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Platelets are a major source of microRNAs (miRNAs) in blood. Relationships between circulating platelet-derived miRNAs were investigated to elucidate their significance as biomarkers. Total miRNAs in serum were analyzed using the 3D-Gene miRNA Oligo chip. Among 22 miRNAs that are included in platelets and play functional roles, sufficient miRNA levels for comparison were detected for 11 miRNAs (let-7b-5p, miR-16-5p, miR-17-5p, miR-24-3p, miR-107, miR-126-3p, miR-150-3p, miR-191-5p, miR-197-3p, miR-223-3p, and miR-326). Among 55 pairs prepared by these miRNAs, relatively strong correlations (Spearman's correlation coefficient >0.8) were shown between miRNAs of 7 pairs including let-7b-5p and miR-16-5p, let-7b-5p and miR-17-5p, let-7b-5p and miR-107, miR-16-5p and miR-17-5p, miR-16-5p and miR-107, miR-17-5p and miR-107, and miR-107 and miR-126-3p. In principal component analysis, the first principal component consisted of let-7b-5p, miR-16-5p, miR-17-5p, miR-107, miR-126-3p, and miR-191-5p. These six miRNAs may be useful biomarkers that reflect platelet condition and function.

Alcohol inhibits platelet function, and platelet count is often reduced in individuals with alcohol use disorder. However, the relation of habitual alcohol drinking with platelet count in a general population remains to be determined. The participants were 6508 men (30 ~ 69 years old) who had received annual health checkup examinations, and most of them (98.6%) were nondrinkers or drinkers with an average ethanol intake of less than 66 g per day. Relationships of platelet count with alcohol intake were investigated by using analysis of covariance and multivariate linear and logistic regression analyses. Platelet count was significantly correlated with age, smoking, γ-glutamyl transpeptidase, body mass index and leukocyte count, which were thus used as explanatory variables in the multivariate analyses. Mean platelet counts in light (

In patients with cardiovascular abnormalities or immunological disorders, an increased number of circulating leukocyte-platelet aggregates is observed. Leukocyte-platelet aggregates play an essential role in linking the hemostatic and immune systems. High shear stress and pro-coagulant and pro-inflammatory stimulants are known to activate platelets and promote the formation of aggregates. Pulsatile blood flow under low shear stress can also induce platelet activation in comparatively mild conditions. However, the effect of such events on leukocyte-platelet aggregates has not yet been investigated. To determine whether low shear stress affects the formation of aggregates, we established a simple "inverting rotation" method of inducing periodic changes in the direction of blood flow in combination with low shear stress. We demonstrated that after the inverting rotation treatment for 10-20 min more than 70% of monocytes selectively aggregated with platelets. The formation of monocyte-platelet complexes was inhibited by an anti-CD162 (PSGL-1) monoclonal antibody or a Ca(2+) chelator. The phagocytic activity of monocytes was augmented by inverting rotation, whereas phagocytosis mediated by granulocytes remained unaffected. Interestingly, the formation of monocyte-platelet complexes suppressed the production of pro-inflammatory cytokines such as interleukin (IL)-1β. At the same time, monocyte-platelet complexes augmented the expression of the anti-inflammatory cytokine IL-10. Our results suggest that platelet-bound monocytes show an anti-inflammatory phenotype under low shear stress conditions. Thus, our method provided new insights into the mechanisms of monocyte-platelet aggregate formation and regulation.

The present study was designed to analyze the molecular basis of the intracellular pH-dependent capacitative Ca2+ entry (CCE) of human platelets and megakaryocytic cells, specifically to test the hypothesis that members of the classical transient receptor potential (TRPC) protein family are involved in the CCE pathway that is promoted by intracellular alkalosis. Human platelets as well as the tested megakaryocytic cell lines (CMK cells, MEG-01 cells) and HEK293 cells displayed thapsigargin-induced CCE and responded to monensin with comparable elevation in intracellular pH. Promotion of CCE by monensin-induced intracellular alkalosis, however, was profound in mature platelets, moderate in CMK cells and lacking in MEG-01 cells as well as in HEK293 cells. Analysis of the TRPC expression pattern by immunoblotting revealed that mature platelets and CMK cells express TRPC4 along with TRPC1 and TRPC3, while TRPC4 is lacking in MEG-01 cells. HEK293 cells displayed CCE characteristics as well as lack of TRPC4 expression similar to MEG-01 cells. Over-expression of TRPC4 in HEK293 cells was found to result in a gain of pH-sensitivity of CCE with clearly detectable promotion of CCE in response to monensin. These results suggest that platelet CCE channel complexes contain TRPC4 as a molecular component that determines sensitivity of CCE to intracellular alkalosis.

Intracellular pH is known to increase during agonist-induced platelet activation. In order to elucidate the role of intracellular alkalinization in platelet activation, the effects of NH4Cl, as a tool to induce intracellular alkalinization, on ionomycin-induced platelet activation were investigated. NH4Cl (2.5-10 mM) concentration-dependently induced intracellular alkalinization. Platelet aggregation induced by ionomycin (0.1 microM) was augmented by treatment with NH4Cl (2.5-10 mM). Ionomycin-induced platelet aggregation in the absence of extraplatelet Ca2+, which was markedly attenuated compared to that in the presence of extraplatelet Ca2+, was also augmented by NH4Cl. NH4Cl treatment increased the number of large aggregates after ionomycin stimulation, while it decreased the number of small aggregates. Both transplasmalemmal Ca2+ entry and intracellular Ca2+ release induced by ionomycin were increased by treatment with NH4Cl (10 mM). SKF-96365 (100 microM), an inhibitor of receptor-operated Ca2+ channels, did not affect ionomycin-induced Ca2+ entry but abolished the effect of NH4Cl on Ca2+ entry. Thus, NH4Cl augments receptor-operated Ca2+ channels and intracellular Ca2+ release. These findings suggest that intracellular alkalinization plays a significant role in agonist-induced platelet activation.