Lp-PLA2 Activity and Genotype-Guided Dual Antiplatelet Therapy in Minor Stroke or Transient Ischemic Attack

Abstract

BACKGROUND:

Lp-PLA2 (lipoprotein-associated phospholipase A2) is a sensitive biomarker of vascular inflammation and atherosclerosis. This study evaluated the influence of Lp-PLA2 activity on the efficacy and safety of ticagrelor-aspirin versus clopidogrel-aspirin among patients with minor stroke or transient ischemic attack carrying CYP2C19 loss-of-function alleles.

METHODS:

The CHANCE-2 trial (Clopidogrel in High-Risk Patients With Acute Nondisabling Cerebrovascular Events-II) randomized 6412 patients with minor stroke or transient ischemic attack carrying CYP2C19 loss-of-function alleles to receive ticagrelor-aspirin or clopidogrel-aspirin. This subgroup study included patients with available baseline Lp-PLA2 activity measurements, stratified by the median value of 188.4 nmol/min per milliliter. The primary efficacy and safety outcomes were stroke recurrence and severe or moderate bleeding events within 90 days. Associations between treatment and outcomes were assessed using multivariable Cox proportional hazards models, adjusting for a history of hyperlipidemia.

RESULTS:

A total of 5919 patients were included (mean age, 64.4 years; 33.9% female). Among patients with low Lp-PLA2 activity, ticagrelor-aspirin reduced the 90-day risk of recurrent stroke compared with clopidogrel-aspirin (5.4% versus 7.4%; adjusted hazard ratio, 0.72 [95% CI, 0.54–0.97]). In patients with high Lp-PLA2 activity, no significant difference was observed (6.9% versus 8.2%; adjusted hazard ratio, 0.84 [95% CI, 0.65–1.09]). The P value was 0.45 for the treatment × Lp-PLA2 activity interaction effect on stroke recurrence. The risk of bleeding associated with ticagrelor–aspirin did not differ across Lp-PLA2 activity levels.

CONCLUSIONS:

In patients with minor stroke or transient ischemic attack carrying CYP2C19 loss-of-function alleles, elevated Lp-PLA2 activity did not significantly modify the efficacy or safety of dual antiplatelet therapy. Further research is needed to clarify the potential role of Lp-PLA2 in guiding individualized treatment decisions.

REGISTRATION:

URL: https://www.clinicaltrials.gov; Unique identifier: NCT04078737.

Ischemic cerebrovascular disease poses a significant public health challenge due to its high risk of recurrence, which significantly compromises long-term clinical outcomes.¹ The CHANCE-2 trial (Clopidogrel in High-Risk Patients With Acute Nondisabling Cerebrovascular Events-II) demonstrated that, among patients with acute minor stroke or transient ischemic attack (TIA) carrying CYP2C19 loss-of-function (LOF) alleles, ticagrelor-aspirin significantly reduced the risk of stroke recurrence compared with clopidogrel-aspirin.² Despite these advances in genotype-guided antiplatelet therapies, recurrent ischemic events still occur in ≈6% of patients, indicating an unmet need to identify additional risk factors to inform individualized secondary prevention.²

One area of growing interest involves the contribution of vascular inflammation to cerebrovascular risk.^3,4 Lp-PLA2 (lipoprotein-associated phospholipase A2) is a 45-kDa enzyme secreted by various inflammatory cells, including monocytes/macrophages, T lymphocytes, and mast cells.⁵ It hydrolyzes oxidized low-density lipoprotein phospholipids, generating lysophosphatidylcholine and oxidized nonesterified fatty acids, which are proinflammatory and proapoptotic mediators implicated in necrotic core formation within atherosclerotic plaques.^6–8 Moreover, Lp-PLA2 is highly expressed in vulnerable and ruptured plaques, suggesting a potential role in plaque destabilization.⁴ Consistent clinical evidence reveals that elevated Lp-PLA2 activity independently correlates with an increased risk of ischemic stroke and predicts stroke recurrence, highlighting its potential utility as a prognostic biomarker in cerebrovascular disease.^9,10

Given the role of Lp-PLA2 in vascular inflammation and its association with plaque vulnerability, we hypothesized that elevated Lp-PLA2 activity may modify the clinical response to antiplatelet therapy. This study aimed to investigate the influence of baseline Lp-PLA2 activity levels on the efficacy and safety of ticagrelor-aspirin versus clopidogrel-aspirin in patients with minor stroke or TIA carrying CYP2C19 LOF alleles.

Methods

Data Availability

Data are available from the corresponding author on reasonable request to allow replication of the results and validation of the methods.

Study Design and Patients

This study was a post hoc analysis of the CHANCE-2 trial, a multicenter, randomized, double-blind, parallel-group study designed to evaluate the efficacy and safety of ticagrelor-aspirin compared with clopidogrel-aspirin. The detailed study design has been previously published.^2,11 Between September 23, 2019, and March 22, 2021, a total of 6412 patients were enrolled from 202 hospitals across China. Eligible participants were 40 years of age or older and presented within 24 hours of symptom onset with noncardioembolic minor stroke (National Institutes of Health Stroke Scale score ≤3) or high-risk TIA (ABCD² [age, blood pressure, clinical features, duration, diabetes] score ≥4). All enrolled patients were confirmed to carry at least 1 CYP2C19 LOF allele, categorized as poor metabolizers with 2 variant alleles (*2/*2,*2/*3, or*3/*3) or intermediate metabolizers with a single variant allele (*1/*2 or*1/*3).

Patients were randomly assigned in a 1:1 ratio within 24 hours of symptom onset to receive either ticagrelor (180 mg loading dose on day 1, followed by 90 mg twice daily on days 2–90) or clopidogrel (300 mg loading dose on day 1, followed by 75 mg once daily on days 2–90). All patients also received aspirin (75 mg–300 mg loading dose, followed by 75 mg once daily for 21 days).

The trial protocol received approval from the ethics committee of Beijing Tiantan Hospital (IRB number: KY2019-035-02) and all participating centers. Written informed consent was obtained from all participants or their legal representatives before enrollment. This study was reported following the Strengthening the Reporting of Observational Studies in Epidemiology guidelines.

Measurement of Lp-PLA2 Activity

Fasting venous blood samples were collected on the morning following enrollment. Samples were processed, aliquoted, and transported under cold chain conditions to the central laboratory at Beijing Tiantan Hospital. All specimens were stored at –80 °C until analysis. Lp-PLA2 activity was measured using an automated enzymatic assay (PLAC test for Lp-PLA2 activity; diaDexus Inc, San Francisco, CA) on a Hitachi 7600 analyzer.⁹

Clinical Outcomes

The primary efficacy outcome was recurrent stroke (ischemic or hemorrhagic) within 90 days. Secondary efficacy outcomes included stroke within 30 days, composite of vascular events (stroke, TIA, myocardial infarction, or vascular death), ischemic stroke, and disabling stroke (modified Rankin Scale score ≥2) within 90 days. The primary safety outcome was severe or moderate bleeding within 90 days, as defined by the Global Utilization of Streptokinase and Tissue Plasminogen Activator for Occluded Coronary Arteries criteria.¹² Secondary safety outcomes included any bleeding event and all-cause mortality within 90 days. All clinical events were adjudicated by an independent committee blinded to treatment assignment.

Statistical Analysis

Participants were stratified into 2 groups according to the median level of Lp-PLA2 activity, using a cutoff of 188.4 nmol/min per milliliter. Continuous variables were summarized as mean±SD for normally distributed data and as median with interquartile range for non-normally distributed data. Between-group comparisons were conducted using the independent samples t test or the Wilcoxon rank-sum test, as appropriate. Categorical variables were presented as percentages and compared using the χ² test. The association between dual antiplatelet therapy and clinical outcomes was assessed through multivariable Cox proportional hazards regression models. Hazard ratios and 95% CIs were reported. Interaction analyses were conducted to evaluate whether the treatment effect varied according to Lp-PLA2 activity. Models were adjusted for a history of hyperlipidemia based on a significant between-group difference. The cumulative incidence of stroke recurrence over the 90-day follow-up was estimated through Kaplan-Meier survival analysis, with between-group comparisons conducted with the log-rank test. In cases of multiple events of the same type, only the time to the first event was included in the analysis. Statistical significance was defined as a 2-sided P<0.05. All analyses were performed using SAS software, version 9.4 (SAS Institute, Cary, NC).

Results

Baseline Characteristics

Of the 6412 patients enrolled in the CHANCE-2 trial, 5919 (92.3%) with available Lp-PLA2 activity measurements were included in the present subgroup analysis (Figure 1). Compared with those excluded, included patients were more likely to have a history of hypertension, ischemic stroke or TIA, and prior use of antiplatelet or lipid-lowering therapy. They were also more frequently admitted with acute ischemic stroke, classified as large-artery atherosclerosis according to the Trial of ORG 10172 in Acute Stroke Treatment classification, and had higher ABCD² scores (Table S1). Among the included patients, 2963 (50.1%) were classified as having low Lp-PLA2 activity (≤188.4 nmol/min per milliliter), and 2956 (49.9%) as having high Lp-PLA2 activity (>188.4 nmol/min per milliliter; Table S2). Within each Lp-PLA2 activity subgroup, baseline characteristics were well balanced between the 2 treatment groups. An exception was observed in the high Lp-PLA2 activity group, where a higher proportion of patients in the ticagrelor-aspirin group had a history of hyperlipidemia (Table 1).

Figure 1.

Flowchart of included patients. Lp-PLA2 indicates lipoprotein-associated phospholipase A2; and TIA, transient ischemic attack.

Table 1.

Baseline Characteristics According to Lp-PLA2 Activity Levels and Treatment

Efficacy Outcomes

During the 90-day follow-up period, 413 patients (7.0%) experienced a new stroke, 497 patients (8.4%) had a composite vascular event, 406 patients (6.9%) suffered an ischemic stroke, and 178 patients (3.0%) experienced a disabling stroke. Among those with recurrent stroke, 343 events (83.1%) occurred within the first 30 days.

In patients with low Lp-PLA2 activity, ticagrelor-aspirin was associated with a lower risk of recurrent stroke at 90 days compared with clopidogrel-aspirin (5.4% versus 7.4%; adjusted hazard ratio, 0.72 [95% CI, 0.54–0.97]; Table 2). In contrast, no significant difference was observed between treatment groups in patients with high Lp-PLA2 activity (6.9% versus 8.2%; adjusted hazard ratio, 0.84 [95% CI, 0.65–1.09]; Table 2). The cumulative incidence of recurrent stroke stratified by Lp-PLA2 activity and treatment group was shown in Figure 2 (log-rank, P=0.03). The analysis did not reveal a significant interaction between Lp-PLA2 activity and treatment effect (P for interaction=0.45; Table 2). Similar findings were observed for secondary efficacy outcomes, including strokes within 30 days, composite vascular events, and ischemic stroke, with the exception of disabling stroke (Table 2).

Table 2.

Efficacy and Safety Outcomes of Patients With Different Dual Antiplatelet Therapies Stratified by Lp-PLA2 Activity

Figure 2.

Cumulative probability of recurrent stroke according to Lp-PLA2 (lipoprotein-associated phospholipase A2) activity levels and treatment.

Safety Outcomes

In patients with either high or low Lp-PLA2 activity, the risk of severe or moderate bleeding was comparable between the ticagrelor-aspirin group and the clopidogrel-aspirin group (0.2% versus 0.3% in the high Lp-PLA2 activity group; 0.4% versus 0.5% in the low Lp-PLA2 activity group; P for interaction=0.74; Table 2). A similar trend was observed for all-cause mortality (0.3% versus 0.5% in the high Lp-PLA2 activity group; 0.4% versus 0.6% in the low Lp-PLA2 activity group; P for interaction=0.84; Table 2). Nevertheless, ticagrelor-aspirin was associated with a higher risk of any bleeding compared with clopidogrel-aspirin (5.1% versus 2.4% in the high Lp-PLA2 activity group; 6.2% versus 2.9% in the low Lp-PLA2 activity group; P for interaction=0.98; Table 2).

Discussion

In this post hoc analysis of the CHANCE-2 trial, for patients with acute minor ischemic stroke or TIA carrying CYP2C19 LOF alleles, ticagrelor-aspirin did not significantly reduce the 90-day risk of stroke recurrence compared with clopidogrel-aspirin in those with high Lp-PLA2 activity. Conversely, in patients with low Lp-PLA2 activity, ticagrelor-aspirin was associated with a significant reduction in 90-day stroke recurrence risk relative to clopidogrel-aspirin. The risk of bleeding associated with ticagrelor-aspirin was not influenced by Lp-PLA2 activity.

The differential efficacy observed in this study carries important clinical implications for managing patients with acute ischemic stroke or TIA who carry CYP2C19 LOF alleles and exhibit elevated baseline Lp-PLA2 activity. This subgroup, comprising nearly half of the CHANCE-2 population, may experience limited benefit from current antiplatelet strategies alone. This underscores a critical therapeutic gap, as no approved therapies specifically target elevated Lp-PLA2 activity in ischemic stroke patients.

Pharmacologically, clopidogrel is a prodrug requiring hepatic activation via the CYP2C19 enzyme to inhibit platelet aggregation, whereas ticagrelor is an active agent that directly antagonizes the P2Y12 receptor.¹³ Reduced responsiveness to clopidogrel among carriers of CYP2C19 LOF alleles formed the rationale for the CHANCE-2 trial, which showed superior efficacy of ticagrelor-aspirin over clopidogrel-aspirin in this genetic subgroup.² However, among patients with high Lp-PLA2 activity, the expected benefit of ticagrelor was not evident. One possible explanation is that increased vascular inflammation, as indicated by elevated Lp-PLA2 activity, contributes to a prothrombotic state that attenuates the efficacy of even intensified antiplatelet therapy.¹⁴ Alternatively, recurrent stroke in this subgroup might be driven by non-platelet-mediated mechanisms, such as accelerated atherosclerosis, endothelial dysfunction, or inflammatory plaque destabilization, which would limit the incremental benefit of more potent platelet inhibition.^3,9,15 Given that Lp-PLA2 activity is influenced by age, lifestyle factors, comorbidities, and genetic variability, stratifying stroke patients by Lp-PLA2 levels may facilitate a deeper understanding of the underlying pathophysiological mechanisms and identify potential targets for secondary prevention.⁴

Previous attempts to therapeutically inhibit Lp-PLA2, exemplified by darapladib, a selective oral Lp-PLA2 inhibitor, did not demonstrate a reduction in the risk of ischemic events in patients with acute or stable coronary artery disease.^16,17 These findings might reflect trial design limitations, potential pharmacological interactions (eg, with statins), or inadequate targeting of high-risk inflammatory phenotypes.^16,17 Importantly, patients with acute cerebrovascular events and elevated Lp-PLA2 activity have not been the focus of such interventions. Therefore, the therapeutic relevance of Lp-PLA2 inhibition in stroke prevention remains to be clarified in appropriately selected populations. Future investigations could explore combination regimens integrating antiplatelet therapy with agents targeting vascular inflammation, such as Lp-PLA2 pathway antagonists, to improve outcomes in this high-risk subgroup.

This study has several limitations. First, as a post hoc analysis, the findings are exploratory and should be interpreted with appropriate caution. Second, Lp-PLA2 activity was measured only at baseline, precluding assessment of longitudinal changes and their relationship to clinical outcomes. Third, the study cohort comprised only Chinese patients, potentially limiting generalizability to other populations.

In summary, among patients with minor stroke or TIA carrying CYP2C19 LOF alleles, elevated Lp-PLA2 activity did not significantly modify the efficacy or safety of dual antiplatelet therapy. Although no statistically significant interaction was detected, the observed trend suggests that Lp-PLA2 may warrant further investigation as a potential biomarker for risk stratification and individualized therapy.

Article Information

Acknowledgments

The authors gratefully acknowledge the invaluable contributions of the participants and their families, as well as the investigators and study teams across the 202 centers involved in the CHANCE-2 trial (Clopidogrel in High-Risk Patients With Acute Nondisabling Cerebrovascular Events-II).

Sources of Funding

This study is supported by the grants from National Key Research and Development Program of China (2022YFC2502403, 2022YFC2502404), National Natural Science Foundation of China (U20A20358), Chinese Academy of Medical Sciences Innovation Fund for Medical Sciences (2019-I2M-5-029), Beijing Hospitals Authority Clinical Medicine Development of special funding support (ZLRK202312), and National Science and Technology Major Project (2017ZX09304018).

Disclosures

None.

Supplemental Material

Tables S1–S2