Platelet Activation in type 2 diabetic patients with stable coronary artery disease: insight into their trombotic 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.

à 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.

à 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. à 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.

à 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. à 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