Platelets, known for maintaining blood balance, also participate in antimicrobial defense. Upon severeacute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, platelets become hyperactivated, releasing molecules such as cytokines, granule contents, and bioactive lipids. The key effector biolipids produced by platelets include 12-hydroxyeicosatetraenoic acid (12-HETE) and 12-hydroxyeicosatrienoic acid (12-HETrE), produced by 12-lipoxygenase (12-LOX), and prostaglandins and thromboxane, produced by cyclooxygenase-1. While prostaglandin E2 and thromboxane B2 were previously associated with lung inflammation in severe COVID-19, the role of platelet 12-LOX in SARS-CoV-2 infection remains unclear. Using mice deficient for platelets' 12-LOX, we report that SARS-CoV-2 infection resulted in higher lung inflammation characterized by histopathological tissue analysis, increased leukocyte infiltrates, and cytokine production relative to wild-type mice. In addition, distinct platelet and lung transcriptomic changes, including alterations in NOD-like receptor (NLR) family pyrin domain-containing 1 (NLRP1) inflammasome-related gene expression, were observed. Mass spectrometry lipidomic analysis in 12-LOX-deficient-infected mice revealed significant changes in bioactive lipid content, including reduced levels of 12-HETrE that inversely correlated with disease severity. Finally, platelet 12-LOX deficiency was associated with increased morbidity and lower survival rates relative to wild type (WT) mice. Overall, this study highlights the complex interplay between 12-LOX-related lipid metabolism and inflammatory responses during SARS-CoV-2 infection. The findings provide valuable insights into potential therapeutic targets aimed at mitigating severe outcomes, emphasizing the pivotal role of platelet enzymes in the host response to viral infections.

Limited information exists regarding the impact of interferons (IFNs) on the information carried by extracellular vesicles (EVs). This study aimed at investigating whether IFN-α2b, IFN-β, IFN-γ, and IFN-λ1/2 modulate the content of EVs released by primary monocyte-derived macrophages (MDM). Small-EVs (sEVs) were purified by size exclusion chromatography from supernatants of MDM treated with IFNs. To characterize the concentration and dimensions of vesicles, nanoparticle tracking analysis was used. SEVs surface markers were examined by flow cytometry. IFN treatments induced a significant down-regulation of the exosomal markers CD9, CD63, and CD81 on sEVs, and a significant modulation of some adhesion molecules, major histocompatibility complexes and pro-coagulant proteins, suggesting IFNs influence biogenesis and shape the immunological asset of sEVs. SEVs released by IFN-stimulated MDM also impact lymphocyte function, showing significant modulation of lymphocyte activation and IL-17 release. Altogether, our results show that sEVs composition and activity are affected by IFN treatment of MDM.
© 2024 The Authors.

High platelet reactivity is associated with adverse clinical events and is more frequent in people with diabetes mellitus (DM). To better understand platelet dysfunction in DM, we performed a proteomic analysis in platelets from a matched cohort of 34 people without, and 42 people with type 2 DM. The cohorts were matched by clinical characteristics including age, sex, and coronary artery disease burden. Using high sensitivity unbiased proteomics, we consistently identified over 2,400 intracellular proteins, and detected proteins that are differentially released by platelets from people with diabetes in response to low dose thrombin. Importantly, we identified the endoplasmic reticulum (ER) protein SEC61 translocon subunit beta (SEC61B) was increased in platelets from humans and mice with in vivo hyperglycemia. SEC61B was increased in megakaryocytes in mouse models of diabetes, in association with megakaryocyte ER stress. A rise in cytosolic calcium is a key aspect in platelet activation, and the SEC61 translocon is known to act as a channel for ER calcium leak. We demonstrate that cultured cells overexpressing SEC61B have increased calcium flux and decreased protein synthesis. In accordance, hyperglycemic mouse platelets mobilized more calcium to the cytosol and had lower protein synthesis compared with normoglycemic platelets. Independently, in vitro induction of ER stress increased platelet SEC61B expression and markers of platelet activation. We propose a mechanism whereby ER stress-induced upregulation of platelet SEC61B leads to increased cytosolic calcium, potentially contributing to platelet hyperactivity in people with diabetes. Key Points Platelet SEC61B is increased in hyperglycemia and contributes to increased endoplasmic reticulum (ER) calcium leak Increased ER calcium leak is associated with ER stress and platelet hyperactivity

Platelet activation induces the secretion of proteins that promote platelet aggregation and inflammation. However, detailed analysis of the released platelet proteome is hampered by platelets' tendency to preactivate during their isolation and a lack of sensitive protocols for low abundance releasate analysis. Here, we detail the most sensitive analysis to date of the platelet releasate proteome with the detection of >1300 proteins. Unbiased scanning for posttranslational modifications within releasate proteins highlighted O-glycosylation as being a major component. For the first time, we detected O-fucosylation on previously uncharacterized sites including multimerin-1 (MMRN1), a major alpha granule protein that supports platelet adhesion to collagen and is a carrier for platelet factor V. The N-terminal elastin microfibril interface (EMI) domain of MMRN1, a key site for protein-protein interaction, was O-fucosylated at a conserved threonine within a new domain context. Our data suggest that either protein O-fucosyltransferase 1, or a novel protein O-fucosyltransferase, may be responsible for this modification. Mutating this O-fucose site on the EMI domain led to a >50% reduction of MMRN1 secretion, supporting a key role of EMI O-fucosylation in MMRN1 secretion. By comparing releasates from resting and thrombin-treated platelets, 202 proteins were found to be significantly released after high-dose thrombin stimulation. Complementary quantification of the platelet lysates identified >3800 proteins, which confirmed the platelet origin of releasate proteins by anticorrelation analysis. Low-dose thrombin treatment yielded a smaller subset of significantly regulated proteins with fewer secretory pathway enzymes. The extensive platelet proteome resource provided here (larancelab.com/platelet-proteome) allows identification of novel regulatory mechanisms for drug targeting to address platelet dysfunction and thrombosis.
Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.

Introduction: An active role of platelets in the progression of triple-negative breast cancer (TNBC) cells has been described. Even the role of platelet-derived extracellular vesicles on the migration of MDA-MB-231 cells has been reported. Interestingly, upon activation, platelets release functional mitochondria into the extracellular environment. However, the impact of these platelet-derived mitochondria on the metabolic properties of MDA-MB-231 cells remains unclear. Methods: MDA-MB-231 and MDA-MB-231-Rho-0 cells were co-cultured with platelets, which were isolated from donor blood. Mitochondrial transfer was assessed through confocal microscopy and flow cytometry, while metabolic analyses were conducted using a Seahorse XF HS Mini Analyzer. The mito-chondrial DNA (mtDNA) copy number was determined via quantitative PCR (qPCR) following platelet co-culture. Finally, cell proliferation and colony formation assay were performed using crystal violet staining. Results and Discussion: We have shown that platelet-derived mitochondria are internalized by MDA-MB-231 cells in co-culture with platelets, increasing ATP production, oxygen (O2) consumption rate (OCR), cell proliferation, and metabolic adaptability. Additionally, we observed that MDA-MB-231 cells depleted from mtDNA restore cell proliferation in uridine/pyruvate-free cell culture medium and mitochondrial O2 consumption after co-culture with platelets, indicating a reconstitution of mtDNA facilitated by platelet-derived mitochondria. In conclusion, our study provides new insights into the role of platelet-derived mitochondria in the metabolic adaptability and progression of metastatic MDA-MB-231 TNBC cells.
Copyright © 2024 Cereceda, Cardenas, Khoury, Silva-Pavez and Hidalgo.