PAR-4/Ca2+-calpain pathway activation stimulates platelet-derived microparticles in hyperglycemic type 2 diabetes

Alessandra Giannella,Elisabetta Iori,Claudia Maria Radu,Saula Vigili De Kreutzenberg,Giulio Ceolotto,Fabrizio Fabris,Andrea Benetti,Arianna Cattelan,Angelo Avogaro,Paolo Simioni

doi:10.1186/s12933-021-01267-w

Abstract

BackgroundPatients with type 2 diabetes (T2DM) have a prothrombotic state that needs to be fully clarified; microparticles (MPs) have emerged as mediators and markers of this condition. Thus, we investigate, in vivo, in T2DM either with good (HbA1c ≤ 7.0%; GGC) or poor (HbA1c > 7.0%; PGC) glycemic control, the circulating levels of MPs, and in vitro, the molecular pathways involved in the release of MPs from platelets (PMP) and tested their pro-inflammatory effects on THP-1 transformed macrophages.MethodsIn 59 T2DM, and 23 control subjects with normal glucose tolerance (NGT), circulating levels of CD62E+, CD62P+, CD142+, CD45+ MPs were determined by flow cytometry, while plasma levels of ICAM-1, VCAM-1, IL-6 by ELISA. In vitro, PMP release and activation of isolated platelets from GGC and PGC were investigated, along with their effect on IL-6 secretion in THP-1 transformed macrophages.ResultsWe found that MPs CD62P+ (PMP) and CD142+ (tissue factor-bearing MP) were significantly higher in PGC T2DM than GGC T2DM and NGT. Among MPs, PMP were also correlated with HbA1c and IL-6. In vitro, we showed that acute thrombin exposure stimulated a significantly higher PMP release in PGC T2DM than GGC T2DM through a more robust activation of PAR-4 receptor than PAR-1 receptor. Treatment with PAR-4 agonist induced an increased release of PMP in PGC with a Ca2+-calpain dependent mechanism since this effect was blunted by calpain inhibitor. Finally, the uptake of PMP derived from PAR-4 treated PGC platelets into THP-1 transformed macrophages promoted a marked increase of IL-6 release compared to PMP derived from GGC through the activation of the NF-kB pathway.ConclusionsThese results identify PAR-4 as a mediator of platelet activation, microparticle release, and inflammation, in poorly controlled T2DM.

Highlights

Patients with type 2 diabetes (T2DM) have a prothrombotic state that needs to be fully clarified; microparticles (MPs) have emerged as mediators and markers of this condition
According to the values of glycated hemoglobin (HbA1c), Patient with Type 2 Diabetes Mellitus (T2DM) were divided into two groups, i.e. with good and poor glycemic control
Control subjects showed significantly lower BMI, glucose level, systolic blood pressure, IL-6, Intercellular Adhesion Molecule 1 (ICAM-1), and Vascular Cell Adhesion Molecules-1 (VCAM-1) compared to T2DM (Table 1)

Summary

Introduction

Patients with type 2 diabetes (T2DM) have a prothrombotic state that needs to be fully clarified; microparticles (MPs) have emerged as mediators and markers of this condition. We investigate, in vivo, in T2DM either with good (HbA1c ≤ 7.0%; GGC) or poor (HbA1c > 7.0%; PGC) glycemic control, the circulating levels of MPs, and in vitro, the molecular pathways involved in the release of MPs from platelets (PMP) and tested their pro-inflammatory effects on THP-1 transformed macrophages. Hyperglycemia promotes systemic inflammation and platelet dysfunction in patients with type 2 diabetes (T2DM) [1]. The MPs are a heterogeneous population of membrane vesicles of 100–1000 nm diameter, generated by various stimuli including hyperglycemia, apoptosis, proinflammatory cytokines, oxidative stress, infectious agents from several cell types, of which MPs maintain the surface and cytoplasmic markers [10]. The mechanisms underlying the propensity of hyperglycemia to induce MP release by platelets are mainly unknown

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