New molecular insights into modulation of platelet reactivity in aspirin-treated patients using a network-based approach

Abstract

Platelet reactivity (PR) is variable between individuals and modulates clinical outcome in cardiovascular (CV) patients treated with antiplatelet drugs. Although several data point to a genetic control of platelet reactivity, the genes contributing to the modulation of this phenotype are not clearly identified. Integration of data derived from high-throughput technologies may yield novel insights into the molecular mechanisms that govern platelet reactivity. The aim of this study is to identify candidate genes modulating platelet reactivity in aspirin-treated CV patients using an integrative network-based approach. Patients with extreme high (n = 6) or low PR (n = 6) were selected and 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. Two modules within the network containing 123 and 182 genes were identified. We then specifically assessed the level of miRNAs in these two groups of patients. Among the 12 miRNAs differentially expressed, 2 (miR-135a-5p and miR-204-5p) correlated with PR. The predicted targets of these miRNAs were mapped onto the network, allowing the identification of seven overlapping genes (THBS1, CDC42, CORO1C, SPTBN1, TPM3, GTPBP2, and MAPRE2), suggesting a synergistic effect of these two miRNAs on these predicted targets. Integration of several omics data sets allowed the identification of 2 candidate miRNAs and 7 candidate genes regulating platelet reactivity in aspirin-treated CV patients.

Development of a label‒free nanolc/ms/ms assay for monitoring the changes in the proteomic landscape of thrombin‒activated human platelets

“…PRP were subjected to the whole platelets purification as described previously by other research groups. [30][31][32][33][34][35] Then, platelets were pelleted at 750xg for 10min and twice washed with the Tyrode's Buffer containing 134mM NaCl, 12mM NaHCO 3 , 2.9mM KCl, 0.34mM Na 2 HPO 4 , 1mM MgCl 2 , and 10mM Hepes (pH 7.4). Platelet count Development of a label-free nanolc/ms/ms assay for monitoring the changes in the proteomic landscape of thrombin-activated human platelets 234 Copyright: ©2018 Gonzalez et al Citation: Gonzalez J, Babinska A, Ewul ELV, et al Development of a label-free nanolc/ms/ms assay for monitoring the changes in the proteomic landscape of thrombin-activated human platelets.…”

“…Venous blood was collected from three healthy volunteers, who did not use any medication in the past 10days, as described in the previous published protocols. [30][31][32][33][34][35] The collection of blood from the volunteers was performed conform with the declaration of Helsinki and this part of the study was approved by the ethics review board of the Department of Medicine at SUNY, Downstate Medical Center, Brooklyn, New York, USA. Blood for MS-based proteomics analysis was collected with an open system, anticoagulated with 3.2% trisodium citrate.…”

“…18,51 In addition, these pharmacoproteomic approaches were successfully employed to study the effects of antiplatelet therapy for finding the best management of cardiovascular diseases (CVDs) and other inflammatory diseases. 20,25,32,[40][41][42][48][49][50][51][52][53][54][55][56][57][58] It is very well known that platelets play a pivotal role in the pathophysiological mechanisms underlying many CVDs, especially acute coronary syndromes (ACSs), since platelets are the key players in thrombus formation after atheroma plaque rupture. 29,31,35 .…”

“…Consequently, the recent advances in platelet proteomics applied to the study of CVDs focus primarily on sample preparation, subcellular fractionation, and bioinformatics requirements for proteome analysis of platelets derived from clinical samples. [30][31][32][33][34][35][36][37][38][39][40][41][52][53][54][55][56][57][58] To better understand the platelets' response to thrombin activation and its subsequent engagement of PAR receptors under clinical circumstances, especially when potent thrombin inhibitors can be used to treat the ACS syndrome, we developed and optimized a bottom-up, qualitative and quantitative label-free proteomic research study of human platelets isolated from healthy donors and ex-vivo activated with thrombin, using as reference the proteome of resting platelets. The label-free global proteomics analysis retrieved a total of 924 proteins, representing about 17% from the total proteins reported to date for platelets proteomes.…”

“…The label-free global proteomics analysis retrieved a total of 924 proteins, representing about 17% from the total proteins reported to date for platelets proteomes. [30][31][32][33][34][35][36][37][38][39][40][41]50 . About 50% of the total proteome of thrombin-activated platelets was represented by up-regulated previously validated protein biomarkers involved in the pathways mediating the protease and thrombin activated receptor (PARs) and integrin signaling, the actin and rhoA linked to cytoskeleton signaling, and activated kinases pathways regulating the cellular motility, aggregation, procoagulation, and degranulation.…”