International Journal of Cardiology, 36 (1992) 41-47 0 1992 Elsevier Science Publishers B.V. All rights reserved
CARD10
41 0167-5273/92/$05.00
01464
The effect of antiplatelet therapy on platelet accumulation after experimental angioplasty in the rabbit iliac model David P. Faxon, Lynn Anne Balelli, Timothy Sandborn, Christian Haudenschild, Robert Valeri and Thomas J. Ryan EL:ans Memorial Department of Clinical Research and Department of Medicine. Uniuersity Hospital, Boston Unirlersity Medical Center, The Mallory Institute of Pathology and The Natal Blood Research Laboratory, Boston, MA, USA (Received
23 July 1991; revision
Faxon DP, Balelli LA, Sandborn T, Haudenschild therapy on platelet accumulation after experimental 1992:36:41-47.
accepted
23 December
1991)
C, Valeri R, Ryan TJ. The effect of antiplatelet angioplasty in the rabbit iliac model. Int J Cardiol
Antiplatelet therapy is routinely used in balloon angioplasty. Yet recent clinical trials have not shown these agents to reduce the incidence of restenosis. As this might be due to inadequate antiplatelet effect, this study was designed to evaluate the effectiveness of antiplatelet therapy in reducing platelet accumulation immediately after angioplasty. Sixty-six atherosclerotic rabbits underwent angioplasty of a focal (50-99%) iliac stenosis after pretreatment with an antiplatelet drug. The animals were randomly given aspirin (10 mg/kg orally) (n = 101, aspirin (10 mg/kg intravenously) (n = 7), aspirin (10 mg/kg per OS) plus dipyridamole (25 mg orally) (n = 8), a thromboxane synthetase inhibitor (GS13080, 1 mg/kg/h intravenously) (n = 9) or heparin (500 U/kg intravenously) (n = 9). Platelet accumulation, determined by “Cr-labeled platelet counts 30 min after angioplasty disclosed a significant reduction from control (44.1 + 51.2 platelets/cm x 106) with intravenous aspirin (3.3 f 1.9; p < 0.007) and the thromboxane synthetase inhibitor CGS13080 (15.1 + 16.6, p < 0.04). Aspirin plus dipyridamole reduced platelet accumulation as well (10.4 f 9.8, p = 0.076), while oral aspirin (109.5 -t 240) and heparin (34.7 + 35.5) were ineffective. The results of this study demonstrate that pretreatment with antiplatelet agents can reduce platelet accumulation significantly after experimental angioplasty. However, the wide variability in effectiveness particularly with oral aspirin may have important clinical implications. Further investigation into more effective antiplatelet therapy may help to reduce the incidence of abrupt vessel closure and restenosis. Key words: Restenosis; Antiplatelet
therapy; Angioplasty
Correspondence to: D.P. Faxon, M.D., The University Hospital, Section of Cardiology. 88 East Newton Street, Boston, MA 02118. USA. Grant No. 85-1028 of the American Heart Association.
Introduction Empiric therapy with antiplatelet drugs is routinely used for angioplasty in hopes of preventing
42
immediate abrupt closure of the vessel and reducing the incidence of restenosis [l]. While clinical trials have demonstrated the effectiveness of aspirin in preventing abrupt closure, there is no conclusive evidence that these agents reduce the incidence of restenosis [2-41. We have previously shown that platelet aggregation at the site of experimental angioplasty occurs immediately after the procedure reaching a maximum at 4 h with little further aggregation by 6 h 151. The degree of platelet aggregation is massive and far exceeds that seen with milder degrees of vascular injury. The lack of clinical effectiveness of these agents to prevent restenosis may be due in part to their inability to inhibit the massive platelet accumulation seen with angioplasty or may indicate that alternative mechanisms of restenosis are more important. This study was undertaken to examine what effect antiplatelet drugs have on platelet accumulation immediately following angioplasty in an experimental rabbit model of atherosclerosis.
Methods Aortic and bilateral iliac atherosclerosis was developed in 66 3 kg New Zealand white male rabbits by balloon de-endothelialization and a 2% cholesterol diet as previously described [6]. Briefly, the animal was anesthetized with pen-
tothal intravenously and surgical exposure of the left and right femoral arteries, was accomplished. A 3 French Fogarty catheter was passed to 30 cm and inflated to just approximate the arterial wall. De-endothelialization was accomplished by gradually pulling the catheter down the aorta and iliac vessels. The procedure was repeated three times to ensure complete removal of the endothelium. All animals were placed on a 2% cholesterol diet composed of rabbit chow supplemented with 10% peanut oil. On this diet, cholesterol levels ranged from 1500 to 2000 mg%. After 6 weeks, during which time atherosclerosis was allowed to develop all animals underwent aorto-iliac arteriography by right carotid arteriotomy using a 4Fr Swan Ganz catheter advanced fluoroscopically to the aortic bifurcation. Cineangiographic images were obtained at 30F/s using a single plane Philips angiographic system with a resolution of 3.1 linepairs/mm. Angioplasty was performed only on those iliac stenoses exceeding 50% luminal diameter narrowing as determined on a still frame video image. Through a femoral arteriotomy, a 2.5 mm Gruentzig transluminal angioplasty catheter (USC1 Billerica, MA) was retrogradely passed to the site of greatest iliac stenosis using fluoroscopic guidance (Fig. 1). The balloon size was chosen to be equal in diameter to the least diseased proximal vessel segment. If the artery was
Fig. 1. An example of a focal 50% stenosis in the right iliac (left panel). A 2.5 mm balloon angioplasty catheter is inflated right iliac (middle panel) with a successful reduction in the stenosis to less than 10% (right panel).
in the
43
severely diseased a 2.0 mm angioplasty balloon was chosen. The balloon was inflated three times at 5 atm for 30 s. The catheter was withdrawn, the femoral artery ligated, and angiography repeated. Platelet accumulation was determined by using platelets labeled with 5’Cr as previously described by Adelman et al. [7]. Forty milliliters of blood were obtained from a donor animal and collected into acidified citrate dextrose anticoagulant. After adjustment of the pH to 6.5 with 1 ml of ACD (NIH formula A) per 5 ml of blood the platelets were centrifuged and the pellet was resuspended in Ringer’s citrate dextrose solution and labelled with 100 Ci of Naz51Cr0, (New England Nuclear, Boston, MA) by incubation of 30 min at 37°C. Labelled platelets were then washed twice in platelet-poor plasma and resuspended in platelet-poor plasma for injection into the experimental animal. This method resulted in an average labeling efficiency of 77%. Those animals with adequate stenosis of at least one iliac vessel were injected with 51Crlabelled platelets 30 min prior to angioplasty. Thirty min after angioplasty blood samples were taken to determine specific platelet reactivity. Animals then were sacrificed with pentothal overdose followed by rapid carotid pressure perfusion with a formalin-glutaraldehyde mixture at 100 mmHg. A 2 cm iliac arterial segment identified at the site of angioplasty by reference to the arteriogram was removed for gamma counting. The segments were gently washed but care was taken to avoid dislodgement of platelet and thrombi. The number of platelets adherent to the angioplasty site was derived by dividing the number of gamma counts at the site by the number of counts per blood platelet derived at the time of sacrifice. The number of platelets per 1 cm was calculated by measurement of the length of the segment in cm, divided into the number of total platelets per segment. Due to the platelet studies routine histology was not performed. Animals were randomly given either aspirin (10 mg/kg orally) (n = lo>, aspirin (10 mg/kg intravenously) (n = 7), aspirin (10 mg/kg orally) plus dipyridamole (25 mg orally) (n = 8), a thromboxane synthesis inhibitor (GS 13080 1 mg/kg/h
intravenously) (n = 9) or heparin (500 U/kg intravenously) (n = 9) prior to the procedure. Intravenous aspirin was prepared by suspension of sterile aspirin in a sterile water solution and passage through a millipore filter. All oral drugs were begun 48 h prior to angioplasty and continued thereafter, the last dose given 2 h before the procedure. Donor rabbits followed the same protocol. The intravenous drugs were administered 30 min prior to angioplasty. Twenty-three rabbits served as controls and received no medications before angioplasty. Platelet accumulation was determined 30 min after the procedure as described above. To determine if aspirin was absorbed in the oral aspirin group, salicylate levels and thromboxane Bz levels were measured in 5 additional animals, before and 2 h after oral or intravenous administration of aspirin. The salicylate levels were < 2.0 mg/dl after both intravenous and oral administration. Likewise there was no difference in thromboxane Bz levels falling from 106 & 60 to 39 + 9 pg/O.l ml after oral aspirin and 96 f 12 to 40 + 15 after intravenous aspirin. Cineangiograms or video images obtained before and after angioplasty were viewed by two independent investigators. Caliper measurements were taken of the smallest luminal diameter at the angioplasty site and was compared to the largest lumen diameter just proximal to the stenosis. Only animals demonstrating a 50% or greater stenosis before angioplasty and at least a 20% change in lumen diameter were entered into the study. These changes were chosen to closely mimic clinical practice. Comparison between the groups was done using the non-paired Student f-test. A p value of < 0.05 was considered significant. Means and standard deviations were determined for each intervention. Results Angiography demonstrated significant iliac stenosis ranging from 50-99% in all animals. In most animals only one iliac vessel showed significant enough focal disease to be used in this study. In 25, however, both iliac vessels underwent dilation (6 controls, 6 intravenous aspirin, 0 aspirin
44
TABLE 1 Group Control Oral aspirin Intravenous aspirin Oral aspirin & oral dipyridamole Intravenous thromboxane synthetic inhibitor CGS13080 Intravenous heparin
Dose
n 29
Platelet/cm (lo6 f SD) 44.1+51.2
10 mg/kg
13 109.5&240
lOmg/kg 10 mg/kg 25 mg
13 8
1 mg/kg/h 500 U/kg
3.3f 10.4f
p vs control -
at the angioplasty site when visually inspected prior to platelet counting. In each of the groups a wide variation of platelet accumulation was noted consistent with the known variability of platelet deposition previously reported in this model [4].
NS
1.9 0.007 9.8 0.076
15
15.1* 16.6 0.04
13
34.7k35.5
NS
n = number of vessel segments; platelet/cm = number of platelets/l cm vessel segment accumulated 30 min after angioplasty.
plus dipyridamole, 6 thromboxane synthetase inhibitor, 3 oral aspirin and 4 heparin-treated animals). Angioplasty resulted in a change in the stenosis of more than 20% in all animals by protocol design and paralleled our previous studies. Pretreatment with antiplatelet therapy resulted in a significant reduction in platelet accumulation at the site of angioplasty (Table 1). The control vessel segments had 44.1 k 51 X lo6 platelets/l cm segment 30 min following angioplasty. Intravenous aspirin was most effective in reducing platelet accumulations to 3.26 + 1.92 X lo6 platelets/l cm segment when compared to control (p < 0.007). Oral aspirin plus dipyridamole reduced platelet accumulation less effectively to 10.4 + 9.8 x lo6 platelets/l cm segment (p = 0.076) while the thromboxane synthetase inhibitor reduced platelets to 15.1 + 16.6 X lo6 platelets/l cm segment (p < 0.04). Heparin did not have a significant effect as platelets were reduced to only 33.7 f 35.5 x lo6 platelets/l cm segment (p = NS). Interestingly, oral aspirin was ineffective in this animal model reducing platelets to 37.9 x lo6 platelets/l cm segment (p = 0.16). Arterial segments with the greatest degree of platelet accumulation had obvious thrombi seen
Discussion Antiplatelet drugs are routinely administered before and after percutaneous transluminal coronary angioplasty in hopes of lessening the degree of platelet accumulation at the angioplasty site and thus preventing both abrupt vessel closure and subsequently restenosis [l]. Abrupt vessel closure occurs in 5-10% of patients undergoing coronary angioplasty and is associated with significantly higher incidence of acute myocardial infarction, emergency bypass surgery and death [8]. Restenosis occurs in approximately 25-35% in patients and its incidence has not changed significantly since the introduction of angioplasty more than 10 years ago 191. While clinical trials have shown that antiplatelet therapy can significantly reduce the incidence of abrupt vessel closure these agents have not reduced the incidence of restenosis [2-41. A lack of effectiveness in this setting may be due to an inability of these agents to effectively reduce the marked platelet accumulation at the site of vascular damage [5] or it may be due to the limited role of platelets and thrombi in the process of restenosis. The observations by Nobuyoshi would refute this as thrombus was commonly seen within the first month in patients dying following angioplasty [lo]. Further support of the importance of platelets in the restenosis process comes from the recent report by Ferns demonstrating a 30-40% reduction in neointimal proliferation in a rat model after angioplasty by use of an antibody to platelet derived growth factor [ 111. As shown in our study, the administration of intravenous aspirin and an experimental thromboxane synthetase inhibitor (CGS 13080) were effective in reducing platelet deposition. Aspirin plus dipyridamole was less effective than intravenous aspirin and neither heparin nor orally administered aspirin seemed to reduce platelet numbers significantly.
45
The mechanism of restenosis after angioplasty is not well understood but it seems likely that the events are similar to those described by Ross and Glomset in the injury hypothesis of arteriosclerosis [12]. In accord with this hypothesis experimental studies have shown that following experimental angioplasty the endothelium is damaged and denuded 161and the vessel is stretched and fractured [13-151 inducing platelet adhesion and aggregation and thrombus formation [5,16,17]. Aggregating platelets release various factors including adenosine diphosphate, platelet derived growth factor, platelet factor 4, and thromboxane A, [16]. Platelet derived growth factor may be important in the migration of smooth muscle cells from the media into the intima with subsequent proliferation of these cells in response to paracrine and autocrine mitogens. Thrombus may provide a stimulatory effect as well. Human autopsy material and histological sections of atheromatous material derived from patients during atherectomy procedures indicate that restenosis is primarily due to a proliferative intimal lesion [18-211. It is not unreasonable to speculate that the intense platelet aggregation and thrombosis contribute to the proliferation that creates the restenosis lesion. As mentioned, the finding of thrombi in autopsy specimens following angioplasty and inhibition of neointimal formation with an antibody to platelet derived growth factor underscore the importance of thrombosis in restenosis despite the negative findings in clinical trials to date. Platelet aggregation is maximal in the first 2 h after angioplasty in this model with continued low level accumulation occurring up to 24 h and then returning to baseline by 1 week [5]. In a porcine model of angioplasty, Steele et al. demonstrated a high level of platelet deposition at 24 h with reduction in accumulation thereafter [17]. Because platelet deposition begins immediately as blood flows through the injured vessel, platelet modifying therapy must begin prior to balloon dilatation and have a maximal effect within the first 2 h after angioplasty. Aspirin is known to block platelet cyclooxygenase, thus inhibiting the release reaction by blocking endoperoxide and thromboxane synthesis [22]. PGI, which is formed
from the blood vessel wall, is a potent inhibitor of platelet aggregation which may also be blocked by high doses of aspirin. In the current study oral aspirin did not produce a significant reduction in platelet aggregation at the site of angioplasty, most likely due to a variable effect on platelet aggregation and thrombus formation. One explanation for this variable effect is that oral aspirin may have had a variable gastrointestinal absorption in this animal model, as the more reliable administration of intravenous aspirin resulted in the most marked reduction in platelet aggregation. Also, intravenous aspirin would be expected to have a higher and more rapid tissue level than oral aspirin. While salicylate levels were not measured in these animals, a subsequent study in 5 additional animals utilizing the same dose of oral and intravenous aspirin showed insignificant salicylate levels 2 h later by either method of administration; however, both methods of administration resulted in the reduction in platelet thromboxane B, levels indicating that the drug wa:, absorbed and had an antiplatelet effect. The different pharmacokinetics and tissue effects including inhibition of PGI, might also be important. The variable effect of oral aspirin may be of clinical importance and could be one explanation for the lack of effectiveness of oral aspirin in preventing restenosis in man. Dipyridamole is known to inhibit platelet phosphodiesterase resulting in an increased platelet cyclic AMP level. These elevated levels of cyclic AMP are thought to inhibit platelet adhesion, aggregation and release. Potentiation of the inhibitory effects of platelet aggregation by combining aspirin plus dipyridamole has previously been suggested. However, as shown by our study the combination of aspirin plus dipyridamole was less effective than intravenous aspirin in reducing platelet aggregation. A thromboxane synthetase inhibitor like aspirin prevents the formation of TxA, from arachidonic acid and this also affects platelet aggregation. In the current study this agent was equally effective as aspirin in preventing platelet aggregation of the site of angioplasty. Heparin is well known as an effective short acting anticoagulant by virtue of its effect on antithrombin III. In addition it has a mild antiplatelet effect. By in-
46
hibiting thrombin formation, it may reduce one of the major stimulants for further platelet aggregation. However, this study did not demonstrate a significant effect of heparin alone on prevention of platelet accumulation. Our study is consistent with the findings of Steele et al. who reported and that aspirin, aspirin plus dipyridamole nifedipine were effective in relieving platelet thrombi following balloon angioplasty of normal porcine carotid vessels while verapamil, and sulfinpyrazone were not [23]. In addition, this same group has shown that high doses of heparin can also decrease acute platelet-thrombus deposition following angioplasty in the pig, but was less effective than a specific thrombin inhibitor recombinant Hirudin [24]. These studies would emphasize the importance of inhibition of thrombin in reduction of platelet accumulation and suggests that combined therapy with both an antiplatelet agent and a thrombin inhibitor, may provide a more potent means of reducing thrombosis following angioplasty. A recent report from our laboratory by Franklin would also support this concept [251. The results of this study demonstrate that platelet modifying therapy particularly intravenous aspirin, and a thromboxane synthetase inhibitor significantly reduced the number of platelets deposited at the angioplasty site after experimental balloon angioplasty. Both clinical and experimental evidence would suggest that reduction of marked platelet aggregation has been beneficial both in terms of reducing abrupt vessel closure and potentially in reducing restenosis [22,23]. Further investigation into the role of more potent antiplatelet drugs and combined antiplatelet and antithrombin therapy should be actively pursued.
4
5
6
I
8
9
10
I1
12 13
14
15
References Blackshear JL, O’Callaghan WG, Califf RM. Medical approaches to prevention of restenosis after coronary angioplasty. J Am Coil Cardiol 1987;9:831-848. Schwartz L, Bourassa MG, Lesperance J et al. Aspirin and dipyridamole in the prevention of restenosis after percutaneous transluminal coronary angioplasty. N Engl J Med 1988;318:1714-1719. White CW, Knudson M, Schmidt D et al. Neither ticlopidine nor aspirin-dipyridamole prevents restenosis post
16
17
18
PTCA: results from a randomized placebo-controlled trial. Circulation 1987;suppl IV:213. Serruys PW, Rutsch W, Heyndrickx et al. Prevention of restenosis after percutaneous transluminal coronary angioplasty with thromboxane A, receptor blockade. Circulation 1991;84:1568-1580. Wilentz JR, Sanborn TA, Haudenschild CC, Valeri CR, Ryan TJ, Faxon DP. Platelet accumulation in experimental angioplasty: time course and relation to vascular injury. Circulation 1987;75:636-642. Faxon DP, Weber VJ, Haudenschild CC, Gottsman SB, McGovern WA, Ryan TJ. Acute effects of transluminal angioplasty in three experimental models of atherosclerosis. Atherosclerosis 1982;2:125-133. Adelman B. Stemerman MB, Merrell D, Handin RJ. The interaction of platelets with aortic subendothelium: Inhibition of adhesion and secretion by prostaglandin I,. Blood 1987;58:198-202. Ellis SG, Roubin GS, King SB III et al. In-hospital cardiac mortality after acute closure after coronary angioplasty: analysis of risk factors from 8.207 procedures. J Am Coil Cardiol 1988;11:211-216. Serruys PW, Luijten KJ, Beatt R et al. Incidence of restenosis after successful coronary angioplasty: a time-related phenomenon: a quantitative angiographic study in 342 consecutive patients at 1, 2, 3 and 4 months. Circulation 1988;77:361-371. Nobuyoshi M, Takeshi K, Hiroto 0 et al. Restenosis after percutaneous transluminal artery angioplasty: pathologic observations in 20 patients. J Am Co11 Cardiol 1991;17: 433-439. Ferns GA, Raines EW, Sprugel KH, Motani AS, Reidy MA, Ross R. Inhibition of neointimal smooth muscle accumulation after angioplasty by an antibody to PDGF. Science 1991;253:1129-1132, Ross R, Glomset GA. The pathogenesis of atherosclerosis. N Engl J Med 1976;296:369-377. Sanborn TA, Faxon DP, Haudenschild CC, Gottsman SB, Ryan TJ. The mechanism of transluminal angioplasty: evidence for aneurysm formation in experimental atherosclerosis. Circulation 1983;68:1136-1140. Block PC, Baughman KL, Pasternack RC, Fallen TJ. Transluminal angioplasty: correlation of morphologic and angiographic findings in an experimental model. Circulation 1980;61:778-785. Casteneda-Zuniga WR, Fomanek T, Tadavarthy M et al. The mechanism of balloon angioplasty. Radiology 1980;135:565-571. Harker LA. Role of platelets and thrombus in mechanisms of acute occlusion and restenosis after angioplasty. Am J Cardiol 1987;60:20B-28B. Steele PM, Cheseboro 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. Essed CE, Van der Brand M, Becker AE. Transluminal coronary angioplasty and early restenosis: fibrocellular occlusion after wall laceration. Br Heart J 1983;49:393-396.
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19 Austin CE, Ratliff MB, Hollman J. Tabei S, Philips DF. Intimal proliferation of smooth muscle cells as an explanation for recurrent coronary artery stenosis after percutaneous transluminal coronary angioplasty. J Am Coil Cardiol 1985;6:369-375. 20 Wailer BF, McManus BM, Gorfinkel HJ et al. Status of the major epicardial coronary arteries 80 to 150 days after percutaneous transluminal coronary angioplasty: analysis of 3 necropsy patients. Am J Cardiol 1983;51:81-84. 21 Johnson DE, Robertson G, Simpson JB. Coronary atherectomy: light microscopy and immunochemical study of excised tissues. Circulation 1988;78(suppl II):83. 22 Packham MA, Mustard JF. Pharmacology of platelet-affecting drugs. Circulation 1980;62(suppl V):26-41. 23 Steele PM, Cheseboro JH, Holmes DR. Badimon L, Fuster
V. Balloon angioplasty in pigs: comparative effects of platelet-inhibitor drugs (abstract). Circulation 1984; 7Otsuppl IIk361. 24 Hews M, Chesebro JH, Penny WJ, Bailey KR, Budimen L, Fuster V. Effect of thrombin inhibition on the development of acute platelet-thrombus depression during angioplasty in pigs. Heparin versus recumbinant hirudin, a specific thrombin inhibitor. Circulation 1989;79:657-664. 25 Franklin S. Currier JW, Cannistra A et al. Warfarin/aspirin combination reduces restenosis after angioplasty in atherosclerotic rabbits. Circulation 1990;82(suppl 111):427. 26 Faxon DP, Sanborn TA, Haudenschild CC, Ryan TJ. Effect of antiplatelet drugs on restenosis following experimental angioplasty. Am J Cardiol 1984:53:72C-76C.
CARD10
41 0167-5273/92/$05.00
01464
The effect of antiplatelet therapy on platelet accumulation after experimental angioplasty in the rabbit iliac model David P. Faxon, Lynn Anne Balelli, Timothy Sandborn, Christian Haudenschild, Robert Valeri and Thomas J. Ryan EL:ans Memorial Department of Clinical Research and Department of Medicine. Uniuersity Hospital, Boston Unirlersity Medical Center, The Mallory Institute of Pathology and The Natal Blood Research Laboratory, Boston, MA, USA (Received
23 July 1991; revision
Faxon DP, Balelli LA, Sandborn T, Haudenschild therapy on platelet accumulation after experimental 1992:36:41-47.
accepted
23 December
1991)
C, Valeri R, Ryan TJ. The effect of antiplatelet angioplasty in the rabbit iliac model. Int J Cardiol
Antiplatelet therapy is routinely used in balloon angioplasty. Yet recent clinical trials have not shown these agents to reduce the incidence of restenosis. As this might be due to inadequate antiplatelet effect, this study was designed to evaluate the effectiveness of antiplatelet therapy in reducing platelet accumulation immediately after angioplasty. Sixty-six atherosclerotic rabbits underwent angioplasty of a focal (50-99%) iliac stenosis after pretreatment with an antiplatelet drug. The animals were randomly given aspirin (10 mg/kg orally) (n = 101, aspirin (10 mg/kg intravenously) (n = 7), aspirin (10 mg/kg per OS) plus dipyridamole (25 mg orally) (n = 8), a thromboxane synthetase inhibitor (GS13080, 1 mg/kg/h intravenously) (n = 9) or heparin (500 U/kg intravenously) (n = 9). Platelet accumulation, determined by “Cr-labeled platelet counts 30 min after angioplasty disclosed a significant reduction from control (44.1 + 51.2 platelets/cm x 106) with intravenous aspirin (3.3 f 1.9; p < 0.007) and the thromboxane synthetase inhibitor CGS13080 (15.1 + 16.6, p < 0.04). Aspirin plus dipyridamole reduced platelet accumulation as well (10.4 f 9.8, p = 0.076), while oral aspirin (109.5 -t 240) and heparin (34.7 + 35.5) were ineffective. The results of this study demonstrate that pretreatment with antiplatelet agents can reduce platelet accumulation significantly after experimental angioplasty. However, the wide variability in effectiveness particularly with oral aspirin may have important clinical implications. Further investigation into more effective antiplatelet therapy may help to reduce the incidence of abrupt vessel closure and restenosis. Key words: Restenosis; Antiplatelet
therapy; Angioplasty
Correspondence to: D.P. Faxon, M.D., The University Hospital, Section of Cardiology. 88 East Newton Street, Boston, MA 02118. USA. Grant No. 85-1028 of the American Heart Association.
Introduction Empiric therapy with antiplatelet drugs is routinely used for angioplasty in hopes of preventing
42
immediate abrupt closure of the vessel and reducing the incidence of restenosis [l]. While clinical trials have demonstrated the effectiveness of aspirin in preventing abrupt closure, there is no conclusive evidence that these agents reduce the incidence of restenosis [2-41. We have previously shown that platelet aggregation at the site of experimental angioplasty occurs immediately after the procedure reaching a maximum at 4 h with little further aggregation by 6 h 151. The degree of platelet aggregation is massive and far exceeds that seen with milder degrees of vascular injury. The lack of clinical effectiveness of these agents to prevent restenosis may be due in part to their inability to inhibit the massive platelet accumulation seen with angioplasty or may indicate that alternative mechanisms of restenosis are more important. This study was undertaken to examine what effect antiplatelet drugs have on platelet accumulation immediately following angioplasty in an experimental rabbit model of atherosclerosis.
Methods Aortic and bilateral iliac atherosclerosis was developed in 66 3 kg New Zealand white male rabbits by balloon de-endothelialization and a 2% cholesterol diet as previously described [6]. Briefly, the animal was anesthetized with pen-
tothal intravenously and surgical exposure of the left and right femoral arteries, was accomplished. A 3 French Fogarty catheter was passed to 30 cm and inflated to just approximate the arterial wall. De-endothelialization was accomplished by gradually pulling the catheter down the aorta and iliac vessels. The procedure was repeated three times to ensure complete removal of the endothelium. All animals were placed on a 2% cholesterol diet composed of rabbit chow supplemented with 10% peanut oil. On this diet, cholesterol levels ranged from 1500 to 2000 mg%. After 6 weeks, during which time atherosclerosis was allowed to develop all animals underwent aorto-iliac arteriography by right carotid arteriotomy using a 4Fr Swan Ganz catheter advanced fluoroscopically to the aortic bifurcation. Cineangiographic images were obtained at 30F/s using a single plane Philips angiographic system with a resolution of 3.1 linepairs/mm. Angioplasty was performed only on those iliac stenoses exceeding 50% luminal diameter narrowing as determined on a still frame video image. Through a femoral arteriotomy, a 2.5 mm Gruentzig transluminal angioplasty catheter (USC1 Billerica, MA) was retrogradely passed to the site of greatest iliac stenosis using fluoroscopic guidance (Fig. 1). The balloon size was chosen to be equal in diameter to the least diseased proximal vessel segment. If the artery was
Fig. 1. An example of a focal 50% stenosis in the right iliac (left panel). A 2.5 mm balloon angioplasty catheter is inflated right iliac (middle panel) with a successful reduction in the stenosis to less than 10% (right panel).
in the
43
severely diseased a 2.0 mm angioplasty balloon was chosen. The balloon was inflated three times at 5 atm for 30 s. The catheter was withdrawn, the femoral artery ligated, and angiography repeated. Platelet accumulation was determined by using platelets labeled with 5’Cr as previously described by Adelman et al. [7]. Forty milliliters of blood were obtained from a donor animal and collected into acidified citrate dextrose anticoagulant. After adjustment of the pH to 6.5 with 1 ml of ACD (NIH formula A) per 5 ml of blood the platelets were centrifuged and the pellet was resuspended in Ringer’s citrate dextrose solution and labelled with 100 Ci of Naz51Cr0, (New England Nuclear, Boston, MA) by incubation of 30 min at 37°C. Labelled platelets were then washed twice in platelet-poor plasma and resuspended in platelet-poor plasma for injection into the experimental animal. This method resulted in an average labeling efficiency of 77%. Those animals with adequate stenosis of at least one iliac vessel were injected with 51Crlabelled platelets 30 min prior to angioplasty. Thirty min after angioplasty blood samples were taken to determine specific platelet reactivity. Animals then were sacrificed with pentothal overdose followed by rapid carotid pressure perfusion with a formalin-glutaraldehyde mixture at 100 mmHg. A 2 cm iliac arterial segment identified at the site of angioplasty by reference to the arteriogram was removed for gamma counting. The segments were gently washed but care was taken to avoid dislodgement of platelet and thrombi. The number of platelets adherent to the angioplasty site was derived by dividing the number of gamma counts at the site by the number of counts per blood platelet derived at the time of sacrifice. The number of platelets per 1 cm was calculated by measurement of the length of the segment in cm, divided into the number of total platelets per segment. Due to the platelet studies routine histology was not performed. Animals were randomly given either aspirin (10 mg/kg orally) (n = lo>, aspirin (10 mg/kg intravenously) (n = 7), aspirin (10 mg/kg orally) plus dipyridamole (25 mg orally) (n = 8), a thromboxane synthesis inhibitor (GS 13080 1 mg/kg/h
intravenously) (n = 9) or heparin (500 U/kg intravenously) (n = 9) prior to the procedure. Intravenous aspirin was prepared by suspension of sterile aspirin in a sterile water solution and passage through a millipore filter. All oral drugs were begun 48 h prior to angioplasty and continued thereafter, the last dose given 2 h before the procedure. Donor rabbits followed the same protocol. The intravenous drugs were administered 30 min prior to angioplasty. Twenty-three rabbits served as controls and received no medications before angioplasty. Platelet accumulation was determined 30 min after the procedure as described above. To determine if aspirin was absorbed in the oral aspirin group, salicylate levels and thromboxane Bz levels were measured in 5 additional animals, before and 2 h after oral or intravenous administration of aspirin. The salicylate levels were < 2.0 mg/dl after both intravenous and oral administration. Likewise there was no difference in thromboxane Bz levels falling from 106 & 60 to 39 + 9 pg/O.l ml after oral aspirin and 96 f 12 to 40 + 15 after intravenous aspirin. Cineangiograms or video images obtained before and after angioplasty were viewed by two independent investigators. Caliper measurements were taken of the smallest luminal diameter at the angioplasty site and was compared to the largest lumen diameter just proximal to the stenosis. Only animals demonstrating a 50% or greater stenosis before angioplasty and at least a 20% change in lumen diameter were entered into the study. These changes were chosen to closely mimic clinical practice. Comparison between the groups was done using the non-paired Student f-test. A p value of < 0.05 was considered significant. Means and standard deviations were determined for each intervention. Results Angiography demonstrated significant iliac stenosis ranging from 50-99% in all animals. In most animals only one iliac vessel showed significant enough focal disease to be used in this study. In 25, however, both iliac vessels underwent dilation (6 controls, 6 intravenous aspirin, 0 aspirin
44
TABLE 1 Group Control Oral aspirin Intravenous aspirin Oral aspirin & oral dipyridamole Intravenous thromboxane synthetic inhibitor CGS13080 Intravenous heparin
Dose
n 29
Platelet/cm (lo6 f SD) 44.1+51.2
10 mg/kg
13 109.5&240
lOmg/kg 10 mg/kg 25 mg
13 8
1 mg/kg/h 500 U/kg
3.3f 10.4f
p vs control -
at the angioplasty site when visually inspected prior to platelet counting. In each of the groups a wide variation of platelet accumulation was noted consistent with the known variability of platelet deposition previously reported in this model [4].
NS
1.9 0.007 9.8 0.076
15
15.1* 16.6 0.04
13
34.7k35.5
NS
n = number of vessel segments; platelet/cm = number of platelets/l cm vessel segment accumulated 30 min after angioplasty.
plus dipyridamole, 6 thromboxane synthetase inhibitor, 3 oral aspirin and 4 heparin-treated animals). Angioplasty resulted in a change in the stenosis of more than 20% in all animals by protocol design and paralleled our previous studies. Pretreatment with antiplatelet therapy resulted in a significant reduction in platelet accumulation at the site of angioplasty (Table 1). The control vessel segments had 44.1 k 51 X lo6 platelets/l cm segment 30 min following angioplasty. Intravenous aspirin was most effective in reducing platelet accumulations to 3.26 + 1.92 X lo6 platelets/l cm segment when compared to control (p < 0.007). Oral aspirin plus dipyridamole reduced platelet accumulation less effectively to 10.4 + 9.8 x lo6 platelets/l cm segment (p = 0.076) while the thromboxane synthetase inhibitor reduced platelets to 15.1 + 16.6 X lo6 platelets/l cm segment (p < 0.04). Heparin did not have a significant effect as platelets were reduced to only 33.7 f 35.5 x lo6 platelets/l cm segment (p = NS). Interestingly, oral aspirin was ineffective in this animal model reducing platelets to 37.9 x lo6 platelets/l cm segment (p = 0.16). Arterial segments with the greatest degree of platelet accumulation had obvious thrombi seen
Discussion Antiplatelet drugs are routinely administered before and after percutaneous transluminal coronary angioplasty in hopes of lessening the degree of platelet accumulation at the angioplasty site and thus preventing both abrupt vessel closure and subsequently restenosis [l]. Abrupt vessel closure occurs in 5-10% of patients undergoing coronary angioplasty and is associated with significantly higher incidence of acute myocardial infarction, emergency bypass surgery and death [8]. Restenosis occurs in approximately 25-35% in patients and its incidence has not changed significantly since the introduction of angioplasty more than 10 years ago 191. While clinical trials have shown that antiplatelet therapy can significantly reduce the incidence of abrupt vessel closure these agents have not reduced the incidence of restenosis [2-41. A lack of effectiveness in this setting may be due to an inability of these agents to effectively reduce the marked platelet accumulation at the site of vascular damage [5] or it may be due to the limited role of platelets and thrombi in the process of restenosis. The observations by Nobuyoshi would refute this as thrombus was commonly seen within the first month in patients dying following angioplasty [lo]. Further support of the importance of platelets in the restenosis process comes from the recent report by Ferns demonstrating a 30-40% reduction in neointimal proliferation in a rat model after angioplasty by use of an antibody to platelet derived growth factor [ 111. As shown in our study, the administration of intravenous aspirin and an experimental thromboxane synthetase inhibitor (CGS 13080) were effective in reducing platelet deposition. Aspirin plus dipyridamole was less effective than intravenous aspirin and neither heparin nor orally administered aspirin seemed to reduce platelet numbers significantly.
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The mechanism of restenosis after angioplasty is not well understood but it seems likely that the events are similar to those described by Ross and Glomset in the injury hypothesis of arteriosclerosis [12]. In accord with this hypothesis experimental studies have shown that following experimental angioplasty the endothelium is damaged and denuded 161and the vessel is stretched and fractured [13-151 inducing platelet adhesion and aggregation and thrombus formation [5,16,17]. Aggregating platelets release various factors including adenosine diphosphate, platelet derived growth factor, platelet factor 4, and thromboxane A, [16]. Platelet derived growth factor may be important in the migration of smooth muscle cells from the media into the intima with subsequent proliferation of these cells in response to paracrine and autocrine mitogens. Thrombus may provide a stimulatory effect as well. Human autopsy material and histological sections of atheromatous material derived from patients during atherectomy procedures indicate that restenosis is primarily due to a proliferative intimal lesion [18-211. It is not unreasonable to speculate that the intense platelet aggregation and thrombosis contribute to the proliferation that creates the restenosis lesion. As mentioned, the finding of thrombi in autopsy specimens following angioplasty and inhibition of neointimal formation with an antibody to platelet derived growth factor underscore the importance of thrombosis in restenosis despite the negative findings in clinical trials to date. Platelet aggregation is maximal in the first 2 h after angioplasty in this model with continued low level accumulation occurring up to 24 h and then returning to baseline by 1 week [5]. In a porcine model of angioplasty, Steele et al. demonstrated a high level of platelet deposition at 24 h with reduction in accumulation thereafter [17]. Because platelet deposition begins immediately as blood flows through the injured vessel, platelet modifying therapy must begin prior to balloon dilatation and have a maximal effect within the first 2 h after angioplasty. Aspirin is known to block platelet cyclooxygenase, thus inhibiting the release reaction by blocking endoperoxide and thromboxane synthesis [22]. PGI, which is formed
from the blood vessel wall, is a potent inhibitor of platelet aggregation which may also be blocked by high doses of aspirin. In the current study oral aspirin did not produce a significant reduction in platelet aggregation at the site of angioplasty, most likely due to a variable effect on platelet aggregation and thrombus formation. One explanation for this variable effect is that oral aspirin may have had a variable gastrointestinal absorption in this animal model, as the more reliable administration of intravenous aspirin resulted in the most marked reduction in platelet aggregation. Also, intravenous aspirin would be expected to have a higher and more rapid tissue level than oral aspirin. While salicylate levels were not measured in these animals, a subsequent study in 5 additional animals utilizing the same dose of oral and intravenous aspirin showed insignificant salicylate levels 2 h later by either method of administration; however, both methods of administration resulted in the reduction in platelet thromboxane B, levels indicating that the drug wa:, absorbed and had an antiplatelet effect. The different pharmacokinetics and tissue effects including inhibition of PGI, might also be important. The variable effect of oral aspirin may be of clinical importance and could be one explanation for the lack of effectiveness of oral aspirin in preventing restenosis in man. Dipyridamole is known to inhibit platelet phosphodiesterase resulting in an increased platelet cyclic AMP level. These elevated levels of cyclic AMP are thought to inhibit platelet adhesion, aggregation and release. Potentiation of the inhibitory effects of platelet aggregation by combining aspirin plus dipyridamole has previously been suggested. However, as shown by our study the combination of aspirin plus dipyridamole was less effective than intravenous aspirin in reducing platelet aggregation. A thromboxane synthetase inhibitor like aspirin prevents the formation of TxA, from arachidonic acid and this also affects platelet aggregation. In the current study this agent was equally effective as aspirin in preventing platelet aggregation of the site of angioplasty. Heparin is well known as an effective short acting anticoagulant by virtue of its effect on antithrombin III. In addition it has a mild antiplatelet effect. By in-
46
hibiting thrombin formation, it may reduce one of the major stimulants for further platelet aggregation. However, this study did not demonstrate a significant effect of heparin alone on prevention of platelet accumulation. Our study is consistent with the findings of Steele et al. who reported and that aspirin, aspirin plus dipyridamole nifedipine were effective in relieving platelet thrombi following balloon angioplasty of normal porcine carotid vessels while verapamil, and sulfinpyrazone were not [23]. In addition, this same group has shown that high doses of heparin can also decrease acute platelet-thrombus deposition following angioplasty in the pig, but was less effective than a specific thrombin inhibitor recombinant Hirudin [24]. These studies would emphasize the importance of inhibition of thrombin in reduction of platelet accumulation and suggests that combined therapy with both an antiplatelet agent and a thrombin inhibitor, may provide a more potent means of reducing thrombosis following angioplasty. A recent report from our laboratory by Franklin would also support this concept [251. The results of this study demonstrate that platelet modifying therapy particularly intravenous aspirin, and a thromboxane synthetase inhibitor significantly reduced the number of platelets deposited at the angioplasty site after experimental balloon angioplasty. Both clinical and experimental evidence would suggest that reduction of marked platelet aggregation has been beneficial both in terms of reducing abrupt vessel closure and potentially in reducing restenosis [22,23]. Further investigation into the role of more potent antiplatelet drugs and combined antiplatelet and antithrombin therapy should be actively pursued.
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References Blackshear JL, O’Callaghan WG, Califf RM. Medical approaches to prevention of restenosis after coronary angioplasty. J Am Coil Cardiol 1987;9:831-848. Schwartz L, Bourassa MG, Lesperance J et al. Aspirin and dipyridamole in the prevention of restenosis after percutaneous transluminal coronary angioplasty. N Engl J Med 1988;318:1714-1719. White CW, Knudson M, Schmidt D et al. Neither ticlopidine nor aspirin-dipyridamole prevents restenosis post
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PTCA: results from a randomized placebo-controlled trial. Circulation 1987;suppl IV:213. Serruys PW, Rutsch W, Heyndrickx et al. Prevention of restenosis after percutaneous transluminal coronary angioplasty with thromboxane A, receptor blockade. Circulation 1991;84:1568-1580. Wilentz JR, Sanborn TA, Haudenschild CC, Valeri CR, Ryan TJ, Faxon DP. Platelet accumulation in experimental angioplasty: time course and relation to vascular injury. Circulation 1987;75:636-642. Faxon DP, Weber VJ, Haudenschild CC, Gottsman SB, McGovern WA, Ryan TJ. Acute effects of transluminal angioplasty in three experimental models of atherosclerosis. Atherosclerosis 1982;2:125-133. Adelman B. Stemerman MB, Merrell D, Handin RJ. The interaction of platelets with aortic subendothelium: Inhibition of adhesion and secretion by prostaglandin I,. Blood 1987;58:198-202. Ellis SG, Roubin GS, King SB III et al. In-hospital cardiac mortality after acute closure after coronary angioplasty: analysis of risk factors from 8.207 procedures. J Am Coil Cardiol 1988;11:211-216. Serruys PW, Luijten KJ, Beatt R et al. Incidence of restenosis after successful coronary angioplasty: a time-related phenomenon: a quantitative angiographic study in 342 consecutive patients at 1, 2, 3 and 4 months. Circulation 1988;77:361-371. Nobuyoshi M, Takeshi K, Hiroto 0 et al. Restenosis after percutaneous transluminal artery angioplasty: pathologic observations in 20 patients. J Am Co11 Cardiol 1991;17: 433-439. Ferns GA, Raines EW, Sprugel KH, Motani AS, Reidy MA, Ross R. Inhibition of neointimal smooth muscle accumulation after angioplasty by an antibody to PDGF. Science 1991;253:1129-1132, Ross R, Glomset GA. The pathogenesis of atherosclerosis. N Engl J Med 1976;296:369-377. Sanborn TA, Faxon DP, Haudenschild CC, Gottsman SB, Ryan TJ. The mechanism of transluminal angioplasty: evidence for aneurysm formation in experimental atherosclerosis. Circulation 1983;68:1136-1140. Block PC, Baughman KL, Pasternack RC, Fallen TJ. Transluminal angioplasty: correlation of morphologic and angiographic findings in an experimental model. Circulation 1980;61:778-785. Casteneda-Zuniga WR, Fomanek T, Tadavarthy M et al. The mechanism of balloon angioplasty. Radiology 1980;135:565-571. Harker LA. Role of platelets and thrombus in mechanisms of acute occlusion and restenosis after angioplasty. Am J Cardiol 1987;60:20B-28B. Steele PM, Cheseboro 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. Essed CE, Van der Brand M, Becker AE. Transluminal coronary angioplasty and early restenosis: fibrocellular occlusion after wall laceration. Br Heart J 1983;49:393-396.
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19 Austin CE, Ratliff MB, Hollman J. Tabei S, Philips DF. Intimal proliferation of smooth muscle cells as an explanation for recurrent coronary artery stenosis after percutaneous transluminal coronary angioplasty. J Am Coil Cardiol 1985;6:369-375. 20 Wailer BF, McManus BM, Gorfinkel HJ et al. Status of the major epicardial coronary arteries 80 to 150 days after percutaneous transluminal coronary angioplasty: analysis of 3 necropsy patients. Am J Cardiol 1983;51:81-84. 21 Johnson DE, Robertson G, Simpson JB. Coronary atherectomy: light microscopy and immunochemical study of excised tissues. Circulation 1988;78(suppl II):83. 22 Packham MA, Mustard JF. Pharmacology of platelet-affecting drugs. Circulation 1980;62(suppl V):26-41. 23 Steele PM, Cheseboro JH, Holmes DR. Badimon L, Fuster
V. Balloon angioplasty in pigs: comparative effects of platelet-inhibitor drugs (abstract). Circulation 1984; 7Otsuppl IIk361. 24 Hews M, Chesebro JH, Penny WJ, Bailey KR, Budimen L, Fuster V. Effect of thrombin inhibition on the development of acute platelet-thrombus depression during angioplasty in pigs. Heparin versus recumbinant hirudin, a specific thrombin inhibitor. Circulation 1989;79:657-664. 25 Franklin S. Currier JW, Cannistra A et al. Warfarin/aspirin combination reduces restenosis after angioplasty in atherosclerotic rabbits. Circulation 1990;82(suppl 111):427. 26 Faxon DP, Sanborn TA, Haudenschild CC, Ryan TJ. Effect of antiplatelet drugs on restenosis following experimental angioplasty. Am J Cardiol 1984:53:72C-76C.
