PLAC-Activity: A Novel Predictive Biomarker of Coronary Heart Disease and Stroke [Communiqué]

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Heart attack and stroke are leading causes of morbidity and mortality in the United States and across the globe.1 These diseases, in addition to markedly altering the quality of life of afflicted patients, account for more than $500 billion annually in health care expenditures and related expenses.2 Ironically, the common underlying pathology of these diseases, atherosclerosis, is among the most preventable diseases in health care.

Atherosclerotic plaques often do not cause pain or other symptoms, and a heart attack or stroke can be the first sign of atherosclerosis in otherwise healthy people. Consequently, risk factors for atherosclerosis have been thoroughly studied. Many risk factors cannot be controlled (ie, age, gender, ethnicity, and other genetic factors). However, several modifiable risk factors include obesity, blood pressure, tobacco use, sedentary lifestyle, and blood lipid levels.

Blood cholesterol, particularly low-density lipoprotein (LDL) cholesterol and non-high-density lipoprotein (non-HDL) cholesterol, are the primary biomarkers used in risk assessment and treatment decisions.3-5 However, multiple studies have found that a substantial residual risk of cardiovascular disease remains present despite achieving desirable LDL cholesterol goals.6,7 For this reason, additional biomarkers of atherosclerotic risk, which improve on LDL and non-HDL cholesterol, are in strong demand.

Lipoprotein-associated Phospholipase A2 and Atherosclerosis
Lipoprotein-associated phospholipase A2 (Lp-PLA2) is a novel biomarker of atherosclerosis and is often referred to by the trademark name “The PLAC Test.” Lp-PLA2 is an enzyme produced mainly by monocytes and macrophages that converts phosphotidylcholine into 2 proinflammatory and proatherogenic molecules: nonesterified fatty acids (NEFA) and lysophosphatidylcholine (LPC). While Lp-PLA2 is highly expressed in atherosclerotic plaques, it also associates with lipoproteins in circulation through noncovalent binding to apolipoproteins such as apolipoprotein B (apoB) and, to a lesser extent, apolipoprotein A.

In contrast to other nonlipid biomarkers, Lp-PLA2 appears to be directly involved with atherosclerotic plaque formation. During atherosclerosis, apoB-containing lipoproteins accumulate in the vascular intima. Some of these lipoproteins become oxidized. Lp-PLA2 converts oxidatively modified phosphotidylcholine into oxidized free fatty acids (oxNEFA) and LPC (see Figure). These products are strong chemoattractants for monocytes and macrophages, which migrate to and accumulate in atherosclerotic plaques. This accumulation of macrophages leads to localized production of Lp-PLA2, which may help to promote the atherosclerotic process.8,9

Figure. Atherosclerotic plaque development

Measurement of Lp-PLA2 in circulation has been pursued in an effort to identify patients with the highest plaque burden−more specifically the highest unstable plaque burden−and, thus, patients with the highest risk of future cardiovascular events. In the majority of published studies, Lp-PLA2 has been shown to strongly associate with coronary and cerebrovascular occlusive disease. However, because Lp-PLA2 is bound in circulation to lipoproteins, especially LDL, it is strongly associated with the concentrations of LDL-C and apoB. Thus, it should be appreciated that the association of Lp-PLA2 with cardiovascular disease events is attenuated in models adjusted for baseline lipid and apolipoprotein concentrations.

Lp-PLA2 Testing: Activity versus Concentration
Clinical Lp-PLA2 measurement is available by 2 Food and Drug Administration (FDA)-approved methods; one measures the protein concentration and the other measures the activity of the enzyme. The first method to be FDA approved in 2005 was an enzyme-linked immunosorbent assay (ELISA) that determines the concentration of Lp-PLA2 in a commercial immunoassay format. Three generations of the commercially available concentration assay have been developed; however, most published studies have used the second-generation assay.10,11 A cutpoint of 200 ng/ml Lp-PLA2 using the ELISA assay is recommended to differentiate patients at higher or lower risk for future cardiovascular events.12,13

The second commercially available assay format approved by the FDA in 2014 measures the enzymatic activity of Lp-PLA2. This method uses colorimetric detection of a labeled substrate of the Lp-PLA2 enzyme. In multivariate analysis with traditional risk factors and C-reactive protein, Lp-PLA2 activity has also been shown to be an independent predictor of coronary heart disease and stroke in the general population.10,14,15 Based on data from the REGARDS multicenter substudy, the clinical decision point of 225 nmol/min/mL activity units showed a significantly increased rate of coronary heart disease among patients with elevated Lp-PLA2 activity (HR 1.54).16

While both Lp-PLA2 concentration and activity assays have been shown to identify patients at higher risk for future cardiovascular events, significant disadvantages have been reported for the concentration assay. For example, large variation in median Lp-PLA2 concentrations among different studies has been noted.17 It has also been suggested that Lp-PLA2 concentration may not correlate with the active enzyme transported within lipoprotein fractions.18 Additionally, the ELISA assay is not dose responsive to treatment with Lp-PLA2 inhibitors such as darapladib.19However, this may be less of a disadvantage given that the inhibitors have failed to live up to their promise as novel cardiovascular disease-event-lowering therapies.

Perhaps the most important disadvantage of the Lp-PLA2 concentration assay is that significant preanalytical issues influencing the reliability of results have been reported. It is concerning that concentrations of Lp-PLA2 could be affected by as much as 50% based on preanalytical sample handling.20,21 All of these factors have limited the clinical adoption of the Lp-PLA2 concentration test.

An appealing solution to the aforementioned challenges is to measure the enzymatic activity of Lp-PLA2. Reports can be found for either equivalent or improved cardiovascular risk prediction using the activity assay compared to the concentration assay. For example, a nested case-control study of more than 32,000 women found that Lp-PLA2 activity was significantly associated with cardiovascular events (HR 1.75; 95% CI 1.09-2.84), even after normalizing for lifestyle, clinical lipid, and inflammatory risk factors.22 In the same study, Lp-PLA2 mass was not significantly associated with cardiovascular events after multivariate adjustment. Several studies have shown that risk assessment by plasma Lp-PLA2 activity is at least equivalent to23-25 or better14,15 than Lp-PLA2 concentration. A meta-analysis of 32 clinical studies determined the relative risk of a 1 standard deviation increase in Lp-PLA2 activity was 1.10 (95% CI 1.05–1.16).10

However, the biggest advantage of the activity assay for Lp-PLA2 may reside in its superiority in analytical performance. Lp-PLA2 activity is significantly more analytically robust than the Lp-PLA2 concentration method because preanalytical processing does not affect activity assay results.28 While gender differences are reported in normal healthy populations, it is unclear whether a gender-specific cutoff will improve risk prediction. Differences in median activity values were reported among different studies; however, the direction of the gender difference is similar and the range of values can be reasonably attributed to unique patient populations.26-28

Lp-PLA2 Activity as a Modifiable Risk Marker
Statin therapy reduces both Lp-PLA2 concentration and activity by 20% to 40%29 even with moderate doses.30 However, Lp-PLA2 is carried in the circulation bound to LDL, and there is some controversy as to the predictive value of Lp-PLA2 activity after initiation of statin therapy.31

A study based on the Justification for the Use of Statins in Primary Prevention: An Intervention Trial Evaluating Rosuvastatin (JUPITER) trial found that baseline Lp-PLA2 activity was significantly associated with increased risk of cardiovascular events among the placebo group but not among patients receiving rosuvastatin, suggesting the risk was attenuated by the intervention.32 However, in patients treated for 1 year Lp-PLA2 activity was not predictive of events among subjects allocated to statin treatment. Finally, Lp-PLA2 activity at baseline did not modify the benefits obtained by statin therapy, indicating that risk prediction is best done in the primary care setting among patients prior to starting lipid-lowering pharmacotherapy.

Clinical utility of Lp-PLA2 activity was also demonstrated among a secondary prevention cohort in the Long-term Intervention with Pravastatin in Ischemic Disease (LIPID) study, a double-blind, placebo-controlled study of pravastatin in patients with stable coronary heart disease.33,34 In this study, Lp-PLA2 activity was not predictive of coronary events in a model containing many baseline risk factors. However, the LIPID study investigators performed an additional analysis and stratified patients according to the change in Lp-PLA2 activity from baseline to 1 year on treatment. They found a relative risk of 1.54 (95% CI 1.16–1.97) for patients with the smallest change (-3 to 3 nmol/min/mL) compared to those with the largest change (≤47 nmol/min/mL). Therefore, prospective clinical trials are warranted to determine whether treatment decisions based on Lp-PLA2 activity result in the reduction of future coronary events.

Lastly, targeting Lp-PLA2 enzyme activity reduction as a treatment strategy to directly reduce events has been evaluated. The efficacy and safety of direct modification of Lp-PLA2 has been studied in 2 large randomized clinical trials using darapladib (GlaxoSmithKline). This drug is an oral, selective inhibitor of Lp-PLA2 enzymatic activity.35,36 Lp-PLA2 concentration and activity were significantly reduced among patients on the darapladib arm compared with placebo. But both studies found that darapladib did not significantly reduce the risk of cardiovascular death, myocardial infarction, or stroke.

Conclusions

Lp-PLA2 is an enzyme involved with atherosclerotic plaque formation. Elevations in plasma Lp-PLA2 concentration and activity are associated with increased risk of cardiovascular disease. Clinically, Lp-PLA2 activity is the preferred biomarker due to improved analytical and preanalytical stability compared with Lp-PLA2 concentration. Based on data currently available, it would be reasonable to use Lp-PLA2 activity as an adjunct risk marker in patients with ambiguous risk profiles by traditional markers, or in patients at target LDL or non-HDL cholesterol with a significant concern for residual risk.

Authored by Jeffrey Meeusen, PhD and Leslie Donato, PhD

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