Thrombosis and Haemostasis

Platelet activation plays a key role in atherogenesis and atherothrombosis. Non-invasive measurement of thromboxane (TX) metabolite excretion, namely 11-dehydro-TXB₂ excretion, in the urines provides biochemical evidence of TX-dependent platelet activation, while avoiding artifactual platelet activation during and after blood sampling. This expertise allowed us to detect persistent TX-dependent platelet activation in a number of acute and chronic conditions associated with increased risk of atherothrombosis, including cardiovascular disease, cerebrovascular diseases, type 1 (T1DM) and type 2 (T2DM) diabetes mellitus, obesity, hypercholesterolemia, hyperhomocysteinemia, hypertension, chronic kidney disease, and chronic inflammatory diseases.

Persistently increased platelet activation in vivo can be monitored through non-invasive measurement of urinary 11-dehydro-thromboxane (TX)B₂ levels. This TX metabolite can be decreased by low-dose aspirin, and by several disease-modifying agents. An incomplete TX metabolite suppression predicts the future risk of cardiovascular events in aspirin-treated patients (from Simeone P, et al. Ageing Res Rev 2018; 48:51-78).

By measuring urinary 11-dH-TXB₂ excretion, our group, in collaboration with other groups,  has recently shown for the first time that TX-dependent platelet activation is high in subjects with a form of prediabetes, namely impaired glucose tolerance (IGT), as in patients with T2DM, and that it further increases over a 36-month median follow-up, especially in the subjects who progress to overt diabetes. These findings may provide a novel rationale for testing the efficacy and safety of antiplatelet therapy at an earlier stage of the disease than currently recommended. We also showed that in subjects with obesity and prediabetes, TX-dependent platelet activation can be mitigated by weight loss achieved with lifestyle programs or antihyperglycemic drugs with an impact on body weight, such as liraglutide.

Increased TX-dependent platelet activation in subjects with IGT and enhanced 11-dehydro-TXB₂ excretion in IGT patients who developed diabetes. Enhanced TXA₂‐dependent platelet activation, as reflected by 11‐dehydro‐thromboxane (TX)B₂ urinary excretion, is comparably abnormal in IGT as in T2DM, and it is persistent over long‐term follow‐up. Baseline TXM excretion was comparable between subjects with IGT and T2DM. During a 36‐month follow‐up, increasing urinary TXM excretion over time was observed in the subjects who progressed to diabetes vs non progressors (from Santilli F, et al. Diabetes Metab Res Rev 2020; 36:e3232).

Bioactive prostaglandin (PG) F₂-like compounds (isoprostanes) are produced from arachidonic acid through a nonenzymatic process of lipid peroxidation, catalyzed by oxygen free radicals on cell membranes and LDL particles. We observed a direct correlation between urinary 8-iso-PGF_2α, marker of in vivo lipid peroxidation, and 11-dehydro-TXB₂ excretion, supporting the hypothesis that persistent platelet activation may, at least in part, be related to enhanced formation of biologically active products of arachidonic acid peroxidation. In clinical settings characterized by accelerated atherothrombosis, we showed that the underlying metabolic abnormality (hyperglycemia, insulin resistance, hypercholesterolemia, hyperhomocysteinemia) is the primary trigger of low-grade inflammation, lipid peroxidation and TX-dependent platelet activation. In these contexts, platelets are potent triggers of inflammation, tissue damage, accelerated atherogenesis and further platelet activation, through the release of a number of proteins, miRNA, microvesicles. Our group studies the complex role of the “platelet releasate” in the pathogenesis of atherothrombosis.

Metabolic abnormalities in type 2 diabetes. The metabolic abnormalities associated with T2DM may affect platelet transcriptome and/or posttranscriptional regulation through intermediate mediators, such as oxidative stress with isoprostane formation, inflammatory molecule production, endothelial dysfunction with circulating endothelial cells and microparticle release, and cross talk between cells with miRNA exchange through circulating microparticles. The variable contribution and still poorly characterized interaction among these mechanisms result in platelet hyperreactivity (from Santilli F, Simeone P, Liani R, Davì G. Platelets and Diabetes. Chapter in: Platelets in Thrombotic and Non-Thrombotic Disorders. Gresele P; 2017, pp.1225-1238).

Other anti-hyperglycemic strategies are currently being tested to investigate the pathophysiological mechanisms underlying their benefits in the prevention of obesity- and diabetes-related complications.

Cardiometabolic effects of liraglutide in obese subjects with prediabetes or diabetes mellitus. By performing a randomized study of liraglutide vs. lifestyle changes to achieve comparable weight loss, we unraveled that liraglutide improves visceral fat loss, β-cell function, lipid peroxidation and platelet activation, as well as memory function, in obese patients with prediabetes or early diabetes mellitus (from Vadini F, et al. Int J Obes (Lond); 2020, Jan 21. Simeone P, et al. Nutrients 2018, Dec 2;10(12). Santilli F, et al. Diabetes Care 2017;40:1556-1564).

Serum TXB₂ is an index ex vivo of the maximal platelet activity that reflects the capacity to generate cyclooxygenase (COX)-1-dependent TXA₂. By studying the recovery rate of platelet COX-1 activity during the 12 to 24h dosing interval of aspirin administration, in aspirin-treated subjects with and without diabetes, we characterized substantial interindividual variability, and this abnormal biochemical phenotype could be completely reversed by aspirin 100 mg given twice daily. Thus, we showed that the variable turnover rate of the drug target represents the main determinant of the interindividual variability in aspirin response.
Along these lines, we performed a subsequent study to characterize, in diabetic vs non-diabetic patients on aspirin 100 mg od, the platelet phenotype, in terms of miRNA profile and proteome, associated with inadequate response to aspirin. We expect to identify platelet or circulating biomarkers of less-than-expected aspirin response, for disease-tailored therapeutic regimens. For this project, the PI has been awarded a Grant from the Italian Ministry of Health. (Figure)

Possible determinants of interindividual variability in response to aspirin. A suboptimal response to aspirin can be due to: (1) an impaired acetylation and inhibition of COX-1, mainly related to decreased absorption of the drug, and to oxidative stress-induced lipid hydroperoxide and peroxynitrite generation; (2) an increase in aspirin-insensitive agonists, related to low-grade inflammation, with increased TXA₂ formation by platelet, or to oxidative stress, with enhanced TX receptor activation by 8-iso-PGF_2α; and (3) an accelerated recovery of COX-1 activity linked to an enhanced platelet turnover (from Santilli F and Simeone P. Intern Emerg Med 2019;14:1217-1231).

Platelets carry messenger RNAs (mRNAs), a large number of micro(mi)RNAs and several proteins involved in miRNA processing (e.g., Dicer, TRBP2, Ago2), which are delivered from MKs. Several studies have reported altered levels of miRNA in platelets from patients with DM. The levels of some miRNA (miR-223, miR-26b, miR-126, mir103, and miR-140) are significantly lower in both platelets and MKs of patients with T2DM. This is likely due to overactivation of calpain, which impairs proteolytic degradation of Dicer resulting in decreased levels of several platelet enriched miRNAs and accumulation of pre-miRNAs, and to altered capacity of MKs to deliver sufficient amount of miRNAs and their precursors into platelets. This altered miRNA profile of platelets results in enhanced platelet function/activation, since miR-223 regulates the expression of platelet P2Y12 receptor, and miR- 26b and miR-140 target the SELP mRNA that encodes P-selectin. Activated platelets transfer miR-126 from the platelet to the plasma compartment, an effect inhibited by low-dose aspirin.

MiRNA formation and altered miRNA profile in diabetes. Platelet hyperreactivity in diabetes mellitus and relationship between altered miRNome in megakaryocytes and platelets (from Santilli F, Simeone P, Liani R. Platelets, Fourth Edition. Chapter 27: The Role of Platelets in Diabetes mellitus. Michelson AD. Elsevier/Academic Press; 2019, pp.469–504).