The activation of platelets following coronary stent implantation has been reported by measuring platelet aggregation and surface receptor expression in circulating blood.1,2 The administration of various pharmacological agents, including adenosine diphosphate (ADP)-receptor antagonists and parenteral glycoprotein (GP) IIb/IIIa inhibitors, reduces aggregation. Clearly, platelet activation is a major factor that affects immediate- and long-term outcome after stenting.3,4 Subacute closure is mediated primarily by the formation of large platelet aggregates bound to the stent surface and restenosis is affected by the release of platelet-derived mitogenic factors after activation.5,6 Platelet activation can be induced by shear forces and direct contact to the stent surface.7,8 The effect of various coatings, including diamond-like carbon,7 silicon-carbide,9 heparin10 and gold,11 has been evaluated. However, little is known about how the stent design itself may result in the activation of platelets as they pass the surface of the implant. Most of the available data have been produced in in vitro studies.7,9–11 As yet, there is no randomized trial in humans evaluating platelet activation following the implantation of stents with two different designs. Therefore, the purpose of the current pilot trial was to compare platelet activation following implantation of the closed-cell NIR stent (Boston Scientific/Scimed, Inc., Maple Grove, Minnesota) versus the open-cell TETRA stent (Guidant Corporation, Temecula, California). An ex vivo study in explanted swine hearts was performed to elucidate differences between the stents with respect to the surface contour of the vessel wall following implantation. The differences in design and resulting pattern of stent expansion could be expected to impart different shear forces on platelets and thus affect activation. Methods Patients. The study was approved by the Investigational Review Board of Sinai Hospital. This pilot trial consisted of 54 consecutive patients who underwent elective coronary artery stenting. Patients with a history of bleeding diathesis, acute myocardial infarction within 48 hours, stroke within 3 months, drug or alcohol abuse, prothrombin time > 1.5 times control, platelet count 4.0 mg/dl were excluded. No patient had previously received thienopyridines or intravenous GP IIb/IIIa blockers. Informed consent was obtained from all patients. The inclusion data were identical to those previously reported in the PRONTO trial.2 The population was randomly assigned to revascularization with either a NIR or TETRA stent. All patients received at least 81 mg aspirin daily for at least 1 week prior to the procedure. On the day of the procedure and qd thereafter, 325 mg of aspirin was given. All patients received a loading dose of clopidogrel (300 mg) in the catheterization laboratory followed by 75 mg daily for the next 30 days. Intravenous unfractionated heparin was administered prior to stent implantation to achieve an activated clotting time > 300 seconds. The stent diameter was recorded according to the manufacturers’ specifications as the maximum final balloon diameter. Stents were deployed at >= 12 atmospheres. All patients were followed in-hospital and for 30 days after discharge. Blood samples. Blood was collected in a vacutainer tube containing 3.8% trisodium citrate, filled to capacity, and then inverted 3–5 times for gentle mixing. Samples were obtained at baseline and at 2 hours, 24 hours, 5 days and 30 days post-stenting. Platelet-rich plasma aggregation. The blood-citrate mixture was centrifuged at 1,200 g for 2.5 minutes. The resulting platelet-rich plasma (PRP) was kept at room temperature for use within 1 hour. The platelet count was determined in the PRP sample and adjusted to 3.5 x 108/ml with homologous platelet-poor plasma. Platelets were stimulated with 5 µmol ADP (Chronolog, Hawerton, Pennsylvania) and aggregation was assessed as previously described using a Chronolog Lumi-Aggregometer (model 560-Ca) with the AggroLink software package.12 Aggregation was expressed as the maximal percent change in light transmittance from baseline using platelet-poor plasma as a reference. Curves were analyzed according to international standards.13 Whole blood aggregation. Whole blood aggregation was determined using a Chronolog device. The whole blood-citrate mixture was diluted 1:1 with 0.5 ml phosphate buffered saline and gently swirled. The sample was allowed to warm to 37 °C for 5 minutes and then transferred to the assay well. The electrode was then placed in the cuvette and the sample stimulated with 1 µg/ml collagen. The change in electrical impedance was recorded as previously described.14 Whole blood flow cytometry. The surface expression of platelet receptors was determined by flow cytometry using the following monoclonal antibodies: CD 41 (GP IIb/IIIa, aIIbb3), CD 31 [platelet/endothelial cell adhesion molecule (PECAM)-1], CD 107a (LAMP-1) and CD 151 (PETA-3) (PharMingen, San Diego, California). The PAC-1 antibody was obtained from Dr. Alan Michelson. This antibody binds to the active aIIbb3 receptor. The CD 41 antibody binds to both active and inactive aIIbb3 receptor. The blood-citrate mixture (50 ml) was diluted with 450 ml Tris buffered saline (TBS; 10 mmol/l Tris, 0.15 mol/l sodium chloride) and mixed by inverting an Eppendorf tube gently 2 times. The corresponding antibody was then added (5 ml) and incubated at 4 °C for 30 minutes. After incubation, 400 ml of 2% buffered paraformaldehyde was added for fixation. The samples were analyzed on a Becton Dickinson FACScan flow cytometer set up to measure fluorescent light scatter as previously described.14 All parameters were collected using 4 decade logarithmic amplification. The data were collected in list mode files and then analyzed. Platelet-leukocyte aggregates (CD 151 + 14) were determined by using dual antibodies for a pan-platelet marker (CD 151) and a monocyte-macrophage marker (CD 14). Antigens were expressed as log mean fluorescence intensity. Ex vivo studies. To determine the effect of stent design on luminal geometry, a casting procedure was performed on explanted hearts from common swine (~ 45 kg). The hearts were collected from a local abbatoir and transported to our laboratory in 0.9% saline solution for immediate study. The coronary artery was cannulated with a standard guiding catheter and sutured at the ostium. The heart was flushed with 0.9% saline solution to remove any residual blood. The vessel was visually inspected and then interrogated with an intravascular ultrasound catheter (40 MHz Atlantis SR, Boston Scientific/Scimed, Inc.) to precisely size the target segment. NIR Royal and TETRA stents were then deployed using a balloon to artery ratio of 1:1 on a straight vessel segment. A solution of 2% agarose (Sigma) was prepared by thorough mixing and heating to boiling in order to fully dissolve the agarose. The mixture was then injected at 37 °C into the stented artery at 100 mmHg until complete filling was achieved. After injection, the preparation was cooled to room temperature for 20 minutes to allow for complete solidification of the gel. Using standard surgical instruments, the artery was then carefully dissected free from the stent-gel cast in order to avoid damage to the stent. The cast was then dyed with food coloring to provide contrast in photography. The cast was photographed with a stereomicroscope and backlit to delineate the stent-artery wall interaction at the margins of the stent. Statistical analysis. Between-treatment comparisons were made at respective time points using t-tests. Data are expressed as means ± standard error, and p Clinical and procedural data. Fifty-four consecutive patients undergoing elective stenting were enrolled. The clinical characteristics are shown in Table 1. There were no major differences in clinical characteristics between the groups. One patient from each group received integrilin prior to stent implantation. Among the 54 eligible patients, twenty-two were randomly assigned to receive the NIR stent (Figure 1). The NIR stent was successfully implanted in 20/22 patients. In the TETRA group, implantation was successful in 31/32 patients. There were no episodes of subacute thrombosis, Q-wave myocardial infarction or death in the 30-day observation period post-stenting. Angiographic data. The two groups were similar with respect to total stent length and number of stents inserted per patient (Table 2). However, in the NIR group, procedure duration was approximately 10 minutes longer and minimal stent diameter was approximately 0.3 mm greater. Platelet aggregation. Baseline aggregation (%) in response to 5 mmol ADP did not differ between the groups (60.0 ± 14.3 for TETRA versus 63.2 ± 12.0 for NIR; p = 0.19). Following stenting, no differences were observed until 30 days when aggregation was 32.3 ± 6.1 in the NIR group versus 44.5 ± 18.9 in the TETRA group (p = 0.02) (Figure 2C). Baseline aggregation in response to collagen (ohms) did not differ between groups (21.8 ± 5.8 for TETRA versus 22.9 ± 5.2 for NIR; p = 0.407) and also did not differ significantly following stent implantation. However, a trend toward reduced aggregation was observed 5 days post-stenting with the NIR stent (21.5 ± 6.4 versus 25.8 ± 5.6; p = 0.09). Platelet surface molecules CD 31. Both groups had similar expression (mean fluorescence intensity) at baseline (125.1 ± 37 for TETRA versus 128 ± 69 for NIR; p = 0.36). At 24 hours post-stenting, patients randomized to the TETRA stent had greater expression (136 ± 48 versus 110 ± 48; p = 0.042) (Figure 2B). No differences were observed at other time points. CD 151. This activation-dependent marker did not differ between groups at baseline (97.6 ± 35.8 for TETRA versus 81.3 ± 45.6 for NIR; p = 0.22). However, enhanced activation was observed in the TETRA group at 24 hours (103.6 ± 45 versus 90.6 ± 30.5 for NIR; p = 0.045) (Figure 2B) and at 30 days (99 ± 33.3 versus 81 ± 31.7; p = 0.032) post-stenting (Figure 2D). CD 107a. No differences were observed between groups at baseline (18.4 ± 5.2 for TETRA versus 19.3 ± 7.1 for NIR; p = 0.332). At 2 hours (Figure 2A) and 24 hours (Figure 2B) post-stent implantation, the expression of CD 107a was less with the NIR stent (18.5 ± 5.0 versus 22.3 ± 12.9 at 2 hours; p = 0.045 and 16.6 ± 4.4 versus 24.4 ± 11.8 at 24 hours; p = 0.03). PAC-1. Baseline expression at this marker did not differ between groups (81.0 ± 50.7 for TETRA versus 78.4 ± 40.6 for NIR; p = 0.35). However, at 30 days, PAC-1 was lower in the NIR group (71.8 ± 30.5 versus 87.8 ± 41.0 in the TETRA group; p = 0.025) (Figure 2D). No differences were observed at other time points. Platelet-leukocyte aggregates (CD 151 + 14). The formation of platelet-leukocyte aggregates was significantly greater at 30 days post-TETRA stent implantation (73.8 ± 35.0 versus 71.8 ± 30.5 for NIR; p = 0.045) (Figure 2D). No differences were observed at baseline or at other time points. CD 41. No differences were observed between groups at baseline (390.9 ± 95 for TETRA versus 400.3 ± 125; p = 0.49) or at other time points in the expression of this molecule. Stent size and platelet markers. In each group, no significant correlation was observed between each of the above markers and minimal stent diameter (Figure 3). Ex vivo studies. Photographs of the stent-gel casts of the coronary arterial lumen are shown in Figures 4A and 4B. The artery stented with the TETRA stent exhibited multiple irregular areas created by invagination of the vessel wall between the struts. This property of the open-cell design was markedly less present in the closed-cell NIR design, which created a smoother stent-vessel wall interface. Discussion The results of the PAST study suggest that the degree of platelet activation following elective coronary artery stenting is dependent on stent design and that it is greater following implantation of an open-cell stent. Enhanced platelet activity was demonstrated using established, sensitive methods to detect the expression of adhesive molecules on the platelet surface.15 GP IIb/IIIa mediates fibrinogen binding. The PAC-1 antibody detects the active conformation of this receptor and is an established marker of stimulated platelets. Other receptors mediate important cell-cell interactions independent of GP IIb/IIIa. During platelet activation, the expression of those adhesive molecules is also increased. CD 31, a member of the immunoglobulin gene family, is present in 8–15,000 copies on the surface and modulates integrin function and p-selectin expression;16 CD 107a, a heavily glycosylated receptor, displays sialyl Lex termini that adhere to selectins present on the surface of multiple cell types;17 and CD 151 is a newly recognized low abundance membrane protein that is a proposed modulator of integrin function.18 Moreover, platelet-leukocyte aggregate formation, mediated by surface-bound p-selectin, is also a sensitive marker of platelet activation.19,20 This study is the first to prospectively examine the relationship of stent design to platelet activation in humans. Conclusions regarding thrombogenicity cannot be made from this study due to the small overall population. However, subacute thrombosis was not observed in any patient. In the PAST study, patient demographics were fairly evenly matched and baseline markers of platelet activity did not differ. Previous investigations have focused on the effect of various antiplatelet and anticoagulant therapies on reducing stent thrombosis.21–23 However, relatively less information is available regarding the intrinsic risk per se with a particular stent design. Investigations of the relationship of stent design and coatings to platelet activation have been performed largely in in vitro models.7,9,11,24 Unquestionably, platelet activation has been demonstrated in humans following stent placement in the coronary artery by measuring changes in platelet surface receptors from the systemic circulation by flow cytometry.1,2 It has also been suggested that enhanced expression of platelet membrane proteins are of prognostic value and are associated with an increased risk of stent thrombosis.25 There are fundamental differences between NIR and TETRA stents. As a closed-cell stent, the NIR cell is bound on all sides by structural stent members (Figure 4). In this design, the junction of each strut pair is joined to another strut pair junction (i.e., there are no unattached junction nodes within the stent structure). In the TETRA stent, the cellular area is not bound on all sides by structural stent members. In a rabbit iliac artery model, Garasic et al. studied different stent designs varying in the number of struts per cross section.8 The stents with the greatest number of struts per cross section, and thus with the most circular lumen, exhibited the least degree of mural thrombosis three days post-implantation. In the closed-cell NIR design, cross-sectional strut number is greater. The NIR stent also creates a more circular lumen with less intimal prolapse than the TETRA stent, as demonstrated in our ex vivo studies (Figure 4). This important property of NIR therefore results in a smoother stent-vessel wall interface. By creating a more favorable surface, less shear can be expected; this may explain the observed lower degree of platelet activation after implantation of the NIR stent. A potential influence on the results of the PAST trial is the slightly smaller minimal stent diameter (~ 0.3 mm) present in the group receiving the TETRA stent. This may be expected to affect shear and thus platelet activation. However, a correlation was not found in the PAST study between stent size and platelet activity. A bias toward greater procedure duration in the NIR group could have been a factor associated with increased adverse outcomes. However, no differences between groups were observed in the study. All patients in the PAST trial received identical anticoagulant and antiplatelet therapy and thus, these factors also could not have influenced the results. In conclusion, in this prospective, randomized pilot trial evaluating the effect of stent design on platelet activation, a closed-cell stent exhibited less subsequent influence on platelet activity than an open-cell stent. Less platelet activation may be explained by the creation of a more circular and smooth stent-vessel wall interface. This observation may be critical in understanding the relation of stent design to the subsequent development of stent thrombosis and restenosis and deserves further investigation. Acknowledgment. The authors would like to thank Ms. Janine Muldowney for her technical excellence in the preparation of this manuscript.
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