Immature platelets and cardiovascular events in patients with stable coronary artery disease

Abstract Many patients with coronary artery disease (CAD) have reduced the effect of aspirin, which may partly be explained by immature platelets. We aimed to investigate whether immature platelet markers can predict cardiovascular events in a large cohort of stable CAD patients. A total of 900 stable CAD patients were included and followed for a median of 3 years. We measured markers of immature platelets (platelet count, immature platelet count, immature platelet fraction, mean platelet volume, platelet distribution width, platelet mass, and thrombopoietin) using automated flow cytometry and studied their relation to cardiovascular events. Our primary endpoint was a composite of acute myocardial infarction (MI), ischemic stroke, and cardiovascular death. A composite of MI, ischemic stroke, stent thrombosis and all-cause mortality was analyzed as a secondary endpoint. We found no difference in immature platelet markers between CAD patients with or without cardiovascular events. Regression analysis using hazards rates showed that markers of immature platelets did not have any predictive value for endpoints (p-values >.05). Markers of immature platelets did not predict future cardiovascular events during a 3-year follow-up period in CAD patients. This suggests that immature platelets measured in a stable phase does not have a major role in predicting future cardiovascular events. Plain Language Summary What is the context? Many patients with coronary artery disease (CAD) have reduced antiplatelet effect of aspirin The reduced antiplatelet effect of aspirin is most likely multifactorial and may partly be explained by immature platelets What is new? In a cohort of 900 stable CAD patients, we measured markers of immature platelets and studied their relation to cardiovascular events during a 3-year follow-up Our study demonstrated that markers of immature platelets did not predict cardiovascular events in our cohort What is the impact? The findings from the present study suggest that immature platelets, measured in a stable phase, do not have a major role in predicting future cardiovascular events in CAD patients


Introduction
][4] Treatment with aspirin is still considered a cornerstone in the secondary prevention of cardiovascular events, 5 yet a considerable proportion of CAD patients have reduced the antiplatelet effect of aspirin implying an increased risk of cardiovascular events. 3The reduced antiplatelet effect is most likely multifactorial, including compliance, genetics, and clinical factors. 6,7Aspirin exerts its effect by irreversible acetylation of the cyclooxygenase enzyme (COX) in platelets, which inhibits the production of thromboxane A 2 and platelet function for the entire platelet lifespan. 8However, the irreversible COX inhibition by aspirin may be counteracted by the continuous production of platelets, known as platelet turnover, which causes the release of platelets into the bloodstream that are not affected by aspirin. 9he production of platelets is regulated by thrombopoietin (TPO) and other compounds. 102][13] In a recent systematic review by our group, a positive association was found between immature platelet markers and the risk of future cardiovascular events in patients with CAD. 14 As we previously have enrolled a large cohort of stable CAD patients, 15 we aimed to investigate whether markers of immature platelet predictor cardiovascular events in this cohort.

Study design and population
A total of 900 stable CAD patients with angiographically documented CAD was included prospectively from 2007 to 2011 from the Western Denmark Heart Registry as previously described. 15All patients were more than 18 years old and treated with a single antiplatelet therapy with 75 mg aspirin once daily.Exclusion criteria were as follows: a) ongoing treatment known to affect platelet function or coagulation other than aspirin, b) platelet count below 120 × 10 9 /L or c) ischemic vascular event, percutaneous coronary intervention or coronary artery bypass grafting within the previous 12 months.Approvals from The Central Denmark Region Committees on Health Research Ethics and the Danish Data Protection Agency were obtained.Patients gave informed written consent before inclusion.The Helsinki Declaration was followed.

Cardiovascular endpoints and follow-up
Patient follow-up has previously been described in detail. 15In brief, national registries were used to assess the incidence of cardiovascular events.The Danish Civil Registration, which assigns a unique 10-digit identification number to Danish citizens at birth and to residents upon immigration, allowed the linking of individual records from different registries. 16ur primary endpoint was the composite of acute non-fatal MI, non-fatal ischemic stroke and cardiovascular death.Our secondary endpoint was a composite of non-fatal MI, nonfatal ischemic stroke, non-fatal stent thrombosis (ST) and allcause mortality.Co-authors SDK and ELG blinded to the results of the platelets parameters independently reviewed endpoints and source documents regarding MI and ST to confirm diagnoses.

Laboratory measurements
Blood samples were obtained with a minimum of stasis in the morning one hour after intake of aspirin 75 mg.The first 3 mL of blood was discarded.Blood samples for measurement of markers of immature platelets (platelet count, immature platelet count Whole blood for TPO measurement was allowed to clot for 30 minutes at room temperature, after which serum was separated by centrifugation at 1000 g for 15 minutes and stored at −80°C until analysis.TPO concentrations were measured in duplicate, and the mean of the two results was used in the statistical analyses.The coefficient of variance was 9%.The TPO analyses were performed using an enzyme-linked immunosorbent assay (ELISA) protocol in accordance with the instructions of the manufacturer (Human Tpo Immunoassay, R&D Systems Europe Ltd., Abingdon, UK).
Plasma creatinine and estimated glomerular filtration rate were measured using Cobas 6000 (Roche Diagnostics, Indianapolis, US).Leukocytes and red blood cells were assessed using automated flow cytometry (Sysmex XE-2100 Norderstedt, Germany).
Serum thromboxane B 2 (TXB2) is considered the pharmacologically most specific analysis to evaluate the effect of aspirin on platelets. 17Serum TXB 2 was determined using ELISA (Cayman Chemical, Ann Arbour, MI, USA) according to the manufacturer's instructions.

Statistical analysis
Data distribution was evaluated by Q-Q plots and histograms.Continuous data are presented as means and standard deviation (SD) or median and interquartile range depending on normality.Categorical data are presented in percentages.To compare differences between unpaired groups, a two-sided t-test, or the Mann-Whitney test was used as appropriate.Multivariable Cox proportional hazards survival regression was used to investigate the effect of markers of immature platelets on the primary and secondary outcomes before and after adjustment for relevant prognostic factors in the study cohort (covariates: age, sex, prior MI, diabetes mellitus, smoking, body mass index, platelet count, and renal function).All analyses were 2-sided, and P-values of < .05were considered statistically significant.Analyses were performed using Stata version 11.0 (StataCorp, TX, USA).

Results and discussion
The study population consisted of 900 stable CAD patients, of which 22% were female, had a median age of 65 years, and 22% were current smokers.These characteristics are shown in Table I.Of the study population, 88% patients had previously experienced MI, 6% had a history of stroke, and 28% had type 2 diabetes mellitus.All patients were on aspirin 75 mg monotherapy and 90% received a statin.The prescribed aspirin treatment was followed by all patients, as demonstrated by low levels of serum TXB 2 , Table I.
Overall, we found no difference in markers of immature platelets between CAD patients with or without cardiovascular events, as shown in Table II.IPF was the only marker that showed a statistically significant difference between CAD patients with or without the primary or secondary event in the follow-up period, Table II.Accordingly, our survival regression analysis using proportional hazards ratio showed that markers of immature platelets overall did not have any predictive value for the primary or secondary endpoint (all p-values > .05),Table II.This was consistent for both crude values and after adjusting for risk factors, such as age, sex, prior MI, diabetes mellitus, smoking, body mass index, platelet count, and renal function.Only IPF had a predictive value for the secondary endpoint with a statistically significant negative crude hazard ratio (HR), but clinically irrelevant difference (crude HR: 0.8 (95%-confidence interval (CI): 0.7;0.98),p = .02),Table II.However, after adjustment, no predictive value of IPF on the secondary endpoint was observed.Platelet count showed a statistically significant positive HR on the secondary endpoint, but no clinically relevant difference was observed (crude HR: 1.0 (95%confidence interval CI: 1.00;1.01),p = .03,adjusted HR: 1.0 (95%-CI: 1.00;1.01),p = .01),Table II.In addition, we found no predictive value of markers of immature platelets for the single endpoints (data not shown).
The findings of the present study do therefore not concur with our recent systematic review demonstrating that increased markers of immature platelets,in particular, MPV, are positively associated with cardiovascular events in CAD patients even after control for confounders. 14This inconsistency could partly be explained by variations in platelet populations, timing of blood sampling, follow-up time and event rates.

Table 1 .
Baseline characteristics of the study population consisting of 900 patients with stable coronary artery disease.

Table II .
Difference in markers of immature platelets in stable coronary artery disease patients with or without the primary or secondary endpoint.