Project description:Coronary artery disease (CAD) is the leading cause of human morbidity and mortality worldwide, underscoring the need to improve diagnostic strategies. Platelets play a major role, not only in the process of acute thrombosis during plaque rupture, but also in the formation of atherosclerosis itself. MicroRNAs are endogenous small non-coding RNAs that control gene expression and are expressed in a tissue and disease-specific manner. Therefore they have been proposed to be useful biomarkers. The aim of this study was to investigate whether differences in miRNA expression levels in platelets can be found (i) between patients with premature CAD and healthy controls and (ii) within healthy controls after and before aspirin and statin administration. In this case-control study we measured expression levels of platelet miRNAs using microarrays from 40 male patients with premature CAD and 40 age- and sex-matched healthy controls. Premature CAD was defined as a cardiac event before the age of 51 years. The patients were selected from the outpatient clinic of the Academic Medical Center (AMC) of Amsterdam, which is specialised in premature CAD. The control cohort was composed of 40 healthy Caucasian male volunteers, who were recruited by advertisement and who were matched with the cases for age and smoking habits. Individuals of this control cohort did not have a history of CVD, nor did they have a positive family history of CVD and they were not allowed to use any medication. Patients and controls were excluded when they suffered from diabetes. Most CAD patients use aspirin and statins as secondary prevention. The influence of these drugs on miRNA profiles is unknown. To assess the possible influence of medication on miRNA expression and to control for medication as confounding factor, we asked 27 volunteers in our control cohort to also use these drugs. We administered simvastatin 40 mg, once daily, for 6 weeks and during the last two weeks we added 100mg of acetyl salicylic acid, once daily. Blood samples including isolated platelets were collected at baseline in the absence of aspirin and statins and after six weeks of medication use. We also assessed platelet function using the Multiplate® Analyzer (Roche) in the absence of aspirin and statin use. In short, 300 µl whole blood was diluted with 300 µl 0.9% saline and stirred for 3 minutes at 37 ºC. Adenosine diphosphate (ADP) was added in a final concentration of 2.5 ?mol/L to initiate platelet aggregation. Aggregation was measured for 6 minutes and was reported in arbitrary aggregation units plotted against time. Also, the area under the aggregation curve (AUC) was measured. All samples were measured in the absence and presence of 200 ?mol/L indomethacin (20 min incubation with blood) to mimic the effect of aspirin use. We calculated the percentage reduction in AUC after incubation with indomethacin as an in vitro measure of the effect of aspirin use on whole blood platelet aggregation.
Project description:The goal of this experiment was to explore how human platelets affect the transcriptional responses of primary human CD14+ blood monocytes to lipopolysaccharide (LPS), and NLRP3 activation with Nigericin. For this purpose, we analyzed the mRNA expression of 770 myeloid-specific transcripts using the nCounter® Nanostring Human Myeloid Innate Immunity Panel v2. We isolated classical monocytes (CD14+CD16−) from the peripheral blood of healthy volunteers. Monocytes were derived from PBMCs using negative selection with the EasySep™ Human Monocyte Isolation Kit from STEMCELLTM Technologies. We modified this process by either including or excluding a platelet-depleting cocktail, creating two groups: \\"Standard Monocytes (StdMo)\\" and \\"Platelet-depleted Monocytes (PdMo).\\" To examine the impact of platelets further, we supplemented PdMos with fresh autologous platelets at a ratio of 50 platelets per monocyte, resulting in a third group, \\"PdMo + Plts.\\" StdMos were prepared according to the standard protocol provided by the EasySep™ Kit. For the PdMos, a Platelet Removal Component (50 µl ml−1) from the kit was used during isolation. We also reconstituted platelet-depleted monocytes with platelets and analyzed platelets alone to determine their specific mRNA contributions. The groups—StdMo, PdMo, PdMo + Plts, and platelets alone—were then exposed to 2 ng/ml of Ultrapure LPS (from E. coli O111:B4) for 4.5 hours, or stimulated with LPS for 3 hours followed by inflammasome activation with Nigericin (10 µM) for 90 min before extracting RNA for analysis.
Project description:To explore the diverse platelet microRNA (miRNA) expression between high platelet reactivity (HPR) and low platelet reactivity (LPR) patients with acute coronary syndromes (ACS), we enrolled a cohort of ACS patients and performed miRNA expression profiling of platelets from four HPR and four LPR patients using human miRNA microarray system. VerifyNow P2Y12 assay was applied to indentify HPR and LPR. Venous blood was drawn from the patients and was centrifuged to prepare platelets. Among the candidate differentially expressed miRNAs, miR-15b expression was further confirmed to be lower in platelets of 22 HPR patients than 17 LPR by quantitative reverse-transcription polymerase chain reaction (RT-qPCR). We enrolled a consecutive cohort of 290 ACS patients and assessed the platelet reactivity using VerifyNow P2Y12 assay. In this study, HPR was defined as M-bM-^IM-%300 platelet reactivity unit (PRU) while LPR <170 PRU. miRNA microarray analysis was performed in platelets of four HPR and four LPR patients with ACS.
Project description:Platelet-rich plasma (PRP) has been obtained by centrifuging whole blood at 160 g for 15 min at room temperature. Platelets have been activated with 2 different physiologic agonists, namely collagen (60ug/mL) or Thrombin Receptor Activating Peptide (TRAP 25uM) under continuous stirring. An aliquot of platelets has been obtained at 120 minutes following collagen and TRAP and then processed to determine mRNA expression profiles. Time 0, indicating samples before any agonist treatment, was used as baseline. Total RNA was extracted using the mirVana PARIS kit (LifeTechnologies), according to manufacturers instruction. Indexed libraries were prepared using 500 ng of total RNA as starting material and sequenced on HiSeq 1500 (Illumina, USA) at a concentration of 8pM for 2x100 plus 7 additional cycles for indexes sequencing. Standard workflow for quality control through RTA and bcl2fastq conversion tool was applied.
Project description:The aim of this study is to identify candidate genes modulating platelet reactivity in aspirin-treated cardiovascular patients using an integrative network-based approach. Platelet reactivity was assessed in 110 cardiovascular patients treated with aspirin 100mg/d by aggregometry using several agonists. Patients with extreme high or low PR were selected for further analysis. Data derived from quantitative proteomic of platelets and platelet sub-cellular fractions, as well as from transcriptomic analysis were integrated with a network biology approach.
Project description:Platelets contain abundant miRNAs, however, the biogenesis pathway of miRNAs in anucleate platelets is unclear. Platelet-rich plasma was diluted in washing buffer and the platelet suspension was centrifuged to isolated pure platelets.Finally, platelets were recovered in suspension buffer at the concentration of 4–5×10^8 platelets/ml. Aliquots of the platelet suspensions were activated in the presence of 2.5 mM CaCl2 with 0ng/ml or 1 ng/ml thrombopoietin (TPO)
Project description:Different inflammatory stimuli contribute to the formation of atherosclerosis. It is hypothesized that although the end result is the same - plaque formation in arterial vessels - the pathogenesis is dependent on the etiology. In particular, platelets will respond differently depending on the inflammatory stimuli and timepoint. Using a microarray and platelet inflammatory function studies, we identified the transcriptional and functional changes that occur early and late with different inflammatory stimuli. ApoE-/- C57BL/6 mice were left untreated (control) or administered oral P. gingivalis (Pg) or intranasal C. pneumoniae (Cp) for 3 weeks, and sacrificed either 1 day (early timepoint) or 9 weeks (late timepoint) after this 3-week period. A separate group of animals was fed a Western Diet (WD) for 9 weeks and then sacrificed. Whole blood samples were collected from each animal into citrate solution and serially centrifuged to produce a pure platelet population. RNA was extracted and pooled from each experimental group and hybridized to Affymetrix Mouse Gene 1.0 ST microarrays.
Project description:Platelets have multiple roles in cancer cell metastasis. In this work we employed exon microarray technology to address platelet gene expression in metastatic non small cell lung cancer versus controls without cancer. We found that 197 of the 200 genes with the most significantly altered expression levels had their expression levels downregulated. Retrospective observational study. Total of 12 samples: 6 controls and 6 lung cancer samples.
Project description:Hibernating mammals undergo a dramatic drop in temperature and blood flow during torpor and must suppress hemostasis to avoid stasis blood clotting. In addition, cold storage of most mammalian platelets induces cold storage lesions, resulting in rapid clearance following transfusion. 13-lined ground squirrels (Ictidomys tridecemlineatus) provide a model to study hemostasis and cold storage of platelets during hibernation because, even with a body temperature of 4-8C, their platelets are resistant to cold storage lesions. We quantified and systematically compared proteomes of platelets collected from ground squirrels at summer (activity), fall (entrance), and winter (topor) to elucidate how molecular-level changes in platelets may support hemostatic adaptations in torpor. Platelets were isolated from squirrel blood collected in June, October, and January. Platelet lysates from each animal were digested with trypsin prior to 11-plex tandem mass tag (TMT) labeling, followed by LC-MS/MS analysis for relative protein quantification. We found over 700 platelet proteins with significant changes over the course of entrance, torpor, and activity – including systems of proteins regulating translation, platelet degranulation, metabolism, complement, and coagulation cascades. We also noted species specific differences in hemostatic, secretory, and inflammatory regulators in ground squirrel platelets relative to human platelets. In addition to providing the first ever proteomic characterization of platelets from hibernating animals, our results support a model whereby systematic changes in metabolic, hemostatic, and other proteins support physiological adaptations in torpor. In addition, our results could translate into better methods to cold store human platelets, increasing their supply and quality for transfusions.
Project description:RNA-sequencing analysis of human platelet polyA-mRNA from a normal male. The goal of this experiment was to identify genes expressed in unstimulated circulating platelets.