Relationship between post-treatment platelet reactivity and ischemic and bleeding events at 1-year follow-up in patients receiving prasugrel

Authors


Laurent Bonello, Department of Cardiology, Hôpital universitaire nord, Chemin des bourrely, 13015 Marseille, France.
Tel.: +33 4 919 68 858; fax: +33 4 919 68 979.
E-mail: laurentbonello@yahoo.fr

Abstract

Summary.  Background:  Post-treatment platelet reactivity (PR) is associated with ischemic and bleeding events in patients receiving P2Y12 receptor antagonists.

Objectives:  We aimed to study the relationship between post-treatment PR after a 60-mg loading dose (LD) of prasugrel and 1-year thrombotic and bleeding events.

Method:  Patients were prospectively included in this multicenter study if they had a successful percutaneous coronary intervention (PCI) for acute coronary syndrome (ACS) and received prasugrel. The platelet reactivity index (PRI) was measured using the Vasodilator-Stimulated Phosphoprotein index (VASP) after a prasugrel LD. Endpoints included the rate of thrombotic events and bleeding events at 1 year.

Results:  Among the 301 patients enrolled, 9 (3%) were lost to follow-up at 1 year. The rates of thrombotic and bleeding events at 1 year were of 7.5% and 6.8%, respectively. Receiver-operating curve (ROC) analysis demonstrated an optimal cut-off value of 53.5% of PRI to predict thrombotic events at 1 year. Using this cut-off value we observed that patients exhibiting high on-treatment platelet reactivity (HTPR) had a higher rate of thrombotic events (22.4% vs. 2.9%; < 0.001). In parallel the optimal cut-off value of PRI to predict bleeding was 16%. Patients with a PRI ≤ 16% had a higher rate of bleeding events compared with those with a PRI > 16% (15.6% vs. 3.3%; < 0.001). In multivariate analysis, the PRI predicted both thrombotic and bleeding events (OR: 1.44, 95% confidence interval [CI]: 1.2–1.72; < 0.001 and OR: 0.75, 95% CI: 0.59–0.96; = 0.024 [respectively, per 10% increase]).

Conclusion:  Platelet reactivity measurement after a prasugrel LD predicts both ischemic and bleeding events at 1 year follow-up for ACS patients undergoing PCI.

Introduction

Platelet reactivity (PR) inhibition is critical to prevent thrombotic events in acute coronary syndrome (ACS) patients undergoing percutaneous coronary intervention (PCI). In fact, the addition of a P2Y12 receptor antagonist to aspirin has dramatically reduced the risk of thrombotic events including stent thrombosis [1]. Several studies have demonstrated a strong association between high on-treatment platelet reactivity (HTPR) after a clopidogrel loading dose (LD), as measured by various platelet assays, and post-PCI thrombotic events such as cardiovascular death, myocardial infarction (MI) or stent thrombosis [2]. The Vasodilator-Stimulated Phosphoprotein (VASP) index is a specific and reproducible test to assess P2Y12 receptor blockade [3]. While the relationship between PR and thrombotic events is well established, a potential link between PR and bleedings has recently emerged. Sibbing et al. [4] observed, in a prospective trial including patients treated with PCI, an association between a high level of PR inhibition and bleeding events. These preliminary findings were confirmed by other investigators showing that excessive PR inhibition after a clopidogrel LD translated into bleedings [5]. Accordingly, in the TRITON thrombolysis in myocardial infarction (TIMI) 38 trial, the higher PR inhibition obtained with prasugrel compared with clopidogrel was associated with a reduced rate of thrombotic events but with higher non-coronary artery bypass graft (CABG)-related TIMI bleedings [6,7]. In fact, more aggressive anti-thrombotic therapy in addition to invasive procedures is responsible for an increase in the rate of bleeding complications. Of importance, these bleeding complications are associated with short- and long-term mortality [8,9].

In a multicenter prospective open label trial, we have recently validated the predictive value of PR for major adverse cardiovascular events (MACE) at 1 month [10]. In this study PR failed to predict bleeding events. We aimed to analyze, in the previously studied cohort of ACS patients, the relationship between platelet reactivity after a prasugrel LD and 1-year thrombotic and bleeding events.

Method

Between February and September 2010, all patients admitted for ACS who underwent successful PCI and received prasugrel therapy were prospectively included in this multicenter trial. The protocol was approved by the local ethic committee and was in agreement with the declaration of Helsinki. Informed consent was obtained for all patients. The details of the protocol have been previously published [10].

Exclusion criteria were failed PCI, cardiac arrest, contraindications to anti-platelet therapy, a platelet count < 100 G L−1, a history of bleeding diathesis and concurrent severe illness with a expected survival of < 1 month, surgery within 1 month or scheduled in the year, age > 75 years old, coumadin or other oral anticoagulant therapy, weight < 60 kg and a history of stroke.

Platelet reactivity measurements

Blood samples for VASP index analysis were drawn by atraumatic venipuncture of the antecubital vein at least 6 h after a prasugrel LD (mean 9 ± 3; range 6–12 h). The initial blood drawn was discarded to avoid measuring platelet activation induced by needle puncture; blood was collected into a Vacutainer containing 3.8% trisodium citrate and filled to capacity. The Vacutainer was inverted 3–5 times for gentle mixing and sent immediately to the hemostasis laboratory. VASP index phosphorylation analysis was performed within 24 h of blood collection by an experienced investigator using Platelet VASP kits (Diagnostica Stago, Asnières, France) [11,12]. Briefly, a citrated blood sample was incubated with prostaglandin E1 (PGE1) or with PGE1 and ADP 10 μm for 10 min and fixed with paraformaldehyde, after which the platelets were permeabilized with non-ionic detergent. Analyzes were performed on an EPICS XL-MCL flow cytometer (Beckman Coultronics, Margency, France), the platelet population was identified from its forward and side-scatter distribution and 10 000 platelets were gated. A platelet reactivity index (PRI) VASP index was calculated from the mean fluorescence intensity (MFI) of samples incubated with PGE1 or PGE1 and ADP according to the formula: PRI = [(MFI(PGE1)-MFI(PGE1+ADP))/MFI(PGE1)] × 100.

Study end-points

The below-mentioned endpoints were pre-specified. The first end-point was the rate of thrombotic events including cardiovascular death, non-fatal MI and stent thrombosis at 1 year. Cardiovascular death was considered as any death with a demonstrable CV cause or any death that is not clearly attributable to a non-CV cause. Stent thrombosis was defined according to the ARC definition (definite and probable) [13]. A non-fatal MI was defined as recurrent ischemic symptoms (> 20 minutes), and/or ECG changes after the initial presentation with an increase ≥ 20% of troponin measured after the recurrent event, with at least one value above the 99th percentile of the reference range [14]. Peri-procedural MIs, as defined in the consensus document, were excluded.

Two other end-points were analyzed at 1 year follow-up: ischemic and bleedings events. Ischemic events were defined as a composite of thrombotic events (as described above) and non-urgent coronary revascularization. Non-urgent coronary revascularizations included all revascularizations at 1 year, either by PCI or CABG for stable angina, a positive stress test or silent ischemia. Stages PCI were excluded. Bleeding events were defined as all major and minor non-CABG-related TIMI bleeding during 1-year follow-up [15].

Data management

Clinical data were prospectively collected at each site. The data for all patients with eventful follow-up were reviewed by an independent adjudication committee whose members were blinded to treatment assignments. Events adjudication was performed separately by two members and in case of disagreement, the opinion of a third member was obtained and the final decision was taken by consensus.

Statistical analyzes

All statistical analyzes were performed using spss 17.0 software (SPSS, Chicago, IL, USA). Continuous variables are expressed as means ± standard deviations (SD) or medians (range [or Inter Quartile Range]). Categorical variables are expressed as frequencies and percentages. Two-sided Mann–Whitney tests were used to compare PRI among patients’ subgroups. The time to thrombotic and bleeding events between subgroups was analyzed using the Kaplan–Meier method. Survival curves were compared using the log-rank test and hazard ratios were calculated using Cox’s regression models. Linear models were used to evaluate the association between patients characteristics associated with time to first event in univariate analysis. Those covariates with a P-value < 0.20 were then entered in the multiple Cox’s regression models. Forward stepwise Cox’s regression models were conducted to select independent prognostic factors. This forward approach was based on the log-likelihood ratio test (entry threshold: P-value < 0.05). PRI was systematically included in the final regression models if not selected by the stepwise approach. Sensitivity and specificity of PRI to predict thrombotic events were calculated at different thresholds in a receiver-operating curve (ROC) analysis. A complementary analysis compared the time to thrombotic events between high on-treatment platelet reactivity (HTPR) and good responders (GR) groups using the Kaplan–Meier method. Survival curves were compared using the log-rank test. All tests were two-sided and the level of significance was P < 0.05.

Results

Baseline demographic, clinical, angiographic and biological characteristics were described previously (Table 1). Three hundred and one patients were enrolled and 42.5% of them had a ST-elevation myocardial infarction (STEMI) on admission. The mean PRI after 60 mg of prasugrel was 34.3 ± 23 (inter-quartile range [IQR]: 15–50.3%).

Table 1.   Baseline demographic, clinical, angiographic and biological characteristics
Mean ± SD (IQR)All patients (n = 301)
  1. IQR, inter quartile range; BMI, body mass index; MI, myocardial infarction; CAD, coronary artery disease; STEMI, ST-elevation myocardial infarction; NSTEMI, non ST-elevation myocardial infarction; UA, unstable angina; DES, drug eluting stent; GP, glycoprotein.

Age58.1 ± 10.4 (51–64)
Gender (m)267 (88.7%)
BMI28.7 ± 13.6 (24.2–30.9)
History of MI57 (18.9%)
Cardiovascular risk factor (%)
 Smoking154 (51.2)
 Hypercholesterolemia146 (48.5)
 Diabetes mellitus70 (23.3)
 Hypertension122 (40.5)
 Family history of CAD64 (21.3)
Clinical setting (%)
 STEMI128 (42.5)
 NSTEMI100 (33.2)
 UA73 (24.3)
Angiography and Intervention
 Number of treated vessels1.2 ± 0.4 (1–1.5)
 Number of stent per patients1.33 ± 0.8 (1–2)
 DES per patients0.7 ± 0.9 (1–2)
 GP IIb/IIIa use44 (14.6%)
Biology
 Leucocytes (G L−1)10.4 ± 3.5 (7.7–14.8)
 Hemoglobin (g dL−1)13.9 ± 1.5 (11.6–15.3)
 Platelets (103 per L)228 ± 68 (164–270)
 Fibrinogen (g l−1)3.7 ± 1.1 (3–4.2)
 Creatinin (μm)86 ± 74
 VASP index (%)34 ± 23 (15–50.3)

We have previously published data regarding the 1-month outcome for this cohort [10].

One-year outcome

Nine patients were lost to follow-up at 1 year. Two patients had a non-cardiac death. The primary thrombotic end-point (four cardiovascular deaths, 11 non-fatal MI and seven stent thrombosis) occurred in 7.5% of the study population (Fig. 1).

Figure 1.

 Kaplan–Meier analysis of thrombotic, ischemic and bleeding events during 1-year follow-up.

Regarding the ischemic endpoint, 21 patients underwent non-urgent coronary revascularization; the rate of the 1-year ischemic end-point was therefore 14%.

Regarding bleedings, 20 major and minor non-CABG TIMI bleedings occurred during the 1-year follow-up period (6.8%) including six TIMI major bleeds. None of these bleeds were fatal (Fig. 1). Access site bleedings were rare: one major and one minor TIMI bleeds.

The mean delay between the start of prasugrel therapy and occurrence of the thrombotic or the bleeding events were of 3.8 ± 3.8 and 3.7 ± 2.4 months, respectively (P = 0.5).

Relationship between platelet reactivity and 1-year outcome

Patients with a thrombotic event during follow-up had a higher PRI compared with those without (55 ± 22 [IQR: 60–77.8] vs. 33 ± 22% [IQR: 11.8–35]; P < 0.001).

ROC analysis was performed to evaluate the predictive value of the PRI for thrombotic events. The area under the curve was of 0.76. The optimal cut-off value for the VASP index to predict CV was 53.5% with a sensitivity of 70% and a specificity of 81.6% (Fig. 2). The 50% threshold mentioned in the consensus document had a sensitivity of 70% and a specificity of 78.3%. Although the vast majority of patients (77.7%) were considered to be good responders, using a 53.5% cut-off value to define HTPR, 65 patients (22.3%) were considered to have an insufficient PR inhibition after a prasugrel LD. Using this threshold of PR to define HTPR, we observed that patients exhibiting HTPR had a higher rate of thrombotic events compared with patients considered as good responders (22.4% vs. 2.9%; P < 0.001).

Figure 2.

 Receiver-operating curve (ROC) analysis of the sensibility and 1-specificity of the Vasodilator-Stimulated Phosphoprotein (VASP) index to predict thrombotic events.

In addition, the rate of ischemic events, which includes non-urgent revascularization, was also significantly higher in patients with HTPR compared with those considered as good responders (26.9% vs. 10.7%; log-rank P = 0.001) (Table 2).

Table 2.   Thrombotic events at 1-year follow-up in HTPR (VASP index ≥ 53.5%) compared with GR (VASP index < 53.5%)
One year outcomeGR (n = 234)HTPR (n = 67)Log-rank P-Value
  1. GR, good responders (VASP index < 53.5%); HTPR, high on-treatment platelet reactivity (VASP index ≥ 53.5%); CV, cardio-vascular; ACS, acute coronary syndrome; MACE, major adverse cardiovascular events.

CV death220.13
Recurrent ACS38< 0.001
Stent thrombosis250.001
Revascularization1830.6
All MACE7 (2.9%)15 (22.4%)< 0.001
All MACE + Revascularization25 (10.7%)18 (26.9%)0.001

In addition, we aimed to analyze the relationship between PRI and bleeding events. Although there was no significant difference between GR and HTPR patients (P = 0.18) regarding non-CABG-related TIMI bleedings, patients with a minor or major bleeding event had a significantly lower PRI compared with those without (21 ± 17 [IQR 15–51] vs. 35 ± 23% [IQR 9–36]; P = 0.008). ROC analysis was performed to evaluate the predictive value of the PRI for bleeding events. The area under the curve was of 0.67. The optimal cut-off value for the PRI to predict bleedings was 16% with a sensitivity of 65% and a specificity of 74.3% (Fig. 3). Using this cut-off value, 27.9% of patients were considered to have a very low PRI. In addition, these patients with a very low PR had a higher rate of bleeding events (15.6% vs. 3.3%; P < 0.001).

Figure 3.

 Receiver-operating curve (ROC) analysis of the sensibility and 1-specificity of the Vasodilator-Stimulated Phosphoprotein (VASP) index to predict bleeding events.

Optimal PR or therapeutic window of PR to prevent thrombotic and bleeding events

We defined an optimal window of PR after a prasugrel LD as a PRI comprised between 16% and 53.5% according to ROC curve analysis. Half of the study population (49.7%) was comprised within this therapeutic window.

We further analyzed the hazard ratio of adverse events (thrombotic and bleeding events) during 1 year follow-up for patients with a PR outside the therapeutic window of PR (PRI ≤ 16 or ≥ 53.5). In this analysis, adjusted for platelet count, these patients had a HR of 2.92 (95% CI: 1.37–6.2; P = 0.006) to suffer an adverse event during 1-year follow-up (Fig. 4).

Figure 4.

 Adverse events rates according to the level of post-prasugrel loading dose platelet reactivity as measured by the Vasodilator-Stimulated Phosphoprotein (VASP) index. In red bleeding events and in black thrombotic events.

Independent predictors of thrombotic and bleeding events at 1 year

In multivariate analysis, the only factor significantly associated with thrombotic events was the PRI with a hazard ratio of 1.44 per 10% increase (95% CI: 1.2–1.72; P < 0.001). Regarding the secondary ischemic endpoint, which included cardiovascular death, non-fatal MI, stent thrombosis and non-urgent coronary revascularizations, the independent predictors were active smoking and creatinine level. In this analysis, the VASP index was not an independent predictor of ischemic events (P = 0.16) (Table 3).

Table 3.   Multivariate predictors of thrombotic, ischemic and bleeding events at 1-year follow-up
 OR95% CI P
  1. VASP, Vasodilator-Stimulated Phosphoprotein.

Thrombotic events
 VASP index (10% increase)1.441.2–1.72<0.001
Ischemic events1.631.09–2.660.02
 VASP index (10% increase)1.10.96–1.260.16
 Active smoking2.741.28–5.850.009
 Creatinine level (> 150 μmol/l)7.542.17–26.120.001
Bleeding events
 VASP index (10% increase)0.750.59–0.960.024
 Hypertension2.721.03–7.190.044
 Platelet count (per 50 × 103 per L decrease)1.701.16–2.480.006

Regarding the bleeding endpoint (major and minor non-CABG TIMI bleedings), the independent predictors were hypertension, platelet count and PRI (Table 3).

Discussion

The present study suggests that the level of PR inhibition after a 60-mg prasugrel LD in ACS patients undergoing PCI predicts thrombotic and bleeding events during 1-year follow-up. These findings are original and of a great potential interest to optimize antiplatelet therapy and further improve the clinical outcome of such patients. Another important finding was that the rate of thrombotic events and bleeding events were similar at 1-year follow-up in this multicenter study. This finding confirms that bleeding complications have become a major clinical issue in contemporary practice for ACS patients undergoing PCI.

The link between platelet reactivity and thrombotic events was fully demonstrated with clopidogrel [2]. In fact, several trials demonstrated a strong relationship between high on-treatment platelet reactivity and thrombotic events such as cardiovascular death and MI [3]. In a previous report analysing 1-month outcome of the present multicenter study, we observed consistently with the literature, a relationship between on-prasugrel platelet reactivity and thrombotic outcome [10]. The present study further demonstrates that this link persists at 1-year follow-up. This finding is in agreement with data demonstrating the long-term predictive value of PR measurement after a P2Y12 receptor antagonist loading dose. Accordingly, in a prospective trial, Parodi et al. [16] demonstrated the predictive value of HTPR for major adverse cardiovascular events during a 2-year follow-up. In addition, the optimal cut-off value to define HTPR using ROC analysis in the present study was similar to that observed with clopidogrel [3]. Such findings are consistent with the fact that prasugrel and clopidogrel share similar active metabolites and therefore mainly differed regarding their respective pharmacodynamic and pharmacokinetic properties [17]. However, PR failed to predict the overall ischemic endpoint which includes non-urgent coronary revascularizations, suggesting that PR inhibition has limited or no impact on in-stent restenosis. The rate of patients exhibiting HTPR after a prasugrel LD may be considered relatively high compared with recent studies but is consistent with the platelet sub-study of the TRITON TIMI 38 Trial [18,19]. Such discrepancies between trials may be related to the different platelet assays used. The VASP index was used in the present study and in the study by Michelson et al. [20] This test is highly specific of the P2Y12 receptor pathway and correlates with the concentration of active metabolites.

Interestingly, in the present study we observed a link between platelet reactivity and non-CABG-related TIMI bleedings at 1-year follow-up. In a previous analysis of this multicenter study, at 1 month this association was not observed [10]. This finding is of potential clinical interest. In fact, our results together with that of Parodi et al. [19] suggest a potential predictive value of PR for significant bleeding events in ACS patients receiving prasugrel. Of importance, in the TRITON TIMI 38 trial one of the main limitations of prasugrel compared with clopidogrel was an excess in non-CABG-related TIMI bleedings [7]. Consistent with the result of the TRITON TIMI 38 trial, we observed a slow but constant increase in the rate of bleeding complications during the 1-year follow-up period which explains the lack of a significant link within the first 30 days. Our findings suggest that PR measurement after a prasugrel LD may identify patients at high risk of bleeding. Such complications have become a major clinical issue because of an increasing incidence and the fact that they have a significant impact on the outcome. In fact, studies demonstrated that patients who bled had a poor short- and long-term outcome, including a higher mortality [8,9]. Our findings are in line with preliminary reports suggesting a link between on-treatment PR and bleeding events. Sibbing [4] using the Multiplate analyzer to assess PR inhibition demonstrated that a low level of PR translated into bleeding events after PCI in patients receiving clopidogrel. Patti et al. [5] also identified excessive PR inhibition after a clopidogrel LD as a key determinant of major bleedings and entry site complications in patients undergoing PCI. The higher risk of bleedings is the main limitation for prasugrel prescription in spite of its superiority over clopidogrel regarding the secondary prevention of thrombotic events. Therefore, PR monitoring may help to identify those who would benefit from this more potent drug compared with clopidogrel without increasing their risk for bleeding complications.

Gurbel [21] was the first to propose the hypothesis of a therapeutic window of PR to prevent both thrombosis and bleedings in ACS patients undergoing PCI. Subsequently, Sibbing et al. [22] observed in a prospective observational study enrolling patients undergoing PCI a therapeutic window of PR. Following these first evidence for a therapeutic window in the clopidogrel era, a recent study confirmed a sweet spot of PR to be reached to prevent both thrombotic and bleeding events [23]. In line with these preliminary studies suggesting an optimal window of PR to be reached with clopidogrel, our study suggests that a similar therapeutic window may exist with prasugrel. Of importance, the potential benefit of platelet reactivity monitoring and potent P2Y12 receptor antagonists may be limited to ACSs [24].

In conclusion, the present study suggests a link between platelet reactivity after a prasugrel LD and both thrombotic and bleeding events during a 1-year follow-up period. These results are in line with the hypothesis of a therapeutic window of PR inhibition to optimize anti-platelet therapy and may highlight the potential clinical value of PR testing and of individualized anti-platelet therapy.

Limitation

The present study included a relatively small number of patients and was powered to evaluate the link between PR and thrombotic events at 1-month follow-up. Therefore, although we observed a statistically significant link between PR and both thrombotic and bleeding events, these results should be considered as hypothesis generating and require confirmation in an adequately powered trial.

Conclusion

Platelet reactivity after a prasugrel LD in patients undergoing PCI for an ACS, predicts both thrombotic and bleeding events during 1-year follow-up. These findings reinforce the hypothesis of a therapeutic window of PR and could be of interest in order to optimize the risk benefit ratio of anti-platelet therapy in ACS patients.

Addendum

Laurent Bonello, conception, design and analysis and interpretation of data; Julien Mancini, Luc Maillard, Philippe Rossi and Bernard Jouve, analysis and interpretation of data; Michel Pansieri, drafting of the manuscript and revising it critically for important intellectual content; Frédéric Collet, Olivier Wittenberg and Jacques Bessereau, drafting of the manuscript; Marc Laine and Gilles Lemesle, revising it critically for important intellectual content; Pierre Michelet, Franck Paganelli and Françoise Dignat-George, drafting of the manuscript and revising it critically for important intellectual content; Françoise Dignat-George and Laurence Camoin-Jau, conception and design and final approval of the manuscript submitted.

Acknowledgements

The authors are grateful to Marie-Anne Prioux and Olfa Helal for their help in study and data management. We would also like to thank Marie, Paul and Emilie Bonello for the time devoted to this study. The present work was supported by a grant from the Assistance publique-Hopitaux de Marseille.

Disclosure of Conflict of Interest

Laurent Bonello received lectures fees from Sanofi, AstraZeneca and Eli Lilly.

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