SARS-CoV-2 spike protein activates NOX2-p66SHC axis via inhibiting SLAMF8 to promote thrombogenesis

SARS-CoV-2 spike protein activates NOX2-p66SHC axis via inhibiting SLAMF8 to promote thrombogenesis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article SARS-CoV-2 spike protein activates NOX2-p66SHC axis via inhibiting SLAMF8 to promote thrombogenesis Quan Liu, XIZI LUO, Nan Liu, Liyan Sui, Siwei Zhang, Mengmeng Wang, and 12 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5572297/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract COVID-19 associated coagulation abnormalities and thrombosis are life-threatening complications after SARS-CoV-2 infection. However, the underlying mechanisms are unclear. Here, we found that SARS-CoV-2 spike (S) protein induced excessive reactive oxygen species (ROS) production, disrupting mitochondrial dynamics and causing endothelial cells damage, thereby promoting thrombogenesis. Mechanistically, the S protein inhibited the expression of signaling lymphocytic activation molecule family 8 (SLAMF8) to induce an upregulation of NADPH oxidase 2 (NOX2) expression and p66SHC phosphorylation. This activation of NOX2-p66SHC axis resulted in a persistent elevation of ROS and mitochondrial dynamics disorder, ultimately leading to endothelial cells injury. SARS-CoV-2 infection also promoted the transformation of endothelial cells into a prothrombotic phenotype by inhibiting the expression of SLAMF8. Furthermore, the single-cell sequencing analysis revealed a negative correlation between SLAMF8 expression and thrombotic activity of endothelial cells in COVID-19 patients. Notably, the overexpression of SLAMF8 reversed the S protein-mediated increase in blood flow obstruction and platelet aggregation observed in mice with ferric chloride-induced thrombosis. These findings suggest a distinct mechanism of the S protein in the pathogenesis of SARS-CoV-2-associated thrombosis, providing novel perspectives and strategies for the prevention, management and treatment of thrombotic complications in individuals with COVID-19 or long COVID. Health sciences/Pathogenesis/Infection Biological sciences/Microbiology/Virology/SARS-CoV-2 SARS-CoV-2 spike protein thrombogenesis SLAMF8 p66SHC Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction The COVID-19 pandemic, caused by the novel coronavirus SARS-CoV-2, has triggered an unprecedented global public health crisis. 1 While the majority of infected individuals experience mild to moderate illness, without the need for specialized treatment, a significant subset of patients faces serious complications. 2 These complications can include coagulopathy, thromboembolism, multiorgan failure, septic shock, and death, with acute symptoms typically lasting only 2 to 3 weeks. 3–6 Alarmingly, approximately 30% of patients develop persistent symptoms that endure for at least three months following the acute phase of COVID-19, leading to conditions referred to as long COVID, post-acute COVID-19 syndrome (PACS), or post-acute sequelae of COVID-19 (PASC). 7 Common symptoms associated with PASC include fatigue, flu-like manifestations, autonomic dysfunction, cognitive impairments, and post-exertional malaise. 8,9 Many pathophysiological processes contribute to the development of PASC, including viral persistence, immune dysregulation, mitochondrial dysfunction, complement system dysregulation, endothelial inflammation, and microbiome dysbiosis. 7,10 These mechanisms culminate in a long-term disorder characterized by thrombotic endothelialitis, hyperactivated platelets, and the formation of fibrin-like microclots. 11,12 Such vascular and coagulation abnormalities impact multiple organ systems and represent a unifying pathway for the diverse symptoms of PASC, highlighting the urgent need for a comprehensive understanding of the mechanisms driving thrombogenesis during SARS-CoV-2 infection. The spike (S) protein of SARS-CoV-2 plays a crucial role in mediating the thrombotic complications associated with the virus. 13 Its presence has been documented in thrombi from COVID-19 patients, and intriguing associations have been noted between the ChAdOx1 nCov-19 (AstraZeneca) vaccine and atypical thrombotic events. 14,15 Furthermore, the S protein may persist in various organs and even in feces for months following infection, raising critical questions about its potential role in the thrombotic complications associated with PASC. 16–18 Notably, the S protein can independently promote thrombosis by binding to endothelial cell surface receptors, such as angiotensin-converting enzyme 2 (ACE2) and cytoskeleton-associated protein 4 (CKAP4). 19,20 However, the precise mechanism by which the S protein induces endothelial cell injury and thrombogenesis remains inadequately understood. In this study, we demonstrate that the SARS-CoV-2 S protein inhibits the expression of SLAMF8, which subsequently leads to an upregulation of NOX2 expression and phosphorylation of p66SHC. This, in turn, causes mitochondrial damage and a persistent elevation of ROS, ultimately causing endothelial cell injury and promoting thrombosis. Importantly, overexpression of SLAMF8 effectively reverses S protein-induced thrombogenesis in vivo. Our findings unveil a novel mechanism by which the S protein exerts pro-thrombotic effects, suggesting potential intervention strategies for addressing SARS-CoV-2 infection and post-acute sequelae of COVID-19 (PASC). Results SARS-CoV-2 S protein promotes thrombogenesis, enhances ROS production and mitochondrial fission in endothelial cells To investigate the mechanisms underlying SARS-CoV-2-associated thrombosis, we used the recombinant SARS-CoV-2 S protein to treat human aortic endothelial cells (HAEC), thereby simulating the effects of SARS-CoV-2 infection (Figure S1A and B). Following exposure to the S protein, we observed a significant upregulation of the endothelial cell injury factor von Willebrand factor (VWF), the adhesion molecules including E-selectin (SELE), P-selectin (SELP), intercellular cell adhesion molecule (ICAM)-1, as well as the thrombotropic factors including the coagulation factor III (TF) and the plasminogen activator inhibitor-1 (PAI-1) at both mRNA and protein levels in HAECs (Figure S1C-E, and Figure 1A). Additionally, the adhesion of monocytes to HAECs was markedly enhanced after the S protein treatment, which is a critical step in the prothrombotic transformation of endothelial cells (Figure 1B). These findings indicate that exposure to the S protein stimulates the secretion of thrombogenic factors from endothelial cells. We also found a significant increase in ROS production in HAECs following S protein exposure (Figure 1C and D). Elevated ROS levels can lead to mitochondrial dysfunction, which is a key contributor to endothelial cell injury. 21 Indeed, we found that treatment with the S protein resulted in a decrease in the mitochondrial membrane potential, a hallmark indicative of mitochondrial dysfunction (Figure 1E, Figure S2A and B). Furthermore, exposure to the S protein caused a reduction in mitochondria length and an increase in mitochondrial fragmentation within endothelial cells (Figure 1F), without affecting overall mitochondrial mass (Figure S2C and D). This suggests that the S protein may induce mitochondrial damage by disrupting the homeostasis of mitochondrial dynamics. Mitochondrial dynamics is a critical physiological process that encompasses both mitochondrial fusion and fission. Mitochondrial fusion is regulated by the outer membrane fusion protein mitofusins 1 and 2 (MFN1 and 2) and the inner membrane fusion protein optic atrophy 1 (OPA1), 22,23 while fission is mediated by dynamin family GTPases, including mitochondrial fission 1 protein (FIS1) and dynamin-related protein 1 (DRP1). 23 Phosphorylation of DRP1 at serine 616 (pDRP1-S616) activates DRP1 and promotes its translocation to mitochondria, thereby facilitating mitochondrial fission. Conversely, phosphorylation at serine 637 (pDRP1-S637) inhibits mitochondrial fission by reducing DRP1's association with the mitochondria. We found that S protein treatment resulted in an upregulation of pDRP1-S616 and a downregulation of pDRP1-S637 and OPA1, leading to shortened mitochondrial length and disrupted mitochondrial dynamics (Figures 1G and S2E). These findings demonstrate that the S protein enhances ROS production to promote mitochondrial fission in endothelial cells, creating a highly conducive environment for thrombogenesis. Inhibition of ROS elevation and mitochondrial fission reverse thrombogenesis induced by S protein To explore whether the surge in ROS serves as a critical driver of mitochondrial damage and the pro-thrombotic phenotype induced by the SARS-CoV-2 S protein, we introduced the ROS scavenger mitoquinone (MitoQ). Our results demonstrated that MitoQ effectively mitigated the ROS levels induced by the S protein (Figure 2A). Additionally, treatment with MitoQ reversed the S protein-induced decrease in mitochondrial membrane potential, reduced mitochondrial fragmentation, and inhibited the expression of mitochondrial fission-related protein DRP1 and FIS1 (Figures S3A, 2B and C). To determine whether restoring mitochondrial function by reducing ROS levels could counteract the endothelial cells injury and thrombotic phenotype elicited by the S protein, we assessed thrombosis-related phenotypic changes in HAECs treated with MitoQ following S protein stimulation. The addition of MitoQ effectively decreased both mRNA and protein levels of critical thrombotic factors in endothelial cells, including coagulation factor III, PAI-1, and adhesion molecules such as ICAM, SELE, SELP, as well as the injury factor VWF induced by the S protein (Figure S3B and C, Figure 2D and E). Furthermore, MitoQ treatment significantly reduced the adhesion of monocytes to HAECs (Figure 2F). Our findings indicate that inhibiting the ROS surge triggered by the S protein can prevent the development of thrombosis by mitigating excessive mitochondrial fission. To further elucidate whether excessive mitochondrial fission induced by the S protein is a key factor in promoting thrombogenesis, we employed the mitochondrial fission inhibitor Midivi-1. Our findings revealed that Midivi-1 effectively reversed the mRNA and protein elevation induced by the S protein, including the thrombotic factor PAI-1 and coagulation factor III, the adhesion factor ICAM-1, as well as SELE and SELP, and the damage factor VWF (Figure S4, Figure 2G and H). Moreover, Midivi-1 treatment reversed the increased adhesion of monocytes to HAECs induced by the S protein (Figure 2I). Collectively, these results demonstrate that inhibiting excessive mitochondrial fission trigged by the S protein can alleviate the pro-thrombotic phenotype in endothelial cells. S protein promotes endothelial cells injury by inhibiting SLAMF8 expression To elucidate the mechanism by which the SARS-CoV-2 S protein promotes thrombogenesis through oxidative stress, we conducted a transcriptome analysis, revealing that the differentially expressed gene (DEGs) were significantly enriched in pathways associated with hydrogen peroxide metabolism and cellular responses to oxygen-containing compounds (Figure S5A-C, and Appendix Table 1). Notably, we identified and validated that the S protein markedly suppressed the expression of SLAMF8 (Figure S5D), a member of the signaling lymphocytic activation molecule (SLAMF) family, which plays a crucial role in regulating immune cell activation and cytokine production. 24 To explore whether SLAMF8 is responsible for the increased ROS levels, mitochondrial damage, and thrombogenesis caused by the S protein, we overexpressed SLAMF8 in HAECs prior to S protein stimulation. The results demonstrated that ectopic expression of SLAMF8 effectively reversed the S protein-induced increase in ROS levels and loss of mitochondrial membrane potential (Figures S5E and 3A). Furthermore, SLAMF8 overexpression attenuated S protein-induced mitochondrial fragmentation (Figure 3B) and inhibited the phosphorylation of DRP1 at serine 616 (pDRP1-S616), while promoting phosphorylation of at serine 637 (pDRP1-S637) and increasing OPA1 expression (Figures 3C and S5F). Additionally, we observed that SLAMF8 overexpression led to the decreased mRNA and protein levels of key thrombotic factors, including coagulation factor III and PAI-1, as well as adhesion molecules such as SELE, SELP, and ICAM-1, along with the damage factor VWF, thereby reducing endothelial cell adhesion (Figure S6A, Figures 3D-F). These findings indicate that the S protein contributes to excessive ROS production and subsequent mitochondrial damage by suppressing SLAMF8, ultimately resulting in endothelial cells injury that promotes thrombosis in context of COVID-19 and long COVID. S protein activates NOX2-p66SHC axis to induce oxidative stress To investigate how the SARS-CoV-2 S protein inhibits SLAMF8 to induce oxidative stress, we focused on NOX2, a membrane-bound protein that generates superoxide anions and plays a pivotal role in ROS production. 25 Elevated ROS levels lead to the phosphorylation of p66SHC through the PKC or ERK/JNK signals, which promotes mitochondrial fission and dysfunction, ultimately resulting in increased production of mitochondrial-derived ROS, thereby creating a vicious cycle of ROS overproduction (Figure 4A). 26,27 We hypothesized that the suppression of SLAMF8 by the S protein could activate the NOX2-p66SHC axis, enhancing ROS production and mitochondrial fission. Our results demonstrated that exposure to the S protein significantly increased NOX2 secretion and p66SHC phosphorylation, while SLAMF8 overexpression effectively reversed these changes (Figures 4B and C). Furthermore, we found that the NOX2 inhibitor gp91ds-tat reduced both p66SHC phosphorylation and ROS generation induced by the S protein (Figures 4D, S6B). Additionally, treatment with gp91ds-tat diminished the expression of adhesion molecules SELE, SELP, and ICAM, as well as reduced PAI-1 levels and coagulation factor III secretion (Figures 4E-G). This treatment also led to decreased monocyte adhesion to endothelial cells (Figure 4H). Collectively, these data indicate that the S protein activates the NOX2-p66SHC cascade by inhibiting SLAMF8, thereby triggering an oxidative stress response that contributes to the pathogenesis of thrombosis. Knockdown of p66SHC abolishes endothelial cells injure and thrombogenesis To elucidate the role of the S protein in promoting the phosphorylation of p66SHC via NOX2 and its subsequent effects on mitochondrial fission, we silenced p66SHC in endothelial cells. Our results demonstrated that p66SHC knockdown effectively prevented the S protein-induced increase in ROS and the associated decline in mitochondrial membrane potential (Figures 5A and B). Consistent with these findings, p66SHC knockdown reversed the alterations in the mitochondrial dynamics-related proteins, including DRP1, FIS1, and OPA1, that were induced by the S protein (Figure 5C). Moreover, p66SHC knockdown mitigated the thrombotic phenotype triggered by the S protein, as evidenced by reduced expression levels of thrombotic factor PAI-1 and coagulation factor III, the adhesion molecules SELE, SELP, and ICAM, as well as endothelial cells’ adhesion ability (Figures 5D-F). Our findings indicate that the S protein caused endothelial cells damage through activating p66SHC to promote thrombogenesis. SARS-CoV-2 infection promotes thrombogenesis by inhibiting SLAMF8 in endothelial cells We further determined whether SARS-CoV-2 infection also promotes oxidative stress and thrombosis in endothelial cells through inhibiting SLAMF8 expression. Our findings revealed a significant decrease in SLAMF8 expression in SARS-CoV-2-infected endothelial cells (Figure 6A). Consistent with the effects observed following the S protein treatment, SARS-CoV-2 infection increased the expression of thrombotic factors, including coagulation factor III and PAI-1, along with the damage factor VWF and adhesion molecules SELE, SELP, and ICAM. Notably, these changes were reversed by SLAMF8 overexpression (Figures 6B-D). Moreover, we observed that SARS-CoV-2 infection led to increased ROS production and mitochondrial fragmentation in HAECs, and these effects were mitigated by SLAMF8 overexpression (Figures 6E, F, and S6C). These results suggest that SARS-CoV-2 infection inhibits SLAMF8 expression, resulting in enhanced ROS overproduction, mitochondrial damage, and the induction of a thrombotic phenotype in endothelial cells. Overexpression of SLAMF8 reverses S protein-induced thrombogenesis in vivo To validate the role of SLAMF8 in S protein-induced thrombogenesis in vivo , we constructed adeno-associated virus expressing human SLAMF8 (pAAV-SLAMF8) and a control virus (pAAV-Ctrl) under the endothelial cell specific Tie1 promoter. Equivalent dose of pAAV-SLAMF8 and pAAV-Ctrl were injected to C57BL/6 mice via the tail vein to achieve specific overexpression of SLAMF8 in endothelial cells. Immunoblotting confirmed a significant enhancement of SLAMF8 expression in the endothelium of mesenteric arteries from pAAV-SLAMF8-injected mice (Fig. 7A). We monitored thrombus formation using a live cell workstation and found that the S protein exacerbated the blood flow obstruction and platelet aggregation in the FeCl 3 -induced thrombus model (Fig. 7B and C), providing evidence that the S protein promotes thromboembolism in vivo . Importantly, overexpression of SLAMF8 in endothelial cells reversed the S protein-induced increase in blood flow obstruction and platelet aggregation (Fig. 7B and C). Additionally, SLAMF8 overexpression reduced the ROS generation caused by the S protein, thereby inhibiting thrombosis in vivo (Fig. 7D). These findings highlight the protective role of SLAMF8 in counteracting the thrombotic consequences of SARS-CoV-2 infection. SLAMF8 expression is negatively correlated with thrombosis risk in COVID-19 patients To further explore the relationship between SLAMF8 and thrombosis in COVID-19 patients, we examined the expression of thrombosis-related genes and SLAMF8 in these individuals. As shown in Figures 8A-C, the expression levels of thrombosis-related genes PAI-1 and SELP were significantly elevated in the pulmonary endothelial cells of COVID-19 patients compared to healthy controls, while SLAMF8 expression was markedly reduced. Furthermore, SLAMF 8 expression exhibited a strong negative correlation (R=-0.58) with the thrombosis score in patients with SARS-CoV-2 infections compared to normal subjects (Figures 8D and E). Discussion Reduced blood flow, endothelial damage, and hypercoagulability are recognozied as primary drivers of thromboembolism. 28 Numerous viruses, including cytomegalovirus (CMV), herpesviruses, influenza A virus (IAV), and coronaviruses such as SARS-CoV, SARS-CoV-2 and Middle East respiratory syndrome CoV (MERS-CoV), are known to trigger thrombosis. This process is typically are characterized by the activation of factor X and the production of tissue factor and VWF from endothelial cells. 29–35 In our study, we found that SARS-CoV-2 enhances thrombogenesis by inducing the expression of tissue factor, VWF, and PAI-1 in endothelial cells. Mechanistically, the S protein of SARS-CoV-2 activates the NOX2-p66SHC axis by inhibiting SLAMF8, leading to persistent ROS elevation, mitochondrial dynamics disruption, endothelial cells injury, and ultimately thrombogenesis. These findings elucidate the underlying mechanisms and identify potential therapeutic targets for thrombotic complications associated with SARS-CoV-2 infection. Recent studies suggest that COVID-19-related thrombosis is largely due to abnormalities in the coagulation system triggered by SARS-CoV-S protein. 13,14 The S protein binds to ACE-2 on platelets, leading to the release of coagulation factors and promoting leukocyte-platelet aggregate formation. 36 Additionally, it competitively inhibits the binding of antithrombin and heparin cofactor II to heparin/HS, resulting in abnormal thrombin activity. 37 The S protein also fosters a thrombogenic environment by activating NF-κB signaling, which induces endothelial inflammation and barrier dysfunction. 38–40 Our study identified that the S protein induces oxidative stress in endothelial cells via the NOX2-p66SHC axis, resulting in a prothrombotic phenotype. Patients with long COVID frequently present with fibrin amyloid microclots, which can cause tissue hypoxia and hinder oxygen exchange. 41,42 This tissue hypoxia stimulates ROS generation, exacerbating oxidative stress. 43 Elevated ROS levels under pathological conditions are crucial for thrombosis development due to their role in mitochondrial damage. 44,45 Our study found that the S protein induces excessive ROS production in endothelial cells, disrupting mitochondrial dynamics and significantly contributing to endothelial cell injury, a key factor in the thrombogenesis. The SLAMF family, comprising members SLAMF1 to SLAMF9, plays critical roles in regulating immune cell activation and cytokine production. 24 SLAMF8, a type I cell surface glycoprotein, modulates the tumor immune microenvironment and is considered a promising therapeutic target for various conditions, including cancer, mastocytosis, acute hepatic injury, and cardiac ischemia/reperfusion injury. 46–52 Mechanistically, SLAMF8 upregulates Toll-like receptor-4 (TLR4) in macrophages to promote inflammatory cytokine secretion, 47,53 and reduces NADPH oxidase subunit phosphorylation, inhibiting macrophage microbicidal activity during bacterial infections. 54 Recent studies have shown that FOXJ2 upregulates SLAMF8 in β2GPI/anti-β2GPI-treated monocytes in antiphospholipid syndrome, where SLAMF8 interacts with TREM1 to stimulate TLR4/NF-κB signaling, inhibit autophagy, and release inflammatory and thrombotic markers. 55 Our study demonstrates SLAMF8 negatively regulates thrombosis induced by SARS-CoV-2 S protein, with a negative correlation between its expression and thrombotic activity in COVID-19 patients. Overexpression of SLAMF8 in endothelial cells counteracts the thrombotic phenotype triggered by the S protein and live virus, highlighting its potential as a therapeutic target for COVID-19-related thrombotic complications. NOX2, an NADPH oxidase, is instrumental in ROS production via electron transport. 56 NOX2-derived ROS stimulate mitochondria to produce additional ROS, a process that is regulated by the adapter protein p66SHC. 26,57 Under physiological conditions, electrons are seamlessly transferred through complexes I-IV of the electron transport chain, ultimately resulting in the formation of water molecules. However, stressors like oxidative stress or UV irradiation cause p66Shc to translocate to mitochondria, redirecting electron transfer pathway and generating superoxide. 58,59 Our study found that inhibiting NOX2 or knocking down p66SHC mitigated the increase in ROS and the upregulation of thrombogenic factors, demonstrating that SLAMF8 is responsible for increasing NOX2 expression and p66SHC phosphorylation induced by the S protein. Current treatments for SARS-CoV-2-associated thrombosis are primarily symptomatic and supportive. 60 However, potential therapies such as sulodexide, triple anticoagulant therapy, Pycnogenol®, and heparin-induced extracorporeal lipoprotein (HELP) apheresis have shown antithrombotic efficacy by improving endothelial function, and reducing inflammatory and oxidative stress markers. 61–64 Our study revealed that overexpression of SLAMF8 in endothelial cells can reverse S protein-mediated blood flow obstruction and platelet aggregation, suggesting SLAMF8 could be a viable target for addressing SARS-CoV-2-induced coagulation anomalies and thrombosis. In summary, our study demonstrates that that the SARS-CoV-2 S protein suppresses SLAMF8 expression, thereby activating the NOX2-p66SHC axis. This activation induces a sustained increase in ROS, disrupts mitochondrial dynamics, and results in endothelial cell damage and thrombosis. These findings reveal a critical molecular mechanism through which the SARS-CoV-2 S protein contributes to endothelial cell thrombosis, offering new insights for the treatment and prevention of coagulation abnormalities in both COVID-19 and long COVID patients. Materials and Methods Antigen, antibodies, plasmids, and drugs The SARS-CoV-2 (2019-nCoV) spike protein (40589-V08B1) was obtained from Sino Biological. The following antibodies were used in our experiments: His (66005-1-IG), OPA1 (27733-1-AP), FIS1 (10956-1-AP), PAI-1 (13801-1-AP), SELP (83947-5-RR), ICAM-1 (10831-1-AP), GAPDH (10494-1-AP), MFN1 (13798-1-AP), MFN2 (12186-1-AP) and Flag (20543-1-AP) from ProteinTech; DRP1 (12957-1-AP), Phospho-DRP1 S616 (ab314755), Phospho-DRP1S637 (ab193216) and SELE (ab180782) from Abcam. CoraLite 594-(RGAR004) and 488-conjugated IgG (GB2AF488) secondary antibodies were also sourced from ProteinTech. The plasmid pAAV-TIEIP-SLAMF8-3×FLAG-P2A-WPRE were purchased from OBiO Biotechnology. Mitoquinone (MitoQ) (S8978) and gp91ds-tat (P1213) were obtained from Selleck, while mitochondrial division inhibitor 1 (Mdivi-1) (HY-15886) was acquired from MCE. Cell culture and transfection Human aortic endothelial cells (HAECs) and THP-1 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) (HyClone) supplemented with 10% fetal bovine serum (BBI), 100 U/ml ampicillin, and 100 µg/ml streptomycin (Sangon). Transfection of plasmids was conducted following the manufacturer’s instructions using Hieff Trans® Polyethylenimine Linear (PEI) (YEASON). For siRNA transfections RNAimax (Invitrogen) was utilized according to the manufacturer’s guidelines. Non-targeting control siRNA and SHC siRNAs were purchased from GenePharma. Monocyte adhesion assay THP-1 cells were harvested and suspended in serum-free RPMI-1640 medium. CFSE fluorescent probes were added to the cell suspension and incubated for 15 min in a CO 2 incubator to label cells. Following the incubation, an equal volume of RPMI-1640 medium containing 20% fetal bovine serum was added to terminate the labeling reaction. CFSE-labeled THP-1 cells were then collected and washed with PBS. The cells were suspended in RPMI-1640 medium containing 10% fetal bovine serum and co-cultured with HAECs in the dark for 4 h. After the co-culture period, unbound THP-1 cells were removed by washing the cells with PBS three to five times. The remaining cells were suspended in an appropriate volume of PBS. The binding of THP-1 cells to HAECs were visualized using a fluorescence microscope (Olympus IX71) allowing for the assessment of monocyte adhesion. Mitochondrial membrane potential detection Mitochondrial membrane potential was detected using the Enhanced Mitochondrial Membrane Potential Assay Kit with JC-1 (Beyotime) following the manufacturer’s instruction. Briefly, cells were cultured in a JC-1 containing medium at 37°C for 20 minutes, after which they were collected and washed twice with PBS. The uptake and distribution of JC-1 within the cells were visualized using a fluorescence microscope. Cellular reactive oxygen species detection Cellular reaction oxygen species (ROS) was measured using the Reactive Oxygen Species Assay Kit (YEASEN) in accordance with the manufacturer’s protocol. Briefly, cells were incubated with the ROS indicator DCFH-DA at 37°C for 20 minutes and subsequently washed three times with PBS. The levels of ROS were analyzed using either fluorescence microscopy or a microplate reader. For ROS staining in mouse tissues, frozen tissue sections were prepared and incubated with the Hydroethidine probe (YEASEN) in the dark for 30 minutes. The sections were then decolorized, washed and mounted. ROS levels in the tissue sections were analyzed with a fluorescence microscope. Immunofluorescent staining Cells were fixed with 4% paraformaldehyde and subsequently permeabilized using 0.5% Triton X-100 and stained with primary antibodies. Afterwards, the cells were incubated with CoraLite 594- or 488-conjugated IgG secondary antibodies. The cell nuclei were stained with DAPI (YEASON). Fluorescence images were acquired and analyzed using a confocal microscope (FV3000, OLYMPUS). Coagulation factor III detection The concentration of coagulation factor III was detected using the Human Coagulation Factor III/Tissue Factor ELISA Kit (Beyotime) according to manufacturer’s instruction. Briefly, cell culture supernatants were added to a 96-well plate coated with human coagulation factor III antibody and incubated for 2 hours. Following incubation, the wells were washed and incubated with biotinylated human coagulation factor III antibodies for an additional hour. After extensive washing (five times), the concentration of coagulation factor III was determined using horseradish peroxidase (HRP)-conjugated streptavidin, with 3,3’,5,5’-tetramethyl-benzidine (TMB) serving as the substrate. NOX2 concentration detection The concentration of human NADPH oxidase 2 (NOX2) was measured using an ELISA kit obtained from Elisa Biotech. Briefly, the cell culture supernatant was added to a 96-well plate coated with human NOX2 antibodies and incubated for 2 hours. After incubation, the wells were washed and then incubated with biotinylated human NOX2 antibodies for an additional hour. Following extensive washing, the concentration of NOX2 was determined using HRP-conjugated streptavidin, with TMB as the substrate for detection. Quantitative real-time PCR (qPCR) Total RNA was isolated using the EasyPure RNA Kit (TransGen) following the manufacturer's instructions. Subsequently, reverse transcription was performed using cDNA Synthesis SuperMix kit (TransGen). All qPCR reactions were conducted using SYBR green PCR master mix (Roche) on a Real-Time PCR Detection System (Applied Biosystems). The qPCR results were analyzed using the comparative threshold cycle (Ct) method, with normalization to an endogenous glyceraldehyde phosphate dehydrogenase (GAPDH) as the reference gene and relative to an experimental control. The fold change in relative expression levels was calculated using the formula 2 −△△Ct . 65 Primer information is provided in Appendix Table 2. RNA sequencing and analysis RNA sequencing was conducted on HAECs exposed to the SARS-CoV-2 S protein and those not exposed. After 24 hours of treatment with SARS-CoV-2 S protein at a concentration of 10 nM, the cells were collected for RNA sequencing at Kidio Bio. Differentially expressed genes (DEGs) between the groups were analyzed using the Deseq2 package in R software, applying the criteria of |log (fold change) |>1 and P < 0.05. Gene ontology and KEGG enrichment analyses were conducted using the cloud analytics platforms provided by Kidio Bio. Immunoblotting analysis Cells were collected, washed with cold PBS, and lysed in a lysis buffer containing 10 mM Tris/Cl pH 7.5, 150 mM NaCl, 2 mM MgCl 2 , 0.5% NP-40 supplemented with a cocktail of protease and phosphatase inhibitors (Thermo Fisher Scientific) at 4 ℃ for 30 minutes. The cell lysates were then centrifuged at 12,000 g for 15 min at 4 ℃, and the protein concentration was quantified using Pierce™ BCA Protein Assay Kits (Thermo Fisher Scientific). For electrophoresis, 20–30 µg of protein from each sample was separated by 8–15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore) using a Trans-Blot System (Bio-Rad). After transfer, the membranes were blocked with 2% BSA (VETEC) in PBS containing 0.2% Tween-20 (PBST) and then incubated with appropriate primary antibody at 4 ℃ overnight. Following washing, the membranes were incubated with HRP-conjugated secondary antibodies, and antibody-antigen complexes were visualized using a chemiluminescent substrate (ECL) (Millipore). Grayscale analysis of the bands was performed using ImageJ software. Viral infection SARS-CoV-2 (BetaCoV/Beijing/IME-BJ05/2020) was isolated from a patient with viral pneumonia in Wuhan China. HAECs were infected with SARS-CoV-2 at a multiplicity of infection (MOI) of 0.01 at 37 ℃ in serum-free DMEM (high glucose) for 1 hour. After the infection period, the infected cells were washed twice with PBS and cultured in DMEM containing 2% FBS for 24 hours to prepare for subsequent experiments. All experiments involving infectious SARS-CoV-2 were performed in a biosafety level 3 (BSL3) laboratory at the Changchun Veterinary Research Institute of Chinese Academy of Agriculture Sciences. Animal experiments All procedures were approved by the Institutional Animal Care and Use Committee at Shengchang Biotechnology Co., LTD with the ethical batch number 2024-04-JLDX-LXZ-091, and conformed to the legal mandates and federal guidelines for the care and maintenance of laboratory animals. C57BL/6 mice (female; 6–8 weeks old; Vital River, China) were maintained and treated under specific pathogen-free conditions. Mice were injected with either pAAV-SLAMF8 or pAAV-Ctrl via the tail vein, and were observed for a duration of 2 weeks. Following this observation period, tissues were collected for subsequent experiments. Thrombosis mouse model The SARS-CoV-2 S protein was administrated to C57BL/6 mice at a dosage of 80 µg/kg via tail vein injection, occurring 10 minutes prior to the induction of thrombosis using ferric chloride (FeCl 3 ). Following anesthesia, a precise incision was made in the abdominal region of the mice to allow for the separation and exposure of the mesentery. A piece of filter paper saturated with ferric chloride solution was then applied to the surface of the mesenteric vein for a duration of 20 minutes. After the application period, the mesentery was repositioned, and the abdominal incision was carefully closed in layered using sutures. The formation of the thrombus was monitored in real-time using a live cell workstation. Hematoxylin and Eosin (H&E) Staining Following the collection of tissue samples from mice, the tissues were immediately immersed in precooled PBS to ensure thorough rinsing. Excess PBS was carefully absorbed using clean filter paper, and the tissues were subsequently fixed in 4% paraformaldehyde for a minimum of 24 hours. After fixation, the tissue underwent a graded dehydration process. Once dehydration was complete, the tissues were embedded in paraffin wax, and 5 µm-thick sections were prepared. These sections were then deparaffinized to remove the wax, followed by staining with hematoxylin and eosin to enhance cellular features. After staining, the sections were dehydrated through a graded series of ethanol, cleared with xylene, and finally mounted using a neutral resin. Once the mounting medium was set, the slides were allowed to air-dry naturally. The dried slides were then prepared for microscopic observation and photography. Clinical correlation analysis The snRNA-Seq data were obtained from the Human Lung Cell Atlas, 66 which includes raw and normalized counts, integrated embedding, cell type annotations and clinical and technical metadata. This data is publicly available for download via cellxgene ( https://cellxgene.cziscience.com/collections/6f6d381a-7701-4781-935c-db10d30de293 ). The expression matrix in “.h5ad” format was downloaded and filtered to isolate endothelial cells from both COVID-19 and normal subjects using the scanpy library. 67 The GMT file for the Human gene set HP_ABNORMAL_THROMBOSIS (M35560) was obtained from the Molecular Signature Database ( www.gsea-msigdb.org ). After converting the expression matrix to a Seurat Object, the AddModuleScore function from the Seurat Package (v4) was employed to calculate module scores. 68 Subsequently, the expression levels of SLAMF8 were plotted against the scores for "Abnormal_thrombosis" using the ggplot2 package. The Pearson coefficient was then calculated to assess the relationship between SLAMF8 expression and the module scores. Statistical analyses Data are presented as the mean ± standard deviation (SD), with “n” representing the number of independent replicates. Statistically significant differences were assessed using one-way or two-way analysis of variance (ANOVA), accompanied by multiple comparisons correction. Significance levels are indicated by asterisks as follows: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, while “ns” denotes non-significant differences. Declarations Acknowledgements This work was supported by grant from Project supported by National Key R&D Program of China (Grant No. 2022YFF1203204), the Joint Funds of the National Natural Science Foundation of China (Grant No. U23A20269), the National Natural Science Foundation of China (Grant No. 82372250, 82302516, and 82341105), and the Medical Innovation Team Project of Jilin University (Grant No. 2022JBGS02). DATA AVAILABILITY The RNA sequencing data for this article are available in the GEO repository with accession number GSE189706. The data supporting the conclusions of this article are included within the article and its additional files or available from the authors upon reasonable request. Competing interests The authors declare no competing interests. References Chan, J.F., Yuan, S., Chu, H., Sridhar, S. & Yuen, K.Y. COVID-19 drug discovery and treatment options. Nat Rev Microbiol 22 , 391-407 (2024). Meijer, S.E. , et al. Persistent COVID-19 in immunocompromised patients-Israeli society of infectious diseases consensus statement on diagnosis and management. Clin Microbiol Infect 30 , 1012-1017 (2024). Poor, H.D. Pulmonary Thrombosis and Thromboembolism in COVID-19. Chest 160 , 1471-1480 (2021). Spyropoulos, A.C. , et al. 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Additional Declarations There is NO Competing Interest. Supplementary Files Appendixtable1.xlsx Appendix Table 1 Appendixtable2.xlsx Appendix Table 2 supplementarymaterials.docx Supplementary materials Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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12:35:36","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5572297/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5572297/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":72910778,"identity":"d74ee2fc-7cfa-4dc2-aa2c-dd00b168266a","added_by":"auto","created_at":"2025-01-03 14:45:02","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":640102,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSARS-CoV-2 S protein promotes thrombogenesis and enhances ROS production and mitochondrial fission in endothelial cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A) \u003c/strong\u003eHuman airway epithelial cells (HAECs) were left untreated (CTR) or treated with varying doses of the S protein as indicated. The levels of coagulation factor III in the supernatants were measured by ELISA.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(B)\u003c/strong\u003e HAECs treated as described in (A) were incubated with the CFSE-labeled THP-1 cells for 45 min. After washing away unbound cells, the adhered THP-1 cells were visualized using fluorescence microscopy. The number of bound THP-1 cells in three randomly selected images was quantified using Image J.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(C\u003c/strong\u003e and \u003cstrong\u003eD)\u003c/strong\u003e HAECs were either untreated or treated with the S protein. Cells were subjected to immunofluorescence using the DCFH-DA probe (green signal indicating ROS levels) and anti-His antibody (red signal for the S protein). Nuclei were counterstained with DAPI in panel (C). Cells were stained with DCFH-DA and detected the fluorescence intensity using a fluorescent microplate reader (D).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(E)\u003c/strong\u003e HAECs were treated as described in (A). The viable cells were stained with the JC-1 probe to detect the mitochondrial membrane potential (MMP). In healthy mitochondria, JC-1 accumulates in the mitochondrial matrix, emitting red fluorescence, while in unhealthy mitochondria, JC-1 exist as monomers, producing green fluorescence. The degree of mitochondria depolarization was measured by calculating the ratio of red to green fluorescence intensity.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(F)\u003c/strong\u003e HAECs, either untreated or treated with S protein for 6 h, underwent immunofluorescence staining with MitoTracker-Red (MTR) and an anti-His antibody, with nuclei counterstained using DAPI. Statistical analysis of the mean branch length of mitochondria was performed using Image J (n ≥ 6).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(G)\u003c/strong\u003e HAECs were either untreated or treated with S protein and subsequently subjected to immunoblotting. The Grayscale analysis of phosphorylated DRP1 relative to total DRP1 is presented. Data are expressed as the mean ± SD of at least three independent experiments, with asterisks indicating significant difference (*\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.01, and ****\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001).\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-5572297/v1/c4240192cfebe3d5f839fb13.png"},{"id":72910021,"identity":"985f15ee-c680-49d5-8354-5154edf76a2c","added_by":"auto","created_at":"2025-01-03 14:37:02","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":805221,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInhibition of ROS elevation and mitochondrial fission reverses the pro-thrombotic phenotype induced by S protein.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A-F)\u003c/strong\u003eHuman airway epithelial cells (HAECs) were incubated with S protein for 6 h, and subsequently treated with either 10 μM of the ROS scavenger MitoQ or DMSO for an additional 6 h. Viable cells were stained with DCFH-DA to assess ROS levels, which were quantified using a fluorescent microplate reader (A). Cells were subjected to immunofluorescence staining with MitoTracker-Red (MTR) and anti-His antibody. Statistical analysis of the mean branch length of mitochondria is presented on the right (n ≥6) (B). The phosphorylation and expression of proteins involved in mitochondrial fission and fusion were analyzed by immunoblotting. Grayscale analysis of phosphorylated DRP1 relative to total DRP1, as well as OPA1 and FIS1 relative to GAPDH, is shown (C). Protein levels of PAI-1, SELE, SELP, and ICAM-1 were analyzed by immunoblotting, with corresponding grayscale statistical analysis relative to GAPDH are displayed (D). The levels of coagulation factor Ⅲ in the supernatants were measured using ELISA (E).\u003cstrong\u003e \u003c/strong\u003eCells were incubated with the CFSE-labeled THP-1 cells for 45 min, and the bound THP-1 cells were visualized using fluorescence microscopy. Statistical analysis of the number of bound THP-1 cells in three randomly selected images was performed using Image J (F).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(G-I) \u003c/strong\u003eHAECs that incubated with S protein for 6 h were treated with either 10 μM of Mdivi-1 or DMSO for an additional 6 h. The levels of coagulation factor Ⅲ were assessed (G). HAECs treated as indicated in (G) underwent immunoblotting to analyze the protein levels of SELP, PAI-1, SELE, and ICAM-1 (H). The adhesion of THP-1 cells to HAECs were evaluated (I).\u003cstrong\u003e \u003c/strong\u003eData are expressed as the mean ± SD of three independent experiments, with asterisks indicating significant differences (*\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001, and ****\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001).\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-5572297/v1/0309ac7130e7eb2b0b215c1a.png"},{"id":72910023,"identity":"bbb214a2-9d1a-4cec-a3e4-ed6f6c86e416","added_by":"auto","created_at":"2025-01-03 14:37:02","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":848669,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eS protein promotes endothelial cells injury by inhibiting SLAMF8 expression.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHuman airway epithelial cells (HAECs) were transfected with Flag-tagged SLAMF8 or control vector plasmids. At 48 hours post-transfection (hpt), cells were either left untreated or treated with S protein (10 nM) for 6 hours.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A) \u003c/strong\u003eViable cells were stained with the fluorescent probe JC-1 to assess mitochondrial membrane potential (MMP).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(B)\u003c/strong\u003e Cells were subjected to immunofluorescence with MitoTracker-Red (MTR) and anti-His antibody. Statistical analysis of the mean branch length of mitochondria is shaown (n ≥6).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(C) \u003c/strong\u003eThe phosphorylation and expression of proteins related to mitochondrial fission and fusion were analyzed by immunoblotting. Grayscale analysis of phosphorylated DRP1 relative to total DRP1 is presented.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(D) \u003c/strong\u003eThe levels of coagulation factor Ⅲ in the supernatants were assessed by ELISA.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(E)\u003c/strong\u003e Protein levels of SELP, PAI-1, SELE, and ICAM-1 were assessed by immunoblotting.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(F) \u003c/strong\u003eThe adhesion of THP-1 cells to HAECs were evaluated. Data are expressed as the mean ± SD of three independent experiments, with asterisks indicating significant differences (*\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001, and ****\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001).\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-5572297/v1/f5f57ed219a2153a380f04f6.png"},{"id":72910030,"identity":"f12ef57f-b08b-4def-b301-af24203a422f","added_by":"auto","created_at":"2025-01-03 14:37:02","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":442171,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eS protein activates NOX2-p66SHC axis to induce oxidative stress.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A) \u003c/strong\u003eSchematic representation of the NOX2-p66SHC-mitochondrial fission signaling pathway.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(B)\u003c/strong\u003e HAECs were transfected with Flag-tagged SLAMF8 or control vector plasmids. At 48 hpt, cells were either left untreated or treated with S protein for 6 h. The levels NOX2 secreted in the cell supernatants were quantified by ELISA.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(C) \u003c/strong\u003eHAECs treated as in described in (B) were subjected to immunoblotting. Grayscale analysis of NOX2 relative to GAPDH, as well as phosphorylated P66SHC (p-P66SHC) relative to total SHC, is shown.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(D-H) \u003c/strong\u003eHAECs were incubated with the NOX2 inhibitor gp91ds-tat for 12 h, followed by treatment with S protein for 6 h. The cells were then analyzed by immunoblotting, with the grayscale analysis of p-P66SHC relative to SHC presented (D). The protein levels of SELP, PAI-1, SELE, and ICAM-1 were also evaluated by immunoblotting, and corresponding grayscale statistical analyses relative to GAPDH are displayed (E-F). The levels of coagulation factor Ⅲ in supernatants were measured (G), the adhesion of THP-1 cells to HAECs were assessed (H). Data are expressed as the mean ± SD of three independent experiments, with asterisks representing significant differences (*\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, and ****\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001).\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-5572297/v1/2ebf6f92387e2df9b6d2c605.png"},{"id":72910025,"identity":"520cba94-1276-4933-ba94-3409a86bbded","added_by":"auto","created_at":"2025-01-03 14:37:02","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":764908,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKnockdown of p66SHC abolishes endothelial cells injure and thrombogenesis.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHuman airway epithelial cells (HAECs) were transfected with either siRNA targeting SHC (si-SHC) or a control siRNA (si-control) to knockdown SHC expression. At 72 hpt, cells were either left untreated or treated with S protein for 6 h.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A) \u003c/strong\u003eViable cells were stained with DCFH-DA to assess ROS levels.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(B) \u003c/strong\u003eCells were stained with JC-1, and the degree of mitochondria depolarization is quantified by measuring by the ratio of red to green fluorescence intensity.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(C)\u003c/strong\u003e The expression of proteins involved in mitochondrial fission (FIS1 and Drp1 S616) and fusion (OPA1) were analyzed by immunoblotting. Grayscale analysis of phosphorylated DRP1 relative to total DRP1, as well as FIS1 and OPA1 relative to GAPDH, is presented.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(D)\u003c/strong\u003e The levels of coagulation factor Ⅲ in the supernatants were measured using ELISA.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(E) \u003c/strong\u003eCells were subjected to immunoblotting with the indicated antibodies, and the grayscale analysis of protein levels relative to GAPDH is shown.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(F) \u003c/strong\u003eThe adhesion of THP-1 cells to HAECs were evaluated. Data are expressed as the mean ± SD of three independent experiments, with asterisks indicating significant differences (*\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.01, and ****\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001).\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-5572297/v1/bec932c6eb834a74fac51a7f.png"},{"id":72910046,"identity":"9e8d91eb-e0a1-447c-9b65-69991ffccc4e","added_by":"auto","created_at":"2025-01-03 14:37:03","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":855542,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSARS-CoV-2 infection promotes thrombogenesis by inhibiting SLAMF8.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A) \u003c/strong\u003eHuman airway epithelial cells (HAECs) were either mock-infected or infected with SARS-CoV-2 at a multiplicity of infection (MOI) of 0.1 for 24 h. The mRNA levels of SLAMF8 were quantifiedusing qPCR.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(B-F) \u003c/strong\u003eHAECs were transfected with Flag-tagged SLAMF8 or control vector plasmids. After 24 h, cells were either mock-infected or infected with SARS-CoV-2 (MOI 0.1) for an additional 24 h. The levels of coagulation factor Ⅲ in the supernatants were assessed using ELISA (B). The mRNA levels of tissue factor (TF), PAI-1, and VWF relative to GAPDH were measured using qPCR (C). Cells were subjected to immunoblotting with the indicated antibodies, and grayscale analysis of the protein levels relative to GAPDH is shown (D). Immunofluorescence staining was performed using DCFH-DA, along with Flag or S protein antibodies to visualize ROS levels (E). Additionally, immunofluorescence staining was conducted with MTR, and Flag or S protein antibodies (F). Data are expressed as the mean ± SD of three independent experiments, with asterisks representing significant differences (*\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, and ****\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001).\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-5572297/v1/258e7da6a3274d7beb0fa372.png"},{"id":72910780,"identity":"ed4b00b8-6b39-479e-9b54-d19c7b13d303","added_by":"auto","created_at":"2025-01-03 14:45:02","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":810705,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOverexpression of SLAMF8 reverses S protein-induced thrombogenesis \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vivo\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e. \u003c/strong\u003eMice were injected via the tail vein with either pAAV-SLAMF8 or pAAV-Ctrl for two weeks, followed by treatment with FeCl\u003csub\u003e3\u003c/sub\u003e 10 minutes after tail vein injection, with or without S protein (80 μg/kg).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A) \u003c/strong\u003eThe expression of Flag-SLAMF8 in mice injected with pAAV-SLAMF8 or pAAV-Ctrl was assessed by immunoblotting.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(B)\u003c/strong\u003e Blood flow changes in the modeling area were monitored using a blood flow meter.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(C)\u003c/strong\u003e Hematoxylin and eosin (HE) staining was performed to detect changes of platelet aggregation in the modeling area.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(D) \u003c/strong\u003eReactive oxygen species (ROS) levels were analyzed using Hydroethidine probe, with ROS visualized in red and cell nucleus in blue. Data are expressed as the mean ± SD of three independent experiments, with asterisks representing significant differences (*\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, and ****\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001).\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-5572297/v1/0741d06a0b8074bcf1745731.png"},{"id":72910035,"identity":"c06e99f3-f3f5-4c30-84c4-e2050eb62524","added_by":"auto","created_at":"2025-01-03 14:37:02","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":162168,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEvaluation of clinical correlation between SLAMF8 and COVID-19 thrombosis.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A-C)\u003c/strong\u003e The\u003cstrong\u003e \u003c/strong\u003emRNA expression levels of \u003cem\u003eSLAMF8 \u003c/em\u003e(A)\u003cem\u003e, PAI-1 \u003c/em\u003e(B) and \u003cem\u003eSELP \u003c/em\u003e(C) in endothelial cells from normal individuals and COVID-19 patients are presented, derived from HLCA dataset.re. Statistical significance was determined using Student’s t-tests, with *\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01, ***\u003cem\u003ep\u003c/em\u003e\u0026lt;0.001\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(D, E) \u003c/strong\u003eCorrelation plots illustrate the relationship between “Abnormal thrombosis” and SLAMF8 expression levels in endothelial cells from both normal and COVID-19 patients. Person correction coefficients are indicated on the plots.\u003c/p\u003e","description":"","filename":"image8.png","url":"https://assets-eu.researchsquare.com/files/rs-5572297/v1/ef9f5ab2fac1d6994a93d0b6.png"},{"id":75353351,"identity":"110babc4-da47-41b6-bd40-b7eaa51968bb","added_by":"auto","created_at":"2025-02-03 16:22:04","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":7173495,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5572297/v1/e450c703-0b60-44e7-ab9d-a13d8736cd88.pdf"},{"id":72910776,"identity":"e90fe6df-5d32-4fef-a641-a9e95ec3b714","added_by":"auto","created_at":"2025-01-03 14:45:02","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":105891,"visible":true,"origin":"","legend":"Appendix Table 1","description":"","filename":"Appendixtable1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5572297/v1/ac1af5852df88c85f1e7e5d8.xlsx"},{"id":72910018,"identity":"e50bfff7-f560-454c-9d7b-dd6fa6e09a5a","added_by":"auto","created_at":"2025-01-03 14:37:02","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":10970,"visible":true,"origin":"","legend":"Appendix Table 2","description":"","filename":"Appendixtable2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5572297/v1/17c6007458cc7ace963818e4.xlsx"},{"id":72910026,"identity":"a7e37c7b-fb70-40a7-b07f-e0e95f6b35e2","added_by":"auto","created_at":"2025-01-03 14:37:02","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":1314914,"visible":true,"origin":"","legend":"Supplementary materials","description":"","filename":"supplementarymaterials.docx","url":"https://assets-eu.researchsquare.com/files/rs-5572297/v1/2d06cdf1aaaa45967493a77f.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"SARS-CoV-2 spike protein activates NOX2-p66SHC axis via inhibiting SLAMF8 to promote thrombogenesis","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe COVID-19 pandemic, caused by the novel coronavirus SARS-CoV-2, has triggered an unprecedented global public health crisis.\u003csup\u003e1\u003c/sup\u003e While the majority of infected individuals experience mild to moderate illness, without the need for specialized treatment, a significant subset of patients faces serious complications.\u003csup\u003e2\u003c/sup\u003e These complications can include coagulopathy, thromboembolism, multiorgan failure, septic shock, and death, with acute symptoms typically lasting only 2 to 3 weeks. \u003csup\u003e3\u0026ndash;6\u003c/sup\u003e Alarmingly, approximately 30% of patients develop persistent symptoms that endure for at least three months following the acute phase of COVID-19, leading to conditions referred to as long COVID, post-acute COVID-19 syndrome (PACS), or post-acute sequelae of COVID-19 (PASC).\u003csup\u003e7\u003c/sup\u003e Common symptoms associated with PASC include fatigue, flu-like manifestations, autonomic dysfunction, cognitive impairments, and post-exertional malaise.\u003csup\u003e8,9\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eMany pathophysiological processes contribute to the development of PASC, including viral persistence, immune dysregulation, mitochondrial dysfunction, complement system dysregulation, endothelial inflammation, and microbiome dysbiosis.\u003csup\u003e7,10\u003c/sup\u003e These mechanisms culminate in a long-term disorder characterized by thrombotic endothelialitis, hyperactivated platelets, and the formation of fibrin-like microclots.\u003csup\u003e11,12\u003c/sup\u003e Such vascular and coagulation abnormalities impact multiple organ systems and represent a unifying pathway for the diverse symptoms of PASC, highlighting the urgent need for a comprehensive understanding of the mechanisms driving thrombogenesis during SARS-CoV-2 infection.\u003c/p\u003e \u003cp\u003eThe spike (S) protein of SARS-CoV-2 plays a crucial role in mediating the thrombotic complications associated with the virus.\u003csup\u003e13\u003c/sup\u003e Its presence has been documented in thrombi from COVID-19 patients, and intriguing associations have been noted between the ChAdOx1 nCov-19 (AstraZeneca) vaccine and atypical thrombotic events.\u003csup\u003e14,15\u003c/sup\u003e Furthermore, the S protein may persist in various organs and even in feces for months following infection, raising critical questions about its potential role in the thrombotic complications associated with PASC.\u003csup\u003e16\u0026ndash;18\u003c/sup\u003e Notably, the S protein can independently promote thrombosis by binding to endothelial cell surface receptors, such as angiotensin-converting enzyme 2 (ACE2) and cytoskeleton-associated protein 4 (CKAP4).\u003csup\u003e19,20\u003c/sup\u003e However, the precise mechanism by which the S protein induces endothelial cell injury and thrombogenesis remains inadequately understood.\u003c/p\u003e \u003cp\u003eIn this study, we demonstrate that the SARS-CoV-2 S protein inhibits the expression of SLAMF8, which subsequently leads to an upregulation of NOX2 expression and phosphorylation of p66SHC. This, in turn, causes mitochondrial damage and a persistent elevation of ROS, ultimately causing endothelial cell injury and promoting thrombosis. Importantly, overexpression of SLAMF8 effectively reverses S protein-induced thrombogenesis \u003cem\u003ein vivo.\u003c/em\u003e Our findings unveil a novel mechanism by which the S protein exerts pro-thrombotic effects, suggesting potential intervention strategies for addressing SARS-CoV-2 infection and post-acute sequelae of COVID-19 (PASC).\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eSARS-CoV-2 S protein promotes thrombogenesis, enhances ROS production and mitochondrial fission in endothelial cells\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate the mechanisms underlying SARS-CoV-2-associated thrombosis, we used the recombinant SARS-CoV-2 S protein to treat human aortic endothelial cells (HAEC), thereby simulating the effects of SARS-CoV-2 infection (Figure S1A and B). Following exposure to the S protein, we observed a significant upregulation of the endothelial cell injury factor von Willebrand factor (VWF), the adhesion molecules including E-selectin (SELE), P-selectin (SELP), intercellular cell adhesion molecule (ICAM)-1, as well as the thrombotropic factors including the coagulation factor III (TF) and the plasminogen activator inhibitor-1 (PAI-1) at both mRNA and protein levels in HAECs (Figure S1C-E, and Figure 1A). Additionally, the adhesion of monocytes to HAECs was markedly enhanced after the S protein treatment, which is a critical step in the prothrombotic transformation of endothelial cells (Figure 1B). These findings indicate that exposure to the S protein stimulates the secretion of thrombogenic factors from endothelial cells.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe also found a significant increase in ROS production in HAECs following S protein exposure (Figure 1C and D). Elevated ROS levels can lead to mitochondrial dysfunction, which is a key contributor to endothelial cell injury.\u003csup\u003e21\u003c/sup\u003e Indeed, we found that treatment with the S protein resulted in a decrease in the mitochondrial membrane potential, a hallmark indicative of mitochondrial dysfunction (Figure 1E, Figure S2A and B). Furthermore, exposure to the S protein caused a reduction in mitochondria length and an increase in mitochondrial fragmentation within endothelial cells (Figure 1F), without affecting overall mitochondrial mass (Figure S2C and D). This suggests that the S protein may induce mitochondrial damage by disrupting the homeostasis of mitochondrial dynamics.\u003c/p\u003e\n\u003cp\u003eMitochondrial dynamics is a critical physiological process that encompasses both mitochondrial fusion and fission. Mitochondrial fusion is regulated by the outer membrane fusion protein mitofusins 1 and 2 (MFN1 and 2) and the inner membrane fusion protein optic atrophy 1 (OPA1),\u003csup\u003e22,23\u003c/sup\u003e while fission is mediated by dynamin family GTPases, including mitochondrial fission 1 protein (FIS1) and dynamin-related protein 1 (DRP1).\u003csup\u003e23\u003c/sup\u003e Phosphorylation of DRP1 at serine 616 (pDRP1-S616) activates DRP1 and promotes its translocation to mitochondria, thereby facilitating mitochondrial fission. Conversely, phosphorylation at serine 637 (pDRP1-S637) inhibits mitochondrial fission by reducing DRP1\u0026apos;s association with the mitochondria. We found that S protein treatment resulted in an upregulation of pDRP1-S616 and a downregulation of pDRP1-S637 and OPA1, leading to shortened mitochondrial length and disrupted mitochondrial dynamics (Figures 1G and S2E). These findings demonstrate that the S protein enhances ROS production to promote mitochondrial fission in endothelial cells, creating a highly conducive environment for thrombogenesis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInhibition of ROS elevation and mitochondrial fission reverse thrombogenesis induced by S protein\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo explore whether the surge in ROS serves as a critical driver of mitochondrial damage and the pro-thrombotic phenotype induced by the SARS-CoV-2 S protein, we introduced the ROS scavenger mitoquinone (MitoQ). Our results demonstrated that MitoQ effectively mitigated the ROS levels induced by the S protein (Figure 2A). Additionally, treatment with MitoQ reversed the S protein-induced decrease in mitochondrial membrane potential, reduced mitochondrial fragmentation, and inhibited the expression of mitochondrial fission-related protein DRP1 and FIS1 (Figures S3A, 2B and C). To determine whether restoring mitochondrial function by reducing ROS levels could counteract the endothelial cells injury and thrombotic phenotype elicited by the S protein, we assessed thrombosis-related phenotypic changes in HAECs treated with MitoQ following S protein stimulation. The addition of MitoQ effectively decreased both mRNA and protein levels of critical thrombotic factors in endothelial cells, including coagulation factor III, PAI-1, and adhesion molecules such as ICAM, SELE, SELP, as well as the injury factor VWF induced by the S protein (Figure S3B and C, Figure 2D and E). Furthermore, MitoQ treatment significantly reduced the adhesion of monocytes to HAECs (Figure 2F). Our findings indicate that inhibiting the ROS surge triggered by the S protein can prevent the development of thrombosis by mitigating excessive mitochondrial fission.\u003c/p\u003e\n\u003cp\u003eTo further elucidate whether excessive mitochondrial fission induced by the S protein is a key factor in promoting thrombogenesis, we employed the mitochondrial fission inhibitor Midivi-1. Our findings revealed that Midivi-1 effectively reversed the mRNA and protein elevation induced by the S protein, including the thrombotic factor PAI-1 and coagulation factor III, the adhesion factor ICAM-1, as well as SELE and SELP, and the damage factor VWF (Figure S4, Figure 2G and H). Moreover, Midivi-1 treatment reversed the increased adhesion of monocytes to HAECs induced by the S protein (Figure 2I). Collectively, these results demonstrate that inhibiting excessive mitochondrial fission trigged by the S protein can alleviate the pro-thrombotic phenotype in endothelial cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eS protein promotes endothelial cells injury by inhibiting SLAMF8 expression\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo elucidate the mechanism by which the SARS-CoV-2 S protein promotes thrombogenesis through oxidative stress, we conducted a transcriptome analysis, revealing that the differentially expressed gene (DEGs) were significantly enriched in pathways associated with hydrogen peroxide metabolism and cellular responses to oxygen-containing compounds (Figure S5A-C, and Appendix Table 1). Notably, we identified and validated that the S protein markedly suppressed the expression of SLAMF8 (Figure S5D), a member of the signaling lymphocytic activation molecule (SLAMF) family, which plays a crucial role in regulating immune cell activation and cytokine production.\u003csup\u003e24\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003eTo explore whether SLAMF8 is responsible for the increased ROS levels, mitochondrial damage, and thrombogenesis caused by the S protein, we overexpressed SLAMF8 in HAECs prior to S protein stimulation. The results demonstrated that ectopic expression of SLAMF8 effectively reversed the S protein-induced increase in ROS levels and loss of mitochondrial membrane potential (Figures S5E and 3A). Furthermore, SLAMF8 overexpression attenuated S protein-induced mitochondrial fragmentation (Figure 3B) and inhibited the phosphorylation of DRP1 at serine 616 (pDRP1-S616), while promoting phosphorylation of at serine 637 (pDRP1-S637) and increasing OPA1 expression (Figures 3C and S5F). Additionally, we observed that SLAMF8 overexpression led to the decreased mRNA and protein levels of key thrombotic factors, including coagulation factor III and PAI-1, as well as adhesion molecules such as SELE, SELP, and ICAM-1, along with the damage factor VWF, thereby reducing endothelial cell adhesion (Figure S6A, Figures 3D-F). These findings indicate that the S protein contributes to excessive ROS production and subsequent mitochondrial damage by suppressing SLAMF8, ultimately resulting in endothelial cells injury that promotes thrombosis in context of COVID-19 and long COVID.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eS protein activates NOX2-p66SHC axis to induce oxidative stress\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate how the SARS-CoV-2 S protein inhibits SLAMF8 to induce oxidative stress, we focused on NOX2, a membrane-bound protein that generates superoxide anions and plays a pivotal role in ROS production.\u003csup\u003e25\u003c/sup\u003e Elevated ROS levels lead to the phosphorylation of p66SHC through the PKC or ERK/JNK signals, which promotes mitochondrial fission and dysfunction, ultimately resulting in increased production of mitochondrial-derived ROS, thereby creating a vicious cycle of ROS overproduction (Figure 4A).\u003csup\u003e26,27\u003c/sup\u003e We hypothesized that the suppression of SLAMF8 by the S protein could activate the NOX2-p66SHC axis, enhancing ROS production and mitochondrial fission. Our results demonstrated that exposure to the S protein significantly increased NOX2 secretion and p66SHC phosphorylation, while SLAMF8 overexpression effectively reversed these changes (Figures 4B and C). Furthermore, we found that the NOX2 inhibitor gp91ds-tat reduced both p66SHC phosphorylation and ROS generation induced by the S protein (Figures 4D, S6B). Additionally, treatment with gp91ds-tat diminished the expression of adhesion molecules SELE, SELP, and ICAM, as well as reduced PAI-1 levels and coagulation factor III secretion (Figures 4E-G). This treatment also led to decreased monocyte adhesion to endothelial cells (Figure 4H). Collectively, these data indicate that the S protein activates the NOX2-p66SHC cascade by inhibiting SLAMF8, thereby triggering an oxidative stress response that contributes to the pathogenesis of thrombosis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eKnockdown of p66SHC abolishes endothelial cells injure and thrombogenesis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo elucidate the role of the S protein in promoting the phosphorylation of p66SHC via NOX2 and its subsequent effects on mitochondrial fission, we silenced p66SHC in endothelial cells. Our results demonstrated that p66SHC knockdown effectively prevented the S protein-induced increase in ROS and the associated decline in mitochondrial membrane potential (Figures 5A and B). Consistent with these findings, p66SHC knockdown reversed the alterations in the mitochondrial dynamics-related proteins, including DRP1, FIS1, and OPA1, that were induced by the S protein (Figure 5C). Moreover, p66SHC knockdown mitigated the thrombotic phenotype triggered by the S protein, as evidenced by reduced expression levels of thrombotic factor PAI-1 and coagulation factor III, the adhesion molecules SELE, SELP, and ICAM, as well as endothelial cells\u0026rsquo; adhesion ability (Figures 5D-F). Our findings indicate that the S protein caused endothelial cells damage through activating p66SHC to promote thrombogenesis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSARS-CoV-2 infection promotes thrombogenesis by inhibiting SLAMF8 in endothelial cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe further determined whether SARS-CoV-2 infection also promotes oxidative stress and thrombosis in endothelial cells through inhibiting SLAMF8 expression. Our findings revealed a significant decrease in SLAMF8 expression in SARS-CoV-2-infected endothelial cells (Figure 6A). Consistent with the effects observed following the S protein treatment, SARS-CoV-2 infection increased the expression of thrombotic factors, including coagulation factor III and PAI-1, along with the damage factor VWF and adhesion molecules SELE, SELP, and ICAM. Notably, these changes were reversed by SLAMF8 overexpression (Figures 6B-D). Moreover, we observed that SARS-CoV-2 infection led to increased ROS production and mitochondrial fragmentation in HAECs, and these effects were mitigated by SLAMF8 overexpression (Figures 6E, F, and S6C). These results suggest that SARS-CoV-2 infection inhibits SLAMF8 expression, resulting in enhanced ROS overproduction, mitochondrial damage, and the induction of a thrombotic phenotype in endothelial cells.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOverexpression of SLAMF8 reverses S protein-induced thrombogenesis \u003cem\u003ein vivo\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo validate the role of SLAMF8 in S protein-induced thrombogenesis \u003cem\u003ein vivo\u003c/em\u003e, we constructed adeno-associated virus expressing human SLAMF8 (pAAV-SLAMF8) and a control virus (pAAV-Ctrl) under the endothelial cell specific Tie1 promoter. Equivalent dose of pAAV-SLAMF8 and pAAV-Ctrl were injected to C57BL/6 mice via the tail vein to achieve specific overexpression of SLAMF8 in endothelial cells. Immunoblotting confirmed a significant enhancement of SLAMF8 expression in the endothelium of mesenteric arteries from pAAV-SLAMF8-injected mice (Fig. 7A). We monitored thrombus formation using a live cell workstation and found that the S protein exacerbated the blood flow obstruction and platelet aggregation in the FeCl\u003csub\u003e3\u003c/sub\u003e-induced thrombus model (Fig. 7B and C), providing evidence that the S protein promotes thromboembolism \u003cem\u003ein vivo\u003c/em\u003e. Importantly, overexpression of SLAMF8 in endothelial cells reversed the S protein-induced increase in blood flow obstruction and platelet aggregation (Fig. 7B and C). Additionally, SLAMF8 overexpression reduced the ROS generation caused by the S protein, thereby inhibiting thrombosis \u003cem\u003ein vivo\u003c/em\u003e (Fig. 7D). These findings highlight the protective role of SLAMF8 in counteracting the thrombotic consequences of SARS-CoV-2 infection.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSLAMF8 expression is negatively correlated with thrombosis risk in COVID-19 patients\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo further explore the relationship between SLAMF8 and thrombosis in COVID-19 patients, we examined the expression of thrombosis-related genes and SLAMF8 in these individuals. As shown in Figures 8A-C, the expression levels of thrombosis-related genes PAI-1 and SELP were significantly elevated in the pulmonary endothelial cells of COVID-19 patients compared to healthy controls, while SLAMF8 expression\u003cem\u003e\u0026nbsp;\u003c/em\u003ewas markedly reduced. Furthermore, SLAMF\u003cem\u003e8\u003c/em\u003e expression exhibited a strong negative correlation (R=-0.58) with the thrombosis score in patients with SARS-CoV-2 infections compared to normal subjects (Figures 8D and E).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eReduced blood flow, endothelial damage, and hypercoagulability are recognozied as primary drivers of thromboembolism.\u003csup\u003e28\u003c/sup\u003e Numerous viruses, including cytomegalovirus (CMV), herpesviruses, influenza A virus (IAV), and coronaviruses such as SARS-CoV, SARS-CoV-2 and Middle East respiratory syndrome CoV (MERS-CoV), are known to trigger thrombosis. This process is typically are characterized by the activation of factor X and the production of tissue factor and VWF from endothelial cells.\u003csup\u003e29\u0026ndash;35\u003c/sup\u003e In our study, we found that SARS-CoV-2 enhances thrombogenesis by inducing the expression of tissue factor, VWF, and PAI-1 in endothelial cells. Mechanistically, the S protein of SARS-CoV-2 activates the NOX2-p66SHC axis by inhibiting SLAMF8, leading to persistent ROS elevation, mitochondrial dynamics disruption, endothelial cells injury, and ultimately thrombogenesis. These findings elucidate the underlying mechanisms and identify potential therapeutic targets for thrombotic complications associated with SARS-CoV-2 infection.\u003c/p\u003e \u003cp\u003eRecent studies suggest that COVID-19-related thrombosis is largely due to abnormalities in the coagulation system triggered by SARS-CoV-S protein.\u003csup\u003e13,14\u003c/sup\u003e The S protein binds to ACE-2 on platelets, leading to the release of coagulation factors and promoting leukocyte-platelet aggregate formation.\u003csup\u003e36\u003c/sup\u003e Additionally, it competitively inhibits the binding of antithrombin and heparin cofactor II to heparin/HS, resulting in abnormal thrombin activity.\u003csup\u003e37\u003c/sup\u003e The S protein also fosters a thrombogenic environment by activating NF-κB signaling, which induces endothelial inflammation and barrier dysfunction.\u003csup\u003e38\u0026ndash;40\u003c/sup\u003e Our study identified that the S protein induces oxidative stress in endothelial cells via the NOX2-p66SHC axis, resulting in a prothrombotic phenotype.\u003c/p\u003e \u003cp\u003ePatients with long COVID frequently present with fibrin amyloid microclots, which can cause tissue hypoxia and hinder oxygen exchange.\u003csup\u003e41,42\u003c/sup\u003e This tissue hypoxia stimulates ROS generation, exacerbating oxidative stress.\u003csup\u003e43\u003c/sup\u003e Elevated ROS levels under pathological conditions are crucial for thrombosis development due to their role in mitochondrial damage.\u003csup\u003e44,45\u003c/sup\u003e Our study found that the S protein induces excessive ROS production in endothelial cells, disrupting mitochondrial dynamics and significantly contributing to endothelial cell injury, a key factor in the thrombogenesis.\u003c/p\u003e \u003cp\u003eThe SLAMF family, comprising members SLAMF1 to SLAMF9, plays critical roles in regulating immune cell activation and cytokine production.\u003csup\u003e24\u003c/sup\u003e SLAMF8, a type I cell surface glycoprotein, modulates the tumor immune microenvironment and is considered a promising therapeutic target for various conditions, including cancer, mastocytosis, acute hepatic injury, and cardiac ischemia/reperfusion injury.\u003csup\u003e46\u0026ndash;52\u003c/sup\u003e Mechanistically, SLAMF8 upregulates Toll-like receptor-4 (TLR4) in macrophages to promote inflammatory cytokine secretion,\u003csup\u003e47,53\u003c/sup\u003e and reduces NADPH oxidase subunit phosphorylation, inhibiting macrophage microbicidal activity during bacterial infections.\u003csup\u003e54\u003c/sup\u003e Recent studies have shown that FOXJ2 upregulates SLAMF8 in β2GPI/anti-β2GPI-treated monocytes in antiphospholipid syndrome, where SLAMF8 interacts with TREM1 to stimulate TLR4/NF-κB signaling, inhibit autophagy, and release inflammatory and thrombotic markers.\u003csup\u003e55\u003c/sup\u003e Our study demonstrates SLAMF8 negatively regulates thrombosis induced by SARS-CoV-2 S protein, with a negative correlation between its expression and thrombotic activity in COVID-19 patients. Overexpression of SLAMF8 in endothelial cells counteracts the thrombotic phenotype triggered by the S protein and live virus, highlighting its potential as a therapeutic target for COVID-19-related thrombotic complications.\u003c/p\u003e \u003cp\u003eNOX2, an NADPH oxidase, is instrumental in ROS production via electron transport.\u003csup\u003e56\u003c/sup\u003e NOX2-derived ROS stimulate mitochondria to produce additional ROS, a process that is regulated by the adapter protein p66SHC.\u003csup\u003e26,57\u003c/sup\u003e Under physiological conditions, electrons are seamlessly transferred through complexes I-IV of the electron transport chain, ultimately resulting in the formation of water molecules. However, stressors like oxidative stress or UV irradiation cause p66Shc to translocate to mitochondria, redirecting electron transfer pathway and generating superoxide.\u003csup\u003e58,59\u003c/sup\u003e Our study found that inhibiting NOX2 or knocking down p66SHC mitigated the increase in ROS and the upregulation of thrombogenic factors, demonstrating that SLAMF8 is responsible for increasing NOX2 expression and p66SHC phosphorylation induced by the S protein.\u003c/p\u003e \u003cp\u003eCurrent treatments for SARS-CoV-2-associated thrombosis are primarily symptomatic and supportive.\u003csup\u003e60\u003c/sup\u003e However, potential therapies such as sulodexide, triple anticoagulant therapy, Pycnogenol\u0026reg;, and heparin-induced extracorporeal lipoprotein (HELP) apheresis have shown antithrombotic efficacy by improving endothelial function, and reducing inflammatory and oxidative stress markers.\u003csup\u003e61\u0026ndash;64\u003c/sup\u003e Our study revealed that overexpression of SLAMF8 in endothelial cells can reverse S protein-mediated blood flow obstruction and platelet aggregation, suggesting SLAMF8 could be a viable target for addressing SARS-CoV-2-induced coagulation anomalies and thrombosis.\u003c/p\u003e \u003cp\u003eIn summary, our study demonstrates that that the SARS-CoV-2 S protein suppresses SLAMF8 expression, thereby activating the NOX2-p66SHC axis. This activation induces a sustained increase in ROS, disrupts mitochondrial dynamics, and results in endothelial cell damage and thrombosis. These findings reveal a critical molecular mechanism through which the SARS-CoV-2 S protein contributes to endothelial cell thrombosis, offering new insights for the treatment and prevention of coagulation abnormalities in both COVID-19 and long COVID patients.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n \u003ch2\u003eAntigen, antibodies, plasmids, and drugs\u003c/h2\u003e\n \u003cp\u003eThe SARS-CoV-2 (2019-nCoV) spike protein (40589-V08B1) was obtained from Sino Biological. The following antibodies were used in our experiments: His (66005-1-IG), OPA1 (27733-1-AP), FIS1 (10956-1-AP), PAI-1 (13801-1-AP), SELP (83947-5-RR), ICAM-1 (10831-1-AP), GAPDH (10494-1-AP), MFN1 (13798-1-AP), MFN2 (12186-1-AP) and Flag (20543-1-AP) from ProteinTech; DRP1 (12957-1-AP), Phospho-DRP1 S616 (ab314755), Phospho-DRP1S637 (ab193216) and SELE (ab180782) from Abcam. CoraLite 594-(RGAR004) and 488-conjugated IgG (GB2AF488) secondary antibodies were also sourced from ProteinTech.\u003c/p\u003e\n \u003cp\u003eThe plasmid pAAV-TIEIP-SLAMF8-3\u0026times;FLAG-P2A-WPRE were purchased from OBiO Biotechnology. Mitoquinone (MitoQ) (S8978) and gp91ds-tat (P1213) were obtained from Selleck, while mitochondrial division inhibitor 1 (Mdivi-1) (HY-15886) was acquired from MCE.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n \u003ch2\u003eCell culture and transfection\u003c/h2\u003e\n \u003cp\u003eHuman aortic endothelial cells (HAECs) and THP-1 cells were cultured in Dulbecco\u0026apos;s modified Eagle\u0026apos;s medium (DMEM) (HyClone) supplemented with 10% fetal bovine serum (BBI), 100 U/ml ampicillin, and 100 \u0026micro;g/ml streptomycin (Sangon). Transfection of plasmids was conducted following the manufacturer\u0026rsquo;s instructions using Hieff Trans\u0026reg; Polyethylenimine Linear (PEI) (YEASON). For siRNA transfections RNAimax (Invitrogen) was utilized according to the manufacturer\u0026rsquo;s guidelines. Non-targeting control siRNA and SHC siRNAs were purchased from GenePharma.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n \u003ch2\u003eMonocyte adhesion assay\u003c/h2\u003e\n \u003cp\u003eTHP-1 cells were harvested and suspended in serum-free RPMI-1640 medium. CFSE fluorescent probes were added to the cell suspension and incubated for 15 min in a CO\u003csub\u003e2\u003c/sub\u003e incubator to label cells. Following the incubation, an equal volume of RPMI-1640 medium containing 20% fetal bovine serum was added to terminate the labeling reaction. CFSE-labeled THP-1 cells were then collected and washed with PBS. The cells were suspended in RPMI-1640 medium containing 10% fetal bovine serum and co-cultured with HAECs in the dark for 4 h. After the co-culture period, unbound THP-1 cells were removed by washing the cells with PBS three to five times. The remaining cells were suspended in an appropriate volume of PBS. The binding of THP-1 cells to HAECs were visualized using a fluorescence microscope (Olympus IX71) allowing for the assessment of monocyte adhesion.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n \u003ch2\u003eMitochondrial membrane potential detection\u003c/h2\u003e\n \u003cp\u003eMitochondrial membrane potential was detected using the Enhanced Mitochondrial Membrane Potential Assay Kit with JC-1 (Beyotime) following the manufacturer\u0026rsquo;s instruction. Briefly, cells were cultured in a JC-1 containing medium at 37\u0026deg;C for 20 minutes, after which they were collected and washed twice with PBS. The uptake and distribution of JC-1 within the cells were visualized using a fluorescence microscope.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\n \u003ch2\u003eCellular reactive oxygen species detection\u003c/h2\u003e\n \u003cp\u003eCellular reaction oxygen species (ROS) was measured using the Reactive Oxygen Species Assay Kit (YEASEN) in accordance with the manufacturer\u0026rsquo;s protocol. Briefly, cells were incubated with the ROS indicator DCFH-DA at 37\u0026deg;C for 20 minutes and subsequently washed three times with PBS. The levels of ROS were analyzed using either fluorescence microscopy or a microplate reader. For ROS staining in mouse tissues, frozen tissue sections were prepared and incubated with the Hydroethidine probe (YEASEN) in the dark for 30 minutes. The sections were then decolorized, washed and mounted. ROS levels in the tissue sections were analyzed with a fluorescence microscope.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\n \u003ch2\u003eImmunofluorescent staining\u003c/h2\u003e\n \u003cp\u003eCells were fixed with 4% paraformaldehyde and subsequently permeabilized using 0.5% Triton X-100 and stained with primary antibodies. Afterwards, the cells were incubated with CoraLite 594- or 488-conjugated IgG secondary antibodies. The cell nuclei were stained with DAPI (YEASON). Fluorescence images were acquired and analyzed using a confocal microscope (FV3000, OLYMPUS).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\n \u003ch2\u003eCoagulation factor III detection\u003c/h2\u003e\n \u003cp\u003eThe concentration of coagulation factor III was detected using the Human Coagulation Factor III/Tissue Factor ELISA Kit (Beyotime) according to manufacturer\u0026rsquo;s instruction. Briefly, cell culture supernatants were added to a 96-well plate coated with human coagulation factor III antibody and incubated for 2 hours. Following incubation, the wells were washed and incubated with biotinylated human coagulation factor III antibodies for an additional hour. After extensive washing (five times), the concentration of coagulation factor III was determined using horseradish peroxidase (HRP)-conjugated streptavidin, with 3,3\u0026rsquo;,5,5\u0026rsquo;-tetramethyl-benzidine (TMB) serving as the substrate.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\n \u003ch2\u003eNOX2 concentration detection\u003c/h2\u003e\n \u003cp\u003eThe concentration of human NADPH oxidase 2 (NOX2) was measured using an ELISA kit obtained from Elisa Biotech. Briefly, the cell culture supernatant was added to a 96-well plate coated with human NOX2 antibodies and incubated for 2 hours. After incubation, the wells were washed and then incubated with biotinylated human NOX2 antibodies for an additional hour. Following extensive washing, the concentration of NOX2 was determined using HRP-conjugated streptavidin, with TMB as the substrate for detection.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\n \u003ch2\u003eQuantitative real-time PCR (qPCR)\u003c/h2\u003e\n \u003cp\u003eTotal RNA was isolated using the EasyPure RNA Kit (TransGen) following the manufacturer\u0026apos;s instructions. Subsequently, reverse transcription was performed using cDNA Synthesis SuperMix kit (TransGen). All qPCR reactions were conducted using SYBR green PCR master mix (Roche) on a Real-Time PCR Detection System (Applied Biosystems). The qPCR results were analyzed using the comparative threshold cycle (Ct) method, with normalization to an endogenous glyceraldehyde phosphate dehydrogenase (GAPDH) as the reference gene and relative to an experimental control. The fold change in relative expression levels was calculated using the formula 2\u003csup\u003e\u0026minus;△△Ct\u003c/sup\u003e. \u003csup\u003e65\u003c/sup\u003e Primer information is provided in Appendix Table\u0026nbsp;2.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\n \u003ch2\u003eRNA sequencing and analysis\u003c/h2\u003e\n \u003cp\u003eRNA sequencing was conducted on HAECs exposed to the SARS-CoV-2 S protein and those not exposed. After 24 hours of treatment with SARS-CoV-2 S protein at a concentration of 10 nM, the cells were collected for RNA sequencing at Kidio Bio. Differentially expressed genes (DEGs) between the groups were analyzed using the Deseq2 package in R software, applying the criteria of |log (fold change) |\u0026gt;1 and P\u0026thinsp;\u0026lt;\u0026thinsp;0.05. Gene ontology and KEGG enrichment analyses were conducted using the cloud analytics platforms provided by Kidio Bio.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\n \u003ch2\u003eImmunoblotting analysis\u003c/h2\u003e\n \u003cp\u003eCells were collected, washed with cold PBS, and lysed in a lysis buffer containing 10 mM Tris/Cl pH 7.5, 150 mM NaCl, 2 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 0.5% NP-40 supplemented with a cocktail of protease and phosphatase inhibitors (Thermo Fisher Scientific) at 4 ℃ for 30 minutes. The cell lysates were then centrifuged at 12,000 g for 15 min at 4 ℃, and the protein concentration was quantified using Pierce\u0026trade; BCA Protein Assay Kits (Thermo Fisher Scientific). For electrophoresis, 20\u0026ndash;30 \u0026micro;g of protein from each sample was separated by 8\u0026ndash;15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore) using a Trans-Blot System (Bio-Rad). After transfer, the membranes were blocked with 2% BSA (VETEC) in PBS containing 0.2% Tween-20 (PBST) and then incubated with appropriate primary antibody at 4 ℃ overnight. Following washing, the membranes were incubated with HRP-conjugated secondary antibodies, and antibody-antigen complexes were visualized using a chemiluminescent substrate (ECL) (Millipore). Grayscale analysis of the bands was performed using ImageJ software.\u003c/p\u003e\n \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e\n \u003ch2\u003eViral infection\u003c/h2\u003e\n \u003cp\u003eSARS-CoV-2 (BetaCoV/Beijing/IME-BJ05/2020) was isolated from a patient with viral pneumonia in Wuhan China. HAECs were infected with SARS-CoV-2 at a multiplicity of infection (MOI) of 0.01 at 37 ℃ in serum-free DMEM (high glucose) for 1 hour. After the infection period, the infected cells were washed twice with PBS and cultured in DMEM containing 2% FBS for 24 hours to prepare for subsequent experiments. All experiments involving infectious SARS-CoV-2 were performed in a biosafety level 3 (BSL3) laboratory at the Changchun Veterinary Research Institute of Chinese Academy of Agriculture Sciences.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e\n \u003ch2\u003eAnimal experiments\u003c/h2\u003e\n \u003cp\u003eAll procedures were approved by the Institutional Animal Care and Use Committee at Shengchang Biotechnology Co., LTD with the ethical batch number 2024-04-JLDX-LXZ-091, and conformed to the legal mandates and federal guidelines for the care and maintenance of laboratory animals. C57BL/6 mice (female; 6\u0026ndash;8 weeks old; Vital River, China) were maintained and treated under specific pathogen-free conditions. Mice were injected with either pAAV-SLAMF8 or pAAV-Ctrl via the tail vein, and were observed for a duration of 2 weeks. Following this observation period, tissues were collected for subsequent experiments.\u003c/p\u003e\n \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e\n \u003ch2\u003eThrombosis mouse model\u003c/h2\u003e\n \u003cp\u003eThe SARS-CoV-2 S protein was administrated to C57BL/6 mice at a dosage of 80 \u0026micro;g/kg via tail vein injection, occurring 10 minutes prior to the induction of thrombosis using ferric chloride (FeCl\u003csub\u003e3\u003c/sub\u003e). Following anesthesia, a precise incision was made in the abdominal region of the mice to allow for the separation and exposure of the mesentery. A piece of filter paper saturated with ferric chloride solution was then applied to the surface of the mesenteric vein for a duration of 20 minutes. After the application period, the mesentery was repositioned, and the abdominal incision was carefully closed in layered using sutures. The formation of the thrombus was monitored in real-time using a live cell workstation.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec26\" class=\"Section3\"\u003e\n \u003ch2\u003eHematoxylin and Eosin (H\u0026amp;E) Staining\u003c/h2\u003e\n \u003cp\u003eFollowing the collection of tissue samples from mice, the tissues were immediately immersed in precooled PBS to ensure thorough rinsing. Excess PBS was carefully absorbed using clean filter paper, and the tissues were subsequently fixed in 4% paraformaldehyde for a minimum of 24 hours. After fixation, the tissue underwent a graded dehydration process. Once dehydration was complete, the tissues were embedded in paraffin wax, and 5 \u0026micro;m-thick sections were prepared. These sections were then deparaffinized to remove the wax, followed by staining with hematoxylin and eosin to enhance cellular features. After staining, the sections were dehydrated through a graded series of ethanol, cleared with xylene, and finally mounted using a neutral resin. Once the mounting medium was set, the slides were allowed to air-dry naturally. The dried slides were then prepared for microscopic observation and photography.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec27\" class=\"Section3\"\u003e\n \u003ch2\u003eClinical correlation analysis\u003c/h2\u003e\n \u003cp\u003eThe snRNA-Seq data were obtained from the Human Lung Cell Atlas,\u003csup\u003e66\u003c/sup\u003e which includes raw and normalized counts, integrated embedding, cell type annotations and clinical and technical metadata. This data is publicly available for download via cellxgene (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://cellxgene.cziscience.com/collections/6f6d381a-7701-4781-935c-db10d30de293\u003c/span\u003e\u003c/span\u003e). The expression matrix in \u0026ldquo;.h5ad\u0026rdquo; format was downloaded and filtered to isolate endothelial cells from both COVID-19 and normal subjects using the scanpy library.\u003csup\u003e67\u003c/sup\u003e The GMT file for the Human gene set HP_ABNORMAL_THROMBOSIS (M35560) was obtained from the Molecular Signature Database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ewww.gsea-msigdb.org\u003c/span\u003e\u003c/span\u003e). After converting the expression matrix to a Seurat Object, the AddModuleScore function from the Seurat Package (v4) was employed to calculate module scores.\u003csup\u003e68\u003c/sup\u003e Subsequently, the expression levels of SLAMF8 were plotted against the scores for \u0026quot;Abnormal_thrombosis\u0026quot; using the ggplot2 package. The Pearson coefficient was then calculated to assess the relationship between SLAMF8 expression and the module scores.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec28\" class=\"Section2\"\u003e\n \u003ch2\u003eStatistical analyses\u003c/h2\u003e\n \u003cp\u003eData are presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD), with \u0026ldquo;n\u0026rdquo; representing the number of independent replicates. Statistically significant differences were assessed using one-way or two-way analysis of variance (ANOVA), accompanied by multiple comparisons correction. Significance levels are indicated by asterisks as follows: *\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, ***\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, ****\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, while \u0026ldquo;ns\u0026rdquo; denotes non-significant differences.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by grant from Project supported by National Key R\u0026amp;D Program of China (Grant No. 2022YFF1203204), the Joint Funds of the National Natural Science Foundation of China (Grant No. U23A20269), the National Natural Science Foundation of China (Grant No. 82372250, 82302516, and 82341105), and the Medical Innovation Team Project of Jilin University (Grant No. 2022JBGS02).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDATA AVAILABILITY\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe RNA sequencing data for this article are available in the GEO repository with accession number GSE189706. 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SCANPY: large-scale single-cell gene expression data analysis. \u003cem\u003eGenome Biol\u003c/em\u003e \u003cstrong\u003e19\u003c/strong\u003e, 15 (2018).\u003c/li\u003e\n\u003cli\u003eHao, Y.\u003cem\u003e, et al.\u003c/em\u003e Integrated analysis of multimodal single-cell data. \u003cem\u003eCell\u003c/em\u003e \u003cstrong\u003e184\u003c/strong\u003e, 3573-3587 e3529 (2021).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"SARS-CoV-2, spike protein, thrombogenesis, SLAMF8, p66SHC","lastPublishedDoi":"10.21203/rs.3.rs-5572297/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5572297/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCOVID-19 associated coagulation abnormalities and thrombosis are life-threatening complications after SARS-CoV-2 infection. However, the underlying mechanisms are unclear. Here, we found that SARS-CoV-2 spike (S) protein induced excessive reactive oxygen species (ROS) production, disrupting mitochondrial dynamics and causing endothelial cells damage, thereby promoting thrombogenesis. Mechanistically, the S protein inhibited the expression of signaling lymphocytic activation molecule family 8 (SLAMF8) to induce an upregulation of NADPH oxidase 2 (NOX2) expression and p66SHC phosphorylation. This activation of NOX2-p66SHC axis resulted in a persistent elevation of ROS and mitochondrial dynamics disorder, ultimately leading to endothelial cells injury. SARS-CoV-2 infection also promoted the transformation of endothelial cells into a prothrombotic phenotype by inhibiting the expression of SLAMF8. Furthermore, the single-cell sequencing analysis revealed a negative correlation between SLAMF8 expression and thrombotic activity of endothelial cells in COVID-19 patients. Notably, the overexpression of SLAMF8 reversed the S protein-mediated increase in blood flow obstruction and platelet aggregation observed in mice with ferric chloride-induced thrombosis. These findings suggest a distinct mechanism of the S protein in the pathogenesis of SARS-CoV-2-associated thrombosis, providing novel perspectives and strategies for the prevention, management and treatment of thrombotic complications in individuals with COVID-19 or long COVID.\u003c/p\u003e","manuscriptTitle":"SARS-CoV-2 spike protein activates NOX2-p66SHC axis via inhibiting SLAMF8 to promote thrombogenesis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-01-03 14:36:57","doi":"10.21203/rs.3.rs-5572297/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"809f62db-7b6d-4776-91c7-a74cd735f8d5","owner":[],"postedDate":"January 3rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":42281672,"name":"Health sciences/Pathogenesis/Infection"},{"id":42281673,"name":"Biological sciences/Microbiology/Virology/SARS-CoV-2"}],"tags":[],"updatedAt":"2025-06-24T09:45:15+00:00","versionOfRecord":[],"versionCreatedAt":"2025-01-03 14:36:57","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5572297","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5572297","identity":"rs-5572297","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}