(Circulation. 1995;92:3323-3330.)
© 1995 American Heart Association, Inc.
Articles |
Tissue Factor Mediates Prolonged Procoagulant Activity on the Luminal Surface of Balloon-Injured Aortas in Rabbits
From the Department of Medicine, Washington University School of Medicine, St Louis, Mo, and Scripps Research Institute, La Jolla, Calif (W.R., T.S.E.).
Correspondence to Dana R. Abendschein, PhD, Cardiovascular Division, Washington University School of Medicine, 660 S Euclid Ave, Campus Box 8086, St Louis, MO 63110.
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Abstract |
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Background Activation of coagulation has been implicated in both acute thrombotic occlusion and restenosis after balloon angioplasty. However, concomitant administration of antithrombotic agents has thus far failed to prevent these complications. Importantly, the factors contributing to procoagulant activity of balloon-injured arteries over time have not been defined. This study was designed to determine the duration of procoagulant activity on the luminal surface of balloon-injured arteries and the relative roles of tissue factor and thrombin in this response.
Methods and Results Abdominal aortas in rabbits were subjected to
repetitive balloon hyperinflations sufficient to disrupt the internal
elastic lamina. Aortas were excised at <1, 2, 4, 8, 16, 24, 48, and 72
hours and 1, 2, and 4 weeks after injury; divided into segments; and
perfused with recalcified human pooled plasma (n=58) or plasma depleted
of vitamin K–dependent coagulation factors (n=27) or first
incubated
with a monoclonal antibody to rabbit tissue factor (n=33) followed by
perfusion with human plasma. Samples of the effluent and plasma
perfusate were collected over 10 minutes and assayed for
fibrinopeptide A (FPA) as an index of the rate of
thrombin-induced fibrin formation. FPA in the effluent from
segments perfused with recalcified plasma, expressed as a percentage of
FPA in the perfusate, was elevated for 16 hours after
balloon-induced injury and exhibited two distinct increases
occurring <1 hour (1297±473%, mean±SD, n=5) and 8
hours
(1052±330%, n=6) after injury (P
.000001 versus
uninjured
vessels). Preincubation of segments at these intervals with an antibody
to tissue factor markedly attenuated the increases in FPA, as did
perfusion of segments with plasma depleted of vitamin K–dependent
coagulation factors, indicating that the observed increases in FPA in
whole plasma did not result from preformed thrombin bound to the
injured vessel wall.
Conclusions Tissue factor–mediated coagulation appears to be primarily responsible for prolonged procoagulant activity of balloon-injured arteries.
Key Words: thrombosis • coagulation • angioplasty
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Introduction |
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TopAbstractIntroductionMethods Results Discussion References |
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Coronary angioplasty is limited by acute thrombotic occlusion in 5% to 10% of patients and restenosis resulting in the recurrence of ischemic symptoms in 30% to 50% of patients.1 2 3 The mechanisms responsible for these complications are unclear, although thrombosis associated with mechanical trauma to the arterial wall has been implicated.4 Nevertheless, administration of currently available antithrombotic agents has failed to prevent either thrombotic occlusion or restenosis after angioplasty5 6 7 8 9 despite some success with these agents in experimental animals.10 11 12 13
Inefficient inhibition of thrombin has been suggested to account for the failure of available antithrombotic agents to attenuate restenosis.14 Importantly, the necessary duration of inhibition has yet to be determined, in part because it is not clear how long the vessel remains procoagulant after injury. Thus, administration of agents for too brief an interval may allow some thrombin to escape inhibition, activate factor V, and thereby enhance local formation of the prothrombinase complex.15 In addition, persistent availability of thrombin could activate factors VIII16 and XI,17 providing synergistic activation of coagulation.
Therapeutic efficacy of antithrombotic agents in previous studies also may have been limited by persistent activation of factors IX and X by the complex of tissue factor and factor VIIa.18 Tissue factor, a constituent primarily of the arterial adventitia,19 also has been identified in the subendothelium20 and atherosclerotic plaques21 and may be exposed by vessel injury.22 When exposed to blood, tissue factor binds factors VII and VIIa, resulting in assembly of the functional tissue factor–factor VIIa complex, which in turn activates factors IX and X.23 24 Factor Xa and its cofactor Va assembled on cell surfaces then convert prothrombin to thrombin.
Tissue factor mRNA and protein also are induced in the wall of vessels after balloon injury,25 but it is not clear to what extent the increased expression of vascular tissue factor contributes to the procoagulant activity of the luminal surface. This study was designed (1) to determine the duration of procoagulant activity on the luminal surface of balloon-injured aortas by ex vivo perfusion of the vessels obtained at different intervals after injury with recalcified human plasma and assay of fibrinopeptide A (FPA) in the effluent as an index of the rate of thrombin-induced fibrin formation and (2) to investigate the relative roles of tissue factor and thrombin in this response.
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Methods |
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TopAbstractIntroductionMethods Results Discussion References |
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Induction of Aortic Injury
All experiments were performed in accordance with the guidelines of the American Physiological Society and were approved by the Animal Studies Committee at Washington University. Abdominal aortas in 62 New Zealand White rabbits (3 to 5 kg) were injured by repetitive balloon hyperinflations by use of a modification of a method described previously.26 Briefly, rabbits were anesthetized with ketamine (50 mg/kg IM) and xylazine (5 mg/kg IM). A 4F Fogarty embolectomy catheter (Baxter Pharmaceuticals) was inserted through a left femoral arteriotomy and advanced 25 cm into the abdominal aorta, verified initially to position the balloon proximal to the renal arteries. The balloon was gently inflated with saline by use of a tuberculin syringe until a wedge position was confirmed. The balloon volume was then decreased by
0.05 mL to permit withdrawal of the catheter to the iliac
bifurcation. The balloon was deflated, and the procedure was repeated
twice. The catheter was removed, the femoral artery was ligated, and
the skin incision was closed with suture. Rabbits in which the aortas
were harvested at intervals >2 hours after injury were allowed to
regain consciousness. Rabbits in the extended survival group (ie, 
24
hours) received a single dose of benzathine penicillin G and procaine
penicillin G (60 000 U/kg IM, Hanford Manufacturing) after the
procedure.
Perfusion of Injured Aortic Segments
The aorta was harvested
at <1, 2, 4, 8, 16, 24, 48, or 72 hours
or 1, 2, or 4 weeks after balloon-induced injury and perfused with
human pooled plasma ex vivo to determine the procoagulant activity
associated with the luminal surface. Rabbits that had regained
consciousness were reanesthetized. The chest and abdomen
were incised longitudinally. The entire abdominal aorta was exposed by
blunt dissection, and all branches were ligated. After the pericardium
was opened, a perfusion catheter was inserted into the ascending aorta
through a left ventriculotomy, and the right atrium was incised.
The aorta was perfused in situ with 500 mL of 0.9% NaCl at 120 mm Hg
pressure to remove blood from the circulation and thereby prevent blood
stasis and coagulation, which in initial experiments were shown to
potentiate procoagulant activity of the luminal surface. The previously
injured aortas were excised, and some were divided into two or three
segments of equivalent length. Segments of uninjured aortas also were
obtained as a control in other rabbits. Segments were washed
gently with PBS.
Vessel segments were attached with suture to pieces of silicone tubing (Technical Products, Inc) so that the cut ends of the vessel were isolated from the perfusate and a consistent length of the vessel wall (1.5 cm) was exposed to perfusate. The vessel was immersed in a tray containing PBS that was incubated in a constant-temperature bath at 35°C. One piece of tubing was connected to a syringe pump (Harvard Pump 22); the other was used to collect effluent samples. To prevent fibrin that could form within the tubing from influencing determinations of procoagulant activity, tubing pieces were replaced daily.
The vessel segments were first equilibrated with PBS perfused at a flow rate of 1 mL/min for 10 minutes. The segments were then perfused with either barium-adsorbed human plasma or recalcified, citrated human pooled plasma purchased from the American Red Cross and verified to have an FPA concentration <35 ng/mL. Frozen plasma aliquots were thawed at 35°C and centrifuged at 2000g for 10 minutes, and the supernatant was recalcified with CaCl2 (final concentration, 25 mmol/L) immediately before the start of the perfusion. The plasma was perfused through the vessel segment at a flow rate of 1 mL/min for 10 minutes. Effluent was collected in 1-mL aliquots in vials containing 100 µL reconstituted FPA anticoagulant containing EDTA, aprotinin, and D-Phe-Pro-Arg chloromethyl ketone (Byk-Sangtec). Samples of the perfusing plasma (1 mL) also were collected before and at the end of the 10-minute perfusion.
In experiments designed to characterize the role of tissue factor in the procoagulant response to vessel injury, a monoclonal antibody to rabbit tissue factor was infused into the vessel, and the tubing pieces were occluded to retain the solution under gentle pressure. The antibody was incubated in the vessel for 15 minutes; then the segment was flushed with PBS followed by perfusion with whole plasma, as described above.
Validation of the Heterologous Perfusion System
To verify
that coagulation induced in human plasma perfused over
aortic segments from rabbits reflects what could be induced in rabbit
plasma, recalcified plasma from rabbits and human volunteers was
incubated with different concentrations of rabbit tissue factor
(thromboplastin, No. T0263, Sigma Chemical Co), and the clotting time
was assayed at 35°C.
Generation of Monoclonal Antibodies to Rabbit Tissue
Factor
The coding sequence for rabbit tissue factor cloned into a
mammalian expression vector27 was transfected into murine
NCTC clone 929 cells (ATCC, CCL1), and colonies that stably expressed
rabbit tissue factor were isolated. Mice (C3H/Hen) were
repeatedly immunized by intraperitoneal injection
of 107 tissue factor–positive cells. Spleen cells from
two mice were pooled for fusion with myeloma P3Ag8.653.1 to generate
hybridomas. Hybridoma culture supernatants were screened for a lack of
binding to untransfected cells and for reactivity with tissue
factor–positive cells, both of which were fixed on microtiter
plates. After repeated single-cell cloning, hybridomas were
analyzed for inhibition of tissue factor function. Hybridoma
RbTF7-3A5 supernatant inhibited the procoagulant activity of rabbit
tissue factor by >90%, and this hybridoma was selected for further
studies. Hybridoma RbTF7-3A5 secreted homogenous IgG2a
monoclonal antibody with a 
light chain.
Purification and Characterization of Monoclonal Antibody to Rabbit
Tissue Factor
Hybridoma RbTF7-3A5 was grown in suspension culture for
antibody
production. Culture supernatant was concentrated 20-fold and
diluted with an equal volume of 1 mol/L glycine, 150 mmol/L NaCl, pH
8.6 (binding buffer), for absorption to a protein A resin. After
loading, the column was washed with 100 mL binding buffer and eluted
with 0.1 mol/L glycine-HCl, pH 3.0. Fractions containing protein based
on OD280 were neutralized with 1 mol/L Tris, pH 8.0. The
buffer was exchanged to Dulbecco's PBS (Bio-Whittaker), and the
antibody solution was sterile filtered (0.22 µm) after concentration.
The antibody was homogenous as determined by SDS-PAGE and Coomassie
staining. The lot used in this study had <0.25 EU/mL endotoxin in the
5.2 mg/mL antibody solution as determined by limulus assay
(Bio-Whittaker). Antibody specificity was determined by Western blot
analysis and incubation of increasing concentrations of the
antibody with rabbit thromboplastin or phospholipid-reconstituted
human tissue factor for 30 minutes on ice followed by determination of
the residual tissue factor activity in a one-stage clotting assay
with recalcified human plasma.
Preparation of Barium Citrate–Adsorbed Plasma
Citrated
human pooled plasma was depleted of vitamin
K–dependent coagulation proteins (factors II, VII, IX, and X) by
adsorption with barium chloride as previously reported.28
Barium chloride (100 mmol/L) was incubated with the plasma at 4°C for
30 minutes, the mixture was centrifuged at 1800g for
15 minutes, and the supernatant was recovered. Additional precipitate
was allowed to form for 30 minutes. The mixture was
recentrifuged, and the supernatant plasma was dialyzed
exhaustively against 0.15 mol/L NaCl and 0.012 mol/L sodium citrate, pH
6.0. Barium-adsorbed plasma (1 mL) prepared with this method and
incubated for 10 minutes at 37°C with 1.0 µL Taipan snake venom
(0.1 mg/mL), a prothrombin activator, generated no FPA,
indicating that prothrombin had been completely removed. To confirm the
absence of factor X, 1.0 µL Russell viper venom (0.3 mg/mL), a factor
X activator, was added to barium-adsorbed plasma (1
mL), and factor Xa activity was determined by addition of 1.0 µL (2.0
mmol/L) of a chromogenic substrate for factor Xa (S-2765,
Chromogenix) to 100 µL of the plasma-venom mixture. No change in
absorbance was detected with a microtiter plate reader (ThermoMax,
Molecular Devices) after 15 minutes, indicating that factor X had been
completely removed. However, incubation of recalcified
barium-adsorbed plasma for 10 minutes at 37°C with human
prothrombin (final concentration, 0.9 µmol/L) and human factor Xa
(final concentration, 2 nmol/L) generated levels of FPA similar to
those obtained with whole plasma, indicating that fibrinogen had not
been removed by adsorption with barium chloride.
Radioimmunoassay for FPA
The concentration of FPA in the
plasma perfusate and
effluent samples was measured with a previously validated
radioimmunoassay (Byk-Sangtec).29 Before assay, bentonite
(400 µL) was added to the plasma sample (200 µL) to remove
potentially cross-reactive fibrinogen and fibrinogen degradation
products. The mixture was centrifuged at 2400g
for 20 minutes, and the supernatant was recovered and assayed for FPA.
The lower limit of detection of FPA with this assay is 1 ng/mL; the
linear range is 1 to 40 ng/mL. Intra-assay variability is
5.7±0.7%. Samples with FPA levels >40 ng/mL were diluted with an FPA
diluent buffer.
ELISA for Prothrombin Fragment 1.2
Levels of prothrombin
fragment 1.2 in the perfused plasma
effluent were assayed by an ELISA (Baxter Diagnostics) with
a monoclonal antibody specific for fragment 1.2 that does not recognize
native prothrombin.30 The antibody was coated on a
microtiter plate and incubated with the plasma sample or purified human
prothrombin fragment 1.2 for 30 minutes. The plate was then washed and
incubated for 10 minutes with a second monoclonal antibody conjugated
to horseradish peroxidase that recognizes an independent epitope on
fragment 1.2. The plate was washed, and 3,3',5,5'
tetramethylbenzadine,
a peroxidase substrate, was added. The reaction was stopped with 1
mol/L sulfuric acid, and the absorbance was measured with a microtiter
plate reader. Concentrations of fragment 1.2 in samples were determined
by comparison with a standard curve obtained from purified human
fragment 1.2.
Histological Analysis
After ex vivo perfusion, the vessel
segments were immersion
fixed in 4% paraformaldehyde for 24 hours. Selected
segments obtained at each interval after vascular injury were embedded
in paraffin and sectioned at 5 µm. Sections were stained with
hematoxylin and eosin and Verhoeff"s–van Gieson stain for
elastic tissue to delineate the internal elastic lamina.
Statistical Analysis
Data are reported as mean±SD.
Comparisons of the FPA and
prothrombin fragment 1.2 concentrations at different time intervals and
between groups were made with ANOVA by use of the Bonferroni method for
multiple comparisons. Significance was defined as
P<.05.
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Results |
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TopAbstractIntroductionMethods Results Discussion References |
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Validation of Perfusion of Rabbit Aortas With Human Plasma
Addition of rabbit tissue factor to either human or rabbit plasma in vitro yielded clotting times that were twice as fast in rabbit compared with human plasma (Fig 1
).
However, clotting times were prolonged similarly in both plasma samples
by serial dilution of the rabbit tissue factor concentration,
indicating comparable activation of coagulation. Thus,
coagulation induced in human plasma perfused over aortic segments
from rabbits reflects that which could be induced by perfusion of
rabbit plasma.
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Duration of Procoagulant Activity After Vessel Injury
A total
of 128 abdominal aortic segments were obtained from 62
rabbits at intervals between 1 hour and 4 weeks after
balloon-induced injury. Segments were excluded from
analysis because of the presence of postmortem clot (n=1),
aortic rupture or dissection with gross thrombus in contact with the
true lumen (n=3), or formation of plasma clot within the perfusion
apparatus (n=6).
Fig 2 shows representative
profiles of
FPA concentrations in the recalcified plasma perfusate and the
effluent from an injured vessel segment. Because FPA in the effluent
after 10 minutes was increased markedly and consistently above
that in the perfusate, vessel perfusions were terminated after
10 minutes. In the remaining results, FPA levels in the 10-minute
effluent samples are expressed as percentages of concentrations in
10-minute perfusate samples because experiments were performed
with several pools of human plasma that exhibited different coagulation
rates.
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Compared with uninjured control aortic segments, the
procoagulant
activity of injured segments was increased immediately after
balloon-induced injury and remained elevated for 16 hours (Fig
3). Peaks of FPA were noted <1 hour
(1297±473%) and 8 hours (1052±330%) after injury
(P.000001 versus control segments).
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Contribution of Tissue Factor Activity to Luminal
Procoagulant Activity
To determine the contribution of tissue
factor–mediated
coagulation to the procoagulant activity of balloon-injured
vessels, additional aortic segments obtained at intervals over the
first 24 hours after injury were incubated with monoclonal antibody to
rabbit tissue factor before perfusion with recalcified plasma. Western
blot analysis showed that the antibody reacted with a
heterogeneous protein of 49 to 50 kD in cells
transfected with rabbit tissue factor cDNA and 45-kD protein from crude
rabbit brain extract (Fig 4A). The same
bands also were detected by a weakly cross-reactive polyclonal
antibody raised against human tissue factor. The monoclonal
antibody to rabbit tissue factor inhibited coagulation of human
plasma initiated by rabbit but not human tissue factor (Fig
4B), indicating that the inhibitory
function of the antibody is not indirectly mediated by the inactivation
of other coagulation factors. In a coagulation assay with rabbit
plasma, rabbit tissue factor function was inhibited by 50% with 10
µg/mL antibody and >95% with 100 µg/mL antibody. The
inhibitory potency of the antibody was found to be
comparable when tested with rabbit tissue factor and purified human
factor VIIa and factor X in a factor Xa generation
assay.31
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The maximally effective concentration of antibody
for inhibition of
procoagulant activity of balloon-injured aortas was defined
empirically by incubation of multiple vessel segments with increasing
concentrations of antibody and comparison of the suppression of FPA
generation (see the Table). A dose-dependent
inhibition of FPA generation was observed with concentrations of
antibody up to 80 µg/mL, in agreement with the inhibitory
potency of antibody in coagulation assays. Preincubation of vessel
segments with maximally inhibitory concentrations of
antibody resulted in marked attenuation of FPA generation to levels not
significantly different from those obtained on uninjured, control
vessels and significantly lower than levels in the effluent from
vessels obtained <1 and 8 hours after injury but not preincubated with
antibody (Fig 5). Preincubation of
vessels with the same concentration of IgG of an irrelevant monoclonal
antibody (creatine kinase–MB) did not attenuate FPA production
(data not shown).
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Assessment of Thrombin Bound to the Luminal Surface
To
determine whether thrombin formed in vivo and associated with
the luminal surface contributed to the procoagulant activity of
balloon-injured vessels, additional aortic segments from the same
rabbits were perfused with barium-adsorbed plasma depleted of
vitamin K–dependent coagulation factors. Under these conditions,
production of FPA reflects the activity of preformed thrombin
because thrombin cannot be generated in the barium-adsorbed plasma.
FPA levels in the effluent from vessels injured 1 to 24 hours
previously were no greater than those observed in the effluent from
uninjured, control vessels and were significantly less than those
observed in the whole-plasma effluent from vessels at
intervals when procoagulant activity was detected (Fig 5
).
Prothrombin Activation as the Source of Procoagulant
Activity
To confirm that prothrombin activation, not preformed
thrombin,
was the primary source of the observed increases in luminal
procoagulant activity, generation of prothrombin fragment 1.2 was
measured in the 10-minute plasma effluent samples from selected
experiments. The concentration of prothrombin fragment 1.2 was
increased in samples from vessels obtained <1 hour after injury, with
a trend for the concentration to increase in samples from vessels
obtained 8 hour after injury (Fig 6).
Importantly, the appearance of prothrombin fragment 1.2 in the effluent
was abolished by preincubation of vessel segments with antibody to
tissue factor (Fig 6), consistent with the role
of tissue factor in mediating the activation of prothrombin and the
procoagulant response at these intervals.
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Confirmation of Deep Vessel Injury
Aortic segments
(n=36) obtained at each time interval after
balloon-induced injury were examined morphologically to determine
the extent of vascular injury. Multiple disruptions of the internal
elastic lamina were evident in each section (Fig 7), consistent
with deep (type
III) vascular injury, as described previously.32
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Discussion |
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TopAbstractIntroductionMethods Results Discussion References |
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The results of this study show that tissue factor–mediated coagulation is primarily responsible for procoagulant activity associated with the luminal surface of balloon-injured aortas in rabbits. Furthermore, injured aortas became relatively noncoagulant within 24 hours after balloon-induced injury (Fig 3
). These
data suggest that tissue factor–mediated
coagulation must be inhibited for up to 24 hours after
balloon-induced arterial injury to prevent thrombosis
that may contribute to acute occlusion, subsequent
restenosis, or both.
Thrombin has previously been implicated as a mediator of procoagulant
activity after vessel
injury33 34 35 36 and the
cellular
responses believed to participate in
restenosis.37 38 39 Increased thrombin
activity
could result from either de novo activation of prothrombin or
association of preformed thrombin with fibrin and extracellular matrix
proteins at the site of vascular injury. We observed with perfusion of
barium-adsorbed plasma through injured aortic segments only a
modest amount of procoagulant activity attributable to
surface-associated thrombin (Fig 5). Thus, the
majority of the procoagulant activity of injured vessels appears to be
accounted for by de novo generation of thrombin, as indicated by
suppression of both prothrombin fragment 1.2, a reliable marker of
prothrombin activation,40 41 and FPA in the effluent
after
preincubation of vessel segments with the antibody to tissue factor
(Figs 5 and 6). Persistent activation of
prothrombin mediated by tissue
factor–dependent pathways may explain why inhibition of thrombin
alone has failed to attenuate restenosis in
patients.6
Our results showing attenuation of procoagulant activity on the luminal
surface of injured vessels preincubated with the antibody to rabbit
tissue factor (Fig 5) agree with studies in vivo in
which thrombosis in stenotic carotid arteries of rabbits was
inhibited by infusion of a similar antibody.42 The FPA we
observed in plasma perfused over vessel segments despite inhibition
with tissue factor antibody may have resulted from modest quantities of
factor Xa or IXa or thrombin associated with the luminal surface.
Tissue factor located in the
subendothelium20 and media exposed by
breaks in the internal elastic lamina probably accounted for the
procoagulant activity observed immediately after injury (Fig
3). The subsequent decline of FPA in the effluent from
vessels after 2 and 4 hours of injury followed by a second peak at 8
hours is consistent with induction of vessel tissue factor mRNA
and protein that has been reported recently in rat aortas after balloon
trauma.25 The origin of induced tissue factor is unclear
but may be smooth muscle cells in the media or monocytes, which are
recruited to the exposed subendothelium and readily
express tissue factor.43
A critical feature of our rabbit preparation was the production
of reproducible deep vascular injury compatible with the extent of
injury observed after clinical coronary
angioplasty.26 Deep vascular injury was defined by
disruption of the internal elastic lamina that exposes circulating
coagulation factors to molecules within the subintima and media. The
technique used was implemented initially with the abdominal aorta
exposed to visually define the maximum inflation pressure of the
balloon that would not result in aortic rupture. Use of this procedure
in intact rabbits resulted in consistent, multiple disruptions
of the internal elastic lamina over the entire length of the abdominal
aorta (Fig 7).
Buffer systems containing purified coagulation factors and synthetic
phospholipid vesicles have been used previously to investigate the
surface-associated assembly of coagulation factors.44
Our ex vivo perfusion model may more closely mimic conditions of
coagulation on an injured vessel in vivo. Perfusion with human plasma
was required to permit use of immunoassays specific for human FPA and
prothrombin fragment 1.2. However, comparable prolongation of clotting
times in rabbit and human plasma containing different concentrations of
rabbit tissue factor (Fig 1) indicates that results
obtained with perfusion of human plasma over rabbit aortas reflect
those that would have been obtained with perfusion of homologous rabbit
plasma.
In summary, our data indicate that balloon-injured vessels remain
highly procoagulant for 24 hours after injury and, importantly,
exhibit a bimodal pattern of procoagulant activity. The initial
increase in procoagulant activity observed in vessels obtained
immediately after injury is consistent with exposure of vessel
tissue factor, which initiates coagulation and formation of thrombin.
The second increase in procoagulant activity observed 8 to 16 hours
after injury also appears to depend primarily on tissue factor and is
consistent with induction of tissue factor mRNA and protein in
the vascular wall in response to injury. Procoagulant activity was
markedly attenuated at each of these intervals by exposure of the
injured surface to antibodies against tissue factor. Accordingly,
inhibition of tissue factor–mediated coagulation during this
discrete interval may be particularly effective for inhibiting local
acute thrombosis that could contribute to subsequent
restenosis.
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Acknowledgments |
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This work was supported in part by NIH grant HL-17646, SCOR in Vascular Diseases, and a Monsanto/Washington University Biomedical Research Grant. We thank John Engelbach and Gwen Mohr for assistance with the animal preparations; Julie Tinker for assistance with ex vivo perfusions; Mary Jane Eichenseer and Ann Guillerman for analyzing FPA and prothrombin fragment 1.2 in samples; Ken Schechtman, PhD, for assistance with the statistical analysis; and Barbara Donnelly for preparation of the manuscript.
Received December 27, 1994; revision received May 4, 1995; accepted July 24, 1995.
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TopAbstractIntroductionMethods Results Discussion References |
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