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Ambient concentrations of carbon monoxide in the vicinity of or inside urban and industrial areas can substantially exceed environmental background levels and can be detrimental to human health and welfare. In this period of analysis (July 2006 – March 2007), the maximum allowable concentration (MAC) was exceeded especially at Mihai Bravu and Cercul Militar. The accompanying diagrams showing the time evolution and charts revealing the spatial distribution of CO ambient air concentrations (based on GIS techniques) can be useful instruments in identifying the potential risk areas, like the important streets in the center of Bucharest.

Invariant NKT (iNKT) cells are a group of innate-like T cells that plays important roles in immune homeostasis and activation. We found that iNKT cells, compared with CD4+ T cells, have significantly higher levels of lipid peroxidation in both mice and humans. Proteomic analysis also demonstrated that iNKT cells express higher levels of phospholipid hydroperoxidase glutathione peroxidase 4 (Gpx4), a major antioxidant enzyme that reduces lipid peroxidation and prevents ferroptosis. T cell-specific deletion of Gpx4 reduces iNKT cell population, most prominently the IFN-γ-producing NKT1 subset. RNA-sequencing analysis revealed that IFN-γ signaling, cell cycle regulation, and mitochondrial function are perturbed by Gpx4 deletion in iNKT cells. Consistently, we detected impaired cytokine production, elevated cell proliferation and cell death, and accumulation of lipid peroxides and mitochondrial reactive oxygen species in Gpx4 knockout iNKT cells. Ferroptosis inhibitors, iron chelators, vitamin E, and vitamin K2 can prevent ferroptosis induced by Gpx4 deficiency in iNKT cells and ameliorate the impaired function of iNKT cells due to Gpx4 inhibition. Last, vitamin E rescues iNKT cell population in Gpx4 knockout mice. Altogether, our findings reveal the critical role of Gpx4 in regulating iNKT cell homeostasis and function, through controlling lipid peroxidation and ferroptosis., (Copyright © 2024 by The American Association of Immunologists, Inc.)

Bacillus anthracis peptidoglycan (PGN) is a major component of the bacterial cell wall and a key pathogen-associated molecular pattern contributing to anthrax pathology, including organ dysfunction and coagulopathy. Increases in apoptotic leukocytes are a late-stage feature of anthrax and sepsis, suggesting there is a defect in apoptotic clearance. In this study, we tested the hypothesis that B. anthracis PGN inhibits the capacity of human monocyte-derived macrophages (MΦ) to efferocytose apoptotic cells. Exposure of CD163+CD206+ MΦ to PGN for 24 h impaired efferocytosis in a manner dependent on human serum opsonins but independent of complement component C3. PGN treatment reduced cell surface expression of the proefferocytic signaling receptors MERTK, TYRO3, AXL, integrin αVβ5, CD36, and TIM-3, whereas TIM-1, αVβ3, CD300b, CD300f, STABILIN-1, and STABILIN-2 were unaffected. ADAM17 is a major membrane-bound protease implicated in mediating efferocytotic receptor cleavage. We found multiple ADAM17-mediated substrates increased in PGN-treated supernatant, suggesting involvement of membrane-bound proteases. ADAM17 inhibitors TAPI-0 and Marimastat prevented TNF release, indicating effective protease inhibition, and modestly increased cell-surface levels of MerTK and TIM-3 but only partially restored efferocytic capacity by PGN-treated MΦ. We conclude that human serum factors are required for optimal recognition of PGN by human MΦ and that B. anthracis PGN inhibits efferocytosis in part by reducing cell surface expression of MERTK and TIM-3., (Copyright © 2024 The Authors.)

Disseminated intravascular coagulation (DIC) is a syndrome triggered by infectious and noninfectious pathologies characterized by excessive generation of thrombin within the vasculature and widespread proteolytic conversion of fibrinogen. Despite diverse clinical manifestations ranging from thrombo-occlusive damage to bleeding diathesis, DIC etiology commonly involves excessive activation of blood coagulation and overlapping dysregulation of anticoagulants and fibrinolysis. Initiation of blood coagulation follows intravascular expression of tissue factor or activation of the contact pathway in response to pathogen-associated or host-derived, damage-associated molecular patterns. The process is further amplified through inflammatory and immunothrombotic mechanisms. Consumption of anticoagulants and disruption of endothelial homeostasis lower the regulatory control and disseminate microvascular thrombosis. Clinical DIC development in patients is associated with worsening morbidities and increased mortality, regardless of the underlying pathology; therefore, timely recognition of DIC is critical for reducing the pathologic burden. Due to the diversity of triggers and pathogenic mechanisms leading to DIC, diagnosis is based on algorithms that quantify hemostatic imbalance, thrombocytopenia, and fibrinogen conversion. Because current diagnosis primarily assesses overt consumptive coagulopathies, there is a critical need for better recognition of nonovert DIC and/or pre-DIC states. Therapeutic strategies for patients with DIC involve resolution of the eliciting triggers and supportive care for the hemostatic imbalance. Despite medical care, mortality in patients with DIC remains high, and new strategies, tailored to the underlying pathologic mechanisms, are needed., (© 2022 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Peptidoglycan (PGN), a polymeric glycan macromolecule, is a major constituent of the bacterial cell wall and a conserved pathogen-associated molecular pattern (PAMP) that triggers immune responses through cytosolic sensors. Immune cells encounter both PGN polymers and hydrolyzed muropeptides during infections, and primary human innate immune cells respond better to polymeric PGN than the minimal bioactive subunit muramyl dipeptide (MDP). While MDP is internalized through macropinocytosis and/or clathrin-mediated endocytosis, the internalization of particulate polymeric PGN is unresolved. We show here that PGN macromolecules isolated from Bacillus anthracis display a broad range of sizes, making them amenable for multiple internalization pathways. Pharmacologic inhibition indicates that PGN primarily, but not exclusively, is internalized by actin-dependent endocytosis. An alternate clathrin-independent but dynamin dependent pathway supports 20-30% of PGN uptake. In primary monocytes, this alternate pathway does not require activities of RhoA, Cdc42 or Arf6 small GTPases. Selective inhibition of PGN uptake shows that phagolysosomal trafficking, processing and downstream immune responses are drastically affected by actin depolymerization, while dynamin inhibition has a smaller effect. Overall, we show that polymeric PGN internalization occurs through two endocytic pathways with distinct potentials to trigger immune responses.

Late-stage anthrax infections are characterized by dysregulated immune responses and hematogenous spread of Bacillus anthracis , leading to extreme bacteremia, sepsis, multiple organ failure, and, ultimately, death. Despite the bacterium being nonhemolytic, some fulminant anthrax patients develop a secondary atypical hemolytic uremic syndrome (aHUS) through unknown mechanisms. We recapitulated the pathology in baboons challenged with cell wall peptidoglycan (PGN), a polymeric, pathogen-associated molecular pattern responsible for the hemostatic dysregulation in anthrax sepsis. Similar to aHUS anthrax patients, PGN induces an initial hematocrit elevation followed by progressive hemolytic anemia and associated renal failure. Etiologically, PGN induces erythrolysis through direct excessive activation of all three complement pathways. Blunting terminal complement activation with a C5 neutralizing peptide prevented the progressive deposition of membrane attack complexes on red blood cells (RBC) and subsequent intravascular hemolysis, heme cytotoxicity, and acute kidney injury. Importantly, C5 neutralization did not prevent immune recognition of PGN and shifted the systemic inflammatory responses, consistent with improved survival in sepsis. Whereas PGN-induced hemostatic dysregulation was unchanged, C5 inhibition augmented fibrinolysis and improved the thromboischemic resolution. Overall, our study identifies PGN-driven complement activation as the pathologic mechanism underlying hemolytic anemia in anthrax and likely other gram-positive infections in which PGN is abundantly represented. Neutralization of terminal complement reactions reduces the hemolytic uremic pathology induced by PGN and could alleviate heme cytotoxicity and its associated kidney failure in gram-positive infections., Competing Interests: Competing interest statement: A.R. was employed by Ra Pharmaceuticals at the time that the work was conducted. Data presented in this report make the subject of a preliminary patent application.

Background: During sepsis, gram-negative bacteria induce robust inflammation primarily via lipopolysacharride (LPS) signaling through TLR4, a process that involves the glycosylphosphatidylinositol (GPI)-anchored receptor CD14 transferring LPS to the Toll-like receptor 4/myeloid differentiation factor 2 (TLR4/MD-2) complex. Sepsis also triggers the onset of disseminated intravascular coagulation and consumptive coagulopathy., Objectives: We investigated the effect of CD14 blockade on sepsis-induced coagulopathy, inflammation, organ dysfunction, and mortality., Methods: We used a baboon model of lethal Escherichia (E) coli sepsis to study two experimental groups (n = 5): (a) E coli challenge; (b) E coli challenge plus anti-CD14 (23G4) inhibitory antibody administered as an intravenous bolus 30 minutes before the E coli., Results: Following anti-CD14 treatment, two animals reached the 7-day end-point survivor criteria, while three animals had a significantly prolonged survival as compared to the non-treated animals that developed multiple organ failure and died within 30 hours. Anti-CD14 reduced the activation of coagulation through inhibition of tissue factor-dependent pathway, especially in the survivors, and enhanced the fibrinolysis due to strong inhibition of plasminogen activator inhibitor 1. The treatment prevented the robust complement activation induced by E coli, as shown by significantly decreased C3b, C5a, and sC5b-9. Vital signs, organ function biomarkers, bacteria clearance, and leukocyte and fibrinogen consumption were all improved at varying levels. Anti-CD14 reduced neutrophil activation, cell death, LPS levels, and pro-inflammatory cytokines (tumor necrosis factor, interleukin (IL)-6, IL-1β, IL-8, interferon gamma, monocyte chemoattractant protein-1), more significantly in the survivors than non-surviving animals., Conclusions: Our results highlight the crosstalk between coagulation/fibrinolysis, inflammation, and complement systems and suggest a protective role of anti-CD14 treatment in E coli sepsis., (© 2020 International Society on Thrombosis and Haemostasis.)

Neutrophils are the most abundant innate cell population and a key immune player against invading pathogens. Neutrophils can kill both bacterium and spores of Bacillus anthracis , the causative anthrax pathogen. Unlike interactions with professional phagocytes, the molecular recognition of anthrax by neutrophils is largely unknown. In this study, we investigated the role of complement C3 deposition on anthrax particles for neutrophil recognition of bacterium and/or its cell wall peptidoglycan, an abundant pathogen-associated molecular pattern that supports anthrax sepsis. C3 opsonization and recognition by complement receptors accounted for 70-80% of the affinity interactions between neutrophils and anthrax particles at subphysiologic temperatures. In contrast, C3 supported up to 50% of the anthrax particle ingestion under thermophysiologic conditions. Opsonin-dependent low affinity interactions and, to a lower extent, opsonin-independent mechanisms, provide alternative entry routes. Similarly, C3 supported 58% of peptidoglycan-induced degranulation and, to a lower extent, 23% of bacterium-induced degranulation. Interestingly, an opsonin independent mechanism mediated by complement C5, likely through C5a anaphylatoxin, primes azurophilic granules in response to anthrax particles. Overall, we show that C3 deposition supports anthrax recognition by neutrophils but is dispensable for pathogen ingestion and neutrophil degranulation, highlighting immune recognition redundancies that minimize the risk of pathogen evasion.

Background: Sepsis triggers dysfunction of coagulation and fibrinolytic systems leading to disseminated intravascular coagulation (DIC) that contributes to organ failure and death. Fondaparinux (FPX) is a synthetic pentasaccharide that binds to antithrombin (AT) and selectively inhibits factor (F) Xa and other upstream coagulation proteases but not thrombin (T)., Objectives: We used a baboon model of lethal Escherichia coli sepsis to investigate the effects of FPX treatment on DIC, organ function, and outcome., Methods: Two experimental groups were studied: (a) E. coli challenge (n = 4); and (b) E coli plus FPX (n = 4). Bacteremia was modeled by intravenous infusion of pathogen (1-2 × 10 10  CFU/kg). Fondaparinux (0.08 mg/kg) was administered subcutaneously, 3 h prior to and 8 h after bacteria infusion., Results: Bacteremia rapidly increased plasma levels of inhibitory complexes of AT with coagulation proteases. Activation markers of both intrinsic (FXIa-AT), and extrinsic (FVIIa-AT) pathways were significantly reduced in FPX-treated animals. Factor Xa-AT and TAT complexes were maximal at 4 to 8 h post challenge and reduced >50% in FPX-treated animals. Fibrinogen consumption, fibrin generation and degradation, neutrophil and complement activation, and cytokine production were strongly induced by sepsis. All parameters were significantly reduced, while platelet count was unchanged by the treatment. Fondaparinux infusion attenuated organ dysfunction, prolonged survival, and saved two of four challenged animals (log-rank Mantel-Cox test, P = .0067)., Conclusion: Our data indicate that FPX-mediated inhibition of coagulation prevents sepsis coagulopathy; protects against excessive complement activation, inflammation, and organ dysfunction; and provides survival benefit in E. coli sepsis., (© 2019 International Society on Thrombosis and Haemostasis.)

Disseminated intravascular coagulation is a frequent manifestation during bacterial infections and is associated with negative clinical outcomes. Imbalanced expression and activity of intravascular tissue factor (TF) is central to the development of infection-associated coagulopathies. Recently, we showed that anthrax peptidoglycan (PGN) induces disseminated intravascular coagulation in a nonhuman primate model of anthrax sepsis. We hypothesized that immune recognition of PGN by monocytes is critical for procoagulant responses to PGN and investigated whether and how PGN induces TF expression in primary human monocytes. We found that PGN induced monocyte TF expression in a large cohort of healthy volunteers similar to lipopolysaccharide stimulation. Both immune and procoagulant responses to PGN involve intracellular recognition after PGN internalization, as well as surface signaling through immune Fcγ receptors (FcγRs). In line with our hypothesis, blocking immune receptor function, both signaling and FcγR-mediated phagocytosis, significantly reduced but did not abolish PGN-induced monocyte TF expression, indicating that FcγR-independent internalization contributes to intracellular recognition of PGN. Conversely, when intracellular PGN recognition is abolished, TF expression was sensitive to inhibitors of FcγR signaling, indicating that surface engagement of monocyte immune receptors can promote TF expression. The primary procoagulant responses to PGN were further amplified by proinflammatory cytokines through paracrine and autocrine signaling. Despite intersubject variability in the study cohort, dual neutralization of tumor necrosis factor-α and interleukin-1β provided the most robust inhibition of the procoagulant amplification loop and may prove useful for reducing coagulopathies in gram-positive sepsis., (© 2019 by The American Society of Hematology.)

Anthrax infections exhibit progressive coagulopathies that may contribute to the sepsis pathophysiology observed in fulminant disease. The hemostatic imbalance is recapitulated in primate models of late-stage disease but is uncommon in toxemic models, suggesting contribution of other bacterial pathogen-associated molecular patterns (PAMPs). Peptidoglycan (PGN) is a bacterial PAMP that engages cellular components at the cross talk between innate immunity and hemostasis. We hypothesized that PGN is critical for anthrax-induced coagulopathies and investigated the activation of blood coagulation in response to a sterile PGN infusion in primates. The PGN challenge, like the vegetative bacteria, induced a sepsis-like pathophysiology characterized by systemic inflammation, disseminated intravascular coagulation (DIC), organ dysfunction, and impaired survival. Importantly, the hemostatic impairment occurred early and in parallel with the inflammatory response, suggesting direct engagement of coagulation pathways. PGN infusion in baboons promoted early activation of contact factors evidenced by elevated protease-serpin complexes. Despite binding to contact factors, PGN did not directly activate either factor XII (FXII) or prekallikrein. PGN supported contact coagulation by enhancing enzymatic function of active FXII (FXIIa) and depressing its inhibition by antithrombin. In parallel, PGN induced de novo monocyte tissue factor expression in vitro and in vivo, promoting extrinsic clotting reactions at later stages. Activation of platelets further amplified the procoagulant state during PGN challenge, leading to DIC and subsequent ischemic damage of peripheral tissues. These data indicate that PGN may be a major cause for the pathophysiologic progression of Bacillus anthracis sepsis and is the primary PAMP behind the pathogen-induced coagulopathy in late-stage anthrax., (© 2018 by The American Society of Hematology.)

We showed that human IgG supported the response by human innate immune cells to peptidoglycan (PGN) from Bacillus anthracis and PGN-induced complement activation. However, other serum constituents have been shown to interact with peptidoglycan, including the IgG-like soluble pattern recognition receptor serum amyloid P (SAP). Here, we compared the abilities of SAP and of IgG to support monocyte and complement responses to PGN. Utilizing in vitro methods, we demonstrate that SAP is superior to IgG in supporting monocyte production of cytokines in response to PGN. Like IgG, the response supported by SAP was enhanced by phagocytosis and signaling kinases, such as Syk, Src, and phosphatidylinositol 3-kinase, that are involved in various cellular processes, including Fc receptor signaling. Unlike IgG, SAP had no effect on the activation of complement in response to PGN. These data demonstrate an opsonophagocytic role for SAP in response to PGN that propagates a cellular response without propagating the formation of the terminal complement complex., (Copyright © 2018 Girton et al.)

Peptidoglycan (PGN), a major component of bacterial cell walls, is a pathogen-associated molecular pattern (PAMP) that causes innate immune cells to produce inflammatory cytokines that escalate the host response during infection. In order to better understand the role of PGN in infection, we wanted to gain insight into the cellular receptor for PGN. Although the receptor was initially identified as Toll-like receptor 2 (TLR2), this receptor has remained controversial and other PGN receptors have been reported. We produced PGN from live cultures of Bacillus anthracis and Staphylococcus aureus and tested samples of PGN isolated during the purification process to determine at what point TLR2 activity was removed, if at all. Our results indicate that although live B. anthracis and S. aureus express abundant TLR2 ligands, highly-purified PGN from either bacterial source is not recognized by TLR2.

Bacterial sepsis triggers robust activation of the complement system with subsequent generation of anaphylatoxins (C3a, C5a) and the terminal complement complex (TCC) that together contribute to organ failure and death. Here we tested the effect of RA101295, a 2-kDa macrocyclic peptide inhibitor of C5 cleavage, using in vitro whole-blood assays and an in vivo baboon model of Escherichia coli sepsis. RA101295 strongly inhibited E. coli- induced complement activation both in vitro and in vivo by blocking the generation of C5a and the soluble form of TCC, sC5b-9. RA101295 reduced the E. coli- induced " oxidative burst," as well as leukocyte activation, without affecting host phagocytosis of E. coli RA101295 treatment reduced plasma LPS content in E. coli- challenged baboons, implying reduced complement-mediated bacteriolysis, whereas treated animals showed slightly improved bacterial clearance during the bacteremic stage compared with controls. Treatment with RA101295 also improved consumptive coagulopathy and preserved endothelial anticoagulant and vascular barrier functions. RA101295 abolished sepsis-induced surges in proinflammatory cytokines and attenuated systemic circulatory and febrile responses, likely reflecting decreased systemic levels of LPS and C5a. Overall, RA101295 treatment was associated with significant organ protection and markedly reduced mortality compared with nontreated controls (four of five animals survived in a 100% lethal model). We therefore conclude that inhibition of C5 cleavage during the bacteremic stage of sepsis could be an important therapeutic approach to prevent sepsis-induced inflammation, consumptive coagulopathy, and subsequent organ failure and death., Competing Interests: Conflict of interest statement: F.L. has received research support from Ra Pharmaceuticals. S.J.D. is employed by Ra Pharmaceuticals. T.E.M. is a consultant for Ra Pharmaceuticals.

Degradation of Gram-positive bacterial cell wall peptidoglycan in macrophage and dendritic cell phagosomes leads to activation of the NLRP3 inflammasome, a cytosolic complex that regulates processing and secretion of interleukin (IL)-1β and IL-18. While many inflammatory responses to peptidoglycan are mediated by detection of its muramyl dipeptide component in the cytosol by NOD2, we report here that NLRP3 inflammasome activation is caused by release of N-acetylglucosamine that is detected in the cytosol by the glycolytic enzyme hexokinase. Inhibition of hexokinase by N-acetylglucosamine causes its dissociation from mitochondria outer membranes, and we found that this is sufficient to activate the NLRP3 inflammasome. In addition, we observed that glycolytic inhibitors and metabolic conditions affecting hexokinase function and localization induce inflammasome activation. While previous studies have demonstrated that signaling by pattern recognition receptors can regulate metabolic processes, this study shows that a metabolic enzyme can act as a pattern recognition receptor. PAPERCLIP., (Copyright © 2016. Published by Elsevier Inc.)

Extracellular histones are mediators of tissue injury and organ dysfunction; therefore they constitute potential therapeutic targets in sepsis, inflammation, and thrombosis. Histone cytotoxicity in vitro decreases in the presence of plasma. Here, we demonstrate that plasma inter-α inhibitor protein (IAIP) neutralizes the cytotoxic effects of histones and decreases histone-induced platelet aggregation. These effects are mediated through the negatively charged glycosaminoglycans (GAGs) chondroitin sulfate and high-molecular-weight hyaluronan (HMW-HA) associated with IAIP. Cell surface anionic glycosaminoglycans heparan sulfate and HA protect the cells against histone-mediated damage in vitro. Surface plasmon resonance showed that both IAIP and HMW-HA directly bind to recombinant histone H4. In vivo neutralization of histones with IAIP and HMW-HA prevented histone-induced thrombocytopenia, bleeding, and lung microvascular thrombosis, decreased neutrophil activation, and averted histone-induced production of inflammatory cytokines and chemokines. IAIP and HMW-HA colocalized with histones in necrotic tissues and areas that displayed neutrophil extracellular traps. Increasing amounts of IAIP-histone complexes detected in the plasma of septic baboons correlated with increase in histones and/or nucleosomes and consumption of plasma IAIP. Our data suggest that IAIP, chondroitin sulfate, and HMW-HA are potential therapeutic agents to protect against histone-induced cytotoxicity, coagulopathy, systemic inflammation, and organ damage during inflammatory conditions such as sepsis and trauma., (© 2015 by The American Society of Hematology.)

Sepsis-induced inflammation of the lung leads to acute respiratory distress syndrome (ARDS), which may trigger persistent fibrosis. The pathology of ARDS is complex and poorly understood, and the therapeutic approaches are limited. We used a baboon model of Escherichia coli sepsis that mimics the complexity of human disease to study the pathophysiology of ARDS. We performed extensive biochemical, histological, and functional analyses to characterize the disease progression and the long-term effects of sepsis on the lung structure and function. Similar to humans, sepsis-induced ARDS in baboons displays an early inflammatory exudative phase, with extensive necrosis. This is followed by a regenerative phase dominated by proliferation of type 2 epithelial cells, expression of epithelial-to-mesenchymal transition markers, myofibroblast migration and proliferation, and collagen synthesis. Baboons that survived sepsis showed persistent inflammation and collagen deposition 6-27 months after the acute episodes. Long-term survivors had almost double the amount of collagen in the lung as compared with age-matched control animals. Immunostaining for procollagens showed persistent active collagen synthesis within the fibroblastic foci and interalveolar septa. Fibroblasts expressed markers of transforming growth factor-β and platelet-derived growth factor signaling, suggesting their potential role as mediators of myofibroblast migration and proliferation, and collagen deposition. In parallel, up-regulation of the inhibitors of extracellular proteases supports a deregulated matrix remodeling that may contribute to fibrosis. The primate model of sepsis-induced ARDS mimics the disease progression in humans, including chronic inflammation and long-lasting fibrosis. This model helps our understanding of the pathophysiology of fibrosis and the testing of new therapies.

Platelet activation frequently accompanies sepsis and contributes to the sepsis-associated vascular leakage and coagulation dysfunction. Our previous work has implicated peptidoglycan (PGN) as an agent causing systemic inflammation in gram-positive sepsis. We used flow cytometry and fluorescent microscopy to define the effects of PGN on the activation of human platelets. PGN induced platelet aggregation, expression of the activated form of integrin αIIbβ3, and exposure of phosphatidylserine (PS). These changes were dependent on immunoglobulin G and were attenuated by the Fcγ receptor IIa-blocking antibody IV.3, suggesting they are mediated by PGN-anti-PGN immune complexes signaling through Fcγ receptor IIa. PS exposure was not blocked by IV.3 but was sensitive to inhibitors of complement activation. PGN was a potent activator of the complement cascade in human plasma and caused deposition of C5b-9 on the platelet surface. Platelets with exposed PS had greatly accelerated prothrombinase activity. We conclude that PGN derived from gram-positive bacteria is a potent platelet agonist when complexed with anti-PGN antibody and could contribute to the coagulation dysfunction accompanying gram-positive infections.

Thrombosis and cardiovascular disease (CVD) represent major causes of morbidity and mortality. Low androgen correlates with higher incidence of CVD/thrombosis. Tissue Factor Pathway Inhibitor (TFPI) is the major inhibitor of tissue factor-factor VIIa (TF-FVIIa)-dependent FXa generation. Because endothelial cell (EC) dysfunction leading to vascular disease correlates with low EC-associated TFPI, we sought to identify mechanisms that regulate the natural expression of TFPI. Data mining of NCBI's GEO microarrays revealed strong coexpression between TFPI and the uncharacterized protein encoded by C6ORF105, which is predicted to be multispan, palmitoylated and androgen-responsive. We demonstrate that this protein regulates both the native and androgen-enhanced TFPI expression and activity in cultured ECs, and we named it androgen-dependent TFPI-regulating protein (ADTRP). We confirm ADTRP expression and colocalization with TFPI and caveolin-1 in ECs. ADTRP-shRNA reduces, while over-expression of ADTRP enhances, TFPI mRNA and activity and the colocalization of TF-FVIIa-FXa-TFPI with caveolin-1. Imaging and Triton X-114-extraction confirm TFPI and ADTRP association with lipid rafts/caveolae. Dihydrotestosterone up-regulates TFPI and ADTRP expression, and increases FXa inhibition by TFPI in an ADTRP- and caveolin-1-dependent manner. We conclude that the ADTRP-dependent up-regulation of TFPI expression and activity by androgen represents a novel mechanism of increasing the anticoagulant protection of the endothelium.

Tissue factor has been recognized as a regulator of tumor angiogenesis and metastasis. The tissue factor gene is selectively expressed in highly invasive breast cancer cells, and the mechanisms regulating tissue factor expression in these cells remain unclear. This study demonstrates that microRNA-19 (miR-19) regulates tissue factor expression in breast cancer cells, providing a molecular basis for the selective expression of the tissue factor gene. Tissue factor protein was barely detectable in MCF-7, T47D, and ZR-75-1 cells (less invasive breast lines) but was expressed at a significantly higher level in MDA-MB-231 and BT-20 cells (invasive breast lines) as assayed by Western blot. The tissue factor gene promoter was activated, and forced expression of tissue factor cDNA was achieved in MCF-7 cells, implying that the 3'-UTR of the tissue factor transcript is responsible for the suppression of tissue factor expression. Bioinformatics analysis predicted microRNA-binding sites for miR-19, miR-20, and miR-106b in the 3'-UTR of the tissue factor transcript. Reporter gene assay using the TF-3'-UTR luciferase reporter construct confirmed that the 3'-UTR negatively regulates gene expression in MCF-7 cells, an effect reversed by deletion of the miR-19-binding site. Application of the miR-19 inhibitor induces endogenous tissue factor expression in MCF-7 cells, and overexpression of miR-19 down-regulates tissue factor expression in MDA-MB-231 cells. RT-PCR analysis using cDNA made from Ago2-immunoprecipitated RNA samples confirmed that Ago2 binds preferentially to tissue factor 3'-UTR in MCF-7 cells, as compared with MDA-MB-231 cells, consistent with the observation that miR-19 levels are higher in MCF-7 cells.

Tissue factor (TF) is the cellular receptor for plasma protease factor VIIa (FVIIa), and the TF-FVIIa complex initiates coagulation in both hemostasis and thrombosis. Cell surface-exposed TF is mainly cryptic and requires activation to fully exhibit the procoagulant potential. Recently, the protein disulfide isomerase (PDI) has been hypothesized to regulate TF decryption through the redox switch of an exposed disulfide in TF extracellular domain. In this study, we analyzed PDI contribution to coagulation using an in vitro endothelial cell model. In this model, extracellular PDI is detected by imaging and flow cytometry. Inhibition of cell surface PDI induces a marked increase in TF procoagulant function, whereas exogenous addition of PDI inhibits TF decryption. The coagulant effects of PDI inhibition were sensitive to annexin V treatment, suggesting exposure of phosphatidylserine (PS), which was confirmed by prothrombinase assays and direct labeling. In contrast, exogenous PDI addition enhanced PS internalization. Analysis of fluorescent PS revealed that PDI affects both the apparent flippase and floppase activities on endothelial cells. In conclusion, we identified a new mechanism for PDI contribution to coagulation on endothelial cells, namely, the regulation of PS exposure, where PDI acts as a negative regulator of coagulation.

Severe sepsis leads to massive activation of coagulation and complement cascades that could contribute to multiple organ failure and death. To investigate the role of the complement and its crosstalk with the hemostatic system in the pathophysiology and therapeutics of sepsis, we have used a potent inhibitor (compstatin) administered early or late after Escherichia coli challenge in a baboon model of sepsis-induced multiple organ failure. Compstatin infusion inhibited sepsis-induced blood and tissue biomarkers of complement activation, reduced leucopenia and thrombocytopenia, and lowered the accumulation of macrophages and platelets in organs. Compstatin decreased the coagulopathic response by down-regulating tissue factor and PAI-1, diminished global blood coagulation markers (fibrinogen, fibrin-degradation products, APTT), and preserved the endothelial anticoagulant properties. Compstatin treatment also improved cardiac function and the biochemical markers of kidney and liver damage. Histologic analysis of vital organs collected from animals euthanized after 24 hours showed decreased microvascular thrombosis, improved vascular barrier function, and less leukocyte infiltration and cell death, all consistent with attenuated organ injury. We conclude that complement-coagulation interplay contributes to the progression of severe sepsis and blocking the harmful effects of complement activation products, especially during the organ failure stage of severe sepsis is a potentially important therapeutic strategy.

Cell exposed tissue factor (TF) is generally in a low procoagulant ("cryptic") state, and requires an activation step (decryption) to exhibit its full procoagulant potential. Recent data suggest that TF decryption may be regulated by the redox environment through the oxidoreductase activity of protein disulfide isomerase (PDI). In this article we review PDI contribution to different models of TF decryption, namely the disulfide switch model and the phosphatidylserine dynamics, and hypothesize on PDI contribution to TF self-association and association with lipid domains. Experimental evidence debate the disulfide switch model of TF decryption and its regulation by PDI. More recently we showed that PDI oxidoreductase activity regulates the phosphatidylserine equilibrium at the plasma membrane. Interestingly, PDI reductase activity could maintain TF in the reduced monomeric form, while also maintaining low exposure of PS, both states correlated with low procoagulant function. In contrast, PDI inhibition or oxidants may promote the adverse effects with a net increase in coagulation. The relative contribution of disulfide isomerization and PS exposure needs to be further analyzed to understand the redox control of TF procoagulant function. For the moment however TF regulation remains cryptic., (Copyright (c) 2009 Elsevier Ltd. All rights reserved.)