Comparison of assays measuring extracellular vesicle-tissue factor in plasma samples: Communication from the ISTH SSC Subcommittee on Vascular Biology.

BACKGROUND: Scientific and clinical interest in extracellular vesicles (EVs) is growing. EVs that expose tissue factor (TF) bind factor VII/VIIa and can trigger coagulation. Highly procoagulant TF-exposing EVs are detectable in the circulation in various diseases, such as sepsis, COVID-19 or cancer. Many in-house and commercially available assays have been developed to measure EV-TF activity and antigen but only a few studies have compared some of these assays. The ISTH SSC Subcommittee on Vascular Biology initiated a multicenter study to compare the sensitivity, specificity and reproducibility of these assays. MATERIALS AND METHODS: Platelet-depleted plasma samples were prepared from blood of healthy donors. The plasma samples were spiked either with EVs from human milk, or EVs from TF-positive and TF-negative cell lines. Plasma was also prepared from whole human blood with or without LPS stimulation. Twenty-one laboratories measured EV-TF activity and antigen in the prepared samples using their own assays representing 18 functional and 9 antigenic assays. RESULTS: There was a large variability in the absolute values for the different EV-TF activity and antigen assays. Activity assays had higher specificity and sensitivity compared to antigen assays. In addition, there was a large intra-assay and inter-assay variability. Functional assays that used a blocking anti-TF antibody or immunocapture were the most specific and sensitive. Activity assays that used immunocapture had a lower coefficient of variation compared to assays that isolated EVs by high-speed centrifugation. CONCLUSION: Based on this multicenter study, we recommend measuring EV-TF using a functional assay in the presence of an anti-TF antibody.

Coagulation and platelet biology at the intersection of health and disease: illustrated capsules of the 11th Symposium on Hemostasis at the University of North Carolina.

The University of North Carolina Symposia on Hemostasis began in 2002, with The First Symposium on Hemostasis with a Special Focus on FVIIa and Tissue Factor. They have occurred biannually since and have maintained the primary goal of establishing a forum for the sharing of outstanding advances made in the basic sciences of hemostasis. The 2024 11th Symposium on Hemostasis will bring together leading scientists from around the globe to present and discuss the latest research related to coagulation factors and platelet biology. In keeping with the tradition of the conference, we expect novel cross-disciplinary collaborations to result from bringing together fundamental scientists and physician-scientists from different backgrounds and perspectives. The aim of these collaborations is to springboard the next generation of important advances in the field. This year's program was designed to discuss Coagulation and Platelet Biology at the Intersection of Health and Disease. The goal is to develop a better understanding of the pathophysiologic mechanisms leading to hemostatic and thrombotic disorders as this understanding is critical for the continued development of safe and efficacious therapeutics. Included in this review article are illustrated capsules provided by our speakers that highlight the main conclusions of the invited talks.

Biomarkers of bleeding and venous thromboembolism in patients with acute leukemia.

BACKGROUND: Coagulopathy and associated bleeding and deep vein thrombosis (DVT) are major causes of morbidity and mortality in patients with acute leukemia. The underlying mechanisms of these complications have not been fully elucidated. OBJECTIVES: To evaluate the associations between biomarker levels and bleeding and DVT in acute leukemia patients. METHODS: We examined plasma levels of activators, inhibitors, and biomarkers of the coagulation and fibrinolytic pathways in patients aged ≥18 years with newly diagnosed acute leukemia compared with those of normal controls. Multivariable regression models were used to examine the association of biomarkers with bleeding and DVT in acute leukemia patients. The study included 358 patients with acute leukemia (29 with acute promyelocytic leukemia [APL], 253 with non-APL acute myeloid leukemia, and 76 with acute lymphoblastic leukemia) and 30 normal controls. RESULTS: Patients with acute leukemia had higher levels of extracellular vesicle tissue factor (EVTF) activity, phosphatidylserine-positive extracellular vesicles, plasminogen activator inhibitor-1, plasmin-antiplasmin complexes, and cell-free DNA and lower levels of citrullinated histone H3-DNA complexes compared with normal controls. APL patients had the highest levels of EVTF activity and the lowest levels of tissue plasminogen activator among acute leukemia patients. There were 41 bleeding and 23 DVT events in acute leukemia patients. High EVTF activity was associated with increased risk of bleeding (subdistribution hazard ratio, 2.30; 95% CI, 0.99-5.31), whereas high levels of plasminogen activator inhibitor-1 were associated with increased risk of DVT (subdistribution hazard ratio, 3.00; 95% CI, 0.95-9.47) in these patients. CONCLUSION: Our study shows alterations in several biomarkers in acute leukemia and identifies biomarkers associated with risk of bleeding and DVT.

Mucin 1 and venous thrombosis in tumor-bearing mice and patients with cancer.

INTRODUCTION: Mucins released from epithelial tumors have been proposed to play a role in cancer-associated thrombosis. Mucin1 (MUC1) is a transmembrane mucin that is overexpressed in a variety of human malignancies, including breast and pancreatic cancer. We analyzed the association of MUC1 and venous thrombosis in a mouse tumor model and in patients with cancer. MATERIALS AND METHODS: We used a human pancreatic cancer cell line HPAF-II that expresses a high level of MUC1. We grew HPAF-II tumors in the pancreas of Crl:NU-Foxn1nu male mice. MUC1 in plasma and extracellular vesicles (EVs) isolated from plasma was measured using an enzyme-linked immunosorbent assay. MUC1 in EVs and venous thrombi from tumor-bearing mice was assessed by western blotting. We measured MUC1 in plasma from healthy controls and patients with stomach, colorectal or pancreatic cancer with or without venous thromboembolism. RESULTS AND DISCUSSION: MUC1 was detected in the plasma of mice bearing HPAF-II tumors and was associated with EVs. MUC1 was present in venous thrombi from mice bearing HFAP-II tumors. Recombinant MUC1 did not induce platelet aggregation. Levels of MUC1 were higher in patients with pancreatic cancer compared with healthy controls. In contrast to the mouse model, MUC1 was present in EV-free plasma in samples from healthy controls and patients with cancer. There was no significant difference in the levels of MUC1 in cancer patients with or without VTE. Our data did not find any evidence that MUC1 contributed to VTE in patients with cancer.

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.

Biomarkers of bleeding and venous thromboembolism in patients with acute leukemia.

Background: Coagulopathy and associated bleeding and venous thromboembolism (VTE) are major causes of morbidity and mortality in patients with acute leukemia. The underlying mechanisms of these complications have not been fully elucidated. Objectives: To evaluate the associations between biomarker levels and bleeding and VTE in acute leukemia patients. Patients/Method: We examined plasma levels of activators, inhibitors and biomarkers of the coagulation and fibrinolytic pathways in patients ≥18 years with newly diagnosed acute leukemia compared to healthy controls. Multivariable regression models were used to examine the association of biomarkers with bleeding and VTE in acute leukemia patients. The study included 358 patients with acute leukemia (29 acute promyelocytic leukemia [APL], 253 non-APL acute myeloid leukemia [AML] and 76 acute lymphoblastic leukemia [ALL]), and 30 healthy controls. Results: Patients with acute leukemia had higher levels of extracellular vesicle (EV) tissue factor (TF) activity, phosphatidylserine-positive EVs, plasminogen activator inhibitor-1 (PAI-1), plasmin-antiplasmin complexes, cell-free DNA and lower levels of citrullinated histone H3-DNA complexes compared to healthy controls. APL patients had the highest levels of EVTF activity and the lowest levels of tissue plasminogen activator among the acute leukemia patients. There were 41 bleeding and 37 VTE events in acute leukemia patients. High EVTF activity was associated with increased risk of bleeding (sHR 2.30, 95%CI 0.99-5.31) whereas high PAI-1 was associated with increased risk of VTE (sHR 3.79, 95%CI 1.40-10.28) in these patients. Conclusions: Our study shows alterations in several biomarkers in acute leukemia and identifies biomarkers associated with risk of bleeding and VTE.

Tissue factor activates the coagulation cascade in mouse models of acute promyelocytic leukemia.

Acute promyelocytic leukemia (APL) is associated with a high risk of bleeding and thrombosis. APL patients have an activated coagulation system, hyperfibrinolysis, and thrombocytopenia. APL cells express tissue factor (TF), a receptor and cofactor for factor VII/VIIa. This study had 2 goals. Firstly, we measured biomarkers of coagulation and fibrinolysis activation as well as platelet counts and bleeding in both mouse xenograft and allograft models of APL. Secondly, we determined the effect of inhibiting TF on the activation of coagulation in these models. We observed increased levels of plasma thrombin-antithrombin complexes (TAT), D-dimer, and plasmin-antiplasmin complexes, reduced platelet counts, and increased tail bleeding in both mouse models of APL. Fibrinogen levels decreased in the xenograft model but not in the allograft model. In contrast, the red blood cell count decreased in the allograft model but not in the xenograft model. Inhibition of APL-derived human TF with an anti-human TF monoclonal antibody reduced the level of TAT, increased platelet count, and normalized tail bleeding in a xenograft model. Inhibition of all sources of TF (APL cells and host cells) in the allograft model with a rat anti-mouse TF monoclonal antibody decreased the levels of TAT but did not affect the platelet count. Our study demonstrates that TF plays a central role in the activation of coagulation in both the xenograft and allograft mouse models of APL. These APL mouse models can be used to investigate the mechanisms of coagulopathy and thrombocytopenia in APL.

Detection of tissue factor-positive extracellular vesicles using the ExoView R100 system.

Background: Tissue factor (TF) is essential for hemostasis. TF-expressing extracellular vesicles (TF+ EVs) are released in pathological conditions, such as trauma and cancer, and are linked to thrombosis. Detection of TF+ EV antigenically in plasma is challenging due to their low concentration but may be of clinical utility. Objectives: We hypthesised that ExoView can allow for direct measurement of TF+ EV in plasma, antigenically. Methods: We utilized the anti-TF monoclonal antibody 5G9 to capture TF EV onto specialized ExoView chips. This was combined with fluorescent TF+ EV detection using anti-TF monoclonal antibody IIID8-AF647. We measured tumor cell-derived (BxPC-3) TF+ EV and TF+ EVs from plasma derived from whole blood with or without lipopolysaccharide (LPS) stimulation. We used this system to analyze TF+ EVs in 2 relevant clinical cohorts: trauma and ovarian cancer. We compared ExoView results with an EV TF activity assay. Results: BxPC-3-derived TF+ EVs were identified with ExoView using 5G9 capture with IIID8-AF647 detection. 5G9 capture with IIID8-AF647 detection was significantly higher in LPS+ samples than in LPS samples and correlated with EV TF activity (R2 = 0.28). Trauma patient samples had higher levels of EV TF activity than healthy controls, but activity did not correlate with TF measurements made by ExoView (R2 = 0.15). Samples from patients with ovarian cancer have higher levels of EV TF activity than those from healthy controls, but activity did not correlate with TF measurement by ExoView (R2 = 0.0063). Conclusion: TF+ EV measurement is possible in plasma, but the threshold and potential clinical applicability of ExoView R100, in this context, remain to be established.

Evaluation of four commercial ELISAs to measure tissue factor in human plasma.

Background: Under pathological conditions, tissue factor (TF)-positive extracellular vesicles (EVs) are released into the circulation and activate coagulation. Therefore, it is important to identify methods that accurately quantitate levels of TF in plasma. Enzyme-linked immunosorbent assays (ELISAs) are a fast and simple method to quantitate levels of proteins. However, there are several specific challenges with measuring TF antigen in plasma including its low concentration and the complexity of plasma. Objectives: We aimed to evaluate the ability of 4 commercial ELISAs to measure TF in human plasma. Methods: We determined the ability of 4 commercial ELISAs (Imubind, Quantikine, Human SimpleStep, and CD142 Human) to detect recombinant human TF (Innovin) (12.5-100 pg/mL), TF-positive EVs isolated from the culture supernatant from a human pancreatic cancer cell line (57 pg/mL), TF in plasma containing low levels of EV TF activity (1.2-2.6 pg/mL) from lipopolysaccharide-stimulated whole blood, and plasma containing high levels of EV TF activity (151-696 pg/mL) from patients with acute leukemia. Results: The CD142 Human ELISA could not detect recombinant TF. Imubind and Quantikine but not Human SimpleStep detected recombinant TF spiked into plasma and TF-positive EVs isolated from the culture supernatant of a human pancreatic cancer cell line. Quantikine and Imubind could not detect low levels of TF in plasma from lipopolysaccharide-stimulated whole blood. However, Quantikine but not Imubind detected TF in plasma from acute leukemia patients with high levels of EV TF activity. Conclusion: Our results indicate that commercial ELISAs have different abilities to detect TF. Quantikine and Imubind could not detect low levels of TF in plasma, but Quantikine detected TF in plasma with high levels of TF.

Mechanisms of cancer-associated thrombosis.

A State-of-the-Art lecture titled "Mechanisms of cancer-associated thrombosis" was presented at the ISTH Congress in 2022. Patients with cancer have a 4-fold to 9-fold increased risk of venous thromboembolism (VTE) compared with the general population, so-called cancer-associated thrombosis (CAT). Different rates of VTE are observed in different types of cancer, suggesting that there are cancer-type specific mechanisms of CAT. We will discuss 4 pathways of CAT: tissue factor, podoplanin, neutrophil extracellular traps, and plasminogen activator inhibitor-1. In addition, specific gene mutations may increase the rate of CAT in some cancer types. A better understanding of these pathways may lead to the identification of biomarkers that can identify patients at risk for VTE and allow the development of new treatments to prevent CAT. Finally, we summarize relevant new data on this topic presented at the 2022 ISTH Congress.

Tissue factor activity of small and large extracellular vesicles in different diseases.

Background: Circulating procoagulant extracellular vesicles (EVs) are increased in diseases, such as cancer, sepsis, and COVID-19. EV tissue factor (TF) activity is associated with disseminated intravascular coagulation in sepsis and venous thrombosis in patients with pancreatic cancer and COVID-19. EVs are commonly isolated by centrifugation at ∼20,000 g. Objectives: In this study, we analyzed the TF activity of 2 EV populations enriched for large and small EVs in patients with either sepsis, pancreatic cancer, or COVID-19. Methods: EVs were isolated from plasma by sequential centrifugation at 20,000 g (large EVs, LEVs) and then 100,000 g (small EVs, SEVs). We analyzed EVs from plasma prepared from whole blood samples from healthy individuals with or without lipopolysaccharide (LPS) stimulation as well as EVs from plasma samples from patients with either sepsis, pancreatic cancer, or COVID-19. TF-dependent (EV-TF activity) and TF-independent factor Xa (FXa) generation of the EVs was measured. Results: LPS increased EV-TF activity in LEVs but not SEVs. Similarly, in 2 patients with sepsis who had EV-TF activity above the background of the assay we observed EV-TF activity in LEVs but not SEVs. Patients with pancreatic cancer or COVID-19 had circulating EV-TF activity in both LEVs and SEVs. Conclusion: We recommend that EVs are isolated from plasma from patients by centrifugation at 100,000 g rather than 20,000 g to obtain a more accurate measure of levels of circulating EV-TF activity.

Decreased Platelet Reactivity and Function in a Mouse Model of Human Pancreatic Cancer.

Cancer patients have increased thrombosis and bleeding compared with the general population. Cancer is associated with activation of both platelets and coagulation. Mouse models have been used to study the dysregulation of platelets and coagulation in cancer. We established a mouse model of pancreatic cancer in which tissue factor-expressing human pancreatic tumors (BxPC-3) are grown in nude mice. Tumor-bearing mice have an activated coagulation system and increased venous thrombosis compared to control mice. We also showed that tumor-derived, tissue factor-positive extracellular vesicles activated platelets ex vivo and in vivo. In this study, we determined the effect of tumors on a platelet-dependent arterial thrombosis model. Unexpectedly, we observed significantly reduced carotid artery thrombosis in tumor-bearing mice compared to controls. In addition, we observed significantly increased tail bleeding in tumor-bearing mice compared to controls. These results suggested that the presence of the tumor affected platelets. Indeed, tumor-bearing mice exhibited a significant decrease in platelet count and an increase in mean platelet volume and percentage of reticulated platelets, findings that are consistent with increased platelet turnover. Levels of the platelet activation marker platelet factor 4 were also increased in tumor-bearing mice. We also observed decreased platelet receptor expression in tumor-bearing mice and reduced levels of active αIIb/ß3 integrin in response to PAR4 agonist peptide and convulxin in platelets from tumor-bearing mice compared with platelets from control mice. In summary, our study suggests that in tumor-bearing mice there is chronic platelet activation, leading to thrombocytopenia, decreased receptor expression, and impaired platelet adhesive function.

Extracellular Vesicle Tissue Factor Activity Assay.

Tissue factor (TF) is a transmembrane receptor for factor (F) VII and FVIIa. The TF/FVIIa complex initiates the coagulation cascade by activating both FIX and FX. TF is released from cells into the circulation in the form of extracellular vesicles (EVs). The level of TF-positive (+) EVs is increased in various diseases, including cancer, bacterial and viral infections, and cirrhosis, and is associated with thrombosis, disseminated intravascular coagulation, disease severity, and mortality. There are two ways to measure TF+ EVs in plasma: antigen- and activity-based assays. Data indicates that activity-based assays have higher sensitivity and specificity than antigen-based assays. This paper describes our in-house EVTF activity assay based on a two-stage FXa generation assay. FVIIa, FX, and calcium are added to the TF+ EV-containing samples to generate FXa in the presence and absence of anti-TF antibody to distinguish TF-dependent FXa generation from TF-independent FXa generation. A chromogenic substrate cleaved by FXa is used to determine the FXa level, while a standard curve generated with a relipidated recombinant TF is used for the determination of the TF concentration. This in-house EVTF activity assay has higher sensitivity and specificity than a commercial TF activity assay.

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.

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.

Evaluation of a new bead-based assay to measure levels of human tissue factor antigen in extracellular vesicles in plasma.

Background: Circulating tissue factor (TF)-expressing extracellular vesicles (EVs) are associated with thrombosis in several diseases, such as coronavirus disease 2019 (COVID-19). Activity assays have higher sensitivity and specificity compared to antigen assays for measuring TF+ EVs in plasma. The MACSPlex Exosome Kit is designed to detect 37 exosomal surface epitopes, including TF, on EVs in plasma using various fluorescently labeled beads. The different EV-bead complexes are detected by flow cytometry. A recent study used the MACSPlex Exosome Kit to measure levels of TF+ EVs in serum from patients with COVID-19. Objectives: To evaluate the ability of the MACSPlex Exosome Kit to detect TF on EVs in plasma. Methods: We measured levels of TF+ EVs isolated from plasma with or without TF detected using our in-house EVTF activity assay and the MACSPlex Exosome Kit. Results: The MACSPlex Exosome Kit gave a very low TF antigen signal (TF bead signal) compared to platelet-derived CD41b+ EVs, which was used as a control. Lipopolysaccharide (LPS) increased levels of EVTF activity but not TF bead signal in four donors. Inhibition of TF reduced levels of EVTF activity but did not affect the TF bead signal in EVs isolated from plasma from LPS-treated blood. Finally, we found no correlation between levels of EVTF activity and TF bead signal in EVs isolated from plasma from ovarian cancer patients (r = .16, P = .62). Conclusion: Our data suggest that the MACSPlex Exosome Kit gives a nonspecific signal for TF and does not have the sensitivity to detect TF+ EVs in plasma.