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(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

Christopher M. Speidel, MD; Paul R. Eisenberg, MD, MPH; Wolfram Ruf, MD; Thomas S. Edgington, MD; Dana R. Abendschein, PhD

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.

	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

	Introduction

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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 angioplasty^{5 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.

	Methods

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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 IgG_2a 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.

	Results

Top Abstract Introduction Methods Results Discussion References

 
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.

View larger version (17K): [in this window] [in a new window]   Figure 1. Plot showing clotting times in rabbit and human plasma mixed with different concentrations of rabbit tissue factor (thromboplastin). Points reflect the average of three mixtures.

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.

View larger version (14K): [in this window] [in a new window]   Figure 2. Plot showing the generation of fibrinopeptide A in the recalcified plasma perfusate and effluent plasma perfused over a rabbit aorta obtained <1 hour after balloon-induced injury.

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).

View larger version (17K): [in this window] [in a new window]   Figure 3. Bar graph showing the generation of fibrinopeptide A (FPA) in recalcified plasma effluent after 10 minutes of perfusion over noninjured (control) rabbit aortas and aortas obtained at intervals after balloon-induced injury. Results are expressed as percentages of FPA in 10-minute perfusate samples. *P<.005, **P<.000001 vs control aortas.

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

View larger version (26K): [in this window] [in a new window]   Figure 4. Characterization of the monoclonal antibody against rabbit tissue factor (TF, RbTF7-3A5). A, Western blot showing the reactivity of RbTF7-3A5 antibody or affinity-purified anti-human tissue factor polyclonal antibody with 2.5x104 cells transfected with rabbit tissue factor cDNA (left, rabbit cells positive for tissue factor) or 100 µg of rabbit brain extract (right) after electrophoretic separation and transfer. The antibodies reacted with a 49-kD protein from cells and a 45-kD protein and a lower-molecular-weight degradation fragment from brain. Slower mobility of the protein from cells probably reflects heterogeneity attributable to glycosylation, as previously shown for expression of human tissue factor in Chinese hamster ovary cells.45 B, Plot showing the functional inhibition of rabbit tissue factor or phospholipid-reconstituted human tissue factor by increasing concentrations of RbTF7-3A5 antibody as determined by a one-stage clotting assay with human plasma.

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).

View this table: [in this window] [in a new window]   Table 1. Inhibition of FPA Generation in the Plasma Effluent After Preincubation of Vessel Segments Obtained 8 Hours After Balloon-Induced Injury With Increasing Concentrations of Tissue Factor Antibody

View larger version (24K): [in this window] [in a new window]   Figure 5. Bar graph showing the generation of fibrinopeptide A in the effluent after 10 minutes from noninjured (control) rabbit aortas and aortas obtained at intervals after balloon-induced injury and either preincubated with an antibody to rabbit tissue factor (TF) followed by perfusion with recalcified plasma or perfused with barium-adsorbed plasma. Results with recalcified whole plasma perfused through other vessel segments (solid bars) are from Fig 3 and shown for comparison. Bars represent the mean±SD for three to seven experiments. *P<.005, **P<.00001 vs segments perfused with whole plasma.

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.

View larger version (29K): [in this window] [in a new window]   Figure 6. Bar graph showing the generation of prothrombin fragment 1.2 in plasma effluent after 10 minutes of perfusion over control aortas and aortas obtained <1 and 8 hours after balloon-induced injury both with and without preincubation with an antibody to tissue factor (TF). *P<.001 vs segments not exposed to antibody.

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

View larger version (190K): [in this window] [in a new window]   Figure 7. Representative microphotographs of an aortic cross section from a rabbit with balloon injury induced 16 hours previously and stained with Van Gieson stain for elastic tissue. Higher magnification (B) of the vessel segment within brackets (A) reveals multiple disruptions of the internal elastic lamina (arrows), consistent with deep vascular injury.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
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 injury^{33 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.

	Acknowledgments

 
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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