Megakaryocyte (MK)-derived miRNAs have been detected in platelets. Here, we analysed the expression of platelet and circulating miR-223, miR-26b, miR-126 and miR-140 that might be altered with their target mRNAs in type 2 diabetes mellitus (DM2). MiRNAs were isolated from leukocyte-depleted platelets and plasma samples obtained from 28 obese DM2, 19 non-DM obese and 23 healthy individuals. The effect of hyperglycaemia on miRNAs was also evaluated in MKs using MEG-01 and K562 cells under hyperglycaemic conditions after 8 hours up to four weeks. Quantitation of mature miRNA, pre-miRNAs and target mRNA levels (P2RY12 and SELP) were measured by RT-qPCR. To prove the association of miR-26b and miR-140 with SELP (P-selectin) mRNA level, overexpression or inhibition of these miRNAs in MEG-01 MKs was performed using mimics or anti-miRNAs, respectively. The contribution of calpain substrate Dicer to modulation of miRNAs was studied by calpain inhibition. Platelet activation was evaluated via surface P-selectin by flow cytometry. Mature and pre-forms of investigated miRNAs were significantly reduced in DM2, and platelet P2RY12 and SELP mRNA levels were elevated by two-fold at increased platelet activation compared to controls. Significantly blunted miRNA expressions were observed by hyperglycaemia in MEG-01 and K562-MK cells versus baseline values, while the manipulation of miR-26b and miR-140 expression affected SELP mRNA level. Calpeptin pretreatment restored miRNA levels in hyperglycaemic MKs. Overall, miR-223, miR-26b, miR-126 and miR-140 are expressed at a lower level in platelets and MKs in DM2 causing upregulation of P2RY12 and SELP mRNAs that may contribute to adverse platelet function.

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Circulating leukocyte-platelet heterophilic aggregates produce procoagulant, oxidative and mitogenic substances, and can cause microembolism in capillaries as well as acute arterial thrombosis. Our aim was to determine if there was any difference in the number of circulating heterophilic aggregates between diabetic patients and controls, if the formation of aggregates correlated with the actual HgbA1c level, duration of diabetes and postprandial rise in serum glucose level, with different vascular complications and whether decreasing postprandial serum glucose had any effect on heterophilic aggregate formation. The number of circulating heterophilic aggregates was measured in 90 diabetic patients (Type 1, 29; Type 2, 61) and in 23 control subjects by a flow-cytometric assay, and the result was given as percentage of the respective leukocyte subsets. There was no significant difference in lymphocyte-platelet and neutrophil-platelet aggregate number in patients and controls; however, there was a significant difference in the percentage of monocyte-platelet aggregates between the diabetic and control group (Type 1, 43.0 +/- 17.8; Type 2, 34.9 +/- 12.5; control, 24.6 +/- 8.2; P < 0.01 and P < 0.5, respectively). Patients with proliferative retinopathy and nephropathy showed the highest number of monocyte-platelet aggregates. No significant correlation was, however, found with HgbA1c. In Type 2 diabetes a non-significant, but remarkable, tendency between elevation of postprandial serum glucose levels and platelet-monocyte aggregate formation was observed and acarbose seemed to be effective in decreasing both. This study provides further support that heterophilic aggregates might have role in the pathogenesis of diabetic vascular complications.

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