Inflammasome Activation Triggers Blood Clotting and Host Death through Pyroptosis.

Inflammasome activation and subsequent pyroptosis are critical defense mechanisms against microbes. However, overactivation of inflammasome leads to death of the host. Although recent studies have uncovered the mechanism of pyroptosis following inflammasome activation, how pyroptotic cell death drives pathogenesis, eventually leading to death of the host, is unknown. Here, we identified inflammasome activation as a trigger for blood clotting through pyroptosis. We have shown that canonical inflammasome activation by the conserved type III secretion system (T3SS) rod proteins from Gram-negative bacteria or noncanonical inflammasome activation by lipopolysaccharide (LPS) induced systemic blood clotting and massive thrombosis in tissues. Following inflammasome activation, pyroptotic macrophages released tissue factor (TF), an essential initiator of coagulation cascades. Genetic or pharmacological inhibition of TF abolishes inflammasome-mediated blood clotting and protects against death. Our data reveal that blood clotting is the major cause of host death following inflammasome activation and demonstrate that inflammasome bridges inflammation with thrombosis.

Circulating tissue factor-positive extracellular vesicles and their association with thrombosis in different diseases.

Tissue factor (TF) is a procoagulant protein released from activated host cells, such as monocytes, and tumor cells on extracellular vesicles (EVs). TF + EVs are observed in the circulation of patients with various types of diseases. In this review, we will summarize the association between TF + EVs and activation of coagulation and survival in different types of diseases, including cancer, sepsis, and infections with different viruses, such as human immunodeficiency virus (HIV), influenza A virus (IAV), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We will also discuss the source of TF + EVs in various diseases. EVTF activity is associated with thrombosis in pancreatic cancer patients and coronavirus disease 2019 patients (COVID-19) and with disseminated intravascular coagulation in cancer patients. EVTF activity is also associated with worse survival in patients with cancer and COVID-19. Monocytes are the major sources of TF + EVs in sepsis, and viral infections, such as HIV, Ebola virus, and SARS-CoV-2. In contrast, alveolar epithelial cells are the major source of TF + EVs in bronchoalveolar lavage fluid in COVID-19 and influenza A patients. These studies indicate that EVTF activity could be used as a biomarker to identify patients that have an increased risk of coagulopathy and mortality.

Measurement of tissue factor-positive extracellular vesicles in plasma: strengths and weaknesses of current methods.

PURPOSE OF REVIEW: This review evaluates the different methods used to measure levels of tissue factor (TF) in plasma and on extracellular vesicles (EVs). Levels of TF-positive (TF+) EVs in blood are increased in a variety of diseases, such as cancer, sepsis, and viral infection, and are associated with thrombosis. Highly sensitive assays are required to measure the low levels of TF+ EVs in blood. RECENT FINDINGS: TF antigen levels in plasma have been measured using standard ELISAs, SimpleStep ELISA technology, and solid-phase proximity ligation assay. Some studies reported the detection of TF+ EVs in plasma by flow cytometry. In addition, TF+ EVs can be captured onto beads and chips using anti-TF antibodies. Several assays have been developed to measure TF activity in EVs isolated from plasma. Importantly, activity-based assays are more sensitive than antigen-based assays as a single TF/FVIIa complex can generate large amounts of FXa. SUMMARY: We recommend isolating EVs from plasma and measuring TF activity using a functional assay in the presence and absence of an anti-TF antibody. We do not recommend using antigen-based assays as these are not sensitive enough to detect the low levels of TF in plasma.

Tissue factor and its procoagulant activity on cancer-associated thromboembolism in pancreatic cancer: Comment by Mackman et al.

The Quantikine® ELISA detects tissue factor in cell lysates and culture supernatants containing extracellular vesicles from tissue factor-expressing cell lines but does not detect low levels of tissue factor antigen in plasma.

Cancer Therapy-Associated Thrombosis.

[Figure: see text].

Patients With COVID-19 Have Elevated Levels of Circulating Extracellular Vesicle Tissue Factor Activity That Is Associated With Severity and Mortality-Brief Report.

OBJECTIVE: Patients with coronavirus disease 2019 (COVID-19) have a high rate of thrombosis. We hypothesized that severe acute respiratory syndrome coronavirus 2 infection leads to induction of TF (tissue factor) expression and increased levels of circulating TF-positive extracellular vesicles (EV) that may drive thrombosis. Approach and Results: We measured levels of plasma EV TF activity in 100 patients with COVID-19 with moderate and severe disease and 28 healthy controls. Levels of EV TF activity were significantly higher in patients with COVID-19 compared with controls. In addition, levels of EV TF activity were associated with disease severity and mortality. Finally, levels of EV TF activity correlated with several plasma markers, including D-dimer, which has been shown to be associated with thrombosis in patients with COVID-19. CONCLUSIONS: Our results indicate that severe acute respiratory syndrome coronavirus 2 infection induces the release of TF-positive EVs into the circulation that are likely to contribute to thrombosis in patients with COVID-19. EV TF activity was also associated with severity and mortality.

Quantification of Viral and Host Biomarkers in the Liver of Rhesus Macaques: A Longitudinal Study of Zaire Ebolavirus Strain Kikwit (EBOV/Kik).

Zaire ebolavirus (EBOV) causes Ebola virus disease (EVD), which carries a fatality rate between 25% and 90% in humans. Liver pathology is a hallmark of terminal EVD; however, little is known about temporal disease progression. We used multiplexed fluorescent immunohistochemistry and in situ hybridization in combination with whole slide imaging and image analysis (IA) to quantitatively characterize temporospatial signatures of viral and host factors as related to EBOV pathogenesis. Eighteen rhesus monkeys euthanized between 3 and 8 days post-infection, and 3 uninfected controls were enrolled in this study. Compared with semiquantitative histomorphologic ordinal scoring, quantitative IA detected subtle and progressive features of early and terminal EVD that was not feasible with routine approaches. Sinusoidal macrophages were the earliest cells to respond to infection, expressing proinflammatory cytokine interleukin 6 (IL6) mRNA, which was subsequently also observed in fibrovascular compartments. The mRNA of interferon-stimulated gene-15 (ISG-15), also known as ISG15 ubiquitin like modifier (ISG15), was observed early, with a progressive and ubiquitous hybridization signature involving mesenchymal and epithelial compartments. ISG-15 mRNA was prominent near infected cells, but not in infected cells, supporting the hypothesis that bystander cells produce a robust interferon gene response. This study contributes to our current understanding of early EVD progression and illustrates the value that digital pathology and quantitative IA serve in infectious disease research.

Circulating Extracellular Vesicle Tissue Factor Activity During Orthohantavirus Infection Is Associated With Intravascular Coagulation.

BACKGROUND: Puumala orthohantavirus (PUUV) causes hemorrhagic fever with renal syndrome (HFRS). Patients with HFRS have an activated coagulation system with increased risk of disseminated intravascular coagulation (DIC) and venous thromboembolism (VTE). The aim of the study was to determine whether circulating extracellular vesicle tissue factor (EVTF) activity levels associates with DIC and VTE (grouped as intravascular coagulation) in HFRS patients. METHODS: Longitudinal samples were collected from 88 HFRS patients. Patients were stratified into groups of those with intravascular coagulation (n = 27) and those who did not (n = 61). We measured levels of circulating EVTF activity, fibrinogen, activated partial prothrombin time, D-dimer, tissue plasminogen activator (tPA), plasminogen activator inhibitor 1 (PAI-1), and platelets. RESULTS: Plasma EVTF activity was transiently increased during HFRS. Levels of EVTF activity were significantly associated with plasma tPA and PAI-1, suggesting that endothelial cells could be a potential source. Patients with intravascular coagulation had significantly higher peak EVTF activity levels compared with those who did not, even after adjustment for sex and age. The peak EVTF activity value predicting intravascular coagulation was 0.51 ng/L with 63% sensitivity and 61% specificity with area under the curve = 0.63 (95% confidence interval, 0.51-0.76) and P = .046. CONCLUSIONS: Plasma EVTF activity during HFRS is associated with intravascular coagulation.

Neutrophils and neutrophil extracellular traps enhance venous thrombosis in mice bearing human pancreatic tumors.

Pancreatic cancer is associated with a high incidence of venous thromboembolism. Neutrophils have been shown to contribute to thrombosis in part by releasing neutrophil extracellular traps (NET). A recent study showed that increased plasma levels of the NET biomarker, citrullinated histone H3 (H3Cit), are associated with venous thromboembolism in patients with pancreatic and lung cancer but not in those with other types of cancer, including breast cancer. In this study, we examined the contribution of neutrophils and NET to venous thrombosis in nude mice bearing human pancreatic tumors. We found that tumor-bearing mice had increased circulating neutrophil counts and levels of granulocyte-colony stimulating factor, neutrophil elastase, H3Cit and cell-free DNA compared with controls. In addition, thrombi from tumor-bearing mice contained increased levels of the neutrophil marker Ly6G, as well as higher levels of H3Cit and cell-free DNA. Thrombi from tumor-bearing mice also had denser fibrin with thinner fibers consistent with increased thrombin generation. Importantly, either neutrophil depletion or administration of DNase I reduced the thrombus size in tumor-bearing but not in control mice. Our results, together with clinical data, suggest that neutrophils and NET contribute to venous thrombosis in patients with pancreatic cancer.

Neutrophil Extracellular Traps: Villains and Targets in Arterial, Venous, and Cancer-Associated Thrombosis

Recent studies have demonstrated a role of neutrophils in both venous and arterial thrombosis. A key prothrombotic feature of neutrophils is their ability to release web-like structures composed of DNA filaments coated with histones and granule proteins referred to as neutrophil extracellular traps (NETs). NETs were discovered over a decade ago as part of our first line of host defense against invading microorganisms. Although NETs have a protective role against pathogens, recent data suggest that an uncontrolled and excessive NET formation within the vasculature may contribute to pathological thrombotic disorders. In vitro studies suggest that NETs promote vessel occlusion by providing a scaffold for platelets, red blood cells, extracellular vesicles, and procoagulant molecules, such as von Willebrand factor and tissue factor. In addition, NET components enhance coagulation by both activating the intrinsic pathway and degrading an inhibitor of the extrinsic pathway (tissue factor pathway inhibitor). NET formation has, therefore, been proposed to contribute to thrombus formation and propagation in arterial, venous, and cancer-associated thrombosis. This review will describe animal and human studies suggesting a role of NETs in the pathogenesis of various thrombotic disorders. Targeting NETs may be a novel approach to reduce thrombosis without affecting hemostasis.

Roles of Coagulation Proteases and PARs (Protease-Activated Receptors) in Mouse Models of Inflammatory Diseases.

Activation of the blood coagulation cascade leads to fibrin deposition and platelet activation that are required for hemostasis. However, aberrant activation of coagulation can lead to thrombosis. Thrombi can cause tissue ischemia, and fibrin degradation products and activated platelets can enhance inflammation. In addition, coagulation proteases activate cells by cleavage of PARs (protease-activated receptors), including PAR1 and PAR2. Direct oral anticoagulants have recently been developed to specifically inhibit the coagulation proteases FXa (factor Xa) and thrombin. Administration of these inhibitors to wild-type mice can be used to determine the roles of FXa and thrombin in different inflammatory diseases. These results can be compared with the phenotypes of mice with deficiencies of either Par1 (F2r) or Par2 (F2rl1). However, inhibition of coagulation proteases will have effects beyond reducing PAR signaling, and a deficiency of PARs will abolish signaling from all proteases that activate these receptors. We will summarize studies that examine the roles of coagulation proteases, particularly FXa and thrombin, and PARs in different mouse models of inflammatory disease. Targeting FXa and thrombin or PARs may reduce inflammatory diseases in humans.

Cancer cell-derived tissue factor-positive extracellular vesicles: biomarkers of thrombosis and survival.

PURPOSE OF REVIEW: Tissue factor (TF) is released from cancer cells and tumors in the form of extracellular vesicles (EVs). This review summarizes our current knowledge of the mechanisms of release of TF-positive EVs (TF+EVs) from cancer cells and the effect of these TF+EVs on cultured endothelial cells. In addition, we will summarize the contribution of TF+EVs to thrombosis in mice, and the association between plasma EVTF activity and venous thrombosis as well as survival of cancer patients. RECENT FINDINGS: The release of TF+EVs from cancer cells is regulated by multiple factors, including hypoxia, epithelial-mesenchymal transition, and various intracellular signaling pathways. Cancer cell-derived, TF+EVs confer procoagulant activity to endothelial cells and induce the expression of adhesion proteins and IL-8. In addition, they contribute to thrombosis by directly activating the coagulation system and by generating thrombin that activates platelets in mouse models. Finally, there is an association between EVTF activity and venous thrombosis in pancreatic cancer patients as well as mortality in cancer patients. SUMMARY: Cancer cell-derived TF+EVs bind to and activate endothelial cells. In addition, they serve as biomarkers of survival of cancer patients and venous thrombosis in pancreatic cancer patients.

Tissue Factor and Cancer: Regulation, Tumor Growth, and Metastasis.

There is a strong relationship between tissue factor (TF) and cancer. Many cancer cells express high levels of both full-length TF and alternatively spliced (as) TF. TF expression in cancer is associated with poor prognosis. In this review, the authors summarize the regulation of TF expression in cancer cells and the roles of TF and asTF in tumor growth and metastasis. A variety of different signaling pathways, transcription factors and micro ribonucleic acids regulate TF gene expression in cancer cells. The TF/factor VIIa complex enhances tumor growth by activating protease-activated receptor 2 signaling and by increasing the expression of angiogenic factors, such as vascular endothelial growth factor. AsTF increases tumor growth by enhancing integrin ß1 signaling. TF and asTF also contribute to metastasis via multiple thrombin-dependent and independent mechanisms that include protecting tumor cells from natural killer cells. Finally, a novel anticancer therapy is using tumor TF as a target to deliver cytotoxic drugs to the tumor. TF may be useful in diagnosis, prognosis, and treatment of cancer.

Cancer-associated pathways and biomarkers of venous thrombosis.

Cancer patients have an increased risk of venous thromboembolism (VTE). In this review, we summarize common and cancer type-specific pathways of VTE in cancer patients. Increased levels of leukocytes, platelets, and tissue factor-positive (TF+) microvesicles (MVs) are all potential factors that alone or in combination increase cancer-associated thrombosis. Patients with lung or colorectal cancer often exhibit leukocytosis. Neutrophils could increase VTE in cancer patients by releasing neutrophil extracellular traps whereas monocytes may express TF. Thrombocytosis is often observed in gastrointestinal, lung, breast, and ovarian cancer and this could decrease the threshold required for VTE. Soluble P-selectin has been identified as a biomarker of cancer-associated thrombosis in a general cancer population and may reflect activation of the endothelium. P-selectin expression by the endothelium may enhance VTE by increasing the recruitment of leukocytes. Studies in patients with pancreatic or brain cancer suggest that elevated levels of PAI-1 may contribute to VTE. Although elevated levels of TF+ MVs have been observed in patients with different types of cancer, an association between TF+ MVs and VTE has been observed only in pancreatic cancer. Podoplanin expression is associated with VTE in patients with brain cancer and may activate platelets. Future studies should measure multiple biomarkers in each cancer type to determine whether combinations of biomarkers can be used as predictors of VTE. A better understanding of the pathways that increase VTE in cancer patients may lead to the development of new therapies to reduce the morbidity and mortality associated with thrombosis.

Evaluation of venous thrombosis and tissue factor in epithelial ovarian cancer.

OBJECTIVE: Ovarian clear cell carcinoma (OCCC) and high grade serous ovarian cancer (HGSOC) are associated with the highest risk of VTE among patients with epithelial ovarian cancer (EOC). Tissue factor (TF) is a transmembrane glycoprotein which can trigger thrombosis. We sought to evaluate if there is an association between VTE and tumor expression of tissue factor (TF), plasma TF, and microvesicle TF (MV TF) activity in this high-risk population. METHODS: We performed a case-control study of OCCC and HGSOC patients with and without VTE. 105 patients who underwent surgery at a tertiary care center between January 1995 and October 2013 were included. Plasma TF was measured with an enzyme-linked immunosorbent assay. A TF-dependent Factor Xa generation assay was used to measure MV TF activity. Immunohistochemical (IHC) analysis was performed to evaluate tumor expression of TF. RESULTS: 35 women with OCCC or HGSOC diagnosed with VTE within 9months of surgery were included in the case group. Those with VTE had a worse OS, p<0.0001, with a greater than three-fold increase in risk of death, HR 3.33 (CI 1.75-6.35). There was no significant difference in median plasma TF level or MV TF activity level between patients with and without VTE. OCCC patients had greater expression of TF in their tumors than patients with HGSOC, p<0.0001. CONCLUSIONS: TFMV activity and plasma TF level were not predictive of VTE in this patient population. Given the extensive expression of TF in OCCC tumors, it is unlikely IHC expression will be useful in risk stratification for VTE in this population.

Antitumor effect of antitissue factor antibody-MMAE conjugate in human pancreatic tumor xenografts.

Tissue factor (TF) triggers the extrinsic blood coagulation cascade and is highly expressed in various types of cancer. In this study, we investigated the antitumor effect of an antibody-drug conjugate (ADC) consisting of an anti-TF monoclonal antibody and monomethyl auristatin E (MMAE). MMAE was conjugated to an anti-human TF or anti-mouse TF antibody using a valine-citrulline linker that could be potentially hydrolyzed by cathepsin B in the acidic environment of the lysosome. The cytotoxic and antitumor effects of the ADCs against four pancreatic cancer cell lines were analyzed. Both the ADC with the anti-human TF antibody and that with the anti-mouse TF antibody were stable under physiological conditions. The anti-human ADC was internalized in TF-expressing human tumor cell lines, followed by effective MMAE release. The half maximal inhibitory concentration (IC50 ) of MMAE was approximately 1 nM for all of the cell lines used. Meanwhile, the IC50 of anti-human ADC was 1.15 nM in the cell lines showing high TF expression, while exceeding 100 nM in the cells showing low TF expression levels. Anti-human ADC with passive and active targeting ability exerted significant suppression of tumor growth as compared to that observed in the saline group (p < 0.01). Also significant tumor growth suppressions were seen at the anti-mouse ADC and control ADC groups compared to the saline group (p < 0.01) due to EPR effect. Because various clinical human cancers express highly amount of TF, this new anti-TF ADC may deserve a clinical evaluation.

Dysregulated hemostasis in acute promyelocytic leukemia.

Acute promyelocytic leukemia (APL) is associated with a high incidence of early death, which occurs within 30 days of diagnosis. The major cause of early death in APL is severe bleeding, particularly intracranial bleeding. Although APL is known to be associated with activation of coagulation, hyperfibrinolysis, and thrombocytopenia, the precise mechanisms that cause bleeding have not yet been elucidated. I propose that a combination of four pathways may contribute to bleeding in APL: (1) tissue factor, (2) the urokinase plasminogen activator/urokinase plasminogen activator receptor, (3) the annexin A2/S100A100/tissue plasminogen activator, and (4) the podoplanin/C-type lectin-like receptor 2. A better understanding of these pathways will identify new biomarkers to determine which APL patients are at high risk of bleeding and allow the development of new treatments for APL-associated bleeding.