© 1998 American Heart Association, Inc.
Original Contributions |
Prolonged Activation of Prothrombin on the Vascular Wall After Arterial Injury
From the Cardiovascular Division, Washington University School of Medicine (A.R.W., C.S., D.R.A., P.R.E.), St Louis, Mo, and the Department of Cardiology, University of Genoa, Italy (G.G.).
Correspondence to Paul R. Eisenberg, MD, MPH, Associate Professor of Medicine, Cardiovascular Division, Campus Box 8086, Washington University School of Medicine, 660 S Euclid, St Louis, MO 63110. E-mail eisenber{at}im.wustl.edu
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Abstract—This study was designed to characterize the relative roles of bound Xa/Va and thrombin activity in vascular wall procoagulant activity after balloon-induced injury and the extent to which intravenous aspirin and heparin attenuate procoagulant activity associated with the vascular wall. Abdominal aortic injury was induced in rabbits by overinflation and multiple passages of a 4F embolectomy catheter. Rabbits were killed 15 minutes or 4, 8, 24, 48, 72, 96, or 120 hours after injury. Aortic segments were incubated ex vivo to define bound procoagulant activity. Thrombin activity bound to the aorta was detected by 4 hours after injury and was most marked over the first 24 hours, as estimated by increases in concentration of fibrinopeptide A during incubation of segments with recalcified barium-adsorbed plasma or activity against the thrombin-synthetic substrate S-2238. Based on comparison with purified human thrombin incubated under the same conditions, a maximum of 0.04 to 0.1 nmol/L per square centimeter of thrombin activity was associated with the vascular wall during the first 24 hours and remained detectable for 72 hours. In contrast, bound Xa/Va complex activity to injured segments was detected within 15 minutes and induced activation of prothrombin added to recalcified barium-adsorbed plasma incubated with injured segments for 96 hours. Aspirin (15 mg/kg) administered 30 minutes before injury attenuated 111In-platelet deposition at 4 hours by 67%, with an associated decrease in bound Xa/Va and thrombin activity at 15 minutes and 4 hours. However, intravenous heparin did not attenuate bound Xa/Va activity at 15 minutes or thrombin activity at 15 minutes and 4 hours. Platelet-dependent bound Xa/Va activity occurs rapidly after arterial injury and may promote thrombin elaboration for up to 96 hours. Bound thrombin activity and de novo thrombin elaboration on the vascular wall may play an important role in the progression of thrombosis and vascular wall remodeling.
Key Words: thrombin • factor Xa • thrombosis • platelets • tissue factor
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The initial response to arterial injury involves platelet adhesion and activation, as well as activation of the coagulation system.1 2 3 The subsequent development of a thrombus is dependent on platelet and fibrin recruitment to the injured vascular wall, which is mediated primarily by local thrombin elaboration. We and others have shown that local thrombin elaboration is dependent on tissue factor–mediated activation of coagulation.4 5 6 Tissue factor, a membrane-associated glycoprotein, serves as a cofactor for factor VIIa–mediated activation of factor X.7 Accumulation of platelets and fibrin at the site of arterial injury, even when thrombus is not grossly apparent, also mediates the progression of thrombosis by binding activated coagulation factors.8 9 We have shown that arterial thrombi induce marked activation of the coagulation system attributable to platelet-associated Xa/Va activity and thrombin bound to fibrin.8 However, the extent to which procoagulant factors bind to the injured vessel wall, particularly in the absence of significant mural thrombus, and the duration of procoagulant activity associated with the vascular wall after arterial injury are not well defined.
In a previous study, we demonstrated that tissue factor exposed by balloon-induced deep injury of the aorta in rabbits promoted activation of the coagulation system for up to 24 hours after injury.5 We also found evidence for binding of enzymatically active thrombin to the vascular wall after injury that contributed to the procoagulant activity in the first 24 hours. However, the contribution of other coagulation factors was not specifically assessed. We hypothesized that the Xa/Va complex expressed by platelets that bind to the vascular wall after injury may be an important determinant of thrombin elaboration.8 10 11 Accordingly, the objectives of this study were to characterize the functional activity of Xa/Va and thrombin associated with the vascular wall after balloon-induced injury to the rabbit aorta and the extent to which the activity of these factors contributes to procoagulant activity over time. In addition, we characterized the extent to which aspirin and heparin attenuate vascular wall-associated procoagulant activity after injury.
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Methods |
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Preparation of Coagulation Factors and Plasma Samples
Purified coagulation factors were prepared in our laboratory in collaboration with Dr Joseph Miletich. Human prothrombin and factor X were isolated from pooled citrated plasma and were purified by affinity chromatography against the other vitamin K–dependent proteins, as previously described.8 12 Prothrombin and factor X preparations were incubated with 5 mmol/L D-Phe-D-Pro-L-Arg chloromethyl ketone (PPACK; Calbiochem) to inhibit trace amounts of contaminating active proteases. These preparations were then extensively dialyzed against 0.02 mol/L HEPES, 0.15 mol/L NaCl, pH 7.4 (HEPES saline), to remove free D-Phe-D-Pro-L-Arg chloromethyl ketone. Thrombin was prepared by activation of prothrombin with Taipan snake venom, followed by purification with mono-S chromatography. Activated factor X (factor Xa) was prepared by incubation of factor X with purified factor X coagulant protein from Russell's viper venom at a 1:1 ratio by weight in HEPES saline containing 0.002 mol/L CaCl2, pH 7.4, at 37°C for 60 minutes. Tick anticoagulant peptide (TAP) was a generous gift from Dr George Vlasuk (Corvas, San Diego, Calif). Recombinant desulfatohirudin was from Ciba-Geigy (Basel, Switzerland).
Pooled, citrated human plasma was purchased from the American Red Cross (St Louis, Mo). Plasma was depleted of vitamin K–dependent factors by addition of 0.1 mol/L BaCl2 at 4°C for 60 minutes (barium-adsorbed plasma). Plasma was centrifuged to separate the precipitate, the supernatant was recovered, and additional BaCl2 precipitate was allowed to form at 4°C. The centrifugation step was repeated and the supernatant plasma was then exhaustively dialyzed against buffer containing 0.15 mol/L NaCl, 0.012 mol/L sodium citrate, pH 6.0, and stored as 10-mL aliquots at -70°C. All plasma samples were rapidly thawed at 37°C immediately before use.
Aortic Injury in the Rabbit
Animal studies were approved by the Animal Studies Committee at
Washington University and conform with the Guide for the Care and
Use of Laboratory Animals (US National Institutes of Health
publication 93–23, revised 1985). New Zealand White rabbits (3 to 4
kg), fed a normal diet, were anesthetized with intramuscular
ketamine (6.5 mg/kg) and xylazine (0.75 mg/kg). The animals
were prepared for surgery as previously
described.5 Briefly, deep arterial
injury was induced with a 4F Fogarty embolectomy catheter (Baxter
Pharmaceuticals) inserted through a left femoral arteriotomy and
advanced into the abdominal aorta proximal to the renal branches. The
balloon was overinflated with saline with use of a tuberculin syringe
to obtain a wedge position, and the balloon volume was then decreased
by approximately 0.05 mL to permit withdrawal of the catheter to the
level of the distal abdominal aorta. The procedure was repeated twice.
Control sham-operated rabbits were subjected to the same surgical
procedure, with the exception that the balloon was not inserted through
the exposed femoral artery.
The rabbits were killed at 15 minutes or 4, 8, 24, 48, 72, 96, or 120 hours after balloon overinflation–induced injury of the aorta. Initially, a midline incision was made under anesthesia, and the abdominal aorta was exposed and collateral branches were ligated. A catheter was then introduced into the ascending thoracic aorta, which was flushed with 500 mL of saline buffer at a pressure of 100 mm Hg before the aorta was excised. These washing conditions were shown in preliminary studies to be extremely stringent and resulted in no visible adherent clot. The aorta was maintained in phosphate-buffered saline until luminal procoagulant activity was assayed, as described below.
Some animals were randomly assigned to receive intravenous infusions of either heparin (heparin sodium, LyphoMed) administered as a bolus (150 U/kg) followed by a continuous infusion (50 U/kg per hour) (n=6) or aspirin (Maggioni-Winthrop) administered as a bolus (15 mg/kg) followed by an infusion (15 mg/kg) for 30 minutes (n=6). In animals given heparin, the activated partial thromboplastin time (aPTT) was measured with a Coag-A-Mate XC automated coagulation timer (Organon Technica). In animals given aspirin, platelet aggregation in response to addition of collagen (5 µg/mL) to platelet-rich plasma was measured with a ChronoLog aggregometer (ChronoLog Corporation).
Characterization of Extent of Arterial Injury
In each animal, a 1.5-cm segment of the injured abdominal aorta
was collected to assess the extent of arterial damage.
Segments were fixed in 4% paraformaldehyde and
embedded in paraffin; serial sections (5 µm) were collected at
100-µm intervals and stained with Verhoeff–van Gieson's stain for
elastic tissue. At least four serial sections from each dilated
arterial segment were examined by two of the investigators
(Drs Abendschein and Eisenberg), who were blinded to the treatment
assignment. The extent of injury was defined as deep (ie, involving the
media) if one or more areas of the internal elastic lamina were
ruptured in each section examined.
Characterization of Procoagulant Activity on the Luminal Surface of
Aortic Segments
The aorta was cut into two or three segments as quickly as
possible after excision from the rabbit. Each aortic segment was
cannulated with silicone elastomer tubing at either end (0.062-in
internal diameter, 0.125-in external diameter; Medical Division, TPI)
such that approximately 1.5 cm of the luminal surface of the segment
was exposed between the ends of the tubing. The area of exposed lumen
was directly measured at the end of each experiment by transsectioning
only the segment between the cannulated ends. Segments were then cut
longitudinally, traced, and digitized, and the luminal area was
quantified with the use of an image analysis program on a
Macintosh PC (Image 1.5, NIH). The cannulated segment was placed in a
water bath at 37°C, and the lumen was flushed with 5 mL of
phosphate-buffered saline. The segment was then sequentially incubated
with one or more of the plasma preparations or chromogenic
substrates described below.
We have previously shown that rabbit thrombin induces proteolysis of human fibrinogen and that compared with human tissue factor, rabbit tissue factor–induced coagulation of human plasma induces similar degrees of thrombin elaboration.5 In addition, we have shown that rabbit Xa/Va activates human prothrombin and that activation is completely abolished with TAP (data not shown).
To characterize thrombin activity associated with the injured vessel
wall, segments were incubated for 30 minutes with either recalcified
(25 mmol/L CaCl2 final concentration)
barium-adsorbed plasma, after which the concentration of
fibrinopeptide A (FPA) was measured to define fibrin
formation induced by bound thrombin, or with 333 µmol/L (final
concentration) S-2238
(H-D-Phe-Pip-Arg-p-nitroaniline;
Chromogenics) in 200 µL of 0.05 mol/L Tris-HCl, 0.175
mol/L NaCl, and 0.002 mol/L CaCl2, pH 7.8, after which
bound thrombin activity was assayed. Proteolysis of S-2238 was measured
by the change in absorbance at 405 nm in an automated microplate reader
at 37°C (ThermoMax Molecular Devices). The activity of thrombin bound
to vessels against the chromogenic substrate was compared
with activity of purified human 
-thrombin against the same substrate
and under the same conditions. To confirm the specificity of the assay
for vascular wall–bound thrombin activity, selected segments were
incubated with either barium-adsorbed plasma or S-2238 in buffer in the
presence of recombinant desulfatohirudin.
To define Xa/Va activity bound to the vessel wall, aortic segments were incubated with recalcified (25 mmol/L CaCl2 final concentration) barium-adsorbed plasma repleted with 0.9 µmol/L human prothrombin, and concentrations of FPA were measured after a 30-minute incubation. Under these conditions, increases in FPA were attributable to both the activity of preformed thrombin associated with the segment (which was determined by incubation without added prothrombin) and thrombin elaborated by activation of the added prothrombin by bound Xa/Va. Accordingly, increases in FPA attributable to Xa/Va activity alone were determined by subtracting the increases in FPA observed after incubation with barium-adsorbed plasma alone from increases observed in the presence of added prothrombin. Bound factor Xa/Va-mediated activation of prothrombin was also assessed in a purified system by incubation of injured segments with 0.9 µmol/L prothrombin in 200 µL of 0.05 mol/L Tris-HCl, 0.175 mol/L NaCl, and 0.002 mol/L CaCl2, pH 7.8. Aliquots of the incubation mixture were removed and assayed for thrombin activity by incubation with 333 µmol/L S-2238 in the same buffer, as described above. To confirm the specificity of the assay for vascular wall–bound Xa/Va activity, selected segments were incubated with barium-adsorbed plasma containing prothrombin in the presence of TAP, a specific Xa inhibitor.13
Selected injured aortic segments were incubated in 200 µL of 0.05 mol/L Tris-HCl, 0.175 mol/L NaCl, and 0.002 mol/L CaCl2, pH 7.8, containing 50 nmol/L purified human factor X to determine whether procoagulants capable of activating factor X are bound to the injured vessel wall (ie, IXa/VIIIa or VIIa/tissue factor). Factor Xa activity was determined after a 30-minute incubation by adding S-2222 (N-benzoyl-L-Ile-Glu-Gly-Arg-p-nitroaniline, Chromogenics) to a final concentration of 450 µmol/L and measuring the change in absorbance at 405 nm.
111In-Platelet Deposition After Arterial
Injury
To characterize platelet deposition after
arterial injury, platelets from separate donor rabbits
were washed and labeled with 111In, as previously
described.14 The radiolabeled platelets were
injected into the rabbits 1 to 2 minutes before the aortic injury, and
the aorta was perfused in situ and excised after 4 hours as described
above. Radioactivity associated with vessel segments was counted with
the use of a gamma counter. The luminal area was directly measured as
described above. The number of platelets per square centimeter
adhering to the aortic segment was calculated from the radioactivity
and platelet count of the injected platelet suspension,
corrected for the luminal area of the injured aortic segment.
111In-labeled platelets were also injected
into control, sham-operated animals to determine the binding of
platelets on the uninjured aorta.
Radioimmunoassay of FPA
Thrombin activity in barium-adsorbed plasma, with or without
added prothrombin, was characterized by elaboration of fibrin in plasma
determined by measuring changes in the concentration of FPA, a peptide
released from fibrinogen by thrombin. Plasma was adsorbed with
bentonite before assay of FPA.15 Concentrations
of FPA were measured with a polyclonal antiserum-based commercial
radioimmunoassay (Byk Sangtee), and results were compared with a
standard curve prepared by use of purified human FPA as previously
described.16
Statistical Analysis
Data are reported as the mean±SE. FPA data were log transformed
before statistical analysis to normalize the distribution of
the data. Comparisons between all conditions were analyzed by
ANOVA (Statview 4, Abacus Concepts) on a Macintosh PC with the use of
Scheffé's test to compare differences between individual time
points; a level of P<.05 was considered indicative of a
significant difference.
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Results |
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Procoagulant Activity Bound to the Vessel Wall Immediately After Arterial Injury
Injury resulting in multiple disruptions of the internal elastic lamina was documented in all animals. FPA was not increased when uninjured segments from sham-operated rabbits were incubated with barium-adsorbed plasma. Thrombin activity associated with the injured vessel wall was not detected in aortic segments acquired 15 minutes after balloon hyperinflations, judging by the lack of significant increase in concentrations of FPA in barium-adsorbed plasma incubated with injured aortic segments compared with concentrations in uninjured segments obtained from control, sham-operated rabbits (Fig 1
). The lack of significant bound
thrombin activity was also confirmed by a negligible proteolysis of the
chromogenic substrate for thrombin when incubated with the
injured segments (Table 1). However,
within the first 15 minutes after injury, significant Xa/Va activity
was associated with the same injured segments, as indicated by marked
elaboration of FPA in barium-adsorbed plasma containing 0.9
µmol/L human prothrombin (P<.0001; see Fig 2) and by activation of prothrombin in
the buffer system (Table 2). There were
no increases in FPA when recalcified barium-adsorbed plasma containing
prothrombin was incubated with uninjured segments. Furthermore, there
was no activation of the same preparation of prothrombin incubated with
uninjured segments in the buffer system containing calcium. These
results indicate that activation of prothrombin required incubation
with injured aortic segments and was not attributable to the presence
of active coagulation proteases in either the prothrombin preparation
or the barium-adsorbed plasma.
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Bound Thrombin Activity Over 120 Hours After Injury
Significant increases in thrombin activity associated with the
vascular wall were detected by 4 hours after aortic injury and
persisted until 24 hours (Fig 1
and Table 1). Increases in FPA were
identical in a subset of segments acquired 4 hours (n=3) after
incubation with nonrecalcified barium-adsorbed plasma (290±98 ng/mL)
compared with incubations in the same plasma in the presence of calcium
(247±66 ng/mL, P=NS). Because increases in FPA did not
require the addition of calcium, they appear to reflect activity of
thrombin bound to the vascular wall in vivo, rather than increases
attributable to de novo elaboration of thrombin during the incubation
with recalcified barium-adsorbed plasma. To further confirm that the
increases in thrombin activity were attributable to thrombin bound to
the injured vascular wall, after the initial incubation with
barium-adsorbed plasma, aortic segments acquired 4 hours after injury
were incubated with 0.1 µmol/L recombinant desulfatohirudin in
0.01 mol/L HEPES, 0.15 NaCl, 2 mmol/L CaCl2,
pH 7.4, for 30 minutes and then reincubated with barium-adsorbed
plasma. Concentrations of FPA in the incubated barium-adsorbed plasma
decreased to 23.4±2.9 ng/mL after incubation with hirudin compared
with 218.1±26.9 ng/mL before hirudin (n=4, P<.01). There
was also a modest but significant increase in concentrations of FPA
attributable to bound thrombin activity on segments acquired at 72
hours (Fig 1). By comparison with increases in FPA induced in the same
plasma incubated under similar conditions with purified human thrombin,
the activity of thrombin associated with the vessel wall was estimated
to be 0.04±0.01 nmol/L per square centimeter at 4 hours, 0.05±0.03
nmol/L per square centimeter at 8 hours, and 0.05±0.02 nmol/L per
square centimeter at 24 hours (Table 1). The concentrations of thrombin
associated with injured vessels 4 hours after injury were somewhat
higher when determined by amidolytic assay and compared with activity
of purified human thrombin (0.1±0.02 nmol/L per square centimeter; see
Table 1). Proteolysis of S-2238 in the amidolytic assay induced by
thrombin associated with the vascular wall in segments acquired 4 hours
after injury was also abolished by preincubation with hirudin.
Bound Xa/Va Activity Over 120 Hours After Injury
Activity of the Xa/Va complex associated with the same injured
aortic segments in which bound thrombin activity was characterized was
detected at 15 minutes and persisted for up to 96 hours after injury,
judging from the activation of prothrombin in recalcified
barium-adsorbed plasma or the buffered system (Fig 2 and Table 2).
Although concentrations of FPA attributable to activation of
prothrombin by bound Xa/Va were increased at 8 hours after injury, they
were not significantly higher than those induced by incubation with
uninjured segments (P=.054). Because we could not directly
quantify the concentrations of bound Xa/Va, we compared Xa/Va activity
at each time point by quantifying thrombin elaboration. During the
30-minute incubation with prothrombin in recalcified barium-adsorbed
plasma, bound Xa/Va activity induced elaboration of approximately
0.06±0.02 and 0.04±0.02 nmol/L thrombin in segments recovered at 4
and 72 hours, respectively, judging from the increases in
concentrations of FPA compared with those induced by purified thrombin
in the same plasma (Table 2). The activity of Xa/Va associated with the
vascular wall was also assayed by incubating segments acquired at 4
hours with prothrombin in the buffer system. Bound Xa/Va activity in
segments obtained at 4 hours induced activation of prothrombin in
buffer, resulting in elaboration of 0.16±0.02 nmol/L thrombin (Table 2).
To confirm that the results of these assays reflect activation of prothrombin by Xa/Va associated with the injured vascular aortic segments, recalcified barium-adsorbed plasma with added prothrombin was incubated with segments obtained 15 minutes and 4 hours after injury in the presence of the factor Xa inhibitor TAP. Concentrations of FPA attributable to Xa/Va activity were reduced in the presence of TAP from 244±25 to 18±1 ng/mL at 15 minutes, and from 300±36 to 26±7 ng/mL at 4 hours (P<.01 for both comparisons). Furthermore, when injured aortic segments were incubated with prothrombin in buffer with 0.1 µmol/L TAP, activation of prothrombin was abolished, 0.18±0.02 nmol/L thrombin elaborated in the absence of TAP compared with 0.003±0.003 nmol/L thrombin with TAP (P<.001, n=4).
Activation of Factor X by Procoagulants Bound to the Injured
Vessel Wall
To determine whether complexes (ie, IXa/VIIIa and tissue
factor/VIIa) that induce activation of factor X were associated with
vessel segments after injury, we incubated segments acquired from
rabbits 4 hours after injury (n=4) with 50 nmol/L factor X in the
buffer system. Minimal, inconsistent activation of factor X was
detected as activity against the substrate S-2222 that was too low to
permit further characterization.
Dependence of Bound Xa/Va Activity on Platelets and Inhibition
by Aspirin
To characterize the dependence of the Xa/Va activity associated
with the vascular wall on the binding of platelets, rabbits were
given intravenous aspirin 30 minutes before injury. This
dose of aspirin decreased collagen-stimulated platelet aggregation
in platelet-rich plasma obtained 20 minutes after bolus
administration by 80% to 100%. Aortas were acquired 15 minutes and 4
hours after the injury (n=6 for each condition), and bound thrombin and
Xa/Va activity were characterized as noted above. In addition,
111In-platelet deposition on the injured
segment was measured at 4 hours after injury in separate groups of
rabbits injured in the same manner with (n=5) or without (n=9)
administration of intravenous aspirin. Treatment with
aspirin markedly attenuated 111In-platelet
deposition at 4 hours compared with that in untreated animals (Fig 3). Aspirin also markedly attenuated the
increases in FPA induced in both barium-adsorbed plasma alone (Fig 4) and plasma containing prothrombin (Fig 5), consistent with a significant
reduction in thrombin and Xa/Va activity bound to the injured vessel
wall.
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Effects of Heparin on Bound Thrombin and Xa/Va Activity
Heparin catalyzes antithrombin III–mediated inhibition of both
thrombin and Xa activity. Therefore, we sought to determine whether
heparin would attenuate the activity of these procoagulants associated
with the injured vessel wall. Heparin was administered as a bolus 30
minutes before injury, followed by a constant infusion until the
rabbits were killed at 4 hours. This dose of heparin prolonged the
activated partial thromboplastin time to >150 seconds.
Thrombin activity associated with the injured vascular wall was
paradoxically increased in segments acquired 15 minutes after injury
(P<.01 compared with injured segments without heparin
treatment) and was not significantly decreased in segments acquired at
4 hours compared with those from untreated rabbits (Fig 4). Heparin
also did not significantly attenuate bound Xa/Va activity at 15
minutes, but Xa/Va activity was significantly decreased by 4 hours
compared with that in injured aortic segments from animals not given
heparin (P<.01; see Fig 5). Heparin did not significantly
attenuate 111In-platelet deposition at 4
hours (n=4; see Fig 3).
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The principal findings of this study were that Xa/Va complex activity associated with the vascular wall is detected immediately after arterial injury and that bound Xa/Va potentially promotes thrombin elaboration for up to 96 hours after injury (Fig 2
).
The activity of Xa/Va associated with the vascular wall appears to be
dependent on the deposition of platelets on the injured aorta,
judging from the marked attenuation that occurs in rabbits given
intravenous aspirin (Figs 3 and 5). We also confirmed
previous findings from our group and others indicating that thrombin
activity is associated with the subendothelium after
endothelial injury, most likely because of specific
binding to matrix glycoproteins or
fibrin.4 17 18 19 However, thrombin activity is not
immediately detectable on the injured vascular wall, and bound thrombin
activity diminishes markedly after 24 hours, although it remains
elevated to a modest extent up to 72 hours after injury (Fig 1). We
also demonstrated that Xa/Va activity detected early after injury on
the vascular wall and bound thrombin activity measured 4 hours after
injury are resistant to heparin–antithrombin III–mediated
inhibition. These results are consistent with previous data
indicating that the Xa in the Xa/Va complex on phospholipid membranes
and in association with arterial thrombi is
resistant to heparin–antithrombin III–mediated
inhibition.8 20 We were surprised to find a
modest but significant increase in thrombin activity associated with
the vessel wall early after injury when heparin was administered, but
this finding is consistent with a potentiation by heparin of
the binding of thrombin to subendothelial matrix and
the resistance of matrix- or fibrin-bound thrombin to antithrombin
III–mediated inhibition.21 22
Role of Platelet-Dependent Xa/Va Activity in Thrombin
Elaboration After Arterial Injury
The importance of platelet- and thrombin-dependent mechanisms
of thrombosis in response to arterial injury has been
extensively characterized.1 2 3 Platelet
adhesion to the arterial wall is one of the earliest events
after endothelial injury and is regulated, at least in
part, by receptor-mediated binding to collagen in the
subendothelial matrix.23 24 25 26 27 28 29 30
Activated platelet membranes are thought to be the primary
site for assembly of the Xa/Va complex because platelets bind Xa
and, when activated, express factor V on their
surface.7 In addition, several investigators have
suggested that monocytes may also play a role in supporting the
procoagulant activity of the Xa/Va complex.31 32
Inhibition of bound Xa/Va activity by aspirin in our study suggests
that platelets are the primary site for assembly of the Xa/Va
complex early after arterial injury in this preparation.
Although aspirin may have effects other than inhibition of platelet
function, the correlation we observed between decreased platelet
accumulation at the site of injury, decreased bound Xa/Va activity, and
local thrombin elaboration suggests that inhibition of platelet
adhesion accounts for these
observations.33 34
The results of this study are consistent with our previous
observations showing that local Xa/Va activity is directly related to
the platelet content of arterial
thrombi.8 35 Reverter et
al36 have also shown that the murine/human
chimeric monoclonal antibody 7E3, which inhibits both platelet
glycoprotein IIb/IIIa and the
vß3 integrin,
attenuates tissue factor–stimulated prothrombin activation. These
results are consistent with the hypothesis that the initiation
and progression of arterial thrombosis is dependent on a
dynamic balance between platelet adhesion/activation, expression of
the prothrombinase complex, elaboration of thrombin, and platelet
aggregation.37
Role of the Tissue Factor Pathway After Arterial Injury
We have previously shown that tissue factor is primarily
responsible for activating coagulation in the first 24 hours after
balloon-induced injury to the rabbit aorta.5
Tissue factor–mediated procoagulant activity was identified by
preincubation of aortic segments with a monoclonal antibody that
inhibits tissue factor. This assay would not be sufficiently sensitive
to detect less potent procoagulant activity, such as that expressed by
the Xa/Va complex, because recalcified citrated plasma clots after 15
to 20 minutes, even when it is not exposed to the injured aorta. With
the assay used in this study, Xa/Va activity associated with the vessel
wall did not induce sufficient activation of the added prothrombin to
result in clotting during the 30-minute incubation. These results
suggest that the extent of Xa/Va activity after injury in this
preparation is substantially less than that we have previously observed
associated with arterial thrombi formed in vivo; the
thrombi induce clotting in less than 15 minutes under similar
conditions.8 10 However, the importance of bound
Xa/Va activity should not be underestimated, because the thrombin
elaborated may markedly potentiate thrombosis by activating the
coagulation cofactors V and VIII and by activating
platelets.3 7
In our preparation of balloon-induced aortic injury, mural thrombus was not observed, but platelet and fibrin deposition occurred. Our previous findings document the observation that conditions which induce more extensive mural vascular injury and platelet-rich thrombosis (ie, high shear, electrical injury) cause considerably more Xa/Va activity to be associated with the thrombus, leading to marked local generation of thrombin.4 8 10 11
Persistence of Bound Xa/Va Activity After Arterial Injury
Data from previous experimental and clinical studies are
consistent with our observation that Xa/Va activity persists
for long periods of time after initial arterial injury. For
example, using assays similar to those we used, Barry et
al19 have shown that a 2-hour infusion of hirudin
attenuates arterial wall–associated thrombin activity at
24 hours but that bound thrombin activity increases by 48 hours.
Although these investigators did not characterize bound Xa/Va activity,
our findings that Xa/Va mediates prothrombin activation between 24 and
96 hours (Fig 3) likely account for the late increase in bound thrombin
activity they observed. Hatton et al17 also
observed that the injured aortic wall elaborated low levels of thrombin
for as long as 10 days, presumably secondary to bound procoagulant
complexes that induced activation of prothrombin. Recently, Hatton and
Ross-Ouellet18 reported that thrombin elaboration
by the injured vascular wall and binding of radiolabeled hirudin
decreased substantially after 24 hours and returned to baseline 7 to 10
days after injury. However, none of these studies have characterized
the specific mechanisms involved in thrombin elaboration. In our
experimental model, in which activation of prothrombin induced by the
injured arterial wall was characterized, we found that
bound Xa/Va may induce considerably more thrombin elaboration than has
previously been appreciated. This may also occur in patients with acute
coronary syndromes, judging from clinical evidence of increased
prothrombin activation for up to 6 months after ischemic events
or infarction.38
Significance of Procoagulant Activity Bound to the
Arterial Wall After Injury
The persistence of Xa/Va and thrombin activity on the vascular
wall for up to 4 days after initial arterial injury
provides a mechanism for prolonged thrombin elaboration that may play a
role in mediating vascular wall remodeling. Thrombin has been shown to
induce vascular smooth muscle cell proliferation in culture. In
experimental models, vascular smooth muscle cell proliferation is
maximal in the first 72 hours after arterial injury, which
is the interval during which we observed that thrombin was elaborated
and bound to the vascular wall. Sarembock et al39
found that even a 2-hour infusion of hirudin attenuated
neointimal hyperplasia after balloon injury of
atherosclerotic femoral lesions in the rabbit, but this did not appear
to be attributable to decreased smooth muscle cell
proliferation.40 However, because direct thrombin
inhibition induces only transient attenuation of bound-thrombin
activity,19 inhibition of the tissue factor
pathway or the Xa/Va complex may be a more effective strategy for
inhibiting neointimal hyperplasia after
arterial injury.41 42 43 44
Our findings are consistent with clinical observations of persistent prothrombin activation in patients with thrombotic complications of coronary artery disease that is not inhibited by heparin or direct thrombin inhibitors.38 45 46 47 The persistence of Xa/Va activity after arterial injury also likely contributes to the increases in thrombin activity that occur after discontinuation of thrombin inhibitors or heparin.48 This phenomenon, recently referred to as "thrombin rebound" in one study, did not occur when patients were concurrently treated with aspirin.48 It is possible that the efficacy of aspirin in attenuating or preventing rebound thrombin activity reflects attenuation of platelet-bound Xa/Va activity.
In summary, the results of this study indicate that the progression of arterial thrombosis is dependent on a dynamic balance between adhesion and activation of platelets to the injured vascular wall, tissue factor/VIIa activation of factor X, and the prolonged expression of platelet-dependent Xa/Va activity on the injured vascular wall. Our data suggest that strategies that attenuate platelet adhesion may have antithrombotic efficacy after arterial injury because they decrease thrombin elaboration. Alternatively, inhibition of the activation of factor X or factor Xa activity would appear to be an attractive means of attenuating local thrombin elaboration. Persistence of Xa/Va-mediated thrombin elaboration and binding of thrombin to the vascular wall may also play an important role in vascular wall remodeling.
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Acknowledgments |
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This work was supported in part by a grant from the National Institutes of Health (1RO1 HL54255–01A1, to Dr Eisenberg), a Grant-in-Aid from the American Heart Association (96013730, to Dr Eisenberg), and a Fellowship from the Societá Italiana di Cardiologia (Dr Ghigliotti). We thank Pam Baum for assistance with the animal preparations, Mary Jane Eichenseer and Matt Ohlendorf for assistance with coagulation assays, Elizabeth Engeszer for editorial assistance, and Ellen Visse for manuscript preparation.
Received March 27, 1997; accepted October 14, 1997.
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