Platelet Activation in Type 2 Diabetic Patients with Stable Coronary Artery Disease: Insights into their Thrombotic Propensity using a Genome-Wide Approach Platelet activation plays a key role in atherothrombosis and coronary artery disease (CAD) and increased in vivo platelet activation has been reported in patients with CAD. Several studies have likewise described the platelet contribution in the onset and progression of vascular complications observed in type-2 diabetes mellitus (T2DM) affected patients. These patients show a higher incidence of cardiovascular (CV) events compared to non-diabetic subjects, associated with a platelet hyperreactive state and reduced platelet responsiveness to aspirin and efficacy of antiplatelet agents. We have recently shown that platelets from patients with stable CAD or with acute coronary syndrome are potentially preconditioned to a different degree of reactivity on the transcriptional level; e.g., they expressed different levels of Tissue Factor (TF), a key protein in the coagulation cascade and in the thrombotic complications of atherosclerosis. In line with this evidence, the general objective of this project is to provide insights into the enhanced thrombotic propensity of CAD patients with T2DM using a genome-wide approach. The relevant hypotheses to be tested, and the corresponding goals, are the following: 1. T2DM-related metabolic unbalance (e.g. insulin resistance, hyperglycemia, dyslipidemia) may influence platelet associated TF expression, thus enhancing the platelet prothrombotic potential in CAD. To investigate this point, we will evaluate platelet-, platelet-leukocyte complexes-, and microparticle- associated TF expression and activity in stable CAD patients with or without T2DM. This will elucidate whether platelet TF may contribute to the enhanced thrombogenicity in diabetes, by connecting coagulation cascade and platelet hyperresponsiveness in this setting. 2. T2DM-related metabolic unbalance, more generally, may affect platelet transcriptome or proteome composition and/or posttranscriptional regulation, thus influencing platelet phenotype and reactivity. To address this point, we will evaluate platelet expression profiles (mRNA, microRNA, and proteins) in patients with T2DM and/or stable CAD. This will allow identifying novel molecular pathways associated with and/or triggering persistent platelet activation in T2DM/CAD patients. Another goal is to identify gene/protein expression signatures that predict phenotype membership, i.e. platelet predictive markers for classifying patients into disease subgroups based on expression levels. 3. Disease-related phenotype changes might alter the pharmacodynamics of platelet response to antiplatelet drugs, blunting the clinical efficacy of preventive anti-thrombotic strategies in a setting at high CV risk. Newly released platelets contain increased levels of RNA and accelerated platelet turnover has been shown to affect the response to antiplatelet therapy. We will test whether cyclooxygenase (COX)-2 and unacetylated COX-1 expression in newly formed platelets might contribute to persistent aspirin-insensitive thromboxane (TX) A2 biosynthesis in T2DM/CAD patients. Determinants of persistent platelet activation despite aspirin treatment with T2DM and/or stable CAD will be explored and transcriptional changes potentially hampering antiplatelet drug response will be sought. This will give insights into the pathways involved in the escape from aspirin inhibition documented in these subjects. To these aims, we will compare a cohort of stable CAD with T2DM to non-diabetic stable CAD patients. To assess the contribution of diabetes per se, a cohort of patients with T2DM without stable CAD at increased CV risk will be used as a second comparator. A group of sex and age matched healthy subjects will be used as ‘baseline’ reference. The experimental plan is divided in four Work-packages (WP) and scheduled as follows: WP1. 1.1 Recruitment of ~120 subjects for each group; 1.2 Separation and storage of cells, sera, and urines. WP2. 2.1 Genomic analysis on a first cohort of patients (≥60) for each group (training set); 2.2 Genomic analysis on a second cohort of patients (≥50) for each group (validation set); 2.3 Proteomic analysis on a cohort of 20 stable CAD patients with T2DM and 20 without T2DM. 2.4 Primary analysis of genome-wide data. WP3. 3.1 Flow-cytometry analysis of platelet activation markers, including TF expression; 3.2 Determination of immature platelets; immunophenotyping of COX-1/2 by flow cytometry to be linked to the assessment of platelet COX-1 inhibition by aspirin; 3.3 Evaluation of platelet activation and responsiveness to aspirin by assessment of the in vivo biosynthesis of TXA2 (urinary TX and non-enzymatic arachidonic acid metabolites) and serum TXB2 generation ex vivo; 3.4 Assessment of plasma biomarkers of inflammation and activation of the coagulation cascade (such as CD40 ligand, esRAGE, CRP, IL-6, von Willebrand factor multimers, fibrinogen, TAT, F1+2). 3.5 Primary analysis of cytometric and biochemical data. WP4. 4.1 Repetition of unreliable measurements and confirmation of gene expression and proteomic data by independent quantitative methods. 4.2 Statistical and data integration analysis and correlations among variables. To these purposes, a multidisciplinary integrated approach will be used, assembling different expertise from the Research teams involved in the project. The research will be realized in collaboration with the Dept. of Medicine and Aging Science, University of Chieti, and the Dept. of Pharmacology, Catholic University of Rome.
|Titolo:||Coordinatore di progetto finanziato da Fondazione CARIPLO (2011-0473) "PLATELET ACTIVATION IN TYPE 2 DIABETIC PATIENTS WITH STABLE CORONARY ARTERY DISEASE: INSIGHTS INTO THEIR THROMBOTIC PROPENSITY USING A GENOME-WIDE APPROACH”|
|Enti collegati:||Dept. of Medicine and Aging Science, Università di Chieti|
Dept. of Pharmacology, Università Cattolica di Roma
|Citazione:||Coordinatore di progetto finanziato da Fondazione CARIPLO (2011-0473) "PLATELET ACTIVATION IN TYPE 2 DIABETIC PATIENTS WITH STABLE CORONARY ARTERY DISEASE: INSIGHTS INTO THEIR THROMBOTIC PROPENSITY USING A GENOME-WIDE APPROACH”(2012 Mar).|
|Appare nelle tipologie:||15 - Attività|