Circulating platelets are responsible for hemostasis and thrombosis but are also primary sensors of pathogens and are involved in innate immunity, inflammation, and sepsis. Sepsis is commonly caused by an exaggerated immune response to bacterial, viral, and fungal infections, and leads to severe thrombotic complications. Among others, the endotoxin lipopolysaccharide (LPS) found in the outer membrane of Gram-negative bacteria is the most common trigger of sepsis. Since the discovery of the expression of the LPS receptor TLR4 in platelets, several studies have investigated the ability of LPS to induce platelet activation and to contribute to a prothrombotic phenotype, per se or in combination with plasma proteins and platelet agonists. This issue, however, is still controversial, as different sources, purity, and concentrations of LPS, different platelet-purification protocols, and different methods of analysis have been used in the past two decades, giving contradictory results. This review summarizes and critically analyzes past and recent publications about LPS-induced platelet activation in vitro. A methodological section illustrates the principal platelet preparation protocols and significant differences. The ability of various sources of LPS to elicit platelet activation in terms of aggregation, granule secretion, cytokine release, ROS production, and interaction with leukocytes and NET formation is discussed.

Background: Platelets can support cancer progression via the release of microparticles and microvesicles that enhance the migratory behaviour of recipient cancer cells. We recently showed that platelet-derived extracellular vesicles (PEVs) stimulate migration and invasiveness in highly metastatic MDA-MB-231 cells by stimulating the phosphorylation of p38 MAPK and the myosin light chain 2 (MLC2). Herein, we assessed whether the pro-migratory effect of PEVs involves the remodelling of the Ca²⁺ handling machinery, which drives MDA-MB-231 cell motility. Methods: PEVs were isolated from human blood platelets, and Fura-2/AM Ca²⁺ imaging, RT-qPCR, and immunoblotting were exploited to assess their effect on intracellular Ca²⁺ dynamics and Ca²⁺-dependent migratory processes in MDA-MB-231 cells. Results: Pretreating MDA-MB-231 cells with PEVs for 24 h caused an increase in Ca²⁺ release from the endoplasmic reticulum (ER) due to the up-regulation of SERCA2B and InsP₃R1/InsP₃R2 mRNAs and proteins. The consequent enhancement of ER Ca²⁺ depletion led to a significant increase in store-operated Ca²⁺ entry. The larger Ca²⁺ mobilization from the ER was required to potentiate serum-induced migration by recruiting p38 MAPK and MLC2. Conclusions: PEVs stimulate migration in the highly metastatic MDA-MB-231 breast cancer cell line by inducing a partial remodelling of the Ca²⁺ handling machinery.

Cell genome integrity is continuously threatened by various sources, both endogenous and exogenous. Oxidative stress causes a multitude of damages, severely affecting cell viability, fidelity of genetic information inheritance, and determining profound alterations in gene expression. Epigenetics represents a major form of gene expression modulation, influencing DNA accessibility to transcription factors and the overall nuclear architecture. When assessing the stress-induced epigenome reprogramming, widely diffused biochemical and molecular approaches commonly fail to incorporate analyses such as architectural chromatin alterations and target molecules precise spatial localization. Unveiling the significance of the nuclear response to the oxidative stress, as well as the functional effects over the chromatin organization, may reveal targets and strategies for approaches aiming at limiting the impact on cellular stability. For these reasons, we utilized potassium bromate treatment, a stressor able to induce DNA damages without altering the cellular microenvironment, hence purely modeling nuclear oxidative stress. By means of high-resolution techniques, we described profound alterations in DNA and histone epigenetic modifications and in chromatin organization in response to the reactive oxygen species.