Phd in EXPERIMENTAL MEDICINE

Thesis title: Metodi diagnostici per l’identificazione dell’over-espressione della proteina MRP-4 come fattore di rischio cardiovascolare in pazienti in trattamento cronico con aspirina

Multidrug Resistance Protein 4 (MRP4), a transporter belonging to the ABCC family, plays a key role in regulating nucleotide homeostasis and modulating platelet responsiveness to antiplatelet agents, particularly aspirin. In this project, the expression of MRP4 was investigated in platelets from patients undergoing chronic aspirin therapy, distinguishing between individuals without cardiovascular events and patients with a history of acute coronary syndrome (ACS). Gene and protein expression analyses revealed a statistically significant increase in both MRP4 mRNA and protein levels in ACS patients compared with clinically stable individuals receiving chronic therapy. The study also explored the possibility of simplifying MRP4 quantification through immunoenzymatic approaches. During platelet solubilization attempts using RIPA buffer, the unexpected detection of MRP4 in platelet-poor plasma prompted further investigation of its plasma distribution. Flow cytometry and microvesicle isolation demonstrated that a fraction of circulating MRP4 is associated predominantly with microvesicles of platelet and erythrocyte origin, and to a lesser extent with exosomes. Plasma MRP4 concentrations were reliably measurable by ELISA both in total plasma and in isolated microvesicles and exosomes, with a significant reduction observed following selective immunodepletion. Functional classification of patients showed that aspirin Non-Responders exhibit higher platelet expression of MRP4 and markedly elevated levels of MRP4-positive microvesicles, both under basal conditions and after platelet activation. From a diagnostic standpoint, ELISA proved to be a reliable and standardizable method for quantifying total plasma MRP4, although it does not allow discrimination among molecular forms or cellular origins. Conversely, flow cytometry enables a more detailed characterization and effectively differentiates aspirin Responders from Non-Responders based on both platelet-associated and vesicle-associated MRP4, highlighting its potential clinical relevance for thrombotic risk stratification. In conclusion, this work underscores the importance of monitoring MRP4 expression as a predictive factor for aspirin resistance and opens new perspectives for the optimization of antiplatelet therapy. These findings contribute to cardiovascular prevention through innovative diagnostic approaches and support the development of precision-medicine strategies employing clinically accessible methodologies to identify aspirin resistance and enhance therapeutic effectiveness.