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(Circulation. 1996;93:1542-1548.)
© 1996 American Heart Association, Inc.

Articles

Neointimal Thickening After Severe Coronary Artery Injury Is Limited by Short-term Administration of a Factor Xa Inhibitor

Results in a Porcine Model

Robert S. Schwartz, MD; Daniel J. Holder, PhD; David R. Holmes, Jr, MD; John P. Veinot, MD; Allan R. Camrud; Michael A. Jorgenson; Robert G. Johnson, Jr, MD, PhD

From the Division of Cardiovascular Diseases and Internal Medicine, Mayo Clinic and Foundation, Rochester, Minn, and Departments of Pharmacology and Biometrics Research, Merck Research Laboratories, West Point, Pa.

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
Background Fibrin- and platelet-rich thrombus formations occur as the initial event after percutaneous transluminal coronary angioplasty. We therefore tested the hypothesis that short-term administration of the recombinant tick anticoagulant peptide (rTAP), a factor Xa inhibitor, would reduce the thickness of neointima at 28 days after injury in a porcine coronary balloon angioplasty model.

Methods and Results Continuous intravenous infusion of rTAP (average dose, 194 µg·kg^-1·min^-1) or placebo (vehicle only) was given to the study pigs for 60 hours. The goal of anticoagulation was to maintain the activated clotting time at 200 seconds. A central venous catheter was inserted 2 days before the procedure. On the day of coronary injury, the animals were administered boluses of rTAP (6.5 mg) and then underwent injury with an oversized metallic coil by standard methods in the right, circumflex, or left anterior descending coronary artery. No significant difference in vascular injury between rTAP and vehicle control was observed after euthanasia at 28 days. Significantly less neointimal thickening occurred in the rTAP-treated animals (thickness, mean±SD: 0.30±0.08 mm) compared with the control (0.48±0.12 mm, P<.001).

Conclusions The specific factor Xa inhibitor rTAP, when given in fully anticoagulant doses for a short duration after coronary artery injury in the porcine model, resulted in a long-term decrease in neointimal thickness. These results implicate thrombin generation in neointimal formation and suggest that administration of a potent antithrombotic for several days immediately after the procedure may influence the long-term outcome of the coronary injury with a decrease in neointimal formation.

Key Words: restenosis • angioplasty • peptides

	Introduction

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PTCA is widely accepted as a therapy for coronary artery disease. Unfortunately, restenosis occurs in up to 50% of cases, caused in part by a fibrocellular neointima, and results from the mechanical vessel injury during the procedure. To date, no pharmacological agent or mechanical intervention reliably limits the formation of this hyperplastic response.^{1 2 3 4 5}

The progression from injury to fully healed tissue in the pig coronary artery consists of three phases, comparable to those suggested in humans. These phases are (1) ongoing formation of mural thrombus rich in platelets and fibrin; (2) cellular recruitment of endothelial cells, lymphocytes, macrophages, and smooth muscle cells; and (3) cellular proliferation and extracellular matrix formation.^{6 7 8} The relationship between the early thrombotic elements and the thickness of eventual neointimal hyperplasia is unclear. This study was therefore designed to test whether effective early inhibition of thrombus by use of the specific factor Xa inhibitor rTAP would affect long-term neointimal thickness. rTAP exhibits potent antithrombotic efficacy in a wide range of experimental models and is equal or superior to heparin and hirudin in its action.^{9 10 11 12 13 14 15 16 17 18} rTAP has the additional potential advantage that it can inhibit thrombin generation at the initiation of the cascade (as opposed to inhibition of thrombin activity) and has activity in the prothrombinase complex.^{19 20 21 22 23}

	Methods

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Coronary Artery Injury
The porcine coronary injury angioplasty model was used in these experiments. It was chosen because (1) the coronary artery size and distribution in the pig are similar to those in humans, (2) the morphology of porcine neointima is similar to that in humans, and (3) injury and neointimal response are easily quantified. The procedure has been described elsewhere.^{6 8 24 25 26} Briefly, after general anesthesia with ketamine (12 mg/kg IM) and xylazine (8 mg/kg IM), a ventral neck cutdown was performed under sterile conditions with 10 mL xylocaine (1%). Two days before coronary injury, a 7F indwelling central venous catheter for chronic infusion was inserted and sutured into the superior vena cava via the right internal jugular vein for access. Pigs were premedicated with one dose of aspirin (650 mg) and long-acting verapamil (120 mg) within 24 hours of the procedure. On the day of the coronary injury, the right or left carotid artery was exposed and an 8F hemostatic sheath was placed for arterial access. The animals were given 100% oxygen through a close-fitting mask. A single bolus of rTAP (6.5 mg, treated group) or heparin (10 000 U in the control group) was administered through the sheath. One dose of antibiotic (flocillin 1 g or cephoxitin 1 g) was given immediately after the procedure in all animals.

A commercial coronary balloon angioplasty catheter containing a tantalum metallic stent was then advanced under fluoroscopic guidance into the proximal half of either the left, right, or circumflex coronary artery. The locations of the arteries are shown in Table 1. Oversizing was achieved at a 1.2 to 1.4 ratio of balloon to artery diameter. Inflation of the balloon deployed the stent into the wall of the coronary artery, resulting in arterial injury. Fluoroscopy with contrast injection immediately after balloon inflation confirmed adequate stent expansion and vessel patency. After the carotid sheath was removed, the carotid artery was ligated, the neck wound was meticulously closed with interrupted sutures, and the animals were returned to their quarters for observation. A vest was placed around the animal with a pocket to house a battery-operated constant infusion pump (Pharmacia) connected to the central venous catheter. The animal was able to move freely and normally. The pigs were fed a standard laboratory chow without lipid or cholesterol supplementation.

View this table: [in this window] [in a new window]   Table 1. Location and Severity of Arterial Stent Injuries1 and Neointima

The animal protocol was approved by the Institutional Animal Care and Use Committee of the Mayo Foundation. Pigs weighed 50 to 65 pounds and were 2 to 4 months of age.

Study Design
Preliminary experiments indicated that an rTAP infusion of 3 µg·kg^-1·min^-1 resulted in elevation of the ACT in the range of 195 to 210 seconds, with a bolus of 6.5 mg rTAP used for loading. The ACT of 200 seconds was selected for rTAP to closely mimic a typical clinical setting. All ACT values were obtained by use of the Hemochron ACT measurement device with standardized kaolin commercial ACT tubes for blood by identical methods. Animals were randomized to either the treatment or the control group. For animals randomized to treatment, 10 minutes after initiation of the neck cutdown, a bolus of rTAP (6.5 mg IV) was given, followed immediately by a constant infusion of rTAP. The dose was adjusted to maintain the ACT at 190 to 200 seconds. The ACT was monitored every 30 minutes for the first 6 hours, then six times per day plus additional times if the dose was altered. aPTT values and plasma rTAP concentrations were obtained an average of four times daily. All control animals received saline infusion. The following daily laboratory tests were done twice daily for the first 3 days: white blood cell count, hemoglobin concentration, platelet count, Na⁺, K⁺, creatinine, Ca²⁺, total protein, albumin, glucose, alkaline phosphatase, AST, bilirubin (total), bilirubin (direct), uric acid, and fibrinogen.

Data Analysis
Twenty-eight days after the procedure, the animals were euthanatized by barbiturate overdose. The hearts were immediately removed and pressure perfusion–fixed (at physiological pressure) for 24 hours with 10% neutral buffered formalin. After fixation, the injured coronary artery segments were carefully dissected free, and sections were made at 2-mm intervals perpendicular to the vessel long axis. The stent fragments were removed. Each section was embedded in paraffin, cut, and stained with hematoxylin-eosin and elastic van Gieson's stains. All histopathologic measurements and observations were performed by an experienced observer using calibrated, computerized digital microscopic planimetry. The observer was blinded with regard to treatment or control group. The vessel injury severity and neointimal response were measured from the van Gieson's–stained sections according to previously described methods.^{6 26} Briefly, an ordinal vessel injury score was obtained at each wire site, and the neointimal thickness was measured. Additional measurements were obtained, consisting of the luminal area of the vessel distal to the site of the injury. These points have been shown to be statistically independent when two or three have been taken from different arteries in the same pig.

Statistical analysis was performed by the Merck Research Laboratories statistics group using linear regression analysis of neointimal thickness on the injury score, with the jackknife technique used to assess variability.^{27 28} By this method, the comparison for study was thus on a "per-pig" basis.

Plasma concentration of rTAP was measured by use of purified human factor Xa and the chromogen substrate Spectozyme Xa as described previously.^{14 16}

Chemicals/Reagents
rTAP was prepared as described previously.^{21 29 30} The protein was >98% homogeneous. The rTAP concentration was determined by quantitative amino acid analysis. The chromogenic substrate Spectozyme Xa was from American Diagnostics. ACT measurements were performed on a dual-channel Hemochron model 801 coagulation system. For the ACT values, both channels were used, so that two blood samples were measured simultaneously and the average value was calculated.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Effects on Neointimal Thickness
In the rTAP group, the 6.5-mg rTAP bolus was followed by a constant infusion with the goal of maintaining the ACT at 200 seconds. Table 2 shows that the average rTAP infusion rate was 194±70 µg·kg^-1·h^-1 and the average ACT 189±26 seconds. The corresponding aPTT values were 29±8 seconds in the rTAP group and 20±3.9 seconds in the controls. Table 3 shows the percentage of time spent by the treated group in each ACT range. Fig 1 shows the time course of rTAP levels and corresponding ACT and aPTT values for each rTAP-treated pig. The minimal effect of rTAP on the aPTT has been discussed previously and probably represents slow binding kinetics of rTAP to the exogenous factor Xa in the assay.^{11 19} The average serum rTAP concentration was 274±59 nmol/L. There were no significant differences in the chemistry parameters monitored, white blood cell count, platelet count, or fibrinogen levels. At the dose of rTAP used, slow bleeding was observed around the chronic catheter sites; this is reflected in the average decreased hemoglobin values (8.7±1.4 versus 10.3±1.3 mg/dL) in the rTAP group.

View this table: [in this window] [in a new window]   Table 2. Parameters for rTAP and Control Groups

View this table: [in this window] [in a new window]   Table 3. Percent of Time At, Below, or Above Specified ACT Level (rTAP Group)

View larger version (29K): [in this window] [in a new window]   Figure 1. Relationship between rTAP levels and ACT and aPTT values for rTAP-treated pigs. rTAP infusion rates were immediately adjusted in response to ACT level, in much the same way that infusions of anticoagulants are managed clinically. "Overshoot" and "undershoot" values are thus noted, as seen in this figure.

A total of 19 coronary lesions were made in eight rTAP animals and 15 lesions in six control animals. The relationship between depth of the arterial injury and neointimal thickness was determined for both rTAP and control groups (Fig 2). The striking feature is the segregation of the rTAP data compared with the control group. The measured neointimal thicknesses are smaller in the rTAP-treated group for the same degree of injury.

View larger version (18K): [in this window] [in a new window]   Figure 2. Relationship between neointimal thickness and depth of arterial injury. Neointimal thickness is proportional to depth of injury (measured as an ordinal injury score; see "Methods") in porcine arterial segments from rTAP-treated () and control () animals. For the same degree of arterial injury, the degree of neointimal thickness was less in the rTAP group. Regression analysis is reported in text. Each point plotted represents one lesion.

Table 4 shows neointimal data both unadjusted and adjusted for injury. The arteries of the treated and control groups did not differ in size. Geometric mean neointimal thickness in the rTAP group was 0.30±0.12 mm, versus 0.48±0.20 mm in the control group. Mean injury scores in the rTAP group (1.98±0.37) did not differ from mean injury scores in the control group (1.98±0.41) (Table 4, P=NS). Since linear regression of the logarithm of neointimal thickness on injury score for the rTAP and control groups gave no evidence of differing slopes, the treatments were compared by use of the difference between the intercepts of their regression lines. This method yields an estimate of a mean difference between rTAP and control groups of -36.2%, which is statistically significantly different from 0% (P<.001), and a 95% confidence interval for the true mean difference of -48.8% to -20.4%.

View this table: [in this window] [in a new window]   Table 4A. Neointimal Thickness (in Millimeters): Comparison of rTAP and Control Vessels Unadjusted for Injury (Estimates of SD Between and SD Within Based on the Restricted Maximum Likelihood [REML] Method)

View this table: [in this window] [in a new window]   Table 4B. Neointimal Thickness Comparison of rTAP and Control Vessels Adjusted for Injury: Log(NIT)=Intercept+bx(Injury Score)+Treatment+Between-Animal Effect+Within-Animal Error (Estimates Based on the REML Method)

No trend was found between average neointimal thickness for each pig and its average ACT. The data on plasma rTAP level showed a possible correlation with rTAP level, shown by linear regression of average neointimal thickness on plasma rTAP level (P=.053 adjusted for injury score; P=.063 unadjusted).

Plots of cumulative percentage injury scores were similar (Fig 3). This enabled neointimal thickness to be represented also as a function of the cumulative percentage. This graph indicates that over the entire range, neointimal thickness was inhibited in the group receiving rTAP. Luminal diameters and areas at sites distal to the injury were measured (Table 4) for controls. The average distal uninvolved diameter and area did not differ between the control and treatment groups. Fig 4 shows representative sections of both groups.

View larger version (15K): [in this window] [in a new window]   Figure 3. Graph of cumulative percentage for injury scores (left) and neointimal thicknesses (right) for rTAP group () and controls (). There was no significant difference between injury scores in the two groups; however, a 36% decrease in neointimal thickness was observed in the rTAP group.

View this table: [in this window] [in a new window]   Table 4C. Mean Injury Score and Dimensions of rTAP and Control Vessels (Estimated Based on the REML Method)

View larger version (241K): [in this window] [in a new window]   Figure 4. Photomicrographs showing representative sections of artery from control groups (circumflex coronary artery, No. 6 in Table 1, top) and rTAP (right coronary artery, No. 13 in Table 1, bottom). These sections were chosen to be presented because they have comparable injury scores (2.3). Although these particular arteries are different sizes, the mean size of all arteries did not differ across groups, documented in Table 4 (elastic van Gieson's stain, x10).

The neointimal area was also measured by calibrated digital planimetry. There was an excellent correlation between neointimal area and neointimal thickness (data not shown). Differences between the neointimal area in the two groups were also highly significant. This decrease in neointimal formation is reflected in the improvement in chronic loss for the rTAP group compared with the control.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
rTAP is a 60-amino-acid polypeptide isolated from the soft tick Ornithodoros moubata.²² It is a highly specific and potent inhibitor of the serine protease factor Xa, with a K_i of 180 pmol/L.¹⁹ An intriguing and potentially clinically useful property is that rTAP can inhibit factor Xa already assembled in the prothrombinase complex. The affinity of rTAP for factor Xa in the prothrombinase complex may be greater than that for the free enzyme. In the presence of factor Va, the K_i of rTAP for factor Xa was measured to be 6 pmol/L.²² Thus, rTAP possesses several significant advantages. rTAP effectively inhibits thrombin generation by binding factor Xa; this mechanism is different from that of heparin, which activates antithrombin III– and heparin cofactor II–mediated inactivation of free thrombin but does not effectively inhibit thrombin bound to fibrin. rTAP also blocks an early step in the cascade and thus inhibits the generation of thrombin rather than the activity of thrombin already present. Finally, rTAP bound to factor Xa is long lasting, suggesting that short-term rTAP therapy may achieve long-term inhibition at the site of arterial injury.³¹

The earliest event after PTCA is the appearance of a platelet- and fibrin-rich thrombus at the site of the injury.⁷ It is possible that thrombin and fibrin formations directly correlate with neointimal thickness. One proposed mechanism is that thrombin is a potent platelet aggregation secretagogue and is therefore responsible for deposition of platelet growth factors at the site of the injury. Thrombin itself has been proposed to be a smooth muscle cell mitogen. In addition, the organizing fibrin clot attracts macrophages, lymphocytes, and neutrophils, all containing factors that may contribute to neointimal formation.

Few large prospective studies of antithrombotics have been reported.^31A Several small clinical restenosis trials have been published. They have been reported as having no impact on neointima.^{30 32 33} However, it is possible that the failure of traditional anticoagulants to limit hyperplasia is a result of their inability to eliminate circulating or clot-bound thrombin. Other possibilities include inadequate dosing, insufficient duration of administration, and inadequate route of administration (ie, subcutaneous versus intravenous).

Beneficial Action of rTAP on Decreasing Neointimal Formation
The rationale for this investigation was to administer a potent antithrombotic at a high dosage to establish whether inhibition of mural thrombus resulted in a quantitative decrease in chronic neointimal thickness. The ACT was chosen as the primary clotting assay because preliminary studies indicated that lower rTAP infusion rates were required over time to maintain a constant serum rTAP concentration and that rTAP is relatively insensitive in the aPTT assay because of slow binding properties. Heparin activity in humans correlates with the ACT; the ACT is now routinely used as a bedside monitor of coagulation status.^{34 35} It was possible to maintain the ACT at roughly 200 seconds with an rTAP infusion and serum rTAP concentration of about 250 nmol/L. When the ACT exceeded 200 seconds for longer than 30 minutes, slow bleeding was observed at the catheter sites.

The metal stent used in this pig model creates a reliable arterial injury that is readily quantified. This is an important aspect of the arterial response, since the extent of injury determines the thickness of neointimal hyperplasia. In the absence of the stent, the degree of neointima formed is highly variable even in the control group, presumably not only because of individual differences in the degree of injury but also because of individual responses in each animal.⁶ Reports of two major clinical studies showed that coronary stents reduce long-term loss of minimal luminal diameter, presumably through gaining a larger initial lumen that forms immediately after angioplasty. These studies strongly suggest that more neointima forms in the stented groups than in the balloon angioplasty groups.^{36 37}

A number of important findings resulted from this study. First, treatment with the potent antithrombotic agent rTAP caused a substantial reduction in neointimal thickness at 28 days. This finding is consistent with previous observations that mural fibrin deposition is an early event in neointimal formation.^{38 39 40} We have previously shown that mural fibrin at the site of arterial injury is colonized first by inflammatory cells and later by smooth muscle cells leading to mature neointima.⁸ Factor Xa and thrombin have both been implicated as mitogens.⁴¹ According to this paradigm of neointimal formation, reduction of the volume of fibrin should lessen the amount of neointima. Recent data suggest that in atherectomy specimens in culture, the degree of thrombus correlates with the amount of outgrowth.⁴² Based on the results of this study, the paradigm is at least partially correct.

The effect of rTAP on neointimal formation has been studied in one other animal model, the rabbit femoral artery atherosclerotic balloon angioplasty model.⁴³ When rTAP was given at very high doses (1.0 mg/kg bolus followed by 1 hour of 2.2 mg·kg^-1·h^-1 and 1 hour of 1.1 mg·kg^-1·h^-1, a total of 2 hours), there was a trend toward less cross-sectional narrowing (P=.07). In this case, the plasma rTAP concentration at the end of the 2-hour infusion was 1054±239 nmol/L.

Clinical Implications
A potentially important finding from this study is that early intervention for less than 3 days after coronary injury can have long-term effects on neointimal hyperplasia. This observation has implications for preventing restenosis, because systemic drug therapy at the time of angioplasty may be able to limit later neointimal hyperplasia. The mean difference in neointimal thickness between the rTAP and control groups was 0.18 mm, corresponding to 0.36 mm in diameter increase. The increase in minimum luminal diameter in the BENESTENT study in the stent group compared with the balloon group was 0.12 mm at follow-up (1.85 mm stent group versus 1.73 mm balloon group).⁴⁴ This apparently small difference in minimum luminal diameter resulted in a marked difference in restenosis rates (33% versus 20% in the stent and balloon groups, respectively). These differences resulted in comparable differences in clinical events. Small mean differences in neointimal thickness may thus have important clinical effects.

Limitations of the Study
The primary conclusion of this study is that rTAP, when given at the indicated dose for 60 hours, effected a decrease in neointima formation and is the first agent to do so unequivocally in this model. Several caveats are necessary. First, the ACT was chosen as the anticoagulant measurement, since the aPTT is insensitive to rTAP dose because of its slow binding kinetics. Clinically, the ACT is being used increasingly to determine anticoagulant status rapidly for procedures such as sheath removal after interventional procedures. Clinical bleeding is more likely when the ACT is >200 seconds for prolonged time periods. The ACT is a parameter with nonspecific, general implications for the overall hemostatic state. This choice was based on empirical clinical observation that this is a comparatively safe level for the short term in terms of few bleeding complications in patients. Previous studies in domestic pigs indicated that ACT levels of 200 seconds for short periods of time are not associated with bleeding. The fact that bleeding was observed when the ACT remained >200 seconds for both rTAP and heparin indicates that this was probably a reasonable choice. Although an rTAP dose of this magnitude has the potential to result in adverse events when given systemically, local (ie, intracoronary) administration might be similarly efficacious.

The stent injury model is well established as a restenosis model for neointima forming after injury. Laceration of the internal elastic lamina uniformly results in neointima. It is uncertain whether arterial injury by balloon angioplasty preceding the stent placement would create additional injury, which might alter the neointimal response and its duration and the subsequent findings of this study.

Finally, the ability to correctly extrapolate the drug results from animal restenosis models to human restenosis studies has not been established. We used the 28-day end point because there is little evidence that additional neointima forms after this time in the pig. Data from the drug studies in the rat carotid angioplasty model have not been predictive of the outcome in human clinical trials.⁴ Extrapolation from the pig coronary model to humans is currently uncertain. This model uses the muscular coronary arteries in a species in which the anatomic distribution is similar to that in humans. Lesions are induced with methods similar or identical to human interventions. Progression and morphology of the porcine neointima are nearly indistinguishable from those in humans. Drugs tested in human clinical trials that have failed to favorably impact restenosis similarly demonstrate little difference in this animal model.

Conclusions
This study used a potent anticoagulant for 60 hours after experimental coronary arterial injury in an established porcine coronary balloon angioplasty model to demonstrate lasting effects at 28 days on neointimal volume. The degree of reduction was enough to have clinically significant effects if comparable reductions could be obtained in patients undergoing angioplasty.

	Selected Abbreviations and Acronyms

 

ACT = activated clotting time aPTT = activated partial thromboplastin time PTCA = percutaneous transluminal coronary angioplasty rTAP = recombinant tick anticoagulant peptide

	Acknowledgments

 
This work was supported by Merck Research Laboratories and the J. Holden DeHaan Foundation. Many thanks to E. Mayer for assay of the rTAP concentrations and to B. Huckle for critical reading of the manuscript. Also thanks to Dale Lehman for furnishing the rTAP.

	Footnotes

 
Reprint requests to Robert S. Schwartz, MD, E-16B, Division of Cardiovascular Diseases, Mayo Clinic, Rochester, MN 55905.

Received July 27, 1995; revision received October 12, 1995; accepted October 16, 1995.

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