key: cord-0930306-4hv68ysp authors: Rosell, Axel; Havervall, Sebastian; von Meijenfeldt, Fien; Hisada, Yohei; Aguilera, Katherina; Grover, Steven P.; Lisman, Ton; Mackman, Nigel; Thålin, Charlotte title: Patients With COVID-19 Have Elevated Levels of Circulating Extracellular Vesicle Tissue Factor Activity That Is Associated With Severity and Mortality—Brief Report date: 2020-12-03 journal: Arterioscler Thromb Vasc Biol DOI: 10.1161/atvbaha.120.315547 sha: 03b05201299bb2345141b4bc58f65465bce36d9b doc_id: 930306 cord_uid: 4hv68ysp 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. S evere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection leads to coronavirus disease 2019 and has caused a global pandemic. Strikingly, patients with COVID-19 have a high rate of thrombotic events. 1 There has been much speculation about the mechanism of thrombosis in patients with COVID-19, including activation of coagulation and platelets, endothelial cell activation, release of neutrophil extracellular traps, inflammation, and activation of the complement system. 2 TF (tissue factor) is the main activator of the coagulation cascade. 3 Two recent reviews speculated that induction of TF expression may play a significant role in COVID-19-related thrombosis. 2, 4 We developed an assay to measure levels of extracellular vesicle (EV) TF activity in plasma. 5 EVs are also referred to as microparticles or microvesicles and are small membrane vesicles released from activated cells. 6 We and others found that circulating EV TF activity is increased in a variety of diseases that are associated with activation of coagulation and thrombosis, including cancer, endotoxemia, and bacterial and viral infection. 7 Importantly, we showed that EV TF activity is increased with several viral infections, including influenza A virus/H1N1 (hemagglutinin type 1 and neuraminidase type 1), Puumala orthohantavirus, Sin Nombre Virus, and Ebola virus. [8] [9] [10] [11] [12] Notably, levels of EV TF activity are associated with thrombosis in patients with pancreatic cancer and patients infected with Puumala orthohantavirus. 9, 13, 14 This suggests that EV TF activity can be used as a biomarker to assess induction of TF expression in patients and thrombotic risk. In this study, we measured levels of circulating EV TF activity in patients with COVID-19 and determined if they were associated with severity and mortality. One hundred patients with COVID-19 admitted to Danderyd Hospital, Stockholm, Sweden between April 9 and June 8, 2020 were included in the study. Patients were diagnosed with COVID-19 using reverse-transcriptase polymerase chain reaction viral detection of oropharyngeal or nasopharyngeal swabs (n=96) or clinical presentation (n=4). Eighty-five patients received anticoagulation (prophylactic low molecular weight heparin, n=57; prophylactic high dose low molecular weight heparin, n=24; oral anticoagulant, n=4; therapeutic anticoagulation, n=0). Twelve patients received glucocorticoids. No antivirals or hydroxychloroquine were used in these patients. Exclusion criteria were age <18 years and thrombosis before blood sampling (1 deep vein thrombosis and 3 pulmonary embolism). Demographic data, comorbidities, medications, respiratory support, and mortality (<45 days from admission) were obtained from medical records. Median age for the patients was 60 (50-69) years (65% male). Patients were divided into 2 groups based on the WHO-OSCI (World Health Organization-Ordinal Scale for Clinical Improvement; https://www.who.int/blueprint/priority-diseases/key-action/COVID-19_Treatment_Trial_Design_Master_ Protocol_synopsis_Final_18022020.pdf). Ninety-six patients had a score of <5 and 4 had a score of ≥5. Due to the low number of patients in the more severe group, we also divided the patients into 2 groups based on the level of respiratory support at the time of sampling: none/low level (≤5 L oxygen on cannula, n=83) and high level of respiratory support (>5 L oxygen on cannula, noninvasive respiratory support or intubation, n=17). Samples from 28 healthy individuals were used as controls with a median age of 71 (71-73) years (79% male). The study complied with the declaration of Helsinki, and informed consent was obtained from all healthy controls and patients or their next-of-kin. The study was approved by the Swedish Ethical Review Board (COMMUNITY study [Covid-19 Biomarker and Immunity] dnr 2020-01653). Blood samples were collected within 7 days of admission via venipuncture (98 patients) or preexisting arterial lines (2 patients) into sodium citrate (9:1 vol/vol) vacuum tubes. Platelet poor plasma was prepared from whole blood within 3 hours of sampling by centrifugation (2000×g, 20 minutes, room temperature) and stored at −80 °C. Blood samples were collected from controls via venipuncture. EV TF activity was measured as described. 5 Briefly, EVs are pelleted from plasma (100 μL) at 20 000g for 15 minutes at 4 °C. EVs are washed to remove coagulation factors in the plasma and resuspended in Hepes buffer saline with BSA. TF activity was measured by adding human factor VIIa After 2 hours of incubation at 37 °C, the reaction was stopped with EDTA and factor Xa measured using a chromogenic substrate (Pefachrome FXa 8595 [final concentration: 0.67 mmol/L] from Enzyme Research Laboratories). The absorbance at 405 nm was measured using a plate reader. We measured levels of a variety of other biomarkers and functional assays described below in a separate study. 15 Prothrombin time and plasma levels of fibrinogen, prothrombin, antithrombin, factor VIII, and D-dimer were measured using an automated coagulation analyzer (STACompact 3, Stago, Breda, the Netherlands). Levels of thrombin-antithrombin complexes were measured using a commercial assay (TAT; Siemens, Erlangen, Germany). We used a commercially available ELISA from R&D systems (Minneapolis, MN) to measure plasma levels of plasminogen activator inhibitor type 1. Plasma levels of plasmin-antiplasmin complexes were measured with a commercially available ELISA (Technozyme, Technoclone, Vienna, Austria). An in-house ELISA with a commercially available polyclonal antibodies against von Willebrand factor (DAKO, Glostrup, Denmark) was used to measure plasma levels of von Willebrand factor. We used the FRETS-VWF73 assay (Peptanova, Sandhausen, Germany) to measure plasma levels of a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 activity. Thrombin generation assay was performed with thrombomodulin using the fluorimetric method as described. 16 We measured the fibrinolytic capacity Levels of EV TF activity were significantly higher in patients with COVID-19 compared with healthy controls (Figure [A] ). It is notable that 2 of the patients had high levels of EV TF activity (3.03 and 3.37 pg/mL). These 2 patients were 2 of 4 patients with WHO-OSCI scores of ≥5 indicating severe disease. We have observed similar high values for EV TF activity in individual patients with pancreatic cancer (0.94, 1.23, 4.4, and 5.5) 18, 19 and in patients with severe influenza virus A/H1N1 infection (3.80±0.9, mean±SD, n=15). 10 We divided the patients into 2 groups based on WHO-OSCI and found that patients with a score of ≥5 had significantly higher levels of EV TF activity than patients with a score of <5 (median, 2. Due to the low numbers of severely ill patients using the WHO-OSIC score, we also stratified patients with respect to respiratory support and observed significantly higher levels of EV TF activity in patients with O 2 >5 L/min compared with those with O 2 ≤5 L/min (Figure [B] ). Furthermore, levels of EV TF activity were associated with mortality with significantly higher levels being observed with patients who died compared with levels in patients who survived (Figure [C] ). In unadjusted Cox regression, EV TF activity had a hazard ratio of 3.4 (95% CI, 1.9-6.0) for death (P<0.001). After adjustment for D-dimer levels, the hazard ratio for death was 4.6 (95% CI, 2.0-10.4) for EV TF activity (P<0.001). These results indicate a strong association between EV TF activity and short-term mortality. Finally, significantly more patients who died had EV TF activity above the threshold of 0.565 pg/mL (6/10, 60%) compared with patients that survived (19/90, 21%; P<0.05, Fisher exact test). Using a cutoff of 0.565 pg/mL, we found that patients with ≥0.565 pg/mL of EV TF activity had significantly higher mortality compared with patients with <0.565 pg/mL ( Figure We would not expect anticoagulants to affect levels of EV TF activity because EV production should not be reduced by treatment with anticoagulants, and EV are isolated from plasma for measurement of TF activity. At present, we do not know the cellular source of the circulating TF+ EVs in patients with COVID-19. We and others cannot detect TF+EVs by flow cytometry. 20 We speculate the TF+EVs are derived from activated monocytes and endothelial cells as well as perivascular cells. 3, 21 We determined if EV TF activity correlated with other biomarkers and coagulation and fibrinolysis assays in patients with COVID-19. EV TF activity was significantly correlated with D-dimer, prothrombin time, and international normalized ratio but not with thrombin-antithrombin complexes (Table) . D-dimer has a considerably longer half-life than thrombinantithrombin complexes and is used clinically. 22 Interestingly, EV TF activity was significantly correlated with the lagtime in the thrombin generation assay but not with other parameters (Table) . The positive correlation between EV TF activity and lagtime is surprising because we have shown that TF decreases lagtime. 23 EV TF activity was significantly correlated with levels of prothrombin, fibrinogen, and antithrombin (Table) . In terms of biomarkers of fibrinolysis, EV TF activity significantly correlated with plasmin-antiplasmin complexes but not with total plasminogen activator inhibitor type 1 or the clot lysis time (Table) . EV TF activity positively correlated with von Willebrand factor levels and negatively correlated with a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (Table) . These results indicate a link between EV TF, coagulation, fibrinolysis, and endothelial activation. Our studies with patients with cancer found a stronger correlation between EV TF activity and D-dimer in patients with pancreatic cancer (r s =0.35 and r s =0.51) compared with a general cancer population (r s =0.145). [24] [25] [26] Importantly, D-dimer has been shown to be associated with thrombosis in patients with COVID-19. 1 The current study suggests that the increase in EV TF activity reflects an induction of TF expression in patients with COVID-19 and release of TF-positive EVs into the circulation. It is likely that TF-positive EVs also contribute to thrombosis in patients with COVID-19. A limitation of the study is that the number of severely ill patients with COVID-19 in our cohort at the time of sample collection was small (n=4). Two of these severely ill patients had high levels of EV TF activity that were similar to the level observed in patients with severe influenza A virus/H1N1 infection. 10 We and others have shown that EV TF activity is associated with mortality in a general cancer population and also in patients with pancreatic cancer. [25] [26] [27] [28] [29] In addition, levels of EV TF activity are associated mortality in severe influenza A/H1N1 infection. 10 The current study ADAMTS13 indicates a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13; AT, antithrombin; CLT, clot lysis time; COVID-19, coronavirus disease 2019; ETP, endogenous thrombin potential; FG, fibrinogen; INR, international normalized ratio; NS, not significant; PAI-1, plasmin activator inhibitor 1; PAP, plasmin antiplasmin; PT, prothrombin time; TAT, thrombin-antithrombin; TG, thrombin generation; and VWF, von Willebrand factor. Prevalence and outcomes of d-dimer elevation in hospitalized patients with covid-19 Coagulation abnormalities and thrombosis in patients infected with SARS-CoV-2 and other pandemic viruses Tissue factor: an essential mediator of hemostasis and trigger of thrombosis Potential role for tissue factor in the pathogenesis of hypercoagulability associated with in COVID-19 Measurement of tissue factor activity in extracellular vesicles from human plasma samples Methodological guidelines to study extracellular vesicles Measurement of microparticle tissue factor activity in clinical samples: a summary of two tissue factor-dependent FXa generation assays Patients with severe orthohantavirus cardiopulmonary syndrome due to Sin Nombre Virus infection have increased circulating extracellular vesicle tissue factor and an activated coagulation system Circulating extracellular vesicle tissue factor activity during orthohantavirus infection is associated with intravascular coagulation Microvesicle tissue factor activity and interleukin-8 levels are associated with mortality in patients with influenza A/H1N1 infection A matched cross-sectional study of the association between circulating tissue factor activity, immune activation and advanced liver fibrosis in hepatitis C infection Quantification of viral and host biomarkers in the liver of rhesus macaques: a longitudinal study of zaire Ebolavirus Strain Kikwit (EBOV/Kik) Update from the laboratory: mechanistic studies of pathways of cancer-associated venous thrombosis using mouse models Tumor-derived tissue factor-positive microparticles and venous thrombosis in cancer patients Prothrombotic changes in patients with COVID-19 are associated with disease severity and mortality Calibrated automated thrombin generation measurement in clotting plasma Synergistic effects of hypofibrinolysis and genetic and acquired risk factors on the risk of a first venous thrombosis Plasma tissue factor may be predictive of venous thromboembolism in pancreatic cancer Effect of chemotherapy and longitudinal analysis of circulating extracellular vesicle tissue factor activity in patients with pancreatic and colorectal cancer Pre-analytical and analytical variables affecting the measurement of plasma-derived microparticle tissue factor activity Oxidative stress product, 4-hydroxy-2-nonenal, induces the release of tissue factor-positive microvesicles from perivascular cells into circulation Labelfree kinetic studies of hemostasis-related biomarkers including D-dimer using autologous serum transfusion Detection of endogenous tissue factor levels in plasma using the calibrated automated thrombogram assay Increased microparticle tissue factor activity in cancer patients with venous thromboembolism Microparticle-associated tissue factor activity, venous thromboembolism and mortality in pancreatic, gastric, colorectal and brain cancer patients Circulating microparticle tissue factor, thromboembolism and survival in pancreaticobiliary cancers Microparticle-associated tissue factor activity: a link between cancer and thrombosis? Microparticle-associated tissue factor activity in cancer patients with and without thrombosis Comparison of microvesicle tissue factor activity in non-cancer severely ill patients and cancer patients We would like to acknowledge the patients who participated in this study and Lena Gabrielsson, Ann-Christin Salomonson, Nina Greilert, and Eva Isaksson at Danderyd Hospital for administration and blood sampling. None.