EXPERIMENTAL STUDY
Inhibition of tissue factor reduces thrombus formation and intimal hyperplasia after porcine coronary angioplasty
Mercè Roqué, MD*,
Ernane D. Reis, MD ,
Valentin Fuster, MD, PhD*,
Adrian Padurean, MD*,
John T. Fallon, MD*  ,
Mark B. Taubman, MD, PhD*  ,
James H. Chesebro, MD* and
Juan J. Badimon, PhD*
* Zena and Michael A. Wiener Cardiovascular Institute, New York, New York, USA
Department of Surgery, Mount Sinai School of Medicine, New York, New York, USA
Department of Pathology, Mount Sinai School of Medicine, New York, New York, USA
Department of Physiology and Biophysics, Mount Sinai School of Medicine, New York, New York, USA
Manuscript received December 28, 1999;
revised manuscript received May 25, 2000,
accepted July 13, 2000.
Reprint requests and correspondence: Dr. Juan J. Badimon, Cardiovascular Institute, The Mount Sinai Medical Center, One Gustave L. Levy Place, Box #1030, New York, New York 10029-6574
juan.badimo{at}mssm.edu
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Abstract
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OBJECTIVES
We investigated the in vivo effects of tissue factor (TF) inhibition with recombinant tissue factor pathway inhibitor (rTFPI) on acute thrombus formation and intimal hyperplasia and the in vitro effects on smooth muscle cell migration and proliferation.
BACKGROUND
Inhibition of TF with TFPI has been shown to reduce intimal hyperplasia in experimental models. However, its effects after coronary angioplasty and the cellular mechanisms involved have not been investigated.
METHODS
Twenty-three swine underwent multivessel coronary angioplasty. Fifteen (n = 25 arteries) were euthanized at 72 h to assess thrombus formation and eight (n = 24 arteries) at 28 days to assess intimal hyperplasia. Animals in the 72-h time point received: 1) human rTFPI (0.5 mg bolus plus 25 µg/kg/min continuous infusion for 3 days) plus heparin (150 IU/kg intravenous bolus) plus acetyl salicylic acid (ASA) (325 mg/day); 2) rTFPI regimen plus ASA and 3) heparin (150 IU/kg intravenous bolus) plus ASA.
RESULTS
On histology the control group had evidence of mural thrombus (area 0.8 ± 0.4 mm2). Treatment with TFPI plus heparin abolished thrombus formation (mean area: 0.0 ± 0.0 mm2, p < 0.05) but was associated with prolonged activated partial thromboplastin time and extravascular hemorrhage. Recombinant TFPI alone inhibited thrombosis without bleeding complications (mean area: 0.03 ± 0.02 mm2, p < 0.05 vs. control). Animals in the 28-day time point received continuous intravenous infusion of rTFPI or control solution for 14 days. Tissue factor pathway inhibitor reduced neointimal formation with mean intimal area of 1.2 ± 0.3 mm2 versus 3.2 ± 0.4 mm2 in the control group; p < 0.01. Recombinant TFPI had no effect on human aortic smooth muscle cell growth but inhibited platelet-derived growth factor BB-induced migration.
CONCLUSIONS
Inhibition of TF with rTFPI can prevent acute thrombosis and intimal hyperplasia after injury. Tissue factor plasma inhibitor may prove useful as an adjunct to intracoronary interventions.
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Abbreviations and Acronyms
| | aPTT | = activated partial thromboplastin time | | ASA | = acetyl salicylic acid | | BSA | = bovine serum albumin | | DMEM | = Dulbecco’s modified essential medium | | FBS | = fetal bovine serum | | IEL | = internal elastic lamina | | I/M | = intima-to-media | | PDGF | = platelet-derived growth factor | | rTFPI | = recombinant tissue factor pathway inhibitor | | SMC | = smooth muscle cell | | TF | = tissue factor | | TFPI | = tissue factor pathway inhibitor |
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Percutaneous transluminal coronary angioplasty is the procedure of choice for treating coronary stenosis—almost one million procedures are performed each year worldwide (1). Many innovations, notably coronary stenting and adjunctive antithrombotic therapy, have made percutaneous coronary interventions safer and more effective, expanding their indications; however, restenosis continues to be an important limitation (2).
Thrombus formation is a frequent feature after arterial injury (3–6). Thrombin generation, platelet activation and fibrin deposition are the main events leading to acute thrombosis (7,8). Inhibition of thrombosis after angioplasty has been associated with a decrease in restenosis (9–12). Thrombin, in addition to being a powerful activator of platelets, induces the release of mitogenic factors, such as platelet-derived growth factor (PDGF) from vascular smooth muscle cells (SMC) (13) and has been implicated in SMC migration and proliferation (14). Inhibition of thrombin generation in vivo is, therefore, considered an important therapeutic target in the sequence of events leading to intimal hyperplasia and restenosis after arterial injury.
Tissue factor (TF) is a membrane-bound glycoprotein that activates the coagulation cascade. Tissue factor and factor VII/VIIa form complexes that catalyze the activation of factors IX and X, leading to thrombin generation (15,16). Balloon injury is associated with rapid induction of TF mRNA and activity throughout the arterial wall (17,18). Tissue factor content of human atherosclerotic plaques correlates with thrombogenicity in an ex vivo perfusion system (19). Therefore, blockade of TF activity may reduce thrombosis.
Tissue factor pathway inhibitor (TFPI) is the principle physiologic inhibitor of the TF-factor VII/VIIa complex and is found mainly in endothelial cells (20). Tissue factor pathway inhibitor therapy has been proven beneficial in deep vein thrombosis (21) in preventing arterial reocclusion after fibrinolysis (22) and in reducing intimal hyperplasia in experimental models of arterial injury (9,10). Using an ex vivo perfusion system we have shown previously that specific inhibition of TF with TFPI reduces thrombogenicity of disrupted human aortic atherosclerotic plaques (23).
In this study we investigated the effects of TF inhibition in a porcine model of coronary angioplasty. Human recombinant TFPI (rTFPI) inhibited acute thrombus formation and reduced intimal hyperplasia. Tissue factor pathway inhibitor had a synergistic anticoagulant effect when given in combination with unfractionated heparin. In addition, TFPI inhibited migration of human SMC in culture. These findings support TF inhibition as a potential therapeutic approach to prevent acute thrombosis and restenosis after coronary interventions.
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Methods
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Animals.
Yorkshire-albino swine, 27 to 32 kg body weight (Animal Biotech Industries, Danboro, Pennsylvania), were housed at the Center for Laboratory Animal Sciences at The Mount Sinai Medical Center, New York, New York, received standard chow (PMI Nutrition International, St. Louis, Missouri) and tap water ad libitum. Procedures and animal care were approved by the Institutional Animal Care and Use Committee and were in accordance with the "Guide for the Care and Use of Laboratory Animals" (24).
rTFPI.
Recombinant tissue factor pathway inhibitor was provided by Chiron Corporation, Emeryville, California. Human rTFPI was expressed in Escherichia coli as a nonglycosylated protein with additional alanine attached to the amino-terminus of wild-type TFPI. The compound was purified by anion exchange chromatography, refolded through a disulfide interchange reaction and resolved from relatively inactive and misfolded molecules on a cation-exchange chromatography column (25). Human rTFPI was dissolved in buffer solution containing 200 mmol/L arginine, 20 mM sodium citrate buffer, 0.01% polysorbate-80 and 150 mM NaCl (pH = 7.2) at a final concentration of 5 mg/mL. The same buffer was used as control solution.
Experimental design.
Effect of TF inhibition on acute thrombus formation
Animals (n = 15) were divided into the following three groups according to treatment: 1) rTFPI, heparin and acetyl salicylic acid (ASA); 2) rTFPI and ASA and 3) control (vehicle solution), heparin and ASA. Recombinant tissue factor pathway inhibitor was administered as an intravenous bolus of 0.5 mg/kg of body weight, 10 min before angioplasty followed by constant infusion of 25 µg/kg/h for 72 h. Unfractionated heparin was given as a single bolus of 150 IU/kg intravenous 10 min before the procedure. Acetyl salicylic acid was given per os, 325 mg/day, beginning 24 h before angioplasty and continuing until euthanasia. The vehicle solution (control) was given as an intravenous bolus before angioplasty, followed by an infusion for 72 h, using a volume equivalent to the rTFPI solution. Animals were euthanized 72 h after angioplasty to analyze acute thrombus formation.
Effect of TF inhibition on intimal hyperplasia
Animals (n = 8) were assigned to two treatment groups: 1) bolus plus constant intravenous infusion of rTFPI for 14 days and ASA and 2) bolus of unfractionated heparin plus intravenous infusion of vehicle solution and ASA as control treatment. All drugs were administered at the same doses specified above. Animals were euthanized 28 days after angioplasty to assess intimal hyperplasia.
Interventional procedures.
For coronary balloon angioplasty, the pigs were sedated initially with ketamine (intravenous, 20 mg/kg body weight) followed by sodium pentobarbital (intravenous, 25 mg/kg), intubated and ventilated mechanically. Anesthesia was maintained with inhaled isoflurane (Forane, Ohmeda Caribe, Guayama, Puerto Rico). The right carotid artery and internal jugular vein were exposed through a median neck incision. Both vessels were cannulated, and each line was tunneled subcutaneously to exit through a posterior neck incision. The carotid cannula was used for blood sampling; the venous cannula was attached to an infusion pump (Harvard Apparatus, Holliston, Massachusetts). Coronary angioplasty was performed by three inflations of a 4.0 x 2.0 mm balloon (Olimpix, Cordis Corp., Miami, Florida) to 8 to 10 atms, as described previously (12). Fluoroscopy-guided angiographic examinations were performed before, during and after percutaneous transluminal coronary angioplasty.
Tissue harvesting and histology.
After anesthesia with ketamine and pentobarbital, the aorta and heart were exposed through a median sternotomy. Animals were then euthanized with an overdose of pentobarbital (Sleepaway, Fort Dodge Laboratories, Fort Dodge, New Jersey). The heart and ascending aorta were excised immediately and perfusion-fixed with 4% paraformaldehyde in phosphate buffered saline. Specimens were kept in fresh fixative solution overnight. Coronary segments of interest were excised, cross-sectioned at 2-mm intervals and processed for paraffin embedding. Sections were cut sequentially and stained with combined Masson elastin trichrome and hematoxylin-eosin.
Morphometry.
Arterial specimens were analyzed by two investigators blinded to the study design. For each coronary specimen the section with maximal luminal narrowing was selected. Degree of arterial injury induced by angioplasty was classified according to an injury score modified from Schwartz et al. (26): 0 = intact endothelium, 1 = endothelial denudation, 2 = internal elastic lamina (IEL) laceration, 3 = IEL and media laceration and 4 = external elastic lamina laceration. All coronary segments analyzed had an injury score greater than or equal to 3. The sections with maximal luminal area occupied by either thrombus (3-day time point) or intimal hyperplasia (28-day time point) were further evaluated by computerized morphometry (Software: NIH Image 1.60). Measurements of luminal area, thrombus area, intimal area, medial area, intima-to-media (I/M) ratio, vessel area and percentage of missing IEL were performed to assess differences in degree of injury between the different treatment groups.
Hematology and drug levels.
Blood samples were obtained at baseline, 10 min after the heparin bolus, 15 min after TFPI or vehicle boluses, immediately after angioplasty and every 24 h until euthanasia. Hematologic values included complete blood count, activated partial thromboplastin time (aPTT), thrombin time, prothrombin time and plasma fibrinogen level (Diagnostica Stago, American Bioproducts, Parsipanny, New Jersey). Tissue factor pathway inhibitor plasma levels were measured in a sandwich immunoassay. The assay uses a monoclonal antibody directed against the first Kunitz domain of human rTFPI for capture and a fluorescein-labeled polyclonal antibody to rTFPI for detection. All samples were assayed in triplicate. The lower limit of quantitation was 40 ng/mL.
Cell culture studies.
Human SMC were obtained from the ascending aorta of cardiac transplant donors and grown using an explant technique: the adventitia was removed carefully and the media was cut into 1-cm2 segments and incubated in culture dishes with collagenase and Dulbecco’s modified essential medium (DMEM) containing 20% fetal bovine serum (FBS), 100 U/mL of penicillin and 100 µg/mL of streptomycin. Cells were incubated at 37°C with 5% CO2. Smooth muscle cell lineage was confirmed by positive  -actin staining (M851, Dako, Carpinteria, California) in more than 90% of the cells. All experiments were performed using passages 3 to 5 of cells grown in 10% FBS. To analyze growth cells were plated in 12-well dishes at a density of 5 x 103 cells/well, in the presence or absence of rTFPI (250 nM). Fresh rTFPI was added with each medium change every 48 h. DNA synthesis was determined by 3H-thymidine incorporation. Cells were plated in 12-well plates (5 x 103 cells/well) and incubated for 24 h in DMEM supplemented with 1% FBS. Smooth muscle cells were then washed in phosphate buffered saline and incubated for 24 h with 1 µCi of 3H-thymidine in DMEM plus 10% FBS in the presence or absence of rTFPI. Cells were precipitated with 10% trichloroacetic acid for 60 min at 4°C, solubilized with 0.1N NaOH and radioactivity measured in a liquid scintillation counter. Experiments were done in triplicate, using quadruplicate wells.
Vascular SMC migration assay.
A modified Boyden chamber with a polycarbonate filter with 8-µm pores (Nucleopore, Cabin John, Maryland) was used to measure SMC migration. The filter was coated with collagen, 0.1 mg/mL (Vitrogen 100, Centrix, California) in 0.2 M acetic acid, 24 h before the experiment. To induce chemotaxis, PDGF-BB (20 ng/mL) in DMEM plus 0.2% bovine serum albumin (BSA) was placed in the lower chamber; 0.2% BSA without PDGF was used as negative control. Fifty µL of SMC (2 x 105 cells/mL) in DMEM, containing different concentrations of TFPI, were loaded in the wells of the upper chamber (four wells per condition). After a 6-h incubation at 37°C in 5% CO2, cells were gently scraped from the upper surface of the filter, and cells on the lower surface of the membrane were fixed with methanol and stained with Diff Quik (Baxter, Largo, Florida). The number of migrating cells was determined by counting four high-power fields (x 200) in the central area of each well. Results are expressed as mean percentage of migrating cells after subtracting the negative control (BSA), averaging data from six separate experiments.
Statistical analysis.
Numerical data are expressed as mean ± standard error of the mean. Comparisons of data among the different groups were performed using one-way analysis of variance and Bonferroni’s test for multiple comparisons among treatment groups, as well as unpaired Student t test as appropriate. A p value <0.05 was considered significant.
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Results
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Effect of TF inhibition on acute thrombus formation.
Data from coronary specimens harvested 72 h after angioplasty are summarized in Table 1. The control group had a mean mural thrombus area of 0.8 ± 0.4 mm2 (Fig. 1, A and B). In the group treated with rTFPI plus ASA plus bolus of heparin, the thrombus area was zero (p < 0.05 versus control), but the specimens displayed severe bruising and hemorrhage of surrounding tissues (Fig. 1, C and D). In this group hematomas in the surgically intervened areas, such as the groin, were observed frequently (not shown). In the group treated with rTFPI plus ASA (without heparin), the thrombus area was 0.03 ± 0.02 mm2 (p < 0.05 versus control; Fig. 1, E and F); no bleeding complications were noted.
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Figure 1 The histology of porcine coronary arteries 72 h after angioplasty. (A and B) coronary sections of vehicle-treated animals showing the presence of mural thrombus (red arrows). (C and D) sections of recombinant tissue factor pathway inhibitor plus heparin-treated animals. Note the hemorrhagic area surrounding the coronary wall (yellow arrows). (E and F) sections of recombinant tissue factor pathway inhibitor alone-treated animals, with no mural thrombosis and no periarterial hemorrhage. Note the comparable degrees of injury, with disruption of the internal elastic lamina in all sections. (All sections photographed at 40x).
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Effect of TF inhibition on intimal hyperplasia.
Specimens harvested 28 days after angioplasty were analyzed for intimal hyperplasia. Coronary artery sections in the control group (n = 12) had a mean intimal area of 3.3 ± 0.4 mm2, mean I/M ratio of 3.9 ± 0.7 mm2 and luminal narrowing of 61 ± 3% (Fig. 2 and 3, A to C). The rTFPI-treated group (n = 12) had a mean intimal area of 1.2 ± 0.4 mm2, mean I/M ratio of 1.5 ± 0.5 and luminal narrowing of 34 ± 5% (p < 0.05 vs. control; Fig. 2 and 3, D to F).
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Figure 2 The morphometry of coronary arteries from control and rTFPI-treated swine. (A) Standardized intimal area or intima-to-media ratio of control and rTFPI-treated animals. (B) percentage luminal narrowing in both treatment groups, control and rTFPI. I/M = intima-to-media; rTFPI = recombinant tissue factor pathway inhibitor.
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Figure 3 The proliferative response in porcine coronary arteries 28 days after angioplasty. Representative coronary sections of control (A to C) and recombinant tissue factor pathway inhibitor-treated (D to F) animals. Note the internal elastic lamina disruption in all sections and larger amounts of intimal hyperplasia in the sections from controls compared with tissue factor pathway inhibitor-treated group. (All sections photographed at 40x.)
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rTFPI plasma levels and hematology.
The plasma levels of rTFPI after bolus and infusion are shown in Figure 4. Recombinant TFPI alone induced a modest prolongation of the aPTT (Fig. 5), as well as a two-fold increase in thrombin time. No changes in prothrombin time or fibrinogen levels were observed in any of the different treatment groups. Administration of TFPI plus heparin increased the aPTT markedly. No bleeding complications were noted in animals receiving rTFPI plus ASA or in the control group. The rTFPI plus heparin bolus regimen caused a high incidence of hematomas in the groin and other areas of surgical manipulation, in addition to severe bruising and gross hemorrhage in the pericoronary tissues of all animals (not shown).
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Figure 4 Plasma levels of TFPI. Plasma levels of TFPI at different times before and after angioplasty during continuous intravenous administration of recombinant TFPI. PTCA = percutaneous transluminal coronary angioplasty; TFPI = tissue factor pathway inhibitor.
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Figure 5 Activated partial thromboplastin time monitoring during TFPI treatment. Activated partial thromboplastin time values at baseline and different times over a 72-h period after angioplasty for the three treatment groups (UFH plus ASA, heparin bolus plus aspirin; UFH plus ASA plus TFPI, heparin bolus plus ASA plus TFPI; ASA plus TFPI, ASA plus rTFPI). Note the synergistic anticoagulant effect of TFPI and heparin compared with the modest increase in aPTT elicited by TFPI alone. Long-dash line = UFH+ASA; solid line = UFH+ASA+TFPI; short-dash line = ASA+TFPI. aPTT = activated partial thromboplastin time; ASA = acetyl salicylic acid; PTCA = percutaneous transluminal coronary angioplasty; TFPI = tissue factor pathway inhibitor; UFH = unfractionated heparin.
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rTFPI inhibits SMC migration.
Recombinant TFPI, at a concentration of 250 nM, did not inhibit serum-induced 3H-thymidine incorporation (8,388 ± 1,433 cpm vs. 6,508 ± 172 cpm in cells grown in 10% FBS; p = NS) or cell number in cultured SMC (Fig. 6). In contrast, rTFPI inhibited PDGF-inducible SMC migration in a dose-dependent fashion (Fig. 7).
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Figure 6 The effect of tissue factor pathway inhibitor on smooth muscle cell proliferation. Seven-day growth curve of human aortic smooth muscle cells in the presence or absence of rTFPI. Short-dash line = 1% FBS; solid line = 10% FBS; long-dash line = 10% FBS + rTFPI. FBS = fetal bovine serum; rTFPI = recombinant tissue factor pathway inhibitor.
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Figure 7 The effect of TFPI on smooth muscle cell migration. Concentration-dependent inhibition of human aortic smooth muscle cell migration by recombinant TFPI. All concentrations expressed as nM. BSA = bovine serum albumin; HPF = high power field; TFPI = tissue factor pathway inhibitor.
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Discussion
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This study reports on the effects of rTFPI in a porcine model of coronary angioplasty. The results further support the concept that inhibition of thrombin and acute thrombus formation can reduce the development of intimal hyperplasia and provide new evidence for a synergistic anticoagulant effect of rTFPI and unfractionated heparin.
Tissue factor is thought to be critical in initiating thrombotic events after plaque rupture—either spontaneous or induced mechanically and, thereby, is likely to have a significant role in promoting intimal hyperplasia leading to restenosis after injury (19,27–32). Therefore, specific inhibition of TF is a potential therapeutic approach to prevent clinical events subsequent to coronary interventions. The antithrombotic efficacy of rTFPI has been shown in models of venous thrombosis in rabbits (21) and arterial thrombosis in dogs, where it inhibited reocclusion after thrombolysis with tPA (22,33). In this study inhibition of TF activity by rTFPI abolished acute thrombus formation after coronary angioplasty in a porcine model.
Synergistic effect of rTPI and heparin.
The association of rTFPI with heparin had a synergistic anticoagulant effect and resulted in a high incidence of bleeding in the injured areas. The plasma levels of TFPI measured in the acute phase after angioplasty in this study were within the therapeutic range used in previous studies (9,19). Heparin is known to induce the release of TFPI from endothelial cells in a concentration- and time-dependent fashion (34–36). However, it is unlikely that this is the only responsible mechanism for the observed synergistic effect because the levels of heparin-releasable TFPI reported in previous studies are rather insignificant (50 to 100 ng/mL) (36) when compared with the levels of TFPI obtained in our study (2.7 to 3.2 µg/mL). Recombinant TFPI administered in combination with aspirin only induced a two-fold increase in aPTT ratio without periprocedural thrombotic complications. These observations are of relevance because rTFPI is a powerful antithrombotic with potential clinical application in the near future. Routine use of heparin during percutaneous coronary interventions appears unnecessary when rTFPI is used or should be kept at a minimum dose to ensure adequate anticoagulation without bleeding complications.
TF inhibition reduces intimal hyperplasia.
In this study TF inhibition with rTFPI administered for 14 days after coronary angioplasty reduced intimal hyperplasia. The formation of a platelet-rich thrombus at the site of vascular injury may serve as a matrix for SMC migration and proliferation because thrombin is a growth factor for SMC (14). In this context TF inhibition and, subsequently, thrombin generation by rTFPI has been shown previously to reduce the development of intimal hyperplasia after angioplasty in other experimental models such as the pig carotid artery (9) and rabbit femoral artery (10). A factor Xa inhibitor (recombinant tick anticoagulant peptide) also has been shown to inhibit intimal hyperplasia in the pig coronary (11).
rTFPI inhibits SMC migration.
To further define the mechanism of action of rTFPI, we also studied the effects of rTFPI on SMC migration and proliferation. This study demonstrates that rTFPI inhibits PDGF-BB-induced human SMC migration in a dose-dependent fashion, whereas it does not interfere with serum-induced SMC growth. The beneficial effect of rTFPI has been attributed largely to the inhibition of acute thrombus formation after injury. Our findings indicate that, in addition to its antithrombotic properties and the extended benefit of acute thrombus inhibition on intimal hyperplasia, rTFPI may reduce neointimal thickening, in part, by direct effects on SMC migration. It should be noted that no effect on SMC proliferation was seen in culture.
The effect of TFPI on PDGF-mediated SMC migration is intriguing. Tissue factor pathway inhibitor has not been shown to interact directly with PDGF or its receptors, nor is it known to bind to cell-surface molecules other than TF. It is thus likely that the inhibition of SMC migration is due to the interaction between TFPI and TF. One possibility is that the binding of TFPI transduces an intracellular signal, which inhibits migration. Although several studies have suggested that TF may be involved in cytoplasmic signaling (37), this remains controversial. Alternatively, SMC migration may be regulated, in part, by a product of TF catalytic activity such as thrombin or Xa and, therefore, may be altered by inhibiting TF activity. Sato et al. (38) found that the TF VIIa complex stimulates SMC migration. The mechanisms for this finding remain to be determined. We have reported previously that the expression of active TF on the SMC surface is transient and peaks 4 to 6 h after PDGF stimulation (39). Under the conditions of this study, SMC were exposed to PDGF as a chemoattractant for 6 h, well within the time frame for the surface expression of newly synthesized TF. It is thus possible that the migratory effect of PDGF on SMC is mediated through the synthesis of TF. It should be noted that Sato and colleagues (40) did not find that TFPI inhibited SMC migration, perhaps due to differences in the SMC (their study used rabbit SMC, whereas ours were human) or the conditions under which they were cultured.
Conclusions.
In summary using a porcine model of coronary angioplasty, we demonstrated that specific inhibition of the TF pathway, interfering with in vivo generation of thrombin, prevents not only acute thrombus formation but also neointimal formation. In addition to its antithrombotic effect, TFPI also may exert its inhibitory effect on intimal hyperplasia by impairing SMC migration. This novel therapeutic approach may be beneficial in avoiding the complications associated with percutaneous coronary interventions although special care should be taken regarding the synergistic anticoagulant potential of TFPI and heparin.
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Acknowledgments
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The authors wish to thank Eduardo Acampado, Maria Rossikhina, Veronica Gulle and Ameera Ali for their technical assistance.
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Footnotes
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Supported, in part, by a grant-in-aid from the Spanish Heart Association to Dr. Roqué.
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References
|
|---|
- Bittl JA. Advances in coronary angioplasty. N Engl J Med. 1996;335:1290–1302[Free Full Text]
- Bauters C, Hubert E, Prat A, et al. Predictors of restenosis after coronary stent implantation. J Am Coll Cardiol. 1998;31:1291–1298[Abstract/Free Full Text]
- Losordo DW, Rosenfield K, Pieczek A, Baker K, Harding M, Isner JM. How does angioplasty work? Serial analysis of human iliac arteries using intravascular ultrasound. Circulation. 1992;86:1845–1858[Abstract/Free Full Text]
- Nath FC, Muller DW, Ellis SG. Thrombosis of a flexible coil coronary stent: frequency, predictors and clinical outcome. J Am Coll Cardiol. 1993;21:622–627[Abstract]
- Steele PM, Chesebro JH, Stanson AW, et al. Balloon angioplasty: natural history of the pathophysiologic response to injury in a pig model. Circ Res. 1985;57:105–112[Abstract/Free Full Text]
- Groves MH, Rathbone-Kinlough LR, Richardson M, Jorgensen L, Moore S, Mustard FJ. Thrombin generation and fibrin formation following injury to rabbit neointima. Lab Invest. 1982;46:605–612[Medline]
- Sarembock IJ, Gertz SD, Gimple LW, Owen RM, Powers ER, Roberts WC. Effectiveness of recombinant desulphatohirudin in reducing restenosis after balloon angioplasty of atherosclerotic femoral arteries in rabbits. Circulation. 1991;84:232–243[Abstract/Free Full Text]
- Fuster V, Falk E, Fallon JT, Badimon L, Chesebro JH, Badimon JJ. The three processes leading to post PTCA restenosis: dependence on the lesion substrate. Thromb Haemost. 1995;74:552–559[Medline]
- Oltrona L, Speidel CM, Recchia D, Wickline SA, Eisenberg PR, Abendschein DR. Inhibition of tissue factor-mediated coagulation markedly attenuates stenosis after balloon-induced arterial injury in minipigs. Circulation. 1997;96:646–652[Abstract/Free Full Text]
- Jang Y, Guzman LA, Lincoff M, et al. Influence of blockade at specific levels of the coagulation cascade on restenosis in a rabbit atherosclerotic femoral artery injury model. Circulation. 1995;92:3041–3050[Abstract/Free Full Text]
- Schwartz RS, Holder DJ, Holmes DR, et al. Neointimal thickening after severe coronary artery injury is limited by short-term administration of a factor Xa inhibitor. Results in a porcine model. Circulation. 1996;93:1542–1548[Abstract/Free Full Text]
- Gallo R, Padurean A, Toschi V, et al. Prolonged thrombin inhibition reduces restenosis after balloon angioplasty in porcine coronary arteries. Circulation. 1998;97:581–588[Abstract/Free Full Text]
- Golden MA, Au YP, Kirkman TF, et al. Platelet-derived growth factor activity and mRNA expression in healing vascular grafts in baboons. Association in vivo of platelet-derived growth factor mRNA and protein with cellular proliferation. J Clin Invest. 1991;87:406–414[Medline]
- Fager G. Thrombin and proliferation of vascular smooth muscle cells. Circ Res. 1995;77:645–650[Free Full Text]
- Nemerson Y. Tissue factor: then and now. Thromb Haemost. 1995;74:180–184[Medline]
- Broze GJ Jr. Tissue factor pathway inhibitor and the revised theory of coagulation. Annu Rev Med. 1995;46:103–112[CrossRef][Medline]
- Gertz SD, Fallon JT, Gallo R, et al. Hirudin reduces tissue factor expression in neointima after balloon injury in rabbit femoral and porcine coronary arteries. Circulation. 1998;98:580–587[Abstract/Free Full Text]
- Marmur J, Rossikhina M, Guha A, et al. Tissue factor is rapidly induced in arterial smooth muscle after balloon injury. J Clin Invest. 1993;91:2253–2259[Medline]
- Toschi V, Gallo R, Lettino M, et al. Tissue factor modulates the thrombogenicity of human atherosclerotic plaques. Circulation. 1997;95:594–599[Abstract/Free Full Text]
- Abumiya T, Enjyoji K, Kokawa T, Kamikubo Y, Kato H. An antitissue factor pathway inhibitor (TFPI) monoclonal antibody recognized the third Kunitz domain (K3) of free-form of TFPI but not lipoprotein-associated forms in plasma. J Biochem. 1995;118:178–182[Abstract/Free Full Text]
- Holst J, Lindblad B, Bergqvist D, et al. Antithrombotic effect of recombinant truncated tissue factor pathway inhibitor (TFPI 1-161) in experimental venous thrombosis—a comparison with low molecular weight heparin. Thromb Haemost. 1994;71:214–219[Medline]
- Abendschein DR, Meng YY, Torr-Brown S, Sobel BE. Maintenance of coronary patency after fibrinolysis with tissue factor pathway inhibitor. Circulation. 1995;92:944–949[Abstract/Free Full Text]
- Badimon JJ, Lettino M, Toschi V, et al. Local inhibition of tissue factor reduces the thrombogenicity of disrupted human atherosclerotic plaques. Effects of tissue factor pathway inhibitor on plaque thrombogenicity under flow conditions. Circulation. 1999;99:1780–1787[Abstract/Free Full Text]
- National Research Council. Guide for the Care and Use of Laboratory Animals. Washington, DC: National Academy Press; 1996.
- Wun TC, Kretzmer KK, Girard TJ, Miletich JP, Broze GJ Jr. Cloning and characterization of a cDNA coding for the lipoprotein-associated coagulation inhibitor shows that it consists of three tandem Kunitz-type inhibitory domains. J Biol Chem. 1988;263:6001–6004[Abstract/Free Full Text]
- Schwartz RS, Murphy JG, Edwards WD, Camrud AR, Vliestra RE, Holmes DR. Restenosis and the proportional neointimal response to coronary artery injury: results in a porcine model. J Am Coll Cardiol. 1992;19:267–274[Abstract]
- Marmur J, Thiruvikraman S, Fyfe B, et al. Identification of active tissue factor in human coronary atheroma. Circulation. 1996;94:1226–1232[Abstract/Free Full Text]
- Gori AM, Pepe G, Attanasio M, et al. Tissue factor reduction and tissue factor pathway inhibitor release after heparin administration. Thromb Haemost. 1999;81:589–593[Medline]
- Moor E, Hamsten A, Karpe F, Bavenholm P, Blombäch M, Silveira A. Relationship of tissue factor pathway inhibitor activity to plasma lipoproteins and myocardial infarction at a young age. Thromb Haemost. 1994;71:707–712[Medline]
- Misumi K, Ogawa H, Yasue H, et al. Comparison of plasma tissue factor levels in unstable and stable angina pectoris. Am J Cardiol. 1998;81:22–26[CrossRef][Medline]
- Soejima H, Ogawa H, Yasue H, et al. Heightened tissue factor associated with tissue factor pathway inhibitor and prognosis in patients with unstable angina. Circulation. 1999;99:2908–2913[Abstract/Free Full Text]
- Falciani M, Gori AM, Fedi S, et al. Elevated tissue factor and tissue factor pathway inhibitor circulating levels in ischemic heart disease patients. Thromb Haemost. 1998;79:495–499[Medline]
- Haskel EJ, Torr SR, Day KC. Prevention of arterial reocclusion after thrombolysis with recombinant lipoprotein-associated coagulation inhibitor. Circulation. 1991;84:821–827[Abstract/Free Full Text]
- Lupu C, Poulsen EM, Roquefeuil S, Westmuckett AD, Kakkar VV, Lupu F. Cellular effects of heparin on the production and release of tissue factor pathway inhibitor in human endothelial cells in culture. Arterioscler Thromb Vasc Biol. 1999;19:225–262
- Soejima H, Ogawa H, Yasue H, et al. Plasma tissue factor pathway inhibitor and tissue factor antigen levels after administration of heparin in patients with angina pectoris. Thromb Res. 1999;93:17–25[CrossRef][Medline]
- Altman R, Scazziota A, Rouvier J. Efficacy of unfractionated heparin, low-molecular weight heparin and both combined for releasing total and free tissue factor pathway inhibitor. Haemostasis. 1998;28:229–235[CrossRef][Medline]
- Ott I, Fischer EG, Miyagi Y, Mueller BM, Ruf W. A role for tissue factor in cell adhesion and migration mediated by interaction with actin-binding protein 280. J Cell Biol. 1998;140:1241–1253[Abstract/Free Full Text]
- Sato Y, Asada Y, Marutsuka K, Hatakeyama K, Sumiyoshi A. Tissue factor induces migration of cultured aortic smooth muscle cells. Thromb Haemost. 1996;75:389–392[Medline]
- Schecter AD, Giesen PL, Taby O, et al. Tissue factor expression in human arterial smooth muscle cells: TF is present in three cellular pools after growth factor stimulation. J Clin Invest. 1997;100:2276–2285[Medline]
- Sato Y, Asada Y, Marutsuka K, Hatakeyama K, Kamikubo Y, Sumiyoshi A. Tissue factor pathway inhibitor inhibits aortic smooth muscle cell migration induced by tissue factor/factor VIIa complex. Thromb Haemost. 1997;78:1138–1141[Medline]
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