Inhibition of Tissue Factor–Mediated Coagulation Markedly Attenuates Stenosis After Balloon-Induced Arterial Injury in Minipigs
Background Exposure and upregulation of tissue factor in the wall of balloon-injured arteries may result in prolonged activation of coagulation contributing to restenosis. This study was designed to determine whether brief or more prolonged inhibition of tissue factor–mediated coagulation with tissue factor pathway inhibitor (TFPI) attenuates neointimal formation and luminal stenosis after balloon-induced arterial injury.
Methods and Results The carotid artery of minipigs fed an atherogenic diet was injured by repetitive balloon hyperinflations, a procedure that rapidly yields complex, plaque-like neointimal lesions and high-grade luminal stenosis. Recombinant TFPI (rTFPI) was administered intravenously beginning 15 minutes before balloon injury as either a high dose (0.5 mg/kg bolus and 100 μg·kg−1·min−1) for 3 hours (n=7) or 24 hours (n=6) or as a low dose (0.5 mg/kg and 25 μg·kg−1·min−1) for 24 hours (n=6). Control animals received intravenous heparin (100 U·kg−1·h−1) for 3 hours (n=6) or 24 hours (n=7) or aspirin (5 mg/kg PO) followed by heparin for 24 hours (n=7). Luminal stenosis, assessed histologically 4 weeks after injury, was 73±17% and 76±18% (mean±SEM) in animals that received rTFPI or heparin for 3 hours, respectively. In contrast, luminal stenosis was only 11±12% and 6±3% in pigs given high and low doses, respectively, of rTFPI for 24 hours compared with 46±22% in pigs given heparin for 24 hours and 40±19% in those given both heparin and aspirin (P<.0002).
Conclusions Inhibition of tissue factor–mediated coagulation during the first 24 hours after deep arterial injury appears to be particularly effective for attenuating subsequent neointimal formation and stenosis.
The efficacy of coronary angioplasty is limited by a 30% to 50% incidence of restenosis that occurs within 6 months after the intervention.1 The mechanisms responsible for restenosis remain unclear, but thrombin has been implicated because it promotes platelet-rich thrombosis and because it serves as a potent mitogen for vascular SMCs, both components of the restenotic neointima.2 Nevertheless, conventional anticoagulation with heparin and even potent, direct inhibition of thrombin with hirudin administered during and for several days after angioplasty have failed to prevent restenosis in patients.3 4 5
Antithrombin agents may have failed to attenuate restenosis because of incomplete local inhibition of thrombin generated immediately after vascular injury. Recently, we6 and others7 have shown that intravenous administration of recombinant hirudin in dosages that increased partial thromboplastin time more than twofold for 3 hours after balloon-induced injury to the carotid or coronary arteries in minipigs decreased neointimal formation and luminal stenosis 1 month later. However, frequent bleeding complications observed in patients given more modest dosages of hirudin imply that higher intravenous dosing levels will not be acceptable for clinical use.8
The efficacy of antithrombin agents for attenuating restenosis may have been limited also by persistent activation of factors IX and X by the complex of tissue factor and VIIa. Activated factors IX and X, in turn, activate prothrombin to thrombin. Tissue factor is a membrane-bound glycoprotein expressed constitutively by cells primarily in the vascular adventitia9 that is also associated with matrix in the subendothelium10 and in atherosclerotic plaques11 and is expressed by monocytes/macrophages and endothelial cells after their activation by various agonists.12 13 14 15 In experimental preparations of deep arterial injury simulating angioplasty, exposure of tissue factor on the luminal surface of vessels is responsible for initiation of procoagulant activity leading to thrombus formation.16 17 18 Furthermore, tissue factor is upregulated in the injured vessel wall,19 which we have shown recently16 to lead to a bimodal pattern of prolonged procoagulant activity on the luminal surface over the first 24 hours after injury.
Inhibition of tissue factor/VIIa as well as factor Xa in vivo is mediated by TFPI, a 276-amino-acid glycoprotein produced by and bound to the surface of endothelium.20 Heparin and thrombin displace TFPI from endothelium,21 22 but it is not clear that circulating levels of endogenous TFPI increase sufficiently during angioplasty to inhibit procoagulant activity at the site of injury on the basis of the high incidence of restenosis despite administration of heparin.3 4 The present study was designed to determine whether inhibition of tissue factor–mediated coagulation with pharmacological doses of rTFPI administered as either 3-hour or 24-hour infusions can attenuate stenosis after balloon hyperinflation–induced injury of the carotid arteries in minipigs fed an atherogenic diet, a preparation that we have shown causes formation of complex, atherosclerosis-like lesions containing infiltrations of SMCs, macrophages, and thrombus analogous to that in human atherosclerotic plaques and restenotic lesions.23
Procedures involving animals were conducted according to the guiding principles of the American Physiological Society and were approved by the Animal Studies Committee at Washington University. Male Yucatan minipigs (Charles River Laboratories) weighing 16 to 18 kg were fed an atherogenic diet containing 4% cholesterol and 30% fat (Purina Test Diet 5748 M-A).23 After verification that serum cholesterol levels had exceeded 300 mg/dL (generally observed 2 weeks after the start of the atherogenic diet), the pigs were sedated with ketamine (22 mg/kg IM), acepromazine (1.1 mg/kg IM), and atropine (0.05 mg/kg IM). Anesthesia was induced with pentobarbital (20 mg/kg IV) and the trachea was intubated, but the pigs were allowed to breathe spontaneously unless ventilation became depressed. The external jugular veins were exposed aseptically, and catheters were placed in each and advanced into the vena cava for infusion of anticoagulant agents and fluids and for collection of blood samples. A femoral artery was exposed, an 8F catheter sheath was inserted, and a bolus of heparin (200 U/kg IV) was administered to prevent clot formation in the catheters.
The pigs were randomly assigned to one of six intravenous antithrombotic regimens (Table 1⇓): high-dose rTFPI for 3 or 24 hours; low-dose rTFPI for 24 hours; or, as control groups, additional heparin for 3 hours or 24 hours with or without aspirin. The rTFPI and heparin solutions were administered with an infusion pump (Life Care Pump Model 4P, Abbott Laboratories). Human rTFPI was the full-length, 34-kD protein derived from Escherichia coli.24 The high dose of rTFPI (100 μg·kg−1·min−1) was chosen because it inhibited reocclusion of coronary arteries after fibrinolysis in dogs.25 The lower dose of rTFPI (25 μg·kg−1·min−1) was selected to perturb hemostasis minimally.
Fifteen minutes after the start of the infusion of antithrombotic agents, a baseline angiogram of the carotid arteries was obtained. A balloon catheter (Proflex 5; 8 mm×2 cm, Mallinckrodt Inc) was then advanced into the left carotid artery to the level of the second and third cervical vertebrae, and the balloon was inflated five times to a pressure of 8 atm for 30 seconds with 60 seconds between inflations, which we and others23 26 have shown induces rupture of the IEL and deep injury to the media. Angiograms obtained during balloon inflations were compared with the baseline angiograms to measure the ratio of diameter of the balloon to that of the vessel as an index of the adequacy of hyperinflations. After removal of the balloon catheter, another angiogram was obtained to verify patency of the injured carotid artery. The catheter sheath was then removed, the femoral artery was occluded, and the animals were allowed to recover from anesthesia.
Patency of the injured vessel was assessed after 48 hours by transcutaneous ultrasound (Hewlett-Packard Sonos 1500 scanner with a 7.5-MHz linear array transducer). One month after balloon-induced injury, patency was reassessed angiographically and the arteries were prepared for analysis of luminal stenosis as described below.
Hematologic Assays, Bleeding Time, and Blood Levels of rTFPI and Cholesterol
Venous blood samples were obtained for analysis of aPTT, PT, and rTFPI levels before the bolus of heparin (baseline) and serially for up to 48 hours after the start of infusions of additional antithrombotic agents. Bleeding time and hematocrit were measured at baseline and 3 and 24 hours after the start of infusions of antithrombotic agents. Serum cholesterol levels were measured in the fasted animals before the start of the atherogenic diet, at the time of balloon-induced carotid injury, and 4 weeks after balloon injury.
The aPTT and PT were measured in citrated plasma with a manual method described previously27 because the turbidity in some samples caused by hyperlipemia interfered with conventional, automated assays. Briefly, after the reagents for assay of PT (0.1 mL Simplastin Excel, international sensitivity index of 2.05, Organon Teknika) or aPTT (0.1 mL Automated aPTT Reagent and 0.1 mL CaCl2, Organon Teknika) were added to 0.1 mL plasma, a 20-gauge needle with a small burr on the tip was slowly dipped and withdrawn repeatedly from the solution until a strand of fibrin was observed attached to the needle. The time from addition of reagent to the first appearance of the fibrin strand was taken to be the clotting time. Results with the manual and automated (Coag-A-Mate XM, Organon Teknika) methods applied to normolipemic samples were comparable (manual PT, 14.3±0.3 seconds; automated PT, 13.2±0.3 seconds; n=6; manual aPTT, 14.5±0.7 seconds; automated aPTT, 18.6±0.3 seconds; n=6).
Bleeding time was measured by the methods of Sawada et al.28 Briefly, an incision was made through the edge of the ear near the apex with a No. 10 Bard-Parker blade. Care was taken to avoid the central ear artery and visible veins on the posterior surface of the ear. The width of the incision was controlled by inserting the blade perpendicular to the ear to a depth of 5 mm. The ear was immersed in a beaker of isotonic saline that had been warmed to 37°C, and the time elapsed between the puncture and the cessation of any visible flow of blood from the incision was considered to be the bleeding time.
Plasma rTFPI levels were assayed by a particle concentration fluorescence immunoassay described previously.25 Serum cholesterol was measured spectrophotometrically at 37°C with a coupled enzyme system (AC5-12 reagent, Schiapparelli Biosystems, Inc) and a Gemeni centrifugal analyzer (Schiapparelli).
Analysis of Luminal Stenosis
The region of the injured carotid artery with the smallest apparent luminal diameter was identified angiographically and its location marked on the skin. The artery was perfused in situ with 300 mL 0.9% NaCl followed by 500 mL 4% paraformaldehyde at a constant pressure of 120 mm Hg via the angiographic catheter positioned proximally in the brachiocephalic artery and with a ligature placed around the artery containing the catheter to prevent retrograde flow. The perfusion-fixed carotid artery was then excised, and a 1-cm segment from the site of stenosis identified angiographically was placed in fixative for 24 hours. The segment was embedded in paraffin and cut through its entirety at a thickness of 5 μm, and sets of sections collected every 100 μm were stained with hematoxylin and eosin and with Verhoeff’s–van Gieson’s stain for elastic tissue.
Low-power microscopic images of sections exhibiting the smallest luminal diameters and stained for elastic tissue (to facilitate identification of the IEL) were digitized with a Nikon Optiphot-2 microscope with a CCD camera attached to a Macintosh IIci computer outfitted with a NuVista frame-grabber board. The cross-sectional areas of the lumen and neointima were planimetered by tracing the margin of the lumen and IEL. Percent luminal obstruction was then calculated as 1 minus the area of the lumen divided by the area within the IEL multiplied by 100.
Analysis of rTFPI Pharmacokinetics In Vivo
In two additional cholesterol-fed, anesthetized minipigs, rTFPI was administered as a bolus (0.5 mg/kg IV), and blood samples were obtained serially for assay of rTFPI. Plasma profiles of rTFPI were analyzed by nonlinear regression and the modified Gauss-Newton method of residuals in which exponential terms are sequentially peeled off. Profiles from both animals fit a biexponential equation of the form C=Ae−at+Be−bt, where C is the concentration of rTFPI at time t (minutes) and A and B are intercept values (t=0) extrapolated from a and b, the first-order elimination constants.
Results are expressed as mean±SEM. ANOVA with a repeated-measures design was used to assess time-dependent changes in cholesterol levels. For hemostatic variables, time-dependent changes were analyzed with a growth curve model (PROCMIXED procedure, SAS/STAT Software: changes and enhancements through release 6.11, 1996) to accommodate for missing data in some experiments. Because luminal stenosis within some treatment groups was not normally distributed as indicated by values >2 SD from the mean, a nonparametric Wilcoxon test was used to compare the severity of luminal stenosis between groups. A value of P≤.05 was considered significant.
Fifty-two animals were subjected to the carotid injury procedure. Thirteen (25%) were excluded: 9 died after the procedure (8 had been treated with heparin or the combination of heparin and aspirin and appeared to succumb to bleeding complications), 2 did not exhibit persistent elevations of serum cholesterol >300 mg/dL over the 4 weeks after arterial injury, and 2 did not exhibit disruption of the IEL at the site of previous balloon hyperinflations. None of the remaining 39 animals that completed the protocol exhibited occlusion of the injured carotid artery assessed ultrasonically after 48 hours or angiographically after 4 weeks. However, each of these animals was confirmed to have multiple and extensive disruptions of the IEL at the site of previous balloon-induced carotid injury. The balloon-to-vessel ratio averaged 1.20±0.06 (n=27) and was not different between treatment groups.
Mean serum cholesterol levels were significantly increased at the time of arterial injury and 4 weeks later compared with baseline values (Table 2⇓). However, the levels did not differ between groups.
Administration of the high dose of rTFPI or heparin for 3 hours after carotid arterial injury was associated with marked luminal stenosis 4 weeks later that did not differ between groups (73±17% for rTFPI and 76±18% for heparin) (Fig 1⇓). Treatment for 24 hours with heparin (46±22%) or the combination of heparin and aspirin (40±19%) resulted in inconsistent reductions in luminal stenosis compared with 3-hour administration of agents (Figs 1⇓ and 2⇓). However, 24-hour administration of both high and low doses of rTFPI was associated with significant reductions in luminal stenosis (11±12% for the high dose and 6±3% for the low dose) compared with all other treatment groups (P<.0002). In fact, all but one pig given the high dose of rTFPI for 24 hours exhibited <1% luminal stenosis (Figs 1⇓ and 2⇓). The remaining animal had a stenosis of 61%, resulting in a higher mean value for the group. Interestingly, the neointimal lesion in this animal was much different in appearance from the lesions from any other animal, exhibiting a loose connective tissue–like composition.
Plasma aPTT and PT levels in animals given 24-hour infusions of antithrombotic agents are shown in Fig 3⇓. Similar data were obtained in animals treated for 3 hours (data not shown). In both high-dose and low-dose rTFPI–treated animals, aPTT was increased significantly above baseline levels for only the first hour after the bolus (P<.0001). In contrast, aPTT remained 2 to 4 times above baseline levels throughout the administration of heparin or heparin and aspirin (P<.0001). PT was also increased over the first hour after the bolus of rTFPI (P<.05). It returned toward baseline within the second hour in animals given the low-dose infusion of rTFPI and persisted at levels between 1.9 and 2.5 times baseline over the 24-hour infusion. In contrast, animals given the high dose of rTFPI exhibited a second, progressive rise of PT beginning at 3 hours to >10 times baseline by 24 hours (P<.0008). The increase of PT in heparin-treated animals was more modest (P<.006).
Bleeding time increased markedly compared with baseline in animals given the high dose of rTFPI (P<.001) and in those given the combination of heparin and aspirin (Fig 4⇓). Bleeding time was not increased significantly in animals given either the low dose of rTFPI or heparin alone. Hematocrit did not change over 48 hours in any of the treatment groups (data not shown).
rTFPI Pharmacokinetics and Blood Levels
The average clearance of rTFPI after a bolus intravenous injection in two pigs was 8.6 mL·min−1·kg−1, with a t1/2 for the α-phase of 1.3 minutes and a t1/2 for the β-phase of 28.8 minutes. Plasma levels during constant intravenous infusions of rTFPI showed a time-dependent increase that reached an apparent maximum concentration of 6.0±4.9 μg/mL after 6 hours in animals given the low dose and 14.3±6.2 μg/mL after 24 hours in animals given the high dose of rTFPI (Fig 5⇓).
The results show for the first time in a large-animal preparation of deep arterial injury that rTFPI, the physiological inhibitor of factor Xa and tissue factor/VIIa, administered intravenously for 24 hours after vascular injury significantly attenuates subsequent formation of neointima and luminal stenosis (Figs 1⇑ and 2⇑). In marked contrast, administration of rTFPI for only 3 hours did not reduce luminal stenosis 1 month later compared with heparin-treated control animals. This implies that inhibition of tissue factor–mediated coagulation induced by initial exposure of tissue factor present in the vessel wall is not sufficient to prevent the formation of neointima. The benefit of a 24-hour administration of rTFPI is consistent with recent observations of increased synthesis of tissue factor in the wall of balloon-injured arteries19 and the resulting increase of procoagulant activity on the luminal surface over the first 24 hours after injury.16 Thus, administration of rTFPI during a critical, early interval when tissue factor is elaborated and available on the luminal surface appears particularly effective for attenuating the formation of neointima leading to stenosis.
The mechanism responsible for attenuation of stenosis in rTFPI-treated animals probably involved reduced local thrombin generation secondary to inhibition of tissue factor/VIIa and factor Xa. Thrombin may promote the formation of neointima by multiple mechanisms, including the activation of platelets, which release mitogens that in turn activate SMCs in the media29 ; conversion of fibrinogen to fibrin, forming thrombus that some have speculated produces a bioabsorbable matrix into which medial SMCs migrate30 ; and stimulation of SMCs by binding to thrombin receptors that are upregulated after vascular injury.31 However, inhibition of thrombin as an approach to limit neointimal formation and stenosis requires high dosages of intravenous antithrombins in experimental animals6 7 that will not be practical to implement clinically. In addition, inhibition of thrombin has failed to yield marked and consistent reductions in luminal stenosis after angioplasty,3 4 5 as confirmed by our results with heparin (Fig 1⇑). Our results with rTFPI support the hypothesis that inhibition of thrombin generation by direct inhibition of “upstream” coagulation factors is more efficient than attempting to inhibit thrombin already formed. Results similar to ours have been reported with direct inhibitors of either factor Xa or VIIa given to rabbits or pigs after balloon-induced arterial injury.32 33 34 A potential advantage of rTFPI is that it inhibits both factor Xa and the generation of factor Xa mediated by tissue factor/VIIa, although direct inhibition of factor Xa may have been short-lived, judging from the transient elevation of aPTT after the bolus injection of rTFPI (Fig 3⇑).
Another mechanism that may account, in part, for reduced stenosis with rTFPI treatment is inhibition of coagulation protease–mediated stimulation of SMCs. Factor Xa has been shown to stimulate mitogenesis in cultured vascular SMCs,35 36 whereas tissue factor binding to VIIa on several cell types has been observed to induce a cytosolic calcium signal that may alter the cell cycle.37 Whether direct inhibition by rTFPI of either factor Xa or tissue factor/VIIa associated with SMCs affects mitogenesis in vivo remains to be elucidated.
It is surprising that a 24-hour infusion of rTFPI was sufficient to attenuate subsequent neointimal thickening, because previous studies have shown that thrombin is elaborated on the luminal surface for >24 hours.16 38 However, because rTFPI inhibits generation of factor Xa, which forms a prothrombinase complex on platelets and consequently inhibits local thrombin generation, the tendency of the luminal surface to accumulate thrombus and recruit inflammatory cells such as monocytes may be actively decreased, thereby accelerating passivation of the surface. An effect of rTFPI on monocyte accumulation may be particularly important in our experimental preparation, because hypercholesterolemia increases the adherence of monocytes and their expression of tissue factor severalfold.23 39 40
Although the higher dose of rTFPI was associated with somewhat less luminal stenosis in the majority of animals compared with the low dose (Fig 1⇑), it was also associated with both progressively increasing PT and bleeding times that exceeded baseline levels by at least fourfold (Figs 3⇑ and 4⇑). This appeared to result from marked accumulation of rTFPI in the circulation (Fig 5⇑). In contrast, the lower dose of rTFPI did not exhibit marked accumulation in the circulation and did not result in either profound elevations of PT or bleeding time, suggesting that it may be within a clinically acceptable range. Importantly, the plasma level of rTFPI after 24 hours in animals given the low dose was in the range of 2 μg/mL, which has been shown in other studies to reflect a pharmacological concentration that inhibits coronary reocclusion after fibrinolysis25 and death after lethal challenge with E coli–induced sepsis in baboons.41 Transient elevations of aPTT after bolus doses of rTFPI (Fig 3⇑) probably resulted from prior administration of heparin, which releases endogenous TFPI from endothelium21 and most likely competes with rTFPI for binding to heparan sulfate on endothelium, providing a higher circulating concentration sufficient to inhibit intrinsic coagulation in the assay. Whether synergy between coadministered heparin and rTFPI contributes to the effect on neointimal formation remains to be defined.
Our animal preparation involved deep and extensive vascular damage similar to that observed after angioplasty-induced plaque rupture in human coronary arteries.23 Deep injury of the arterial wall was confirmed by multiple disruptions of the IEL and a balloon-to-vessel ratio (1.20±0.06) analogous to what others have reported as associated with deep injury to the media.26 One limitation of our study is that we induced injury to a normal artery. Because previous balloon injury and atherosclerosis induce expression of vascular tissue factor,11 19 it is possible that the effects of rTFPI on neointimal formation and stenosis were overestimated in normal arteries. Additional experiments with angioplasty of atherosclerosis-like plaques are needed to confirm the effect of rTFPI on neointimal formation in the presence of upregulated vascular tissue factor.
Platelet deposition and thrombus formation are involved in the complex process leading to restenosis after balloon angioplasty of human coronary arteries.2 Tissue factor exposure and elaboration on the luminal surface of vessels and factor X activation on the surface of platelets appear to play pivotal roles in thrombus formation16 42 as well as the generation of thrombin that contributes to SMC proliferation and migration and secretion of extracellular matrix.30 31 32 Our results show that inhibition of tissue factor–mediated coagulation with rTFPI administered over the first 24 hours after vessel injury is particularly effective for attenuating neointimal formation and stenosis. Considering that a low pharmacological dose of rTFPI also appears to perturb hemostasis minimally, it warrants further study as an approach to attenuate restenosis clinically.
Selected Abbreviations and Acronyms
|aPTT||=||activated partial thromboplastin time|
|IEL||=||internal elastic lamina|
|rTFPI||=||human recombinant tissue factor pathway inhibitor|
|SMC||=||smooth muscle cell|
|TFPI||=||tissue factor pathway inhibitor|
|VIIa||=||activated factor VII|
This study was supported in part by grants HL-42950 and HL-17646, SCOR in Vascular Diseases, from the National Institutes of Health; an Established Investigator Award from the American Heart Association (Dr Wickline); a Postdoctoral Fellowship from the American Heart Association (Dr Recchia); and a Monsanto/Washington University Biomedical Research Grant (Dr Abendschein). The authors thank Gerald Galluppi, PhD, at Monsanto/Searle for providing rTFPI; Mark Palmier, PhD, at Monsanto for analyzing the rTFPI levels in blood samples; Ken Schechtman, PhD, for assistance with the statistical analysis; Pamela Baum and John Engelbach for technical assistance; and Barbara Donnelly for preparation of the manuscript.
Presented in part at the 67th Scientific Sessions of the American Heart Association, Dallas, Tex, November 14-17, 1994, and published in abstract form (Circulation. 1994;90[suppl I]:I-344).
- Received May 20, 1996.
- Revision received January 16, 1997.
- Accepted January 23, 1997.
- Copyright © 1997 by American Heart Association
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