Haemostatic imbalance following carrageenan-induced rat paw oedema
Carla Cicalaa,⁎, Silvana Morellob, Alessio Alfieria, Valentina Velleccoa,
Stefania Marzoccob, Giuseppina Autoreb
aDipartimento Farmacologia Sperimentale, Università degli Studi di Napoli “Federico II”, via D. Montesano 49, 80 131 Napoli, Italy
bDipartimento di Scienze Farmaceutiche, Università di Salerno, Via Ponte Don Melillo, 84084 Fisciano (SA), Italy
Received 2 March 2007; received in revised form 30 July 2007; accepted 6 August 2007
Available online 14 August 2007
Carrageenan-induced rat paw oedema is a widely used model to investigate the physiopathology of an acute local inflammation. Recently,
much attention has been focused on the link between haemostasis and inflammation, and on the impact that inflammation might have on
thrombotic events. It is known that the systemic response to inflammation is the “acute phase reaction” that represents a highly complex reaction
of the organism to a variety of injuries, aimed to restore homeostasis; one important feature of the acute phase reaction is the hepatic synthesis of
proteins involved in the coagulation cascade. Much attention has been focused on the role that systemic inflammation might have on thrombotic
events, while there is not much information on the role played by an acute local inflammation on haemostasis, that can lead toward a pro-
thrombotic state. The present study was conducted to evaluate the haemostatic balance in the early and the late phase of carrageenan-induced rat
paw oedema; i.e. at 3 h, when paw inflammation is maximally expressed, and 24 h following carrageenan injection, when there is an almost
complete absence of local inflammatory symptoms. We found that in inflamed animals, 24 h following oedema induction, there was an increase in
plasma fibrinogen levels, antithrombin III activity and serum interleukin-6 levels, concomitant to a shortened prothrombin time and to an increased
platelet responsiveness to ADP. Furthermore, in inflamed tissues at 3 h there was an increase in antithrombin III proteic expression. Our results
demonstrate that a haemostatic imbalance occurs following carrageenan-induced rat paw oedema.
© 2007 Elsevier B.V. All rights reserved.
Keywords: Carrageenan; Haemostasis; Fibrinogen; Platelet aggregation; Antithrombin III
Both experimental and clinical evidence have shown that
thrombosis and atherosclerosis might be associated to an
inflammatory reaction, suggesting an important link between
inflammation and haemostasis (Jurado and Ribeiro, 1999; Khatun
et al., 1999; Esmon, 2003; Strukova, 2006). Indeed, inflammation
initiates clotting, decreases the activity of natural anticoagulant
mechanisms and impairs the fibrinolytic system. On the other
hand, proteases involved in coagulation system contribute to
inflammation not only by promoting fibrin formation at the site of
injury, but also by stimulating several cell functions (Cicala and
Cirino, 1998; Cirino et al., 2000; Esmon, 2005). Anticoagulants
may be beneficial in limiting inflammation triggered by
coagulation, not only by inhibiting coagulation proteases, but
also by limiting inflammatory cell activation (Roemisch et al.,
2002; Wiedermann and Romisch, 2002; Strukova, 2006).
An important contribution to haemostatic changes observed
following inflammation is given by the “acute phase reaction”,
that represents a highly complex reaction of the organism to a
variety of injuries, aimed to restore homeostasis. One important
feature of the “acute phase reaction” is the hepatic synthesis of
proteins involved in the coagulation cascade (Baumann and
et al., 2002). However, changes in the haemostatic balance
caused by the “acute phase reaction” may lead to an increased
risk of thrombosis, both in human and in experimental animals
(Plesca et al., 1995; Cucuianu et al., 1996; Robson et al., 1996).
There is evidence that fibrinogen, an acute phase reactant that is
increased under inflammatory conditions, is an important link
between inflammation and haemostasis; high fibrinogen levels
have been associated with an increased risk of thrombotic
diseases (Ernst, 1993; Hernandez et al., 2000; Esmon, 2005).
Available online at www.sciencedirect.com
European Journal of Pharmacology 577 (2007) 156–161
⁎Corresponding author. Tel.: +39 081678455; fax: +39 081678403.
E-mail address: email@example.com (C. Cicala).
0014-2999/$ - see front matter © 2007 Elsevier B.V. All rights reserved.
Carrageenan-induced oedema in the rat paw is a widely
used model, developing in about 6 h, to investigate the phys-
iopathology of an acute local inflammation, and to test the
potential anti-inflammatory of new molecules (Morris, 2003).
Up to now, identified mediators involved in this model of
acute inflammation are biogenic amines; bradykinin; prosta-
glandins (Di Rosa et al., 1971; Di Rosa and Willoughby,
1971); nitric oxide (Salvemini et al., 1996) and, more recently,
hydrogen sulphide has been shown to be involved (Bhatia
et al., 2005). We have demonstrated that carrageenan-induced
rat paw oedema is inhibited by systemic administration of the
thrombin inhibitor, hirulog, outlining a possible involvement
of thrombin in the development of oedema (Cirino et al.,
Carrageenan-induced rat paw oedema remains localized in the
injection area. Little is known about the systemic reaction to the
local injection of carrageenan in the rat. In the past, studies carried
out in order to evaluate the systemic response to carrageenan
oedema induction in rats have only analyzed platelet function,
without taking in account any coagulation parameter (Srivastava
that this local acute inflammation may have on the haemostatic
might represent a factor risk for pro-thrombotic events, in the
present study we evaluated the haemostatic balance in the early
and the late phase of carrageenan-induced rat paw oedema; i.e. at
3 h, when paw inflammation is maximally expressed, and 24 h
following carrageenan injection, when there is an almost
complete absence of local inflammatory symptoms.
2. Materials and methods
ADP, aprotinin, bovine serum albumin (BSA), carrageenan,
trisodium citrate were purchased from SIGMA (Italy). Dithio-
threitol (DTT), phenyl-methylsulphonyl fluoride (PMSF),
leupeptin were purchased from ICN Pharmaceutical, S.r.l.
(Milan, Italy). Bradford reagent was purchased from Bio-Rad
Laboratories (Segrate, MI). Polyclonal antibody to antithrombin
III was purchased from DPC, Biermann (Germany). Anti-rabbit
IgG conjugated to horseradish peroxidase (HRP-anti-rabbit
IgG) was purchased from Dako (Denmark). Enhanced chemi-
luminescence's system (ECL) was from Amersham Pharmacia
Biotech (Milan, Italy).
2.2. Carrageenan oedema
Male Wistar rats (200–250 g; Harlan Nossan, Italy) were
slightlyanaesthetizedwithenflurane andoedemawas inducedby
injecting in the left hind paw carrageenan 1% w/v (100 μl). Paw
volume was measured at the time zero and each hour for 6 h; and
Italian D.L. no. 116 of 27 January 1992 and associates guidelines
inthe EuropeanCommunities CouncilDirective of24November
2.3. Blood cell count
At 3 and 24 h after induction of oedema, or vehicle injection,
blood was withdrawn by cardiac puncture, anticoagulated with
trisodium citrate 3.8% (ratio 1:9) and utilized for cell count, by
using a cell counter CELL DYN 610 (SEQUOIA TURNER).
2.4. Coagulation parameters
An aliquot of blood withdrawn 3 and 24 h following oedema
induction, was centrifuged at 650×g for 15 min and the plasma
obtained was utilized to evaluate prothrombin time, activated
partial thromboplastin time and fibrinogen content by a coagul-
ometer (KoaguLab MJ Ortho Diagnostic Systems, Raritan, NJ).
Quantitative determination of antithrombin III activity was
performed by a chromogenic assay Unitest™ (Unicorn Diagnos-
tics Ltd, London UK). Briefly, thrombin is added to a plasma
dilution containing antithrombin III in the presence of heparin.
After an initial incubation period, residual thrombin is determined
with a thrombin specific chromogenic substrate. The residual
thrombin activity is inversely proportional to the antithrombin III
concentration.Reference antithrombinIII activitywas 0.90UI/ml.
2.5. Interleukin-6 assay
Serum samples obtained from rat blood 3 and 24 h following
oedema-induction were subjected to interleukin-6 (IL-6) levels
determination using the rat specific DuoSet ELISA kit (ELISA;
DuoSet R&D Systems, UK), according to the manufacturer's
2.6. Platelet preparation and aggregation
Blood was withdrawn by cardiac puncture after 3 and 24 h
following the injection of carrageenan or of vehicle, as described
as previously described (Cicala et al., 1996). Platelet count in
platelet-rich plasma was adjusted to 3×105platelets/μl with
autologous platelet-poor plasma. Platelet aggregation to ADP (3,
10 and 30 μM) was monitored in an Elvi 840 light transmission
aggregometer by measuring changes in turbidity of 0.25 ml
2.7. Determination of antithrombin III protein by Western blot
3 and 24 h after oedema induction, from each rat soft tissue
was removed from individual inflamed paws. The same tissue
samples were also removed from non-inflamed animals. Tissues
were homogenised with the Polytron homogenizer (3 cycles of
10 s at the maximum speed) in a lysis buffer containing: MgCl2,
2 mM; sodium ortovanadate, 100 μM; β glycerophosphate,
50 mM; EGTA, 1 mM; DTT, 1 mM; PMSF, 1 mM; leupeptin,
10 μg/ml and aprotinin, 10 μg/ml. After centrifugation at
157 C. Cicala et al. / European Journal of Pharmacology 577 (2007) 156–161
650 ×g at 4 °C for 10 min, the supernatants were collected and
centrifuged at 10,000 ×g for 30 min at 4 °C. The protein
concentration was measured by Bradford assay using BSA as a
standard. Membrane proteins (30 μg) were briefly boiled and
loaded on 12% sodium dodecyl sulphate (SDS)-polyacrilamide
gel electrophoresis (PAGE) and transferred onto nitrocellulose
membranes for 45 min at 250 mA.
Non-specific antibody binding to the membrane was blocked
with 5% non-fat milk and 0.1% BSA in PBS-Tween 20 (0.1%,
v/v) for 1 h at room temperature. Membranes were washed with
PBS-Tween 20 (0.1%, v/v) at 5 min intervals for 30 min and
then incubated with a polyclonal antibody to antithrombin III
(1:2000 in PBS-Tween 20 0.1%, v/v, containing 5% non-fat
milk and 0.1% BSA) overnight at 4 °C. Detection blots were
washed with PBS-Tween 20 (0.1%, v/v) at 5 min intervals for
30 min and incubated with HRP-anti-rabbit IgG (1:10,000) for
2 h at 4 °C. The immunoreactive bands were visualised using an
enhanced chemiluminescence's system (ECL).
2.8. Statistical analysis
All data are expressed as mean ± S.E.M. and analyzed with
one way analysis of variance (ANOVA) followed by Dunnett's
test. A value of Pb0.05 was considered significant.
3.1. Blood cell count and coagulation parameters
Injection of carrageenan in the rat paw induced an oedema
reaching its maximum after 3 h and steadily decreasing over 6 h.
At 24 h, the oedema was almost absent. Blood cell analysis
showed that in animals treated with carrageenan, either after 3 h
or 24 h before, total white blood cell number was unchanged
compared to control animals; however the percentage of
granulocytes was significantly increased at 3 h returning to
control value 24 h thereafter. There was no difference in red
blood cell and platelet count between control and inflamed
animals (Fig. 1; Table 1A).
24 h following carrageenan-induced oedema, plasma fibrino-
gen content was significantly increased compared to the value
obtained from non-inflamed animals. At the same time point,
prothrombin time was significantly reduced compared to control
Conversely, prothrombin time, activated partial thromboplastin
injection. Plasma antithrombin III activity was significantly in-
creased on samples obtained 24 h following carrageenan
injection, while it was unchanged on samples at 3 h (Table 1B).
3.2. Platelet aggregation assay
Rat platelets obtained 24 h following carrageenan-induced
oedema showed a response to ADP increased compared to
platelets obtained from non-inflamed animals (Fig. 2).
3.3. IL-6 levels
IL-6 serum levels were significantly increased at 24 h
following oedema induction, but not at 3 h (Fig. 3).
3.4. Western blot analysis
In carrageenan injected paws obtained 3 h following oedema
induction there was an increased antithrombin III proteic
expression compared to the non-injected paws and to paws
obtained 24 h following oedema induction (Fig. 4).
The present study was conducted in order to evaluate the
haemostatic balance in the early and the late phase of
carrageenan-induced rat paw oedema.
Differences in the haemostatic balance between 3 and 24 h
following oedema induction were clearly observed, demon-
strating that a systemic reaction occurs in response to the local
inflammation, that seems to be consistent with an “acute phase
response”. Indeed, 24 h following injection of carrageenan in
Fig. 1. Time course of carrageenan-induced oedema in the rat paw. Carrageenan
(1% w/v; 100 μl) was injected in the rat paw at time zero. Paw volume was
evaluated at the time zero and each hour for 6 h following oedema injection and
at 24 h. Oedemawas evaluatedby the difference of paw volumeat each hourand
basal value (n=8).
Rat blood cell count following carrageenan-induced paw oedema
TreatmentWBC 103/μl Gran % Lymph %RBC 106/μl PLT 103/μl
Cell count were evaluated on blood obtained from rats at 3 and 24 h following carrageenan-induced oedema. Control rats received only vehicle injection in the paw.
WBC, white blood cells; Gran, granulocytes; Lymph, lymphocytes, RBC, red blood cells; PLT, platelets. Data represents means±S.E.M.aPb0.001 vs. control.
(ANOVA followed by Dunnett's test; n=6–8).
158C. Cicala et al. / European Journal of Pharmacology 577 (2007) 156–161
the rat paw, there was an increase in fibrinogen plasma levels,
that was not observed at 3 h. Fibrinogen is an acute phase
reactant whose plasma levels are increased under inflammatory
situations and also a clotting factor (Glenn, 1969; Degen, 1999;
Ceciliani et al., 2002; Gruys et al., 2005). Here, we demonstrate
an increase in plasma fibrinogen only evident when there was
no more sign of local inflammation; this suggests that following
carrageenan-induced oedema in rat paw fibrinogen might be
produced as a an acute phase protein.
High fibrinogen levels have been associated with an
increased risk of thrombotic diseases, with a multifactorial
mechanism involving also an increased platelet aggregability,
an important factor for the genesis of vascular lesion (Ross
et al., 1974; Meade et al., 1985; Ernst, 1993; Hernandez et al.,
2000; Steinhubl and Moliterno, 2005). Based on these
observations, we also evaluated platelet aggregation. We
found an increased platelet aggregability in response to ADP
at 24 h following oedema induction but not at 3 h. Furthermore,
we observed that the increase in fibrinogen plasma levels was
also paralleled by an increase in IL-6 serum levels, a cytokine
known to act as an inducer of acute phase reactants and as a
mediator of the crosstalk between inflammation and coagulation
(Baumann and Gauldie, 1994; Kerr et al., 2001).
The activated partial thromboplastin time and prothrombin
time assays were performed, as non-specific tests of the
haemostatic system, on plasma samples obtained 3 and 24 h
following oedema induction. Activated partial thromboplastin
time reflects activation of the intrinsic and common coagulation
and common coagulation pathways. We found that 24 h
following oedema induction,prothrombintime but not activated
partial thromboplastin time was significantly shortened com-
pared to control values and to values obtained at 3 h, the peak
time of inflammation. The shortening of prothrombin time value
observed, without any change of activated partial thromboplas-
tin time value, could only reflect the increase in coagulation
factors involved in the extrinsic pathway (factors VII and tissue
factor). Although it is known that inflammation causes tissue
factor generation, and hence increased activation of factor VII
(Rapaport and Rao, 1995; Esmon 2003; Chu, 2005), we did not
measure plasma tissue factor levels in our model, thus whether
factor generation needs further investigation.
Antithrombin III is a natural thrombin inhibitor; a reduction
of its activity in systemic inflammatory disorders, such as sepsis
timeand coagulation factors consumption(Esmon, 2003,2005).
3 h but increased 24 h following induction of oedema. An
increase in antithrombin III activity has already been described
to take place during inflammation, paralleled by a down-
regulation of protein C system (Plesca et al., 1995; Cucuianu et
al., 1996), and also during hepatitis A (De Jonge et al., 1995).
The increased antithrombinIII activity concomitant to a reduced
prothrombin time value suggest that even though there is an
increased production of coagulation factors, likely tissue factor,
due to the systemic response to the local inflammation, the
extrinsic coagulation pathway is not activated and there is no
coagulation factor consumption. Thus, the increased antithrom-
bin III activity might represent a defence mechanism able to
prevent intravascular fibrin deposition. Thus, we hypothesise
that following carrageenan-induced rat paw oedema, an
defence to limit thrombin activity and the consequent fibrin
Fig. 2. Aggregation to ADP of platelets obtained from inflamed animals.
Aggregation to ADP (3, 10 and 30 μM) was evaluated on rat platelets obtained
3 h (grey bar) and 24 h (crosshatched bar) following carrageenan-induced
oedema. Control animals (open bar) received only vehicle (distilled water,
100 μl) injection in the paw.⁎Pb0.05 and⁎⁎Pb0.01 (ANOVA followed by
Dunnett's test) vs. control (open bars; n=15–20).
Fig. 3. Serum IL-6 levels from inflamed rats. IL-6 levels were measured by an
bar) following carrageenan oedema induction.⁎⁎Pb0.01 vs. CTRL (ANOVA
followed by Dunnett's test; n=8).
Rat coagulation parameters evaluated following carrageenan-induced paw
Treatment PT sec APTT secFibrinogen mg/dl AT III %
Coagulation parameters were evaluated on plasma obtained from rats 3 and 24 h
following carrageenan-induced oedema. Control rats received only vehicle
injection in the paw. PT, prothrombin time; APTT, activated partial
thromboplastin time; AT III, antithrombin III. All data represent means±S.E.
M (n=6–8). AT III activity was evaluated as % activity normalized to control
activity value (0.586 UI/ml).aPb0.05;bPb0.01 andcPb0.001 vs. control.
dPb0.01 vs. 3 h.
159 C. Cicala et al. / European Journal of Pharmacology 577 (2007) 156–161
formation. In this respect, it is known that thrombin plays a role
in the development of carrageenan-induced rat paw oedema.
Systemic administration of the thrombin inhibitor, hirulog,
inhibits oedema development (Cirino et al., 1996). Furthermore,
it is known that in the inflamed paw there is local platelet and
fibrin deposition (Vincent et al., 1978). Interestingly, we found
that antithrombin III proteic expression was increased locally, in
the inflamed tissues at 3 h but not at 24 h, when inflammation
was dampened out. It is possible that this antithrombin III over-
expression at the inflamed site might participate to modulate
local fibrin deposition.
Itisworthconsideringthatour findingsare inconsistentwitha
late systemic effect of carrageenan due to absorption from the
paw. Firstly, because it is known that the inflammatory effect of
absorption rate (Di Rosa, 1972). Furthermore, because systemic
exposure to carrageenan causes disseminated intravascular
coagulation (Fowler et al., 1977; Thomson and Fowler, 1981).
It is known that disseminated intravascular coagulation is cha-
racterised by consumption of coagulation factors, thus reduction
of fibrinogen levels and prolongation of both prothrombin time
and activated partial thromboplastin time, and reduced platelet
number (Esmon, 2003, 2005); all features very different from the
haemostatic changes that we have observed.
In conclusion, taken together our results show for the first
time that following carrageenan-induced rat paw oedema there
are changes in blood parameters that are suggestive of an acute
phase reaction leading to a haemostatic imbalance. Thus, we
suggest that even a local acute inflammation that is resolved in
about 6 h triggers a systemic reaction, mainly characterized by
high fibrinogen levels, increased platelet reactivity, a shortened
state and further highlight the important link between inflam-
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Fig. 4. Panel A: Detection of antithrombin III (AT III) by Western blot analysis
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