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(Circulation. 1997;95:594-599.)
© 1997 American Heart Association, Inc.

Articles

Tissue Factor Modulates the Thrombogenicity of Human Atherosclerotic Plaques

Vincenzo Toschi, MD; Richard Gallo, MD; Maddalena Lettino, MD; John T. Fallon, MD, PhD; S. David Gertz, MD, PhD; Antonio Fernandez-Ortiz, MD, PhD; James H. Chesebro, MD; Lina Badimon, PhD; Yale Nemerson, MD, PhD; Valentin Fuster, MD, PhD; Juan J. Badimon, PhD

the Cardiovascular Biology Research Laboratory, The Cardiovascular Institute, Mount Sinai School of Medicine, New York, NY; Department of Anatomy and Embryology (S.D.G.), The Hebrew University–Hadassah Medical, Jerusalem, Israel; Servicio de Cardiologia (A.F.-O.), Hospital San Carlos Madrid, Spain; and Cardiovascular Research Center (L.B.), CSIC-Hospital Santa Cruz y San Pablo–UAB, Barcelona, Spain.

Correspondence to Juan J. Badimon, PhD, Cardiovascular Biology Research Laboratory, The Cardiovascular Institute (Box 1030), Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, NY 10029.

	Abstract

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Background The thrombogenicity of a disrupted atherosclerotic lesion is dependent on the nature and extent of the plaque components exposed to flowing blood together with local rheology and a variety of systemic factors. We previously reported on the different thrombogenicity of the various types of human atherosclerotic lesions when exposed to flowing blood in a well-characterized perfusion system. This study examines the role of tissue factor in the thrombogenicity of different types of atherosclerotic plaques and their components.

Methods and Results Fifty human arterial segments (5 foam cell–rich, 9 collagen-rich, and 10 lipid-rich atherosclerotic lesions and 26 normal, nonatherosclerotic segments) were exposed to heparinized blood at high shear rate conditions in the Badimon perfusion chamber. The thrombogenicity of the arterial specimens was assessed by ¹¹¹In-labeled platelets. After perfusion, specimens were stained for tissue factor by use of an in situ binding assay for factor VIIa. Tissue factor in specimens was semiquantitatively assessed on a scale of 0 to 3. Platelet deposition on the lipid-rich atheromatous core was significantly higher than on all other substrates (P=.0002). The lipid-rich core also exhibited the most intense tissue factor staining (3±0.1 arbitrary units) compared with other arterial components. Comparison of all specimens showed a positive correlation between quantitative platelet deposition and tissue factor staining score (r=.35, P<.01).

Conclusions Our results show that tissue factor is present in lipid-rich human atherosclerotic plaques and suggest that it is an important determinant of the thrombogenicity of human atherosclerotic lesions after spontaneous or mechanical plaque disruption.

Key Words: thrombosis • platelets • tissue factor • atherosclerosis • plaque

	Introduction

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Thrombus formation on a disrupted atherosclerotic lesion plays a fundamental role in the development of acute coronary syndromes and the progression of atherosclerosis.^{1 2} Angiography, angioscopy, and necropsy studies have shown thrombi on ulcerated or fissured atherosclerotic plaques in patients with myocardial infarction, unstable angina, and sudden cardiac death.^{3 4 5 6 7 8} The thrombogenicity of atherosclerotic lesions is determined by the nature and extent of the plaque component exposed to flowing blood together with local rheological and systemic blood factors. We⁹ have recently demonstrated that platelet deposition and thrombus formation on the lipid-rich atheromatous core exposed to flowing blood is up to sixfold greater than that on other substrates, including collagen-rich matrix.

TF is a small-molecular-weight glycoprotein that initiates the extrinsic clotting cascade and is considered a major regulator of coagulation, hemostasis, and thrombosis. TF forms a high-affinity complex with coagulation factors VII and VIIa; TF-VIIa complex activates factors IX and X, which in turn leads to thrombin generation.^{10 11}

TF antigen has been demonstrated in endarterectomy specimens from patients with carotid atherosclerosis¹² and, more recently, in some atherectomy specimens from culprit lesions in patients with unstable coronary syndromes.¹³ This suggests a potential role for this protein in the thrombotic complication of atherosclerosis after plaque disruption. Our group has developed a novel approach for the in situ identification of TF by using Dig-FVIIa.¹⁴

The present study correlates platelet deposition on human atherosclerotic plaques and normal vessel components exposed to flowing blood with the content of TF on the surface of the exposed arterial substrate. The aim of this study was to clarify the causative role of TF in the thrombogenicity of disrupted atherosclerotic plaques.

	Methods

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Preparation of Human Arterial Specimens and Classification
Different types of human atherosclerotic plaques and normal arterial segments were obtained from human aortas at autopsy within 24 hours of death. Lesions were selected according to the following macroscopic criteria: (1) fatty streaks, characterized by slightly raised yellow dots or streaks; (2) fibrous plaques, characterized by raised, pearly white lesions without a soft core; and (3) atheromatous lipid-rich core, characterized by raised white or yellow-white plaques containing a soft yellow core of toothpastelike consistency, separated from the lumen by a fibrous cap. Macroscopically normal aortic segments were also selected. Specimens were cut into segments (2.8x0.8 cm) and kept frozen at -70°C.

To mimic the in vivo situation of plaque disruption and to directly expose the internal components of the atherosclerotic plaque, the superficial layers of the plaque were removed as previously described.⁹ In all instances, the exposed substrates were part of the intimal layer of the diseased vessel (above the internal elastic lamina). Special care was taken to avoid gross irregularities on the surface. Normal tunica media was prepared by peeling off the intimal layer with a thin portion of subjacent media starting from a corner of the aortic segment, as previously described.⁹

Perfusion Chamber
The acrylic perfusion chamber used in the present study has been described elsewhere.^{15 16} The arterial segment to be tested was placed in the chamber with a portion of the wall (25 mm in length) replacing a part of the tubular flow channel (1 mm in diameter), permitting the surface of each specimen to be directly exposed to the flowing blood. Human atherosclerotic plaques were exposed to flowing blood at a flow rate of 10 mL/min for 5-minute perfusion periods. This experimental flow condition in this laminar perfusion system gives a theoretically calculated local shear rate of 1690/s (Reynolds number, 60; average blood velocity, 21.2 cm/s) and corresponds to the local rheological conditions that develop in a mild to moderately stenotic coronary artery. Our previous work¹⁵ demonstrated that these rheological conditions result in consistent levels of platelet deposition.

Experimental Procedure
Yorkshire albino pigs (body weight, 30±2 kg) were used as blood donors. All procedures were performed in accordance with the Mount Sinai School of Medicine and American Heart Association guidelines on research animal use. Pigs were initially sedated with ketamine (20 mg/kg body weight) followed by sodium pentobarbital (25 mg/kg IV). Anesthesia was maintained by infusion of pentobarbital as needed. An ex vivo extracorporeal perfusion system was set up as previously described.^{15 16} In brief, the catheterized carotid artery was connected by polyethylene tubing to the input of the acrylic chamber. The output of the chamber was connected to a peristaltic pump with interchangeable heads calibrated to maintain the selected blood flow (Masterflex model 7013). Blood that passed through the chamber was recirculated back into the animal by the external jugular vein. After carotid cannulation, baseline blood samples were taken for hematocrit, red blood cells, platelet count, fibrinogen levels, and aPTT measurement. The animals then received standard heparin (50 IU/kg bolus followed by continuous infusion of 50 IU/kg per hour). This regimen of administration achieved an aPTT ratio of 1.5±0.03x the baseline values (mean±SE). The specimens were mounted in the chamber and initially perfused with PBS (0.01 mol/L, pH 7.4, 37°C) for 60 seconds. Blood was subsequently perfused through the chamber at 10 mL/min for 5 minutes. At the end of the blood perfusions, PBS was again passed through the chamber for 30 seconds to wash away unattached cells and plasma proteins. All the perfusions were performed at 37°C by placing the chamber in a water bath. Hematocrit, platelet count, and fibrinogen levels were constant throughout each perfusion and were similar in all perfusions.

Radioactive Labeling of Platelets
Autologous platelets were labeled with ¹¹¹In-tropolone in plasma and reinjected 16 to 24 hours before the perfusion experiments, as previously described.^{15 16 17} Labeling efficiency was 65±4%. The mean injected activity was 254.3±19.6 µCi with 2.8x10⁶±0.4x10⁶ reinjected indium-labeled platelets/µL in a total of 4 mL autologous platelet-poor plasma.

Measurement of Platelet Deposition
At the end of each perfusion, the arterial segments were removed and fixed overnight in 4% paraformaldehyde in 0.1 mol/L PBS, pH 7.4. Segments were individually counted in a -well counter (Packard Auto-Gamma 5650). The number of platelets deposited on each specimen was calculated by blood activity (counts) and platelet counts in blood and normalized to surface area. Values are expressed as number of platelets/cm².^{15 16 17}

Histological Evaluation
All specimens were routinely processed for paraffin embedding, with sectioning performed parallel to the direction of the flow. Exposed segments were step sectioned every 100 µm, and 5-µm sections were placed onto lysine-coated slides. Sections were deparaffinized, rehydrated, and stained with hematoxylin-eosin and trichrome techniques. Histological evaluation verified the initial macroscopic classification of lesion type.⁹ The gross classification of the specimens performed before blood perfusion was later confirmed by microscopic examination of the specimens.

In Situ Dig-FVIIa Binding
The presence of TF in the perfused human atherosclerotic and normal arterial segments was assessed by a newly developed in situ binding assay based on the high affinity between TF and factor VIIa. The assay involves the use of Dig-FVIIa.¹⁴ Positive digoxigenin staining indicates the existence of TF:FVIIa complex, the catalytic complex for activation of factor X. In brief, rehydrated sections were placed in TBS containing 5 mmol/L Ca²⁺ (pH 7.5). Sections were then incubated with 50 nmol/L Dig-FVIIa in TBS containing 5 mmol/L Ca²⁺ at 37°C for 2 hours. Sections were washed with TBS and fixed with 4% paraformaldehyde in TBS for 5 minutes. Sections were rinsed in TBS and incubated with a 1:1000 dilution of sheep Fab anti-digoxigenin conjugated with horseradish peroxidase at 37°C for 1 hour. Sections were again washed with TBS and reacted with 3,3'-diaminobenzidine (digoxigenin-3-0-methylcarbonyl--aminocaproic; Biogenex) for 10 minutes. Slides were washed in distilled water and dehydrated, coverslips were put on, and the slides were observed microscopically.

Dig-FVIIa staining controls were performed on selected specimens. Negative controls included replacement of Dig-FVIIa with unlabeled factor VIIa, staining with Dig-FVIIa in the presence of a 10-fold excess of unlabeled factor VIIa, and preincubation of sections with a polyclonal antibody to TF before Dig-FVIIa staining (see Fig 1).

View larger version (130K): [in this window] [in a new window]   Figure 1. Photomicrographs of Dig-FVIIa–staining controls. To validate the staining, the following controls were performed on sequential sections of the same specimen. A, Dig-FVIIa staining; B, specimen incubated with FVIIa alone (negative control); C, Dig-FVIIa incubated in the presence of polyclonal anti-TF antibody; and D, Dig FVIIa staining in the presence of 10-fold concentrations of unlabeled FVIIa. These photomicrographs demonstrate the specificity of the in situ staining method because the staining for Dig-FVIIa is absent when replaced by factor VIIa and markedly reduced by either an excess of unlabeled factor VIIa during incubation or preincubation of sections with anti-TF antibody.

The TF content of each specimen beneath the thrombus was semiquantified by assigning an AU score of 0 (no staining) through 3 (the most staining) to each of the Dig-FVIIa–stained sections without knowledge of the quantitative platelet deposition data. The TF score for each specimen was determined by three different investigators, and their scores were averaged.

Hematologic Parameters
Number of blood cells, hematocrit, platelet number, and platelet size distribution were determined with the use of a system 9018 Serono cell analyzer equipped with a veterinarian software program to allow blood cell counting of different animal species. Monitoring of the aPTTs and plasma fibrinogen levels was determined with the use of an ST4 automated clotter and the corresponding specific kits (America Diagnostica) according to the manufacturer's instructions.

Statistical Analysis
All statistical analyses were performed with the use of Statview II software (Abacus Concept, Inc) on a Macintosh 7100/66AV microcomputer. Results are expressed as mean±SE unless otherwise stated. A value of P<.05 was considered significant. Between-group analyses were performed by one-factor ANOVA followed by Fisher's protected least-squares difference and Scheffe's F test to assess specific group differences.

	Results

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Histological Analysis of Atherosclerotic Lesions
A total of 50 human arterial segments were analyzed. Histological evaluation confirmed the initial macroscopic classification of the lesions. Twenty-four were atherosclerotic plaques: 5 foam cell–rich, 9 collagen-rich, and 10 atheromatous lipid-rich core lesions. The remaining 26 arterial segments were normal (nonatherosclerotic) arterial components: 15 intimal, 5 medial, and 6 adventitial layers.

Platelet Thrombus Formation on the Different Types of Human Atherosclerotic Plaques
The results of thrombus formation on the different substrates exposed to flowing blood are shown in Fig 2. Thrombus formation on atheromatous core was significantly higher (712±213x10⁶ platelets/cm²) than on all other substrates (adventitia, 203±58 platelets/cm²; tunica media, 134±41; foam cell–rich matrix, 97±46; collagen matrix, 96±16; and normal intima, 79±16x10⁶; P=.0002). Of the normal, nonatherosclerotic components of the vessel wall, adventitia had the highest platelet deposition, subendothelium had the lowest, and tunica media had an intermediate thrombogenicity. These observations suggest a direct relationship between the severity of injury and acute thrombotic response in nonatherosclerotic arterial substrates.

View larger version (22K): [in this window] [in a new window]   Figure 2. Platelet deposition and TF activity score. Platelet deposition data are expressed as mean±SE; TF staining intensity is expressed as the average of scores assigned by the independent observers. Note the positive correlation between platelet thrombus formation and TF score on the exposed human substrates (P<.01). INT indicates normal intima; Coll, collagen-rich matrix; Foam, foam cell–rich matrix; TM, normal tunica media; ADV, adventitia; and LRC, lipid-rich core. *P=.0002 by ANOVA.

Morphological and Dig-FVIIa Binding on the Different Types of Human Atherosclerotic Plaques
Representative sections stained for Dig-FVIIa binding and the corresponding trichrome-stained sections of human atherosclerotic lesions are shown in Fig 3. Among all the studied atherosclerotic lesions, those containing a lipid-rich core exhibited the most intense TF staining (3±0.1 AU) compared with both normal and arteriosclerotic segments (adventitia, 2±0.1 AU; foam cell–rich matrix, 2±0.01; tunica media, 1±0.3; collagen matrix, 1±0.001; and normal intima, 1±0.1). Adventitia had the highest score for intensity of TF staining of all the normal, nonatherosclerotic specimens. In addition, when all available specimens were analyzed microscopically, the amount of Dig-FVIIa staining increased with the progression of atherosclerotic lesions from foam cell rich with predominantly intracellular lipids to the more advanced lesions characterized by abundant cholesterol crystals (Fig 3). Finally, statistical analysis of all specimens showed a positive correlation between the number of platelets deposited and TF score (P<.01) (Fig 2).

View larger version (270K): [in this window] [in a new window]   Figure 3. Representative low-power photomicrographs of three different human aortic segments exposed to flowing blood. A, C, and E show connective tissue stains of normal aorta (A), aorta with fibrous atherosclerotic plaque (C), and aorta with lipid-rich atherosclerotic lesion (E). B, D, and F show adjacent sections of the same specimens stained for TF (brown) by use of Dig-FVIIa. The most intense staining is present in the lipid-rich core, where extracellular staining surrounds and highlights the cholesterol crystals (F). Positive adventitial staining is seen in B and D. Because we used the same magnification in each of the photomicrographs, aortic adventitia is not visible in E and F.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Arterial injury activates the intrinsic pathway of blood coagulation to maintain the integrity of the vascular system. In addition, the extrinsic pathway of the coagulation is also initiated by exposure of flowing blood to TF, the cellular "receptor" and cofactor for factor VII, leading, through thrombin generation and fibrin deposition, to thrombus formation.

The present study provides evidence of TF involvement in platelet deposition and thrombus formation on disrupted human atherosclerotic lesions exposed to flowing blood in a well-controlled perfusion system. Under the same experimental conditions, we previously demonstrated that the lipid-rich atheromatous core from human atherosclerotic plaques is the most thrombogenic substrate, and platelet deposition on this substrate is up to sixfold higher than on other types of lesions, including adventitia and collagen-rich matrix.⁹ This observation has important pathophysiological and clinical implications. Histopathologic data show that the so-called "vulnerable plaques," the most prone to rupture, are characterized by an eccentric, lipid-rich core usually separated from flowing blood by a thin fibrous cap. Disrupted atherosclerotic lesions are often associated with thrombus formation on their luminal surface. This type of lesion is responsible for triggering acute coronary syndromes.^{3 4 5 6 18 19} By contrast, collagen-rich fibrotic plaques are more stable, and resultant thrombotic complications are often the result of a decreased blood flow due to the stenosis of the vessel.^{1 2 20 21}

In the present study, the inner components of human atherosclerotic plaque were artificially exposed to flowing blood to mimic spontaneous plaque disruption. We found that the lipid-rich core is a more potent stimulus for thrombus formation than fibrous, collagen-rich lesions. This does not lessen the importance of the collagen matrix, which, after plaque disruption, may also activate platelet deposition and the coagulation cascade.^{22 23}

In contrast to our data, van Zanten et al,²⁴ using arterial cross sections mounted in a rectangular perfusion chamber, reported strong platelet deposition on collagen-rich connective tissue and adventitia with little reactivity to the lipid core of the plaque. The lack of platelet deposition on lipid-rich atherosclerotic plaques observed by those investigators may relate to their different experimental conditions. One possible reason for the discrepancies between the latter and our results and those clinically observed may be the anticoagulation (citrate or low-molecular-weight heparin with or without hirudin) and sample manipulation used by van Zanten et al, which may induce differences in thrombin generation and/or function in their perfusion system. In fact, they found that platelet deposition was significantly reduced by antibodies to von Willebrand factor but was not affected by RGDW peptides. We²⁵ recently demonstrated that local thrombin generation plays an important role in platelet deposition and thrombus growth on areas of severe vessel wall damage. We found that hirudin added to heparinized blood reduced platelet deposition to the severely injured wall but not to collagen-coated slides. In addition, hirudin added to citrated blood did not further decrease platelet deposition, which suggests that local thrombin generation plays a role in platelet deposition in heparinized conditions and that thrombin activity is completely blocked in citrated blood by chelating calcium. Furthermore, platelet deposition on collagen fibrils is not mediated by thrombin,²⁴ which suggests other biochemical mechanisms for platelet interaction with this substrate.

In the present study, we also observed that the components of the nonatherosclerotic arterial wall were significantly less thrombogenic than the lipid-rich core of atherosclerotic plaque. Among these normal structures, adventitia appeared to be the most thrombogenic and intima the least, and tunica media showed an intermediate degree of thrombogenicity. These findings agree with previous data from animal models^{26 27} that indicate that the degree of thrombus formation and its persistence after balloon angioplasty depend on the depth of the vascular injury induced by the procedure, which suggests that the thrombogenicity of deeper vascular structures plays an important role in acute reocclusion and late restenosis after coronary interventions.

The exact mechanism(s) responsible for the thrombogenic properties of the various atherosclerotic plaque components, and particularly of the lipid-rich core, is still uncertain. Lipids alone (crystalline structures, phospholipids, or soft lipids) or cellular degradation products in the core were proposed as possible activators of the hemostatic system.²⁸ Among cellular-derived products, TF has been proposed as a major candidate.¹⁰ Previous studies^{29 30} showed that thrombogenicity in a flow system of cultured fibroblasts, smooth muscle cells, and their elaborated matrices is related to TF activity and that the prothrombotic properties of these cells can be blocked by antibodies against TF. Expression of TF antigen by vascular cells has also been identified by immunohistochemistry in both normal and atherosclerotic human vessels,^{12 13 31 32} especially in the necrotic core surrounding cholesterol clefts in atherosclerotic plaques from human carotid endarterectomy specimens.¹² However, no evidence has been presented of the ability of the TF protein to trigger coagulation. By the in situ assay used in this study, we demonstrated that Dig-FVIIa binding sites are highly expressed in the relatively acellular lipid core of atherosclerotic lesions, and a strong correlation exists between Dig-FVIIa staining and platelet deposition on both normal and diseased components of human plaque exposed to flowing blood. Thus, we provide evidence that the increased thrombogenicity of atheromatous lesions, disrupted spontaneously or by angioplasty, may be mediated by TF.

The origin of the TF found in the lipid-rich core of atherosclerotic lesions is poorly understood. It has been suggested that monocyte/macrophage–type cells such as foam cells play a crucial role in the early formation of atherosclerotic plaque and its evolution into a lesion prone to disruption.^{33 34 35 36} The expression of TF by monocytes and macrophage-derived foam cells has been recently demonstrated in atherosclerotic lesions,¹² and TF-mediated procoagulant activity was shown in adherent human peripheral blood monocytes under flow conditions.³⁷ Moreover, the expression of functional TF by human monocytes is stimulated by oxidized low-density lipoproteins present in atheromatous plaque.³⁸ These observations suggest that TF found in the lipid-rich core of atherosclerotic lesions is largely derived from macrophages present in the plaque.³⁹ However, we cannot rule out the possible contribution of smooth muscle cells and even endothelial cells.

Finally, our work has important therapeutic implications. Inhibition of the TF pathway of coagulation by specific anti-TF antibodies,^{40 41} factor VIIa inhibitors,⁴² or the recombinant form of its physiological inhibitor, TF pathway inhibitor, was shown to be effective for prevention of arterial thrombosis in several animal models.^{43 44} Such approaches may offer promising new tools for preventing reocclusion after successful thrombolysis or thrombotic occlusion in patients with unstable angina and after percutaneous coronary angioplasty.

	Selected Abbreviations and Acronyms

 

aPTT = activated partial thromboplastin time AU = arbitrary units Dig-FVIIa = digoxigenin-labeled recombinant human factor VIIa TBS = Tris buffer TF = tissue factor

	Acknowledgments

 
We gratefully acknowledge Dr Adrian Padurean for his technical expertise in the preparation of the animal models and Veronica Gulle for her skillful help in the processing and staining of the specimens.

	Footnotes

 
Dr Toschi's current address is Dipartimento di Ematologia e Trafusionale, Ospedale S. Carlo Borromeo, Via Pio II, 320153 Milano, Italy. Dr Lettino's current address is Primario Divisione di Cardiologia e UCIC, Ospedale Maggiore Policlinico, Via F. Sforza, 35, 20100 Milano, Italy.

Received May 15, 1996; revision received September 9, 1996; accepted September 12, 1996.

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