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Keywords:

  • antiplatelet drug resistance;
  • clopidogrel;
  • endothelial function;
  • percutaneous coronary intervention;
  • periprocedural myocardial infarction

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Addendum
  9. Disclosure of conflict of interests
  10. References

Summary.  Background: Percutaneous coronary intervention (PCI) modulates platelet reactivity (PR). Objectives: To assess: (i) the impact of coronary interventions on periprocedural variations (Δ) of PR; (ii) whether ΔPR correlates with periprocedural myocardial infarction (PMI); and (iii) the mechanisms of these variations in vitro. Methods and results: We enrolled 65 patients on aspirin (80–100 mg day−1) and clopidogrel (600 mg, 12 h before PCI): 15 with coronary angiography (CA group), 40 with PCI (PCI group), and 10 with rotational atherectomy plus PCI (RA group). PR was assessed by ADP, high-sensitivity ADP and thrombin receptor activator peptide 6 tests prior to, immediately after and 24 h after the procedure. E-selectin and ICAM-1 were assessed prior to and immediately after the procedure. In vitro, PR was measured during pulsatile blood flow at baseline, after balloon inflation and after stent implantation in six porcine carotid arteries and five plastic tubes. PR declined in the CA group, but significantly increased in the PCI and RA groups immediately postprocedure, and decreased to baseline at 24 h. ΔPR increased across the three groups (P < 0.0001). In the PCI group, ΔPR was directly related to total inflation time (r = 0.435, P = 0.005) and total stent length (r = 0.586, P < 0.001). The change in E-selectin significantly and inversely correlated with ΔPR (P < 0.001). No correlation was found with sICAM-1. PR increased significantly more in patients with PMI than in patients without PMI (P = 0.013). In vitro, platelet activation was observed in the presence of carotid arteries but not in the presence of plastic tubes. Conclusions: Despite dual antiplatelet therapy, PCI affected platelet function proportionally to procedural complexity and the extent of vascular damage.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Addendum
  9. Disclosure of conflict of interests
  10. References

Optimal platelet inhibition is crucial to prevent procedural thrombotic complications and recurrent ischemic events in patients undergoing percutaneous coronary intervention (PCI) [1,2]. A 600-mg loading dose of clopidogrel has been established as a pretreatment strategy in stable angina patients undergoing PCI [3,4]. However, wide interindividual variability in clopidogrel responsiveness hampers its protective effect, and contributes to higher thrombogenic risk in patients with high residual platelet reactivity (PR) [5]. Genetic, cellular and clinical factors contribute to the suboptimal platelet response to clopidogrel [5]. We have recently demonstrated that the extent and severity of coronary atherosclerosis is an additional patient-related factor that is significantly associated with high PR and impaired response to clopidogrel [6].

A coronary interventional procedure by itself is another important determinant of PR [7–13], which may significantly vary in the periprocedural setting. In particular, significant platelet activation has been associated with aggressive coronary interventions, such as rotational atherectomy (RA) [14]. However, it is unclear whether variations in PR are related to the introduction of devices (i.e. catheters, balloons, stents, etc.) into the coronary circulation, rather than being a consequence of the vascular damage induced by balloon inflation and stent implantation.

The primary aim of our study was to assess the impact of coronary interventions on periprocedural variations in PR after a 600-mg clopidogrel loading dose in patients undergoing elective PCI. We used two reference groups of patients undergoing either coronary angiography (CA) without any intervention (negative control) or RA (positive control).

The secondary aims were: (i) to evaluate whether related periprocedural variations in PR have an impact on periprocedural myocardial infarction (PMI); and (ii) to explore the underlying mechanisms of periprocedural variations in PR by using an in vitro PCI model of the porcine carotid artery.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Addendum
  9. Disclosure of conflict of interests
  10. References

Patient population

Between October 2009 and April 2010, consecutive thienopyridine-naive patients aged between 18 and 85 years with suspected/established stable coronary artery disease (CAD) scheduled for diagnostic and/or interventional coronary procedures were recruited. Patients were divided in three groups: those undergoing CA alone (CA group), those undergoing PCI with stent implantation (PCI group), and those undergoing RA plus PCI (RA group). All patients received a 600-mg loading dose of clopidogrel at least 12 h before the procedure, and were receiving chronic treatment with aspirin (80–100 mg day−1). In patients receiving PCI, a 75 mg day−1 maintenance dose of clopidogrel was started on the day after the procedure. Exclusion criteria were acute coronary syndrome within 30 days, use of glycoprotein IIb/IIIa inhibitors, treatment with oral anticoagulant drugs, platelet count of < 70 × 109 L−1, high bleeding risk (active internal bleeding, history of hemorrhagic stroke, intracranial neoplasm, arteriovenous malformation or aneurysm, ischemic stroke in the previous 3 months), coronary artery bypass surgery in the previous 3 months, liver disease, and moderate to severe renal failure (glomerular filtration rate of < 60 mL min−1). The study complied with the Declaration of Helsinki, and was approved by the local ethics committee. Written informed consent was obtained from all patients enrolled.

Catheterization laboratory

All interventional procedures in the catheterization laboratory were performed according to standard techniques. Procedural anticoagulation consisted of an intravenous bolus of weight-adjusted unfractionated heparin (100 U kg−1). PCI success was defined as a reduction in percentage diameter stenosis of < 30% in the presence of Thrombolysis In Myocardial Infarction flow grade ≥ 2 in the main vessel and all side branches ≥ 2 mm in diameter.

Blood sampling

Blood samples were collected at three time points: (i) in the catheterization laboratory, after arterial sheath insertion and immediately before heparin administration; (ii) immediately postprocedure; (iii) and 24 h after the procedure, but before the administration of the clopidogrel maintenance dose. To assess the potential effect of weight-adjusted unfractionated heparin administered at the beginning of the coronary intervention, in 10 patients an additional blood sample was measured 5 min after heparin administration. Blood was collected into a 3-mL tube containing 200 U mL−1 hirudin (Dynabyte Medical, Munich, Germany), and samples were stored at room temperature for at least 30 min before testing.

PR

PR was measured with the Multiplate Analyzer (Dynabyte Medical), a whole blood platelet function test based on multiple electrode platelet aggregometry [15]. After 300 μL of whole blood had been diluted with 300 μL of 0.9% NaCl solution and stirred for 3 min in the test cuvettes at 37 °C, 6.4 μmol L−1 ADP (ADP test), or 6.4 μmol ADP + 9.4 nmol prostaglandin E1 (high sensitivity ADP [hs-ADP] test) or 32 μmol L−1 thrombin receptor activator peptide 6 (TRAP test) was added, and the increase in electrical impedance was recorded over a period of 6 min. The mean values of two independent determinations are expressed as aggregation units (AU).

Adhesion molecules

Blood samples were also taken immediately before and after the procedure for the measurement of soluble E-selectin (sE-selectin) and soluble ICAM-1 (sICAM-1) with commercially available Quantikine Immunoassay kits (R&D Systems, Minneapolis, MN, USA).

Periprocedural myocardial necrosis

High-sensitivity troponin T (hs-TnT) (Roche Diagnostics, Mannheim, Germany) was determined in blood samples taken before, 8 h after and 24 h after intervention. PMI was defined as a postprocedural increase in hs-TnT of more than three times the 99th percentile of the upper reference limit (14 ng mL−1) for patients with baseline negative myocardial necrosis markers, according to the Joint European Society of Cardiology/American College of Cardiology Foundation/American Heart Association/World Heart Federation task force consensus statement on the redefinition of myocardial infarction for clinical trials on coronary intervention [16]. In patients with increased baseline levels of hs-TnT, a subsequent increase of > 50% of the baseline value fulfilled the criteria for PMI [17,18].

Animal experimental set-up and procedures

Animal in vitro experiments were carried out with a generic constitutive model of passive arterial mechanical behavior under load, as previously described [19,20]. Porcine carotid arteries and porcine blood were obtained from the local slaughterhouse. Carotid arteries were chosen for the following reasons: (i) to dissect out the vascular contribution to changes in PR, without any influence from the surrounding myocardium; (ii) manipulation of carotid arteries is simple, and the risk of injuring the vessel wall is very low; (iii) the size of porcine carotid arteries enabled us to perform standard angioplasty procedures with the same devices used for coronary interventions in humans; and (iv) unlike coronary arteries, carotid arteries do not have side branches, which would have made it impossible to have a closed circuit, which is essential for our aim of evaluating the interaction between circulating blood and the vascular wall. The blood was stored in plastic bottles, and unfractionated heparin (1 IU mL−1) was added immediately after collection. After removing connective/adipose tissue from the arterial samples, we isolated segments of unstretched length 40–60 mm, and 2.5–3.5 mm in lumen diameter. Two polypropylene connectors were inserted at the proximal and distal ends of each arterial segment and fixed with a tie-wrap. All experiments were performed within 4 h of sample collection. The arterial segments were placed into an in vitro set-up (Fig. 1), and mounted between two plastic tubes immersed in a bath filled with phosphate-buffered saline (Sigma-Aldrich, St Louis, MO, USA). The arterial segments were stretched to obtain a 20% increase in length. The plastic tubes were connected to silicone rubber tubes with polypropylene connectors. On one side of the bath, the rubber tube was connected to a pressure pump; on the other side of the bath, it was connected to a 200-mL blood reservoir. With another silicone rubber tube, the blood was transported from the reservoir back to the pump, forming a closed circuit. A pressure transducer (P10EZ; Becton-Dickinson, St Niklaas, Belgium) was positioned between the pump and the bath at the proximal site, for measurement of absolute pressure waveforms during the experiment. Through a Y-connector, angioplasty materials (guidewires, and balloon and stent catheters) could be advanced into the lumen of the arterial segments in the set-up.

image

Figure 1.  Schematic representation of the experimental set-up of the animal in vitro experiments. AU, aggregation units; hs-ADP, high-sensitivity ADP; TRAP, thrombin receptor activator peptide 6; CA group, coronary angiography alone; PCI group, percutaneous coronary intervention with stent implantation; RA group, rotational atherectomy plus percutaneous coronary intervention.

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Figure 2 shows the experimental design. All experiments were performed at room temperature. After the arterial segment had been placed in the set-up, a balloon catheter of 3.0 × 18 mm (Apex; Boston Scientific, Nanterre, France) over a metallic guidewire (Hi-Torque Balance Middle Weight Universal; Abbott Vascular, Diegem, Belgium) was positioned in the middle of the artery. An ultrasound scanner with a linear array probe (Esaote Europe, Maastricht, the Netherlands) was used to visualize the position of the balloon in the artery. Pulsatile blood flow with a frequency of 60 beats min–1 was then activated through the circuit. After 10 min, a 1-mL blood sample was drawn from the outflow (baseline sample) of the experimental set-up. The balloon catheter was then inflated until complete occlusion of the arterial lumen was achieved. Five consecutive inflations of 10 s each were carried out, and the balloon catheter was then removed from the set-up. One-milliliter blood samples were taken immediately after the balloon inflations (T1 sample) and 10 min later (T2 sample). Next, a 3.0 × 18-mm stent (Pro-Kinetic; Biotronik, Berlin, Germany) was advanced into the artery and deployed with 10-s balloon inflation. After implantation, the stent was post-dilated with four additional balloon inflations, each lasting 10 s. One-milliliter blood samples were taken immediately after stent post-dilatation (T3 sample) and 10 min later (T4 sample). ADP-induced (6.4 μmol L−1 ADP) PR was measured on blood samples (within 3 min of collection) drawn at each time point by the means of the Multiplate Analyzer (Dynabyte Medical). A set of sham experiments was performed with the same experimental design for collected blood and a silicone rubber tube instead of the porcine carotid artery (control group). We performed a total of six experiments using collected blood and six carotid arteries from six pigs (artery group), and a total of five sham experiments using collected blood from five of the six pigs (control group).

image

Figure 2.  Flowchart of the animal in vitro experiments. In the “artery” group, angioplasty was performed on porcine carotid arteries (n=6). In the “control group”, angioplasty was performed on silicone rubber tubes (n=5). AU, aggregation units; hs-ADP, high-sensitivity ADP; TRAP, thrombin receptor activator peptide 6.

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Statistics

On the assumption that PR measured with the ADP test in patients pretreated with 600 mg of clopidogrel was 50 AU, and with the expectation of a 15% increase in PR after PCI with a standard deviation (SD) of 30% at both time points (baseline and post-PCI), at least 36 patients were needed to detect such a difference with a power of 80% at a two-sided alpha of 0.05. Therefore, we aimed to recruit a total of 40 patients in the PCI group. Continuous variables are expressed as mean ± SD or as median (interquartile range), as appropriate. Categorical variables are reported as frequencies and percentages. Normal distribution was tested with the Kolmogorov–Smirnov test. Comparisons between continuous variables were performed with the Student t-test or the Mann–Whitney test. One-way anova was used to compare PR between different groups. The Kruskall–Wallis test was used to compare non-normally distributed variables between different groups. Bonferroni correction was used for post hoc multiple comparisons. All tests were corrected for repeated measures, where appropriate. A two-way anova for repeated measures followed by pairwise comparisons was used to detect changes in PR levels over time in the different study groups. The Pearson test was used to assess correlations between normally distributed variables, and the Spearman test was used to assess correlations between non-normally distributed variables. Comparisons between categorical variables were evaluated with the Fisher exact test or the Pearson chi-square test, as appropriate. Statistical analysis was performed with stata/ic 10 (STATA Corp., College Station, TX, USA), and P-values of < 0.05 (two-tailed) were considered to be significant.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Addendum
  9. Disclosure of conflict of interests
  10. References

Patient population

A total of 65 patients were enrolled in the study. Of these, 15 underwent CA alone (CA group), 40 underwent PCI (PCI group), and 10 underwent PCI plus RA (RA group). The clinical and procedural characteristics are listed in Tables 1 and 2, respectively. No significant differences in demographics, previous clinical history or baseline medications were noticed between the three groups. The RA group presented more frequently with type B2/C lesions and a larger diameter of the implanted stent than the PCI group. Procedure duration increased across the three groups. Procedure success was achieved in all patients undergoing PCI.

Table 1.   Clinical characteristics
 CA group (n = 15)PCI group (n = 40)RA group (n = 10) P-value
  1. CA group, coronary angiography alone; PCI group, percutaneous coronary intervention with stent implantation; RA group, rotational atherectomy plus percutaneous coronary intervention; SD, standard deviation.

Age (years), mean ± SD69 ± 1166 ± 1272 ± 90.280
Male, n (%)10 (67)28 (70)7 (70)0.970
Body mass index, mean ± SD24.9 ± 2.827.9 ± 4.327.5 ± 5.30.064
Diabetes mellitus, n (%)2 (13)17 (43)4 (40)0.116
Hypertension, n (%)6 (40)24 (60)5 (50)0.381
Hyperlipidemia, n (%)9 (60)33 (83)7 (70)0.205
Current smoker, n (%)3 (20)4 (10)4 (40)0.072
Previous myocardial infarction, n (%)2 (13)13 (33)0 (0)0.057
Previous coronary intervention, n (%)8 (53)26 (65)3 (30)0.145
Previous bypass surgery, n (%)4 (27)4 (10)1 (10)0.340
Baseline medications, n (%)
 Statin12 (80)32 (80)6 (60)0.385
 Calcium channel blocker1 (7)4 (10)2 (20)0.556
 Proton pump inhibitor4 (27)10 (24)3 (30)0.948
Aspirin dose (mg), n (%)
 8013 (87)36 (90)9 (90)0.858
 1002 (13)4 (10)1 (10)
Table 2.   Procedural characteristics
 CA group (n = 15)PCI group (n = 40)RA group (n = 10) P-value
  1. CA group, coronary angiography alone; PCI group, percutaneous coronary intervention with stent implantation; RA group, rotational atherectomy plus percutaneous coronary intervention; IQR, interquartile range; SD, standard deviation.

Diseased vessels (n), mean ± SD1.6 ± 1.51.6 ± 0.71.8 ± 0.90.783
Vascular access, n (%)
 Femoral14 (99)40 (100)10 (100)1.000
 Radial1 (1)
Sheath size, n (%)
 6 French15 (100)32 (80)2 (20)< 0.001
 7 French8 (20)8 (80)
Treated vessel (n), mean ± SD1.1 ± 0.31.1 ± 0.30.975
Multivessel intervention, n (%)4 (10)1 (10)1.000
Target vessel, n (%)   
 Left anterior descending 18 (41)4 (36)0.828
 Left circumflex 5 (11)2 (18)
 Right coronary artery 21 (48)5 (45)
Lesion type B2/C, n (%)21 (47)11 (100)0.001
Bifurcation lesions, n (%)7 (16)2 (18)1.000
Chronic total occlusions, n (%) 9 (20)1 (9)0.667
Predilatation, n (%)32 (73)11 (100)0.184
Use of stent, n (%)44 (100)11 (100)1.000
Use of drug-eluting stent, n (%)33 (74)11 (100)0.096
Stents implanted (n), mean ± SD1.5 ± 0.91.8 ± 1.00.407
Stent diameter (mm), mean ± SD3.1 ± 0.43.5 ± 0.60.018
Total stent length (mm), median (IQR)23 (12–132)35 (12–68)0.543
Postdilatation, n (%)25 (57)9 (82)0.174
Total inflation time (s), mean ± SD1.6 ± 1.22.5 ± 2.20.097
Maximal inflation pressure (atm), mean ± SD15.8 ± 3.216.2 ± 2.20.700
Contrast medium (mL), mean ± SD166 ± 43304 ± 139389 ± 2130.001
Procedure duration (min), mean ± SD32.7 ± 10.366.0 ± 38.897.6 ± 51.8< 0.001

PR

The results of the ADP, hs-ADP and TRAP tests are shown in Table 3. At baseline, PR was similar between the three study groups. PR tended to decline over time in the CA group. In both the PCI and RA groups, PR increased immediately postprocedure, and decreased towards baseline levels at 24 h. In those 10 patients within the PCI group in whom PR was also measured 5 min after heparin administration, no significant difference in PR was observed before and after heparin administration (ADP test, 39 ± 24 vs. 41 ± 22 AU, P = 0.543; hs-ADP test, 26 ± 19 vs. 25 ± 18 AU, P = 0.866; TRAP test, 85 ± 15 vs. 84 ± 17 AU, P = 0.881).

Table 3.   Platelet reactivity
 CA group (n = 15)PCI group (n = 40)RA group (n = 10)Between-group P-value
  1. CA group, coronary angiography alone; PCI group, percutaneous coronary intervention with stent implantation; RA group, rotational atherectomy plus percutaneous coronary intervention; AU, aggregation units; hs-ADP, high-sensitivity ADP; TRAP, thrombin receptor activator peptide 6; SD, standard deviation.

ADP test (AU)
 Before, mean ± SD41 ± 2647 ± 2543 ± 260.618
 After, mean ± SD38 ± 2353 ± 2461 ± 290.048
 24 h, mean ± SD34 ± 1940 ± 2152 ± 230.108
 Within-group P-value0.138< 0.0010.002 
hs-ADP test (AU)
 Before, mean ± SD25 ± 1729 ± 1928 ± 190.746
 After, mean ± SD24 ± 1635 ± 2245 ± 270.065
 24 h, mean ± SD18 ± 1526 ± 1634 ± 180.045
 Within-group P-value0.006< 0.0010.009 
TRAP test (AU)
 Before, mean ± SD82 ± 4190 ± 3383 ± 240.693
 After, mean ± SD69 ± 3794 ± 30108 ± 250.006
 24 h, mean ± SD65 ± 3682 ± 3382 ± 160.189
 Within-group P-value0.0030.012< 0.001 

Periprocedural variations in PR

Periprocedural variations (defined as the difference between immediately after the procedure and preprocedure) in PR (ΔPR) are shown in Fig. 3. With the ADP test, ΔPR was − 2.7 ± 7.4 AU in the CA group, 6.0 ± 12.4 AU in the PCI group, and 18.6 ± 7.7 AU in the RA group (anova, P < 0.0001). With the hs-ADP test, ΔPR was − 1.0 ± 5.5 AU in the CA group, 6.4 ± 10.1 AU in the PCI group, and 16.7 ± 10.4 AU in the RA group (anova, P < 0.0001). With the TRAP test, ΔPR was − 13.9 ± 16.3 AU in the CA group, 4.1 ± 21.6 AU in the PCI group, and 24.9 ± 11.2 AU in the RA group (anova, P < 0.0001).

image

Figure 3.  Periprocedural variations (defined as the difference between immediately after the procedure and preprocedure) in platelet reactivity (ΔPR). Baseline data points are reported in Table 3. Comparisons between the three groups was performed with anova with Bonferroni correction for multiple analysis. hs-ADP, high-sensitivity ADP; TRAP, thrombin receptor activator peptide 6; CA group, coronary angiography alone; PCI group, percutaneous coronary intervention with stent implantation; RA group, rotational atherectomy plus percutaneous coronary intervention.

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Procedural parameters and variations in PR

In the PCI group, ΔPR showed significant correlations with total inflation time (Fig. 4A) and total stent length (Fig. 4B). ΔPR also moderately correlated with total procedure time (ADP test, r = 0.326, P = 0.040; hs-ADP test, r = 0.322, P = 0.045; TRAP test, r = 0.575, P < 0.001). No significant correlation between ΔPR and any other procedural parameter was found.

image

Figure 4.  Percutaneous coronary intervention (PCI) features and variations in platelet reactivity. (A, B) Correlation of the periprocedural variations (defined as the difference between immediately after the procedure and pre-procedure) in platelet reactivity (ΔPR) with total inflation time (A) and total stent length (B) in the PCI group. Correlation between the variables was performed with the Pearson test. hs-ADP, high-sensitivity ADP; TRAP, thrombin receptor activator peptide 6.

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Adhesion molecules

The levels of sE-selectin and sICAM-1 are shown in Table 4. Periprocedural variations in sE-selectin levels were significantly different across the three study groups (anova, P < 0.0001). In the PCI group, periprocedural variations in sE-selectin levels significantly and inversely correlated with ΔPR (ADP test, = − 0.398, P = 0.004; hs-ADP test, r = − 0.480, P < 0.001; TRAP test, r = − 0.496, P < 0.001). Moreover, periprocedural variations in sE-selectin levels significantly and inversely correlated with total inflation time (r = − 0.469, P = 0.002) and total stent length (r = − 0.403, P = 0.009). This association remained significant after adjustment for procedural variables for the hs-ADP test (P = 0.032), but not for the ADP and TRAP tests. Periprocedural variations in sICAM-1 levels were not significantly different across the three study groups (anova, P = 0.196). No correlation was found between periprocedural variations in sICAM-1 levels and ΔPR or procedural parameters.

Table 4.   Adhesion molecules
 CA group (n = 15)PCI group (n = 40)RA group (n = 10)
  1. CA group, coronary angiography alone; PCI group, percutaneous coronary intervention with stent implantation; RA group, rotational atherectomy plus percutaneous coronary intervention; SD, standard deviation; sE-selectin, soluble E-selectin; sICAM-1, soluble ICAM-1.

sE-selectin (ng/mL)
 Before, mean ± SD24.6 ± 8.729.0 ± 12.739.1 ± 15.9
 After, mean ± SD24.2 ± 8.525.0 ± 11.631.1 ± 12.3
 Within-group P-value0.160< 0.001<0.001
sICAM-1 (ng/mL)
 Before, mean ± SD209.8 ± 49.0206.0 ± 58.2206.6 ± 45.9
 After, mean ± SD187.2 ± 48.8174.4 ± 49.3168.2 ± 44.8
 Within-group P-value<0.001<0.0010.006

PMI

Baseline levels of hs-TNT were 8.96 (3.00–10.96) ng mL−1 in the CA group, 8.90 (4.04–24.88) ng mL−1 in the PCI group, and 6.58 (3–13.83) ng mL−1 in the RA group (Kruskall–Wallis, P = 0.282).

The periprocedural increases in hs-TnT (defined as the difference between postprocedure and preprocedure) levels were 1.16 (0.00–1.82) ng mL−1 in the CA group, 14.63 (4.53–42.27) ng mL−1 in the PCI group, and 33.52 (23.97–130.07) ng mL−1 in the RA group (Kruskall–Wallis, P < 0.0001). PMI occurred in no patients in the CA group, in 17 patients (43%) in the PCI group, and in five patients (50%) in the RA group. In the PCI group, a significant correlation was found between the periprocedural increase in hs-TnT and ΔPR (ADP test, rho = 0.462, P = 0.003; hs-ADP test, rho = 0.631, P < 0.001; TRAP test, rho = 0.386, P = 0.014). Moreover, ΔPR was significantly higher in patients with PMI than in those without PMI (ADP test, 11.5 ± 12.73 vs. 1.87 ± 10.61, P = 0.013; hs-ADP test, 12.0 ± 11.6 vs. 6.3 ± 2.3, P = 0.002; TRAP test, 14.8 ± 25.6 vs. − 1.7 ± 14.4, P = 0.013; Fig. 5). This association remained significant after adjustment for procedural variables (i.e. total inflation time, total stent length, and procedure time; P < 0.05 for all tests).

image

Figure 5.  Platelet reactivity and periprocedural myocardial injury. Periprocedural variations (defined as the difference between immediately after the procedure and preprocedure) in platelet reactivity (ΔPR) in patients with and without periprocedural myocardial infarction in the percutaneous coronary intervention group. hs-ADP, high-sensitivity ADP; PMI, periprocedural myocardial infarction; TRAP, thrombin receptor activator peptide 6. Comparison was performed with an unpaired t-test.

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Animal experiments

At baseline, PR measured with the ADP test was 34 ± 16 in the artery group and 48 ± 42 in the control group (P = 0.450; Fig. 6A). In the artery group, PR varied significantly over time (anova, P = 0.033), with a progressive increase after balloon dilatation (T1) and stent implantation (T3). No significant variations in PR were observed in the control group over time (anova, P = 0.452). The two-way anova showed a significant interaction between experimental groups and timings (P = 0.012). Percentage variations in PR as compared with the baseline in the two groups are shown in Fig. 6B.

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Figure 6.  Results of the in vitro studies. (A, B) Absolute levels of platelet reactivity at the different time points (A) and percentage variations in platelet reactivity as compared with baseline (B) in the two study groups. AU, aggregation units.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Addendum
  9. Disclosure of conflict of interests
  10. References

In the present study, we found that (i) PR increases with more extensive and aggressive coronary interventions (i.e. with multiple stenting or rotational atherectomy), predisposing stable angina patients to suboptimal platelet inhibition, despite the currently recommended loading dose of clopidogrel; (ii) periprocedural variations in PR are specifically linked to the degree of vascular damage and endothelial dysfunction induced by coronary interventions; and (iii) procedure-related platelet activation is associated with an increased risk of myonecrosis.

PR and coronary procedures

PR, despite ongoing dual antiplatelet therapy, is increased early after the procedure as compared with 24 h later in patients undergoing elective PCI with stent implantation [7,9,13]. However, the non-availability of PR at baseline in these studies prevents establishment of the relative contribution of the coronary intervention. Our results suggest that, unlike coronary angiograms, both PCI and PCI plus RA result in a significant increase in PR immediately after the procedure as compared with baseline; at 24 h, PR tends to decrease towards baseline levels. In addition, we searched for a mechanistic explanation for these periprocedural variations in PR. In our study, the degree of PCI-related platelet activation was proportional to the complexity of the procedure. Moreover, PR was higher after RA and complex PCI procedures, and was related to procedural factors such as total inflation time, total stent length, and procedure time.

Platelet activation and vascular damage

Platelet activation after complex PCI procedures has been previously linked to the increased shear stress induced by the use of coronary devices such as those used for RA [21,22]. Whether the vascular damage occurring during PCI might also play a role in platelet activation remained unclear. In an in vitro model of angioplasty, we found that PR increased only when angioplasty and stent implantation was performed in the presence of an arterial conduit (i.e. porcine carotid artery). In contrast, when the same procedures were performed on plastic tubes, no significant variations in PR were observed. These findings suggest that flow turbulence and shear stress induced by the procedures are not sufficient to induce platelet activation, and underscore the importance of the interaction between the damaged vessel and circulating platelets.

These in vitro findings are corroborated in patients by a significant decrease in the levels of the endothelial biomarkers sE-selectin and sICAM-1 observed after PCI. In addition, an inverse correlation was found between sE-selectin levels and PR, supporting the concept that increased PR is partly mediated by the damaged vascular endothelium. In particular, the more aggressive the procedures (i.e. with RA), the more pronounced this biomarker reduction, suggesting that endothelial dysfunction potentially occurs post-PCI, in line with previous studies [8,10–12,23]. In the context of coronary atherosclerosis, a close relationship between platelets and the endothelium has been described: intact endothelium normally prevents platelet adhesion, whereas, in the presence of endothelial damage and activation, selectins mediate the first loose contact between circulating platelets and the vascular endothelium [24–26]. This interaction represents the first step of vascular thrombosis, which is predominantly mediated by endothelial cells, and does not require platelets to be already activated [24,25,27]. Our study suggests that similar events might occur during coronary interventions. In fact, the occurrence of coronary endothelial injury post-PCI (as suggested by a significant reduction in the sE-selectin level) was proportional to the extent of coronary manipulation. This was paralleled by an increase in PR, despite pretreatment with aspirin and a 600-mg loading dose of clopidogrel. Unlike for sE-selectin, sICAM-1 reduction did not correlate with platelet activation, probably because sICAM-1 primarily mediates the interaction between endothelial cells and leukocytes rather than platelets, and is mostly involved in inflammation rather than thrombosis [28].

Clinical implications of periprocedural platelet activation

The impact of baseline PR on periprocedural myonecrosis has been well established [29–32]. In addition to this, we have found that platelet activation induced by PCI can play a role that is even more important in determining postprocedural myocardial injury. In fact, the higher the increase in PR post-PCI, the higher the increase in hs-TnT.

Our finding of transient platelet activation immediately after the procedure has another important clinical implication, especially for linking residual PR assessed post-PCI with future clinical outcomes. This finding might help to explain the negative results of the Gauging Responsiveness with A VerifyNow assay – Impact on Thrombosis And Safety (GRAVITAS) trial [33]. This study was aimed at demonstrating, among patients with high PR after PCI, that the use of high-dose clopidogrel, as compared with standard-dose clopidogrel, could improve clinical outcomes at follow-up. The fact that PR was measured 12–24 h post-PCI might have led to incorrect patient selection. In fact, at least some of the patients classified as ‘non-responders’ (i.e. with high on-treatment PR) could have been reclassified had platelet function been assessed pre-PCI or at least 24 h later.

Finally, the notion of an increase in periprocedural PR proportionally with the complexity of PCI, and with a subsequent increased risk of myocardial injury, supports the use of more effective platelet inhibition strategies in complex interventional procedures. This is corroborated by a recent analysis of the Enhanced Suppression of the Platelet Glycoprotein IIb/IIIa Receptor with Integrilin Therapy (ESPRIT) trial [34], which demonstrated an adjunctive beneficial effect of glycoprotein IIb–IIIa inhibitors (in terms of reduction of early and late risk of complications after PCI) in patients undergoing more complex procedures (i.e. higher number of stents implanted, and greater total stent length).

Limitations

In our study, only patients with stable CAD were enrolled, and therefore we cannot extend these observations to patients with acute coronary syndrome. Similarly, all study patients received a 600-mg loading dose of clopidogrel; therefore, these results might not be applicable to patients receiving chronic clopidogrel treatment or taking different P2Y12 receptor inhibitors. Although the number of patients included in our study was adequate for the assessment of the primary endpoint, we acknowledge that, owing to the limited sample size, clinical read-outs of our findings warrant further investigation and confirmation in significantly larger patient populations. Moreover, only one platelet function test was used, although different pathways of platelet activation were explored. No clinically validated thresholds for high PR are available for the platelet function test used in this study. All patients were uniformly treated with an 80–100-mg aspirin load. This was associated with arachidonic acid-induced platelet aggregation that was similar between the three groups and within the three time points of the protocol (i.e. pre-PCI, post-PCI, and 24 h) (data not shown). However, because a low dose of aspirin was used (as clinically recommended), we are not able to report on the impact that higher doses of aspirin might have had on platelet aggregation during coronary intervention. Measurements of PR at 24 h postprocedure were performed after administration of the maintenance dose of clopidogrel; this could be responsible for lower levels of PR in the CA group than those measured in the catheterization laboratory. Moreover, an impact of emotional stress and iodinated contrast medium on PR during the catheterization cannot be excluded. Although changes in adhesion molecule levels could be considered as a marker of endothelial activation, no direct assessment of vascular damage was performed. Moreover, adhesion molecules were not measured in the in vitro model. Finally, we acknowledge the fact that PR assessed in the porcine carotid artery model, despite reliably providing information on the functional capacity of PR, might not necessarily reflect in vivo platelet activation. Therefore, these results should be interpreted with caution.

Conclusions

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Addendum
  9. Disclosure of conflict of interests
  10. References

Coronary interventions significantly affect platelet function: the more complex the procedure, the larger the transient changes in PR. Damage to the vascular wall and endothelium activation seem to be the underlying mechanisms. Furthermore, patients experiencing a larger periprocedural increase in PR are also exposed to a higher risk of PMI. This study supports the need for more aggressive antiplatelet strategies when complex coronary procedures are planned.

Addendum

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Addendum
  9. Disclosure of conflict of interests
  10. References

F. Mangiacapra and E. Barbato: concept and design, analysis and/or interpretation of data, critical writing or revision of the intellectual content, and final approval of the version to be published; J. Bartunek, N. Bijnens, A. Peace, K. Dierickx, E. Bailleul, and L. Di Serafino: concept and design, analysis and/or interpretation of data, and analysis and/or interpretation of data; S. A. Pyxaras, A. Fraeyman, P. Meeus, and M. Rutten: analysis and/or interpretation of data; B. De Bruyne, W. Wijns, and F. van de Vosse: critical writing or revision of the intellectual content.

Disclosure of conflict of interests

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Addendum
  9. Disclosure of conflict of interests
  10. References

The authors state that they have no conflict of interest.

References

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  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Addendum
  9. Disclosure of conflict of interests
  10. References
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