After successful percutaneous transluminal coronary angioplasty (PTCA), 25–40% of patients develop a restenosis . Activation of coagulation during PTCA can play a role in promoting acute complications and, later, restenosis. Thrombin plays an important role in the activation of platelets and the coagulation system and also reduces the early thrombotic response to injury [2,3]. Selective inhibition of thrombin has been shown to cause a reduction in the occurrence of formation of neointima after various types of injury to the peripheral artery in animals [4–6].
Antithrombin is an endogenous protein (of molecular mass 58 000) that, together with heparin or heparin-like glucoseaminoglycans, inhibits thrombin effectively. In addition, it exerts an inhibitory effect on several other coagulation factors . There is also evidence suggesting that it exerts an anti-inflammatory effect [8–10]. Antithrombin has a high affinity for subendothelium and for glucoseaminoglycans exposed after injury of tissue . This might enhance the antithrombotic action at the site of an arterial injury. Frebelius et al.  have shown that inhibition of thrombin with antithrombin on injured aortic segments was more effective than was inhibition with heparin.
To overcome problems associated with insufficient action of drugs at the target, the concept of local delivery has been developed. With local delivery it is possible to achieve a high concentration of drugs in the arterial wall and in surrounding tissue at the time of the injury . In a previous study, we showed that local delivery of antithrombin attenuates deposition of platelets after balloon injury to porcine coronary vessels . It has been demonstrated that decreasing formation of thrombus results in a decrease in proliferation of neointima after coronary angioplasty . Antithrombin in supraphysiologic systemic doses was effective at reducing restenosis after balloon injury in atherosclerotic porcine coronary vessels . The aim of the present study was to investigate the effects on various arterial wall layers 4 weeks after overstretching injury and local delivery of antithrombin to the coronary vessel in pigs.
Methods and material
This investigation conforms to the Guide for the care and use of laboratory animals published by the USA's National Institute of Health (NIH publication 85-23, revised in 1985). Approval from the Göteborg animal ethics committee was obtained. Sixteen normally fed Swedish landrace swine with an average weight of 30.8 kg (range 28.2–35.0 kg) aged 2–3 months were used. The animals were administered 325 mg aspirin orally once a day 3 days prior to catheterization
The pigs were sedated with an intramuscular injection of Ketalar (50 mg/ml ketamine; Parke Davis, Morris Plains, New Jersey, USA). After 10–15 min a venous canula was inserted and an intravenous injection of a combination of Stressnil (Janssen Pharmaceutical, Beerse, Belgium) and Hypnodil (Lancaster Synthesis, Morecombe, UK) was administered to achieve anesthesia and analgesia. To avoid postoperative complications, the pigs were not intubated. High doses of oxygen were administered. Anesthesia was maintained by intermittent injection of Stressnil and Hypnodil. Prior to catheterization, a bolus dose of 2 mg/kg lidocain was administered intravenously.
A sheath was inserted into the left carotid artery and a bolus dose of 200 U/kg heparin was administered. Angiography of the left coronary artery was then performed using Philips fluoroscopy equipment (Philips Medical Systems, Eindhoven, The Netherlands) and sodium ioxaglat contrast agent. The size of the mid-left anterior descending artery (LAD) segment was estimated by a semiquantitative method involving use of calipers. The guiding catheter served as a reference. For overstretching injury and local delivery a Transport catheter (Scimed Corporation, Maple Grove, Minnesota, USA) was used. The catheter consists of a conventional PTCA balloon, which is used for dilatation of the vessel. The balloon is covered by a membrane with approximately 30 pores 250 μm in diameter, through which a drug can be delivered.
A deep vessel-wall injury was inflicted on the mid-LAD using a balloon : artery diameter ratio of 1.5. The balloon was inflated three times for 15 s using 6–10 atm of pressure. It was then kept in the same position and, after approximately 2 min, the inner balloon was inflated to 3 atm and local delivery was performed. In a randomized, blinded fashion, a local delivery of 2.5 ml of either 50 U/ml antithrombin (Pharmacia Upjohn, Stockholm, Sweden; group A, n = 8) or 2 mg/ml albumin (Pharmacia Upjohn; group B, n = 8) was administered over an average duration of 31 s (range 25–40 s) using 3 atm pressure. The procedure was repeated after 2–4 min (i.e. the pigs were administered either 250 U antithrombin or 10 mg albumin). Intracoronary administration of nitroglycerine was performed if spasms occurred. The dose of antithrombin was chosen as the highest feasible using standard concentration in isotonic solution. Furthermore, to minimize the risk of ventricular fibrillation, it was desirable to limit the duration of delivery. To reduce the risk of widespread dissections, the volume administered was kept to 5 ml in total. Immediately after the procedure, the final result was assessed by a control session of coronary angiography. The balloon, guiding catheter and sheath were then removed, the neck wound was closed, and the pigs were allowed to awaken. We administered 1 g of cloxacillinnatrium (Astra, Södertälje, Sweden) intramuscularly to prevent postoperative infection. The animals were then put in stalls and fed normal grain. We administered 325 mg aspirin once a day throughout the study period.
After 31.7 ± 2.9 days, the animals were killed with an overdose of Stressnil and potassium chloride. The LAD was fixed by in-situ antegrade perfusion with 4% formaldehyde for 10 min. The injured area was identified by analyzing the angiogram recorded at the time of angioplasty. The vessel was carefully harvested and the injured segment was removed together with a 2 mm-long uninjured segment at each end. The fixed specimens were cut in half and the proximal and distal segments were dehydrated in a graded series of alcohol–xylene solutions and imbedded in paraffin while maintaining orientation. Sections 5 mm long were obtained from two levels of each proximal and distal segment, corresponding to the middle 5 mm of the injured area. These sections were stained with a slightly modified Russel–Movat pentachrome stain , which clearly reveals growth of neointima and the internal and external elastic laminae. On video-registered images of one section from each of the four levels, the following structures were traced interactively (Fig. 1
): the outer contour of the adventitia, the outer elastic laminae with rupture if rupture had occurred, the inner elastic laminae with rupture if it had occurred, the media, and the neointima. Using computer-assisted image analysis (KS400 Zeiss; Zeiss, Jens, Germany), the areas of each vascular wall layer as well as percentage area stenosis, defined as 100 [1 – lumen area/(lumen area + area of neointima)], were calculated. To evaluate injury, the percentage rupture of the internal and external elastic laminae of the entire circumference was measured. A primary analysis was performed for the section with the smallest lumen from each pig. Since this could be sensitive to variation within the 2 cm-long injured segment, a secondary analysis was performed on the mean of the four prepared sections from each pig. The investigator performed these analyses without knowledge of which treatment had been applied to each pig.
Data are presented as means ± SD. A Mann–Whitney U test was used to compare histomorphometric parameters of these two groups. P < 0.05 was considered statistically significant.
The LAD of 16 pigs were dilatated and treated by local delivery of antithrombin or placebo. No animal died during follow-up. Immediately after the procedure normal flow was maintained in all vessels. No dissection was seen. There was no significant difference between the groups concerning baseline and procedural characteristics (Table 1
). The time that elapsed between performance of the procedure and killing of pigs was equal for these two groups. A deep vessel-wall injury was found in all vessels. As a measure of injury, the percentage rupture of the internal elastic lamina was measured and found to be similar for these two groups, namely 26.0 ± 7.4% for pigs in the antithrombin group versus 25.2 ± 8.0% for pigs in the control group (NS). The area of the neointima, but not that of the media, correlated significantly to the extent of the rupture of the internal elastic lamina (2a, Fig. 2b
). The mean area stenosis was 36.5 ± 14.9% for pigs in the antithrombin group versus 35.4 ± 16.2% for pigs in the control group (NS). The results of the histomorphometric measurements for the section with the minimal lumen area are presented in Fig. 3
. We found no significant difference between the groups. Histomorphology revealed that wall-layer thicknesses in pigs in these two groups were also similar when the means of all four sections for each pig for these two groups were analyzed (Fig. 4
), although, if anything, the area of media tended to be larger for pigs in the antithrombin group (1.06 ± 0.24 versus 0.89 ± 0.13 mm2). To further assess this tendency, we performed a cell count in the media. The densities of nuclei were 107.8 ± 9.8/100 000 pixels2 for pigs in group A and 114.8 ± 12.4/100 000 pixels2 for pigs in group B (NS).
Thrombus occurs early at the site of injury after balloon angioplasty . The role of thrombin as an important contributor to occurrence of this phenomenon is well known . Thrombin also mediates proliferation and migration of smooth muscle cells . Authors of several studies have demonstrated that thrombin inhibitors can inhibit platelet-mediated formation of thrombus after balloon angioplasty, irrespective of whether they are administered locally or systemically [13,20–22]. The specific thrombin inhibitors recombinant desulfatohirudin and bivalirudin have also proven effective at inhibiting restenosis in experimental models [4–6,23]. These facts support the theory that activation by thrombin and formation of thrombus contribute to formation of neointima and narrowing of lumen after coronary angioplasty. It has also been suggested that inflammatory elements influence formation of neointima after coronary placement of stents in pigs .
Antithrombin is an effective inhibitor of thrombin in the presence of heparin and of heparin-like aminoglucans . It binds to injured subendothelium  and results of recent studies indicate that it also has anti-inflammatory properties [8–10]. Antithrombin inhibits proliferation of cultured human smooth muscle cells . The half-life of its biologic activity is relatively long, 3–8 days, which could decrease rebound activation of coagulation below that associated with other thrombin inhibitors . In a previous study we demonstrated that delivery of 250 U antithrombin locally to the coronary vessel wall significantly attenuated deposition of platelets after balloon injury, compared with placebo. It was also shown that antithrombin is retained in the media 1 h after delivery .
In this study we investigated effects 4 weeks after local delivery of antithrombin and balloon injury to the porcine coronary vessel. We found no significant difference between effects on percentage area stenosis and vessel-wall morphology for the two groups. A trend toward a larger area of media in pigs in the antithrombin group was seen. Possible explanations for this lack of effect are discussed below.
Thrombin need not play the key role in the process of restenosis
In clinical trials both desulfatohirudin and bivalirudin have been tested in connection with PTCA in patients with unstable angina [27,28]. Both substances were demonstrated to cause clear reductions in incidence of acute events but no sustained effects. Results of experimental studies have been ambiguous. In a study by Sarembrock et al.  desulfatohirudin reduced restenosis in a double-injury model of atherosclerotic femoral arteries in the rabbit. This effect, however, can in part be explained by the fact that three arteries in the control group were totally occluded, versus none in the desulfatohirudin group. This could represent an acute, rather than a long-term, effect. Furthermore, long-term subcutaneous infusion of desulfatohirudin into cholesterol-fed rabbits did not inhibit proliferation of neointima in the subclavian artery after angioplasty . The role of organization of thrombus as a contributor to formation of neointima has been questioned .
Too short-lasting an effect
The pharmacokinetics of antithrombin that has been delivered locally to coronary arteries has thus far not been investigated; however, retention of agents administered locally to the vessel wall has been a problem. Fram et al.  showed that locally delivered heparin is very rapidly released from the vessel wall during the first 60 min after its delivery and that only very small amounts persisted 7 days after delivery.
The importance of sustained inhibition of thrombin was demonstrated by Gallo et al. , who found that activation of thrombin occurred 2 weeks after coronary angioplasty in pigs, and showed that intravenous administration of desulfatohirudin during the same period significantly reduced restenosis compared with that in controls . This was further emphasized by Thome et al. , who demonstrated that early plus late administration of desulfatohirudin to atherosclerotic rabbits reduced restenosis more than did early administration alone. These results indicate that sustained inhibition of thrombin is of importance in order to obtain an effect on restenosis.
Dosing and route of administration
We know from our previous work that, with this dose of antithrombin and this route of administration, deposition of platelets after balloon injury is attenuated, and antithrombin can be detected in the media 1 h after delivery of a drug . However, the drug is found only in the most superficial layers of the media. Proliferation and migration of smooth muscle cells and production of extracellular matrix contribute to restenosis and all vessel-wall layers take part in this process . It would therefore be desirable to infiltrate the entire vessel wall with the drug. Furthermore, the anti-inflammatory properties of antithrombin are probably dose-dependent and recent experimental results have suggested that systemic administration of 250–500 U/kg is needed in order to obtain the anti-inflammatory effects of antithrombin [9,10]. Local delivery has proven effective at achieving high concentrations of drugs at the site of angioplasty . However, from our previous trial, we know that, with this method, the greater part of the drug leaks out into the circulating blood, giving a rise in plasma concentration of antithrombin of approximately 10% . Ali et al.  demonstrated that inhibition of coronary restenosis is achieved with systemic administration of 125 U/kg antithrombin to atherosclerotic swine. This dose resulted in a rise in functional activity of antithrombin to 250–300% above normal. These facts indicate that a higher concentration and dose of antithrombin than were used in this study, more efficient local delivery, or both, would be needed in order to achieve an effect on remodeling of the vessel wall.
Limitations of our study
This study was performed on healthy, juvenile pigs, so it is possible that the results are not applicable to human atherosclerotic vessels. This model, however, is widely used and accepted and closely mimics the proliferative portion of human restenosis . In this study, the pigs were killed after 4 weeks. Restenosis in humans is known to continue for up to 6 months after PTCA, which is why it can also be argued that changes may occur after the first month in a pig model. It has, however, been demonstrated that, in a model using the juvenile pig, the proliferation halts after approximately 4 weeks . Consequently this model is widely used for the study of restenosis [5,16,23].
The number of pigs in this study was small and hence the possibility of a type-II statistical error is evident. The magnitude of any such effect was, however, not known which is why this study was designed as pilot study. The result showed that, if anything, there was a tendency toward thicker wall layers after treatment with antithrombin. Thus, the possibility of a sample-size effect was deemed unlikely.
In conclusion, the same dose and route of administration of antithrombin that had previously proven effective at reducing deposition of platelets after balloon injury did not in this study affect the process of remodeling after deep balloon injury to porcine coronary vessels. Too low a dose, ineffective delivery, and insufficient retention of the drug may be invoked to explain this lack of effect.
We would like to thank Mikko Klangh RN, Maggie Evaldsson (medical laboratory technician), and Sten Holm PhD for their valuable assistance.
1. Popma JJ , Topol EJ . Factors influencing restenosis
after coronary angioplasty
. Am J Med 88: 1990; 1N–16N.
2. Chesebro J , Badimon L , Fuster V . Importance of antithrombin
therapy during coronary angioplasty
. J Am Coll Cardiol 17: 1991; 96B–100B.
3. Walters TK , Gorog DA , Wood RFM . Thrombin generation following arterial injury is a critical initiating event in the pathogenesis of the proliferative stages of the atherosclerotic process. J Vasc Res 31: 1994; 173–177.
4. Sarembock IJ , Gertz SD , Gimple LW , Owen RM , Powers ER , Roberts WC . Effectiveness of recombinant desulphatohirudin (CGP 39393) in reducing restenosis
after balloon angioplasty
of atherosclerotic femoral arteries in rabbits. Circulation 84: 1991; 232–243.
5. Abendschein DR , Recchia D , Meng Yuan Yuan , Oltrona L , Wickline SA , Eisenberg PR . Inhibition of thrombin attenuates stenosis after arterial injury in minipigs. J Am Coll Cardiol 28: 1996; 1849–1855.
6. Sarembrock IJ , Gertz SD , Thome LM , McCoy KW , Ragosta M , Maraganore JM et al. Effectiveness of hirulog in reducing restenosis
after balloon angioplasty
of atherosclerotic femoral arteries in rabbits. J Vasc Res 33: 1996; 308–314.
7. Beresford CH , Owen MC . Minireview—antithrombin
III. Int J Biochem 22: 1990; 121–128.
8. Nishino A , Suzuki M , Ohtani H , Motohashi O , Umezawa K , Nagura H et al. Thrombin may contribute to the pathophysiology of central nervous system injury. J Neurotrauma 10: 1993; 167–179.
9. Okada Y , Zuo XJ , Marchevsky AM , Nicolaidou E , Toyoda M , Matloff JM . Antithrombin
III treatment improves parameters of acute inflammation in highly histoincompatible model of rat lung allograft transplantation. Transplantation 67: 1999; 526–528.
10. Dickneite G , Leithauser B . Influence of antithrombin
III on coagulation and inflammation in porcine septic shock. Arterioscler Thromb Vasc Biol 19: 1999; 1566–1572.
11. Nydahl S , Frebelius S , Swedenborg J . Inhibition of thrombin on subendothelium. Eur J Surg Res 21: 1989; 287–295.
12. Frebelius S , Hedin U , Swedenborg J . Thrombogenicity of the injured vessel wall—role of antithrombin
and heparin. Thromb Haemost 1: 1994; 147–153.
13. Meyer BJ , Fernandez-Ortiz A , Mailhac A , Falk E , Badimon L , Michael AD et al. Local delivery
of Hirudin by a double-balloon perfusion catheter prevents mural thrombosis and minimizes platelet deposition after angioplasty
. Circulation 90: 1994; 2474–2480.
14. Scherstén F , Björnheden T , Emanuelsson H , Wiklund O , Grip L . Local delivery
inhibits platelet deposition after balloon injury in a porcine coronary model. Coron Artery Dis 9: 1998; 823–829.
15. Unterberg C , Sandrock D , Nebendahl K , Buchwald AB . Reduced acute thrombus formation results in decreased neointimal proliferation after coronary angioplasty
. J Am Coll Cardiol 26: 1995; 1747–1754.
16. Ali MN , Mazur W , Kleiman NS , Rodgers GP , Abukhalil JM , French BA et al. Inhibition of coronary restenosis
III in atherosclerotic swine. Coron Artery Dis 7: 1996; 851–861.
17. Fuster V , Falk E , Fallon JT , Badimon L , Cheseboro JH , Badimon JJ . The three processes leading to post PTCA restenosis
: Dependence on the lesion substrate. Thromb Haemost 14: 1995; 552–559.
18. Eidt JF , Allison P , Noble S , Ashton J , Golino P , McNatt J et al. Thrombin is an important mediator of platelet aggregation in stenosed canine coronary arteries with endothelial injury. J Clin Invest 84: 1989; 18–27.
19. McNamara CA , Sarembrock IJ , Grimple LW , Coughlin SR , Owens GK . Thrombin stimulates proliferation of cultured rat aortic smooth muscle cells by a proteolytically activated receptor. J Clin Invest 91: 1993; 94–98.
20. Mitchel JF , Azrin A , Fram DB . Inhibition of platelet deposition and lysis of intracoronary thrombus during balloon angioplasty
using hydrogel-coated balloons. Circulation 90: 1994; 1979–1988.
21. Azrin MA , Mitchel JF , Fram DB . Decreased platelet deposition and smooth muscle cell proliferation following intramural heparin delivery with hydrogel-coated balloons. Circulation 90: 1994; 433–441.
22. Buchwald AB , Sandbrock D , Unterberg C , Ebbecke M , Nebendahl K , Lüders S et al. Platelet and fibrin deposition on coronary stents in minipigs: effect of hirudin versus heparin. J Am Coll Cardiol 21: 1993; 249–254.
23. Gallo R , Padurian A , Toschi V , Bischler J , Fallon JT , Cheseboro J et al. Prolonged thrombin inhibition reduces restenosis
after balloon angioplasty
in porcine coronary arteries. Circulation 97: 1998; 581–588.
24. Miller DD , Karim MA , Edwards WD , Schwartz RS . Relationship of vascular thrombosis and inflammatory leukocyte infiltration to neointimal growth following porcine artery stent placement. Atherosclerosis 124: 1996; 145–155.
25. Hedin U , Frebelius S , Sanches J , Ryjski M , Swedenborg J . Antithrombin
III inhibits thrombin-induced proliferation in human arterial smooth muscle cells. Arterioscler Thromb 14: 1994; 254–260.
26. Schwarts RS , Bauer KA , Rosenberg RD , Kavanaugh EJ , Davies DC , Bogdanoff DA . Clinical experience with antithrombin
III concentrate in treatment of congenital and acquired deficiency of antithrombin
. Am J Med 87: 1989; 53S–60S.
27. Serruys PW , Herrman J-PR , Simon R , Rutsch W , Bode C , Laarman G-J et al. Comparison of Hirudin with Heparin in the prevention of restenosis
after coronary angioplasty
. N Engl J Med 333: 1995; 757–763.
28. Bittl JA , Strony J , Brinker JA , Ahmed AH , Meckel CR , Chaitman BR et al. Treatment with bivalirudin (Hirulog) as compared with heparin during coronary angioplasty
for unstable or post infarction angina. N Engl J Med 333: 1995; 764–769.
29. Hadoke PW , Wadsworth RM , Wainwright CL , Butler KD , Giddings MJ . Subcutaneous infusion of r-hirudin does not inhibit neointimal proliferation after angioplasty
of the subclavian artery in cholesterol-fed rabbits. Coron Artery Dis 7: 1996; 599–608.
30. Maeng M , Grönning Olesen P , Emmertsen NC , Thorwst M , Toftegaard Nielsen T , Östergaard Kristensen B et al. Thrombus organization plays no major role in late neointimal formation after angioplasty
in porcine coronary arteries. Cardiovasc Pathol 8: 1999; 121–123.
31. Fram DB , Mitchel JF , Azrin MA , Chow MS , Waters DD , McKay RG . Local delivery
of heparin to balloon angioplasty
sites with a new angiotherapy catheter: Pharmacokinetics and effect on platelet deposition in the porcine model. Cathet Cardiovasc Diagn 41: 1997; 275–286.
32. Thome LM , Gimple LW , Bachhuber BG , McNamara CA , Gertz SD , Powers ER et al. Early plus delayed hirudin reduces restenosis
in the atherosclerotic rabbit more than early administration alone: potential implications for dosing of antithrombin
agents. Circulation 98: 1998; 2301–2306.
33. Schwartz RS , Murphy JG , Edwards WD , Camrud AR , Vliestra RE , Holmes DR . Restenosis
after balloon angioplasty
practical proliferative model in porcine coronary arteries. Circulation 82: 1990; 2190–2200.
34. Hardhammer PA , van Beusekom HM , Emanuelsson HU , Hofma SH , Albertsson PA , Verdouw PD et al. Reduction in thrombotic events with heparin-coated Palmaz–Schatz stents in normal porcine coronary arteries. Circulation 93: 1996; 423–430.