The diverse repertoire of cell surface glycans, generated by the coordinated activity of glycosyltransferases and glycosidases, encodes critical biological information that is interpreted by glycan-binding proteins including galectins. Galectin-1 (GAL1), a member of this family, plays key roles across multiple hallmarks of cancer, including angiogenesis and immune evasion, driving resistance to anti-angiogenic and immunotherapeutic strategies through glycosylation-dependent mechanisms. Here, we first review the contribution of GAL1-glycan interactions to therapeutic resistance in cancer, with a particular focus on anti-angiogenic therapies and immunotherapy, and discuss the central role of glycosyltransferases in shaping these responses. While the biosynthesis of 'GAL1-permissive' glycans has been extensively characterized, the contribution of post-synthetic glycan remodeling to GAL1-driven therapeutic resistance remains uncertain. To explore mechanisms underlying GAL1-mediated resistance, we investigated whether tumor- or stromal-derived sialidases (NEU1 or NEU3) modulate sensitivity to vascular endothelial growth factor (VEGF)-targeted therapies by unmasking GAL1binding glyco-epitopes. In the second part of the study, we present original in vivo experiments using gainand loss-of-function approaches, demonstrating that, at least in our experimental settings, sialidases do not contribute to resistance to anti-VEGF treatment. Finally, bioinformatic analyses of patient datasets revealed differential regulation of GAL1, as well as specific glycosyltransferases, in patients responding or not to antiVEGF or anti-PD-1 therapies. Collectively, these findings indicate that glycosyltransferases, particularly MGAT5, GCNT1, and ST6GAL1, coordinately shape the GAL1-specific glycome in settings of therapeutic resistance, whereas glycan remodeling by endogenous sialidases does not play a major role. Whether sialidases influence GAL1-dependent functions in other contexts remains to be explored.

Glycan-binding proteins (GBPs), including Galectins, sialic acid–binding immunoglobin-type lectins (Siglecs), and C-type lectin receptors (CLRs), are key regulators of immune cell development, activation, differentiation, trafficking, and homeostasis. By interpreting glycan-encoded information, these lectins shape immune responses across T, B, and myeloid cell lineages. Galectins, primarily soluble β-galactoside-binding proteins, function through both extracellular and intracellular mechanisms. Siglecs, expressed on various immune cells, recognize sialoglycans and initiate immunoregulatory signaling, while CLRs detect diverse glycans on pathogens and host cells and thereby contribute to immune sensing and modulation. These glycosylation-dependent pathways serve as integrative hubs that couple intrinsic immune programs with environmental and intracellular cues. In this review, we discuss cellular mechanisms by which GBPs govern immune cell fate under homeostatic physiologic conditions, and we emphasize both conserved and context-dependent roles. Moreover, we address how glycan–GBP dysregulation contributes to immune dysfunction and pathology and how targeting these endogenous glycoimmune checkpoints may open new avenues for immunotherapy.

Chronic hemophilic synovitis (CHS), driven by hemosiderin-laden macrophages from recurrent hemarthrosis, is a major cause of joint damage in hemophilia. Platelet-rich plasma (PRP) is a promising regenerative therapy for joint diseases. This study investigated PRP’s ability to modulate macrophage polarization from a pro-inflammatory (M1) to a pro-resolving, tissue-repairing (M2) phenotype in CHS. We analyzed synovial fluid (SF) from CHS patients (N = 22), both pre- and post-PRP treatment. Ex vivo analysis revealed a predominant M1 profile with an increased proportion of CD11+CD14+CD64hi compared with CD206+ or CD163+ M2 macrophages in CHS SF. In vitro experiments showed that CHS SF skewed monocyte-derived macrophages toward an M1 inflammatory program, evaluated by flow cytometry, qPCR, and ELISA. However, adding PRP significantly modulated the pro-inflammatory macrophage program, promoting an M2 tissue repair profile. Furthermore, a random forest machine learning algorithm, applied to public scRNAseq data, confirmed PRP’s macrophage reprogramming effect. Functional assays also showed increased TGF-β secretion and macrophage fusion when challenged with neutrophil extracellular traps (NETs). A small patient follow-up cohort treated with intra-articular PRP showed similar results, including normalization of cellular content and reduced CD64/CD206 expression. These findings indicate that PRP treatment effectively shifts SF-associated M1 macrophages to an M2-like phenotype, highlighting its potential as a therapeutic strategy for CHS.

Yellow fever (YF), caused by the Yellow Fever Virus (YFV), is a disease endemic in South America and Africa with clinical manifestations ranging from fever to fatal organ failure and hemorrhagic complications. The role of the endothelium in the pathogenesis of YFV is not completely understood. To investigate the effects of YFV infection on human endothelial cells (EC), human umbilical vein endothelial cells (HUVEC) and human microvascular endothelial cells (HMEC-1) were infected with the YFV 17DD strain. Viral infection, cell death, cell adhesion, adhesion molecules, cytokines, von Willebrand factor (VWF), nitric oxide (NO), tissue factor (TF), interferon-I, clotting time and signaling pathways were analyzed by plaque assays, immunofluorescence, cytometry, ELISA, RT-qPCR, DAF-FM DA probe, Griess reaction and Western blot. The results showed that HUVEC and HMEC-1 were susceptible to YFV infection according to virus titer and presence of intracellular dsRNA, which induced an increase in apoptosis at 3- and 7-days post-infection (pi), respectively. At earlier time points (4-48 h pi), infected EC exhibited upregulation of E-selectin and ICAM-1, secretion of IL-1β, IL-6 and VWF, decreased eNOS mRNA, NO production, clotting time, and increased TF and interferon-I mRNA levels, as well as increased adhesion of platelets and leukocytes to EC. Mechanistically, YFV infection led to activation of the NF-κB and MAPK signaling pathways. We conclude that YFV infection triggers a proinflammatory and prothrombotic response in EC that likely contributes to the vascular dysfunction and hemorrhagic manifestations observed in YF. These findings point to potential targets for therapeutic intervention.

Hematopoiesis- the formation of blood cell components- continually replenishes the blood system during embryonic development and postnatal lifespans. This coordinated process requires the synchronized action of a broad range of cell surface associated proteins and soluble mediators, including growth factors, cytokines and lectins. Collectively, these mediators control cellular communication, signalling, commitment, proliferation, survival and differentiation. Here we discuss the role of galectins - an evolutionarily conserved family of glycan-binding proteins - in the establishment and dynamic remodelling of hematopoietic niches. We focus on the contribution of galectins to B and T lymphocyte development and selection, as well as studies highlighting the role of these proteins in myelopoiesis, with particular emphasis on erythropoiesis and megakaryopoiesis. Finally, we also highlight recent findings suggesting the role of galectin-1, a prototype member of this protein family, as a key pathogenic factor and therapeutic target in myelofibrosis. Through extracellular or intracellular mechanisms, galectins can influence the fate and function of distinct hematopoietic progenitors and fine-tune the final repertoire of blood cells, with critical implications in a wide range of physiologically vital processes including innate and adaptive immunity, immune tolerance programs, tissue repair, regeneration, angiogenesis, inflammation, coagulation and oxygen delivery. Additionally, positive or negative regulation of galectin-driven circuits may contribute to a broad range of blood cell disorders.

Snake venoms are intricate mixtures of enzymes and bioactive factors that induce a range of detrimental effects in afflicted hosts. Certain Viperids, including Bothrops jararacussu, harbor C-type lectins (CTLs) known for their modulation of a variety of host cellular responses. In this study, we isolated and purified BjcuL, a CTL from B. jararacussu venom and investigated its impact on endothelial cell behavior, contrasting it with human galectin-1 (Gal-1), a prototype member of the galectin family with shared β-galactoside-binding activity. We found that BjcuL binds to human dermal microvascular endothelial cells (HMECs) in a concentration- and carbohydrate-dependent fashion and reprograms the function of these cells, favoring a pro-inflammatory and pro-coagulant endothelial phenotype. In light of the quest for universal antagonists capable of mitigating the harmful consequences of snake venoms, BjcuL emerges as a promising target to be blocked in order to regulate pathological endothelial cell responses.

Monocytes (Mo) are highly plastic myeloid cells that differentiate into macrophages after extravasation, playing a pivotal role in the resolution of inflammation and regeneration of injured tissues. Wound-infiltrated monocytes/macrophages are more pro-inflammatory at early time points, while showing anti-inflammatory/pro-reparative phenotypes at later phases, with highly dynamic switching depending on the wound environment. Chronic wounds are often arrested in the inflammatory phase with hampered inflammatory/repair phenotype transition. Promoting the tissue repair program switching represents a promising strategy to revert chronic inflammatory wounds, one of the major public health loads. We found that the synthetic lipid C8-C1P primes human CD14+ monocytes, restraining the inflammatory activation markers (HLA-DR, CD44, and CD80) and IL-6 when challenged with LPS, and preventing apoptosis by inducing BCL-2. We also observed increased pseudo-tubule formation of human endothelial-colony-forming cells (ECFCs) when stimulated with the C1P-macrophages secretome. Moreover, C8-C1P-primed monocytes skew differentiation toward pro-resolutive-like macrophages, even in the presence of inflammatory PAMPs and DAMPs by increasing anti-inflammatory and pro-angiogenic gene expression patterns. All these results indicate that C8-C1P could restrain M1 skewing and promote the program of tissue repair and pro-angiogenic macrophage.

Background:CD34+ cells, megakaryocytes (MKs), and platelets express toll-like receptors (TLRs) that enable these cells to amplify the host innate immune response. However, the role of TLR7/TLR8 activation in megakaryopoiesis has not yet been investigated. Objectives:We evaluated the effect of coxsackievirus B3 (CVB3) and synthetic TLR7/TLR8 agonists on the development of human MKs and production of platelets. Methods:CD34+ cells from human umbilical cord were inoculated with CVB3 or stimulated with synthetic TLR7/TLR8 agonists and then cultured in the presence of thrombopoietin. Results:CD34+ cells, MK progenitor cells, and mature MKs expressed TLR7 and TLR8, and exposure to CVB3 resulted in productive infection, as determined by the presence of viral infectious particles in culture supernatants. Cell expansion, differentiation into MKs, MK maturation, and platelet biogenesis were significantly reduced in CD34+-infected cultures. The reduction in MK growth was not due to an alteration in cellular proliferation but was accompanied by an increase in cellular apoptosis and pyroptosis. Impairment of MK generation and maturation of viable cells were also associated with decreased expression of transcription factors involved in these processes. These effects were completely abrogated by TLR7 but not TLR8 antagonists and mimicked by TLR7 but not TLR8 agonists. CVB3 infection of CD34+ cells increased the immunophenotype of MKs characterized as CD148+/CD48+ or CD41+/CD53+ cells. Conclusion:These data suggest a novel role of TLR7 in megakaryo/thrombopoiesis that may contribute to a better understanding of the molecular basis underlying thrombocytopenia and the immunologic role of MKs in viral infection processes.

Reduced expression of a platelet protein protects against thrombosis during chronic immobilization.

Hemolytic uremic syndrome (HUS) is the most common cause of acute renal failure in the pediatric population. The etiology of HUS is linked to Gram-negative, Shiga toxin (Stx)-producing enterohemorrhagic bacterial infections. While the effect of Stx is focused on endothelial damage of renal glomerulus, cytokines induced by Stx or bacterial lipopolysaccharide (LPS) and polymorphonuclear cells (PMNs) are involved in the development of the disease. PMN release neutrophil extracellular traps (NETs) to eliminate pathogens, although NETs favor platelets (Plts) adhesion/thrombus formation and can cause tissue damage within blood vessels. Since thrombus formation and occlusion of vessels are characteristic of HUS, PMN–Plts interaction in the context of Stx may promote netosis and contribute to the endothelial damage observed in HUS. The aim of this study was to determine the relevance of netosis induced by Stx in the context of LPS-sensitized Plts on endothelial damage. We observed that Stx2 induced a marked enhancement of netosis promoted by Plts after LPS stimulation. Several factors seemed to promote this phenomenon. Stx2 itself increased the expression of its receptor on Plts, increasing toxin binding. Stx2 also increased LPS binding to Plts. Moreover, Stx2 amplified LPS induced P-selectin expression on Plts and mixed PMN–Plts aggregates formation, which led to activation of PMN enhancing dramatically NETs formation. Finally, experiments revealed that endothelial cell damage mediated by PMN in the context of Plts treated with LPS and Stx2 was decreased when NETs were disrupted or when mixed aggregate formation was impeded using an anti-P-selectin antibody. Using a murine model of HUS, systemic endothelial damage/dysfunction was decreased when NETs were disrupted, or when Plts were depleted, indicating that the promotion of netosis by Plts in the context of LPS and Stx2 plays a fundamental role in endothelial toxicity. These results provide insights for the first time into the pivotal role of Plts as enhancers of endothelial damage through NETs promotion in the context of Stx and LPS. Consequently, therapies designed to reduce either the formation of PMN–Plts aggregates or NETs formation could lessen the consequences of endothelial damage in HUS.

The COVID-19 pandemic, caused by SARS-CoV-2, had its first cases identified in late 2019 and was considered a clinical pandemic in March 2020. In March 2022, more than 500 million people were infected and 6,2 million died as a result of this disease, increasingly associated with changes in human hemostasis, such as hypercoagulation. Numerous factors contribute to the hypercoagulable state, and endothelial dysfunction is the main one, since the activation of these cells can strongly activate platelets and the coagulation system. In addition, there is a dysregulation of the renin-angiotensin system due to the SARS-CoV-2 takeover of the angiotensin converting enzyme 2, resulting in a strong immune response that could further damage the endothelium. Thrombus formation in the pulmonary microvasculature structure in patients with COVID-19 is an important factor to determine the severity of the clinical picture and the outcome of this disease. This review describes the hemostatic changes that occur in SARS-CoV-2 infection, to further improve our understanding of pathogenic mechanisms and the interaction between endothelium dysfunction, kallikrein-kinins, renin angiotensin, and the Coagulation/fibrinolysis systems as underlying COVID-19 effectors. This knowledge is crucial for the development of new effective therapeutic approaches, attenuating the severity of SARS-CoV-2’s infection and to reduce the deaths.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been responsible for the severe pandemic of acute respiratory disease, coronavirus disease 2019 (COVID-19), experienced in the 21st century. The clinical manifestations range from mild symptoms to abnormal blood coagulation and severe respiratory failure. In severe cases, COVID-19 manifests as a thromboinflammatory disease. Damage to the vascular compartment caused by SARS-CoV-2 has been linked to thrombosis, triggered by an enhanced immune response. The molecular mechanisms underlying endothelial activation have not been fully elucidated. We aimed to identify the proteins correlated to the molecular response of human umbilical vein endothelial cells (HUVECs) after exposure to SARS-CoV-2, which might help to unravel the molecular mechanisms of endothelium activation in COVID-19. In this direction, we exposed HUVECs to SARS-CoV-2 and analyzed the expression of specific cellular receptors, and changes in the proteome of HUVECs at different time points. We identified that HUVECs exhibit non-productive infection without cytopathic effects, in addition to the lack of expression of specific cell receptors known to be essential for SARS-CoV-2 entry into cells. We highlighted the enrichment of the protein SUMOylation pathway and the increase in SUMO2, which was confirmed by orthogonal assays. In conclusion, proteomic analysis revealed that the exposure to SARS-CoV-2 induced oxidative stress and changes in protein abundance and pathways enrichment that resembled endothelial dysfunction.

Archaebacterias are considered a unique source of novel biomaterials of interest for nanomedicine. In this perspective, the effects of nanoarchaeosomes (ARC), which are nanovesicles prepared from polar lipids extracted from the extreme halophilic Halorubrum tebenquinchense, on human umbilical vein endothelial cells (HUVEC) were investigated in physiological and under inflammatory static conditions. Upon incubation, ARC (170 nm mean size, −41 mV ζ) did not affect viability, cell proliferation, and expression of intercellular adhesion molecule-1 (ICAM-1) and E-selectin under basal conditions, but reduced expression of both molecules and secretion of IL-6 induced by lypopolysaccharide (LPS), Pam3CSK4 or Escherichia coli. Such effects were not observed with TNF-α or IL-1β stimulation. Interestingly, ARC significantly decreased basal levels of von Willebrand factor (vWF) and levels induced by all stimuli. None of these parameters was altered by liposomes of hydrogenated phosphatidylcholine and cholesterol of comparable size and concentration. Only ARC were endocytosed by HUVEC and reduced mRNA expression of ICAM-1 and vWF via NF-ĸB and ERK1/2 in LPS-stimulated cells. This is the first report of the anti-inflammatory effect of ARC on endothelial cells and our data suggest that its future use in vascular disease may hopefully be of particular interest.

Platelets are circulating anucleated cytoplasmic fragments with a major role in hemostasis and thrombosis. However, in the past few decades, it has become clear that they also have a major role in inflammatory processes. Platelets can contribute to inflammation directly by secreting proinflammatory molecules or indirectly by recognizing and scavenging invading microbes. In addition, platelets modulate key functions of both endothelial and immune cells, which results in an amplification of both the initiation as well as the resolution of the inflammatory and immune response. In this mini-review, the role of platelets in inflammation and two recent main articles about the so-called inflammatory hemostasis and the contribution of platelets to macrophage extracellular traps release, are critically analyzed.

Platelets have a well-recognized role in hemostasis and thrombosis, and they are important amplifiers of inflammation and innate immune responses. The formation of DNA extracellular traps (ETs) is a complex cellular mechanism, which occurs in response to microbial infections and sterile inflammation, and results in the release of DNA complexed with histones and various granular proteins. ETs were first discovered in neutrophils (NETs); however, it is now accepted that other leukocytes, including eosinophils (EETs) and monocytes/macrophages (MoETs/METs), can also generate them. Moreover, several types of ETs have been described. Increasing evidence has demonstrated that platelets modulate the formation of ETs. This review summarizes recent findings about the physiopathological role of platelets in the formation of ETs during infection and future perspectives in the field.

The same as liposomes, archaeosomes are nanoparticles (NPs), made of archaeolipids employed as drug delivery systems. Up to date, the effect of archaeosomes on the vascular endothelium, critical data for its admission to the clinic, remains largely unknown. In this study we analyzed the effect of new archaeosomes prepared from a hyperhalophilic strain from Patagonia Argentina on human umbilical vein endothelial cells (HUVECs) under physiological and inflammatory conditions and compare it with that of conventional liposomes. Although none of the NPs affected the viability and expression of ICAM-1 and E-selectin under basal conditions, the archaeosomes reduced the expression of both molecules and the secretion of IL-6 induced by LPS and Pam3CSK4, an effect not observed with TNF- α and associated with an inhibition in the activation of the NF-kB and ERK1/2 pathway. None of these parameters were modified by the liposomes. Similarly, only archaeosomes were endocytosed by HUVECs.Our data reveal an important capacity of these archaeosomes to decrease endothelial activation andsuggest that loaded with anti-inflammatory drugs, they could magnify their activity on inflamed endothelium, their research in vasculopathies being of special interest.

Despite the recent therapeutic advances, ischemic stroke remains one of the leading causes of death and permanent disability worldwide. Although the thrombus itself is the primary target of pharmacological thrombolysis, surprisingly, little is known about the composition of thrombi that causes ischemic stroke. Neutrophil extracellular traps (NETs) have been implicated in thrombosis [[1]Thålin C. Hisada Y. Lundström S. Mackman N. Wallén H. Neutrophil extracellular traps.Arterioscler Thromb Vasc Biol. 2019 Sep; 39: 1724-1738Crossref PubMed Scopus (179) Google Scholar]. NETs are DNA fibers decorated with histones and microbicidal proteins and are recognised as an effective strategy of innate immune cells to fight against infections [[2]Sollberger G. Tilley D.O. Zychlinsky A. Neutrophil extracellular traps: the biology of chromatin externalization.Dev Cell. 2018 Mar; 44: 542-553Summary Full Text Full Text PDF PubMed Scopus (170) Google Scholar]. Besides their immunological role, NETs also contribute to both the venous and arterial thrombosis. NETs provide a scaffold for platelets and red blood cells that form a complex to promote the coagulation cascade. Therefore, the uncontrolled formation of NETs is increasingly accepted as another pathogenic mechanism of thrombotic events, not only during infectious diseases but also under sterile inflammatory conditions such as myocardial infarction, deep vein thrombosis, cancer-related thrombosis and stroke [[1]Thålin C. Hisada Y. Lundström S. Mackman N. Wallén H. Neutrophil extracellular traps.Arterioscler Thromb Vasc Biol. 2019 Sep; 39: 1724-1738Crossref PubMed Scopus (179) Google Scholar]. Currently, little is known about the presence of NETs in ischemic thrombus due to stroke. Studies in mice models and humans show that NETs are formed during an acute ischemic stroke (AIS) and that they are involved in brain-blood barrier damage as well as in the mechanical and pharmacological resistance of thrombolysis induced by tissue plasminogen activator [[3]Jiménez-Alcázar M. Kim N. Fuchs T. Circulating extracellular DNA: cause or consequence of thrombosis?.Semin Thromb Hemost. 2017 Sep 30; 43: 553-561Crossref PubMed Scopus (51) Google Scholar]. The pathogenic mechanisms involved in vessel occlusion mediated by NETs formation during stroke were further described in this issue of EBioMedicine, by Zhou and colleagues [[4]Zhou P. Li T. Jin J. et al.Interactions between neutrophil extracellular traps and activated platelets enhance procoagulant activity in acute stroke patients with ICA occlusion.EBioMed. 2020; (Access available from)https://doi.org/10.1016/j.ebiom.2020.102671Summary Full Text Full Text PDF Scopus (67) Google Scholar]. The authors found increased levels of phosphatidylserine (PS) entrapped in the NETs, activated platelets, and PS + platelet microparticles (PMP) in the carotid lesion site (CLS) of patients with AIS. They also demonstrated that plasma from the CLS triggers PS exposure on neutrophil membranes and activates platelets that drive NETs formation. Moreover, the authors show that the DNA scaffold supports the binding of PS + PMP that together with the PS exposed on the NET allow binding of coagulation factors that catalyses thrombin generation and the formation of fibrin. Although previous studies have shown that platelets promote the NET generation [[5]Carestia A. Kaufman T. Schattner M Platelets: new bricks in the building of neutrophil extracellular traps.Front Immunol. 2016; 7: 271Crossref PubMed Scopus (101) Google Scholar] and PMP expressing PS are involved in thrombus formation [[6]Zaldivia M.T.K. McFadyen J.D. Lim B. Wang X. Peter K Platelet-derived microvesicles in cardiovascular diseases.Front Cardiovasc Med. 2017; 21: 4Google Scholar], this study reveals that the crosstalk between platelets, NETs, PMP and particularly PS in the local area of the carotid lesion might be critical for vessel occlusion. It has been shown that NETs induce endothelial injury. However, most of the available information comes from animal models or in vitro studies using cells from healthy subjects [[7]Folco E.J. Mawson T.L. Vromman A. Bernardes-Souza B. Franck G. Persson O. et al.Neutrophil extracellular traps induce endothelial cell activation and tissue factor production through interleukin-1α and cathepsin G.Arterioscler Thromb Vasc Biol. 2018; 38: 1901-1912Crossref PubMed Scopus (161) Google Scholar,[8]Saffarzadeh M. Juenemann C. Queisser M.A. et al.Neutrophil extracellular traps directly induce epithelial and endothelial cell death: a predominant role of histones.Hartl D. PLoS One. 2012; 7: e32366Crossref PubMed Scopus (812) Google Scholar]. Zhou et al., found that NETs from the CLS of patients with AIS trigger endothelial cell activation, increase their procoagulant activity and induce cell injury due to the proteolytic activity of metalloprotease-9 parenthesis should be deleted and replaced by metalloprotease-9 and elastase on endothelial cell junctions. While studies on endothelial cells are extremely important to understanding the pathophysiology of atherothrombotic diseases, it is essential to note that the study of Zhou et al., was performed using human umbilical endothelial cells which may not fully represent the in vivo situation. Despite many advances in our understanding of ischemic stroke, cryptogenic strokes remain a diagnostic and therapeutic challenge. Atherothrombosis is a multifactorial process that involves the intricate participation of different cells and molecules. The authors describe a novel mechanism for hypercoagulability and acute thrombotic complications in AIS patients with carotid lesions, in which PS and NETs appear to have a destructive role. Neutrophils have been traditionally recognized as major mediators of a deleterious inflammatory response in AIS, but their potential as a therapeutic target remains unexplored. Their ability to form NETs, recruit platelets and PMP through PS expression appears to be a novel and promising target for therapeutic intervention. However, several questions remain to be answered. What triggers NET formation in the microenvironment of the CLS? Neutrophils are not the only immune cell capable of triggering NET formation [[9]Pertiwi K.R. de Boer O.J. Mackaaij C. et al.Extracellular traps derived from macrophages, mast cells, eosinophils and neutrophils are generated in a time-dependent manner during atherothrombosis.J Pathol. 2019; 247: 505-512Crossref PubMed Scopus (87) Google Scholar], so what could be the role of DNA traps released by other inflammatory cells during AIS? The levels of nuclear as well as mitochondrial DNA are increased in stroke patients [[10]Tsai N.-.W. Lin T.K. Chen S.D. et al.The value of serial plasma nuclear and mitochondrial DNA levels in patients with acute ischemic stroke.Clin Chim Acta. 2011; 412: 476-479Crossref PubMed Scopus (113) Google Scholar]. We do not know yet whether both types of DNA traps exert similar prothrombotic and coagulation activities. Is the PS exposure on the NET an “eat me signal” for macrophages? Which risk factor is more important to control in order to treat or prevent ischemic stroke: the formation of NETs, PS externalization, platelets and/or endothelial activation? One, two or a little of each factor? Embolic stroke of an unknown source is caused by embolic disease and associated with an elevated risk of recurrent ischemic strokes and clinically silent cerebral ischemic lesions. Are there any differences in NETs or PS expression between thrombi with different etiology? These and many other questions are still yet to be answered. We look forward to more basic and clinical studies that would facilitate in solving this intriguing puzzle. The author apologizes to the many researchers whose work is not specifically referenced due to space limitations. MS is supported by FONCyT grants 2016/1470 and 2017/1188 Interactions between neutrophil extracellular traps and activated platelets enhance procoagulant activity in acute stroke patients with ICA occlusionNETs played a pivotal role in the hypercoagulability of stroke patients. Strategies that prevent NET formation may offer a potential therapeutic strategy for thromboembolism interventions. 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Essential thrombocythemia (ET) is comprised among chronic myeloproliferative neoplasms (MPN) and is caused by driver mutations in JAK2, CALR, and MPL, which lead to megakaryocyte proliferation and prominent thrombocytosis. Thrombosis remains the main cause of morbidity in ET and is driven by the interplay between blood cells, the endothelium, the clotting cascade, and host-derived inflammatory mediators. Platelet activation plays a key role in the thrombotic predisposition, although the underlying mechanisms remain poorly defined. In addition to their role in hemostasis, platelets participate in innate immunity and inflammation owing to the expression of toll-like receptors (TLR), which recognize inflammatory signals, triggering platelet functional responses. Considering the impact of inflammation on ET procoagulant state, we assessed the contribution of TLR2 and TLR4 to platelet hemostatic and inflammatory properties in ET patients, by using Pam3CSK4 and lipopolysaccharide (LPS) as specific TLR2 and TLR4 ligands, respectively. TLR2 ligation induced increased surface translocation of α-granule-derived P-selectin and CD40L, which mediate platelet interaction with leukocytes and endothelial cells, respectively, and higher levels of dense granule-derived CD63 in patients, whereas PAC-1 binding was not increased and LPS had no effect on these platelet responses. Platelet-neutrophil aggregate formation was elevated in ET at baseline and after stimulation of both TLR2 and TLR4. In addition, ET patients displayed higher TLR2- and TLR4-triggered platelet secretion of the chemokine RANTES (CCL5), whereas von Willebrand factor release was not enhanced, revealing a differential releasate pattern for α-granule-stored inflammatory molecules. TLR-mediated hyperresponsiveness contrasted with impaired or preserved responses to classic platelet hemostatic agonists, such as TRAP-6 and thrombin. TLR2 and TLR4 expression on the platelet surface was normal, whereas phosphorylation of downstream effector ERK1/2 was higher in patients at baseline and after incubation with Pam3CSK4, which may partly explain the enhanced TLR2 response. In conclusion, exacerbated response to TLR stimulation may promote platelet activation in ET, boosting platelet/leukocyte/endothelial interactions and secretion of inflammatory mediators, overall reinforcing the thromboinflammatory state. These findings highlight the role of platelets as inflammatory sentinels in MPN prothrombotic scenario and provide additional evidence for the close intertwining between thrombosis and inflammation in this setting.