Antithrombotic Potential of Polymer-Coated Stents Eluting Platelet Glycoprotein IIb/IIIa Receptor Antibody
Background Monoclonal anti-rabbit platelet glycoprotein (GP) IIb/IIIa antibody (AZ1) was adsorbed onto cellulose polymer–coated intracoronary stents to enhance their thromboresistance. We evaluated the antithrombotic efficacy of AZ1 antibody–eluting stents.
Methods and Results Twenty-three polymer-coated stents with AZ1 antibody bound by passive adsorption (AZ1-eluting) were compared with 23 control polymer-coated stents adsorbed with either no antibody (base-polymer, n=12) or isotype-matched irrelevant antibody (anti-CMV–eluting, n=11) by implantation into balloon-damaged, flow-reduced iliac arteries of New Zealand White rabbits. In 13 animals (acute group), flow measurements were made with transit-time flow probes and platelet adhesion was ascertained by use of 111In-labeled autologous platelets. In the other 10 animals (chronic group), stent occlusion was assessed macroscopically after they were killed 28 days after stenting. Arteries with AZ1-eluting stents had significantly less platelet deposition (15.8±4.5×107) than either base-polymer (32.1±4.3×107) or anti-CMV–eluting (35.2±8.8×107) controls (ANOVA, P<.0001). Compared with base-polymer or anti-CMV–eluting controls, arteries with AZ1-eluting stents showed a marked reduction in cyclic blood flow variation (P<.0001) and a significantly greater mean blood flow 2 hours after stent deployment (P<.0001). There was a significant improvement in the patency rate of AZ1-eluting stents compared with controls at both 2 hours (92% versus 46%, P=.034) and 28 days (100% versus 40%, P=.015).
Conclusions Platelet GP IIb/IIIa antibody eluting from polymer-coated stents reduces platelet deposition, improves blood flow, virtually abolishes cyclic flow variation, and improves patency rates after stent implantation in a rabbit iliac artery model. Its potential for reducing stent-related thrombosis in humans warrants further evaluation.
Thrombotic stent occlusion, although reduced in incidence in recent years, remains a significant problem particularly after failed angioplasty (bailout)1 2 3 4 and in small vessels.3 4 These situations generally necessitate systemic anticoagulation, which prolongs hospital stay and increases the incidence of vascular complications.5 6 There is therefore intense interest in reducing the thrombogenicity of intracoronary stents, because eliminating the need for systemic anticoagulation would considerably widen the applicability of this technique. One approach to this is to use the stent itself as a vehicle eluting potent antiplatelet agents.7 8
We reasoned that a potent antiplatelet agent eluting from the stent could passivate adherent platelets on the injured endothelial surface and thereby reduce thrombus formation after stent deployment. To test this hypothesis, we evaluated the antithrombotic efficacy of antiplatelet GP IIb/IIIa antibody eluting from cellulose polymer–coated stents. Using polymer-coated stent wires passively adsorbed with AZ1, we assessed the elution kinetics of AZ1 antibody during in vitro perfusion. Subsequently, we used a previously developed in vivo rabbit iliac artery model of stent thrombosis9 to evaluate the effects of antibody-eluting polymer-coated stents on cyclic blood flow variation, platelet deposition, and stent occlusion rates.
Radioactive iodine (125I) was purchased from Amersham Life Science. All other chemicals were purchased from Sigma Chemical Co unless otherwise stated. The murine monoclonal antibody AZ1 has been characterized previously by Azrin et al.10 The isotype-matched irrelevant murine monoclonal antibody (anti-CMV) was purified from ascites kindly provided by the Central Public Health Laboratory (Colindale, London) by affinity chromatography using protein A–sepharose.11 AZ1 and anti-CMV antibodies were labeled with 125I by the Iodo-gen method.12 All animal procedures were conducted in accordance with the Animals [Scientific Procedures] Act 1986 under license from the Home Office, London.
Cellulose polymer–coated stainless steel stent wires and stents (12 mm long×3 mm expanded diameter) were supplied by Cook Inc. The coating consists of a cellulose polymer that is dissolved in acetone and applied as a 10% solution to standard (0.16-mm diameter) stainless steel stent wire resulting in a 0.19-mm-diameter cellulose polymer–coated stainless steel wire.
AZ1 Antibody Adsorption to Polymer-Coated Stent Wire
Polymer-coated stainless steel stent wire segments (10 mm long) were immersed in a solution of AZ1 antibody (specific activity, 30 μCi/mg) diluted to 0.1, 0.5, or 1.0 mg/mL in coating buffer (0.01 mol/L sodium phosphate/0.145 mol/L sodium chloride, pH 7.2) at 37°C. Antibody solutions were contained in 1.5 mL polypropylene (Eppendorf) tubes, and wire segments were placed vertically and totally immersed in each solution. One, 4, 12, 24, and 48 hours after immersion, wires were removed from each solution and rinsed 3 times with 5 mL PBS, and antibody binding was quantified by counting the radioactivity associated with each wire. Ten wire segments were assessed at each time point for each concentration.
AZ1 Antibody Elution From Stent Wire In Vitro
Stent wire segments were immersed in 1-mg/mL solutions of AZ1 antibody diluted in coating buffer at 37°C for 24 hours as described above. Baseline antibody binding to wires was ascertained by counting the radioactivity of the wires. The wires (n=12) were then perfused continuously at 10 mL/min in a closed-loop circuit with PBS containing 1% BSA. Wires were housed in glass chambers, diameter 2.0 mm, connected to a manifold device (adapted from a multiple manifold dispenser, Labindustries) to ensure equal flow across all channels. The PBS was pumped through the circuit with a peristaltic pump (Watson-Marlow 302S). Sterile silicone tubing (3-mm bore, Fisons) was used to carry the perfusate to the chambers housing stent wires. After 30 minutes and 1, 2, 4, 6, and 12 hours, all 12 wires were removed from the perfusion circuit, counted in a gamma well counter to quantify the amount of antibody remaining bound to each, and then returned to the perfusion circuit. This was repeated every 12 hours for 14 days. The perfusing solution (total volume, 250 mL) was changed routinely every 48 hours.
Antithrombotic Efficacy of AZ1-Eluting Stents
Twenty-three New Zealand White male rabbits (median weight, 3.9 kg; range, 3.5 to 4.7 kg) received aspirin 6 mg·kg−1·d−1 administered in drinking water for 5 days before operation. Thirteen animals (acute group) had 17 mL of blood collected (2 hours before operation) into 3 mL of acid citrate-dextrose by cannulation of an ear artery for 111In labeling of platelets.13 All animals were anesthetized with Hypnorm 0.3 mL/kg IM (fentanyl citrate, 0.315 mg/mL and fluanisone, 10 mg/mL) and inhaled halothane and oxygen. Animals were placed on a heating pad (38°C) and had continuous intraoperative monitoring of rectal temperature, heart rate, and respiratory rate.
AZ1-eluting and anti-CMV–eluting stents were prepared by immersion of expanded stents into 1-mg/mL solutions of each antibody (under the conditions detailed in “AZ1 Antibody Adsorption,” above) for 24 hours before implantation.
Acute Group (n=13)
One hour before induction of anesthesia, autologous 111In-labeled platelets (3 mL suspension, 0.15 to 0.27 mCi) were injected via an ear vein. Each femoral artery was exposed through a groin incision, which was extended cranially to expose the common iliac artery and abdominal aorta. A 3-mm-diameter, noncompliant coronary angioplasty balloon catheter was advanced under direct vision via a femoral arteriotomy to the proximal common iliac artery, where it was inflated to 8 atm for 60 seconds. The balloon inflation was repeated three times to induce deep arterial injury. A 12-mm-long, 3-mm-diameter Gianturco-Roubin cellulose polymer–coated stainless steel stent was then deployed at the site of arterial injury (deploying pressure, 6 atm). Thereafter, ligation of the superficial femoral artery at the arteriotomy site was used to reduce blood flow through the stent. All animals had an AZ1 antibody–eluting, polymer-coated stent implanted in one vessel and an identical control polymer-coated stent with either no active agents (base polymer, n=7) or anti-CMV antibody (n=6) eluting from it implanted in the contralateral vessel.
After stent implantation, flow measurements were made continuously with transit-time perivascular flow probes (Transonics Inc) placed immediately distal to the stents. The following end-point definitions were used: (1) total stent occlusion: reduction in flow to <0.5 mL/min for >10 minutes; and (2) flow variation: transient reduction in flow to ≤0.5 mL/min (with restoration to flow >0.5 mL/min within 10 minutes). Two hours after the second stent was deployed, animals were killed with an intravenous overdose of pentobarbitone (140 mg/kg). Arteries were then flushed with PBS and perfusion-fixed in situ with 4% formaldehyde (Pearce Laboratories) and 0.1% glutaraldehyde. A 22-mm segment of stented vessel (12-mm stent and 5-mm proximal and distal segments) was removed, and platelet deposition was quantified by counting of the 111In activity of vessels, blood specific activity, and platelet count of each animal.
Chronic Group (n=10)
In 10 animals (chronic group), dissection was limited to exposure of the superficial femoral artery and extraperitoneal exposure of the inguinal ring via a midline lower abdominal incision. All had balloon damage to the proximal common iliac arteries before stent deployment as detailed above. However, balloon and subsequent stent positioning was undertaken with the inguinal ligament used as a landmark: the distal end of the balloon was advanced ≈1.5 cm beyond the inguinal ring.
Animals received no anticoagulant or antiplatelet therapy after operation. All were killed 28 days after stent deployment. Stented vessels were then carefully dissected free of surrounding adventitial connective tissue and examined macroscopically for evidence of thrombotic occlusion.
After perfusion-fixation (as for the acute group), vessels were carefully divided into two halves transversely along the midpoint of the stented segment. One half was further sectioned at 5-mm intervals perpendicular to the vessel long axis, and residual stent wire fragments were carefully removed. Each section was then paraffin-embedded before further sectioning and staining with hematoxylin-eosin and van Gieson's elastic tissue stains.
Intimal and medial dimensions of stented arteries from 7 of the 10 animals killed after 28 days were then measured by an observer blinded to the stent type in each vessel. A computerized morphometry system consisting of a Zeiss Axioskop microscope (fitted with a color video camera), a digitizing pad, and an IBM-compatible computer with KS100 Imaging system software (Kontron Elektronik GmbH) was used. The intima was defined as extending from the vessel lumen to the internal elastic lamina, and the media from the internal to the external elastic lamina. The long axis of each arterial section was determined visually, and each was divided into eight equal segments by lines joining the midpoint of the long axis to the adventitia. At points at which these lines intersected the internal or external elastic lamina of the tissue section, perpendiculars were drawn to the luminal surface or internal elastic lamina to determine the intimal or medial thickness, respectively. Mean intimal and medial thickness was thus obtained from eight values for each arterial section. Two noncontiguous sections (≥5 mm apart) were examined for each vessel.
Immunological Response to Antibody-Eluting Stents
Serum samples were available for 5 of the 10 animals in the chronic group 28 days after stent implantation and were analyzed by an ELISA for detecting rabbit antibodies to mouse IgG. Serum samples from 7 untreated rabbits were used as controls.
Polystyrene microtiter plates (Immulon 3, Dynatech) were coated with 100 μL/well AZ1 antibody diluted to 10 mg/L in coating buffer (0.01 mol/L sodium phosphate/0.145 mol/L sodium chloride, pH 7.2). The plates were kept at 4°C for at least 16 hours before use. Assay wells were emptied of storage buffer and washed three times with PBS/T. Aliquots (100 μL) of rabbit serum samples diluted 1:5 with PBS/T were pipetted into duplicate wells, and the plates were incubated at 37°C for 1 hour. Plates were then washed three times with PBS/T, and 100 μL of goat anti-rabbit IgG–horseradish peroxidase conjugate (product code P0448, Dako) diluted 2000-fold in PBS/T containing 1% normal mouse serum was added to each well. The plates were incubated at 37°C for 1 hour. After three further washes with PBS/T, 100 μL of o-phenylenediamine dihydrochloride substrate solution (10 mg o-phenylenediamine dihydrochloride in 25 mL 0.05 mol/L sodium phosphate/0.03% [wt/vol] sodium perborate, pH 5.0) was added to each well, and the plates were incubated at room temperature for 10 minutes. One hundred microliters of 2 mol/L H2SO4 was then added to each well, and the absorbances of the wells were read at 492 nm by an automated microplate reader.
Data are presented either as mean±SD or as proportions. For continuous data, differences between AZ1-eluting, base-polymer, and anti-CMV–eluting stents were compared by one-way ANOVA. Where there was a significant difference between the groups, multiple comparisons between groups were made by a modified t test (Bonferroni correction). Differences in proportions were analyzed with Yates' corrected χ2 test. Significance was defined as P<.05.
AZ1 Antibody Adsorption to Polymer-Coated Stent Wire
AZ1 antibody binding to stent wires was evaluated at three different concentrations (0.1, 0.5, and 1.0 g/L). There was an increase in the amount of antibody bound in relation both to immersion time of stent wires and also to antibody concentration (Fig 1⇓). More than 95% of the final amount bound at each concentration was adsorbed within 24 hours; maximal antibody binding was therefore defined as the amount of agent bound to stent wires after 24 hours of immersion in a 1-mg/mL solution. By this method, maximal antibody binding (per 10-mm wire) was 104.3±1.3 ng for the 1-mg/mL solution, 80.5±2.7 ng for the 0.5-mg/mL solution, and 58.3±1.2 ng for the 0.1-mg/mL solution.
The increase in antibody bound to wires in relation to its concentration in immersing solutions suggests an element of uptake of protein solution within the polymer, because surface adsorption is likely to be constant for the antibody concentrations used in this experiment. Thus, for a 10-mm-long wire segment, the differences in maximum binding between the three solutions suggest uptake of 0.05±0.004 μL of antibody solution by the polymer, 93.3±7.38% of the polymer volume.
AZ1 Antibody Elution From Stent Wire In Vitro
Curve-fitting of data obtained from elution experiments using a computerized scientific data analysis package (Prism, GraphPad Software Inc) demonstrated biexponential elution of AZ1 antibody from stent wires, with an initial rapid washoff followed by a slower exponential reduction in the amount of antibody persisting on stent wires (Fig 2⇓). After 14 days of continuous perfusion with PBS+1% BSA, almost 40% of the amount of AZ1 originally adsorbed remained bound to wires.
Antithrombotic Efficacy of AZ1-Eluting Stents
Baseline iliac artery blood flow, both before and after femoral artery ligation, was well matched for arteries with the three stent types and is shown in Table 1⇓. The overall reduction in blood flow after superficial femoral artery ligation was 56.5±6.0% and was similar for arteries implanted with AZ1 antibody–eluting stents (56.2±5.68%), base-polymer stents (58.9±8.22%), and anti-CMV–eluting stents (54.5±2.72%) (ANOVA, P=.41). Deep arterial injury (ruptured internal elastic lamina) was confirmed histologically in all stented vessels.
Cyclic blood flow variation (gradual reduction in flow to ≤0.5 mL/min followed by abrupt restoration to flow >0.5 mL/min within 10 minutes) occurred in all 13 arteries implanted with control (base-polymer and anti-CMV–eluting) stents, compared with only 1 of 13 arteries implanted with AZ1 antibody–eluting stents (P<.0001). The cyclic flow variation frequency (number of episodes of flow variation per hour) for the three stent types is shown in Fig 3⇓ and was significantly reduced in arteries implanted with AZ1 antibody–eluting stents compared with controls (ANOVA, P<.0001). Mean blood flow through vessels patent after 2 hours was significantly higher in arteries with AZ1 antibody–eluting stents (6.65±2.29 mL/min) compared with base-polymer controls (0.77±0.15 mL/min, P=.0002) and anti-CMV–eluting controls (0.87±0.25 mL/min, P=.0002). The difference in flow between base-polymer and anti-CMV–eluting stents was not statistically significant. Blood flow through stented vessels patent after 2 hours (as a percentage of post–femoral ligation basal flow) is shown in Fig 4⇓.
Platelet deposition in each stented artery (22-mm segment) was significantly reduced in arteries implanted with AZ1 antibody–eluting stents (15.77±4.47×107 platelets) compared with vessels with both base-polymer controls (32.08±4.33×107 platelets, P<.0001) and anti-CMV–eluting controls (35.17±8.8×107 platelets, P<.0001), Fig 5⇓. Two hours after stent implantation, 12 (92.3%) of 13 vessels implanted with AZ1-eluting stents were patent, compared with 6 (46.2%) of 13 vessels with control stents (3 of 7 base-polymer and 3 of 6 anti-CMV–eluting stents), P=.034.
All 10 animals made an uneventful postoperative recovery, and there were no signs of limb ischemia or paralysis during the 28-day observation period. After the animals were killed, occlusive thrombus was present in 3 of 5 arteries with base-polymer stents and 3 of 5 arteries with anti-CMV–eluting controls, compared with none of the 10 vessels with AZ1 antibody–eluting stents (P=.015).
Arterial Patency (Acute and Chronic Groups)
There was a highly significant improvement in the overall patency rate of AZ1 antibody–eluting stents (22 [95.7%] of 23) compared with both base-polymer (5 [41.7%] of 12) and anti-CMV–eluting (5 [45.5%] of 11) controls, P=.0004.
Neointimal formation was observed in all stented vessels examined; mean±SD intimal thickness was 0.12±0.04 mm for arteries with AZ1-eluting stents and 0.10±0.05 mm for vessels with control stents (P=.32). Details of intimal and medial thickness according to stent type are shown in Table 2⇓.
Immunological Response to Antibody-Eluting Stents
All 5 animals for which serum samples were available 28 days after stent implantation appeared to develop anti-mouse antibodies. Mean absorbance (492 nm) for 1:5 dilutions of sera was 1.64±0.23, compared with 0.62±0.48 for similar dilutions of sera from untreated rabbits, P=.001.
In this study, we show that monoclonal antibody to the platelet GP IIb/IIIa receptor, when eluting from cellulose polymer–coated stents, effectively inhibits platelet aggregation in the stent microenvironment, thus reducing thrombus formation, improving blood flow and arterial patency rates, and inhibiting cyclic blood flow variation. These results suggest that such antibody-eluting stents may reduce or even eliminate the need for systemic anticoagulation.
Platelet GP IIb/IIIa Antibody–Eluting Stents
In the present study, there was a >50% reduction in platelet deposition 2 hours after stents eluting platelet GP IIb/IIIa antibody were deployed in a deep-arterial-injury, flow-reduced rabbit iliac artery model compared with control stents eluting either irrelevant antibody or no active agents. Furthermore, platelet aggregation in the stent microenvironment was strikingly inhibited by the eluting GP IIb/IIIa antibody, as evidenced by the almost total abolition of cyclic blood flow variation in these vessels. Cyclic flow variations are caused by repetitive accumulation and dislodgement of platelet aggregates in stenosed, intima-damaged vessels14 and may be abolished by antiplatelet agents.15 16 17 In previous work, however, we found that cyclic flow variations occurring after stent implantation in a flow-reduced, deep-arterial-injury rabbit iliac artery model were not inhibited by aspirin, confirming the need for more potent antiplatelet therapy to reduce vascular stent-related thrombosis.9 Reduction in platelet adhesion and aggregation in the present study also translated into a substantial (>90%) reduction in occlusion rates of vessels with AZ1 antibody–eluting stents compared with controls in this stringent model of stent thrombosis.
Antagonists of the platelet GP IIb/IIIa receptor inhibit platelet aggregation irrespective of the metabolic pathway initiating platelet aggregation18 and are known to be effective in humans, particularly in unstable angina and elective coronary intervention.19 20 21 Systemically administered monoclonal antiplatelet GP IIb/IIIa antibody has also been shown to reduce abrupt vessel closure after high-risk coronary angioplasty,22 but at the expense of a twofold to threefold increase in the risk of bleeding complications.22 23 Recent identification of a high level of surface expression of the platelet fibrinogen receptor as a strong independent predictor of subacute stent occlusion24 also suggests that potent antiplatelet therapy may be particularly effective in preventing stent thrombosis. The most significant limitation of GP IIb/IIIa antagonists remains the risk of bleeding complications associated with their systemic use. The present study evaluated the efficacy of local blockade of platelet GP IIb/IIIa receptors at the site of arterial disruption.
Extent of GP IIb/IIIa Receptor Blockade
In patients undergoing coronary angioplasty, it has been suggested that more than 80% of GP IIb/IIIa receptor blockade is required to significantly reduce platelet aggregation.25 In support of this is the finding that ischemic complications after high-risk coronary angioplasty were prevented more effectively in patients receiving a prolonged (12-hour) intravenous infusion of the monoclonal antibody c7E3 (directed against human platelet GP IIb/IIIa receptors) than in those receiving only a bolus dose in the prospective randomized EPIC study.22
In the present study, the amount of AZ1 antibody delivered by a 12-mm-long stent (1.15±0.11 μg) would clearly be insufficient to block 80% of GP IIb/IIIa receptors on all circulating platelets. However, the amount of available antibody (≈4.62×1012 molecules) would be sufficient to block the receptors on ≈108 platelets, assuming that each platelet had 4.62×104 receptors. Thus, the most likely mechanism of benefit of such a small amount of antibody, delivered locally, is blockade of the GP IIb/IIIa receptors on the first layer of platelets adhering to the injured endothelium and stent surface. This would lead to “passivation” of the adherent platelets and reduce further platelet recruitment.
Accumulation of a platelet monolayer after balloon-induced endothelial denudation has been demonstrated by other workers.26 Additionally, studies estimating platelet adhesion on endothelial or intima-injured rabbit aortas suggest a level of ≈50 000 platelets/mm2.27 The stented arterial surface area in the present studies (12×3-mm stent) is 113 mm2. Thus, ≈5.65×106 platelets may be expected to adhere initially, representing a >17-fold excess of available antibody to passivate the surface-expressed GP IIb/IIIa receptors on this adherent layer of platelets. Additionally, the available antibody would also suffice to inhibit the internal pool of GP IIb/IIIa receptors, because it has been postulated that the internal pool of receptors may be mobilized to join the surface membrane and restore platelet aggregability.28
Advantages of Passively Adsorbed GP IIb/IIIa Antibody
Passive adsorption of drugs to polymeric surfaces is a simple procedure, effective in a number of situations. Local delivery of antithrombin agents passively adsorbed to hydrogel-coated angioplasty balloons, for example, has been shown to effectively decrease early platelet deposition in porcine angioplasty models.29 30 In diagnostic systems such as ELISA, passive adsorption of proteins is routinely used for coating of antibody or antigen onto microtiter plate wells.31 32 Although stents have a smaller surface area and therefore carry smaller amounts of drug than angioplasty balloons, they have the potential advantage of being able to elute the drug over a longer period. Antiplatelet GP IIb/IIIa antibody may be particularly useful in this respect. In the present studies, adsorption of AZ1 antibody increased in relation to its concentration in the solution used for immersing stent wires, suggesting an element of uptake of protein solution by the polymer itself. This may explain the prolonged persistence of antibody on wires during in vitro perfusion.
Surface modification by elution of preadsorbed potent antiplatelet agents can thus enhance the thromboresistance of stents. An alternative is drug immobilization by covalent bonding; this leads to surface passivation, reducing platelet adhesion.33 One example is the heparin-bonded polymer-coated stent currently undergoing clinical trials (Benestent II study). Data from the pilot study indicate that no stent thrombosis occurred despite progressive reduction in systemic anticoagulation and eventual replacement with antiplatelet agents.34 It is noteworthy, however, that the patients in this study all had de novo lesions in relatively large (reference vessel diameter, 3.05 to 3.22 mm) native coronary arteries34 and therefore a low risk of stent thrombosis. Additionally, no control group of patients receiving uncoated stents or polyamine-coated stents without bonded heparin was assessed. Thus, it is unclear whether the Benestent II results can be directly attributed to the heparin-coated stent. Furthermore, heparin is likely to be ineffective in more thrombogenic situations, such as bailout and small-vessel stenting, because fibrin-bound thrombin is relatively resistant to inactivation by heparin35 and heparin is locally neutralized by factors secreted from activated platelets.36 Stents eluting direct thrombin inhibitors or potent antiplatelet agents may thus prove to be more effective in the presence of clot-bound thrombin.
Drug-eluting stents may have other potential benefits, particularly if used to deliver a combination of antithrombotic and antiproliferative therapy.37 Some inhibitors of the platelet integrin GP IIb/IIIa may, for instance, inhibit smooth muscle cell proliferation and migration by cross-reacting with the αvβ3 integrin (vitronectin receptor) present on smooth muscle cells.38 39 In the present study, platelet GP IIb/IIIa antibody–eluting stents did not significantly affect neointimal proliferation. This finding contradicts recent suggestions that the monoclonal antibody c7E3, directed against the human platelet integrin GP IIb/IIIa, may reduce clinical restenosis when administered during and after coronary angioplasty (EPIC trial).40 However, the amount of GP IIb/IIIa (AZ1) antibody delivered by stents in the present study was considerably less than the doses of c7E3 antibody administered systemically in the EPIC trial. Furthermore, c7E3 is relatively nonspecific, and its ability to bind to other integrins such as the vitronectin receptor (αvβ3) may be important for inhibiting neointimal proliferation.18 In contrast, AZ1 antibody has no action against the vitronectin receptor. Further studies are needed to establish the importance of such cross-reactivity in reducing neointimal proliferation.
The adsorption of AZ1 antibody to stent wires did not achieve a plateau at the maximum concentration (1 mg/mL) used in the present studies; it is therefore possible that greater amounts of antibody could be adsorbed if higher concentrations were used. The elution kinetics of passively adsorbed antibody from stents were not studied in vivo, where they may prove to be quite different from the in vitro kinetics. However, our in vitro perfusion studies used a buffer at blood pH with BSA added to mimic the effect of plasma proteins that would be adsorbed onto an artificial surface introduced into the circulation, potentially displacing any bound drugs.41 We did not use blood for these studies because it was technically impractical; furthermore, it is unlikely that blood cells would be viable under the conditions of the elution experiment.
An additional limitation is that platelet deposition was studied only at a single time point in vivo. However, blood flow measurements were made continuously up to the defined study end point, allowing dynamic evaluation of platelet aggregation. We used a deep-arterial-injury, reduced-flow model for this study. This is a particularly stringent model of thrombosis, allowing a high (>50%) total occlusion rate for vessels with control stents. It is possible that this model would exaggerate the benefit of locally eluting antiplatelet agents; there is, however, no ideal model to study thrombosis in vivo, and our model has the advantage of being reproducible and simple. Flow reduction may allow greater persistence of antibody on the stent, thereby exaggerating its benefits; however, we have found no significant change in the elution rate of antibody from stent wires with perfusing flow rates ranging from 5 to 50 mL/min in vitro (unpublished observations).
These promising results indicate that the monoclonal platelet GP IIb/IIIa receptor antibody AZ1, when passively adsorbed to and eluting from a cellulose polymer–coated coronary stent, significantly reduces platelet deposition, almost completely eliminates cyclic blood flow variation, and improves mean blood flow and arterial patency rates in a rabbit iliac artery model of stent thrombosis. Coronary stents eluting GP IIb/IIIa antibody directed against human platelets may be effective in eliminating the need for systemic anticoagulation after deployment in humans, particularly in high-risk situations such as bailout and small-vessel stenting, and warrant further evaluation.
Selected Abbreviations and Acronyms
|AZ1||=||anti–rabbit platelet GP IIb/IIIa antibody|
|PBS/T||=||PBS containing 0.05% (vol/vol) Tween 20|
Dr Aggarwal was supported by project grants (93/109, 95/167) from the British Heart Foundation. Dr Ireland is supported by the British Heart Foundation, and Dr de Bono is a British Heart Foundation Professor. Professor Ezekowitz was supported by a Veterans Administration Merit Review Grant.
Presented in part at the 68th Scientific Sessions of the American Heart Association, Anaheim, Calif, November 13-16, 1995, and published in abstract form (Circulation. 1995;92[suppl I]:I-488).
- Received May 14, 1996.
- Revision received July 3, 1996.
- Accepted July 11, 1996.
- Copyright © 1996 by American Heart Association
Morice MC, Amor M, Benveniste E, Bunouf P, Cribier A, Labrunie P, Masquet C, Petiteau PY. Coronary stenting without coumadin phase II, III, IV, V: predictors of major complications. Circulation. 1995;92(suppl I):I-795. Abstract.
Serruys PW, de Jaegere P, Kiemeneij F, Macaya C, Rutsch W, Heyndrickx G, Emanuelsson H, Marco J, Legrand V, Materne P, Belardi J, Sigwart U, Colombo A, Goy JJ, van den Heuvel P, Delcan J, Morel M, for the Benestent Study Group. A comparison of balloon-expandable-stent implantation with balloon angioplasty in patients with coronary artery disease. N Engl J Med. 1994;331:489-495.
Wong SC, Baim DS, Schatz RA, Teirstein PS, King SB III, Curry RC Jr, Heuser RR, Ellis SG, Cleman MW, Overlie P, Hirshfeld JW, Walker CM, Litvack F, Fish D, Brinker JA, Buchbinder M, Goldberg S, Chuang YC, Leon MB, for the Palmaz-Schatz Stent Study Group. Immediate results and late outcomes after stent implantation in saphenous vein graft lesions: the multicenter U.S. Palmaz-Schatz stent experience. J Am Coll Cardiol. 1995;26:704-712.
Schwartz RS, Murphy JG, Edwards WD, Holmes DR. Bioabsorbable, drug-eluting, intracoronary stents: design and future applications. In: Sigwart U, Frank GI, eds. Coronary Stents. Berlin, Germany: Springer Verlag; 1992:135-154.
Lambert TL, Dev V, Rechavia E, Forrester JS, Litvack F, Eigler NL. Localized arterial wall drug delivery from a polymer-coated removable metallic stent: kinetics, distribution, and bioavailability of forskolin. Circulation. 1994;90:1003-1011.
Aggarwal RK, More RS, Ezekowitz MD, de Bono DP, Gershlick AH. Stent-related thrombosis: a deep arterial injury, reduced flow model. Thromb Haemost. 1995;73:1331. Abstract.
Azrin MA, Ling FS, Chen Q, Pawashe A, Migliaccio F, Homer R, Todd M, Ezekowitz MD. Preparation, characterization, and evaluation of a monoclonal antibody against the rabbit platelet glycoprotein IIb/IIIa in an experimental angioplasty model. Circ Res. 1994;75:268-277.
Folts J. An in vivo model of experimental arterial stenosis, intimal damage and periodic thrombosis. Circulation. 1991;83(suppl IV):IV-3-IV-14.
Folts JD, Crowell EB, Rowe GG. Platelet aggregation in partially obstructed vessels and its elimination with aspirin. Circulation. 1976;54:365-370.
Bush LR, Campbell WB, Buja M, Tilton GD, Willerson JT. Effects of the selective thromboxane synthetase inhibitor dazoxiben on variations in cyclic blood flow in stenosed canine coronary arteries. Circulation. 1984;69:1161-1170.
Yao SK, McNatt J, Benedict CR, Rosolowsky M, Anderson HV, Cui K, Maffrand JP, Campbell WB, Buja LM, Willerson JT. ADP plays an important role in mediating platelet aggregation and cyclic flow variations in vivo in stenosed and endothelium-injured canine coronary arteries. Circ Res. 1992;70:39-48.
Simoons ML, de Boer MJ, van den Brand MJBM, van Miltenburg AJM, Hoorntje JCA, Heyndrickx GR, van der Wieken LR, de Bono D, Rutsch W, Schaible TF, Weisman HF, Klootwijk P, Nijssen KM, Stibbe J, de Feyter PJ, and the European Cooperative Study Group. Randomized trial of a GPIIb/IIIa platelet receptor blocker in refractory unstable angina. Circulation. 1994;89:596-603.
Konstantopoulos K, Kamat SG, Schafer AI, Banez EI, Jordan R, Kleiman NS, Hellums JD. Shear-induced platelet aggregation is inhibited by in vivo infusion of an anti-glycoprotein IIb/IIIa antibody fragment, c7E3 Fab, in patients undergoing coronary angioplasty. Circulation. 1995;91:1427-1431.
Tcheng JE, Harrington RA, Kottke-Marchant K, Kleiman NS, Ellis SG, Kereiakes DJ, Mick MJ, Navetta FI, Smith JE, Worley SJ, Miller JA, Joseph DM, Sigmon KN, Kitt MM, du Me´e CP, Califf RM, Topol EJ, for the IMPACT Investigators. Multicenter, randomized, double-blind, placebo-controlled trial of the platelet integrin glycoprotein IIb/IIIa blocker integrelin in elective coronary intervention. Circulation. 1995;91:2151-2157.
Aguirre FV, Topol EJ, Ferguson JJ, Anderson K, Blankenship JC, Heuser RR, Sigmon K, Taylor M, Gottlieb R, Hanovich G, Rosenberg M, Donohue TJ, Weisman HF, Califf RM, for the EPIC Investigators. Bleeding complications with the chimeric antibody to platelet glycoprotein IIb/IIIa integrin in patients undergoing percutaneous coronary intervention. Circulation. 1995;91:2882-2890.
Tcheng JE, Ellis SG, George BS, Kereiakes DJ, Kleiman NS, Talley D, Wang AL, Weisman HF, Califf RM, Topol EJ. Pharmacodynamics of chimeric glycoprotein IIb/IIIa integrin antiplatelet antibody Fab 7E3 in high-risk coronary angioplasty. Circulation. 1994;90:1757-1764.
Groves HM, Kinlough-Rathbone RL, Mustard JF. Development of nonthrombogenicity of injured rabbit aortas despite inhibition of platelet adherence. Arteriosclerosis. 1986;6:189-195.
Kleiman NS, Raizner AE, Jordan R, Wang AL, Norton D, Mace KF, Joshi A, Coller BS, Weisman HF. Differential inhibition of platelet aggregation induced by adenosine diphosphate or a thrombin receptor-activating peptide in patients treated with bolus chimeric 7E3 Fab: implications for inhibition of the internal pool of GPIIb/IIIa receptors. J Am Coll Cardiol. 1995;26:1665-1671.
Azrin MA, Mitchel JF, Fram DB, Pedersen CA, Cartun RW, Barry JJ, Bow LM, Waters DD, McKay RG. Decreased platelet deposition and smooth muscle cell proliferation after intramural heparin delivery with hydrogel-coated balloons. Circulation. 1994;90:433-441.
Kemeny DM. The solid-phase support and the coating antigen or antibody. In: Kemeny DM. A Practical Guide to ELISA. Oxford, UK: Pergamon Press; 1991:31-44.
Serruys PW, Emanuelsson H, van der Giessen W, Lunn AC, Kiemeney F, Macaya C, Rutsch W, Heyndrickx G, Suryapranata H, Legrand V, Goy JJ, Materne P, Bonnier H, Morice MC, Fajadet J, Belardi J, Colombo A, Garcia E, Ruygrok P, de Jaegere P, Morel MA, on behalf of the Benestent-II Study Group. Heparin-coated Palmaz-Schatz stents in human coronary arteries: early outcome of the Benestent-II pilot study. Circulation. 1996;93:412-422.
Weitz JI, Hudoba M, Massel D, Maraganore J, Hirsh J. Clot-bound thrombin is protected from inhibition by heparin-antithrombin III but is susceptible to inactivation by antithrombin III-independent inhibitors. J Clin Invest. 1990;86:385-391.
Lane DA, Denton J, Flynn AM, Thunberg L, Lindahl U. Anticoagulant activities of heparin oligosaccharides and their neutralization by platelet factor 4. Biochem J. 1984;218:725-732.
Lincoff AM, Topol EJ, Ellis SG. Local drug delivery for the prevention of restenosis: fact, fancy and future. Circulation. 1994;90:2070-2084.
Matsuno H, Stassen JM, Vermylen J, Deckmyn H. Inhibition of integrin function by a cyclic RGD-containing peptide prevents neointima formation. Circulation. 1994;90:2203-2206.
Topol EJ, Califf RM, Weisman HF, Ellis SG, Tcheng JE, Worley S, Ivanhoe R, George BS, Fintel D, Weston M, Sigmon K, Anderson KM, Lee KL, Willerson JT, on behalf of the EPIC investigators. Randomised trial of coronary intervention with antibody against platelet IIb/IIIa integrin for reduction of clinical restenosis: results at six months. Lancet. 1994;343:881-886.