Platelet microRNAs Research Articles

Platelet microRNAs as Potential Novel Biomarkers for Antiplatelet Therapy with P2Y12 Inhibitors and Their Association with Platelet Function.

Patients with acute coronary syndrome (ACS) undergoing percutaneous coronary intervention (PCI) require dual antiplatelet therapy (DAPT). However, the response to treatment can vary considerably. Certain platelet microRNAs (miRs) are suspected to predict DAPT response and influence platelet function. This study aimed to analyze selected miRs' expressions and compare them among patients treated with different P2Y12 inhibitors while assessing their association with platelet activity and turnover parameters. We recruited 79 ACS patients post-PCI treated with clopidogrel, ticagrelor, or prasugrel, along with 18 healthy volunteers. Expression levels of miR-126-3p, miR223-3p, miR-21-5p, miR-197-3p, and miR-24-3p, as well as immature platelet fraction (IPF) and ADP-induced platelet reactivity, were measured and compared between groups. Analyses revealed significantly lower expressions of miR-126-3p, miR-223-3p, miR-21-5p, and miR-197-3p in patients treated with ticagrelor, compared to clopidogrel (fold changes from -1.43 to -1.27, p-values from 0.028 to 0.048). Positive correlations were observed between platelet function and the expressions of miR-223-3p (r = 0.400, p = 0.019) and miR-21-5p (r = 0.423, p = 0.013) in patients treated with potent drugs. Additionally, miR-24-3p (r = 0.411, p = 0.012) and miR-197-3p (r = 0.333, p = 0.044) showed correlations with IPF. The identified platelet miRs hold potential as biomarkers for antiplatelet therapy. (ClinicalTrials.gov number, NCT06177587).

Platelet miRNA Expression in Patients with Sticky Platelet Syndrome

Abstract Sticky platelet syndrome (SPS) is a disorder with familial occurrence and autosomal dominant trait characterized by platelet hyperaggregability in response to a low concentration of adenosine diphosphate (ADP) and/or epinephrine (EPI). The etiology of SPS may be associated with platelet microRNAs (miRNAs), which are considered as potential biomarkers of platelet function and antiplatelet therapy. We were monitoring the expression of platelet miRNAs in patients with laboratory diagnosed SPS and healthy controls. We have found a statistically significant increased expression of both miR-423-5p and miR-338-3p as well as a statistically significant decreased expression of miR-425-5p between the group of patients with diagnosed SPS type II and the group of healthy controls, which seems to be an interesting issue for a further research.

Left ventricular assist devices promote changes in the expression levels of platelet microRNAs.

MicroRNAs (miRs) emerged as promising diagnostic and therapeutic biomarkers in cardiovascular diseases. The potential clinical utility of platelet miRs in the setting of left ventricular assist device (LVAD) support is unexplored. We prospectively measured the expression levels of 12 platelet miRs involved in platelet activation, coagulation, and cardiovascular diseases in LVAD patients by quantitative real-time polymerase chain reaction. Data were longitudinally measured before LVAD implant and after 1, 6, and 12 months of LVAD support, and compared with those measured in healthy volunteers (controls). In silico analysis was also performed to identify pathways targeted by differentially expressed miRs. Data from 15 consecutive patients and 5 controls were analyzed. Pre-implant expression levels of platelet miR-126, miR-374b, miR-223, and miR-320a were significantly different in patients vs. controls. The expression levels of platelet miR-25, miR-144, miR-320, and miR-451a changed significantly over the course of LVAD support; in silico analysis revealed that these miRs are implicated in both cardiac- and coagulation-associated pathways. Furthermore, the patients who suffered from bleeding (n = 5, 33%) had significantly higher pre-implant expression levels of platelet miR-151a and miR-454 with respect to the patients who did not. The same miRs were also differentially expressed in bleeders following LVAD implantation early before the clinical manifestation of the events. This study provides a proof-of-concept evidence of significant modulation of platelet miRs expression driven by LVADs. The possible existence of a platelet miRs signature predictive of the development of bleeding events warrants further validation studies.

Circulating and Platelet MicroRNAs in Cardiovascular Risk Assessment and Antiplatelet Therapy Monitoring.

Micro-ribonucleic acids (microRNAs) are small molecules that take part in the regulation of gene expression. Their function has been extensively investigated in cardiovascular diseases (CVD). Most recently, miRNA expression levels have been suggested as potential biomarkers of platelet reactivity or response to antiplatelet therapy and tools for risk stratification for recurrence of ischemic evens. Among these, miR-126 and miR-223 have been found to be of particular interest. Despite numerous studies aimed at understanding the prognostic value of miRNA levels, no final conclusions have been drawn thus far regarding their utility in clinical practice. The aim of this review is to critically appraise the evidence on the association between miRNA expression, cardiovascular risk and on-treatment platelet reactivity as well as provide insights on future developments in the field.

Platelet microRNAs inhibit primary tumor growth via broad modulation of tumor cell mRNA expression in ectopic pancreatic cancer in mice.

We investigated the contributions of platelet microRNAs (miRNAs) to the rate of growth and regulation of gene expression in primary ectopic tumors using mouse models. We previously identified an inhibitory role for platelets in solid tumor growth, mediated by tumor infiltration of platelet microvesicles (microparticles) which are enriched in platelet-derived miRNAs. To investigate the specific roles of platelet miRNAs in tumor growth models, we implanted pancreatic ductal adenocarcinoma cells as a bolus into mice with megakaryocyte-/platelet-specific depletion of mature miRNAs. We observed an ~50% increase in the rate of growth of ectopic primary tumors in these mice compared to controls including at early stages, associated with reduced apoptosis in the tumors, in particular in tumor cells associated with platelet microvesicles—which were depleted of platelet-enriched miRNAs—demonstrating a specific role for platelet miRNAs in modulation of primary tumor growth. Differential expression RNA sequencing of tumor cells isolated from advanced primary tumors revealed a broad cohort of mRNAs modulated in the tumor cells as a function of host platelet miRNAs. Altered genes comprised 548 up-regulated transcripts and 43 down-regulated transcripts, mostly mRNAs altogether spanning a variety of growth signaling pathways–notably pathways related to epithelial-mesenchymal transition—in tumor cells from platelet miRNA-deleted mice compared with those from control mice. Tumors in platelet miRNA-depleted mice showed more sarcomatoid growth and more advanced tumor grade, indicating roles for host platelet miRNAs in tumor plasticity. We further validated increased protein expression of selected genes associated with increased cognate mRNAs in the tumors due to platelet miRNA depletion in the host animals, providing proof of principle of widespread effects of platelet miRNAs on tumor cell functional gene expression in primary tumors in vivo. Together, these data demonstrate that platelet-derived miRNAs modulate solid tumor growth in vivo by broad-spectrum restructuring of the tumor cell transcriptome.

Screening Analysis of Platelet miRNA Profile Revealed miR-142-3p as a Potential Biomarker in Modeling the Risk of Acute Coronary Syndrome.

Transcriptome analysis constitutes one of the major methods of elucidation of the genetic basis underlying the pathogenesis of various diseases. The post-transcriptional regulation of gene expression is mainly provided by microRNAs. Their remarkable stability in biological fluids and their high sensitivity to disease alteration indicates their potential role as biomarkers. Given the high mortality and morbidity of cardiovascular diseases, novel predictive biomarkers are sorely needed. Our study focuses for the first time on assessing potential biomarkers of acute coronary syndrome (ACS) based on the microRNA profiles of platelets. The study showed the overexpression of eight platelet microRNAs in ACS (miR-142-3p; miR-107; miR-338-3p, miR-223-3p, miR-21-5p, miR-130b-3p, miR-301a-3p, miR-221-3p) associated with platelet reactivity and functionality. Our results show that the combined model based on miR-142-3p and aspartate transaminase reached 82% sensitivity and 88% specificity in the differentiation of the studied groups. Furthermore, the analyzed miRNAs were shown to cluster into two orthogonal groups, regulated by two different biological factors. Bioinformatic analysis demonstrated that one group of microRNAs may be associated with the physiological processes of platelets, whereas the other group may be linked to platelet–vascular environment interactions. This analysis paves the way towards a better understanding of the role of platelet microRNAs in ACS pathophysiology and better modeling of the risk of ACS.

Bioinformatics analysis of tumor-educated platelet microRNAs in patients with hepatocellular carcinoma.

Background: Hepatocellular carcinoma (HCC) is one of the most prevalent malignancies that seriously threaten global health. The primary reason for its grim prognosis is the lack of sensitive tools for early diagnosis. The purpose of the present study was to apply bioinformatics analysis to explore tumor-educated platelet (TEP) microRNA (miRNA) expression and its potential diagnostic utility in HCC.Methods: Twenty-five HCC patients and 25 healthy controls were included. RNA sequencing was utilized to screen miRNA alterations in platelets derived from HCC patients (n=5) and controls (n=5). Gene set enrichment analysis was performed to analyze the targeted mRNAs of differentially expressed miRNAs by using the Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) databases, aiming at main functions and pathways, respectively. We then verified the selected platelet miRNAs in another cohort by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) amplification.Results: A total of 250 differentially expressed miRNAs were identified, among which 111 were down-regulated and 139 were up-regulated. The functional enrichment analysis of differentially expressed miRNAs suggested that their target genes were involved primarily in pathways related to HCC. Expression levels of miR-495-3p and miR-1293 were further validated by qRT-PCR, which yielded results consistent with the sequencing analysis. The area under the receiver operating characteristic (ROC) curve of miR-495-3p and miR-1293 as diagnostic tests for HCC were 0.76 and 0.78, respectively.Conclusion: TEP miRNAs such as miR-495-3p and miR-1293 were differentially expressed in HCC patients, and may be involved in the pathophysiology of HCC.

Platelet miR-107 Participates in Clopidogrel Resistance after PCI Treatment by Regulating P2Y12

Introduction: High platelet reactivity (HPR) caused by clopidogrel tolerance is an adverse reaction of acute coronary syndrome (ACS) patients who receive clopidogrel antiplatelet therapy after percutaneous coronary intervention (PCI) surgery. Platelet microRNA (miRNA) is related to platelet reactivity. This study explored the mechanism of platelet miRNA in regulating platelet reactivity. Methods: We recruited 50 ACS/PCI patients and divided them into the HPR group (P2Y12 reaction units [PRU] ≥300) and the LPR group (PRU < 170) according to the PRU through the VerifyNow P2Y12 assay. P2Y12-related miRNAs were screened by TargetScan, miRWalk, and Gene Expression Omnibus. The expressions of P2Y12 and miRNAs in the HPR group and the LPR group were determined by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Pearson correlation analysis was used to determine the correlation between P2Y12 and miRNAs. The interactions between P2Y12 and miR-107 were predicted by TargetScan and verified by dual-luciferase reporter assay. The regulation of miR-107 mimic or inhibitor on P2Y12 expression was detected by qRT-PCR and Western blot. Results: There were 22 patients in the LPR group and 28 patients in the HPR group. PY212 was highly expressed in the HPR group compared with the LPR group. We screened the P2Y12-related miRNAs (miR-145-5p, miR-4701-3p, miR-107, and miR-15b-5p), but only miR-107 and miR-15b-5p expressions were downregulated in the HPR group and were negatively correlated with PY212 expression. P2Y12 was the target gene of miR-107. PY212 expression was inhibited by miR-107 overexpression but suppressed by miR-107 silencing. Conclusion: Platelet miR-107 participated in clopidogrel resistance in ACS/PCI patients by regulating P2Y12 expression.

Horizontal MicroRNA Transfer by Platelets - Evidence and Implications.

For decades, platelets have been known for their central role in hemostasis and their ability to release bioactive molecules, allowing inter-platelet communication and crosstalk with the immune system and vascular cells. However, with the detection of microRNAs in platelets and platelet-derived microvesicles (MVs), a new level of inter-cellular regulation was revealed. By shedding MVs from their plasma membrane, platelets are able to release functional microRNA complexes that are protected from plasma RNases. Upon contact with macrophages, endothelial cells and smooth muscle cells platelet microRNAs are rapidly internalized and fine-tune the functionality of the recipient cell by post-transcriptional reprogramming. Moreover, microRNA transfer by platelet MVs allows infiltration into tissues with limited cellular access such as solid tumors, thereby they not only modulate tumor progression but also provide a potential route for drug delivery. Understanding the precise mechanisms of horizontal transfer of platelet microRNAs under physiological and pathological conditions allows to design side-specific therapeutic (micro)RNA delivery systems. This review summarizes the current knowledge and the scientific evidence of horizontal microRNA transfer by platelets and platelet-derived MVs into vascular and non-vascular cells and its physiological consequences.

MicroRNA expression profiles analysis of apheresis platelets treated with vitamin B2 and ultraviolet-B during storage

Whether platelet (PLT) microRNA (miRNA) profiles are affected by pathogen reduction technology (PRT) using vitamin B2 and ultraviolet-B (VB2-PRT) remains unclear. Samples from VB2-PRT-treated (experimental group, E_) and untreated (control group, C_) apheresis PLTs were taken on days 1, 3 and 5 of storage, designated as E_1, E_3, E_5, C_1, C_3 and C_5, respectively. The miRNA expression profiles were assessed by DNA Nano Ball (DNB) sequencing technology, and verified by quantitive real-time fluorescence quantitative PCR (qRT-PCR). Compared with the expression profiles of PLT miRNAs, 3895 miRNAs were identified in the E_ groups while 4106 were in the C_ groups. There were 487 significant differentially expressed miRNAs in E_1 vs C_1 group, including 220 upregulated and 287 downregulated, such as miR-146a-5p and let-7b-5p. There were 908 significant differentially expressed miRNAs in E_3 vs C_3 group, including 297 upregulated and 611 downregulated, such as miR-142-5p and miR-7-5p. There were 229 significant differentially expressed miRNAs in E_5 vs C_5 group, including 80 upregulated and 149 downregulated, such as miR-3529-3p and miR-451a. These differentially expressed miRNAs had been suggested to have functional roles in energy homeostasis, cell communication, proliferation, migration and apoptosis. GO analysis showed a significant enrichmen in relevant biological process categories as receptor activity, signal transduction, cell transport, motility and chemotaxis. The significantly enriched KEGG pathway of predicted target genes was Glycosaminoglycan biosynthesis in E_ vs C_ groups. These new observation could provide insights on the understanding of change of miRNA profiles of PLT treated with VB2-PRT.

The Platelet microRNA Profile of Kawasaki Disease: Identification of Novel Diagnostic Biomarkers.

Challenging diagnosis and unknown etiology of Kawasaki disease (KD) increase the coronary artery lesions incidence. microRNAs (miRNAs) are the most promising biomarkers because of their stability in peripheral blood and noninvasive measurement procedure, whose potential utility have been proved in cancers. To explore the utility of differentially expressed (DE) miRNAs as early diagnostic markers, 44 patients (25 incomplete KD and 19 complete KD) and 31 febrile controls were recruited for small RNA sequencing. From all the 1922 expressed miRNA, 210 DE miRNAs were found between KD and febrile control groups. Though platelet miRNA profiles of complete KD incomplete KD were much similar through cluster analysis, the DE miRNAs were not identical. Eight DE miRNAs were validated by real-time quantitative PCR (qRT-PCR) in complete or incomplete KD groups using a normalizer, miR-126-3p, which was identified by geNorm and NormFinder tools. The expression level of miRNAs continuous changed over time was observed and the function analysis showed the potential role of miRNAs as therapeutic biomarkers. Additionally, the prediction model for KD showed a sensitivity of 78.8% and a specificity of 71.4%, respectively. This study used small RNA sequencing to identify miRNA biomarkers KD diagnosis based on a large sample size. Our findings shine a light on the understanding of molecular pathogenesis of KD and may improve the accuracy of KD diagnosis and prognosis in clinical.

Our First Experience with Magnetic Separation of Platelets for Analyses of Platelet MicroRNA in Patients with Sticky Platelet Syndrome

Abstract Introduction: Sticky platelet syndrome (SPS) is referred to as a platelet hyperaggregability triggered by low concentrations of platelet agonists adenosine diphosphate (ADP) and/or epinephrine (EPI). Platelet aggregation with other inducers (collagen, arachidonic acid, ristocetin, and thrombin) remains within a normal range. MicroRNAs (miRNAs) are small, non-coding RNA molecules that play an important role in post-transcriptional regulation of protein expression. More recently, several studies show that the platelets are an abundant source of miRNAs and that the miRNA expression profiles within platelets correlate with the platelet reactivity. Aim: The principle objective of this article is to describe the method which we developed for the preparation of the pure platelet samples and report the results of this method. These final pure platelet samples are intended to be the first step for the platelet miRNA testing. Methods: The blood samples from 50 subjects were examined in the study. Then, the platelet rich plasma (PRP) samples obtained by centrifugation of the patient blood samples were used for our experiments. Subsequently, the erythrocytes and leucocytes remaining in PRP sample were magnetically labelled by CD45 Microbeads and CD235a Microbeads. After incubation the PRP sample passed through the magnetic separation system and the magnetically labelled cells (erythrocytes and leucocytes) were retained within the column of separator. The number of cells in the final PRP samples was measured by the blood cell analyser. Results and conclusion: We successfully developed and optimized the effective and reproducible method for magnetic separation of platelets, resulting in the leukocyte-depleted and erythrocyte-depleted platelet samples, which can be used for further genetic analyses.

Platelet microRNA expression and association with platelet maturity and function in patients with essential thrombocythemia

Essential thrombocythemia (ET) is characterized by persistently elevated platelet counts and an increased risk of thromboembolic events. Dysregulated expression of small noncoding microRNAs (miRNAs) have been shown in ET and may influence platelet maturity and function in ET patients. In this study, we included 22 ET patients and 19 healthy controls to investigate the expression of 12 platelet miRNAs previously reported to be dysregulated in ET. Further, we investigated the correlation between the expression of selected miRNAs and platelet maturity and platelet function. Total RNA was isolated from platelets, and expression analyses were performed using TaqMan quantitative PCR (qPCR). Mean platelet volume (MPV) and immature platelet count and -fraction (IPC and IPF) were measured using the Sysmex XE-5000 automated haematology system. Platelet function was investigated by multiple electrode aggregometry (agonists: arachidonic acid (AA), thrombin-receptor-activating-peptide (TRAP) and adenosine diphosphate (ADP)), while platelet activation was determined by multi-colour flow cytometry (antibodies: bound-fibrinogen, CD63 and P-selectin (CD62p), agonists: AA, TRAP and ADP). We showed that miR-9 and miR-490 were significantly upregulated in ET patients compared with healthy controls (p-values < 0.01), while miR-10a, miR-28, miR-126, miR-155, miR-221, miR-222, miR-223 and miR-431 were significantly downregulated in ET patients (all p-values < 0.001). A significant positive correlation was observed between miR-431 and MPV, IPC and IPF (all p-values < 0.05). The expression of miR-126 was negatively correlated with platelet aggregation induced by AA and TRAP (p < 0.05). In addition, we found the expression of miR-9 and miR-490 to be negatively correlated with the percentage of fibrinogen-, CD63- and P-selectin- positive platelets using TRAP as agonist (p < 0.05). In conclusion, our data indicate that platelet microRNAs may play a role in ET and that specific microRNAs are correlated with platelet maturity and platelet function.

Course of platelet miRNAs after cessation of P2Y12 antagonists.

Circulating platelet micro-RNAs (miRNAs) may be used to monitor platelet function during dual antiplatelet therapy (DAPT). Aim of the study was to measure plasma levels of specific miRNAs (miRNA-223, -150, -21 and -126) after physician-driven cessation of chronic P2Y12 inhibition and to study differences in the expression levels of these miRNAs between the different oral P2Y12 inhibitors clopidogrel, prasugrel and ticagrelor, respectively. Patients with coronary artery disease (CAD) on DAPT maintenance dose (including aspirin 100mg OD, plus clopidogrel 75mg OD, or prasugrel 10mg OD, or ticagrelor 90mg BID) were prospectively enrolled before cessation of the P2Y12-inhibitor therapy. MiRNA-223, -150, -21 and -126 were determined at baseline (=last day of P2Y12-inhibitor intake) and 10, 30 and 180days thereafter. Cessation of P2Y12-inhibitor therapy did not significantly change miRNA levels. However, in ticagrelor-treated patients, miRNA levels were significantly increased at baseline (miRNA-223 and -21), day 10 (miRNA-223, -150, -21, -126) and day 30 (miRNA-223, -150, -21, -126) as compared to prasugrel, and at day 10 (miRNA-150 and -21) and day 30 (miRNA-150) as compared to clopidogrel (all P<0.05). At day 180, only miRNA-126 levels differed significantly with respect to the P2Y12 inhibitor used (P<0.05). After adjustment for confounders, choice of P2Y12-inhibitor was the strongest predictor of miRNA levels (P<0.001), while cessation of P2Y12-inhibitor therapy did not significantly impact miRNA levels. In patients with CAD, ticagrelor intake is associated with increased levels of platelet miRNAs as compared to clopidogrel and prasugrel. Platelet miRNAs are not useful to monitor platelet function after cessation of P2Y12 inhibitors.

Platelet microRNAs and vascular injury.

Vascular smooth muscle cell (VSMC) phenotype switching from a contractile state to a synthetic phenotype has been implicated in intimal remodeling during vascular injury. While multiple studies have focused on dedifferentiation of VSMCs, prevention of VSMC-mediated excessive repair remains poorly understood. In this issue of the JCI, Zeng et al. identified a mechanism by which platelet-derived microRNA-223 (miRNA-223) reverses VSMC dedifferentiation. The authors show that suppression of proliferation occurs after platelet internalization by VSMCs. Moreover, they demonstrate that miRNA-223 inhibits dedifferentiation and intimal hyperplasia in diabetic mice by decreasing PDGFRβ expression in VSMCs. Together, these results identify platelet-derived miRNA-223 as a potential therapeutic target in vascular injury.

The Platelet microRNA Profile of Kawasaki Disease: Identification of Novel Diagnostic and Therapeutic Biomarkers

The Platelet microRNA Profile of Kawasaki Disease: Identification of Novel Diagnostic and Therapeutic Biomarkers

Platelet microRNAs in hypertensive patients with and without cardiovascular disease.

Platelets contain abundant microRNAs (miRs) that regulate gene expression and protein synthesis and may reflect platelet activation. We assessed platelet levels of miR-223, miR-126, and miR-22 in 82 patients with essential hypertension and 28 healthy individuals, using real-time reverse transcription polymerase chain reaction, and evaluated their relation with the patients' clinical profile. Hypertensives had significantly lower platelet miR-22 and miR-223 levels (97.6 ± 170.3 in hypertensives versus 193.8 ± 228.9 in normotensives, p = 0.011, for miR-22; 91.3 ± 154.1 in hypertensives versus 189.9 ± 266.3 in normotensives, p = 0.022, for miR-223). Significant differences in platelet miR levels were also observed between hypertensives who had cardiovascular disease and those who did not (4.1 ± 3.6 versus 75.1 ± 85.2 for miR-126, 24.3 ± 62.9 versus 122.8 ± 187.9 for miR-22, and 10.1 ± 10.4 versus 119.3 ± 169.0 for miR-223, respectively; p < 0.001 for all). In addition, we found a significant negative correlation with systolic blood pressure (SBP) (r = -0.43, p < 0.001, for miR-22; r = -0.47, p < 0.001, for miR-223 in hypertensives; and r = -0.54, p < 0.001, for miR-126). Finally, receiver operating characteristic analysis showed that platelet miR levels were also strong prognostic markers for cardiovascular disease in these patients. In conclusion, platelet miR-22 and miR-223 levels are reduced according to the hypertension status and they are negatively correlated with SBP levels. Platelet miR levels are also related to the presence of overt cardiovascular disease in this population. Further studies are needed to elucidate the exact role of platelet miRs in platelet function and their utility as novel biomarkers of atherothrombotic risk in those patients.

Specific platelet microRNA signature for dementia with Lewy bodies as promising biomarker

Dementia with Lewy bodies (DLB) shows overlapping features with Alzheimer disease (AD) leading to its misdiagnosis and hindering its adequate treatment. It is well established that microRNAs play an important role in neurodegeneration and they can be found in brain and the central nervous system. Since 30 years ago, the presence of RNA (mRNA and microRNA) in platelets has attracted attention and different mRNA expression patterns have been reported to be altered in human diseases. Our main objective was to identify disease specific mRNA and miRNA biosignatures through the analysis of platelet rich plasma (PRP) obtained from DLB patients (n = 31), AD patients (n = 10) and age-matched control individuals (n = 31).After isolation of mRNA and miRNA from PRP, we analyzed SNCA and GBA mRNA expression levels in DLB patients and controls by reverse-transcription real time PCR; next generation sequencing was applied for miRNA discovery in the same samples.22 miRNAs were differentially expressed and selected to be validated by real time qPCR. From those, 9 were considered as potential biomarkers for DLB being evaluated in a group of AD patients and independent groups of control and DLB individuals. Interestingly, miRNAs levels seemed to correlate with clinical symptoms and to correlate inversely with SNCAmRNA expression in DLB patients.These results, although replication and follow-up with larger, carefully characterized cohorts are needed, represent an integrated miRNA and mRNA profile in platelet rich plasma that is likely to provide non-invasive biomarkers for the differential diagnosis of DLB versus AD.

Abstract 477: Platelet microRNAs Crosstalk with Vascular Endothelial Cells in Diabetes

Platelets are major source of extracellular miRNAs (miRs) constituting the plasma miR-pool. Here we generated miR-profile of leukocyte depleted platelets (LDP) isolated from type-2 diabetic (T2DM) or healthy subjects and investigated mechanism of miRNA mediated vascular crosstalk. LDPs were obtained by leukocyte filtration of platelet rich plasma isolated from blood withdrawn from T2DM (6 males, age: 54.8±7.6 yr; HbA1c: 8.9±0.6%; BMI: 30.9±2.8kg/m2) and healthy subjects (10 males, age: 49.2±6.8yr; HbA1c: 5.3±0.2%; BMI: 26.6±3.8kg/m2). MiR-profile was generated using total RNA isolated from T2DM or healthy LDPs. Students t-test applied to compare groups and p≤0.05 considered significant. Among all detected 735 miRs (cutoff&lt;29 Ct), 19.5% miRs were depleted, whereas 7.5% showed &gt;2 fold increase in T2DM-LDPs. Compared to healthy LDPs (n=10), depletion of 14 platelet-enriched miR candidates, including miR-21 and -22, were validated by RTPCR in individual T2DM-LDPs (n=11) irrespective of aspirin intake or presence of coronary heart disease.Ex-vivo thrombin stimulation of washed healthy platelets increased the release of miRNA into cellular-supernatants and confocal microscopy confirmed the cellular uptake of platelet released miRNA-carrying microparticles into human coronary artery endothelial cells (ECs) within 2-24h. In addition, EC overexpression of miR-21 or -22 decreased cell proliferation, whereas miR-22 also attenuated their tube forming capacity. MiR-21 or -22 modulations inversely regulated gene expression of their common predicted target, the cell-cycle regulator gene- CDK-6. MiR concentrations were also measured in particulate debris and plasma derived from coronary aspirates retrieved during stent implantation from 3 T2DM or 4 non-diabetic patients. In line, higher miR-21 or -22 ratios in plasma to particulate debris in T2DM vs non-diabetic aspirates indicated miR release from particulate debris into coronary plasma. This in turn provides relatively richer miR-21 or -22 microenvironment to the vicinal vascular ECs. T2DM platelets show distinct miR-profile compared to healthy platelets and depletion of miRNAs may represent their active secretion from diabetic platelets. Altered coronary microenvironment may lead to altered cellular responses to injury in diabetic vessels.

Platelet microRNA for predicting acute myocardial infarction.

Acute myocardial infarction (AMI) is one of the leading causes of morbidity and mortality worldwide, while early diagnosis still represents an upmost priority. While platelet activation is critical for AMI pathogenesis, the role of platelet microRNAs (pmiRNAs) as biomarkers for AMI is unclear. Furthermore, correlations between the levels of pmiRNAs and indices of platelet activity are also unknown. Expression of platelet miR-1, miR-21, miR-126, miR-150 and miR-223 were prospectively assessed in 20 ST-segment elevation myocardial infarction (STEMI) patients, and 40 healthy volunteers. Platelet reactive units (PRU) were assessed with cartridge analyzer, and vasodilator-stimulated phosphoprotein (VASP) was measured by flow cytometry. There were no significant changes in pmiR-1 expression. Expressions of pmiR-21 and pmiR-126 were decreased, while pmiR-150 and pmiR-223 were increased in STEMI patients when compared to controls (all p < 0.01). However, only pmiR-126 exhibited correlation with plasma cardiac troponin I (r = - 0.556, p = 0.011) in STEMI. There was no correlation between pmiRNAs with PRU or VASP during admission, or at 48h post-stenting. Among tested pmiRNAs, pmiR-126 may serve as a potential novel biomarker for STEMI, while pmiR-1, pmiR-21, pmiR-150, and pmiR-223 were not particularly useful. Moreover, since assessed pmiRNA expression did not correlate well with platelet activity indices their potential diagnostic utility is quite limited.