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From the Cardiovascular Biology Research Laboratory (R.G., A.P., V.T.,
J.B., J.J.B.), the Cardiovascular Institute (J.T.F., J.H.C., V.F.), and the
Department of Pathology (J.T.F.), Mount Sinai School of Medicine, New York,
NY.
Correspondence to Dr Juan J. Badimon, Cardiovascular Biology Research Laboratories, Zena and Michael A. Wiener, Cardiovascular Institute, One Gustave L. Levy Place, Box 1030, New York, NY 10029-6574. E-mail jbadimo{at}smtplink.mssm.edu
Methods and Results—Thirty-six pigs received three different
regimens of hirudin: bolus (1 mg/kg), short-term (bolus+0.7 mg/kg per
day for 2 days), and long-term (bolus+0.7 mg/kg per day for 14 days).
The results on neointima formation at 4 weeks after
coronary angioplasty were compared with the control group (100
IU heparin/kg bolus). Hirudin was continuously administered for 2 weeks
through an infusion pump. In vivo thrombin generation was persistently
increased up to 2 weeks after angioplasty. Inhibition of thrombin
activity for 14 days reduced luminal narrowing by 40% (58±3% versus
35±3%; P<.001). No differences were observed among
the bolus and short-term hirudin groups and the control group.
Conclusions—Our results indicate that there is a continued,
marked thrombin generation that lasts for at least 2 weeks after PTCA.
Administration of r-hirudin for 2 weeks significantly reduces
neointima formation after PTCA. This observation, if
extrapolated to humans, could explain the lack of effect on
restenosis observed in the clinical trials with antithrombin
agents despite the clear benefits on reducing acute thrombotic
complications after PTCA. Therefore an adequate and prolonged
administration of thrombin inhibitors is needed to
"passivate" the thrombogenic substrate (disrupted
arterial wall) and achieve full benefit of this therapeutic
approach.
Thrombin is generated in large amounts at the site of injury after
balloon angioplasty and is amplified by formation of the prothrombinase
complex both in humans and animals.3 4 5 Exposure
of tissue factor in the atherosclerotic plaque to flowing blood leads
to increased thrombin generation, resulting in platelet- and
fibrin-rich thrombus formation.3 6 7 The
important role of thrombin in generating acute platelet-rich
thrombus and vascular healing after arterial injury is well
documented.8 9 10 11 Thrombin is also the most potent
activator of platelets, and its expression is
upregulated in smooth muscle cells after severe arterial
injury. Thrombin also mediates several cellular activities involved in
the restenosis process, including smooth muscle cell migration
and proliferation.12
Hirudin is a potent, specific, direct thrombin inhibitor.
It is highly effective at preventing acute platelet-rich thrombosis
after deep arterial injury.9 10 11
r-Hirudin reduced restenosis after balloon angioplasty in
atherosclerotic femoral arteries in rabbits.13
The results obtained in minipigs after short-term administration of
r-hirudin are controversial; one group reported significant inhibition
of neointimal thickening after carotid
angioplasty,14 whereas another report found no
effect on neointimal formation.15
However, two large clinical trials showed acute reduction in clinical
events but no long-term benefit in reducing restenosis during
short-term administration of hirudin (HELVETICA trial) and hirulog
(Hirulog Angioplasty Study).16 17 In addition,
combined analysis of the results of GUSTO IIb and TIMI 9b
trials suggests a modest but significant reduction of 13%
(P=.026) in the incidence of (re)infarction with hirudin
versus heparin in patients with an acute coronary syndrome
(Reference 1818 and data presented at the 1996 AHA Scientific
Sessions). Likewise, GUSTO IIb showed a significant 62% reduction in
death or myocardial infarction (P<.001) after 24 hours of
hirudin administration but only a mild effect at 30 days (10%
reduction; P<.058). Thus short-term hirudin does not appear
to protect against long-term processes of thrombosis and proliferation
that involves thrombin activation and arterial plaque
disruption.
We hypothesized that to achieve the full benefit of direct thrombin
inhibition, an adequate and prolonged administration of thrombin
inhibitor is required to passivate the injured vascular
wall. Therefore this study was designed to test whether effective and
maintained inhibition of thrombin activity for 2 weeks would reduce
long-term luminal narrowing after coronary balloon
angioplasty.
Recombinant Hirudin
Experimental Design
Hirudin-Bolus Group
Short-term Hirudin Group
Long-term Hirudin Group
The effects of the hirudin administration on neointima
formation after coronary angioplasty were evaluated at 4 weeks
after the coronary intervention and compared with the
heparin-treated group that serves as control.
Installation of Infusion Pump
Coronary Balloon Angioplasty
Blood Sampling and Hematological Parameters
Determination of Plasma Levels of Thrombin-Hirudin Complex
Because of the requirement of hirudin for the determination of THC, we
were only able to study thrombin generation during the 2-week period of
hirudin administration. However, in humans, directional changes of
prothrombin fragment F1.2 and THC were similar to the ones reported
here.25
To assure that the dosage of hirudin could bind all the thrombin being
generated, we measured the plasma levels of free hirudin at different
time points after PTCA. Measurements were achieved by use of a specific
ELISA (American Stago). The plasma levels of free hirudin achieved by
its continuous administration was an average of 2500 ng/mL blood. This
level of hirudin was in excess to neutralize all the thrombin being
generated as a consequence of the coronary angioplasty and was
not a limiting factor for total thrombin inhibition.
Fixation, Harvesting, and Pathological Examination of Injured
Vessels
Histomorphometric Analysis
The degree of arterial injury induced during the
angioplasty was classified according to an injury score, modified from
Schwartz et al,26 defined as follows: 0,
endothelium intact, no injury; 1,
endothelium denuded, internal elastic lamina intact; 2,
internal elastic lamina lacerated, media exposed but not lacerated; 3,
internal elastic lamina lacerated, media visibly lacerated but external
elastic lamina intact; and 4, external elastic lamina lacerated, large
laceration of media extending through the external elastic lamina.
Only those segments with an injury score
The following parameters were measured: LA=luminal area,
area of arterial lumen; MA=medial area, original medial
layer encircled by the internal elastic lamina (IEL) and external
elastic lamina (EEL); VA=vessel area, total arterial area
encircled by the EEL; OM=length of the outer media; MT=medial thickness
(average of five measurements); and IA=intimal area, area occupied by
the neointima.
The neointima was further subdivided into three distinct
areas that were clearly visible on direct histological
examination. These three areas were defined as residual thrombus,
submedial hematoma, and fibrocellular hyperplasia (see Fig 1
From these measurements the following parameter was
calculated
Statistical Analysis
Hematological Parameters
Thrombin-Hirudin Complex
Luminal Stenosis After Arterial Injury
Histomorphometry
Overall, the inhibition of thrombin activity for 14 days after PTCA by
hirudin resulted in a 40% reduction in luminal narrowing in comparison
to the control group (from 58.3±3.5% to 35.1±3.2%, respectively;
P<.005) (Fig 6A
The earliest event after balloon angioplasty in the pig is the
appearance of a platelet- and fibrin-rich thrombus at the site of
injury.1 2 9 10 14 15 22 26 27 The mural thrombus
may act as a growth factor–rich structure with the release of the
platelet
The important role of thrombin as a major agonist for platelet
activation and thrombosis is well established. More recently, thrombin
has also been identified as a growth-promoting factor for smooth muscle
cells, and its modulator role in vessel wall matrix composition has
been reported.12 28 29 With the advent of
specific and potent direct thrombin inhibitors such as
hirudin, the thrombin-mediated effects on restenosis after PTCA
can be investigated.
The importance of thrombin inhibition for reducing acute thrombotic
complications early after severe arterial injury was well
illustrated by two clinical trials using r-hirudin and one of its
analogues, hirulog, as antithrombin probes. Thrombin inhibition
significantly reduced the incidence of acute cardiac events during
active drug administration. However, no impact on late
restenosis was observed.16 17 Hirudin is
a specific and direct inhibitor of thrombin activity, not
thrombin generation. Therefore, soon after the infusion ceases, no
further effect could be expected if thrombin is still being generated.
The marked and continued generation of thrombin for at least 2 weeks
after balloon angioplasty in our model illustrates the importance of
duration of treatment. Therefore, insufficient duration of
administration (2 to 3 days in the longest human trial), inadequate
route of administration (subcutaneous versus intravenous)
to maintain adequate blood levels, or both may explain the lack of
positive results for prevention of late restenosis in these
clinical studies.
We previously showed that thrombin inhibition with hirudin reduces
acute fibrin deposition at lower doses (aPTT, 1.7 times control) in a
porcine model of angioplasty. When higher doses (aPTT >2 times
control) were used, acute mural thrombosis was totally abolished and
platelet–vessel wall interaction was reduced to a
monolayer.9 10
Further evidence supporting the hypothesis of thrombin as a major
mediator of restenosis comes from studies using
hirudin13 and recombinant tick anticoagulant
peptide (rTAP) in the cholesterol-fed rabbit femoral artery
model.30 TAP, by binding and inhibiting factor
Xa, also inhibits thrombin generation at the initiation of the
coagulation cascade. In the first study, hirudin was administered to
rabbits for only 2 hours after balloon angioplasty. The authors found a
trend toward reduced cross-sectional narrowing. The effectiveness of a
prolonged inhibition of antithrombotic activity was initially suggested
by Schwartz et al31 in the pig model after
metallic stent implantation. The authors demonstrated a decrease in
neointimal thickening in coronary arteries after
administration of TAP for 5 days.
The importance of the experimental model for studying therapeutic
interventions to reduce restenosis is a critical factor. It is
clearly emphasized by a recent study comparing the effects of thrombin
inhibition by hirudin in three animal models (rats, rabbits, and
minipigs). This study showed that short-term hirudin administration
reduced neointima formation in the rabbit model, but no
inhibition of neointimal growth was seen after short-term
administration in rats and minipigs.15
The most often-used animal models to study experimental
restenosis after arterial injury are rats, rabbits,
and pigs.32 33 34 35 Among the different animal models
available to study neointima thickening after
arterial injury, the pig coronary model appears to
be the most relevant to human coronary restenosis. The
porcine model shares similar cardiovascular
anatomy and physiology and therefore may provide results more
predictive of responses in humans.36 37 38 39 40 On this
basis, the animal model selected for the present study was balloon
angioplasty of the pig coronary arteries.
In our study we addressed the effects of direct and specific inhibition
of thrombin activity by using r-hirudin on neointimal
formation after coronary angioplasty in the pig. The periods of
antithrombin treatment were selected to cover (a) the acute
thrombogenicity (bolus), (b) short term (2 days) to mimic the human
studies, and (c) long term (2 weeks) on the basis of a previous study
indicating that the thrombogenicity of an injured artery lasts up to 11
days after PTCA.21
Thrombin is the most potent activator of platelets in
humans. Hirudin has the greatest binding affinity of any antithrombin
agent (kD 2x10 for recombinant hirudin). The
dose of hirudin used in this study was based on our previous studies
showing that a 1 mg/kg bolus followed by a 2-hour infusion prevented
thrombus formation after balloon
angioplasty.9 10 11
The oversized balloon injury model used in this study creates a
reproducible and reliable arterial damage that is similar
to that observed in humans. The degree of arterial injury
induced at the time of angioplasty is a major predictor of the
neointimal response.41 42 Our
experimental model of coronary angioplasty causes deep
arterial injury and medial tears with exposure of the
external elastic lamina. As shown in the Table
Our data support the hypothesis that acute thrombus formation
after angioplasty contributes to the deposition of new mass in the
vessel wall and to luminal narrowing. By direct
histological analysis of the stained
coronary segments, we showed that organizing thrombus,
submedial hematoma, and fibrocellular hyperplasia are all present 4
weeks after angioplasty. The presence of these elements contributes to
the overall luminal narrowing. Thrombus may also provide a matrix for
cellular elements to invade and proliferate. In addition, it
constitutes a reservoir for active substances such as
platelet-derived growth factors, thrombin, and fibrin(ogen), which
have all been shown to influence cellular
proliferation.43 44 Therefore, the observed
inhibitory effect of r-hirudin on restenosis might be exerted via two
different mechanisms. First, by directly reducing the overall size of
acute thrombus, and indirectly through an inhibitory effect of cell
proliferation as a consequence of a reduction in the release of growth
factors originated during the process of thrombus formation (PDGF,
etc).
Additional support for the efficacy of adequate antithrombotic therapy
for prevention of restenosis may also be extrapolated from the
clinical results of the recent EPIC and EPILOG
trials.45 46 These studies suggest that the
inhibition of acute thrombotic complications after PTCA by use of the
specific antagonist of the IIb/IIIa platelet receptor
complex, Rheo-Pro, is associated with a reduced rate of "clinical
restenosis." Such observations are based on clinical
symptomatology, since angiography was not included in the initial
design of the study.
Our results in the porcine model indicate that there is continued and
marked generation of thrombin for at least 2 weeks after balloon
angioplasty. This finding suggests that after balloon injury thrombin
generation continues for a longer period of time than initially
thought. If this observation is extrapolated to humans, it could
explain the lack of effect on restenosis observed in clinical
trials with antithrombin agents despite their significant effect on
reducing acute thrombotic complications of PTCA. Therefore the effects
of an effective and prolonged inhibition of thrombin on
restenosis after PTCA in humans deserve to be studied.
In conclusion, our results clearly indicate that to achieve the full
benefits of the therapeutic treatment with direct thrombin
inhibitors, an appropriate dosage and prolonged
administration of these agents is needed to "passivate" the
thrombogenic substrate (injured arterial wall) and achieve
the full benefits of this therapeutic approach.
Received July 16, 1997;
revision received September 15, 1997;
accepted September 30, 1997.
© 1998 American Heart Association, Inc.
Basic Science Reports
Prolonged Thrombin Inhibition Reduces Restenosis After Balloon Angioplasty in Porcine Coronary Arteries
Abstract
Top
Abstract
Introduction
Methods
Results
Discussion
References
Background—Arterial
injury after percutaneous transluminal coronary
angioplasty (PTCA) triggers acute thrombus formation and thrombin
generation. Hirudin, a potent and direct thrombin
inhibitor, prevents thrombus formation after
arterial injury. Two large clinical trials showed marked
reduction in acute clinical events but no long-term benefits in
reducing restenosis during short-term administration of
thrombin inhibitors. Our hypothesis is that adequate,
maintained thrombin inhibition, by inhibiting all the
thrombin-dependent mechanisms, will reduce neointima
formation after PTCA.
Key Words: restenosis • thrombin inhibition • angioplasty
Introduction
Top
Abstract
Introduction
Methods
Results
Discussion
References
Despite its
widespread use, the effectiveness of coronary balloon
angioplasty is limited by the high rate of restenosis. Attempts
to modify the restenotic process have yielded uniformly
disappointing results. Repeated failure to reduce the incidence of
restenosis reflects a basic lack of understanding of the
pathogenesis of the vascular response to injury. Balloon angioplasty
causes severe vascular injury, as evidenced by the significant fracture
of the atherosclerotic plaque. Similarly, there are areas of
endothelial denudation, intimal disruption, and
necrosis of smooth muscle cells. Restenosis is a reparative
process that is activated in response to injury induced by
balloon angioplasty. Mural thrombus formation occurs immediately after
injury and is followed by smooth muscle cell activation, migration,
proliferation, and increased synthesis of extracellular matrix that
tends to cease within the first 3 months.1 2
Methods
Top
Abstract
Introduction
Methods
Results
Discussion
References
Animal Species
This study was performed with Yorkshire Albino pigs (weight, 27
to 32 kg). All animals were purchased from a single local farm.
Intervention procedures and animal handling were approved by the Mount
Sinai School of Medicine animal management program, which is accredited
by the American Association for the Accreditation of Laboratory Animal
Care (AALAC). The AALAC meets NIH standards as set forth in the
"Guide for the Care and Use of Laboratory Animals" (DHHS
Publication No. [NIH] 85 to 23, Revised 1985) according to the PHS
"Policy on Humane Care and Use of Laboratory Animals for Awardee
Institutions" and the AHA "Guidelines for the Use of Animals in
Research."
Hirudin is a potent anticoagulant derived from the European
leech Hirudo medicinalis. Hirudin is a single polypeptide of
65 amino acids that binds by its carboxy terminus to the substrate
recognition site of thrombin and by its amino terminus to the catalytic
center of thrombin.19 In the present study,
we used recombinant desulfatohirudin (r-hirudin; GCP-39393; Revasc,
Ciba-Geigy Corp), which is identical to its natural version except for
a missing sulfate group on the tyrosine-63.20
The experiment consisted of four groups: a control group (n=10
animals; 22 coronary arteries) treated with a single
intravenous bolus of heparin given 15 minutes before the
balloon angioplasty and three different groups being treated with
different regimens of r-hirudin–treated animals. The regimens of
hirudin administration were as follows.
This group (n=9 animals; 17 coronary arteries) received
an intravenous bolus (1 mg/kg) given 15 minutes before the
balloon angioplasty.
This group (n=8 animals; 12 coronary arteries) received
an intravenous bolus (1 mg/kg) given 15 minutes before the
balloon angioplasty followed by a continuous intravenous
infusion of 0.7 mg hirudin/kg per hour maintained for 2 days through an
implantable infusion pump. The administration of hirudin for 2 days was
selected to mimic the design of the human studies in which the mean
length of administration of hirudin was 2.1 days.
This group (n=9 animals; 15 coronary arteries) received
an intravenous bolus (1 mg/kg) 15 minutes before the
balloon angioplasty followed by a continuous intravenous
infusion of 0.7 mg hirudin/kg per hour maintained for 14 days through
an implantable infusion pump. The 2-week time period was selected on
the basis of our previous studies indicating that the thrombogenicity
of an injured artery, evaluated as deposition of indium-labeled
platelets, lasts for up to 11 days after carotid angioplasty in the
pig.21
Pigs were premedicated with ketamine (15 mg/kg IM) and
then deeply anesthetized with pentobarbital (25 mg/kg),
intubated, and mechanically ventilated with room air. Continuous
monitoring of the ECG and arterial pressure was performed.
The right carotid artery and internal jugular vein were exposed after a
medial incision on the anterior surface of the neck. Both vessels were
cannulated with separate 0.8-mm Teflon fluorinated ethylene
polypropylene tubing. After the vessels were cannulated, the opposite
end of each tube was tunneled subcutaneously, exiting on the dorsal
side of the neck. The carotid cannula was used as a blood sampling
port. The venous cannula was secured to a Synchromed 8615 miniature
infusion pump with an internal 18-mL reservoir (Medtronic Inc). The
pump was secured to the animals with a specially designed vest. On the
2nd day (short-term) or 14th day (long-term) after balloon angioplasty,
the pump was removed and both arterial and venous cannulas
were sealed.
After the infusion pump was completely installed and
homeostasis ensured, a 4-cm-long 8F introducer sheath was placed into
the right carotid artery. At this point, the animals serving as
controls were anticoagulated with heparin, and hirudin was administered
to the three experimental groups. An appropriate PTCA catheter (8F) was
inserted through the right carotid artery and advanced under
fluoroscopic guidance (Phillips BV-24) into the left and right
coronary arteries for angioplasty of the left anterior
descending coronary artery, left circumflex artery, and right
coronary artery, respectively. Coronary angioplasty was
performed by three inflations at 10 atm of a 4.0x2.0-mm angioplasty
balloon (Cordis Corp). This angioplasty procedure resulted in
overstretching of the artery, causing severe arterial
injury. Angiographic examinations were performed before, during, and
after PTCA, and x-ray films were taken at each intervention. After
the angioplasty procedure, all animals were allowed to recover and
returned to the pens until their predetermined time for euthanasia. All
animals were fed a normal diet (Purina Farm Chow) with free access
to water and were allowed to move freely within their pens.
Blood samples were drawn through the internal jugular
cannula immediately before anticoagulation, 15 minutes after
anticoagulation, and immediately after balloon angioplasty. Subsequent
blood sampling was performed on days 2, 6, 10, 14, and 28 after balloon
angioplasty. aPTT times were determined by timed assays with a
Diagnostica Stago 7 System.
None of the commercially available assays for the determination
of thrombin generation cross-react with the porcine model. Plasma
levels of THC as a molecular probe for thrombin generation in vitro and
in vivo has been previously validated in the
pig.22 An ELISA was developed for the
determination of THC in porcine plasma. The assay is a modification of
the one developed by Bichler et al.23 The assay
involves the use of a monoclonal antibody to the porcine THC that binds
selectively to the thrombin component of the THC. Antithrombin
antiserum was filtered through a prothrombin-Sepharose column, and the
unbound fraction was further purified by affinity
chromatography on porcine thrombin-Sepharose.
Antihirudin immunoglobulin G (IgG ) from sheep (kindly provided by R.
Maschler, GEN, Munich, Germany) was purified by affinity
chromatography as described.24
The ELISA plates (Immunolon F) were coated with rabbit anti-porcine
thrombin-hirudin IgG. The solid-phase antithrombin antibody binds
selectively to the thrombin component of the THC. Hirudin bound to
thrombin is recognized by a sheep anti-hirudin IgG and detected by
rabbit anti-sheep peroxidase conjugate. The absorbance is then at 405
nm and compared with the standard curve. The detection limit of the
thrombin-hirudin ELISA is 600 pg of THC/mL of plasma. This assay for
thrombin generation is based on levels of THC formed by the 1:1
stoichiometric binding of hirudin:thrombin.
Animals were euthanatized on day 28 after PTCA. At that time,
the animals were again deeply anesthetized with
ketamine and pentobarbital and fully heparinized (100 units/kg
IV). The aorta and heart were exposed through a median thoracotomy, and
the animals were euthanatized by overdoses of pentobarbital. The heart
and ascending aorta were immediately excised, and a large-bore catheter
was inserted retrogradely into the ascending aorta. The
coronary arteries were flushed with 1 L of
physiological buffer (0.1 mol/L PBS containing
heparin and papaverine, pH 7.4) followed by perfusion fixation with 1 L
cold (4°C) 4% paraformaldehyde in 0.1 mol/L PBS, pH
7.4. All coronary perfusions were performed at 100 mm
Hg. After perfusion fixation, the heart was placed in 1 L of fresh
fixative and allowed to stand overnight at 4°C. The coronary
segments of interest were excised and cross-sectioned perpendicular to
the long axis of the artery at 2-mm intervals. Arterial
segments were dehydrated in ethanol and xylene, embedded in paraffin at
59°C, sectioned (4 µm), and stained by combined Masson-elastin
method. Histological sections were analyzed as
described below.
All 2-mm coronary segments were analyzed by two
experienced observers blinded to the treatment group. For each
coronary artery that underwent angioplasty, the section with
the most extensive injury-induced response was identified and
evaluated. 
3 were retained for further
analysis. The segment most narrowed by plaque (ie, that section
with the narrowest lumen) was then further evaluated by
computer-assisted histomorphometry. Photomicrographs were captured on
Adobe Photoshop 6.0 and transformed to NIH Image 1.6 for quantitative
analysis. All procedures were performed on a Power Macintosh
8100/100. 
). Residual thrombus was defined as
thrombus or remnants of an original thrombus induced at the time of
angioplasty and that was undergoing organization. Thrombus under a
medial flap or dissection was referred to as a hematoma. Fibrocellular
hyperplasia was defined as those areas comprised solely of cellular
elements and extracellular matrix, as seen on microscopic
analysis of stained tissues.
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Figure 1. Photomicrograph of a typical coronary
section 4 weeks after PTCA, indicating the three distinct processes
that contribute to neointima thickening. IEL indicates
internal elastic lamina; EEL, external elastic lamina.
To ensure that potential differences between groups were due to
treatment and not to differences in the severity of
arterial injury, a damage index score and percentage of
missing EEL score was determined for every section studied. The damage
index was calculated as the ratio between the total vessel area (VA),
and the area of the circle formed by the length of outer media measured
directly, assuming that there is no loss of medial tissue. The
percentage of missing IEL was calculated as the difference between the
IEL of a circle derived from the measured EEL and medial thickness, and
the IEL measured directly by computerized analysis.
Data are presented as number of coronary
segments in each experimental group and expressed as mean±SEM. Areas
are expressed as mm2. The statistical
significance of differences between the normal and treated groups was
determined with a two-way ANOVA followed by an unpaired Student's
t test to evaluate two-tailed levels of significance, and
multiple regression analysis was used for the evaluation of
variable dependence. Differences were considered significant at
P<.05.
Results
Top
Abstract
Introduction
Methods
Results
Discussion
References
Arterial Injury Induced by the Angioplasty
Procedure
All coronary segments analyzed for intimal
proliferation after angioplasty were histologically
characterized by disruption of the IEL, with laceration of the tunica
media and exposure of the EEL. As shown in the Table, the degree of
arterial injury induced by the coronary
intervention was similar for all the groups.
View this table:
[in a new window]
Table 1. Degree of Injury
Activated Partial Thromboplastin Time
The aPTT ratio for the control group (heparin bolus) was 2.3±0.2
and 1.9±0.3 immediately before and after PTCA, respectively. In all
the hirudin-treated groups, the administration of the bolus induced a
similar prolongation of the aPTT ratio (2.1±0.2) as the one induced by
heparin in the control group. The coronary intervention was
associated with a reduction in the prolongation of the aPTT value after
the administration of the bolus of hirudin (1.64±0.2 versus 2.1±0.2
after the bolus). The aPTT ratios achieved at the time of PTCA and at
different time points during the 14 days of continuous administration
in the long-term hirudin-treated animals are shown in Fig 2. Even though the initial aim of the
study was to maintain an aPTT ratio among 2 and 3 times control, as
seen in Fig 2 the antithrombin regimen used attained slightly higher
aPTT ratios. No bleeding complications were observed in any of the
animals for the duration of the study.
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Figure 2. Values of aPTTs achieved at the time of PTCA and
at different time points during the administration of the thrombin
inhibitor r-hirudin. Results expressed as aPTT ratio vs.
baseline.
Plasma levels of THC were determined and served as a marker for
thrombin generation. The corresponding values of THC in the long-term
hirudin group are reported in Fig 3. A
significant increase in plasma levels of THC was observed immediately
after balloon angioplasty. This observation clearly indicates that the
arterial injury inflicted by the coronary
intervention is associated with a surge in thrombin generation.
Increased thrombin generation persisted for at least 14 days after
balloon angioplasty. Given that the longest hirudin administration was
stopped on the 14th day after intervention, determination of THC was
not possible thereafter.
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Figure 3. Plasma levels of THC at different time points
during the administration of r-hirudin. Results are expressed as pg/mL
of blood. The results clearly indicate that PTCA is associated with a
significant, maintained thrombin generation that persists for at least
14 days after balloon angioplasty.
When the animals were euthanatized (4 weeks after angioplasty),
all groups showed similar coronary artery sizes, assessed by
the vascular area encircled by the EEL. The analysis of the
percentage of lumen narrowing indicated a statistically significant
larger lumen in the 2-week hirudin-treated group compared with the
other groups (P>.001). No differences were observed among
the heparin-control group, the bolus-hirudin, and 2-day hirudin groups
(Fig 4). These results indicate that the
significant reduction observed in the luminal area after angioplasty it
is due to the development of neointima rather than to
remodeling or vascular shrinkage because the vessel area, as defined by
the area encircled by the EEL, was similar in all groups.
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Figure 4. Percent of luminal narrowing at 4 weeks after
balloon angioplasty for the control and the three hirudin-treated
groups. (x±SEM; *P<.001).
Representative photomicrographs of
histological sections from the control and 2-week
hirudin-treated groups are shown in Fig 5. These figures show a significant
intimal thickening in the coronary arteries of the control
animals. The newly formed proliferative tissue, characterized by
spindle-shaped cells, filled medial tears extending between adjacent
medial areas and encroaching into the lumen. On immunohistochemistry,
the majority of these cells stained positive for 
-actin, indicating
their smooth muscle cell origin (data not shown). Fig 6 depicts the values of the three major
contributors (residual thrombus, submedial hematoma, and fibrocellular
hyperplasia) to luminal narrowing in these two groups. Bolus and
short-term administration of hirudin did not induce any differences
compared with the heparin control group.
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[in a new window]
Figure 5. Representative photomicrographs of
combined Masson-elastin–stained histological sections
from control (left) and 2-week hirudin-treated animals (right).
View larger version (24K):
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Figure 6. Contribution of the residual thrombus (B),
submedial hematoma (C), and fibrocellular hyperplasia (D) to the total
luminal narrowing (A) at 4 weeks after balloon angioplasty in the
control and 2-week hirudin-treated animals (x±SEM;
*P<.05). 
). The inhibition of thrombin activity
achieved by the 2-week continuous infusion of hirudin induced a
significant reduction (P<.01) in the contribution of
residual thrombus to luminal narrowing from 10.9±2.1% in the control
animals to 4.2±1.0% (Fig 6B). The contribution of submedial hematoma
to luminal narrowing was also reduced by the long-term antithrombin
treatment from 6.2±1.4% in the control animals to 4.1±1.1% in the
treated animals (P<.05) (Fig 6C). There was also a
significant reduction in the fibrocellular component of the
neointima from 41.3±2.3% to 26.8±3.1%, respectively
(P<.01) (Fig 6D).
Discussion
Top
Abstract
Introduction
Methods
Results
Discussion
References
This study shows that effective and sustained inhibition of
thrombin activity after PTCA significantly reduces the formation of
intimal thickening after PTCA. Our observations strongly support the
hypothesis that thrombin plays an important role in mediating the
restenosis process. Furthermore, these results emphasize the
importance of the dosage and duration of administration of hirudin in
achieving optimal and adequate antithrombin therapy. -granule contents during platelet aggregation and
thrombus formation. Platelets contain platelet-derived growth
factor and transforming growth factor-ß, among other growth and
mitogenic factors. In addition, the presence of a mural
thrombus may also contribute to late neointimal thickening
and restenosis. This process may involve the organization of
the thrombotic mass and release of growth and mitogenic
factors and cytokines. These factors in turn stimulate smooth
muscle cell migration and proliferation, which leads to intimal
hyperplasia and extracellular matrix production. Thus it is
possible that initial thrombus formation correlates with later
neointimal thickening. , the degree of injury
did not differ between coronary arteries taken from the two
groups. Therefore, the observed differences in neointima
formation cannot be attributed to different levels of injury.
Selected Abbreviations and Acronyms
aPTT
=
activated partial thromboplastin time
PTCA
=
percutaneous transluminal coronary angioplasty
THC
=
thrombin-hirudin complex
Acknowledgments
This work was supported by grants from the National Institutes
of Health P50 HL-54469 (J.J.B.) and a grant from Ciba-Geigy Corp
(Summit, NJ). We are most grateful to Eduardo Acampado for his superb
technical support and Veronica Gulle for her skill at processing the
pathological specimens. We are also grateful to Russell Jenkins, Hector
Lopez, and Gladys Vega for their assistance in the handling and
maintenance of the animals.
References
Top
Abstract
Introduction
Methods
Results
Discussion
References
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