A platelet lipidomics signature in patients with COVID-19.

Ischemic cardiovascular and venous thromboembolic events are a frequent cause of death in severe COVID-19 patients. Platelet activation plays a key role in these complications, however platelet lipidomics have not been studied yet. The aim of our pilot investigation was to perform a preliminary study of platelet lipidomics in COVID-19 patients compared to healthy subjects. Lipid extraction and identification of ultrapurified platelets from eight hospitalized COVID-19 patients and eight age- and sex-matched healthy controls showed a lipidomic pattern almost completely separating COVID-19 patients from healthy controls. In particular, a significant decrease of ether phospholipids and increased levels of ganglioside GM3 were observed in platelets from COVID-19 patients. In conclusion, our study shows for the first time that platelets from COVID-19 patients display a different lipidomics signature distinguishing them from healthy controls, and suggests that altered platelet lipid metabolism may play a role in viral spreading and in the thrombotic complications of COVID-19.

What is the context? Besides respiratory system involvement, venous thromboembolism is a severe complication of COVID-19, largely due to the strong derangement of hemostasis, with platelets playing a central role.Great attention has recently been devoted to lipid alterations in COVID-19, both because viruses by reprogramming cellular lipid metabolism remodel lipid membranes to fuel their replication, and because the COVID-19-associated cytokine storm may affect cell/plasma lipidomic signatures.Lipidomics studies in COVID-19 patients have been performed mainly in plasma and serum.To the best of our knowledge, platelet lipidomics have not been examined despite the central role played by platelets in COVID-19 complications.What is the aim of the study?The aim of our pilot study was to preliminarily explore whether platelet lipidomics is altered in COVID-19 patients compared to age- and sex-matched healthy subjects, analyzing lipidomic profile of ultrapurified platelets.What are the results of our study? Our study shows for the first time that platelets from COVID-19 patients display a different lipidomics signature distinguishing them from healthy controls.Ether phospholipids and, intriguingly, two phytoceramides were lower, while ganglioside GM3 was higher in COVID-19 samples compared to healthy controls.What is the impact?Despite the small number of COVID-19 patients enrolled, recognized as a limitation of our study, we show, for the first time, that platelets from COVID-19 patients present a different lipidomics signature and suggest that altered platelet lipid metabolism may play a significant role in viral spreading and in the thrombotic complications of COVID-19.

Thromboinflammation: From Atherosclerosis to COVID-19.

The activating interplay of thrombosis and inflammation (thromboinflammation) has been established as a major underlying pathway, driving not only cardiovascular disease but also autoimmune disease and most recently, COVID-19. Throughout the years, innate immune cells have emerged as important modulators of this process. As the most abundant white blood cell in humans, neutrophils are well-positioned to propel thromboinflammation. This includes their ability to trigger an organized cell death pathway with the release of decondensed chromatin structures called neutrophil extracellular traps. Decorated with histones and cytoplasmic and granular proteins, neutrophil extracellular traps exert cytotoxic, immunogenic, and prothrombotic effects accelerating disease progression. Distinct steps leading to extracellular DNA release (NETosis) require the activities of PAD4 (protein arginine deiminase 4) catalyzing citrullination of histones and are supported by neutrophil inflammasome. By linking the immunologic function of neutrophils with the procoagulant and proinflammatory activities of monocytes and platelets, PAD4 activity holds important implications for understanding the processes that fuel thromboinflammation. We will also discuss mechanisms whereby vascular occlusion in thromboinflammation depends on the interaction of neutrophil extracellular traps with ultra-large VWF (von Willebrand Factor) and speculate on the importance of PAD4 in neutrophil inflammasome assembly and neutrophil extracellular traps in thromboinflammatory diseases including atherosclerosis and COVID-19.

The role of platelets, neutrophils and endothelium in COVID-19 infection.

INTRODUCTION: COVID-19 is associated to an increased risk of thrombosis, as a result of a complex process that involves the activation of vascular and circulating cells, the release of soluble inflammatory and thrombotic mediators and blood clotting activation. AREAS COVERED: This article reviews the pathophysiological role of platelets, neutrophils, and the endothelium, and of their interactions, in the thrombotic complications of COVID-19 patients, and the current and future therapeutic approaches targeting these cell types. EXPERT OPINION: Virus-induced platelet, neutrophil, and endothelial cell changes are crucial triggers of the thrombotic complications and of the adverse evolution of COVID-19. Both the direct interaction with the virus and the associated cytokine storm concur to trigger cell activation in a classical thromboinflammatory vicious circle. Although heparin has proven to be an effective prophylactic and therapeutic weapon for the prevention and treatment of COVID-19-associated thrombosis, it acts downstream of the cascade of events triggered by SARS-CoV-2. The identification of specific molecular targets interrupting the thromboinflammatory cascade upstream, and more specifically acting either on the interaction of SARS-CoV-2 with blood and vascular cells or on the specific signaling mechanisms associated with their COVID-19-associated activation, might theoretically offer greater protection with potentially lesser side effects.

Emerging Role of Neutrophil Extracellular Traps in Gastrointestinal Tumors: A Narrative Review.

Neutrophil extracellular traps (NETs) are extracellular fibrous networks consisting of depolymerized chromatin DNA skeletons with a variety of antimicrobial proteins. They are secreted by activated neutrophils and play key roles in host defense and immune responses. Gastrointestinal (GI) malignancies are globally known for their high mortality and morbidity. Increasing research suggests that NETs contribute to the progression and metastasis of digestive tract tumors, among them gastric, colon, liver, and pancreatic cancers. This article explores the formation of NETs and reviews the role that NETs play in the gastrointestinal oncologic microenvironment, tumor proliferation and metastasis, tumor-related thrombosis, and surgical stress. At the same time, we analyze the qualitative and quantitative detection methods of NETs in recent years and found that NETs are specific markers of coronavirus disease 2019 (COVID-19). Then, we explore the possibility of NET inhibitors for the treatment of digestive tract tumor diseases to provide a new, efficient, and safe solution for the future therapy of gastrointestinal tumors.

P2Y12 Inhibition Suppresses Proinflammatory Platelet-Monocyte Interactions.

BACKGROUND: Monocyte-platelet aggregates (MPAs) represent the crossroads between thrombosis and inflammation, and targeting this axis may suppress thromboinflammation. While antiplatelet therapy (APT) reduces platelet-platelet aggregation and thrombosis, its effects on MPA and platelet effector properties on monocytes are uncertain. OBJECTIVES: To analyze the effect of platelets on monocyte activation and APT on MPA and platelet-induced monocyte activation. METHODS: Agonist-stimulated whole blood was incubated in the presence of P-selectin, PSGL1, PAR1, P2Y12, GP IIb/IIIa, and COX-1 inhibitors and assessed for platelet and monocyte activity via flow cytometry. RNA-Seq of monocytes incubated with platelets was used to identify platelet-induced monocyte transcripts and was validated by RT-qPCR in monocyte-PR co-incubation ± APT. RESULTS: Consistent with a proinflammatory platelet effector role, MPAs were increased in patients with COVID-19. RNA-Seq revealed a thromboinflammatory monocyte transcriptome upon incubation with platelets. Monocytes aggregated to platelets expressed higher CD40 and tissue factor than monocytes without platelets (p < 0.05 for each). Inhibition with P-selectin (85% reduction) and PSGL1 (87% reduction) led to a robust decrease in MPA. P2Y12 and PAR1 inhibition lowered MPA formation (30 and 21% reduction, p < 0.05, respectively) and decreased monocyte CD40 and TF expression, while GP IIb/IIIa and COX1 inhibition had no effect. Pretreatment of platelets with P2Y12 inhibitors reduced the expression of platelet-mediated monocyte transcription of proinflammatory SOCS3 and OSM. CONCLUSIONS: Platelets skew monocytes toward a proinflammatory phenotype. Among traditional APTs, P2Y12 inhibition attenuates platelet-induced monocyte activation.

The impact of neutrophil extracellular traps in coronavirus disease - 2019 pathophysiology.

Coronavirus disease 2019 (COVID-19), which is caused by novel coronavirus-2019 (nCoV-2019), is a highly contagious disease with high mortality and morbidity risk. Infected people may suffer from respiratory infections, which may be more progressive in patients with a defective immune system and underlying medical problems. In this regard, the cells involved in the innate immune system, play a decisive role in disease progression and complication development. Pathogen entrapment is the critical role of neutrophil extracellular traps (NETosis). This process involves the widespread release of fibrous structures by the stimulant-activated neutrophils. These fibrous structures are composed of cytosolic proteins and granular contents brought together by a network of released chromatins. This network can inhibit the spread of pathogens by their entrapment. Moreover, NETosis damage the host by producing toxic agents and triggering thrombosis. Therefore, this phenomenon may act as a double-edged sword. Regarding the rapid expansion of COVID-19, it is crucial to examine the involvement of NETosis in infected patients. This study aims to discuss NETosis participation to show its probable association with increased risk of thrombogenicity and help develop new therapeutic approaches in the battle against this viral disease.

Coagulation potential and the integrated omics of extracellular vesicles from COVID-19 positive patient plasma.

Extracellular vesicles (EVs) participate in cell-to-cell communication and contribute toward homeostasis under physiological conditions. But EVs can also contribute toward a wide array of pathophysiology like cancer, sepsis, sickle cell disease, and thrombotic disorders. COVID-19 infected patients are at an increased risk of aberrant coagulation, consistent with elevated circulating levels of ultra-high molecular weight VWF multimers, D-dimer and procoagulant EVs. The role of EVs in COVID-19 related hemostasis may depend on cells of origin, vesicular cargo and size, however this is not well defined. We hypothesized that the procoagulant potential of EV isolates from COVID-19 (+) patient plasmas could be defined by thrombin generation assays. Here we isolated small EVs (SEVs) and large EVs (LEVs) from hospitalized COVID-19 (+) patient (n = 21) and healthy donor (n = 20) plasmas. EVs were characterized by flow cytometry, Transmission electron microscopy, nanoparticle tracking analysis, plasma thrombin generation and a multi-omics approach to define coagulation potential. These data were consistent with differences in EV metabolite, lipid, and protein content when compared to healthy donor plasma isolated SEVs and LEVs. Taken together, the effect of EVs on plasma procoagulant potential as defined by thrombin generation and supported by multi-omics is enhanced in COVID-19. Further, we observe that this effect is driven both by EV size and phosphatidyl serine.

Toll-Like Receptor 4-Dependent Platelet-Related Thrombosis in SARS-CoV-2 Infection.

BACKGROUND: SARS-CoV-2 is associated with an increased risk of venous and arterial thrombosis, but the underlying mechanism is still unclear. METHODS: We performed a cross-sectional analysis of platelet function in 25 SARS-CoV-2 and 10 healthy subjects by measuring Nox2 (NADPH oxidase 2)-derived oxidative stress and thromboxane B2, and investigated if administration of monoclonal antibodies against the S protein (Spike protein) of SARS-CoV-2 affects platelet activation. Furthermore, we investigated in vitro if the S protein of SARS-CoV-2 or plasma from SARS-CoV-2 enhanced platelet activation. RESULTS: Ex vivo studies showed enhanced platelet Nox2-derived oxidative stress and thromboxane B2 biosynthesis and under laminar flow platelet-dependent thrombus growth in SARS-CoV-2 compared with controls; both effects were lowered by Nox2 and TLR4 (Toll-like receptor 4) inhibitors. Two hours after administration of monoclonal antibodies, a significant inhibition of platelet activation was observed in patients with SARS-CoV-2 compared with untreated ones. In vitro study showed that S protein per se did not elicit platelet activation but amplified the platelet response to subthreshold concentrations of agonists and functionally interacted with platelet TLR4. A docking simulation analysis suggested that TLR4 binds to S protein via three receptor-binding domains; furthermore, immunoprecipitation and immunofluorescence showed S protein-TLR4 colocalization in platelets from SARS-CoV-2. Plasma from patients with SARS-CoV-2 enhanced platelet activation and Nox2-related oxidative stress, an effect blunted by TNF (tumor necrosis factor) α inhibitor; this effect was recapitulated by an in vitro study documenting that TNFα alone promoted platelet activation and amplified the platelet response to S protein via p47phox (phagocyte oxidase) upregulation. CONCLUSIONS: The study identifies 2 TLR4-dependent and independent pathways promoting platelet-dependent thrombus growth and suggests inhibition of TLR4. or p47phox as a tool to counteract thrombosis in SARS-CoV-2.

Elevated Serum Amyloid A Levels Contribute to Increased Platelet Adhesion in COVID-19 Patients.

Coronavirus disease-19 (COVID-19) patients are prone to thrombotic complications that may increase morbidity and mortality. These complications are thought to be driven by endothelial activation and tissue damage promoted by the systemic hyperinflammation associated with COVID-19. However, the exact mechanisms contributing to these complications are still unknown. To identify additional mechanisms contributing to the aberrant clotting observed in COVID-19 patients, we analyzed platelets from COVID-19 patients compared to those from controls using mass spectrometry. We identified increased serum amyloid A (SAA) levels, an acute-phase protein, on COVID-19 patients' platelets. In addition, using an in vitro adhesion assay, we showed that healthy platelets adhered more strongly to wells coated with COVID-19 patient serum than to wells coated with control serum. Furthermore, inhibitors of integrin aIIbß3 receptors, a mediator of platelet-SAA binding, reduced platelet adhesion to recombinant SAA and to wells coated with COVID-19 patient serum. Our results suggest that SAA may contribute to the increased platelet adhesion observed in serum from COVID-19 patients. Thus, reducing SAA levels by decreasing inflammation or inhibiting SAA platelet-binding activity might be a valid approach to abrogate COVID-19-associated thrombotic complications.

COVID-19 patient fibrinogen produces dense clots with altered polymerization kinetics, partially explained by increased sialic acid.

BACKGROUND: Thrombogenicity is a known complication of COVID-19, resulting from SARS-CoV-2 infection, with significant effects on morbidity and mortality. OBJECTIVE: We aimed to better understand the effects of COVID-19 on fibrinogen and the resulting effects on clot structure, formation, and degradation. METHODS: Fibrinogen isolated from COVID-19 patients and uninfected subjects was used to form uniformly concentrated clots (2 mg/ml), which were characterized using confocal microscopy, scanning electron microscopy, atomic force microscopy, and endogenous and exogenous fibrinolysis assays. Neuraminidase digestion and subsequent NANA assay were used to quantify sialic acid residue presence; clots made from the desialylated fibrinogen were then assayed similarly to the original fibrinogen clots. RESULTS: Clots made from purified fibrinogen from COVID-19 patients were shown to be significantly stiffer and denser than clots made using fibrinogen from noninfected subjects. Endogenous and exogenous fibrinolysis assays demonstrated that clot polymerization and degradation dynamics were different for purified fibrinogen from COVID-19 patients compared with fibrinogen from noninfected subjects. Quantification of sialic acid residues via the NANA assay demonstrated that SARS-CoV-2-positive fibrinogen samples contained significantly more sialic acid. Desialylation via neuraminidase digestion resolved differences in clot density. Desialylation did not normalize differences in polymerization, but did affect rate of exogenous fibrinolysis. DISCUSSION: These differences noted in purified SARS-CoV-2-positive clots demonstrate that structural differences in fibrinogen, and not just differences in gross fibrinogen concentration, contribute to clinical differences in thrombotic features associated with COVID-19. These structural differences are at least in part mediated by differential sialylation.

The role of neutrophil extracellular traps in acute lung injury.

Acute lung injury (ALI) is a heterogeneous inflammatory condition associated with high morbidity and mortality. Neutrophils play a key role in the development of different forms of ALI, and the release of neutrophil extracellular traps (NETs) is emerging as a common pathogenic mechanism. NETs are essential in controlling pathogens, and their defective release or increased degradation leads to a higher risk of infection. However, NETs also contain several pro-inflammatory and cytotoxic molecules than can exacerbate thromboinflammation and lung tissue injury. To reduce NET-mediated lung damage and inflammation, DNase is frequently used in preclinical models of ALI due to its capability of digesting NET DNA scaffold. Moreover, recent advances in neutrophil biology led to the development of selective NET inhibitors, which also appear to reduce ALI in experimental models. Here we provide an overview of the role of NETs in different forms of ALI discussing existing gaps in our knowledge and novel therapeutic approaches to modulate their impact on lung injury.

Evidence of SARS-CoV-2 spike protein on retrieved thrombi from COVID-19 patients.

The pathophysiology of COVID-19-associated coagulopathy is complex and not fully understood. SARS-CoV-2 spike protein (SP) may activate platelets and interact with fibrin(ogen). We aimed to investigate whether isolated SP can be present in clots retrieved in COVID-19 patients with acute ischemic stroke (by mechanical thrombectomy) and myocardial infarction. In this pilot study, we could detect SP, but not nucleocapsid protein, on platelets of COVID-19 patients' thrombi. In addition, in all three COVID-19 thrombi analyzed for molecular biology, no SARS-CoV-2 RNA could be detected by real-time polymerase chain reaction. These data could support the hypothesis that free SP, besides the whole virus, may be the trigger of platelet activation and clot formation in COVID-19.

Cellular and Molecular Aspects of Blood Cell-Endothelium Interactions in Vascular Disorders.

In physiology and pathophysiology the molecules involved in blood cell-blood cell and blood cell-endothelium interactions have been identified. Platelet aggregation and adhesion to the walls belonging to vessels involve glycoproteins (GP), GP llb and GP llla and the GP Ib-IX-V complex. Red blood cells (RBCs) in normal situations have little interaction with the endothelium. Abnormal adhesion of RBCs was first observed in sickle cell anemia involving vascular cell adhesion molecule (VCAM)-1, α4ß1, Lu/BCAM, and intercellular adhesion molecule (ICAM)-4. More recently RBC adhesion was found to be increased in retinal-vein occlusion (RVO) and in polycythemia vera (PV). The molecules which participate in this process are phosphatidylserine and annexin V in RVO, and phosphorylated Lu/BCAM and α5 laminin chain in PV. The additional adhesion in diabetes mellitus occurs due to the glycated RBC band 3 and the advanced glycation end-product receptors. The multiligand receptor binds advanced glycation end products (AGEs) or S100 calgranulins, or ß-amyloid peptide. This receptor for advanced glycation end products is known as RAGE. The binding to RAGE-activated endothelial cells leads to an inflammatory reaction and a prothrombotic state via NADPH activation and altered gene expression. RAGE blockade is a potential target for drugs preventing the deleterious consequences of RAGE activation.

Insights Into Immunothrombotic Mechanisms in Acute Stroke due to Vaccine-Induced Immune Thrombotic Thrombocytopenia.

During the COVID-19 pandemic, vaccination is the most important countermeasure. Pharmacovigilance concerns however emerged with very rare, but potentially disastrous thrombotic complications following vaccination with ChAdOx1. Platelet factor-4 antibody mediated vaccine-induced immune thrombotic thrombocytopenia (VITT) was described as an underlying mechanism of these thrombotic events. Recent work moreover suggests that mechanisms of immunothrombosis including neutrophil extracellular trap (NET) formation might be critical for thrombogenesis during VITT. In this study, we investigated blood and thrombus specimens of a female patient who suffered severe stroke due to VITT after vaccination with ChAdOx1 in comparison to 13 control stroke patients with similar clinical characteristics. We analyzed cerebral thrombi using histological examination, staining of complement factors, NET-markers, DNase and LL-37. In blood samples at the hyper-acute phase of stroke and 7 days later, we determined cell-free DNA, myeloperoxidase-histone complexes, DNase activity, myeloperoxidase activity, LL-37 and inflammatory cytokines. NET markers were identified in thrombi of all patients. Interestingly, the thrombus of the VITT-patient exclusively revealed complement factors and high amounts of DNase and LL-37. High DNase activity was also measured in blood, implying a disturbed NET-regulation. Furthermore, serum of the VITT-patient inhibited reactive oxygen species-dependent NET-release by phorbol-myristate-acetate to a lesser degree compared to controls, indicating either less efficient NET-inhibition or enhanced NET-induction in the blood of the VITT-patient. Additionally, the changes in specific cytokines over time were emphasized in the VITT-patient as well. In conclusion, insufficient resolution of NETs, e.g. by endogenous DNases or protection of NETs against degradation by embedded factors like the antimicrobial peptide LL-37 might thus be an important factor in the pathology of VITT besides increased NET-formation. On the basis of these findings, we discuss the potential implications of the mechanisms of disturbed NETs-degradation for diagnostic and therapeutic approaches in VITT-related thrombogenesis, other auto-immune disorders and beyond.

Markers of endothelial cell activation are associated with the severity of pulmonary disease in COVID-19.

Severe coronavirus disease-19 (COVID-19) is characterized by vascular inflammation and thrombosis. We and others have proposed that the inflammatory response to coronavirus infection activates endothelial cells, leading to endothelial release of pro-thrombotic proteins. These mediators can trigger obstruction of the pulmonary microvasculature, leading to worsening oxygenation, acute respiratory distress syndrome, and death. In the current study, we tested the hypothesis that higher levels of biomarkers released from endothelial cells are associated with worse oxygenation in patients with COVID-19. We studied 83 participants aged 18-84 years with COVID-19 admitted to a single center. The severity of pulmonary disease was classified by oxygen requirement, including no oxygen requirement, low-flow oxygen, high-flow nasal cannula oxygen, mechanical ventilation, and death. We measured plasma levels of two proteins released by activated endothelial cells, von Willebrand Factor (VWF) antigen and soluble P-Selectin (sP-Sel), and a biomarker of systemic thrombosis, D-dimer. Additionally, we explored the association of endothelial biomarker levels with the levels of pro-inflammatory cytokine and chemokines, and vascular inflammation biomarkers. We found that levels of VWF, sP-sel, and D-dimer were increased in individuals with more severe COVID-19 pulmonary disease. Biomarkers of endothelial cell activation were also correlated with proinflammatory cytokines and chemokines. Taken together, our data demonstrate increased levels of VWF and sP-selectin are linked to the severity of lung disease in COVID-19 and correlated with biomarkers of inflammation and vascular inflammation. Our data support the concept that COVID-19 is a vascular disease which involves endothelial injury in the context of an inflammatory state.

Anti-Inflammatory Effects and Decreased Formation of Neutrophil Extracellular Traps by Enoxaparin in COVID-19 Patients.

Neutrophil Extracellular Traps (NETs) are a contributing factor of vascular thrombosis and alveolar damage in COVID-19 patients. As enoxaparin is currently used to inhibit vascular thrombosis, this study aimed to investigate whether enoxaparin also reduced inflammation and NETs in COVID-19 patients. Patients with COVID-19 infection were classified into three groups: mild, moderate, and severe (n = 10 for all groups). Plasma was collected from patients and healthy donors (n = 10). Neutrophils isolated from healthy controls were incubated with COVID-19 or healthy plasma, and with or without enoxaparin pretreatment in vitro. Neutrophils and plasma isolated from patients treated with enoxaparin were also investigated. The levels of inflammatory cytokines and NET products such as dsDNA, NE, MPO-DNA and Histone-DNA complexes in plasma and supernatants were measured using immunofluorescence staining and ELISA kits. The expression of inflammatory signaling genes by neutrophils (RELA, SYK, ERK and PKC) was measured using real-time qPCR. The levels of NET products were elevated in the plasma of COVID-19 patients, particularly in the severe group (p < 0.01). Moreover, plasma from the severe group enhanced NET formation (p < 0.01) from neutrophils in vitro. Enoxaparin pretreatment in vitro decreased plasma-induced NETs in a dose-dependent manner and down-regulated the expression of inflammatory genes (p < 0.05). Patients treated with prophylactic enoxaparin showed lower inflammatory cytokine levels and expression of inflammatory genes (p < 0.05). Increased NETs were associated with the severity of COVID-19 infection, particularly in patients with severe pneumonia, and could be used as biomarkers to assess disease severity. Enoxaparin pretreatment inhibited NETs and reduced the expression of inflammatory cytokines, and these effects mostly persisted in patients treated with prophylactic enoxaparin.

Platelet-monocyte interaction amplifies thromboinflammation through tissue factor signaling in COVID-19.

Accumulating evidence into the pathogenesis of COVID-19 highlights a hypercoagulability state with high risk of life-threatening thromboembolic complications. However, the mechanisms of hypercoagulability and their link to hyperinflammation remain poorly understood. Here, we investigate functions and mechanisms of platelet activation and platelet-monocyte interactions in inflammatory amplification during SARS-CoV-2 infection. We used a combination of immunophenotyping, single-cell analysis, functional assays, and pharmacological approaches to gain insights on mechanisms. Critically ill patients with COVID-19 exhibited increased platelet-monocyte aggregates formation. We identified a subset of inflammatory monocytes presenting high CD16 and low HLA-DR expression as the subset mainly interacting with platelets during severe COVID-19. Single-cell RNA-sequencing analysis indicated enhanced fibrinogen receptor Mac-1 in monocytes from patients with severe COVID-19. Monocytes from patients with severe COVID-19 displayed increased platelet binding and hyperresponsiveness to P-selectin and fibrinogen with respect to tumor necrosis factor-α and interleukin-1ß secretion. Platelets were able to orchestrate monocyte responses driving tissue factor (TF) expression, inflammatory activation, and inflammatory cytokines secretion in SARS-CoV-2 infection. Platelet-monocyte interactions ex vivo and in SARS-CoV-2 infection model in vitro reciprocally activated monocytes and platelets, inducing the heightened secretion of a wide panel of inflammatory mediators. We identified platelet adhesion as a primary signaling mechanism inducing mediator secretion and TF expression, whereas TF signaling played major roles in amplifying inflammation by inducing proinflammatory cytokines, especially tumor necrosis factor-α and interleukin-1ß. Our data identify platelet-induced TF expression and activity at the crossroad of coagulation and inflammation in severe COVID-19.

A central role for amyloid fibrin microclots in long COVID/PASC: origins and therapeutic implications.

Post-acute sequelae of COVID (PASC), usually referred to as 'Long COVID' (a phenotype of COVID-19), is a relatively frequent consequence of SARS-CoV-2 infection, in which symptoms such as breathlessness, fatigue, 'brain fog', tissue damage, inflammation, and coagulopathies (dysfunctions of the blood coagulation system) persist long after the initial infection. It bears similarities to other post-viral syndromes, and to myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS). Many regulatory health bodies still do not recognize this syndrome as a separate disease entity, and refer to it under the broad terminology of 'COVID', although its demographics are quite different from those of acute COVID-19. A few years ago, we discovered that fibrinogen in blood can clot into an anomalous 'amyloid' form of fibrin that (like other ß-rich amyloids and prions) is relatively resistant to proteolysis (fibrinolysis). The result, as is strongly manifested in platelet-poor plasma (PPP) of individuals with Long COVID, is extensive fibrin amyloid microclots that can persist, can entrap other proteins, and that may lead to the production of various autoantibodies. These microclots are more-or-less easily measured in PPP with the stain thioflavin T and a simple fluorescence microscope. Although the symptoms of Long COVID are multifarious, we here argue that the ability of these fibrin amyloid microclots (fibrinaloids) to block up capillaries, and thus to limit the passage of red blood cells and hence O2 exchange, can actually underpin the majority of these symptoms. Consistent with this, in a preliminary report, it has been shown that suitable and closely monitored 'triple' anticoagulant therapy that leads to the removal of the microclots also removes the other symptoms. Fibrin amyloid microclots represent a novel and potentially important target for both the understanding and treatment of Long COVID and related disorders.

Impact of COVID-19 on thrombus composition and response to thrombolysis: Insights from a monocentric cohort population of COVID-19 patients with acute ischemic stroke.

BACKGROUND: Resistance to fibrinolysis, levels of procoagulant/antifibrinolytic neutrophil extracellular traps (NETs), and the severity of acute ischemic stroke (AIS) are increased by COVID-19. Whether NETs are components of AIS thrombi from COVID-19 patients and whether COVID-19 impacts the susceptibility of these thrombi to thrombolytic treatments remain unknown, however. OBJECTIVES: We aimed to characterize AIS thrombi from COVID-19 patients by immunohistology and to compare their response to thrombolysis to that of AIS thrombi from non-COVID-19 patients. PATIENTS/METHODS: For this monocentric cohort study, 14 thrombi from COVID-19 AIS patients and 16 thrombi from non-COVID-19 patients, all recovered by endovascular therapy, were analyzed by immunohistology or subjected to ex vivo thrombolysis by tissue-type plasminogen (tPA)/plasminogen. RESULTS: COVID-19 AIS thrombi were rich in neutrophils and contained NETs, but not spike protein. Thrombolysis assays revealed a mean resistance profile to tPA/plasminogen of COVID-19 AIS thrombi similar to that of non-COVID-19 AIS thrombi. The addition of DNase 1 successfully improved thrombolysis by potentiating fibrinolysis irrespective of COVID-19 status. Levels of neutrophil, NETs, and platelet markers in lysis supernatants were comparable between AIS thrombi from non-COVID-19 and COVID-19 patients. CONCLUSIONS: These results show that COVID-19 does not impact NETs content or worsen fibrinolysis resistance of AIS thrombi, a therapeutic hurdle that could be overcome by DNase 1 even in the context of SARS-CoV-2 infection.

Janus Kinase Signaling Pathway and Its Role in COVID-19 Inflammatory, Vascular, and Thrombotic Manifestations.

Acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) infection continues to be a worldwide public health crisis. Among the several severe manifestations of this disease, thrombotic processes drive the catastrophic organ failure and mortality in these patients. In addition to a well-established cytokine storm associated with the disease, perturbations in platelets, endothelial cells, and the coagulation system are key in triggering systemic coagulopathy, involving both the macro- and microvasculatures of different organs. Of the several mechanisms that might contribute to dysregulation of these cells following SARS-CoV-2 infection, the current review focuses on the role of activated Janus kinase (JAK) signaling in augmenting thrombotic processes and organ dysfunction. The review concludes with presenting the current understanding and emerging controversies concerning the potential therapeutic applications of JAK inhibitors for ameliorating the inflammation-thrombosis phenotype in COVID-19 patients.