Differential Expression of Tissue Factor Protein in Directional Atherectomy Specimens From Patients With Stable and Unstable Coronary Syndromes
Background Tissue factor (TF) is a cell membrane–associated protein that catalyzes the rate-limiting step of the extrinsic coagulation pathway, which is the major source of thrombin production in vivo. To explore the potential role that TF may play in ischemic coronary syndromes, directional coronary atherectomy specimens were tested for the presence of TF protein using immunohistochemical techniques.
Methods and Results Frozen sections from atherectomy specimens in 61 patients were examined for TF expression using an IgG murine monoclonal antibody against human TF. Patients were classified according to their admission diagnosis as having either an unstable or a stable coronary syndrome. An unstable coronary syndrome was defined as either angina pectoris occurring at rest or post–myocardial infarction (<1 week) angina. Stable coronary syndromes included patients with stable, progressive, and new-onset (<6 weeks) angina without rest pain. TF was detected in 15 (43%) of 35 patients with unstable coronary syndromes versus only 3 (12%) of 26 patients with stable coronary syndromes (odds ratio, 5.7; 95% confidence interval, 1.3 to 24.3; P=.018). Within the subgroup of patients with unstable coronary syndromes, TF was detected in 14 (60%) of 25 patients with de novo lesions versus only 1 (10%) of 10 patients with a restenosis lesion (P<.02). An additional 8 patients with stable coronary syndromes due to a restenosis lesion were also negative for TF. Therefore, the overall incidence of TF expression was only 6% (1 of 18) in restenosis lesions compared with 33% (14 of 43) in de novo lesions (P<.03).
Conclusions This study provides the first description of TF protein expression in human coronary artery lesions in vivo. Tissue factor was readily detected in de novo lesions in patients with unstable coronary syndromes, suggesting a role for TF in the pathogenesis of this disease process. Conversely, TF was rarely detected in patients with restenosis lesions even if the resulting clinical presentation was an unstable coronary syndrome. These results may have implications for the management of patients with unstable angina from de novo lesions and patients with ischemic symptoms from a restenosis lesion.
Tissue factor (TF) is a cell membrane–associated glycoprotein that serves as the primary initiator of the extrinsic coagulation cascade.1 TF binds to and acts as an essential cofactor for plasma protease factor VII, and the resulting complex activates factors IX and X, which leads to the major source of thrombin production in vivo.2 Therefore, TF is a critical protein in the activation of the coagulation cascade and may play an important role in the pathogenesis of ischemic coronary syndromes by mediating intravascular thrombosis.
Immunohistochemical analysis and in situ hybridization studies have demonstrated high levels of TF protein and mRNA in the adventitia of normal human blood vessels such as internal mammary arteries, aorta, and saphenous veins.3 4 Although TF protein is variably present in the media of some normal arteries, TF protein and mRNA are consistently undetectable in the intima and endothelium.3 4 Wilcox et al4 found that atherosclerotic plaques from human carotid endarterectomy specimens demonstrated islands of TF antigen staining surrounding cholesterol clefts and necrotic cores, while some foam cells, monocytes, and mesenchymal-like intimal cells in these plaques were positive for TF mRNA. Likewise, studies in several different cell types have shown that TF expression can be induced and upregulated by agents or physiological mediators that may play a role in atherosclerosis and arterial injury.5 6 7
At present, the frequency and the pattern of expression of TF in diseased human coronary arteries are unknown, and this information is necessary to begin to understand the role that TF may play in different coronary syndromes. The purpose of this study was therefore to determine the presence of TF expression in directional atherectomy specimens from patients undergoing revascularization for symptomatic coronary artery disease.
The study included 61 patients who underwent directional coronary atherectomy at Duke University Medical Center between July 1991 and June 1992 or at Duke University and the Durham VA Medical Center between October 1993 and June 1994 from whom adequate tissue was available. In 50 patients, the atherectomy was performed as the initial percutaneous revascularization strategy and in 11 patients, it was performed as a “salvage” procedure immediately after an unsuccessful angioplasty (n=8) or threatened vessel closure (n=3). Patients with total coronary occlusions or unequivocal angiographic coronary thrombus before the intervention were not included in this study. Investigations were conducted under protocols approved by the institutional review boards of both institutions.
Pertinent information from patient histories and procedural outcomes was extracted from the Duke Databank for Cardiovascular Disease after the tissue analysis was complete. Patients were classified according to their admission diagnosis as having either an unstable or a stable coronary syndrome. An unstable coronary syndrome was defined as either angina pectoris occurring at rest or post–myocardial infarction (<1 week) angina. Stable coronary syndromes included patients with stable, progressive, and new-onset (<6 weeks) angina without rest pain. A restenosis lesion was defined when an atherectomy was performed (>1 week, <6 months) after an interventional procedure at the same site.
Atherectomy specimens were immediately removed from the cutter housing and immersed in ice-cold 4% paraformaldehyde (Fisher Scientific Co). After 2 to 4 hours, the samples were transferred to 30% sucrose–phosphate-buffered saline solution (PBS), embedded in OCT compound (Miles Scientific Division), snap-frozen in liquid nitrogen, and stored at −70°C. The samples were cryosectioned (6 μm) onto silane-coated microscope slides, which were quickly placed in cold acetone for 2 minutes and stored at −70°C. Samples of human skin were handled in a similar manner.
Immunohistochemistry was performed using a murine monoclonal antibody against human tissue factor (TF9-9C3), as previously described.3 Briefly, slides were thawed and dehydrated in PBS. Blocking solution (10% horse serum) was applied for 30 minutes at room temperature. The TF antibody was diluted to a concentration of 0.1 μg/mL in 10% horse serum and applied for 60 minutes at 37°C in a humidified chamber. This was followed sequentially by incubation with biotinylated anti-mouse IgG and ABC reagent according to manufacturer’s specifications (Vectastain ABC kit, Vector Laboratories, Inc). Levamisole was added to block endogenous alkaline phosphatase activity, and immune complexes were localized using the chromogenic alkaline phosphatase substrate Vector Red (Vector Laboratories, Inc). The sections were counterstained with hematoxylin, dehydrated, and mounted with Permount (Fisher Scientific). Samples were tested in duplicate on separate days. In all experiments, a sample of human skin was included as a positive control, and the adjacent section was handled in parallel with a nonsense murine IgG monoclonal antibody as a negative control. Slides were reviewed independently by two different observers (B.H.A., S.M.D.) without knowledge of the clinical status. The results were expressed dichotomously as positive or negative. Histological thrombus was identified by hematoxylin-eosin stains, as previously described, and verified by trichrome stains.8 For statistical analysis, the Fisher’s exact or χ2 test was used to compare the frequency of expression of TF in the patient populations.
The patients’ demographic data are summarized in the Table⇓. The study included 35 patients who presented with unstable coronary syndromes and 26 patients with stable coronary syndromes. The group with stable coronary syndromes included new-onset angina (n=2), progressive angina (n=17), and stable angina (n=7). The frequency of restenosis lesions was similar in both groups. The mean time from the prior angioplasty for the entire restenosis group was 2.4±0.3 months (mean±SEM).
Two representative lesions that demonstrated TF staining are shown in Fig 1⇓. Overall, TF was detected in 15 (43%) of 35 patients with unstable coronary syndromes versus only 3 (12%) of 26 patients with stable coronary syndromes (odds ratio, 5.7; 95% confidence interval, 1.3 to 24.3; P=.018) (Fig 2⇓, left). Within the subgroup of patients with unstable coronary syndromes, TF was detected in 14 (60%) of 25 patients with a de novo lesion versus only 1 (10%) of 10 patients with a restenosis lesion (P<.02) (Fig 2⇓, middle). An additional 8 patients with stable coronary syndromes due to a restenosis lesion were also negative for TF, for an overall incidence of only 6% (1 of 18) in restenosis lesions compared with 33% (14 of 43) in de novo lesions (P<.03) (Fig 2⇓, right).
Clinical and histological features that may have been related to TF expression were examined. This study included some patients in whom atherectomy was performed as a “salvage” procedure after balloon angioplasty or as a procedure to treat a vein graft stenosis (Table⇑). The 11 salvage lesions included 6 patients with unstable coronary syndromes and 5 with stable coronary syndromes. The incidence of TF expression was 3 (27%) of 11, with two of the three positives found in patients with an unstable coronary syndrome. The 7 vein graft lesions included 5 patients with unstable and 2 with stable coronary syndromes. The incidence of TF expression was 3 (43%) of 7, with two of the three positives found in patients with unstable coronary syndromes. These small groups appeared to be representative of the larger study population and were included in the analysis. The time interval from the onset of symptoms until atherectomy, for the unstable coronary syndrome group, was similar for the TF-positive (5.3±1.0; range, 1 to 12 days) and TF-negative patients (4.9±0.9; range, 1 to 10 days). Also, the frequency of histological thrombus was similar in the TF-positive and TF-negative patients, 33% versus 37%, respectively.
Four patients had an abrupt closure after the atherectomy procedure. One occurred in the catheterization laboratory; one occurred 12 hours after and two occurred 48 hours after the procedure. All lesions were de novo native coronary arteries and were TF positive. Two patients had stable and two had unstable coronary syndromes. Therefore, 4 (22%) of the 18 patients with immunohistochemical staining for TF had an abrupt closure. Although the number of events was small, the association of TF expression with abrupt closure was highly significant (P≤.006).
TF is the primary initiator of intravascular thrombosis and thrombin production in vivo.1 2 However, the precise role that TF plays in the pathogenesis of ischemic coronary syndromes is unknown. This study provides the first analysis of TF protein expression in human coronary artery lesions in vivo. The major findings of this study are that TF is frequently present in de novo lesions in patients with unstable coronary syndromes and that TF is rarely detected in restenosis lesions regardless of the patient’s clinical presentation.
The frequent expression of TF in de novo lesions in patients with unstable coronary syndromes suggests that TF plays a central role in the pathogenesis of this disease process. These findings may be particularly important, with the emergence of several new potent antithrombotic agents.9 Our results suggest that patients with unstable coronary syndromes from de novo lesions may particularly benefit from the use of direct thrombin or TF pathway inhibitors. Conversely, although the absence of TF expression in restenosis lesions does not rule out TF as a potential cause of angioplasty restenosis, our results suggest that restenosis lesions have a low thrombotic potential regardless of the patient’s clinical presentation. These findings may have implications for the potential use of adjunctive pharmacological therapies or interventional treatment strategies to treat restenosis lesions.
The results of this study also may provide evidence that mechanisms other than thrombus formation can be involved in the pathogenesis of unstable coronary syndromes. Angioscopic, angiographic, and pathological studies in patients with unstable angina have consistently identified a significant (20% to 50%) fraction of patients who lack evidence of plaque rupture or thrombus formation.10 11 12 A similar percentage of the patients in our study with unstable coronary syndromes and de novo lesions had no immunohistochemical TF protein expression. Flugelman et al13 studied directional coronary atherectomy specimens from de novo lesions in patients with unstable angina compared with a group with ischemic coronary symptoms from angioplasty restenosis lesions and concluded that smooth muscle cell proliferation may lead to plaque expansion and luminal narrowing, resulting in the clinical syndrome of unstable angina pectoris. Indeed, a variety of different mediators may be involved in the pathogenesis of unstable coronary syndromes.14 TF expression, however, may serve as a useful marker to differentiate mechanisms for unstable coronary syndromes.
Although the number of events in our study was small, all 4 patients who suffered an abrupt closure after directional atherectomy were TF positive. Likewise, animal models have suggested that TF may play a role in angioplasty complications. Pawashe et al15 found that antibodies to TF inhibited arterial thrombosis in a rabbit balloon injury model. Marmur et al7 demonstrated that balloon injury in a rat aorta induced a 10-fold increase in TF coagulant activity and an upregulation of mRNA in less than 2 hours. In our study, TF expression was similar in the primary atherectomy and “salvage” atherectomy groups. However, the time from initial balloon inflation to tissue removal, which was estimated to be 90 minutes from review of the catheterization report, may have been too early to detect an increase in the immunohistochemical protein expression.
Several limitations of this study must be considered. First, sampling error may occur in studies that use coronary atherectomy samples. It is unlikely, however, that sampling error can account for the magnitude of the differences noted in this study. Second, we tested for immunohistochemically detectable TF, and a procoagulant assay may be more sensitive.3 However, the enzyme assay has limitations, including the inability to localize the pattern of TF expression and an even greater potential than immunohistochemistry for sampling error if the specimens are divided. The lesions treated by directional atherectomy may be a skewed group because in general, they are in large vessels, have a proximal location, and lack angiographic thrombus. Caution is necessary before generalizing some of these findings to other coronary lesions.
This study was supported in part by an Initial Career Development Grant from the Duke Heart Center (B.H.A.).
- Received November 7, 1994.
- Accepted December 11, 1994.
- Copyright © 1995 by American Heart Association
Wilcox JN, Smith KM, Schwartz SM, Gordon D. Localization of tissue factor in the normal vessel wall and in the atherosclerotic plaque. Proc Natl Acad Sci U S A. 1989;86:2839-2843.
Bevilacqua MP, Pober JS, Majeua GR, Cotran RS, Gimbrone MA Jr. Recombinant tumor necrosis factor induces procoagulant activity in cultured human vascular endothelium: characterization and comparison with the actions of interleukin 1. Proc Natl Acad Sci U S A. 1986;83:4533-4537.
Grabowski EF, Zuckerman DB, Nemerson Y. The functional expression of tissue factor by fibroblasts and endothelial cells under flow conditions. Blood. 1993;81:3265-3270.
Marmur JD, Rossikhina M, Guha A, Fyfe B, Friedrich V, Mendlowitz M, Nemerson Y, Taubman MB. Tissue factor is rapidly induced in arterial smooth muscle after balloon injury. J Clin Invest. 1993;91:2253-2259.
Lefkovits J, Topol EJ. Direct thrombin inhibitors in cardiovascular medicine. Circulation. 1994;90:1522-1536.
Kragel AH, Reddy SG, Wittes JT, Roberts WC. Morphometric analysis of the composition of coronary arterial plaques in isolated unstable angina pectoris with pain at rest. Am J Cardiol. 1993;66:562-567.
Flugelman MY, Virmani R, Correa R, Yu ZX, Farb A, Leon MB, Elami A, Fu YM, Casscells W, Epstein SE. Smooth muscle cell abundance and fibroblast growth factors in coronary lesions of patients with nonfatal unstable angina, a clue to the mechanism of transformation from the stable to the unstable clinical state. Circulation. 1993;88:2493-2500.
Willerson JT, Golino P, Eidt J, Campbell WB, Buja LM. Specific platelet mediators and unstable coronary artery lesions, experimental evidence and potential clinical implications. Circulation. 1989;80:198-205.
Pawashe AB, Golino P, Ambrosio G, Migliaccio F, Ragni M, Pascucci I, Chiariello M, Bach R, Garen A, Konigsbert WK, Ezekowitz MD. A monoclonal antibody against rabbit tissue factor inhibits thrombus formation in stenotic injured rabbit carotid arteries. Circ Res. 1994;74:56-63.