Effect Of Platelet MicroRNA - 15b On Platelet Reactivity In Patients With Acute Coronary Syndrome After Intervention

Platelet microRNAs relating to interindividual platelet reactivity in patients with acute coronary syndromes undergoing percutaneous coronary interventionBackground:Platelet P2Y12 receptor antagonists play a central role in antiplatelet therapy among patients with acute coronary syndromes (ACS) and those undergoing percutaneous coronary intervention (PCI). However, patients exhibit wide interindividual variability in the magnitude of on-clopidogrel platelet reactivity, with high/low platelet reactivity (HPR/LPR), conferring increased risk of ischemic/bleeding events. Genetic factors have been a major focus in a large amount of investigations. It has been confirmed that cytochrome P450 (CYP) 2C19*2 loss of function carrier is associated with HPR and increased occurrence of thrombotic complications. However, CYP2C19 single nucleotide polymorphisms (SNPs) alone explain only 5-12% of the interindividual variability of platelet reactivity; other genetic factors remain to be clarified. Previous studies have validated the existence of microRNAs (miRNAs) in platelets and suggest that they may play an important role in regulating platelet reactivity.Objective:To investigate the expression pattern of platelet miRNAs in patients with ACS undergoing PCI and to identify the platelet miRNAs relating to interindividual platelet reactivity.Methods:Consecutive 290 ACS patients with were enrolled in the cohort if they had undergone PCI and were on dual antiplatelet therapy of aspirin and clopidogrel. Platelet reactivity was evaluated with VerifyNow P2Y12 assay on the morning of day 1 (12-24 hours) and day 2 (36-48 hours) after PCI. Patients who presented consistent LPR (<170 PRU) or HPR (>300 PRU) on both day 1 and day 2 were recruited into HPR Group or LPR Group. Leukocyte-depleted platelets (LDPs) were prepared and miRNA expression profiles of LDP samples from four LPR and four HPR patients were performed using Agilent human miRNA microarray system. The candidate miRNAs analyzed from microRNA microarray were further validated in a larger LDP sample of 17 LPR and 22 HPR patients by quantitative reverse-transcription polymerase chain reaction (RT-qPCR).Results:We enrolled a cohort of 290 patients undergoing PCI,21 patients were defined as LPR and 26 as HPR according to the platelet function testing. A total of 420 miRNAs were positively detected in platelets of all eight patients. Among these miRNAs, we found miR-145 and miR-15b were differentially expressed between patients with LPR and HPR (P<0.05). Platelet miR-15b expression was confirmed to be 1.4-fold increased in patients with LPR compared with HPR by RT-qPCR (P=0.020). After adjustment for risk factors, we found that platelet miR-15b expression was independently associated with LPR (odds ratio,118.10 [95% confidence interval,3.40 to 4102.96], P=0.008).Conclusions:Platelet miR-15b is negatively correlated with platelet reactivity in ACS patients undergoing PCI. miR-15b may be involved in regulating platelet reactivity.MicroRNA-15b promotes megakaryocyte apoptosis via targeting Bcl-2Background:Platelet microRNA-15b (miR-15b) is expressed at higher levels in low platelet reactivity (LPR) than high platelet reactivity (HPR) among patients with acute coronary syndromes (ACS) undergoing percutaneous coronary intervention (PCI). Bcl-2 is proved to be a target gene for miR-15b and miR-15b enhances apoptosis by suppressing Bcl-2 protein expression in tumor cells. However, the function of miR-15b in platelets is not clarified. The megakaryocytic cell line MEG-01 is often used for platelet miRNA experiments.Objective:To explore the rate of cell apoptosis, Bcl-2 and procaspase-3 protein expression in MEG-01 cells after transfected with miR-15b mimics or inhibitors.Methods:MEG-01 cells were transfected with miR-15b mimics or inhibitors by RNAiMAX reagents. Cells were harvested after 36 hours for western blot analysis or flow cytometric measurement of apoptotic events.Results:miR-15b mimic reduced 24% Bcl-2 expression and miR-15b inhibitor elevated 1.8-fold in Bcl-2 expression in MEG-01 cells (P=0.018; P=0.019). Besides, we observed that miR-15b mimic induced a 42% knockdown of procaspase-3 expression and miR-15b inhibitor elevated 1.3-fold in procaspase-3 expression (P=0.006; P=0.021). miR-15b mimic was also found to induce 2.3-fold increase in Annexin V and PI stained apoptotic events (P=0.002), and 2.2-fold increase in Δψm-depolarized cells (P=0.001).Conclusions:miR-15b promotes megakaryocyte apoptosis via targeting Bcl-2, suggesting a pro-apoptotic role of miR-15b in platelets.The association between platelet apoptosis and platelet reactivity in patients with acute coronary syndromes undergoing percutaneous coronary interventionBackground:Platelet microRNA-15b (miR-15b) is expressed at higher levels in low platelet reactivity (LPR) than high platelet reactivity (HPR) among patients with acute coronary syndromes (ACS) undergoing percutaneous coronary intervention (PCI). miR-15b promotes megakaryocyte apoptosis via targeting Bcl-2, suggesting a pro-apoptotic role of miR-15b in platelets. However, the association between platelet apoptosis and platelet reactivity is not clarified.Objective:To investigate the association between platelet apoptosis and platelet reactivity in patients with acute coronary syndromes undergoing percutaneous coronary interventionMethods:Consecutive 51 ACS patients with were enrolled in the cohort if they had undergone PCI and were on dual antiplatelet therapy of aspirin and clopidogrel. Platelet reactivity was evaluated with VerifyNow P2Y12 assay and platelet apoptosis was assessed by flow cytometry after PCI. To explore the influence of enhanced platelet apoptosis on platelet reactivity, we used ABT-737, a Bcl-2 inhibitor, to induce platelet apoptosis in ACS patients undergoing PCI for 2 hours at 37℃ in vitro. Light transmittance aggregometer was used to measure platelet aggregation of apoptosis-induced platelets. Leukocyte-depleted platelets (LDPs) were prepared for western blot analysis.Results:Among 51 ACS patients undergoing PCI, platelet reactivity was significantly correlated with decreased rate of platelet apoptosis (r=-0.643, P<0.001). In the linear regression analysis, the proportion of platelet apoptosis was independently associated with platelet reactivity (standardized coefficients p=-0.643, P<0.001). Using ROC curve analysis, we demonstrated that the rate of platelet apoptosis was able to distinguish between patients with PRU ≥240 and PRU <240. The area under the curve was 0.715 (95% confidence interval,0.564 to 0.866, P=0.015) and the optimal cut-off value was evaluated to be 6.64% (sensitivity 70.8%, specificity 65.0%) for platelet apoptosis to predict PRU≥240. After incubating with platelet-rich plasma from ACS patients undergoing PCI, ABT-737 promoted platelet apoptosis at the concentration of 2.5μM, 5μM and 10μM, reaching 11.4%,19.9% and 46.8% apoptotic events in platelets, respectively (P<0.001). ABT-737 treatment decreased platelet aggregation in a dose-dependent manner, resulting in 42.4%,38.5% and 29.6% of maximal platelet aggregation (P<0.001). Patients with high platelet reactivity (HPR) showed 2.3-fold and 1.6-fold higher levels of platelet Bcl-2 and procaspase-3 proteins than low platelet reactivity (LPR) (P=0.003, P=0.003).Conclusions:Platelet reactivity is inversely correlated with the rate of platelet apoptosis among ACS patients undergoing PCI and induced platelet apoptosis lowers platelet reactivity in vitro. Platelet miR-15b negatively regulates platelet reactivity in ACS patients undergoing PCI by promoting platelet apoptosis. Platelet apoptosis may represent a novel antiplatelet target to overcome high platelet reactivity for PCI treatment.