Ticagrelor overcomes high platelet reactivity in patients with acute myocardial infarction or coronary artery in-stent restenosis: a randomized controlled trial

High on-treatment platelet reactivity (HTPR) is accompanied by an increased risk of adverse outcomes. Direct comparison of the antiplatelet effects between ticagrelor and high-dose clopidogrel has not yet been reported in acute myocardial infarction (AMI) or coronary artery in-stent restenosis (ISR) patients with HTPR. Consecutive patients with AMI or coronary artery ISR treated with standard-dose clopidogrel (75 mg/day) were screened with the VerifyNow assay, defining HTPR as P2Y12 reaction units (PRUs) >208. Of the 102 screened patients, 48 (47.06%) patients with HTPR were randomly assigned to either ticagrelor (180 mg/90 mg twice daily) or high-dose clopidogrel (150 mg/day) for 24 hours. Baseline characteristics and mean PRUs were similar in both groups. After 24 hours, ticagrelor was associated with a significantly lower platelet reactivity than high-dose clopidogrel (44.38 ± 40.26  vs. 212.58 ± 52.34 PRU, P < 0.05). No patient receiving ticagrelor exhibited HTPR, whereas 15 (62.50%) patients after treatment with high-dose clopidogrel remained HTPR (P < 0.05). During the follow-up (mean, 138.42 ± 53.59 days), no patient exhibited a major bleeding event in either treatment group. In conclusion, in patients with AMI or coronary artery ISR exhibiting HTPR after standard clopidogrel treatment, ticagrelor is significantly more effective compared with high-dose clopidogrel in overcoming HTPR.

consistent inhibition of platelet aggregation 18 . The Response to Ticagrelor in Clopidogrel Non-responders and Responders and Effect of Switching Therapies Study (RESPOND) demonstrated that ticagrelor therapy overcame the problem of HTPR during clopidogrel therapy in patients with stable coronary disease 19 . However, the prevalence of HTPR in Chinese patients with AMI or coronary artery in-stent restenosis (ISR) and the beneficial impact of ticagrelor compared with high-dose clopidogrel in overcoming HTPR are still unknown.
Here, we report the first prospective, randomised trial to be conducted in China to determine the antiplatelet effects of ticagrelor versus high-dose clopidogrel (150 mg) in AMI or coronary artery ISR patients with HTPR.

Method
Patient population and study design. We performed a prospective, randomised, single-center, single-blind study to compare the antiplatelet effects of ticagrelor versus high-dose clopidogrel (150 mg) in AMI or coronary artery ISR patients with HTPR. From April 2014 to November 2014, patients aged between 20 and 80 years hospitalised for ST Segment Elevation Myocardial Infarction (STEMI), non-(N-STEMI), or coronary artery ISR who were on aspirin and clopidogrel therapy were prospectively monitored for platelet reactivity (PR). Restenosis was defined as at least 50% in-stent diameter stenosis at the follow-up angiogram. Patients meeting all the inclusion criteria were consecutively enrolled into the study and treated and followed per the protocol in order to minimize selection bias. Exclusion criteria were (1) contraindication to antiplatelet therapy; (2) haemoglobin < 10 g/dL or platelet count < 100 000/ mm 3 ; (3) active bleeding and bleeding diatheses; (4) stroke within 6 months; (5) aspartate aminotransferase or alanine aminotransferase level > 2 times the upper normal limit; (6) chronic renal failure (creatinine clearance < 30 mL/min); and (7) active peptic ulceration or gastrointestinal bleeding. The study was performed in accordance with ethical principles that have their origin in the Declaration of Helsinki and are consistent with ICH/Good Clinical Practice. Informed consents were signed and obtained by all the patients. All experimental protocol was approved by the Ethics Committee of Changhai Hospital, Second Military Medical University. The trial was registered at Chinese Clinical Trial Registry. Trial registration No. was ChiCTR-RCS-14004303 and the date of registration was January 2, 2014.
A flow chart diagram of the study is shown in Fig. 1. Patients received a loading dose of 600-mg of clopidogrel for at least 6 hours, 300-mg of clopidogrel at least 12 hours, or were receiving chronic clopidogrel therapy (75 mg daily for ≥ 7 days) before the PR measurement. Patients received maintenance dose of 100 mg, once a day of aspirin and 75 mg, once a day of clopidogrel. In patients undergoing percutaneous coronary intervention (PCI), PR measurement was carried out after PCI. If patients received tirofiban or heparin therapy during PCI, tirofiban had to be discontinued for at least 48 hours, and heparin or low-molecular-weight heparin had to be discontinued for at least 12 hours before measurement of PR. Patients with HTPR (as defined subsequently) were randomized in a 1:1 ratio to receive either a high-dose clopidogrel of 150 mg daily or a loading-dose ticagrelor of 180 mg followed by 90 mg twice daily using a computer-generated randomisation table. A study nurse enrolled the participants and assigned participants to their groups. A second PR measurement was carried out 24 hours later in patients who received an alternate treatment. Patients who exhibited HTPR after being treated with high-dose clopidogrel were switched to 90 mg ticagrelor twice daily, and a third PR measurement was performed 24 hours after administration of ticagrelor.
Clinical end points. Endpoints were pre-specified in the study protocol and statistical analysis plan.
The primary end point of the study was PR after 24 hours of administration of ticagrelor versus high-dose clopidogrel, as determined by VerifyNow P2Y12 assay. The secondary end points included the rate of HTPR (defined as > 208 PRU). A clinical follow-up by telephone interviews was obtained at 1, 3, and 6 months and bleeding (major, minor, or minimal according to Thrombolysis in Myocardial Infarction [TIMI] criteria), and major adverse cardiovascular events (cardiovascular death, myocardial infarction, stent thrombosis, and stroke) were evaluated during the follow-up term.
Blood sample collection. Blood samples were collected in 2 vacuum tubes (Becton-Dickinson, Franklin Lakes, NJ) containing 3.2% trisodium citrate, in which the first tube was discarded to avoid spontaneous platelet activation. The second tube was gently inverted 3 to 5 times to ensure complete mixing of the anticoagulant, and the blood sample was analysed within 4 hours for rapid platelet-function assay.
Determination of HTPR. VerifyNow-P2Y12 (Accumetrics, San Diego, California) is a whole blood, rapid platelet function assay designed to measure the inhibition of clopidogrel on the P2Y12 receptor. Technical details of the platelet function assay were previously described [20][21] . Results were expressed as P2Y12 reaction units (PRUs), baseline value (BASE), and percent inhibition. The percent inhibition is calculated as follows: ([BASE-PRU]/BASE) × 100. A PRU of >208 was defined as HTPR or clopidogrel non-responder, linking the cut-off point to ischemic event occurrences 22 . Statistical analysis. For sample size calculation, we hypothesized that ticagrelor 90 mg, twice daily would result in a PR absolute difference of 50 PRU compared with clopidogrel 150 mg (with the assumption that the within-patient standard deviation of the response variable is 50 PRU), based on previously published data 19,23 . Choosing a power of 90% and a 2-sided alpha-level of 0.05, at least 46 patients in total (23 for each group) were required to reach statistical significance on the basis of the preceding assumptions. Continuous variables are reported as mean ± standard deviation (SD) and categorical variables are presented as frequencies and percentages. Continuous variables were compared by the unpaired Student t-test and categorical variables by the chi-square test or Fisher exact test. Analyses were performed using SPSS version 20.0 statistical software (SPSS Inc., Chicago, Illinois); P-value of < 0.05 were considered statistically significant.

Results
Baseline characteristics. Of the 102 (STEMI 63.73%, NSTEMI 16.67%, and ISR 19.61%) consecutive patients who were on the standard dose of clopidogrel therapy, 48 (47.06%) were identified to have HTPR, and randomly assigned to treatment with either high-dose clopidogrel (n = 24) or ticagrelor (n = 24). The demographics, clinical details, and concomitant medications were well balanced across the 2 treatment groups (Table 1).
Laboratory characteristics. There were no significant differences between treatment groups with regard to laboratory characteristics ( Table 2). After the standard dose of clopidogrel therapy, PR measured by the VerifyNow P2Y12 assay did not differ significantly in both groups (all P > 0.05).
Angiographic characteristics. Of the 48 randomised patients, 47 (97.92%) patients underwent PCI and no patient received any revascularization. There were no significant differences in the angiographic characteristics between the 2 groups (Table 3).
Platelet response to treatment modification. In the ticagrelor-treated group, the PRU level in patients with HTPR was significantly reduced from 269.04 ± 34.78 to 44.38 ± 40.26, 24 hours after administration of ticagrelor (P < 0.001; Fig. 2A Table 3. Angiographic characteristics.

Discussion
To our knowledge, this is the first study in China to compare the effects of ticagrelor versus high-dose clopidogrel on platelet inhibition in AMI or coronary artery ISR patients with HTPR. The major findings of the study are (1) HTPR while on clopidogrel treatment was a common phenomenon found in about one half (47.06%) in our population; (2) the antiplatelet effect of ticagrelor was obviously greater than high-dose clopidogrel therapy in patients with HTPR; and (3) all patients treated with ticagrelor had PR below 208 PRU cut-off point, whereas more than half of patients (62.50%) receiving high-dose clopidogrel remained non-responsive to clopidogrel. A number of studies have shown that the level of platelet inhibition achieved by clopidogrel varies considerably between individuals 24 . It has been estimated that nearly 4% to 30% of the patients exhibit low or no response to clopidogrel loading and maintenance therapy 6,8,25,26 . The antiplatelet effect of clopidogrel has been shown to be dose dependent, and has been examined to improve responses by increasing the dose 11,27 . However, persistent presence of HPPR was apparent despite high-dose of clopidogrel, and the clinical benefit is less clear. Compared with administration of clopidogrel 75 mg in patients with high-risk type 2 diabetes mellitus, clopidogrel 150 mg is associated with enhanced antiplatelet effects, but enhanced PR continues to persist in 60% of the patients on the 150-mg regimen 28 . In addition, the Gauging Responsiveness with A VerifyNow assay-Impact on Thrombosis And Safety (GRAVITAS) trial did not show any reduction in the incidence of death from cardiovascular causes, nonfatal MI, or ST with high-dose clopidogrel (600 mg LD, 150 mg/day MD) compared with standard-dose clopidogrel (no additional LD, 75 mg/day MD) in 2214 patients with HTPR after PCI with drug-eluting stents 29 .
Inter-individual variability in response to clopidogrel has prompted the development of novel P2Y12 inhibitors such as ticagrelor. Ticagrelor is the first oral P2Y12 receptor antagonist that blocks ADP-induced platelet aggregation in a reversible manner 18 . Unlike clopidogrel, it does not require activation via CYP450 enzymes because it is an active drug. In recent studies, ticagrelor has resulted in more potent inhibition of ADP-induced platelet aggregation than clopidogrel 18,30 . Bliden reported that compared with clopidogrel, ticagrelor was rapidly and consistently associated with a very low (0%-8%) prevalence of HTPR assessed with the VerifyNow assay 30 . In the PLATO trial, in patients with acute coronary syndrome (ACS), ticagrelor compared with clopidogrel significantly reduced the adverse event rate, without an increase in bleeding risk 31 . Notably, ticagrelor has been shown to be superior to high loading dose of clopidogrel in terms of reversing non-responsiveness to clopidogrel in patients with stable coronary artery disease 19 .
In the PLATO trial, the antiplatelet effects of ticagrelor in ACS patients being nonresponsive to clopidogrel are unknown. Also, the present study population differs from the RESPOND study that enrolled patients with AMI or coronary artery ISR; the clinical setting in which hyper-reactive platelets may be more considerable and the risk for ischemic complications is higher compared with stable coronary artery disease. Indeed, in the present study, 48 (47.06%) patients with HTPR were identified according to the VerifyNow system, a finding obviously significant than the previous reports in which the prevalence of clopidogrel to non-responsiveness was about 28% 8,19 . Other factors contributing to the high prevalence of HTPR in our population may include the definitions of PR cut-offs and the amount of clopidogrel loading dose. Nevertheless, whether the high rate of HTPR is related to the differences between races of human beings, large clinical trials might be needed to further proven.
High PR in patients with AMI has been shown predict the PCI angiographic success and recurrent atherothrombotic events 32 . Thus, the early and strong platelet inhibition seems to be of outmost importance, particularly in a population with high risk of ischemic events. Our study provides the first laboratory evidence that ticagrelor, compared with a high-dose clopidogrel of 150 mg/day may significantly eliminate HTPR in patients with AMI or coronary artery ISR. In the study, baseline rates of HTPR were 47.06%, which was reduced to 0 by switching from clopidogrel to ticagrelor compared with high-dose clopidogrel (31.25%). For the patients who were still resistant to high-dose clopidogrel, the level of PR was also decreased to below 208 PRU after switching to ticagrelor (P < 0.05). The results from this study suggest that in the presence of HTPR, switching to ticagrelor is a more reliably effective option for inhibiting platelet function compared with increasing the dose of clopidogrel. These findings have important clinical implications for guiding antiplatelet therapy in patients with high risk of cardiovascular events who are non-responsiveness to clopidogrel.
In recent years, an increasing number of studies have used VerifyNow P2Y12 assay to assess the antiplatelet effect of clopidogrel, and its relation to ischemic event occurrence during clopidogrel therapy has been demonstrated 33 . In our study, the change in PR was measured using VerifyNow P2Y12 assay as a point-of care test, with non-responder status predefined at > 208 PRU and < 85 PRU as thresholds to bleeding outcome [34][35] . In the present study, ticagrelor suppressed PR to a very low level (44.38 ± 40.26 PRU), which is far below the previous thresholds associated with bleeding risk, but without observing a major bleeding event. The low incidence of bleeding might be contributed to the sample size of the present study that is too small to assess the relation of PR with a bleeding event. However, the rate of bleeding in our study is in line with that was observed in the PLATO substudy, showing a relative low incidence of the bleeding event in Chinese patients 16 . Moreover, the data showed that overall bleeding events were similar in either treatment group, indicating that the benefits of ticagrelor were not associated with the increased rates of bleeding events compared with high-dose clopidogrel. Ticagrelor was more frequently accompanied by other mild side effects such as dyspnea and diarrhoea. This study had small sample size, therefore, large-scale randomised clinical trials and sufficient duration are required to confirm our findings, and to fully evaluate clinical outcomes, such as the incidence of stent thrombosis and major bleeding. Because of poor correlation between different tests of platelet function 36 , only single test of platelet function have been used in our study. However, the VerifyNow assay is most widely accepted test to assess the antiplatelet effects of P2Y12 receptor inhibitors, and has been shown to be useful in assessing the antiplatelet effects for identifying HTPR after clopidogrel or ticagrelor therapy 24,33,37 .

Conclusions
In conclusion, the present results provide clinical evidence that, in patients with AMI or coronary artery ISR exhibiting HTPR after standard clopidogrel treatment, ticagrelor therapy is more effective than high-dose clopidogrel (150 mg) in reducing the PR and HTPR rate. The possible clinical benefits of this regimen require further validation.