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Regular Article
VASCULAR BIOLOGY

Intraperitoneal administration of activated protein C prevents

postsurgical adhesion band formation
Peyman Dinarvand,1 Seyed Mahdi Hassanian,1 Hartmut Weiler,2 and Alireza R. Rezaie1
1

Department of Biochemistry and Molecular Biology, St. Louis University School of Medicine, Saint Louis, MO; and 2Blood Research Institute, Blood Center
of Wisconsin, Milwaukee, WI

Postsurgical peritoneal adhesion bands are the most important causes of intestinal
obstruction, pelvic pain, and female infertility. In this study, we used a mouse model of
adhesion and compared the protective effect of activated protein C (APC) to that of the
APC exhibits anticoagulant,
Food and Drug Administrationapproved antiadhesion agent, sodium hyaluronate/
antifibrinolytic, and
carboxymethylcellulose (Seprafilm) by intraperitoneal administration of either APC or
antiinflammatory properties.
Seprafilm to experimental animals. Pathological adhesion bands were graded on day 7,
Intraperitoneal administration
and peritoneal fluid concentrations of tissue plasminogen activator (tPA), D-dimer,
of APC effectively prevents
thrombinantithrombin complex, and cytokines (IL-1b, IL-6, interferon-g, tumor necropostsurgical adhesion band
sis factor-a, transforming growth factor-b1) were evaluated. Inflammation scores were
formation.
also measured based on histologic data obtained from peritoneal tissues. Relative to
Seprafilm, intraperitoneal administration of human APC led to significantly higher reduction of postsurgical adhesion bands. Moreover,
a markedly lower inflammation score was obtained in the adhesive tissues of the APC-treated group, which correlated with significantly
reduced peritoneal concentrations of proinflammatory cytokines and an elevated tPA level. Further studies using variants of human APC
with or without protease-activated receptor 1 (PAR1) signaling function and mutant mice deficient for either endothelial protein C receptor
(EPCR) or PAR1 revealed that the EPCR-dependent signaling activity of APC is primarily responsible for its protective activity in this model.
These results suggest APC has therapeutic potential for preventing postsurgical adhesion bands. (Blood. 2015;125(8):1339-1348)

Key Points

Introduction
Peritoneal adhesion bands are pathological brous tissues that join
intraabdominal and intrapelvic organs to each other and to the
abdominal wall. These bands develop in 67% to 93% of patients
undergoing a general surgical operation and up to 97% of patients
undergoing an open gynecological pelvic operation.1,2 Peritoneal
adhesion bands are the most important causes of intestinal obstruction,3
pelvic pain,4 and female infertility.5 Many antiadhesive agents such
as antiinammatory drugs, antibiotics, brinolytic agents, and solid
barriers have been used with limited success for the prevention of
postsurgical peritoneal adhesion bands. Selected agents including
sodium hyaluronate/carboxymethylcellulose (Sepralm) and oxidized regenerated cellulose (Interceed) have been approved by the
Food and Drug Administration (FDA) and are considered the gold
standards for the prevention of postsurgical adhesion bands.6-10
These agents physically prevent adjacent tissues from contacting
each other, thereby reducing the probability of adhesion band
formation. A limitation of using these physical barriers is their sitespecic nature, which requires the surgeon to predict where adhesions may occur in order to place these barriers accordingly.
Despite being a common sequel of wound healing process after
abdominal surgery, the cellular and molecular mechanisms responsible
for the formation of brous adhesion bands remain poorly understood.
Injury to peritoneal mesothelial cells after surgery results in secretion of

brin-rich exudates that constitute a suitable environment for migration

and proliferation of underlying broblasts as a normal physiological
response during the wound healing process.11-14 A delicate balance
between brinolysis and cellular growth at this stage is essential to
prevent the formation of adhesion bands. Impaired brinolysis can
be associated with excess cellular growth, brosis, and deposition of
collagen-rich extracellular matrix that leads to pathological adhesion
bands. During peritoneal injury, mesothelial and broblast cells secret
a signicant amount of the probrotic cytokine transforming growth
factor (TGF)-b to the peritoneal cavity, thereby regulating different
phases of the wound healing process (ie, inammation, cellular
migration, proliferation, angiogenesis, and tissue remodeling).11-14 It
has been hypothesized that aberrant regulations of inammation, brinolysis, and TGF-b signaling pathways make key contributions to
the formation of postsurgical adhesion bands.14-16 In this study, we
hypothesized that intraperitoneal administration of the anticoagulant
and antiinammatory protease activated protein C (APC) may inhibit
postsurgical adhesion band formation. APC is an approved drug for
treating adults with severe sepsis,17 although it has been recently pulled
off the market by the manufacturer (Eli Lilly) due to its apparent lack
of benecial effect in a follow-up clinical study.18 Nevertheless,
numerous in vitro and in vivo studies have rmly established potent
antiinammatory, cytoprotective, and probrinolytic properties for

Submitted October 28, 2014; accepted January 2, 2015. Prepublished online

as Blood First Edition paper, January 9, 2015; DOI 10.1182/blood-2014-10609339.

The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked advertisement in accordance with 18 USC section 1734.

P.D. and S.M.H. contributed equally to this study.

The online version of this article contains a data supplement.

BLOOD, 19 FEBRUARY 2015 x VOLUME 125, NUMBER 8

2015 by The American Society of Hematology

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BLOOD, 19 FEBRUARY 2015 x VOLUME 125, NUMBER 8

APC in various injury model systems.19-24 APC, upon interaction with

a vascular cell surface receptor, termed endothelial protein C receptor
(EPCR), activates the G-protein coupled receptor, protease-activated
receptor 1 (PAR1), thereby eliciting protective antiinammatory and
cytoprotective signaling responses.20,25,26 We tested our hypothesis in
a mouse model of abdominal surgery, whereby we directly administered
recombinant APC or its different variants possessing only anticoagulant
or antiinammatory activity24,26 to the peritoneal organs of wild-type
and mutant mice decient in either EPCR or PAR127-29 prior to closing
the abdominal incision. Analyses of these results suggest that APC
exhibits a markedly higher efcacy in preventing postsurgical peritoneal adhesion band formation in comparison with the FDA-approved
Sepralm. We demonstrate that the EPCR-dependent signaling activity
of APC is primarily responsible for its protective properties in this model.
Thus, APC holds the potential to be therapeutically used as a novel
antiadhesive drug during general surgery.

2 methods described by Nair et al.31 and Leach et al.6 (see supplementary Figure 1 and supplementary Table 1 for the grading systems). The
average adhesion score frequency based on these systems for 10 mice
per group is presented in Figure 1A-B. Analysis of data according to
these 2 scoring systems suggests that APC inhibits adhesion bands to
a signicantly greater extent than Sepralm. Indeed, no adhesion band
was visible in mice treated with APC after 7 days. By contrast, most of
adhesion bands remained unaffected in the Sepralm-treated group
over the 7-day observation period (Figure 1A-B). Furthermore, no
mortality or complications (ie, bleeding) were observed in any of the
experimental animals. The efcacy of several different concentrations of APC (10, 25, 50, 100, and 200 mg/kg) was evaluated and the
APC concentration of 25 mg/kg was determined to be only slightly
less active than the 50 mg/kg dose, which yielded the maximal protective effect in preventing adhesions. Increasing the concentration
of APC to .50 mg/kg did not result in any additional protective
effect (Figure 1C-D).

Materials and methods

APC ameliorates peritoneal inflammation

Animals

Peritoneal uid concentrations of different cytokines were determined by enzyme-linked immunosorbent assay at 4 time points of
0 hours, 6 hours, 24 hours, and at 1-week postoperative (Figure 2).
At the 6-hour time point, the IL-1 level was elevated greater than
sixfold in the control and treatment groups and neither Sepralm
nor APC had statistically signicant effects in the cytokine level
(Figure 2A). The IL-1 level gradually declined and reached near the
baseline level 1-week postoperative (Figure 2A). By contrast, the
concentration of IL-6 was elevated in both control and Sepralmtreated groups (Figure 2B). Although APC markedly inhibited the
IL-6 level 6 hours postoperative, Sepralm had minimal effect on the
IL-6 level for the duration of the experiment (Figure 2B). Similarly,
APC effectively inhibited tumor necrosis factor (TNF)-a, thus decreasing its secretion to a near baseline level at all 3 time points
examined (Figure 2C). By contrast, the TNF-a level remained high
in the Sepralm group for 1-week postoperative (Figure 2C). Although the Sepralm had no effect on the expression level of TGF-b,
APC inhibited its secretion at both 6-hour and 24-hour time points
(Figure 2D). APC also inhibited the expression of interferon (IFN)-g,
however, Sepralm had no signicant effect on the IFN-g expression
level (Figure 2E). The concentration of tissue plasminogen activator
(tPA) in peritoneal uid was markedly elevated in the APC group at
6 hours postoperative (Figure 2F), whereas Sepralm had no effect
on the tPA level for the duration of the experiment (Figure 2F). These
results suggest that APC infusion, in contrast to Sepralm, inhibited
postsurgical proinammatory responses associated with adhesion
band formation.

C57BL/6 mice were purchased from Jackson Laboratory. Transgenic EPCRd/d

and PAR12/2 male mice have been previously described.27-29 Animals were
backcrossed onto an inbred C57BL/6 background. All experiments involving
animals were performed according to the guidelines for Care and Use of
Laboratory Animals approved by Saint Louis University.
Experimental design
Eight-week-old C57BL/6 mice, weighing 25 to 30 g, were used for surgery in this
study as described.30 Animals were randomly divided into 7 groups of 10 mice
per group: group 1 had control animals with surgical abrasion plus saline
(1 mL) as vehicle; group 2 had surgical abrasion plus sodium hyaluronate/
carboxymethylcellulose (Sepralm, Genzyme Biosurgery Corporation, Cambridge,
MA) as a gold standard; group 3 had surgical abrasion plus 50 mg/kg recombinant APC-wild type (APC-WT) (in 1 mL saline) administered intraperitoneally; group 4 had surgical abrasion plus 50 mg/kg APC-2Cys; group 5 had
surgical abrasion plus 50 mg/kg APC-E170A; group 6 had surgical abrasion
plus 50 mg/kg of APC-S195A; and group 7 had surgical abrasion plus therapeutic unfractionated heparin (100 U/mouse). The optimal concentration of
APC was determined by evaluating the effect of increasing concentrations of
APC (10, 25, 50, 100, 200 mg/kg) APC on adhesion band formation (n 5 10
mice for each group). The effect of APC on adhesion bands was evaluated
using EPCR-decient and PAR1 knockout mice. In this case, after surgery,
3 groups of 10 mice/group were treated with vehicle, Sepralm, and APCWT (50 mg/kg) as described above. Based on the weights of mice (25-30 g),
the doses of APC ranged from 1.25 to 1.50 mg per mouse in all experiments.
The surgical treatment was performed under a general anesthesia inhalant
containing ethoxyethane (ether) and intraperitoneal injection of 3 mg/kg ketamine hydrochloride as described.10 All animal cares and procedures were performed according to the guidelines approved by the ethical committee of Saint
Louis University.
Surgical methods, grading adhesion bands, and
statistical analysis
Surgical methods, grading adhesion bands, histologic evaluation, and statistical
analysis are described in the supplemental Materials available on the Blood
Web site.

Results
APC infusion prevents adhesion band formation

Grades of intraperitoneal adhesion bands (frequency and distribution)

in experimental mice were measured 7 days postoperative according to

Analysis of mRNA levels of proinflammatory cytokines

Adhesion bands and peritoneal tissues surrounding them for all mice
were isolated one week post-surgery and the effect of APC and
Sepralm on the RNA levels of different cytokines were evaluated
(Figure 3). As negative controls, a part of peritoneum from normal
mice (no adhesion bands) in the mock surgery group was also removed and analyzed. Similar to saline treated group, the messenger
RNA (mRNA) levels of vascular cell adhesion molecule-1, intercellular adhesion molecule-1, and E-selectin were markedly elevated
in the peritoneal tissues in the Sepralm-treated group, however, APC
effectively inhibited the expression levels of all 3 cell adhesion molecules (Figure 3A-C). Moreover, APC, but not Sepralm, inhibited the
mRNA levels of monocyte chemotactic protein-1 (Figure 3D) and matrix

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Figure 1. Analysis of postsurgical adhesion grades

for Seprafilm- and APC-treated groups. (A) Adhesion
grades for 10 mice (supplemental Table 1) based on
Nair et als31 scale. (B) Adhesion grades for 10 mice
(supplemental Table 1) based on Leach et als6 scale.
Analysis of APC concentration dependence of adhesion grades based on Nair et als31 (C) and Leach
et als6 (D) scoring systems.

metalloproteinase-2 (Figure 3E). APC and to a lesser extent Sepralm

downregulated the mRNA levels of matrix metalloproteinase-9
(Figure 3F), E-cadherin (Figure 3G), and plasminogen activator

inhibitor 1 (PAI-1) (Figure 3H), however, only APC could reverse

injury-mediated downregulation of the mRNA level of the tissue
inhibitor of metalloproteinase 2 (TIMP-2) (Figure 3I).

Figure 2. Analysis of peritoneal fluid concentrations of cytokines and tPA. The concentrations of IL-1b (A), IL-6 (B), TNF-a (C), TGF-b1 (D), IFN-g (E), and tPA (F) for all
groups were measured by enzyme-linked immunosorbent assays. The data are shown as mean 6 standard deviation (SD). *P , .05 and **P , .01 compared with salinetreated vehicle at each time point. NS, nonsignificant.

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Figure 3. Analysis of mRNA expression of peritoneal tissues. Real-time quantitative polymerase chain reaction was used to analyze the mRNA expression levels
in peritoneal adhesion bands of normal (no surgery), saline-treated (vehicle), Seprafilm-treated, and APC-WT-treated mice: (A), vascular cell adhesion molecule-1;
(B), intercellular adhesion molecule-1; (C), E-selectin; (D), monocyte chemotactic protein-1; (E), matrix metalloproteinas-2; (F), matrix metalloproteinas-9; (G),
E-cadherin; (H), PAI-1; (I), TIMP-2. ICAM-1, intercellular adhesion molecule-1; MCP 1, monocyte chemotactic protein-1; MMP 2, matrix metalloproteinase-2; MMP 9,
matrix metalloproteinase-9; VCAM-1, vascular cell adhesion molecule-1. Data are shown as mean 6 SD, n 5 5. *P , .05 and **P , .01 compared with saline-treated
vehicle.

Adhesion band formation and postsurgical inflammation are

inhibited by a signaling-selective APC variant

To understand the mechanism by which APC inhibits postsurgical

adhesion band formation, grades of postsurgical intraperitoneal
adhesions were measured in mice treated with 2 different APC
variants, one that only has the signaling function (APC-2Cys) and
the other that has normal anticoagulant but defective PAR1 signaling
function (APC-E170A).24 Analysis of adhesion grades suggests that
APC-2Cys (APC variant possessing only signaling function) inhibits
adhesion band formation to a similar extent as APC-WT (Figure 4A-B;
supplemental Table 1). Minimal adhesion bands were observed in
mice treated with either APC-WT or APC-2Cys in a week-long study
(Figure 4A-B). On the other hand, the APC-E170A derivative, which
has normal EPCR binding and anticoagulant activity, but possesses
no PAR1-dependent signaling function,24 exhibited signicantly
diminished protective activity (Figure 4A-B). The catalytically inactive APC-S195A mutant had no protective activity (Figure 4A-B),
suggesting that the catalytic activity of APC is required for its protective function.

Similar to APC-WT, the signaling procient APC-2Cys derivative

effectively inhibited the IL-6 level 6-hour postoperative (Figure 4C).
Interestingly, there was also a modest decrease in the IL-6 level in the
APC-E170A group (Figure 4C). However, similar to Sepralm group
(Figure 2B), the cytokine level remained elevated with the APC-S195A
group 1-week postoperative. Similar to APC-WT, APC-2Cys effectively inhibited TNF-a, thus decreasing its secretion to a near baseline
level at all 3 time points examined (Figure 4D). By contrast, the TNF-a
level remained high with both APC-E170A and APC-S195A groups
for 1-week postoperative (Figure 4D). Similar to APC-WT, APC-2Cys
dramatically inhibited the secretion of TGF-b at both 6-hour and
24-hour time points (Figure 4E). APC-E170A also exhibited a modest
TGF-b inhibitory effect (6 hour and 24 hour), however, APC-S195A
had no effect in preventing surgery-mediated peritoneal TGF-b expression (Figure 4E). APC-WT and APC-2Cys exhibited similar inhibitory activity proles toward IFN-g, thus reducing its expression
level in peritoneal uid (Figure 4F). The results suggest that the inhibition of adhesion bands and peritoneal inammation require the
signaling but not the anticoagulant activity of APC.

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Figure 4. Analysis of adhesion grades and peritoneal fluid concentrations of cytokines for signaling-selective APC variants. (A) Adhesion grades for 10 mice
(supplemental Table 1) based on Nair et als31 scale. (B) Adhesion grades for 10 mice (supplemental Table 1) based on Leach et als6 scale. The concentrations of IL-6 (C),
TNF-a (D), TGF-b1 (E), IFN-g (F) for all groups were measured by enzyme-linked immunosorbent assays. The data are shown as mean 6 SD. *P , .05 and **P , .01
compared with saline-treated vehicle at each time point.

Both APC-WT and APC-2Cys effectively suppress inflammation

scores in peritoneal tissues

Hematoxylin and eosin staining was conducted to compare the inltration of inammatory cells to peritoneal tissues. The results in 2
magnications for different groups: saline control (A), Sepralm (B),
APC-WT (C), APC-2Cys (D), APC-E170A (E), and APC-S195A
(F) are presented in Figure 5. The inammation scores, evaluated by
2 assessors in a double-blind study based on the histologic data are
presented in Figure 5G-H. In the saline-treated control group, many
inammatory cells were recruited to peritoneal tissues and some
microabscesses could be observed (Figure 5A). The same cell types,
but to a lesser extent, could be detected in the photomicrographs of
the Sepralm group (Figure 5B). In contrast to both the control and
Sepralm groups, the number of inammatory cells was markedly
decreased and essentially no microabscesses could be observed in the
APC-WT and APC-2Cys groups (Figure 5C-D). There were only a
few scattered leukocytes in the APC-WT and APC-2Cys groups.
Inammation scores, as determined in a double-blind study on a scale
of 0 to 3,10,32 are presented in Figure 5G. Statistically signicant

differences were observed between mice receiving Sepralm, APCWT, APC-2Cys, and APC-E170A derivatives from those receiving
saline or APC-S195A. In comparison with the Sepralm and APCE170A groups, a signicant decrease of the inammation score was
observed for APC-WT and APC-2Cys groups (Figure 5H). The total
number of cells in the peritoneal cavity of experimental mice was
determined to be 145 800/mL in untreated controls, 148 500/mL in the
Sepralm group, and 25 600 in the APC group, clearly showing the
dramatic inhibitory effect of APC on migration of inammatory cells
to the peritoneal cavity in response to injury. The signicantly lower
inammation scores of APC (also APC-2Cys) are consistent with their
dramatic inhibitory effect on the expression levels of proinammatory
cytokines as demonstrated above in Figures 2 and 4.
Anticoagulant and direct profibrinolytic effects of APC do not
contribute to the inhibition of adhesion band formation

None of the APC variants exhibited a signicant modulatory effect on

the coagulation cascade (ie, thrombin generation) as judged from levels
of thrombin-antithrombin (TAT) complex in plasma samples measured

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BLOOD, 19 FEBRUARY 2015 x VOLUME 125, NUMBER 8

DINARVAND et al

100m

100m

100m

50m

50m

50m

100m

100m

100m

50m

50m

50m

Figure 5. Peritoneal tissue histology in 2 different magnifications. Representative images are derived from saline-treated control group (A1, A2), Seprafilm group
(B1, B2), APC-WT group (C1, C2), APC-2Cys group (D1, D2), APC-E170A group (E1, E2), and APC-S195A (F1, F2). The numerical scoring of inflammation based on
histology (G) is shown in panel (H).

at different time points (Figure 6A). To further determine whether

the inhibition of coagulation cascade makes any contribution to postsurgical adhesion band formation, the protective effect of therapeutic
unfractionated heparin (100 U/mouse) was compared with the effects of
Sepralm and APC derivatives. Analysis of adhesion bands, according
to the same scoring systems, revealed that heparin has no effect on the
adhesion band formation (Figure 6B).
Interestingly, the concentration of tPA in peritoneal uids was
markedly elevated in both APC-WT and APC-2Cys groups 6 hours
postoperative (Figure 6C). Similar to results presented above for
cytokines, APC-E170A also modestly increased tPA secretion
(Figure 6C). To further evaluate the effect of brinolysis on adhesion
band formation, peritoneal and plasma D-dimer levels of experimental
animals were measured at 3 different time points. D-dimer levels in
peritoneal uids were signicantly elevated after 6 hours and 24 hours
postoperative in both APC-WT and APC-2Cys groups (Figure 6D), but
there were no differences in plasma D-dimer levels between different
groups for the duration of the experiments (Figure 6E). To determine
whether the probrinolytic effect of APC, exerted via neutralization
of PAI-1,21 contributed to its antiadhesive effect, the reactivity of
APC derivatives with PAI-1 was analyzed. APC-WT and APC-E170A
exhibited similar second-order rate constants of 1 3 103 M21s21,
however, APC-2Cys exhibited ;10-fold lower reactivity with PAI-1

(k2 5 1 3 102 M21s21), suggesting that direct interaction of APC with

PAI-1 may not contribute to its protective function.
Effect of APC on adhesion bands in EPCR-deficient and PAR1
knockout mice

To determine whether the EPCR-dependent activation of PAR1 by

APC is required for its protective activity, the same postsurgical
adhesion experiments were conducted in genetically altered EPCRdecient (EPCRd/d) mice, which express a greatly diminished amount
of the receptor (,10% of cell-surface expression) and in PAR12/2
knockout mice. Analysis of adhesion grades according to Nair et al31
and Leach et al6 scoring systems indicated that EPCR is required for the
protective activity of APC as evidenced by the APC-treated EPCRd/d
mice exhibiting no improvement in their adhesion band scores
(Figure 7A; supplemental Table 2). The same modest protective effect
that was observed for Sepralm in wild-type mice was also observed in
EPCRd/d mice, supporting the reliability of the adhesion band scoring
systems. By contrast to EPCRd/d mice, APC inhibited adhesion bands
in PAR12/2 knockout mice, although the extent of the protective effect
was rather lower than that observed in wild-type mice (Figure 7H;
supplemental Table 3), suggesting that APC cleavage of PAR1 makes
some contribution to its protective effect. However, the bulk of the

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Figure 6. Analysis of coagulation and fibrinolytic markers in peritoneal fluids and plasma and the effect of heparin on adhesion band formation. (A) The
concentrations of TAT complex for all groups were determined by an enzyme-linked immunosorbent assay. (B) The effect of therapeutic unfractionated heparin
(100 U/mouse) on adhesion grades (10 mice/group) was compared with the effects of APC and Seprafilm using Nair et als31 and Leach et als6 scoring systems. (C) The
concentrations of tPA in peritoneal fluids of different treatment groups were determined by an enzyme-linked immunosorbent assay. The concentrations of D-dimer in
peritoneal fluids (D) or in plasma (E) for all groups were determined by an enzyme-linked immunosorbent assay. The data are shown as mean 6 SD; NS, nonsignificant.
*P , .05 and **P , .01 compared with saline-treated vehicle at each time point.

protective effect of APC is mediated through EPCR-dependent but

PAR1-independent mechanisms. Unlike in wild-type mice, APC
had minimal effect on cytokines (Figure 7B-F) or tPA (Figure 7G)
levels in EPCRd/d mice. Moreover, similar to adhesion grade scoring
systems, APC exhibited an activity prole in PAR12/2 knockout
mice that mirrored the wild-type mice, although the extent of the
protective activity of APC in inhibiting proinammatory cytokine
secretion or enhancing tPA expression in PAR12/2 knockout mice
was signicantly decreased (Figure 7H-N).

Discussion
In this study, we have demonstrated that APC can effectively prevent
postsurgical adhesion band formation in the peritoneal cavity of experimental animals. APC is an anticoagulant serine protease that
downregulates the clotting cascade through the proteolytic degradation
of factors Va and VIIIa.19-21 In addition to its anticoagulant activity,
APC also possesses potent antiinammatory and cytoprotective
properties when it binds EPCR to activate PAR1, thereby inhibiting NF-kB and other proinammatory signaling pathways.20,25,26
APC also prevents inammation-mediated edema formation through
the Rac1-dependent upregulation of the tight junction proteins
that are responsible for maintaining the integrity of the vascular
endothelium.20,25,26,33 In light of well-documented clinical ndings
that postsurgical adhesion bands are caused by injury-mediated
inammatory and wound healing processes,11-16 we reasoned that
the intraperitoneal administration of APC after surgery may prevent

adhesion bands through the inhibition of inammatory responses.

Thus, we compared the efcacy of APC to Sepralm, as an FDAapproved gold standard in preventing postsurgical adhesion bands in
our model system. In agreement with our hypothesis, we discovered
that APC is markedly more effective than Sepralm in preventing
adhesion band formation after abdominal surgery. Unlike Sepralm,
which had a marginal effect on the expression proles of cytokines,
APC inhibited the secretion of IL-6, TNF-a, IFN-g, and TGF-b by
peritoneal tissues into the abdominal cavity. Interestingly, APC also
markedly upregulated the expression of the brinolytic protease,
tPA, in peritoneal tissues. These results are consistent with another
study showing that recombinant APC can inhibit intraperitoneal
adhesions in a rat model.34 However, this previous study did not
provide any insight into the mechanisms through which APC inhibits
peritoneal adhesions.
The observation that TGF-b was rapidly and highly induced after
surgery suggests that TGF-b, through regulation of tissue remodeling,
angiogenesis, and extracellular matrix deposition/turn over, plays a key
(patho)physiological role during early hours of the surgery-mediated
injury. As a brosis factor, TGF-b also plays an important role in
regulating brinolysis and it is a potent inducer of PAI-1, which can
inhibit brinolysis through inactivation of tPA.15 The activation of
coagulation can also lead to brin deposition after injury and trauma
mediated by the surgery. Although the natural brinolytic pathway can
catalyze the clearance of brin, the unregulated inammatory responses
and signaling through TGF-b can result in excessive brin deposition
and tissue brosis, thereby leading to formation of a pathological brin
matrix on abdominal organs, tissues, and the lining of the peritoneal
cavity. This can result in inltration of broblasts and excessive

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BLOOD, 19 FEBRUARY 2015 x VOLUME 125, NUMBER 8

Figure 7. Analysis of adhesion scores and peritoneal cytokine levels of EPCRd/d and PAR12/2 knockout mice. Adhesion scores for 10 EPCRd/d mice (supplemental
Table 3) based on both Nair et als31 and Leach et als6 scales (A). The concentrations of IL-1b (B), IL-6 (C), TNF-a (D), TGF-b1 (E), IFN-g (F), and tPA (G) for all EPCRd/d
mice groups were measured by enzyme-linked immunosorbent assays. Adhesion scores for 10 PAR12/2 knockout mice (supplemental Table 4) based on both Nair et als31
and Leach et als6 scales (H). The concentrations of IL-1b (I), IL-6 (J), TNF-a (K), TGF-b1 (L), IFN-g (M), and tPA (N) for all PAR12/2 knockout mice groups were measured by
enzyme-linked immunosorbent assays. *P , .05 compared with saline-treated vehicle.

deposition of collagen, thereby fortifying the matrix and facilitating the

tight adherence of surrounding peritoneal tissues to each other and/or to
the abdominal cavity. The most likely sources of TGF-b are peritoneal
broblasts and activated macrophages, which can be recruited into the
peritoneal cavity after surgery-mediated tissue injury. In support of this
hypothesis, it has been shown that surgical-induced hypoxia can recruit
macrophages to sites of injury and hypoxic treatments of peritoneal
broblasts and macrophages can signicantly increase expression
levels of TGF-b by these cells.16 These surgical-induced inammatory
events are the primary cause of postsurgical adhesion bands and it was
interesting to discover that all of these inammatory responses were
inhibited by APC as evidenced by the protease potently inhibiting
expression of TGF-b and other cytokines during the early hours after
the surgery. However, the inhibition of TGF-b by a specic monoclonal antibody did not result in complete inhibition of adhesion bands
(data not shown), suggesting that the pathogenesis of the disease is
multifactorial and that TGF-b signaling is only partially responsible for
adhesion band formation.
The observation that APC induced the expression of tPA suggests
that the protease can also upregulate brinolysis through enhancing
plasminogen activation apart from its direct anticoagulant activity,
which can inhibit brin deposition through inhibition of thrombin

generation. In support of a protective role for enhanced brinolytic

activity of APC, the concentration of D-dimer was signicantly
increased in the peritoneal uids of APC-treated but not Sepralmtreated groups. APC is also known to neutralize PAI-1,21 particularly
when the inhibitor is bound to vitronectin,35 which is expected to be
abundant in the extracellular matrix of abdominal tissues during the
wound healing process. However, the direct neutralization of PAI-1 by
APC did not appear to play a signicant role in enhancing brinolysis
because APC-2Cys, which inhibited adhesion bands similar to APCWT, had a 10-fold lower reactivity with PAI-1, but APC-E170A,
which had signicantly diminished protective activity exhibited normal
reactivity with PAI-1. Based on these data, we propose that APCmediated downregulation of PAI-1 and TGF-b expression, together
with the upregulation of tPA expression by peritoneal tissues, all
mediated by the cellular signaling function of the protease, contributes to enhanced peritoneal brinolysis. It is worth noting that some
protective role for tPA in inhibiting peritoneal adhesion formation
has also been previously demonstrated.36
The results with the genetically altered mice, either decient for
EPCR or devoid of PAR1, together with those of APC derivatives
suggest that the EPCR-dependent signaling function of APC is essential
for the ability of APC to prevent postsurgical adhesion band formation.

From www.bloodjournal.org by guest on July 25, 2016. For personal use only.
BLOOD, 19 FEBRUARY 2015 x VOLUME 125, NUMBER 8

This is evidenced by the observation that APC-2Cys, which has

dramatically decreased anticoagulant activity, exhibited a normal protective activity in both in vitro and in vivo assays; however, APC did
not exhibit any activity in the same assays in EPCRd/d mice. Unlike
interaction with EPCR, which was required for the protective function
of APC, the activation of PAR1 by APC only contributed to the
protective activity of the protease, but it was not strictly required for
its antiadhesive function because APC signicantly inhibited both
cytokine generation and adhesion band formation in PAR12/2
knockout mice. A previous study, investigating the cytoprotective
function of another nonanticoagulant APC in the LPS-induced mouse
endotoxemia, which used the same EPCRd/d and PAR12/2 knockout
mice, also noted that some EPCR-dependent benecial effects of APC
do not require PAR1.22 It is possible that in addition to PAR1, the
EPCR-dependent activation of another receptor (in particular, PAR3)
by APC is required for its full protective activity. In support of this
hypothesis, it has been reported that the EPCR-dependent noncanonical cleavage of PAR3 at Arg-41 by APC also contributes to
its cytoprotective function.37,38 Interestingly, APC-E170A, which has
normal anticoagulant and EPCR-binding properties but cannot activate PAR1, exhibited partial protective activity in both inhibiting
cytokine secretion and adhesion band formation. Further studies will
be required to determine whether the partial protective activity of
APC-E170A in wild-type mice and that of APC-WT in PAR12/2
knockout mice is due to activation of PAR3 by these proteases.
The observations that the plasma TAT and D-dimer levels were
essentially identical for all groups suggest that the direct anticoagulant activity of APC plays a minimal role in the cytoprotective
function of the protease in this model. This is consistent with heparin
not exhibiting any protective effect. Nevertheless, the catalytic
activity of APC was required for its function because the APCS195A mutant, which can also bind EPCR normally but has no
catalytic activity, did not exhibit any protective property. Taken
together, these results suggest that APC effectively prevents postsurgical adhesion band formation by enhancing brinolysis and
inhibiting inammatory mediators during the early days after the
surgery through its EPCR-dependent signaling mechanism that is
only partially dependent on PAR1 activation.

APC INHIBITS ADHESION BAND FORMATION

1347

The results presented above strongly suggest that APC may be

therapeutically developed as a safe and effective drug for preventing
postoperative adhesion band formation in humans. APC has already
been therapeutically used for treating adults with severe sepsis for more
than a decade.17,18 The major drawback of APC was determined to be
its enhancement of bleeding incidence in ;2% of the treated patients.17
This effect of APC as a drug for preventing/treating postsurgical
adhesion bands is not expected to be a factor because unlike with severe
sepsis, in which APC is injected intravenously, the drug is administered
intraperitoneally after surgery and a much lower dose of APC is required to effectively prevent adhesions. This proposal is supported
by the observation that intraperitoneal administration of APC did not
affect the coagulation cascade as determined by the analysis of the
plasma TAT complex levels in all experimental animals.

Acknowledgments
The authors thank Audrey Rezaie for proofreading the manuscript.
This work was supported by a grant from the National Institutes
of Health National Heart, Lung, and Blood Institute (HL 101917 and
HL 62565 to A.R.R.).

Authorship
Contribution: P.D. and S.M.H. designed and performed experiments; P.D. analyzed data; H.W. provided key reagents; and A.R.R.
supervised the project and wrote the manuscript.
Conict-of-interest disclosure: P.D. and A.R.R. are coinventors
on a patent application pertaining to the methods using APC for
preventing adhesion. The remaining authors declare no competing
nancial interests.
Correspondence: Alireza R. Rezaie, Department of Biochemistry
and Molecular Biology, St. Louis University School of Medicine,
1100 S Grand Blvd, St. Louis, MO 63104; e-mail: rezaiear@slu.edu.

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2015 125: 1339-1348

doi:10.1182/blood-2014-10-609339 originally published
online January 9, 2015

Intraperitoneal administration of activated protein C prevents

postsurgical adhesion band formation
Peyman Dinarvand, Seyed Mahdi Hassanian, Hartmut Weiler and Alireza R. Rezaie

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