(Circulation. 1995;92:3041-3050.)
© 1995 American Heart Association, Inc.
Articles |
Influence of Blockade at Specific Levels of the Coagulation Cascade on Restenosis in a Rabbit Atherosclerotic Femoral Artery Injury Model
From the Department of Cardiology and the Joseph J. Jacobs Center for Thrombosis and Vascular Biology, The Cleveland (Ohio) Clinic Foundation (Y.J., L.A.G., A.M.L., M.G.-W., F.F., S.G.E., E.J.T.); Zymogenetics Inc, Seattle, Wash (C.E.H., D.W.C.); and Novo Nordisk, Gentofte, Denmark (M.E.).
Correspondence to Eric J. Topol, MD, Department of Cardiology, The Cleveland Clinic Foundation, 9500 Euclid Ave, Cleveland, OH 44195-5066.
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Abstract |
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 Top Abstract IntroductionMethods Results Discussion References |
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Background The relation among the coagulation cascade, its individual proteins, and the response to vascular injury is largely undefined. We have evaluated the effect of four probes that block specific levels of coagulation cascade on neointimal hyperplasia in the atherosclerotic rabbit arterial injury model.
Methods and Results Focal femoral atherosclerosis was induced by air-desiccation injury and hypercholesterolemic diet in 48 New Zealand White rabbits, followed by balloon angioplasty. Active-site inactivated factor VIIa (DEGR-VIIa), which blocks the binding of factor VIIa to tissue factor, was administered (n=12 arteries) by intravenous bolus (1 mg/kg) at the time of balloon angioplasty and followed by infusion of 50 µg · kg-1 · h-1 for 3 days; for the control (n=13 arteries), 150 U heparin was injected as bolus and followed by infusion of saline at 50 µL · kg-1 · min-1. Recombinant tissue factor pathway inhibitor (TFPI), which binds factor Xa and inhibits the tissue factor–factor VIIa complex and factor Xa, was given as a 1 mg/kg bolus followed by 15 µg · kg-1 · min-1 infusion for 3 days (n=17 arteries). Recombinant tick anticoagulant peptide (TAP; n=15 arteries) and hirudin (n=14 arteries), which block factor Xa and thrombin, respectively, were administered as a 1 mg/kg bolus followed by 5 µg · kg-1 · min-1 infusion for 3 days. These three groups had their own controls (n=14 arteries). There were no differences among treatment groups in preangioplasty and postangioplasty minimal luminal diameter (MLD) by angiography. The mean MLD 21 days after balloon angioplasty was significantly different between control and DEGR-VIIa–treated groups (0.74±0.25 and 1.24±0.27 mm, respectively; P=.0001) and between the TFPI-treated group and others (0.88±0.21 mm for control, 0.97±0.22 mm for hirudin-treated, 0.98±0.14 mm for TAP-treated, and 1.32±0.21 mm for TFPI-treated arteries; P=.0001 by ANOVA). By quantitative histological analysis, the ratio of neointimal cross-sectional area compared with the area of internal elastic lamina in the DEGR-VIIa–treated group was significantly less than control (0.48±0.12 versus 0.67±0.12, P=.0001), and the ratio of neointimal cross-sectional area to the area demarcated by the internal elastic lamina of the TFPI-treated group was significantly reduced compared with the other groups (0.46±0.20 for TFPI-treated, 0.67±0.15 for hirudin-treated, 0.61±0.15 for TAP-treated, and 0.64±0.13 for control groups; P=.003).
Conclusions Treatment with DEGR-VIIa or TFPI for 3 days in this rabbit atherosclerotic injury model reduced angiographic restenosis and decreased neointimal hyperplasia compared with controls. These findings highlight the importance of early initiators of the extrinsic coagulation pathway, especially factor VII and tissue factor, in the response to arterial injury.
Key Words: restenosis • coagulation • vasculature • angioplasty
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Introduction |
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TopAbstractIntroductionMethods Results Discussion References |
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Thrombosis appears to play a role in restenosis, but the precise mechanisms and extent have not been precisely defined.1 2 3 4 5 Thrombin generation response to an arterial injury occurs primarily through activation of the extrinsic pathway as a result of exposure of tissue factor, one of the cell membrane receptor proteins.6 7 8 9 This mechanism is amplified in the atherosclerotic artery subject to injury from exposure of subendothelium, which has more abundant tissue factor than the normal artery.10 Exposed tissue factor binds to factor VIIa, an essential step in the initiation of the coagulation pathway.6 8 In the presence of the appropriate membrane environment, this complex efficiently converts the plasma coagulation zymogens factor X and factor IX to their active enzyme forms, thus serving as the initiators of both intrinsic and extrinsic coagulation pathways and ultimately generating thrombin.
Previous studies that showed a reduction of restenosis with TAP, a factor Xa inhibitor,4 and recombinant hirudin, a direct thrombin inhibitor,5 in the rabbit atherosclerotic model suggested that appropriate blocking of thrombin generation or thrombin directly might be a clinically useful strategy to limit restenosis after percutaneous coronary intervention.
We hypothesized that blocking different levels of the coagulation cascade may affect the restenotic process differently. Therefore, we compared the efficacy of four agents—DEGR-VIIa, which inhibits the initiation of extrinsic pathway by blocking factor VIIa binding to tissue factor; recombinant TFPI, which blocks factor Xa and tissue factor–factor VIIa complex activity; recombinant TAP; and recombinant hirudin—on prevention of neointimal hyperplasia in the rabbit atherosclerotic model.
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Methods |
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TopAbstractIntroductionMethods Results Discussion References |
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Animal Model
The air-drying model originally described by Fishman with modifications by Sarembock et al11 was used. Fifty-two male New Zealand White rabbits (3.8±0.4 kg) were anesthetized by injection of 5 mg/kg IM xylazine (Butler Comp) and 35 mg/kg IM ketamine (Dodge Lab). Both proximal femoral arteries were exposed by cut down below the inguinal ligament with proximal and distal ligatures. The isolated segments were cannulated with a 30-gauge needle. A vent was created by needle puncture. The isolated segments were flushed with saline to clear residual blood and desiccated by air infused at 80 mL/min for 8 minutes. After they were air-dried, the isolated segments were again flushed with saline, and the ligatures were removed. Hemostasis was maintained with nonocclusive local pressure. The segments were demarcated with metal clips. The day after surgery, the rabbits were placed on a diet of 1% cholesterol and 6% peanut oil for 1 month until balloon angioplasty. The serum cholesterol level in this model ranges from 800 to 2000 mg/dL in our laboratory (unpublished data). The rabbits were maintained according to Animal Welfare Act specifications,12 and all surgical procedures were performed with sterile techniques and general anesthesia.
Drug Preparation and Groups
In the present study, two sets of
experiments (control
versus DEGR-VIIa and then control versus TFPI, TAP, or
hirudin) were performed separately because drugs were available during
different time periods. The rabbits were randomly assigned to the study
drug with a randomization computer program (EXCEL,
Microsoft Corp) during each set of experiments, and the operator for
balloon injury and the investigator who analyzed angiographic
and histological data were blinded to treatment
allocation (Fig 1
). The two control groups (n=8 rabbits
for each control group) received a single bolus of heparin (150 U/kg)
to prevent thrombus formation during the angioplasty procedures
followed by a normal saline (50
µL · kg-1 · h-1) infusion
for 3
days by volumetric pump (Harvard Apparatus, model
55-5920).
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All drugs were administered as an intravenous bolus
injection 10 minutes before angioplasty and intravenously
infused continuously for 3 days as a maintenance dose. The
group treated with DEGR-VIIa was compared with controls,
and the groups treated with TFPI, TAP, and hirudin were compared with a
second set of controls (Fig 2).
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DEGR-VIIa was obtained from Zymogenetics Inc. Recombinant wild-type human factor VIIa was purified from culture media of a transfected baby hamster kidney cell line as previously described.13 Recombinant factor VIIa was inactivated by sequential addition of dansyl-Glu-Gly-Arg chloromethyl ketone (DEGRck, Calbiochem) at 0, 1, 3, 7, and 24 hours. In total, a fivefold molar excess of DEGRck was added, and the solution was allowed to incubate at room temperature. Inactivation of factor VIIa was considered complete when residual factor VIIa activity was <1% as measured in a factor VIIa–specific coagulation assay.14 15 Unreacted DEGRck was separated from DEGR-VIIa by ion exchange chromatography. The DEGR-VIIa solution was adjusted to a concentration of 3.12 mg/mL in 10 mmol/L glycine buffer, pH 8.0, 150 mmol/L NaCl, and 10 mmol/L CaCl2, sterile-filtered, and stored at -80°C until use. Eight rabbits received DEGR-VIIa as a 1-mg/kg IV bolus followed by infusion of 50 µg · kg-1 · h-1 for 3 days.
TFPI was supplied by Monsanto Co. The preparation of this agent was described previously.16 Nine rabbits were treated with a 1 mg/kg bolus followed by infusion of 15 µg · kg-1 · min-1 TFPI for 3 days. Recombinant TAP and hirudin were obtained from Corvas International and Ciba-Geigy Pharmaceuticals, respectively, and prepared as previously described.17 18 Eight rabbits in the TAP group and 9 in the hirudin group received a 1-mg/kg bolus followed by 5 µg · kg-1 · min-1 TAP or hirudin infused for 3 days.
One rabbit in the DEGR-VIIa group died 2
days after balloon
angioplasty owing to strangulation by the swivel-tether system, and
1 rabbit in the hirudin group died 3 days after balloon angioplasty at
the time of catheter removal as a result of anesthesia.
Therefore, 8 rabbits in each control group (n=13, 14 arteries), 7 in
the DEGR-VIIa group (n=12 arteries), 8 in the TAP and
hirudin groups (n=15 and 14 arteries, respectively), and 9 in the TFPI
group (n=17 arteries) completed these experiments (Fig
1
).
In Vitro Inhibition of Factor VIIa Binding by
DEGR-VIIa
A cell-surface chromogenic assay was developed
as described15 19 to measure the efficacy of
DEGR-VIIa in blocking factor VIIa binding to
cell surface tissue factor and the subsequent conversion of factor X to
Xa on monolayers of rat smooth muscle cells. The smooth
muscle cells, cultured from aortic explants,20 were plated
into 96-well culture dishes at a concentration of 4000 cells per well
in 200 µL per well of DMEM culture (Bio-Whitacar) supplemented with
1% FCS and maintained in these media for 5 days at 37°C in 5%
CO2. At the time of assay, 110 µL culture media was
removed, and increasing concentrations of factor VIIa or a
constant amount of factor VIIa (100 nmol/L) in combination
with increasing amounts of DEGR-VIIa was added to wells.
Both factor VIIa and DEGR-VIIa were diluted
with HEPES buffer (10 mmol/L HEPES, pH 7.4, 137 mmol/L NaCl, 4 mmol/L
KCl, 5 mmol/L CaCl2, 11 mmol/L glucose, and 0.1%
BSA), and 10 µL of 10x stock solution was added to the cells. The
cells were incubated with the test compounds for 2 hours at 37°C and
then washed with HEPES buffer. A 200-nmol/L solution of factor X (50
µL) in Tris buffer (25 mmol/L Tris, pH 7.4; 150 mmol/L NaCl; 2.7
mmol/L KCl; 5 mmol/L CaCl2; and 0.1% BSA) was then added
to each well. After 5 minutes at room temperature, 25 µL of 0.5-mol/L
EDTA was added to stop the factor X to Xa conversion.
Twenty-five microliters per well of 0.8-mol/L S-2222 (Kabi
Pharmacia), a factor Xa–specific chromogenic
substrate, was added, and the absorbance at 405 nm was read after 40
minutes in a Thermomax microplate reader (Molecular Device Corp).
Balloon Angioplasty
Anesthesia was induced as above and
maintained
throughout the procedure with additional intramuscular injections of
ketamine and xylazine. Through a midline neck incision, the
right internal jugular vein was isolated by blunt dissection, and the
distal end was ligated. A silicone elastomer (Silastic) tube (PE-160)
was introduced into the right internal jugular vein. A subcutaneous
tunnel was created to pass the Silastic tube to the dorsal surface of
the neck. This tube was connected to the Harvard infusion pump through
a 20-gauge single catheter fluid swivel with tether (Harvard
Apparatus, model 56-7461), which allowed the rabbits to
move freely during drug infusion. The right common carotid artery was
isolated by blunt dissection, and the distal end was ligated. Through
an arteriotomy, a 5F introducer was placed and advanced to the junction
of the aortic arch. Blood was drawn for determination of hemostatic
parameters and drug levels. Lidocaine (20 mg) was injected
intra-arterially. A control aortoiliofemoral angiogram
was performed through a 5F Berman catheter (Arrow Co) positioned above
the aortic bifurcation with 3 to 4 mL Hypaque-76 (Sanofi Winthrop
Pharmaceuticals) injected over 3 seconds by hand.
After removal of the Berman catheter, a 0.014-in guide wire was introduced in the descending aorta and positioned above the aortic bifurcation. Under fluoroscopic guidance, a 2.5-mm balloon catheter (Advanced Cardiovascular System) was introduced and advanced over the guide wire and positioned across the stenosis demarcated by previously placed metallic clips. The balloon was inflated to 6 atm for 60 seconds with a hand indeflator. Three inflations were performed with 60-second intervals between inflations. This procedure was performed in both femoral arteries if both were patent.
After balloon dilatation, the angioplasty catheter was withdrawn, and the Berman catheter was reintroduced to a position 3 cm above the aortic bifurcation. To minimize spasm, 20 mg lidocaine was given intra-arterially. A postprocedure angiogram was performed as described above. A 1-cm grid was positioned at the level of the femoral artery to calculate the actual diameter. The catheter was then removed. The right carotid artery was ligated with 3-0 silk, and the wound was sutured by layers. Ampicillin (1 mg/kg IM) was injected, and acetaminophen (10 mg/kg) was given orally. After the 3-day continuous infusion of the study drugs, the Silastic tube was removed under the anesthetic conditions described above after inspection to confirm that correct positioning within the internal jugular vein had been maintained.
Coagulation Parameters
PT and plasma drug levels were
measured in
DEGR-VIIa– and TFPI-treated groups before balloon
angioplasty, 1 hour after bolus injection, and 3 days after balloon
angioplasty immediately before completion of the drug infusion. Plasma
levels of agents were determined as described below. aPTT and plasma
drug levels were measured in TAP- and hirudin-treated groups before
balloon angioplasty, 1 hour after bolus injection, and 3 days after
balloon angioplasty immediately before completion of the drug infusion.
Plasma samples for PT, aPTT, and drug level were collected in 3.8%
sodium citrate (9:1 vol/vol) from the rabbit central ear artery and
centrifuged at 2000 rpm for 15 minutes.
For PT measurements from
DEGR-VIIa rabbits and their
control group, a standard clotting assay was used. Test rabbit plasma
(25 µL) was added to 150 µL TBS (20 mmol/L Tris, pH 7.4, and 150
mmol/L NaCl). The samples were mixed and added to an Electra 800
Automated Coagulation Timer (Medical Laboratories Automation). After
incubation, 200 µL thromboplastin preparation (Sigma Chemical Co)
containing 25 mmol/L CaCl2 was added to the plasma
preparations. A concentration of thromboplastin that gave a clotting
time of 
20 seconds in the control rabbit plasma was selected.
For PT measurements from the TFPI group and its control, assessments were performed on the ST4 coagulation timer (Diagnostica Stago) with thromboplastin (Innovin, Boxter Diagnostics Inc). For aPTT measurements from the TAP, hirudin, and control groups, assessments were performed on the ST4 coagulation timer with Dade Actin FSL activated PTT reagent (Baxter Healthcare Co).
Drug Level Measurements
Analysis of
DEGR-VIIa antigen
An ELISA was used to determine the
concentration of
DEGR-VIIa in plasma samples from the
DEGR-VIIa–treated rabbits. The assay uses a
monoclonal-polyclonal sandwich format. An anti–human factor
VIIa monoclonal antibody (obtained from Walt Kisiel,
University of New Mexico) was added to 96-well microtiter dishes, 100
µL per well, at 2.0 µg/mL in 0.1 mol/L carbonate buffer, pH 9.6.
The antibody was incubated overnight at 4°C, and the plates
subsequently were washed with washing buffer (PBS, pH 7.4, and 0.05%
Tween 20). Blocking of nonspecific binding sites was achieved by adding
200 µL per well of blocking buffer (PBS, pH 7.4, 0.05% Tween 20, and
1% BSA) for 2 hours at 37°C and washing the plates with wash buffer.
After blocking the plates, a twofold dilution series of
DEGR-VIIa, ranging from 20 to 0.027 ng/mL and
diluted into blocking buffer, was added to the test wells to generate a
standard curve. Dilutions of test rabbit plasma, 1:100 to 1:4000 in
blocking buffer, was similarly applied to the test wells, 100 µL per
well. Nonimmune rabbit plasma was used as a negative control. The
plates were incubated for 1 hour at 37°C and washed. A rabbit
anti–human factor VIIa polyclonal antibody (Walt
Kisiel, University of New Mexico) diluted with blocking buffer was
added to the wells for an additional 1 hour at 37°C. The wells were
washed and then incubated with goat anti-rabbit IgG conjugated with
horseradish peroxidase (Sigma Chemical Co). The wells were incubated 1
hour at 37°C, washed, and incubated with 100 µL per well of
substrate solution (0.42 mg/mL 0-phenylenediamine
dihydrochloride in 0.2 mol/L citrate buffer, pH 5.0, and 0.3%
H2O2). The concentration of
DEGR-VIIa in the plasma was determined by comparing the
absorbance in the test samples to the DEGR-VIIa standard
curve.
Immunoassay of TFPI
Standards and plasma
samples were diluted in a buffer containing
PBS, 10 mg/mL BSA, and 0.05% Tween 20, pH 7.2. Sample or standard (40
µL) was incubated at ambient temperature for 1 hour in the presence
of 15 µL of a 0.25% suspension of a monoclonal
anti–TFPI-IgG–bound
carboxylpolystyrene particles.21 Afterward, 30 µL of 10
µg/mL FITC-labeled rabbit-derived TFPI-Sepharose–purified
polyclonal anti-human TFPI IgG was added. Standards and samples
were then incubated for 1 additional hour. TFPI concentration was
measured by standard and sample comparative fluorescence
concentration on a PANDEX PCFIA analyzer.22
Measurement of TAP and Hirudin
Citrated plasma
samples for measurement of TAP and hirudin
levels were centrifuged at 132 000 rpm for 5 minutes to
eliminate lipid particles from plasma. Measurement of plasma levels of
TAP was attempted as previously described.17 Detection of
hirudin levels was attempted by measurement of the inhibition of
purified thrombin amidolytic activity in diluted samples. Briefly, the
assay was conducted by combining in appropriate wells of a Corning
microtiter plate 50 µL HBSA (10 mmol/L HEPES, pH 7.5, 150 mmol/L
sodium chloride, and 0.1% BSA), 50 µL citrated plasma sample diluted
1/1000 in HBSA or standard inhibitor diluted in HBSA, and
50 µL purified human 
-thrombin (3000 U/mg specific activity,
Enzyme Research Lab Inc) diluted in HBSA. After a 30-minute incubation
at ambient temperature (23°C), 50 µL chromogenic
substrate (Pefachrome t-PA, Pentapharm Ltd) was added to the wells,
yielding a concentration of 300 µmol/L (five times Km)
and a final volume of 200 µL. The initial velocity of
chromogenic substrate hydrolysis was measured by the change
in absorbance at 405 nm with a Thermo Max Kinetic Microplate
Recorder (Molecular Devices) over a 5-minute period in which <5%
of the added substrate was used. The plasma level of hirudin was
calculated with a standard curve of purified and quantified
inhibitor made up in control, homologous citrated plasma
covering a broad concentration range followed by dilution to 1:1000
with HBSA. Under these conditions, the IC50 for inhibition
of purified -thrombin (final concentration, 0.25 nmol/L) by the
respective inhibitor in diluted plasma was not
significantly different from that in HBSA alone. The limit of
inhibitor detection in this assay is 75 nmol/L.
Follow-up, Sacrifice, and Pressure Perfusion
Three weeks
after angioplasty, a follow-up angiogram was
performed as described above through the left carotid artery 5 minutes
after intra-arterial injection of 30 mg/kg of
papaverine (Eli Lilly and Co) to prevent arterial spasm.
Through a vertical lower abdominal incision, the distal aorta was then
isolated and tied off proximally, and a perfusion cannula was inserted
above the aortic bifurcation. Intravascular ultrasound studies have
indicated that arteries fixed with Histochoice (Amresco, Inc) do not
undergo substantial shrinkage or retraction compared with their
antemortem in vivo dimensions (J. Vince, PhD, unpublished data,
Department of Biomedical Engineering, Cleveland [Ohio] Clinic
Foundation, March 1995). The distal aorta was flushed with 50 mL saline
followed by in vivo fixation with 500 mL Histochoice infused over 15
minutes at 120 mm Hg. Once the perfusion fixation was started, the
rabbits were sacrificed with overdose of 3 mL Nembutal (Abbott
Laboratories). A 2.5-cm segment of treated femoral artery was excised
bilaterally, with care taken to mark proximal and distal ends. The
tissue was preserved in Histochoice for light microscopy.
Data Analysis
Angiographic Analysis
All
angiographic measurements were made with electronic
calipers.23 The 35-mm cineframe selected for
analysis was mounted in a film holder and projected through
a Tagarno projector. The reference artery was selected as the
proximal part of the femoral artery. MLD and reference artery size were
measured with electronic calipers by two operators in a blinded
fashion, and mean values from both operators were reported. Before
analysis of the baseline angiogram, calibration of the 1-cm
square to screen units was performed with a 1-cm marking grid placed at
the level of the femoral artery. Percent stenosis of luminal
narrowing was calculated with luminal diameter and reference artery
sizes.
Histological Analysis
Approximately 1.5 cm
of the femoral artery segments was
harvested and cut in serial 2-mm sections from the proximal to the
distal end and color coded to identify their relative location to one
another. Duplicate slides were stained with hematoxylin and eosin and
elastic van Gieson's stain. Morphometric analyses were
performed with the Image-1 system (Universal Imaging Co) to measure the
cross-sectional area of the lumen and internal and external elastic
membranes. After visual observation of all sections by light
microscopy, three segments with the greatest extent of
neointimal hyperplasia were selected for quantification.
The neointimal cross-sectional area was calculated by
subtracting the cross-sectional area of lumen from that demarcated
by the internal elastic membrane. The ratio of neointimal
cross-sectional area to the area demarcated by the internal elastic
membrane was calculated. The maximal and mean values for the three
segments were reported for each artery.
Statistical Analysis
Angiographic and morphometric data were
reported as the number
of femoral arteries in each experimental group and expressed as
mean±SD. To compare the DEGR-VIIa–treated group with
the
control group, data were analyzed with unpaired Student's
t test to evaluate two-tailed levels of significance. To
compare hirudin-, TAP-, and TFPI-treated groups with their control, the
factorial ANOVA test was performed with 95% significance to compare
the angiographic and histological data among
experimental groups.
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Results |
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TopAbstractIntroductionMethods Results Discussion References |
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Cell Surface Factor Xa Chromogenic Assay for DEGR-VIIa Efficacy
Fig 3
shows a
dose-dependent increase in
chromogenic activity when increasing amounts of factor
VIIa were added to the rat smooth muscle cells. The
increase in absorbance is a direct measure of the level of factor
Xa generated in the wells and its subsequent cleavage of
the chromogenic substrate S-2222. When the mixture of
DEGR-VIIa and 100 nmol/L factor VIIa was added
to the cells, there was a dose-dependent decrease in
chromogenic activity. An equimolar ratio of
DEGR-VIIa to factor VIIa inhibited 95% of
the chromogenic activity. Even at a 10-fold lower level of
DEGR-VIIa, there was still significant inhibition in
the generation of factor Xa chromogenic
activity.
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). When the mixture of DEGR-VIIa with 100 nmol/L
factor VIIa was added to the cells, there was a
dose-dependent decrease in the chromogenic activity
(
). An equimolar ratio of DEGR-VIIa to factor
VIIa showed to inhibit
Coagulation Parameters and Drug Levels
There were no bleeding
complications in any of the study groups.
The coagulation parameters in all the treated groups showed
therapeutic prolongations that were >1.5 times baseline for aPTT and
PT at 1 hour after bolus injection. However, by the end of the 3-day
infusion (before termination of drug infusion), the coagulation
parameters returned nearly to baseline in all treatment
groups except the TFPI group, in which PT was still prolonged to >1.5
times control (Table 1). In the two control groups, when
a heparin bolus was administered immediately before angioplasty to
prevent acute thrombotic occlusion, aPTT was prolonged to >1.5 times
baseline at 1 hour after bolus injection, while PT was not
affected.
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The mean TFPI plasma level concentration was undetectable at baseline, 1026±528 ng/mL 1 hour after bolus injection, and 2023±573 ng/mL at the end of third infusion day. At baseline (preinjection), the plasma level of DEGR-VIIa was measured at 15±23 ng/mL, representing baseline variations in light absorbance of control plasma. The mean DEGR-VIIa plasma concentration was 3883±1133 ng/mL 1 hour after bolus injection and 314±224 ng/mL at the end of third day of infusion. The drug levels of TAP and hirudin could not be measured owing to turbidity of the hyperlipemic samples despite centrifugation. However, aPTT levels in both groups 1 hour after bolus injection showed that drug levels were fully antithrombotic.17 24
Angiographic Data
Two rabbits died within 3 days of balloon
angioplasty. Two
additional arteries (1 control and 1 DEGR-VIIa–treated
artery) were found to be occluded immediately after balloon angioplasty
and recanalized by 21 days. These two arteries were not included in the
angiographic analysis; a total of 83 arteries were used for the
angiographic analysis.
Table 2 summarizes the
angiographic data. In all
groups, MLD increased significantly after balloon injury, without
significant differences among groups either before or after balloon
angioplasty. In the DEGR-VIIa–treated group, the mean MLD
21 days after balloon angioplasty was significantly greater than
control (1.24±0.27 versus 0.74±0.25, P=.0001).
When the
21-day mean MLD values were compared with the pre–balloon
angioplasty mean MLD values, there was a significant reduction in
control rabbits (0.74±0.25 versus 0.92±0.26, P<.05),
while a significantly larger lumen was found in the
DEGR-VIIa–treated arteries (1.24±0.27 versus
0.98±0.20,
P<.05). Among the control and hirudin-, TAP-, and
TFPI-treated groups, the mean MLD 21 days after angioplasty was
significantly greater for rabbits receiving TFPI than other treatment
groups (1.32±0.21 versus 0.88±0.21, 0.97±0.22,
0.98±0.14,
P<.0001). When the 21-day mean MLD values were compared
with pre–balloon angioplasty mean MLD values, there were no
significant differences in the control and hirudin- and TAP-treated
groups, but a significantly larger lumen was maintained in TFPI-treated
vessels (1.32±0.21 versus 1.00±0.21, P<.05).
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The late loss of luminal diameter (the difference between
post–balloon angioplasty and 21-day MLD) was significantly less in
the arteries treated with DEGR-VIIa than in control
arteries (0.18±0.29 versus 0.69±0.30, P=.004)
and in the
arteries treated with TFPI than in control or hirudin- or TAP-treated
arteries (0.08±0.22 versus 0.48±0.25, 0.29±0.18,
0.33±0.25,
P=.001; Fig 4).
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The percent stenosis
(calculated from the ratio of MLD to
reference artery size) showed the same trends as the late loss of
luminal diameter (Table 3). The MLD of the reference
artery was 1.88±0.27 mm, and there were no significant differences
among the six different groups. The percent stenosis values 21
days after balloon angioplasty were significantly lower in the
DEGR-VIIa– (37±9 versus 54±13,
P=.006) and
TFPI-treated arteries than their corresponding control groups (33±11
versus 57±12, P=.0001).
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Histological Data
The ratio of neointimal cross-sectional
area to
the area subtended by the internal elastic lamina was expressed as both
a mean derived from all three tissue sections analyzed and a
maximum derived from the slice exhibiting the most severe
neointimal proliferation. Table 4 gives the
mean and maximal ratios of neointimal cross-sectional
area calculated by computerized planimetry. For both indexes, the
DEGR-VIIa–treated group had a significantly smaller value
than the control group. Similarly, the TFPI-treated group showed a
significant reduction in the ratio of neointimal area to
the area subtended by the internal elastic lamina compared with other
groups (Fig 5). In both phases of the study, the
absolute neointimal areas in the DEGR-VIIa–
and TFPI-treated groups were significantly less than in the control
groups, whereas the other treated groups were not different from the
controls (Fig 6).
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Discussion |
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TopAbstractIntroductionMethods Results Discussion References |
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These experiments systematically assessed the effect of blockade at four different levels of coagulation cascade on the effect on neointimal hyperplasia in the rabbit atherosclerotic vascular injury model. Our findings show that a prolonged infusion of either DEGR-VIIa or TFPI significantly reduced neointimal formation as assessed histologically and angiographically. This study supports the hypothesis that appropriate inhibition of tissue factor–factor VIIa complex formation will result in reduced restenosis and decreased neointimal hyperplasia after balloon injury in this animal model. In contrast, sustained blockade of coagulation factors lower in the cascade was ineffective in limiting intimal regrowth.
Coagulation Proteins and Restenosis
The possible role of
coagulation proteins in the
restenosis processes has been suggested by their immediate
activation after arterial injury. Several studies have
shown stimulatory effects of coagulation proteins on smooth muscle cell
proliferation in
vitro1 25 26 27 and a
reduction in
restenosis with direct thrombin or factor Xa
inhibitors in vivo.4 5 Thrombogenicity of the
vessel wall after balloon injury is increased immediately, and
deposition of platelets in the injured area peaks in the first few
days.28 29 There are several lines of evidence that
short-term treatment with antagonists of the
platelet glycoprotein IIb/IIIa receptor30
or thrombin inhibitors5 31 can reduce the
thrombogenicity of the vessel wall and shorten the period of active
thrombosis. However, the effect of passivation or reduced
thrombogenicity of vessel wall on restenosis is still not
well understood.
Thrombin, which is a final product of the coagulation cascade, is a multifunctional protein. In addition to the hemostatic action of thrombin, this molecule is a potent mitogen for smooth muscle cells.25 26 27 Several recent reports indicate that thrombin stimulates smooth muscle cells to release platelet-derived growth factor,32 basic fibroblast growth factor33 and transforming growth factor–ß1.34 Thus, thrombin appears to be an important mediator in the restenotic process. Factors X and Xa also possess stimulatory effects on smooth muscle cell proliferation.1 Activation of the tissue factor gene in rat smooth muscle cells after balloon injury was reported by Marmur et al.35 Tissue factor gene activity in the media persisted for >24 hours after balloon injury, possibly allowing continuous activation of coagulation proteins.
In the rabbit model, the proliferation of smooth muscle cells begins early, reaching a peak 72 hours after injury.36 Data from the present study suggest that blocking of the coagulation pathway with doses sufficient to prevent thrombin generation for the first few days might be effective in preventing restenosis in this animal model. Although Sarembock et al5 and Ragosta et al4 demonstrated the preventive effects of a 2-hour infusion of high-dose (1 mg/kg bolus followed by 2.2 mg/kg for the first hour and 1.1 mg/kg for the second hour) TAP and hirudin on the restenosis process in the same model as used in the present study, the high incidence of serious bleeding complications in patients even with a 0.6-mg/kg bolus of hirudin suggests that this dosing level may not be acceptable for clinical use.37 38 The dosages of TAP and hirudin used in the present study are less than half of those used by Sarembock et al5 and Ragosta et al.4 However, even at these lower doses, a prolongation of aPTT to more than twice baseline, an acceptable therapeutic range,17 24 was achieved 1 hour after bolus injection for both agents. Furthermore, in the present study we maintained therapy directed at factor Xa or thrombin for 72 hours, which is substantially longer than in previous experiments.4 5 These findings in aggregate suggest that although inhibition of factor Xa or thrombin may reduce restenosis, substantially more potent effects are demonstrated by more proximal extrinsic pathway inhibition.
Differences in Extrinsic Pathway
Inhibitors
The active enzyme factor VIIa is produced by
limited
proteolysis of factor VII to a two-chain form that usually
circulates at a concentration of about 4 ng/mL with a half-life of
150 minutes in humans.39 DEGR-VIIa was
developed by active-site modification of the coagulation factor
VIIa. Factor VIIa is reacted with
dansyl-Glu-Gly-Arg chloromethyl ketone, which forms an irreversible
covalent bond to the active site of factor VIIa and blocks
its catalytic activity. The half-life of DEGR-VIIa in
baboons was reported to be 120±10 minutes.40
DEGR-VIIa binds tissue factor with fivefold to sevenfold
more affinity than native factor VIIa in the cell surface
chromogenic assay, rendering the
DEGR-VIIa–tissue factor complex enzymatically inactive and
thus inhibiting the conversion of factor IX to IXa and
factor X to Xa. Lindahl et al40 demonstrated
80% reduction of platelet accumulation in endarterectomized baboon
aortic segments with 1.0-mg bolus injection of
DEGR-VIIa.
Tissue factor pathway inhibitor, which is a Kunitz-type protease inhibitor previously called extrinsic pathway inhibitor, lipoprotein-associated coagulation inhibitor, or tissue factor inhibitor, requires the presence of factor X to inhibit activity of tissue factor–factor VIIa complex in a two-step stoichiometric reaction.41 42 In the first step, TFPI binds to the active site of factor Xa, with formation of a TFPI–factor Xa complex with inhibited factor Xa activity. In the second step, a TFPI–factor Xa complex binds to a complex of tissue factor–factor VIIa and forms a quaternary factor Xa–TFPI–factor VIIa–tissue factor complex that inhibits factor VIIa–tissue factor catalytic activity. Several studies have demonstrated that recombinant TFPI effectively inhibits the activation of the coagulation cascade by tissue factor.43 44 45 The clearance of recombinant TFPI in healthy rabbits after intravenous bolus injection showed a biphasic pattern, with half-lives of 2.3 and 79 minutes.46
Relation of Lipid and Extrinsic Pathway of Coagulation
Cascade
Several studies have demonstrated an increase in factor
VIIa and tissue factor activity in the
hyperlipidemic
state.47 48 49 50
Mitropoulos49 showed that factor VIIa activity
increased threefold in rabbits fed a
hypercholesterolemic diet. Lesnik et
al50 showed tissue factor expression in
cholesterol-loaded macrophages to be sensitive
to stimulation (12-fold) by bacterial
lipopolysaccharide, indicating induced tissue factor
procoagulant activity by cholesterol loading. Oxidized LDL,
which is abundant in this animal model,51 plays an
important role in the pathogenesis of
atherosclerosis52 53 and induces the
expression and secretion of tissue factor in
endothelial cells54 55 and
monocytes.56 Therefore, binding of increased factor
VIIa and tissue factor could potentially activate
the coagulation cascade and generate thrombin
continuously,57 suggesting a significant role of factor
VIIa and tissue factor in this animal model. Several lines
of evidence link continuous thrombin generation to
arteriosclerosis.58 59 60
The failure of infusions of hirudin and TAP to prolong the aPTT by 3 days and DEGR-VIIa to increase the PT value at 3 days might be due to continuous inactivation of these drugs by binding to generated tissue factor, factor Xa, or thrombin in this atherosclerotic rabbit model. The plasma levels of TFPI showed an accumulating phenomenon in this study. The recombinant form of TFPI used in this study was obtained from human TFPI cDNA.46 Most of the human TFPI circulates bound to lipoproteins, primarily LDL,61 62 which are abundant in this animal model. Therefore, the accumulation of plasma TFPI level in this study suggests that the increased lipoproteins resulting from the hypercholesterolemic diet might affect the circulating time of recombinant TFPI.
Limitations
Data from any animal model can be extrapolated
only with caution
to restenosis after human percutaneous
transluminal coronary angioplasty. However, the atherosclerotic
rabbit model is known to have several features in common with the human
restenotic responses, including the prominent role of
thrombus formation and
organization.4 5 11
The
reproducibility of the rabbit atherosclerotic model was
demonstrated previously in the control groups reported in several
articles,4 5 although there may be considerable
variability in response. There were statistically insignificant
variations in changes of MLD and absolute neointimal areas
between the two control groups. The changes in MLD between 21 days and
immediately after balloon angioplasty were 0.69±0.30 mm in one control
group and 0.48±0.25 mm in the other control group
(P=.53;
Fig 3). This magnitude of difference between the two control
groups in
our experiments is consistent with that observed in the two
sets of data4 5 (0.77±0.61 and 0.5±0.4
mm in MLD)
published from another group of investigators with considerable
experience with the same injury model.
The dosages of TAP and hirudin
were chosen on the basis of the
antithrombotic and clearance data from healthy rabbits and clinical
applicability in
humans.17 24 37 38 63
Walenga et
al63 demonstrated 75% reduction of clot formation in
the rabbit venous stasis thrombosis model with 5-µg/kg bolus
injection of hirudin. Because the antithrombotic potency of TAP and
hirudin have been demonstrated to be nearly equivalent on a
per-milligram basis,64 65 we chose the same dose of
TAP as for hirudin in this study. We attempted to measure the plasma
levels of TAP and hirudin after intravenous injection with
previously described methods,17 but accurate levels could
not be obtained because of high lipid concentration in the samples.
Although correct positioning of the drug infusion catheter had been
confirmed at the time of removal of the swivel system in every rabbit,
the inability to obtain TAP and hirudin levels does not allow us to
confirm that a therapeutic dosage was sustained for 3 days, and the
negative results may have been due in part to an insufficient infusion
dose. Although this study failed to demonstrate the preventive effect
of factor Xa or thrombin inhibition on
neointimal hyperplasia, it cannot be excluded that a
beneficial effect would have been obtained at dosages sufficient to
prolong aPTT for 3 days.
As discussed, hypercholesterolemia in this animal model may substantially augment factor VIIa and tissue factor activity and its contribution to the restenotic process. Therefore, the effect of extrinsic pathway inhibitors on restenosis in this model might be exaggerated compared with non–cholesterol-fed animal models or humans.
Conclusions
Blocking of initiation of the extrinsic pathway
at the level of
tissue factor with DEGR-VIIa or TFPI for 3 days resulted in
less restenosis by angiography and neointimal
formation by histology at 21 days after balloon angioplasty compared
with controls. Although a favorable effect on restenosis
was not obtained in TAP- or hirudin-treated groups in this study,
further study will be needed to determine an effective dosage of these
agents that may influence the restenotic process. This
study supports the hypothesis that the initiators of extrinsic pathway
of coagulation, factor VIIa and tissue factor, play a key
role in the pathogenesis of the response to arterial
injury.
|
Selected Abbreviations and Acronyms |
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Acknowledgments |
|---|
We wish to thank Ciba-Geigy Pharmaceuticals for providing the r-hirudin in this experiment, George P. Vlasuk (Corvas International Inc) for providing technical assistance and TAP, and Gerald R. Galluppi (Monsanto Corp) for providing TFPI and information regarding its pharmacological properties.
Received April 12, 1995; revision received June 22, 1995; accepted June 25, 1995.
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References |
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H. Kato Regulation of Functions of Vascular Wall Cells by Tissue Factor Pathway Inhibitor: Basic and Clinical Aspects Arterioscler. Thromb. Vasc. Biol., April 1, 2002; 22(4): 539 - 548. [Abstract] [Full Text] [PDF]
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 A. H.M Moons, M. Levi, and R. J.G Peters Tissue factor and coronary artery disease Cardiovasc Res, February 1, 2002; 53(2): 313 - 325. [Abstract] [Full Text] [PDF]
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 P. Golino, P. Cirillo, P. Calabro', M. Ragni, D. D'Andrea, E. V. Avvedimento, F. Vigorito, N. Corcione, F. Loffredo, and M. Chiariello Expression of exogenous tissue factor pathway inhibitor in vivo suppresses thrombus formation in injured rabbit carotid arteries J. Am. Coll. Cardiol., August 1, 2001; 38(2): 569 - 576. [Abstract] [Full Text] [PDF]
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C. Patterson, G. A. Stouffer, N. Madamanchi, and M. S. Runge New Tricks for Old Dogs : Nonthrombotic Effects of Thrombin in Vessel Wall Biology Circ. Res., May 25, 2001; 88(10): 987 - 997. [Abstract] [Full Text] [PDF]
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 P. Zoldhelyi, Z.-Q. Chen, H. S. Shelat, J. M. McNatt, and J. T. Willerson Local gene transfer of tissue factor pathway inhibitor regulates intimal hyperplasia in atherosclerotic arteries PNAS, March 27, 2001; 98(7): 4078 - 4083. [Abstract] [Full Text] [PDF]
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 U. Rauch, J. I. Osende, V. Fuster, J. J. Badimon, Z. Fayad, and J. H. Chesebro Thrombus Formation on Atherosclerotic Plaques: Pathogenesis and Clinical Consequences Ann Intern Med, February 6, 2001; 134(3): 224 - 238. [Abstract] [Full Text] [PDF]
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J. I. Weitz and J. Hirsh New Anticoagulant Drugs Chest, January 1, 2001; 119(1_suppl): 95S - 107S. [Full Text] [PDF]
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M. Roque, E. D. Reis, V. Fuster, A. Padurean, J. T. Fallon, M. B. Taubman, J. H. Chesebro, and J. J. Badimon Inhibition of tissue factor reduces thrombus formation and intimal hyperplasia after porcine coronary angioplasty J. Am. Coll. Cardiol., December 1, 2000; 36(7): 2303 - 2310. [Abstract] [Full Text] [PDF]
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A. Siegbahn, M. Johnell, C. Rorsman, M. Ezban, C.-H. Heldin, and L. Ronnstrand Binding of factor VIIa to tissue factor on human fibroblasts leads to activation of phospholipase C and enhanced PDGF-BB-stimulated chemotaxis Blood, November 15, 2000; 96(10): 3452 - 3458. [Abstract] [Full Text] [PDF]
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 P. Maderna, C. Godson, G. Hannify, M. Murphy, and H. R. Brady Influence of lipoxin A4 and other lipoxygenase-derived eicosanoids on tissue factor expression Am J Physiol Cell Physiol, October 1, 2000; 279(4): C945 - C953. [Abstract] [Full Text] [PDF]
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J. M. Waugh, J. Li-Hawkins, E. Yuksel, M. D. Kuo, P. N. Cifra, P. R. Hilfiker, R. Geske, M. Chawla, J. Thomas, S. M. Shenaq, et al. Thrombomodulin Overexpression to Limit Neointima Formation Circulation, July 18, 2000; 102(3): 332 - 337. [Abstract] [Full Text] [PDF]
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H. Holschermann, R. M. Bohle, H. Schmidt, H. Zeller, L. Fink, U. Stahl, H. Grimm, H. Tillmanns, and W. Haberbosch Hirudin Reduces Tissue Factor Expression and Attenuates Graft Arteriosclerosis in Rat Cardiac Allografts Circulation, July 18, 2000; 102(3): 357 - 363. [Abstract] [Full Text] [PDF]
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D. Hasenstab, H. Lea, C. E. Hart, S. Lok, and A. W. Clowes Tissue Factor Overexpression in Rat Arterial Neointima Models Thrombosis and Progression of Advanced Atherosclerosis Circulation, June 6, 2000; 101(22): 2651 - 2657. [Abstract] [Full Text] [PDF]
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 E. Camerer, E. Gjernes, M. Wiiger, S. Pringle, and H. Prydz Binding of Factor VIIa to Tissue Factor on Keratinocytes Induces Gene Expression J. Biol. Chem., February 25, 2000; 275(9): 6580 - 6585. [Abstract] [Full Text] [PDF]
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P. Zoldhelyi, J. McNatt, H. S. Shelat, Y. Yamamoto, Z.-Q. Chen, and J. T. Willerson Thromboresistance of Balloon-Injured Porcine Carotid Arteries After Local Gene Transfer of Human Tissue Factor Pathway Inhibitor Circulation, January 25, 2000; 101(3): 289 - 295. [Abstract] [Full Text] [PDF]
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A. Lundell, A. B. Kelly, J. Anderson, M. Marijianowski, J. J. Rade, S. R. Hanson, and L. A. Harker Reduction in Vascular Lesion Formation by Hirudin Secreted From Retrovirus-Transduced Confluent Endothelial Cells on Vascular Grafts in Baboons Circulation, November 9, 1999; 100(19): 2018 - 2024. [Abstract] [Full Text] [PDF]
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X. Han, T. J. Girard, P. Baum, D. R. Abendschein, and G. J. Broze Jr Structural Requirements for TFPI-Mediated Inhibition of Neointimal Thickening After Balloon Injury in the Rat Arterioscler. Thromb. Vasc. Biol., October 1, 1999; 19(10): 2563 - 2567. [Abstract] [Full Text] [PDF]
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J. St. Pierre, L.-Y. Yang, K. Tamirisa, D. Scherrer, P. De Ciechi, P. Eisenberg, E. Tolunay, and D. Abendschein Tissue Factor Pathway Inhibitor Attenuates Procoagulant Activity and Upregulation of Tissue Factor at the Site of Balloon-Induced Arterial Injury in Pigs Arterioscler. Thromb. Vasc. Biol., September 1, 1999; 19(9): 2263 - 2268. [Abstract] [Full Text] [PDF]
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T. Nishida, H. Ueno, N. Atsuchi, R. Kawano, Y. Asada, Y. Nakahara, Y.-i. Kamikubo, A. Takeshita, and H. Yasui Adenovirus-Mediated Local Expression of Human Tissue Factor Pathway Inhibitor Eliminates Shear Stress–Induced Recurrent Thrombosis in the Injured Carotid Artery of the Rabbit Circ. Res., June 25, 1999; 84(12): 1446 - 1452. [Abstract] [Full Text] [PDF]
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E. Shinoda, Y. Yui, R. Hattori, M. Tanaka, R. Inoue, T. Aoyama, Y. Takimoto, Y. Mitsui, K. Miyahara, Y. Shizuta, et al. Tissue Factor Pathway Inhibitor-2 Is a Novel Mitogen for Vascular Smooth Muscle Cells J. Biol. Chem., February 26, 1999; 274(9): 5379 - 5384. [Abstract] [Full Text] [PDF]
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Y. Ueda, M. Kitakaze, M. Imakita, H. Ishibashi-Ueda, T. Minamino, H. Asanuma, T. Ozaki, E. Imamura, T. Kuzuya, and M. Hori Glycoprotein IIb/IIIa Antagonist FK633 Could Not Prevent Neointimal Thickening in Stent Implantation Model of Canine Coronary Artery Arterioscler. Thromb. Vasc. Biol., February 1, 1999; 19(2): 343 - 347. [Abstract] [Full Text] [PDF]
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S. D. Gertz, J. T. Fallon, R. Gallo, M. B. Taubman, S. Banai, W. L. Barry, L. W. Gimple, Y. Nemerson, S. Thiruvikraman, S. S. Naidu, et al. Hirudin Reduces Tissue Factor Expression in Neointima After Balloon Injury in Rabbit Femoral and Porcine Coronary Arteries Circulation, August 11, 1998; 98(6): 580 - 587. [Abstract] [Full Text] [PDF]
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A. Lafont and D. Faxon Why do animal models of post-angioplasty restenosis sometimes poorly predict the outcome of clinical trials? Cardiovasc Res, July 1, 1998; 39(1): 50 - 59. [Full Text] [PDF]
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D. W. Courtman, S. M. Schwartz, and C. E. Hart Sequential Injury of the Rabbit Abdominal Aorta Induces Intramural Coagulation and Luminal Narrowing Independent of Intimal Mass : Extrinsic Pathway Inhibition Eliminates Luminal Narrowing Circ. Res., May 19, 1998; 82(9): 996 - 1006. [Abstract] [Full Text] [PDF]
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G. Ghigliotti, A. R. Waissbluth, C. Speidel, D. R. Abendschein, and P. R. Eisenberg Prolonged Activation of Prothrombin on the Vascular Wall After Arterial Injury Arterioscler. Thromb. Vasc. Biol., February 1, 1998; 18(2): 250 - 257. [Abstract] [Full Text] [PDF]
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E. Dai, M. Stewart, B. Ritchie, N. Mesaeli, S. Raha, D. Kolodziejczyk, M. L. Hobman, L. Y. Liu, W. Etches, N. Nation, et al. Calreticulin, a Potential Vascular Regulatory Protein, Reduces Intimal Hyperplasia After Arterial Injury Arterioscler. Thromb. Vasc. Biol., November 1, 1997; 17(11): 2359 - 2368. [Abstract] [Full Text]
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U. Orvim, R. M. Barstad, L. Orning, L. B. Petersen, M. Ezban, U. Hedner, and K. S. Sakariassen Antithrombotic Efficacy of Inactivated Active Site Recombinant Factor VIIa Is Shear Dependent in Human Blood Arterioscler. Thromb. Vasc. Biol., November 1, 1997; 17(11): 3049 - 3056. [Abstract] [Full Text]
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L. Oltrona, C. M. Speidel, D. Recchia, S. A. Wickline, P. R. Eisenberg, and D. R. Abendschein Inhibition of Tissue Factor–Mediated Coagulation Markedly Attenuates Stenosis After Balloon-Induced Arterial Injury in Minipigs Circulation, July 15, 1997; 96(2): 646 - 652. [Abstract] [Full Text]
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B. B. Sorensen, E. Persson, P.-O. Freskgard, M. Kjalke, M. Ezban, T. Williams, and L. V. M. Rao Incorporation of an Active Site Inhibitor in Factor VIIa Alters the Affinity for Tissue Factor J. Biol. Chem., May 2, 1997; 272(18): 11863 - 11868. [Abstract] [Full Text] [PDF]
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V. Toschi, R. Gallo, M. Lettino, J. T. Fallon, S. D. Gertz, A. Fernandez-Ortiz, J. H. Chesebro, L. Badimon, Y. Nemerson, V. Fuster, et al. Tissue Factor Modulates the Thrombogenicity of Human Atherosclerotic Plaques Circulation, February 4, 1997; 95(3): 594 - 599. [Abstract] [Full Text]
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P. R. Moreno, V. H. Bernardi, J. Lopez-Cuellar, J. B. Newell, C. McMellon, H. K. Gold, I. F. Palacios, V. Fuster, and J. T. Fallon Macrophage Infiltration Predicts Restenosis After Coronary Intervention in Patients With Unstable Angina Circulation, December 15, 1996; 94(12): 3098 - 3102. [Abstract] [Full Text]
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R. Singh, S. Pan, C. S. Mueske, T. Witt, L. S. Kleppe, T. E. Peterson, A. Slobodova, J.-Y. Chang, N. M. Caplice, and R. D. Simari Role for Tissue Factor Pathway in Murine Model of Vascular Remodeling Circ. Res., July 6, 2001; 89(1): 71 - 76. [Abstract] [Full Text] [PDF]
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H. Kato Regulation of Functions of Vascular Wall Cells by Tissue Factor Pathway Inhibitor: Basic and Clinical Aspects Arterioscler. Thromb. Vasc. Biol., April 1, 2002; 22(4): 539 - 548. [Abstract] [Full Text] [PDF]
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