key: cord-0740633-2lcw6w6n
authors: von Meijenfeldt, Fien A.; Havervall, Sebastian; Adelmeijer, Jelle; Lundström, Annika; Rudberg, Ann‐Sofie; Magnusson, Maria; Mackman, Nigel; Thalin, Charlotte; Lisman, Ton
title: Prothrombotic changes in patients with COVID‐19 are associated with disease severity and mortality
date: 2020-12-06
journal: Res Pract Thromb Haemost
DOI: 10.1002/rth2.12462
sha: e232577fb3964f4d8c6dc96eb194dc31b6adfb01
doc_id: 740633
cord_uid: 2lcw6w6n

BACKGROUND AND AIMS: Patients with severe coronavirus disease 2019 (COVID‐19) are at significant risk of thrombotic complications. However, their prothrombotic state is incompletely understood. Therefore, we measured in vivo activation markers of hemostasis, plasma levels of hemostatic proteins, and functional assays of coagulation and fibrinolysis in plasma from patients with COVID‐19 and determined their association with disease severity and 30‐day mortality. METHODS: We included 102 patients with COVID‐19 receiving various levels of respiratory support admitted to general wards, intermediate units, or intensive care units and collected plasma samples shortly after hospital admission. RESULTS: Patients with COVID‐19 with higher respiratory support had increased in vivo activation of coagulation and fibrinolysis, as reflected by higher plasma levels of d‐dimer, thrombin‐antithrombin, and plasmin‐antiplasmin complexes as compared to patients with no to minimal respiratory support and healthy controls. Moreover, the patients with COVID‐19 with higher respiratory support exhibited substantial ex vivo thrombin generation and lower ex vivo fibrinolytic capacity, despite higher doses of anticoagulant therapy compared to less severely ill patients. Fibrinogen, factor VIII, and von Willebrand factor levels increased, and ADAMTS13 levels decreased with increasing respiratory support in patients with COVID‐19. Low platelet count; low levels of prothrombin, antithrombin, and ADAMTS13; and high levels of von Willebrand factor were associated with short‐term mortality. CONCLUSIONS: Severe COVID‐19 is associated with prothrombotic changes with increased in vivo activation of coagulation and fibrinolysis, despite anticoagulant therapy.

Patients with coronavirus disease 2019 are at increased risk of venous thromboembolism (VTE). 1 In particular, patients with COVID-19 admitted to intensive care units (ICUs) exhibit high incidences of VTE (approximately 10%-30%) 2-4 compared to patients with COVID-19 in general wards (VTE incidences of approximately 6%). [5] [6] [7] Notably, most of these patients were receiving standard-or higher-dose thromboprophylactic therapy at the time of VTE diagnosis. The most commonly reported thrombotic complication is pulmonary embolism (PE), with a 5-to 6-fold higher incidence in patients with COVID-19 compared to the general ICU population on thromboprophylaxis. 8 A recent cohort study reported an absolute incidence of 20% of PE in 107 consecutive patients with admitted to the ICU compared to 6.1% and 7.5% in a matched general ICU patient cohort and influenza-positive ICU cohort, respectively, admitted to the same ICU a year earlier. 9 Importantly, postmortem studies have demonstrated abundant microthrombi in pulmonary and extrapulmonary vascular beds 10-12 that contain fibrin, platelets, neutrophils, and neutrophil extracellular traps (NETs). 13 These thrombi have been proposed to contribute to lung injury, progression of disease, and multiple organ failure. Given that symptomatic deep vein thrombosis (DVT) is much less commonly observed than PE in patients with COVID-19, 2 it has been proposed that the thrombotic events are in fact not emboli but rather primary pulmonary thromboses. 14 The pathophysiological mechanisms related to the increased risk of thrombotic complications in patients with COVID-19 are incompletely understood. It has been postulated that the massive activation of the immune system in response to COVID-19 and the associated cytokine storm results in endothelial damage, formation of NETs, platelet activation, and hyperactivation of coagulation. 13, 15 Specifically, activation of endothelial cells by the cytokine storm and possibly by direct infection with severe acute respiratory syndrome coronavirus 2 might play an important role in dysregulation of hemostasis by increasing vascular permeability and promoting recruitment, binding, and activation of immune cells. 16, 17 The hemostatic status of patients with COVID-19 has not been studied extensively, but alterations in hemostatic proteins seem more profound with increasing severity of disease. 18, 19 Clinical studies have reported abnormalities in conventional coagulation tests, such as a prolonged prothrombin time (PT) and thrombocytopenia in a proportion of patients, that were associated with increased disease severity. 15 In addition, extremely high levels of d-dimer and other fibrin degradation products have been found in patients with COVID-19 and have been associated with death. 20, 21 Several studies demonstrated a hypercoagulable profile by whole blood thromboelastography with conflicting results on the presence of characteristics of disseminated intravascular coagulation. 22, 23 Moreover, recent reports demonstrated significant ex vivo thrombin generation in the majority of patients with COVID-19 despite anticoagulant therapy, suggesting that before anticoagulation, patients are profoundly hypercoagulable. 18, 19, 24 This hypercoagulable state is also detected in rotational thromboelastography studies using the EXTEM, which contains a heparin-neutralizing agent. 25 In addition, a hypofibrinolytic state has been demonstrated using whole blood thromboelastography, tissue plasminogen activator-modified thromboelastography, and a plasma-based clot lysis test. 18, 19, 26 Here, we assessed the hemostatic status of 102 patients with COVID-19 on admission in relation to disease severity based on level of respiratory support and level of care and 30-day mortality. We assessed the hemostatic status of these patients by measuring in vivo activation markers of hemostasis, plasma levels of hemostatic proteins, and functional assays that determine ex vivo thrombin generation and fibrinolytic capacity.

We prospectively included 102 patients with COVID-19 admitted to Danderyd Hospital, Stockholm, Sweden, between April 9 and June 8, 2020. All patients were diagnosed with COVID-19 based on reverse-transcriptase polymerase chain reaction (RT-PCR) viral RNA detection of nasopharyngeal or oropharyngeal swabs or clinical presentation. Inclusion was conducted consecutively provided that research personnel were available. Exclusion criteria were age < 18 years, and four patients were diagnosed with VTE (three PEs, one DVT) before blood sampling and received full-dose anticoagulant treatment, and were therefore excluded from this study. Demographic data, comorbidities, medications, and clinical variables including respiratory support and 30-day mortality were obtained from medical records. Patients were divided into groups based on respiratory support at the time of blood sampling (no respiratory support, ≤5 L of oxygen on nasal cannula or mask, and higher respiratory support that comprised > 5 L of oxygen on nasal cannula or mask, noninvasive respiratory support, and intubation).

Level of respiratory support and oxygen concentration were set at the discretion of the treating physician. Thirty-day mortality was defined as mortality within 30 days from admission to the hospital.

Notably, 16 patients (18%) admitted to the general ward did not receive anticoagulants at the time of blood sampling, since these patients were included early in the COVID-19 pandemic, and clear hospital guidelines on enhanced anticoagulant treatment in this patient population had not yet been established. As shown in Table 1 Symptom duration, days 10 (6-14) 14 (9) (10) (11) (12) (13) (14) (15) (16) .146 9 (6-14) 11 (7) (8) (9) (10) (11) (12) (13) (14) 13 (9) (10) (11) (12) (13) (14) (15) .144

Days between admission and blood sampling

.021

Routine laboratory values 

Blood samples were collected up to 7 days (median, 2 [2-3] days) after hospital admission. Peripheral blood was drawn either via venipuncture or from preexistent arterial lines into 3.2% sodium citrate (9:1 vol/vol) vacuum tubes. Platelet-poor plasma was prepared from citrated whole blood following centrifugation within 2 hours from sampling for 20 minutes at 2000 g at room temperature and stored at − 80˚C until further analyses. Plasma preparation from patients and healthy individuals was performed by the same research nurse following the same protocol. Notably, the time between blood sampling and last dose of anticoagulation was not standardized.

Platelet and white blood cell (WBC) counts, C-reactive protein (CRP), and creatinine and lactate assays were performed by the Laboratory following the manufacturer's instructions, with the exception of a centrifugation at 10 000 g for 10 minutes of plasma after it was thawed as described previously. 28 We analyzed the following parameters: endogenous thrombin potential (ETP), representing the total enzymatic work performed by thrombin during the time that it was active; peak (thrombin); lag time, defined as the time to reach onesixth of the peak thrombin concentration; and velocity index (slope between end of lag time and peak thrombin).

Anti-Xa levels were determined on an automated coagulation analyzer (STACompact 3, Stago) using Heparin LRT (HYPHEN BioMed, Nodia, Amsterdam, the Netherlands). Note: The results are presented as median (interquartile range). Comparisons between the two groups were made using the student t-test or Mann-Whitney U test, as appropriate.

Abbreviations: ADAMTS13, a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13; ETP, endogenous thrombin potential; PAI-1, plasminogen activator inhibitor type 1; PAP, plasmin-antiplasmin; PT, prothrombin time; TAT, thrombin-antithrombin; VWF, von Willebrand factor. healthy controls. Plasma prothrombin and antithrombin levels did not differ between patients and healthy controls. However, a proportion of patients had clearly decreased prothrombin and antithrombin levels;

23 (23%) patients had prothrombin levels < 75%, and 14 (14%) patients had antithrombin levels < 75%, and most of these patients (20/23 for low prothrombin and 11/14 for antithrombin) were admitted to general wards. Markers of in vivo activation of coagulation and fibrinolysis, d-dimer, TAT, and PAP all were significantly higher in patients with COVID-19 compared to controls. d-dimer and PAP levels, but not TAT levels, were associated with CRP (R 2 = 0.11, P < .01; R 2 = 0.086, P < .01; R 2 = 0.001, P = .76, respectively). In addition, thrombomodulinmodified thrombin generation was higher in patients compared to controls (despite anticoagulation). Ex vivo fibrinolytic potential was decreased in patients compared to healthy controls. 

Finally, we assessed severity of disease by level of respiratory support. Patients with a higher level of respiratory support had similar PT and platelet counts, and increased factor VIII and VWF plasma levels in comparison with patients who required no or minimal respiratory support, as shown in Table 3 . ADAMTS13 levels were lower in patients with respiratory support compared to patients without respiratory support. Levels of prothrombin and antithrombin did not differ between patients with different levels of respiratory support.

Fibrinogen levels increased significantly with increasing respiratory support. PAI-1 levels did not differ between patients with different levels of respiratory support. Markers of in vivo activation of coagulation, d-dimer, and TAT were higher in patients with higher respiratory support, but PAP levels did not differ (P = .051). As for ex vivo thrombin generation, ETP, peak thrombin, and velocity index did not statistically differ based on respiratory support. However, these results are confounded by higher anti-Xa levels in these patients (Table 1) . Lag times increased with increasing respiratory support and correlated with the PT (r 2 = 0.247, P < .001). Clot lysis time was longer in patients who received higher respiratory support compared to patients who received no pulmonary support.

Patients with COVID-19 who died within 30 days from hospital admission had prolonged PT, lower platelet counts, similar factor VIII plasma levels, elevated VWF, and decreased ADAMTS13 plasma levels at hospital admission in comparison to patients who were alive at 30-day follow up ( 

We axis, 18, [30] [31] [32] [33] in vivo and ex vivo activation of coagulation, 18, 19, 25, [34] [35] [36] and in vivo and ex vivo fibrinolytic status. [18] [19] [20] 26 We first assessed disease severity according to level of care and found, in line with existing literature, 18 indicate that other drug classes, notably direct oral anticoagulants, may be more effective. However, the concept of heparin resistance in COVID-19 has been challenged. 39 We also demonstrated that hemostatic parameters became progressively more prothrombotic with increasing respiratory support, with the exception of prothrombin and antithrombin, platelet pneumonia 47 , but more in-depth studies are certainly indicated.

In conclusion, we demonstrate that patients with COVID-19

have increased in vivo activation of coagulation and fibrinolysis despite anticoagulant therapy and derangements in various hemostatic proteins, which were more profound proportional to severity of disease as assessed by respiratory support. Importantly, ex vivo thrombin generation was higher compared to controls, and similar in patients in general wards and patients in higher levels of care, despite significantly higher doses of LMWH in patients in higher levels of care, suggesting that current anticoagulant strategies are insufficient. Whether laboratory tests, such as thrombin generation tests or whole blood thromboelastography, may be useful in guiding anticoagulant treatment requires further study. In addition, low platelet count; low prothrombin, antithrombin, and ADAMTS13 levels; and high levels of VWF on admission were associated with 30-day mortality. Together, these results substantiate the hypothesis that hyperactivation of hemostasis might play a role in the progression of lung injury in COVID-19 by formation of intra-and extrapulmonary clots.

The authors declare no conflict of interest. 

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Prothrombotic changes in patients with COVID-19 are associated with disease severity and mortality