COU254, a specific 3-carboxamide-coumarin inhibitor of coagulation factor XII, does not protect mice from acute ischemic stroke.

RESEARCH Open Access
COU254, a specific 3-carboxamide-coumarin
inhibitor of coagulation factor XII, does not
protect mice from acute ischemic stroke
Peter Kraft1, Tobias Schwarz1, Lionel Pochet2, Guido Stoll1, Christoph Kleinschnitz1*
Abstract
Background: Anticoagulation is an important means to prevent from acute ischemic stroke but is associated with
a significant risk of severe hemorrhages. Previous studies have shown that blood coagulation factor XII (FXII)-
deficient mice are protected from pathological thrombus formation during cerebral ischemia without bearing an
increased bleeding tendency. Hence, pharmacological blockade of FXII might be a promising and safe approach to
prevent acute ischemic stroke and possibly other thromboembolic disorders but pharmacological inhibitors
selective over FXII are still lacking. In the present study we investigated the efficacy of COU254, a novel
nonpeptidic 3-carboxamide-coumarin that selectively blocks FXII activity, on stroke development and post stroke
functional outcome in mice.
Methods: C57Bl/6 mice were treated with COU254 (40 mg/kg i.p.) or vehicle and subjected to 60 min transient
middle cerebral artery occlusion (tMCAO) using the intraluminal filament method. After 24 h infarct volumes were
determined from 2,3,5-Triphenyltetrazoliumchloride(TTC)-stained brain sections and functional scores were assessed.
Hematoxylin and eosin (H&E) staining was used to estimate the extent of neuronal cell damage. Thrombus
formation within the infarcted brain areas was analyzed by immunoblot.
Results: Infarct volumes and functional outcomes on day 1 after tMCAO did not significantly differ between
COU254 pre-treated mice or untreated controls (p > 0.05). Histology revealed extensive ischemic neuronal damage
regularly including the cortex and the basal ganglia in both groups. COU254 treatment did not prevent
intracerebral fibrin(ogen) formation.
Conclusions: COU254 at the given concentration of 40 mg/kg failed to demonstrate efficacy in acute ischemic
stroke in this preliminary study. Further preclinical evaluation of 3-carboxamide-coumarins is needed before the
antithrombotic potential of this novel class of FXII inhibitors can be finally judged.
Introduction
Thromboembolic occlusion of intracerebral vessels is
responsible for the majority of ischemic strokes [1]. Stu-
dies on the early use of anticoagulant drugs (e.g.
heparin) in cerebral ischemia failed to demonstrate over-
all benefit in that reduced lesion progression was coun-
terbalanced by an increase in hemorrhages [2]. In
addition, long-term anticoagulation for secondary pre-
vention of cardioembolic stroke, mainly accomplished
by warfarin prescription, is inevitably associated with
increased bleeding-related morbidity and mortality [3].
Hence, identification of novel targets for more effective
and safer anticoagulation in patients with imminent
stroke is badly needed.
In the classical “waterfall model” of blood coagulation
the formation of a fibrin thrombus can be initiated by
two distinct pathways, the extrinsic and the intrinsic
pathway [4]. Both cascades consist of a series of proteo-
lytic reactions involving trypsin-like serine proteases [5].
Fibrin formation via the extrinsic pathway occurs when
tissue factor (TF), located on cell membranes in the
subendothelium of a vessel, forms a complex with acti-
vated coagulation factor VII (FVIIa) after endothelial
injury [6]. According to the original assumption, the
* Correspondence: christoph.kleinschnitz@mail.uni-wuerzburg.de
1Department of Neurology, Josef-Schneider-Str. 11, University Clinic of
Wuerzburg, Germany
Kraft et al. Experimental & Translational Stroke Medicine 2010, 2:5
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© 2010 Kraft et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited.

intrinsic pathway is initiated when coagulation factor
XII (FXII) becomes activated on a negatively charged
surface followed by successive activation of factor XI
(FXI) and factor IX (FIX) [7]. FXII has long been con-
sidered to be dispensable for clot formation because
humans with hereditary FXII deficiency suffer from
neither spontaneous nor injury-related abnormal bleed-
ings [8,9]. This concept was recently called into question
by studies demonstrating that FXII-deficient mice are
profoundly protected from pathological thrombus for-
mation in different models of arterial thrombosis but,
like FXII-deficient humans, do not show impaired
hemostasis [10,11]. Consequently, it was anticipated that
the use of FXII inhibitors would be associated with rela-
tively low rates of therapy-related hemorrhages, the
major clinical complication associated with current
anticoagulant therapies [1]. Indeed, wild-type mice trea-
ted with D-Pro-Phe-Arg chloromethyl ketone (PCK),
which blocks the amidolytic activity of FXIIa, and sub-
jected to ischemic stroke afterwards, developed less ves-
sel occlusive thrombi in the cerebral microvasculature
but did not show increased bleeding tendencies [11].
However, PCK is not selective over FXII and also inter-
acts with other components of the plasmatic coagulation
cascade [12].
Pochet and co-workers recently described the synth-
esis of new 3-carboxamide-coumarins which are the first
selective nonpeptidic inhibitors of FXIIa [12]. COU254
is a member of this novel class of FXII inhibitors. In the
present study we assessed the effect of COU254 on
stroke development, intracerebral fibrinogen clotting
and post stroke functional outcome in mice.
Methods
Animal experiments
A total of 26 mice were used in this study. Animal
experiments were approved by legal state authorities
(Bezirksregierung of Unterfranken) and conducted
according to the recommendations for research in basic
stroke studies [13]. Focal cerebral ischemia was induced
in 6-8-weeks old male C57Bl/6 mice (Harlan Winkel-
mann, Borchen, Germany) by 60 min transient middle
cerebral artery occlusion (tMCAO) as described [11,14].
Mice were anesthetized with 2.5% isoflurane (Abbott,
Wiesbaden, Germany). Following a midline skin incision
in the neck, the proximal common carotid artery and
the external carotid artery were ligated and a standar-
dized silicon rubber-coated 6.0 nylon monofilament
(6021; Doccol Corp., Redlands, CA, USA) was inserted
and advanced via the right internal carotid artery to
occlude the origin of the right MCA. The operator was
blinded to the treatment groups and operation time per
animal did not exceed 15 minutes. The intraluminal
suture was left in situ for 60 minutes. Then animals
were re-anesthetized and the occluding monofilament
was withdrawn to allow reperfusion.
COU254 dissolved in 25% DMSO was administered
intraperitoneally (i.p.) 2 h before the induction of
tMCAO at a dosage of 40 mg/kg bodyweight. Vehicle-
treated control mice receiving 25% DMSO without
COU254 served as controls. The i.p. route of adminis-
tration was chosen because i.v. application was afflicted
with significant acute toxicity and mortality in prelimin-
ary experiments.
Determination of infarct size and histology
Edema-corrected infarct volumes were quantified by pla-
nimetry from 2,3,5-Triphenyltetrazoliumchloride (TTC)-
stained brain sections 24 h after ischemic stroke as
described [11,14,15]. For morphological assessment, par-
affin embedded brains were stained with hematoxylin
and eosin (H&E).
Protein extraction and Western blot analysis
Following TTC staining cortices were dissected from
formalin-fixed brain slices and homogenized in RIPA
buffer (25 mM Tris pH 7.4, 150 mM NaCl, 1% NP40)
containing 2% SDS. The samples were incubated for 20
min at 100°C followed by incubation at 60°C for 2 h
[16]. After that, tissue lysates were centrifuged at 15.000
× g for 20 min at 4°C and supernatants were used for
BCA protein assay and subsequent Western blot
analysis.
The total lysates were treated with SDS-PAGE loading
buffer (final conc. 65 mM Tris, 5% 2-mercaptoethanol,
3% SDS, 10% glycerol) at 95°C for 5 min. 30 μg of total
protein were electrophoresed and transferred to a PVDF
membrane. After blocking for 1 h with blocking buffer
(5% nonfat dry milk, 50 mM Tris-HCl pH 7.5, 150 mM
NaCl, 0.05% Tween-20) membranes were incubated
with the primary antibody at 4°C over night at the fol-
lowing dilutions: anti-Fibrinogen (cross-reactive against
fibrin) pAb 1:500 (Acris Antibodies) and anti-Actin
mAb 1:10,000 (Dianova). After a washing step with
TBS-T (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.05%
Tween-20), membranes were incubated for 1 h with
HRP-conjugated donkey anti-rabbit IgG (for Fibrinogen)
or donkey anti-mouse IgG (for Actin) at a dilution of
1:5000 and were finally developed using ECLplus (GE
Healthcare).
Assessment of functional outcome
24 h after tMCAO the modified Bederson score [17] was
used to determine global neurological function accord-
ing to the following scoring system: 0, no deficit; 1, fore-
limb flexion; 2, decreased resistance to lateral push; 3,
unidirectional circling; 4, longitudinal spinning; 5, no
movement. Motor function and coordination were
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evaluated by the grip test [18]. For this test, the mouse
was placed midway on a string between two supports
and rated as follows: 0, falls off; 1, hangs onto string by
one or both forepaws; 2, as for 1, and attempts to climb
onto string; 3, hangs onto string by one or both fore-
paws plus one or both hindpaws; 4, hangs onto string
by fore- and hindpaws plus tail wrapped around string;
5, escape (to the supports). Neurological scores were
always assessed by an independent and blinded
investigator.
Laser-Doppler flowmetry
Laser-Doppler flowmetry (Moor Instruments, Axmin-
ster, U.K.) was used in some animals (n = 3/group) to
monitor regional cerebral blood flow (rCBF) in the
MCA territory (6 mm lateral and 2 mm posterior from
bregma) [19].
Statistics
Data are expressed as mean ± standard deviation (SD).
For statistical analysis, PrismGraph 4.0 software package
(La Jolla, CA, USA) was used. Infarct volumes and neu-
rological scores were analyzed using the non-parametric
Mann Whitney test. Laser Doppler flowmetry data were
compared by 1-way ANOVA followed by Bonferroni
post hoc test. P-values < 0.05 were considered to be sta-
tistically significant.
Results
The transient middle cerebral artery filament occlusion
model (tMCAO) was used to induce focal cerebral
ischemia in mice [11,14,15]. After advancing the fila-
ment to the origin of the MCA the decrease in rCBF
was similar between control mice and COU254-treated
mice (5.4 ± 7.5% of baseline levels vs. 11.5 ± 5.2% of
baseline levels; p > 0.05) (Figure 1). Ten minutes after
reperfusion rCBF in the MCA territory was reconsti-
tuted to >70% of baseline levels and again did not signif-
icantly differ between treated and untreated mice (70.0
± 10.3% of baseline levels vs. 74.3 ± 4.2% of baseline
levels; p > 0.05) (Figure 1). Taken together, these find-
ings exclude any significant rCBF alterations related to
COU254 or vehicle treatment and prove that MCA
occlusion and reperfusion were sufficient in our model.
As a next step, we determined infarct sizes and the
extent of neuronal damage in control mice and mice
treated with COU254. 24 h after tMCAO no significant
differences in infarct volumes were observed between
the two groups as revealed by 2,3,5-Triphenyltetrazo-
liumchloride (TTC) staining and successive infarct pla-
nimetry (101.5 ± 31.4 mm3 vs. 110.0 ± 27.2 mm3; p >
0.05) (Figure 2a). In line with these findings, H&E stain-
ing confirmed widespread ischemic neurodegeneration
in both groups which regularly expanded to the basal
ganglia and the neocortex (Figure 2a). Detailed analysis
of the neurological status using two different functional
scores also could not reveal any beneficial effects of
COU254 in acute ischemic stroke (Bederson score: 2.8 ±
1.4 vs. 2.4 ± 1.8; p > 0.05; grip test: 2.0 ± 1.4 vs. 2.5 ±
1.9; p > 0.05) (Figure 2b). Finally, no differences in
thrombus formation within the infarcted brain areas of
COU254-treated mice or controls were detectable by
immunoblot (Figure 2c).
Discussion
Unexpectedly, the selective nonpeptidic FXIIa inhibitor
COU254 could not ameliorate ischemic brain damage
after tMCAO in mice.
COU254 belongs to a new group of recently described
3-carboxamide-coumarins which represent the first
Figure 1 rCBF does not differ between COU254-treated mice and controls after tMCAO. Determination of regional cerebral blood flow
(rCBF) using Laser Doppler flowmetry before the occlusion of the middle cerebral artery (baseline), 10 min after the occlusion (ischemia) and
again 10 min after the removal of the filament (reperfusion) in COU254-treated mice and vehicle-treated controls (n = 3/group). No significant
differences in rCBF were observed between the two groups. 1-way ANOVA, Bonferroni post hoc test, ns = not significant.
Kraft et al. Experimental & Translational Stroke Medicine 2010, 2:5
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selective inhibitors of FXIIa [12]. In contrast to conven-
tional FXII inhibitors it does not interfere with other
components of the contact activation system or plas-
matic coagulation cascade potentially involved in stroke
development such as kallikrein, FXa or the TF/FVIIa
complex [15]. Mechanistically, COU254 mediates anti-
FXII activity through the formation of an acyl enzyme
instead of an alkyl enzyme as observed with thrombin
and 6-chloromethyl ester coumarins [12]. Several
reports have meanwhile highlighted the role of FXII in
pathological thrombogenesis [20]. FXII-deficient mice
were protected against arterial thrombosis, collagen-
induced venous thromboembolism and ischemic stroke
[10,11]. Importantly, FXII-/- mice display normal hemos-
tasis and consequently FXIIa inhibition was not asso-
ciated with increased bleeding complications supporting
the intriguing hypothesis that hemostasis and thrombo-
sis are two mechanistically different processes [20].
Hence, FXIIa is considered an attractive target for phar-
macological inhibitors designed to treat or prevent
thromboembolic disorders. Such a safe therapy might be
particularly advantageous for the treatment of acute
Figure 2 COU254 does not improve outcome after experimental stroke in mice. Infarct size and functional outcome in COU254-treated
mice and controls (vehicle) 24 h after 60 min transient middle cerebral artery occlusion (tMCAO). (a) (top) Representative 2,3,5-
Triphenyltetrazoliumchloride (TTC)-stained coronal brain sections from the two animal groups. Ischemic infarctions appear white and regularly
include the neocortex and basal ganglia as confirmed by hematoxylin and eosin (H&E) staining (bar represents 250 μm). (bottom) Infarct
volumes on day 1 after tMCAO in COU254-treated mice and vehicle-treated controls as determined by planimetry (n = 10/group). Non-
parametric Mann Whitney test, ns = not significant. (b) Neurological Bederson score and grip test score on day 1 after tMCAO in COU254-
treated mice and vehicle-treated controls. In line with the results on infarct volumes, no significant functional differences became apparent
between the treatment groups. Non-parametric Mann Whitney test, ns = not significant. (c) Accumulation of fibrin(ogen) in the infarcted (+) and
contralateral (-) cortices of COU254-treated mice or vehicle-treated controls. Fibrinogen clotting 24 h after ischemia was analyzed by
immunoblotting. Two representative immunoblots of each group are shown.
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ischemic stroke, where the conventional anticoagulants
used against stroke progression or recurrence are inheri-
tably associated with increased bleeding-related morbid-
ity and mortality [1].
We recently established D-Pro-Phe-Arg chloromethyl
ketone (PCK) as FXIIa inhibitor in experimental stroke
[11]. Mice treated with PCK immediately before the
occlusion of the middle cerebral artery (MCA) devel-
oped smaller infarcts and less severe neurological defi-
cits compared to controls. Moreover, the formation of
fibrin within the infarcted brains was significantly
reduced. Although PCK irreversibly inhibits the amido-
lytic activity of FXIIa it is not specific over FXII. Rather,
PCK has been shown to block other components of the
contact activation system or plasmatic coagulation cas-
cade bearing the potential risk of undesired adverse
effects [12]. Moreover, the peptidic structure and the
alkylating behavior of the chloromethyl function prevent
the application of PCK as oral anticoagulant. Natural
anticoagulant proteins displaying anti-FXIIa activity
were also reported, e.g. from leguminous plants [21],
hematophagous insects [22-24], helminth parasites [25]
and bacteria [26]. Again, despite their proven efficacy,
all these proteins were generally not selective over blood
coagulation proteases.
Several reasons might account for the negative results
in present study. Besides “true” inefficacy of COU254 in
acute experimental stroke related for example to the
relatively low FXIIa inhibitory potency of COU254 com-
pared to PCK [12] technical limitations could have been
responsible. The anti-FXIIa activity of COU254 has only
been established from in vitro dose-response curves so
far and pharmacodynamic or pharmacokinetic data on
COU254 in animals, especially rodents, are lacking.
Moreover, the optimum dosage or route of application
of COU254 in mice is yet unknown as is the ideal time
point of administration during the course of ischemic
stroke. Because human and mouse FXII share a high
degree of sequence homology and the established
human FXII inhibitors usually also block murine FXII
[10,11], species specific differences of COU254 mode of
action between humans and rodents seem unlikely but
cannot be completely ruled out.
In summary, 3-carboxamide-coumarins represent a
promising new class of selective FXII inhibitors but
further preclinical evaluation of these compounds in
animal models is clearly needed before any firm conclu-
sions on their antithrombotic potential can be drawn.
Acknowledgements
The expert technical assistance of Melanie Glaser and Daniela Urlaub is
highly appreciated. This work was supported by the Deutsche
Forschungsgemeinschaft (SFB 688 TP B1 and A13).
Author details
1Department of Neurology, Josef-Schneider-Str. 11, University Clinic of
Wuerzburg, Germany. 2Department of Pharmacy, Drug Design and Discovery
Center, FUNDP, University of Namur, Belgium.
Authors’ contributions
All authors have read and approved the final manuscript.
PK operated the animals, assessed the functional scores and interpreted the
data. TS performed the immunoblots. LP provided COU254 and finalized the
manuscript. GS and CK conceived the experiments, funded the project and
wrote the manuscript.
Competing interests
The authors declare that they have no competing interests.
Received: 8 January 2010
Accepted: 15 February 2010 Published: 15 February 2010
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