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

Abstract

Background 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. 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. Methods In 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. Results We 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. Conclusions These results identify PAR-4 as a mediator of platelet activation, microparticle release, and inflammation, in poorly controlled T2DM.

Hypofibrinolysis in type 2 diabetes and its clinical implications: from mechanisms to pharmacological modulation

“…An altered platelet membrane ultrastructure with apoptotic morphology and membrane has been reported in diabetic patents [ 43 ], along with enhanced platelet-derived microparticle (PMP) formation [ 44 ]. It has been recently demonstrated that under chronic hyperglycemia, protease-activated receptor 4 promotes release of PMPs through a Ca 2+ -calpain dependent mechanism [ 45 ]. Fibrin clots formed in the presence of microparticles have been shown to be less susceptible to tPA-induced fibrinolysis in healthy subjects [ 46 ], and this can be most likely extrapolated to diabetic patients.…”

The Role of Platelets in Diabetic Kidney Disease

“…Gianella et al [ 167 ] demonstrated for the first time that PAR-4 as a mediator of platelet activation promoted the release of activated PMVs through a calcium calpain-dependent mechanism and the expression of PAR-4 is upregulated by chronic hyperglycemia in poorly controlled (HbA1c > 7.0%) T2DM patients. PAR-4 also had a pro-inflammatory effect on the release of IL-6 from macrophages treated with platelets from poorly controlled patients.…”

Integrative biology of extracellular vesicles in diabetes mellitus and diabetic complications

“…Exosomal calpain 2 can cleave the ectodomain of the insulin receptor and thus impair insulin action, providing a credible link between calpain 2, exosomes, and T2D etiology 248 . Moreover, activation of calpain 1 and 2 contributes to accelerated atherothrombosis development in T2D by regulating different substrates in platelets and ECs 177 - 181 . Since MVs loaded with elevated calpain 1 can be delivered to ECs and induce vascular inflammation 180 , 181 , MVs might contribute to the phenotypes mentioned above.…”

“…Moreover, activation of calpain 1 and 2 contributes to accelerated atherothrombosis development in T2D by regulating different substrates in platelets and ECs 177 - 181 . Since MVs loaded with elevated calpain 1 can be delivered to ECs and induce vascular inflammation 180 , 181 , MVs might contribute to the phenotypes mentioned above.…”