key: cord-316938-64jxtg9y authors: Blasi, Annabel; von Meijenfeldt, Fien A.; Adelmeijer, Jelle; Calvo, Andrea; Ibañez, Cristina; Perdomo, Juan; Carlos Reverter, Juan; Lisman, Ton title: In vitro hypercoagulability and ongoing in vivo activation of coagulation and fibrinolysis in COVID‐19 patients on anticoagulation date: 2020-08-06 journal: J Thromb Haemost DOI: 10.1111/jth.15043 sha: doc_id: 316938 cord_uid: 64jxtg9y BACKGROUND: COVID‐19 is associated with a substantial risk of venous thrombotic events, even in the presence of adequate thromboprophylactic therapy. OBJECTIVES: We aimed to better characterize the hypercoagulable state of COVID‐19 patients in patients receiving anticoagulant therapy. METHODS: We took plasma samples of 23 patients with COVID‐19 who were on prophylactic or intensified anticoagulant therapy. Twenty healthy volunteers were included to establish reference ranges. RESULTS: COVID‐19 patients had a mildly prolonged prothrombin time, high VWF levels and low ADAMTS13 activity. Most rotational thromboelastometry parameters were normal, with a hypercoagulable maximum clot firmness in part of the patients. Despite detectable anti‐Xa activity in the majority of patients, ex vivo thrombin generation was normal, and in vivo thrombin generation elevated as evidenced by elevated levels of thrombin‐antithrombin complexes and D‐dimers. Plasma levels of activated factor VII were lower in patients, and levels of the platelet activation marker soluble CD40 ligand were similar in patients and controls. Plasmin‐antiplasmin complex levels were also increased in patients despite an in vitro hypofibrinolytic profile. CONCLUSIONS: COVID‐19 patients are characterized by normal in vitro thrombin generation and enhanced clot formation and decreased fibrinolytic potential despite the presence of heparin in the sample. Anticoagulated COVID‐19 patients have persistent in vivo activation of coagulation and fibrinolysis, but no evidence of excessive platelet activation. Ongoing activation of coagulation despite normal to intensified anticoagulant therapy indicates studies on alternative antithrombotic strategies are urgently required. Patients with COVID-19 have a profound risk for venous thrombotic events. Particularly in patients admitted to an intensive care unit (ICU), rates of deep vein thrombosis and pulmonary embolism are exceedingly high, even in the presence of pharmacological thromboprophylaxis [1, 2] . In addition to macrovascular thrombotic events, microvascular thrombosis has been proposed to contribute to disease progression, with pulmonary clots contributing to respiratory failure [3] [4] [5] , and clots in other vascular beds to multiple organ failure [6, 7] . Anticoagulant treatment has been shown to reduce mortality, perhaps due to reduction of microvascular thromboses [8] . The high thrombosis risk in COVID-19 patients has been linked to a hypercoagulable state that has not been well-defined. The in vivo hyperactivation of coagulation appears to be linked to a massive inflammatory response coupled with increases in acute phase proteins including fibrinogen [9] , and involvement of neutrophil extracellular traps (NETs) [10] , which are newly recognized actors in thrombosis. Routine hemostasis tests show mild prolongations in prothrombin time (PT) and activated partial thromboplastin time (APTT), and mild thrombocytopenia in some patients, but massively elevated levels of D-dimer in many patients [9] , that appear to have prognostic value [11] . Whole blood thromboelastography has demonstrated a hypercoagulable profile [12, 13] , and one of these studies concluded that the COVID-19 coagulopathy does not have elements of typical disseminated intravascular coagulation (DIC) as has been suggested by others [13] . Notably, increased (major) bleeding complications have been described in patients, especially in the critically ill, suggesting a fragile balance in hemostatic status of these patients [14] , although others have demonstrated bleeding risks comparable to patients with non-COVID-19 acute respiratory syndromes [15] . In a large academic medical center in Barcelona, Spain, to which approximately 600 patients were admitted at the peak of the COVID-19 pandemic, the initial reports on thrombosis and hypercoagulability and their own observations led to an intensified thromboprophylactic regimen for part of the admitted COVID-19 patients, particularly those with more advanced disease. Although an anticoagulant protocol was instituted, during the period of our study this protocol was poorly adhered to and individualized decisions on anticoagulant dosing were taken. We aimed to study the effects of this individualized anticoagulant therapy on the hemostatic status of these patients. This article is protected by copyright. All rights reserved We included 23 patients that were admitted with COVID-19 (which was confirmed by polymerase chain reaction) to Hospital Clínic Barcelona, Spain, in April 2020. Almost all patients received the low-molecular-weight heparin (LMWH) enoxaparin. Ethical approval from the Medical Ethical Committee Hospital Clínic Barcelona (2020/0371) was obtained. All patients, or in the case of incapacity their consultee, gave informed consent or assent, respectively, for participation in this study. Twenty healthy controls were included to establish reference values for the various assays performed. Exclusion criteria for healthy controls were age below 18 years, pregnancy, hereditary thrombophilia or hemophilia, use of anticoagulant medications, history of venous thromboembolic events, and blood (product) transfusion up to seven days prior to inclusion. Citrated blood samples were taken 4 [2 -6] days after admission to the hospital (9 [6 -13] days after onset of symptoms) on either a general ward or ICU by venipuncture or from dedicated arterial lines. In all patients, anticoagulation was started on admission. Blood samples were either used immediately for rotational thromboelastometry (ROTEM) measurements or processed to platelet-poor plasma within 30 minutes of the blood draw by double centrifugation at 2500g for 15 minutes, and subsequently stored at -80 degrees Celsius until used for analyses. Complete blood cell counts, and creatinine, total bilirubin, c-reactive protein (CRP) were measured as part of routine clinical care by the Centre de Diagnòstic Biomèdic at the Hospital Clínic Barcelona. We measured PT, APTT, international normalised ratio (INR), prothrombin, antithrombin, fibrinogen, and D-dimer on an automated coagulation analyzer (STACompact 3, Stago, Breda, The Netherlands) using reagents and protocols from the manufacturer. Von Willebrand factor (VWF) plasma levels were determined with an in-house enzyme-linked immunosorbent assay (ELISA) using commercially available polyclonal antibodies against VWF (DAKO, Glostrup, Denmark). Plasma activity of a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13) was measured using the FRETS-VWF73 assay (Peptanova, Sandhausen, Germany). Levels of VWF and ADAMTS13 in pooled normal plasma were set at 100%, and values obtained in test plasmas were expressed as a percentage of pooled normal plasma. Plasminogen activator inhibitor type 1 (PAI-1) levels were quantified by commercially available ELISA from R & D systems (Minneapolis, MN, USA). Cell-free DNA was quantified This article is protected by copyright. All rights reserved using the Quant-iT PicoGreen dsDNA assay kit (Fisher Scientific, Landsmeer, the Netherlands) as described previously [16] . The concentration of myeloperoxidase(MPO)-DNA complexes in plasma was determined by enzyme-linked immunosorbent assay (ELISA), as previously described [17] . ROTEM analyses were performed on a ROTEM sigma according to the manufacturers' instructions. Thrombin generation (TGA) was performed using commercially available reagents containing recombinant tissue factor (final concentration: 5 pM), phospholipids (final concentration: 4 µM), and soluble thrombomodulin (the concentration of which is not revealed by the manufacturer) (Thrombinoscope B.V., Maastricht, The Netherlands). Anti-activated factor X (anti-Xa) levels were measured on an automated analyzer (ACL 300 TOP) using Heparin LRT (Hyphen Biomed, Amsterdam, the Netherlands). Plasma fibrinolytic potential was estimated by studying lysis of a tissue factor-induced clot by exogenous tissue plasminogen activator (tPA) by monitoring changes in turbidity during clot formation and subsequent lysis as described previously [19] . Samples that were still clotted at 3 hours after the start of the experiment were arbitrarily assigned a clot lysis time (CLT) of 180 minutes. Plasma levels of thrombin-antithrombin (TAT) complexes, prothrombin fragment 1+2 (F1+2), plasmin-antiplasmin (PAP) complexes, and soluble CD40 ligand were quantified by commercially available ELISAs (Siemens, Erlangen, Germany for TAT and F1+2, Technoclone, Vienna, Austria for PAP, and R&D systems, Bio-techne, United Kingdom for sCD40L). Plasma levels of activated factor VII were quantified using the Hemoclot factor VIIa kit (Hyphen Biomed, Neuville-sur-Oise, France). Statistical analyses were performed using GraphPad Prism version 8.3.1 (San Diego, CA, USA). The results were presented as numbers (percentages) for categorical variables and medians [interquartile ranges] for continuous variables. Test results were compared between COVID-19 patients and healthy controls, and between patients admitted to the ICU and the ward, using the Mann-Whitney U test. Relations between laboratory parameters were made by simple linear regression. P-values < 0.05 were considered statistically significant. We studied 23 patients of whom 12 were admitted to the ICU, and 11 were on general wards. None of the ward patients later had to be admitted to the ICU. Three ICU patients developed thrombotic complications; 1 pulmonary embolism, 1 myocardial infarction, 1 distal ischemia of the fingers. General patient characteristics are shown in Table 1 . Most ICU patients received higher anticoagulant doses compared to ward patients; Table 1 ). Routine diagnostic hemostasis tests and plasma levels of markers for NETs are shown in Table 2 . Compared to healthy controls, patients had a prolonged prothrombin time and INR, which are largely explained by decreased FVII levels that strongly correlated with the prothrombin time (r 2 =0.56, p<0.0001). In addition, patients had a decreased platelet count, elevated fibrinogen levels, slightly decreased levels of prothrombin and antithrombin, high levels of VWF and FVIII, and low levels of ADAMTS13. Of note, 4 patients had prothrombin and antithrombin levels <25%, and two other patients had ADAMTS13 levels <10%. These results are consistent with a mild consumption coagulopathy with a thrombogenic VWF profile. Plasma levels of PAI-1 were approximately 3.7 times higher in COVID-19 patients compared to controls. Markers of NETs were modestly elevated in patients compared to controls, and did not differ between patients that were or were not admitted to ICU, which may argue against a key role of NETs in the pathogenesis of COVID-19 associated sequelae as was suggested previously. Indeed, unlike previously described [10] , NET markers in our cohort did not correlate with C-reactive protein, D-dimer, platelet count, or use of mechanical ventilation. We next studied hemostatic potential of COVID-19 patients by 3 global tests (Table 3) . ROTEM parameters were largely within the normal range, except for elevated maximum clot firmness (MCF) in extem, intem, and fibtem in 6, 8, and 11 patients, respectively. A limitation of these analyses was that normal ranges were not locally established, but taken from the ROTEM user manual. Of note, ROTEM extem and fibtem reagents contain polybrene, which neutralizes heparin present in many of these samples. Thrombomodulin-modified thrombin generation was preserved on a group level, but individual patients were clearly hyper-or hypocoagulable. The patient samples that generated little thrombin contained high levels of LMWH as evidenced by anti-Xa activity assays, whereas hypercoagulable samples generally had low to undetectable anti-Xa This article is protected by copyright. All rights reserved activity although one patient had marked thrombin generation even in the presence of high anti-Xa levels. ETP and peak thrombin levels were inversely correlated with anti-Xa levels (r 2 =0.16, p=0.055 and r 2 =0.20, p=0.03, without 1 clear outlier these correlations became much stronger r 2 =0.54, p<0.0001 and r 2 =0.50, p=0.0002). Samples taken from patients admitted to the ICU generated substantially less thrombin, but this was directly related to much higher anti-Xa levels in samples taken from patients on the ICU compared to ward patients ( Table 3) . As samples were not taken at specific time points relative to the last LMWH injection, and since there was a substantial difference in dosing and timing of LMWH administration between ICU and ward, this likely explains the difference in anti-Xa and thrombin generating capacity between intensive care and ward patients. Plasma CLT was higher in COVID-19 patients compared to healthy controls, but similar between patients on ICU and ward. Five patients had substantially elevated CLT (>100 min). Two of these had underlying liver disease and may have been hypofibrinolytic related to decompensating liver disease as we have described previously [20] , the other 3 were all admitted to the ICU, and hypofibrinolysis is a common feature of patients that are critically ill. Viscoelastic tests have shown evidence of fibrinolytic shutdown in a quarter of COVID-19 patients and fibrinolytic shutdown was associated with thrombosis [21] . However, the definition of hypofibrinolysis using viscoelastic tests is not straightforward as 'no lysis' is in fact part of the reference range [22] . We found 'no lysis' (defined as CLT >180 min) in only 3 patients (13%), whilst our plasma-based test detects 'no lysis' in a much larger proportion of other patient populations (notably post-surgery [23] , and patients with acute liver failure (73.5%) [24] ). This suggests that a true fibrinolytic shutdown is rare in COVID-19, which is also evidenced by highly elevated D-dimer and plasmin-antiplasmin complexes (see below). Importantly, LMWH decreases CLT across physiologically relevant concentrations [25] , which may be why plasma clot lysis was only mildly impaired in our patients. Thus, COVID-19 patients have somewhat elevated clot formation, likely related to hyperfibrinogenemia, normal thrombin generating capacity despite the presence of LMWH, and hypofibrinolysis despite the presence of LMWH. This article is protected by copyright. All rights reserved In vivo markers of activation of coagulation are shown in Table 4 . TAT and PAP complex levels are strongly elevated in patients with COVID-19, indicating ongoing thrombin and plasmin generation in COVID-19 patients despite anticoagulation with LMWH. Interestingly, TAT and PAP levels were not different between patients admitted to the ICU or to the general ward, while D-dimer levels were substantially higher in ICU compared to ward patients. This may indicate that ICU patients have a higher clot burden, which may be primarily intrapulmonary clots [26, 27] , despite similar procoagulant activity. This might be explained by decreased (local) anticoagulant capacity in ICU patients, perhaps related to increased endothelial injury that decreases availability of thrombomodulin and endogenous heparinoids. Surprisingly, F1+2 levels were not increased in COVID-19 patients compared to controls, and we have no explanation for the discrepancy between TAT and F1+2 levels. D-dimer and TAT levels were not correlated to anti-Xa levels, but F1+2 levels were inversely correlated with anti-Xa levels (r 2 =0.22, p=0.03). VIIa levels were lower in patients, which may point to consumption of VIIa similar to what we have previously observed in patients during the acute phase of deep vein thrombosis [28] . Indeed, VIIa levels strongly positively correlated with zymogen VII levels (r 2 =0.59, p=<0.0001). Soluble CD40 ligand levels were similar between patients and controls, suggesting COVID-19 patients are not characterized by excessive platelet activation. Taken together, our data confirm a hypercoagulable status of enhanced thrombin generating capacity, enhanced ex vivo clot formation likely related to hyperfibrinogenemia, and a decreased ex vivo fibrinolytic capacity in patients with COVID-19. Interestingly, despite normal to intensified anticoagulant treatment, in vivo activation of coagulation and fibrinolysis was evident and independent of anti-Xa levels, whereas in vitro activation of coagulation proportionally decreased as a function of anti Xa-levels. Our observations that the hypercoagulable profile is more pronounced in the sicker patients are in line with the hypothesis that activation of coagulation, particularly in the pulmonary circulation, is a key feature of COVID-19 and may contribute to progression of disease [26] . These data suggest that low-therapeutic anticoagulant regimens are often insufficient to downregulate coagulation activation in COVID-19 patients, and call for assessment of alternative or intensified antithrombotic strategies. However, careful individual patient assessment (especially in the critically-ill) is warranted, given the increased bleeding risk that is associated with COVID-19. This article is protected by copyright. All rights reserved This article is protected by copyright. All rights reserved This article is protected by copyright. All rights reserved Anti-Xa, anti-activated factor X; CLT, clot lysis time. This article is protected by copyright. All rights reserved Incidence of thrombotic complications in critically ill ICU patients with COVID-19 Incidence of venous thromboembolism in hospitalized patients with COVID-19 Complement associated microvascular injury and thrombosis in the pathogenesis of severe COVID-19 infection: a report of five cases Microvascular COVID-19 lung vessels obstructive thromboinflammatory syndrome (MicroCLOTS): an atypical acute respiratory distress syndrome working hypothesis Pulmonary Vascular Endothelialitis, Thrombosis, and Angiogenesis in Covid-19 The emerging spectrum of cardiopulmonary pathology of the coronavirus disease 2019 (COVID-19): Report of 3 autopsies from Houston, Texas, and review of autopsy findings from other United States cities Autopsy Findings and Venous Thromboembolism in Patients With COVID-19 Anticoagulant treatment is associated with decreased mortality in severe coronavirus disease 2019 patients with coagulopathy COVID19 coagulopathy in Caucasian patients Neutrophil extracellular traps in COVID-19 Accepted Article This article is protected by copyright. All rights reserved Abnormal coagulation parameters are associated with poor prognosis in patients with novel coronavirus pneumonia The procoagulant pattern of patients with COVID-19 acute respiratory distress syndrome Hypercoagulability of COVID-19 patients in Intensive Care Unit. A Report of Thromboelastography Findings and other Parameters of Hemostasis COVID and Coagulation: Bleeding and Thrombotic Manifestations of SARS-CoV2 Infection High risk of thrombosis in patients with severe SARS-CoV-2 infection: a multicenter prospective cohort study Elevated Plasma Levels of Cell-Free DNA During Liver Transplantation Are Associated With Activation of Coagulation Netting neutrophils in autoimmune small-vessel vasculitis Haemostatic Profiles are Similar across All Aetiologies of Cirrhosis Synergistic effects of hypofibrinolysis and genetic and acquired risk factors on the risk of a first venous thrombosis Mixed Fibrinolytic Phenotypes in Decompensated Cirrhosis and Acute-on-Chronic Liver Failure with Hypofibrinolysis in Those With Complications and Poor Survival Accepted Article This article is protected by copyright. All rights reserved Decreased Fibrinolytic Capacity in Cirrhosis and Liver Transplantation Outcomes A sustained decrease in plasma fibrinolytic potential following partial liver resection or pancreas resection Intact thrombin generation and decreased fibrinolytic capacity in patients with acute liver injury or acute liver failure Enhancement of fibrinolytic potential in vitro by anticoagulant drugs targeting activated factor X, but not by those inhibiting thrombin or tissue factor SARS-2 Coronavirus-Associated Hemostatic Lung Abnormality in COVID-19: Is It Pulmonary Thrombosis or Pulmonary Embolism? Re The source of elevated plasma D-dimer levels in COVID-19 infection Decreased plasma levels of activated factor VII in patients with deep vein thrombosis Comparisons between the two groups were made using the Mann-Whitney U test for non-parametric data * Missing data of one (non-ICU) patient for D-dimer and VIIa This article is protected by copyright. All rights reserved Abbreviations: ICU, intensive care unit; TAT, thrombin-antithrombin; F1+2, prothrombin fragment F1+2; VII(a), (activated) blood coagulation factor VII;PAP, plasmin-antiplasmin; sCD40L, soluble CD40 Ligand.