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.

To cite this article: Kappelmayer J, Beke Debreceni I, Vida A, Antal-Szalm as P, Clemetson KJ, Nagy B Jr. Distinct effects of Re-and S-forms of LPS on modulating platelet activation. J Thromb Haemost 2013; 11: 775-8.Sepsis is characterized by a robust cellular response to invading organisms. In the case of Gram-negative bacteria it is mostly governed by lipopolysaccharide (LPS), the main outer surface component, which is a strong stimulator of the immune system [1]. Patients with sepsis frequently show severe thrombocytopenia because of sequestration of activated platelets in the microvasculature [1,2]. In vitro studies [2-8] extensively examined platelet activation induced by a wide range of concentrations of LPS from Escherichia coli strains under rather variable conditions. Surprisingly, the platelet responses gave controversial data, and it was also debated whether LPS directly modulates platelet activation or only in a leukocyte-dependent manner (reviewed in [9]). Various toll-like receptors (TLR 1,2,4,6, 8 and 9) were detected on the platelet surface, but TLR4 was the main receptor for direct interactions of LPS with platelets [2,3,6,9].Certain clinically relevant Gram-negative bacteria produce a highly heterogeneous mixture of total LPS with varying numbers of repeating polysaccharides [10]. It contains diverse proportions of S-LPS, which comprises lipid A moiety, core-oligosaccharide, and O-polysaccharides, and the rough form of LPS (Re-LPS) lacking O-specific chains. Smooth LPS (S-LPS) from E. coli showed discrepancies in activating platelets [4][5][6][7][8], while Re-LPS from Salmonella minnesota (Re595) stimulated TLR4/MD2 expressing mast cells [10], and human monocytes and neutrophils with a potency similar to its parent S-LPS [11]. Thus, we studied for the first time whether Re-LPS (Re595) from S. minnesota alters the levels of various platelet activation markers in platelet-rich plasma (PRP) samples, and compared these data with experiments with the same type of S-LPS from E. coli (O111:B4) used earlier [4][5][6][7][8].First, to evaluate the biological activity of these LPS forms, we tested their procoagulant properties in a recalcified, LPS-stimulated mononuclear cell suspension. Re-LPS and S-LPS (10 lg mL À1 ) significantly (P < 0.05) decreased the mean clotting time (56.0 s and 60.8 s, respectively) compared with the non-activated control (111.7 s) (Fig. 1A). LPS interacted directly with platelets as FITC-labeled Re-LPS binding resulted in significantly increased FL-1 mean fluorescence intensity (MFI) of the positive cells (MFI, 49.7 AE 8.3; P = 0.01) compared with control platelets with unlabeled Re-LPS (10.2 AE 2.4). An even larger increase in MFI (112.4 AE 31) was observed with TRAP (10 lM), as similarly shown [3] where CD62P was also implicated in LPS binding (Fig. 1B). This 'valid' fluorescence was abolished by a 20-fold molar excess of unlabeled Re-LPS (Fig. S1A), and considerably decreased by anti-TLR4 and anti-P-selectin antibodies (Fig. S1B), in agreement with previous results [3]. Nex...

Since the introduction of tyrosine kinase inhibitors, the overall survival of patients with chronic myeloid leukemia has markedly improved. However long term use of these drugs results in various adverse events. Treatment with second generation dasatinib is often complicated by hemorrhagic events. Previous lumi-aggregometry studies have shown impaired platelet function in patients on dasatinib therapy. Dual agonist activated platelets (coated-platelets) are also sensitive indicators of platelet function. We hypothesized that dual activation with convulxin and thrombin of platelets in a flow cytometric assay could be a more sensitive method for detecting platelet dysfunction as compared to single agonist studies used in lumi-aggregometer. Platelets of healthy volunteers incubated with dasatinib as well as platelets from patients on dasatinib therapy were investigated. Low therapeutic plasma level dasatinib concentrations at which a considerable reduction in coated-platelet generation was observed in vitro, did not cause detectable change in platelet aggregation response. Coated-platelet assay and lumi-aggregometry were also investigated at 0, 1 and 4 hours after drug administration in dasatinib treated CML patients. Significant decrease was observed at 1 hour in maximal aggregation by collagen. Although the aggregation curves became normalized by 4 hours, coated-platelet generation was still inhibited in dasatinib treated patients. Nilotinib, another second generation tyrosine kinase inhibitor, had no effect on aggregation and on coated-platelet formation neither in vitro nor in ex vivo samples. At therapeutic plasma levels coated-platelet assay is more sensitive than lumi-aggregometry studies for the demonstration of the inhibitory effect of dasatinib on platelet function.

Background Platelets contain a high amount of potentially active A subunit dimer of coagulation factor XIII (cellular FXIII; cFXIII). It is of cytoplasmic localization, not secreted, but becomes translocated to the surface of platelets activated by convulxin and thrombin (CVX+Thr). Objective To explore the difference in cFXIII translocation between receptor mediated and non‐receptor mediated platelet activation and if translocation can also be detected on platelet‐derived microparticles. Our aim was also to shed some light on the mechanism of cFXIII translocation. Methods Gel‐filtered platelets were activated by CVX+Thr or Ca2+‐ionophore (calcimycin). The translocation of cFXIII and phosphatidylserine (PS) to the surface of activated platelets and platelet‐derived microparticles was investigated by flow cytometry, immunofluorescence, and immune electron microscopy. Fluo‐4‐AM fluorescence was used for the measurement of intracellular Ca2+ concentration. Results Receptor mediated activation by CVX+Thr exposed cFXIII to the surface of more than 60% of platelets. Electron microscopy revealed microparticles with preserved membrane structure and microparticles devoid of labeling for membrane glycoprotein CD41a. cFXIII was observed on both types of microparticles but was more abundant in the absence of CD41a. Rhosin, a RhoA inhibitor, significantly decreased cFXIII translocation. Non‐receptor mediated activation of platelets by calcimycin elevated intracellular Ca2+ concentration, induced the translocation of PS to the surface of platelets and microparticles, but failed to expose cFXIII. Conclusions The elevation of intracellular Ca2+ concentration is sufficient for the translocation of PS from the internal layer of the membrane, while the translocation of cFXIII from the platelet cytoplasm requires additional receptor mediated mechanism(s).

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Drug-eluting stenting (DES) has become a reliable tool for coronary stenting; however, its direct effects on platelet and endothelium function differ from those of bare-metal stenting (BMS). This study involved a periprocedural analysis of various biomarkers of cellular activation after elective DES (Xience(®), Abbott Vascular, Santa Clara, CA, USA) or BMS (Integrity(®), Medtronic, Minneapolis, MI, USA). Forty-nine stable angina patients were recruited: 28 underwent BMS, and 21 received everolimus-eluting stents. Samples were collected (i) prior to stenting, (ii) at 24 hours after procedure, and (iii) after 1 month of dual antiplatelet therapy. Platelet activation was analyzed by surface P-selectin positivity in parallel with plasma levels of soluble P-selectin, CD40L and platelet-derived growth factor (PDGF). Endothelial cell (EC) activation was detected by measuring markers of early (von Willebrand factor) and delayed response (VCAM-1, ICAM-1, E-selectin). Patients were followed for 6 months for the occurrence of restenosis or stent thrombosis. Increased platelet activation was sustained regardless of stent type or antiplatelet medication. Concentrations of most EC markers were more elevated after BMS than after DES. No stent thrombosis was seen, but six BMS subjects displayed restenosis with significantly higher sCD40L (779 [397-899] vs. 381 [229-498] pg/mL; p = 0.032) and sICAM-1 (222 [181-272] vs. 162 [153-223] ng/mL; p = 0.046) levels than in those without complication, while DES patients exhibited significantly decreased PDGF (572 [428-626] vs. 244 [228-311] pg/mL; p = 0.004) after 1 month. Nonresponsiveness to antiplatelet drugs did not influence these changes. In conclusion, the degree of platelet and EC activation suggests that Xience(®) DES may be regarded a safer coronary intervention than Integrity(®) BMS, with a lower risk of in-stent restenosis.

BackgroundMortality in patients with end-stage renal disorders is often a consequence of cardiovascular complications. Renal replacement therapies may contribute to this morbidity by promoting cellular activation. In renal failure patients peripheral blood samples were investigated for platelet and endothelial cell activation markers to compare the effects of haemodiafiltration (HDF) and haemodialysis (HD).MethodsOverall 28 patients were included in the study. Platelet P-selectin and leukocyte - platelet heterotypic aggregates were studied by flow cytometry. Soluble P- and E-selectin values were determined by ELISA, while von Willebrand factor (vWF) antigen levels were measured by immunoturbidimetry. Statistical analysis was done by the SPSS v22 software.ResultsPlatelet surface P-selectin was below 3.0 % in healthy controls, but it was higher during the dialysis after 4 h, 8 % and 14.3 % in HDF and HD, respectively. Monocyte-platelet heterotypic aggregates were significantly elevated after 4 h in both treatments, up to 69.2 % in HDF and to 82.9 % in HD. Soluble P-selectin levels were also significantly elevated by the end of both treatment procedures (p < 0.001), vWF antigen values, however, showed elevation only during HD treatment.ConclusionsThe attenuated platelet activating effects of HDF compared to HD may contribute to a less unfavourable vascular effect in this treatment modality.Electronic supplementary materialThe online version of this article (doi:10.1186/s12882-016-0364-x) contains supplementary material, which is available to authorized users.

Significant platelet reactivity with endothelial damage may occur during percutaneous coronary intervention such as balloon angioplasty and elective stenting in patients with stenotic or occluded arteries in coronary artery disease. Activated platelets express several surface epitopes to aggregate and form heterotypic interactions with leukocytes and endothelial cells, secrete biomarkers to enhance their prothrombotic properties and also generate increased level of microparticles (PMPs). These events may contribute to subacute stent thrombosis induced by the procedure-mediated trauma. Our aim was to study the level of PMPs and other platelet activation markers at an early time point after stenting with bare metal stent in patients on aspirin antiplatelet pre-treatment, and these results were compared to data obtained from subjects with diagnostic catheterization alone. We found that at 15 minutes after the completion of stenting, the levels of PMPs (557 +/- 83/microl versus 325 +/- 42/microl), the platelet P-selectin expression (2.7 +/- 0.3% versus 1.99 +/- 0.1%) and the ratio of platelet-monocyte heterotypic aggregates (48 +/- 4% versus 38 +/- 3%) were significantly (p < 0.05) elevated in patients compared to unstented subjects, but no difference was found in soluble P-selectin values. We suggest that the observed cellular changes are early and sensitive markers to detect the platelet-activating effect of stent implantation.