidence for their active
role in venous thromboembolic disease.12 We propose that
the clinical importance of increased platelet activation is
reflected by a substantially increased incidence of thromboembolisms in IBD.13–18
Continuous thrombin generation is considered a major
reason for an increased risk of thromboembolic events, as
it is a very potent platelet activator. Thrombin activates
platelets via 4 distinct receptors, protease-activated receptors (PAR)-1, PAR-4,19,20 glycoprotein (GP) Ibα and GPV.21–24
PAR1 mediates platelet responses at low concentrations of
thrombin while PAR4 mediates platelet activation only at
high thrombin concentrations.19 PAR-1 and PAR-4 form a
heterodimeric complex20,25 and with GPIbα complementary
units a trimeric receptor complex regulating platelet
activation by thrombin, that enables thrombin to act as a
bivalent or even trivalent functional agonist. Thus, coordinate activation of PAR by subnanomolar thrombin concentrations has been proposed.26 Clinically, there is strong
evidence that PAR mediated platelet activation is significant.27 IBD is associated with altered plasma levels of a
variety of hemostatic biomarkers indicating subclinical
activation of the coagulation system, finally leading to
enhanced generation of thrombin.28
The complex interplay between inflammation and hemostasis results in activated platelets, aggregate formation, and
release of P-selectin. The latter is the most important platelet
“releasate” for the interaction with peripheral blood leukocytes, including monocytes, leading to enhanced leukocyte
activation. High levels of platelet–monocyte aggregates
(PMA) are regarded a very sensitive marker of thromboembolic
disease.29 Indeed, increased levels of PMA have been reported
in a variety of inflammatory diseases29–32 that are associated
with an increased risk for thromboembolic disease, including
IBD.33,34
We assessed the association between inflammation in CD
patients with their platelets' responsiveness to thrombin. We
compared the responsiveness of platelets to two major
thrombin receptors, PAR-1 and − 4, to their specific agonists
(SFLLRN and AYPGKF for selectively activating PAR-1 and
PAR-4, respectively) by platelet aggregation, determination
of the formation of PMA, and levels of P-selectin expression of
patients with active CD, patients in remission and controls.
Thereby we show an enhanced responsiveness specific for
PAR-inducible platelet activation in patients with active CD
compared to patients in remission and controls. These findings
may have the potential to explain the increased susceptibility
for thrombin mediated risk of thromboembolic disease in
patients with active CD.
2. Materials and methods
2.1. Patients and controls
The study complied with the Declaration of Helsinki, was
approved by the Ethics Committee of the Medical University
of Vienna, and all patients and healthy controls gave written
informed consent. We enrolled 42 consecutive patients
with active CD, 43 patients with CD in remission, and 30
age and sex-matched controls with coeliac disease under
gluten-free diet without any symptoms as controls (Table 1).
All participants were outpatients and recruited from the
Department of Internal III, Division of Gastroenterology and
Hepatology, Medical University of Vienna, Vienna, Austria.
All CD patients were older than 18 years and had an established
diagnosis of CD (based on clinical, endoscopic, histological,
and radiological criteria according to European Crohn's and
Colitis Organisation (ECCO) guidelines).35 CD-related data
were collected from chart review and included age at
diagnosis, disease extent and behavior, CD-related surgery,
medication, smoking habits, and disease activity. Extent of
CD and disease behavior were classified according to the
Montreal classification.36 Active disease was defined by a
Harvey–Bradshaw Index (HBI) of N 4.37 CD-related surgery
was defined as bowel resection only. A smoker was defined
as a patient who smoked at least 7 cigarettes weekly for at
least 1 year.38 The diagnosis of coeliac disease was based on
typical morphology of duodenal mucosal biopsy specimens
taken during oesophago-gastroduodenoscopy confirmed by
positive antibodies, according to ESPGHAN criteria.39 Patients
with coeliac disease in long-term remission served as controls.
They were symptom-free on gluten-free diet for at least
one year and were anti-endomysial antibodies (EMA) and
anti-tissue-transglutaminase antibodies — negative at any
time of the study. Exclusion criteria included hereditary
platelet abnormalities, a family or personal history of
bleeding disorders, significant renal dysfunction, malignant paraproteinemias, myeloproliferative disorders, severe
hepatic failure, malignancies, patients with adipose phenotype
(BMI N 30 kg/m2), a major surgical procedure within 3 months
Platelet activation in Crohn’s disease via PAR-1 and PAR-4
497
Table 1 Characteristics of patients with active Crohn's disease, with Crohn's disease in remission and of controls (patients with
coeliac disease under gluten-free diet).
Age (mean ± SD)
Female, n (%)
Age at diagnosis
Average HBI
Smokers
Body mass index (kg/m2; mean ± SD)
EMA
TTG
Treatment
Azathioprine, n (%)
Corticosteroid, n (%)
Infliximab, n (%)
Adalimumab, n (%)
No treatment, n (%)
Duration of GFD (months, median, range)
Disease location, n (%)
CD patients (n = 85)
L1
L2
L3
L4 (+ L1–L3)
Perianal
Behavior, n (%)
B1
B2
B3
Control (n = 30)
CD inactive (n = 43)
CD active (n = 42)
38.3 ± 12.3
20 (66.6)
23.2 ± 18.7
–
3 (10)
22.7 ± 3.5
0 (0)
0 (0)
37.5 ± 10.9
18 (41.9)
25.6 ± 10.3
1.3 ± 1.4
12 (27.9)
23.1 ± 2.7
–
–
38.5 ± 11.7
23 (54.8)
26.4 ± 11.4
11.4 ± 4.9
18 (41.9)
23.5 ± 4.8
–
–
–
–
–
–
136, (12–420)
14 (32.6)
2 (4.6)
10 (23.3)
5 (11.6)
12 (27.9)
–
6 (14.3)
4 (9.5)
2 (4.8)
15 (35.7)
15 (35.7)
–
–
–
–
–
–
4 (10.8)
5 (11.6)
30 (69.7)
4 (9.3)
11 (25.6)
8 (19.0)
8 (19.0)
23 (54.8)
3 (7.1)
21 (50.0)
–
–
–
19 (44.2)
11 (25.6)
13 (30.2)
18 (42.9)
7 (16.7)
17 (40.5)
Unless otherwise stated values are numbers (percentage) or mean ± standard deviation, respectively.
CD, Crohn's disease; HBI, Harvey–Bradshaw Index; EMA, endomysial antibodies; TTG, tissue − transglutaminase antibodies; GFD,
gluten-free diet.
Active CD was defined by a HBI N 4 and quiescent CD was defined by a HBI ≤ 4.
The location and behavior of Crohn's disease was defined according to the Montreal classification: L1: ileal, L2: colonic, L3: ileocolonic,
and L4: isolated upper gastrointestinal tract (L4 is a modifier that can be added to L1–L3 when concomitant upper gastrointestinal disease
is present); B1: nonstricturing and nonpenetrating, or ‘inflammatory’; B2: stricturing or stenosing; B3: penetrating or perforating.
before enrolment, the administration of anti-platelet therapy,
of aspirin or non-steroidal anti-inflammatory drugs,
5-aminosalicylic acid therapy, therapy with vitamin K antagonists (warfarin, phenprocoumon, acenocoumarol) in the
previous 14 days, a platelet count b 100,000 or N 450,000/μl
and a hematocrit b 30%.
2.2. Blood sampling
Blood was drawn by clean venipuncture from an antecubital
vein using a 21-gauge butterfly needle (0.8 × 19 mm;
Greiner Bio-One, Kremsmünster, Austria). Blood samples
were taken by the same physician applying a light tourniquet
as previously described, which was immediately released.40
Samples were mixed adequately by gently inverting the
tubes. The initial 3 mL of blood was used for routine blood
cell counts, and then blood was drawn into a 3.2% sodium
citrate Vacuette tube (Greiner Bio-One; 9 parts of whole
blood, 1 part of sodium citrate 0.109 M/L) for analyses
by flow cytometry, while hirudin-anticoagulated blood
(13 μg/mL) was used for the determinations by whole
blood multiple electrode impedance aggregometry (MEA).
2.3. Multiple electrode aggregometry (MEA)
MEA (Dynabyte, Munich, Germany) was used to specifically
determine the in vitro responsiveness of patients' platelets to
SFLLRN (PAR-1 activation, 32 μM; Bachem, Bubendorf,
Switzerland) and AYPGKF (PAR-4 activation, 662 μM;
Dynabyte). The concentrations of agonists' were used as suggested by the manufacturer. In brief, hirudin-anticoagulated
whole blood was diluted 1:2 with 0.9% NaCl solution and stirred
in the test cuvettes for 3 min at 37 °C. Then agonists were
added and aggregation was continuously recorded for 5 min.
Adhesion of activated platelets to the electrodes led to an
increase of impedance, which was detected for each sensor unit
separately and transformed to aggregation units (AU) that were
plotted against time.
2.4. Determination of platelets adhering to
monocytes (PMA)
Platelets adhering to monocytes were identified as previously published.41 In brief, agonists for PAR-1 (SFLLRN,
7.1 μM; Bachem), PAR-4 (AYPGKF 357 μM; Dynabyte), or
498
W. Schmid et al.
HEPES buffer were added to 5 μl whole blood, diluted in 55 μl
HEPES-buffered saline. After 15 min, monoclonal antibodies
(anti-CD45, clone 2D1-peridinin chlorophyll protein labeled,
Becton Dickinson, B.D., San Jose, CA; anti-CD41 clone P2,
phycoerythrin labeled, Immunotech, Beckman Coulter Fullerton, CA, USA), and anti-CD14 − (clone MФP9, allophycocyanin
labeled, B.D.), or isotype-matched controls were added.
After 20 min, samples were diluted with FACS Lysing solution
and 10,000 CD45+ events were acquired immediately.
Within these events, lymphocytes, granulocytes, and monocytes were identified, based on their CD14 versus side scatter
characteristics. Monocytes were identified as CD14+ and the
CD45 + CD14+ events were subjected to further analyses
for CD45 + CD41+ and CD45 + CD41− events. Data are shown
as geomean fluorescence intensity (MFI). All samples were
evaluated within 15 min after blood withdrawal. The agonists'
concentrations for the determination of PMA were determined
in an independent cohort of 20 healthy individuals by titration
experiments. Suboptimal concentrations were selected and
resulted in at least a MFI of: PMA-SFLLRN 8 (median: 65, range:
8–1000), PMA-AYPGKF 8 (median: 20, range: 8–292).
2.5. Regulation of P-selectin
Platelet activation was analyzed by P-selectin expression,
without and after in vitro addition of the PAR-1 agonist
SFLLRN or the PAR-4 agonist AYPGKF. In brief, whole blood
was diluted with HEPES buffer to obtain 20 × 109/L platelets. The expression of P-selectin was determined without
agonists and after in vitro exposure to the PAR-1 agonist
(SFLLRN, 5.7 μM; Bachem) or the PAR-4 agonist (AYPGKF,
714 μM; Dynabyte). After 15 min, the platelet population
was identified by staining with anti-CD42b (clone HIP1,
allophycocyanin labeled, BD), and P-selectin expression was
determined by the binding of the monoclonal antibody
anti-CD62p (clone CLB-Thromb/6, phycoerythrin-labeled,
Immunotech). The reaction was stopped by adding 500 μl
PBS, and samples were acquired immediately on a FACS
Calibur flow cytometer (B.D.) with excitation by an argon laser
at 488 nm and a red diode laser at 635 nm at a rate of
200–600 events per second. Standard B.D. Calibrite beads were
used for daily calibration of the cytometer. Data are shown
as geomean fluorescence intensity (MFI). The agonists' concentrations for the determination of P-selectin expression were
determined in an independent cohort of 20 healthy individuals
by titration experiments. Suboptimal concentrations were
Table 2
selected and resulted in at least a MFI of: P-selectin-SFLLRN 5
(median: 23, range: 5–109), P-selectin-AYPGKF 6 (median: 29,
range: 6–250).
2.6. Statistical analyses
The primary endpoint was aggregation units measured by
MEA in CD compared to controls. Considering a minimum
expected difference (effect size) of 10 (AU controls = 95 vs.
AU active CD = 105) the projected sample size at a level of
0.05 and a power of 0.8 was at least 30 per group (active CD,
CD in remission, and control group). Secondary endpoints
were levels of platelets adhering to monocytes and surface
P-selectin expression. Data are presented as median with
interquartile range (IQR), unless indicated otherwise. Platelet function data measured by MEA were normally distributed;
therefore we performed t-tests to detect differences of
platelet reactivity. Since the data of the secondary endpoints
indicated a violation of the distributional assumptions for the
t-test, evaluated by the D'Agostino & Pearson omnibus K2 test,
the non-parametric Mann–Whitney-U test was used. Providing
two-tailed significance levels, differences were considered
statistically significant at p b 0.05. Statistical analysis was
performed using SPSS version 17.0 (Chicago, IL, USA).
3. Results
3.1. Clinical and laboratory data
Clinical and laboratory characteristics of the study population are shown in Tables 1 and 2. Compared to controls and
to CD in remission, platelet count, WBC count, fibrinogen,
and CRP were significantly higher in active CD whereas
transferrin saturation was significantly lower (Table 2).
3.2. Platelet reactivity by multiple
electrode aggregometry
In patients with active CD SFLLRN and AYPGKF inducible
platelet reactivity was significantly higher than in controls
(p = 0.0023 for SFLLRN, Fig. 1a; p = 0.0003 for AYPGKF;
Fig. 1b) as well as in CD patients in remission (p = 0.0068
for SFLLRN, Fig. 1a; p = 0.0019 for AYPGKF; Fig. 1b).
No significant difference was observed between controls
Laboratory data and markers of inflammation.
Controls (n = 30)
Laboratory data (median, IQR)
Platelet count (× 109/μl)
216 (203–255)
CRP (nmol/L)
0.14 (0.05-0.28)
WBC count (× 109/μl)
5.82 (5.1-6.9)
Fibrinogen (μmol/L)
311 (297–351)
Transferrin saturation%
23.8 (18.3-30.6)
pa
CD inactive(n = 43)
pa
CD active (n = 42)
pb
0.1788
0.0139
0.0288
0.4599
0.2054
212 (156–245)
0.21 (0.11-0.6)
6.8 (5.9-7.8)
323 (300–367)
20.0 (11.7-30.4)
0.0001
b 0.0001
b 0.0001
0.0018
b 0.0001
294 (231–361)
1.76 (0.48-7.85)
8.8 (7.1-10.8)
485 (379–627)
9.7 (6.2-20.4)
b 0.0001
b 0.0001
0.0017
b 0.0001
0.0026
Values are median values (interquartile range).
a
p values are determined by the Mann–Whitney U test and are calculated from each patient group vs. the control group.
b
p values from CD subgroups (remission vs. active).
Platelet activation in Crohn’s disease via PAR-1 and PAR-4
499
controls (data not shown). The in vitro addition of agonists
for PAR-1 and PAR-4, respectively, resulted in significantly
higher levels of PMA in patients with active CD than in
controls (p b 0.0001 for SFLLRN, Fig. 2a; p = 0.0125 for
AYPGKF, Fig. 2b) and patients in remission (p = 0.0001 for
SFLLRN, Fig. 2a; p = 0.0329 for AYPGKF, Fig. 2b). There
was no difference between controls and CD in remission
(p = 0.2868 for SFLLRN, Fig. 2a; p = 0.7120 for AYPGKF;
Fig. 2b).
3.4. Regulation of P-selectin
Levels of P-selectin expression ex vivo (determined prior to
exogenously added agonists) were not significantly different
Figure 1 a, b: Platelet reactivity by multiple electrode
aggregometry (MEA) in aggregation units (AU) in response to
PAR-1 (SFLLRN) and PAR-4 agonist (AYPGKF) in inactive and
active patients with Crohn's disease and in controls (patients
with coeliac disease under gluten-free diet). Boundaries of the
box show the lower and upper quartile of data, the line inside
the box represents the median. Whiskers are drawn from the
edge of the box to the highest and lowest values that are outside
the box but within 1.5 times the box length. Values outside this
range (outliers) are shown as circles.
and CD patients in remission (p = 0.5788 for SFLLRN, Fig. 1a;
p = 0.2761 for AYPGKF; Fig. 1b).
3.3. PMA formation
Levels of PMA ex vivo (determined prior to exogenously
added agonists) were not significantly different in patients
with active CD, compared to patients in remission and to
Figure 2 a, b: Levels of platelet–monocyte aggregates (PMA)
after in vitro addition of PAR-1 (SFLLRN) or PAR-4 agonist
(AYPGKF) in patients with inactive and active Crohn's disease
and from controls (patients with coeliac disease under gluten-free
diet). Data are shown as geomean fluorescence intensity (MFI).
Design as in Fig. 1.
500
in patients with active CD from controls or from those in
remission or between patients in remission and controls
(data not shown). Levels of P-selectin expression of PAR-1
activated platelets from patients with active CD were
significantly higher than from controls (p = 0.0001; Fig. 3a).
The corresponding data for patients with CD in remission were
also significantly higher compared to controls (p = 0.0022;
Fig. 3a), whereas no difference was observed between the CD
subgroups (p = 0.3294; Fig. 3a). P-selectin expression of PAR-4
activated platelets from patients with active disease was not
Figure 3 a, b: P-selectin expression after in vitro addition of
PAR-1 (SFLLRN) or PAR-4 agonist (AYPGKF) on platelets from
patients with inactive and active Crohn's disease and from
controls (patients with coeliac disease under gluten-free diet).
Data are shown as geomean fluorescence intensity (MFI). Design
as in Fig. 1.
W. Schmid et al.
significantly different from platelets from CD in remission, or
controls, (p = 0.5078 and p = 0.9582; Fig. 3b).
4. Discussion
By studying PAR-1 and PAR-4 inducible platelet activations,
we specifically addressed platelet responsiveness to two
major thrombin receptors in CD patients. Our data indicate
an increased susceptibility of platelets from patients with
active CD for PAR inducible platelet activation resulting in
increased aggregation and increased formation of platelets
adhering to monocytes. These findings may explain the
increased risk for thromboembolic events in patients with
active CD.
Levels of thrombin, the strongest platelet agonist, are
increased in IBD.42 The increased expression of PAR-1 in IBD
patients43 already strongly suggested its role as a risk factor
to induce platelet activation and consecutively thromboembolic disease. We here add another piece of evidence for
the significant role of thrombin inducible platelet activation
by showing an increased susceptibility for PAR mediated
platelet activation in patients with active CD.
We choose whole blood impedance aggregometry for the
evaluation of the aggregation response of platelets, because
MEA is well standardized and data from one laboratory can
easily be compared to those from another one. As minimal
manipulation requirements by MEA are a major advantage,
any artifacts are significantly reduced and all data can
therefore be regarded highly specific. Previous reports
indicated increased platelet aggregation in response to
epinephrine, collagen and ADP in IBD patients.6–8 Our data
complement these findings by showing an enhanced responsiveness specifically for PAR-inducible platelet aggregation.
However, as an overlap of results is seen, other pathways of
platelet activation may be active as well in some patients. The
role of PAR-1 inducible platelet activation is substantiated by
the findings of significant stronger responsiveness to PAR-1
induced expression of P-selectin in patients with active
disease. Activated platelets express P-selectin (CD62P) and
bind rapidly to lymphocytes, monocytes, neutrophils and
endothelium44 via the constitutively expressed P-selectin
glycoprotein ligand-1 (PSGL-1) receptor.45,46 The interaction
between P-selectin and its main receptor PSGL-1 leads
to platelets adhering to leukocytes, which then become
further activated and secrete inflammatory substances,
like chemokines, including RANTES, platelet factor 4 and
pro-angiogenic factors, including VEGF and PDGF.4 Our data
indicate that PMA formation through PAR-1 and PAR-4
mediated platelet activation is high in active CD compared
to controls. In concordance with the impedance aggregometry
data, this marked responsiveness almost completely resolved
in patients with CD in remission. This direct association of the
clinically active disease, reflected by the HBI, with PAR
mediated platelet activation and levels of PMA is of special
importance, since PMA formation is associated with thromboembolic disease.29
An enhanced platelet activation in IBD was shown by
platelet activation markers such as P-selectin and measurements of β-thromboglobulin47,48 and by various reports
showing an elevation of different platelet associated
products, such as platelet factor 4 (PF4) and CD40 ligand
Platelet activation in Crohn’s disease via PAR-1 and PAR-4
(CD40L) in IBD.49,50 This platelet activation could be just
from their active role in inflammation,4,5,51 without a
specific engagement associated with thrombosis. However,
the platelet response to specific stimuli results in diverse
platelet responses, suggesting that platelets can discriminate between different stimuli.52,53 Thus, demonstrating
increased levels of PAR-mediated platelets adhering to
monocytes, a sensitive marker of thromboembolic disease29,30 and inflammation,31,32 bridges inflammation with
thromboembolic disease in active CD.
We show that PAR-1 and PAR-4 inducible platelet
aggregation and levels of PMA formation normalized in CD
patients in remission. However, PAR-1 activated platelets of
patients with CD express higher levels of surface P-selectin
compared to controls, regardless the clinical disease
activity. These findings are in line with previous reports
showing activated platelets irrespective of the inflammatory
status.8,54 One likely explanation for elevated P-selectin
levels is that despite a HBI ≤ 4, subclinical inflammation
persists, leading to high P-selectin expression even in a
population with clinical remission. The question why we, in
contrast to published data,8 could correlate platelet
activation with disease activity remains to be elucidated.
Possible explanations could be different used platelet tests
and agonists as well as different used clinical markers to
define disease activity. Andoh et. al8 used light transmission
aggregometry which requires preparation of platelet rich
plasma. This procedure, in contrast to whole blood MEA, leads
to manipulation and possible in vitro activation of platelets
thereby masking potential pathophysiological effects.
In contrast to PAR-1, PAR-4 mediated activation led to no
significant differences in surface P-selectin expression with
respect to the status of activation or in comparison to
controls. The disparate observation of significant stronger
response of PMA to PAR-4 mediated platelet activation
in patients with active CD, but without such a stronger
response of platelet surface P-selectin expression, may be
due to other receptor–ligand associations for platelet–
monocyte aggregate formation.55 We cannot exclude that
we have underestimated other interactions between platelets and monocytes by focusing on the P-selectin-PSGL1-axis
only.56–58
Our study has some strengths and some limitations. We
show for the first time, with the highly specific and well
standardized whole blood impedance aggregometry (MEA),
significantly higher platelet aggregation via thrombin receptors PAR-1 and PAR-4 in patients with active CD compared
to patients in remission or controls. The choice of coeliac
disease patients as controls could be discussed. However, these
patients were symptom-free on gluten-free diet for at least
12 months and were anti-endomysial antibodies (EMA) and
anti-tissue-transglutaminase antibodies — negative. Furthermore, in a previous study, we demonstrated that patients with
coeliac disease are not at increased risk for venous thromboembolism.17 Usually patients with coeliac disease in long-term
remission do not have any signs of significant inflammation in
the gut or in the serum and are appropriate non-inflammatory
controls. A limitation of this study is the lack of clinical
outcome data with thromboembolic complications. No patient
had any documented thromboembolic event in the medical
history at the time of study entry. However, in this study
we tried to complement current epidemiologic data17,18 on
501
thromboembolic disease in IBD with functional data of primary
hemostasis. Further, our findings may not be generalized
to UC as only platelets from CD patients were studied. It
is however difficult to recruit patients with UC for studies
on platelet activation, as most of these patients are on
5-aminosalycilic acid treatment, which may influence platelet
function.
In conclusion, we demonstrate that PAR responsiveness
is increased in CD and this effect can be correlated to
clinical disease activity of CD. We may provide a biological
foundation for the increased risk of thromboembolic disease
in CD patients, that may translate into new diagnostic and
therapeutic opportunities.
Conflict of interest
Each author disclose that he or she has no commercial
associations (e.g., consultancies, stock ownership, equity
interest, patent/licensing arrangements, etc.) that might
pose a conflict of interest in connection with the submitted
article.
Acknowledgment
We acknowledge the financial support of the “MedizinischWissenschaftlicher Fonds des Bürgermeisters der Bundeshauptstadt
Wien (08035)”. The study sponsors had no role in the study design, in
the collection, analysis and interpretation of data; in the writing of
the manuscript; and in the decision to submit the manuscript for
publication.
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