Revealing the Impaired MEchanisms underneath aspirin low-responder patients with DIabetes mellitus (RIMEDI)

1. State of the art Type 2 diabetes mellitus (T2DM) is a major cardiovascular risk factor. PLT activation plays a key role in atherothrombosis and low-dose aspirin, which induces a permanent inactivation of PLT cyclooxygenase (COX)-1 inhibiting thromboxane (TX) A2 biosynthesis, is less effective in T2DM patients (1-3). The variable turnover rate of the drug target (PLT COX-1) represents the most convincing determinant of the interindividual variability in ASA response (4). Due to the 20-minute half-life of ASA, in the presence of enhanced PLT turnover, newly generated PLT may not be exposed to ASA and their unacetylated COX-1 may yield accelerated recovery of TX in the 12- to 24-hour dosing interval (5). The metabolic abnormalities associated with T2DM, through intermediate mediators such as oxidative stress (OS), may affect PLT mRNA and miRNA profiles. Specific profiles may identify PLT with enhanced reactivity or function, with the potential to escape inhibition by antiplatelet agents. Thus, PLT may affect surrounding cells by transferring miRNA that regulates recipient cell mRNA profile (6). T2DM is associated with OS, with over-production of ROS and reduced antioxidant levels (7). Hyperglycemia and OS increase plasma levels of asymmetric dimethyl arginine (ADMA), an endogenous nitric oxide synthase (NOS) inhibitor, enhancing PLT activation and aggregation (8). Activation of the receptor for advanced glycation end-product (RAGE) as reflected in vivo by decreased circulating soluble form (esRAGE), also leads to ROS generation, and contributes to vascular complications in T2DM (9, 10). In addition, hyperglycemia triggers the release of S100A8/A9, which binds to the RAGE on Kupffer cells, ultimately leading to increased thrombopoietin (TPO) and PLT production, thus linking DM and OS to thrombocytosis (11). OS may induce epigenetic changes by acting on post-transcriptional regulation (OS-responsive miRNA), and vice versa, several miRNAs regulate the expression of OS-related proteins, including Heme Oxygenase-1 (HO-1) (12). HO-1 is the inducible, rate-limiting enzyme of heme degradation and exerts potent anti-inflammatory, antioxidative, and antiapoptotic effects in response to various stressors (13). Prospective studies revealed a strong association between HO-1 polymorphisms, which translate into lower enzyme expression, and cardiovascular risk (14). Additionally, recent studies have shown that heme (HO enzymatic substrate) can mediate human PLT activation and death through ferroptosis, a novel non-apoptotic cell death triggered by OS and lipid peroxidation (15). Both synthetic (Dimethyl fumarate, Apolipoprotein-AI Mimetic Peptide L4F) and natural compounds (curcumin, sulforaphane, resveratrol) can activate Nrf2, the major transcriptional regulator of several antioxidant genes, including HO-1, which controls the intracellular antioxidative response system (16, 17). Based on these premises, we hypothesize that diabetic patients with poor ASA response may display an abnormal PLT phenotype, mechanistically linked to OS extent and potentially ferroptosis, leading to accelerated PLT turnover and escape from ASA. Thus, the present project aims at investigating the role of OS on mechanisms potentially involved in suboptimal ASA response in T2DM, including PLT turnover and PLT heterogeneity in terms of mRNA and miRNA content. To unravel these mec