Coagulation profile of COVID-19 patients admitted to the ICU: An exploratory study

Thiago Domingos Corrêa; Ricardo Luiz Cordioli; João Carlos Campos Guerra; Bruno Caldin da Silva; Roseny dos Reis Rodrigues; Guilherme Martins de Souza; Thais Dias Midega; Niklas Söderberg Campos; Bárbara Vieira Carneiro; Flávia Nunes Dias Campos; Hélio Penna Guimarães; Gustavo Faissol Janot de Matos; Valdir Fernandes de Aranda; Leonardo José Rolim Ferraz

doi:10.1371/journal.pone.0243604

. 2020 Dec 15;15(12):e0243604. doi: 10.1371/journal.pone.0243604

Coagulation profile of COVID-19 patients admitted to the ICU: An exploratory study

Thiago Domingos Corrêa ^1,^#, Ricardo Luiz Cordioli ^1,^*,^#, João Carlos Campos Guerra ², Bruno Caldin da Silva ¹, Roseny dos Reis Rodrigues ¹, Guilherme Martins de Souza ¹, Thais Dias Midega ¹, Niklas Söderberg Campos ¹, Bárbara Vieira Carneiro ¹, Flávia Nunes Dias Campos ¹, Hélio Penna Guimarães ¹, Gustavo Faissol Janot de Matos ¹, Valdir Fernandes de Aranda ², Leonardo José Rolim Ferraz ¹

Editor: Pablo Garcia de Frutos³

PMCID: PMC7737963 PMID: 33320874

Abstract

Background

Coagulation abnormalities in COVID-19 patients have not been addressed in depth.

Objective

To perform a longitudinal evaluation of coagulation profile of patients admitted to the ICU with COVID-19.

Methods

Conventional coagulation tests, rotational thromboelastometry (ROTEM), platelet function, fibrinolysis, antithrombin, protein C and S were measured at days 0, 1, 3, 7 and 14. Based on median total maximum SOFA score, patients were divided in two groups: SOFA ≤ 10 and SOFA > 10.

Results

Thirty patients were studied. Some conventional coagulation tests, as aPTT, PT and INR remained unchanged during the study period, while alterations on others coagulation laboratory tests were detected. Fibrinogen levels were increased in both groups. ROTEM maximum clot firmness increased in both groups from Day 0 to Day 14. Moreover, ROTEM–FIBTEM maximum clot firmness was high in both groups, with a slight decrease from day 0 to day 14 in group SOFA ≤ 10 and a slight increase during the same period in group SOFA > 10. Fibrinolysis was low and decreased over time in all groups, with the most pronounced decrease observed in INTEM maximum lysis in group SOFA > 10. Also, D-dimer plasma levels were higher than normal reference range in both groups and free protein S plasma levels were low in both groups at baseline and increased over time, Finally, patients in group SOFA > 10 had lower plasminogen levels and Protein C than patients with SOFA <10, which may represent less fibrinolysis activity during a state of hypercoagulability.

Conclusion

COVID-19 patients have a pronounced hypercoagulability state, characterized by impaired endogenous anticoagulation and decreased fibrinolysis. The magnitude of coagulation abnormalities seems to correlate with the severity of organ dysfunction. The hypercoagulability state of COVID-19 patients was not only detected by ROTEM but it much more complex, where changes were observed on the fibrinolytic and endogenous anticoagulation system.

Introduction

More than thirteen million people have been diagnosed with coronavirus disease 2019 (COVID-19) worldwide [1] since severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first identified in China in December 2019 [2]. Almost one third of the hospitalized patients with COVID-19 are admitted to the ICU [2–4].

Coagulation abnormalities, mainly thrombotic complications, have been described in COVID-19 patients [5–11]. The incidence of arterial and venous thrombotic complications in COVID-19 patients admitted to the ICU may reach 31% [12]. Indeed, it has been shown that D-dimer and fibrinogen degradation products (FDP) values were higher in patients with most severe SARS-CoV-2 infection than in patients with milder forms of disease [6]. Moreover, Chen et al. have demonstrated that deceased patients with COVID-19 exhibited D-dimer concentrations approximately seven times higher than recovered patients [13]. Additionally, low platelet count was show to be associated with increased risks of more severe forms of disease and increased hospital mortality in COVID-19 patients [10]. Although D-dimer, FDP and platelet count have been used as clinical indicators of SARS-CoV-2 infection severity [14], coagulation abnormalities in COVID-19 patients have not been addressed in depth.

Conventional coagulation tests (CCT) such as prothrombin time (PT), international normalized ratio (INR), thrombin time (TT) and activated partial thromboplastin time (aPTT) are unable to reflect the complexity of hemostatic impairment observed in intensive care unit (ICU) patients [15]. Rotational thromboelastometry (ROTEM) represents a point-of-care test that assess the viscoelastic properties of whole blood, providing a real-time evaluation of clot formation kinetics, i.e., clot formation, stabilization and dissolution, at the bedside [16]. To the best of our knowledge, a comprehensive and longitudinal analysis of the coagulation profile, including CCT, ROTEM, platelet function, fibrinolysis, and endogenous inhibitors of coagulation (antithrombin, protein C and S) have not been described in ICU COVID-19 patients so far.

We hypothesized that ICU patients diagnosed with COVID-19 have a prothrombotic profile that may be explained by an impaired endogenous anticoagulation system. Moreover, the degree of coagulation abnormalities reflects the severity of the disease. Therefore, our objective was to perform a comprehensive longitudinal evaluation of coagulation, fibrinolysis, and endogenous anticoagulation system of patients admitted to the ICU with severe COVID-19.

Materials and methods

Study design and setting

We performed a single center prospective longitudinal study in an ICU of a private tertiary care hospital in São Paulo, Brazil. The study was approved by the Local Ethics Committee at Hospital Israelita Albert Einstein and by Comissão Nacional de Ética em Pesquisa (CONEP) with waiver of informed consent (CAAE: 30175220.3.0000.0071). This study is reported in accordance with the Strengthening the Reporting of Observational studies in Epidemiology (STROBE) statement [17].

Study participants

Thirty patients aged ≥18 years old admitted to the ICU with confirmed diagnosis of COVID-19 were included in this study. Laboratory confirmation of SARS-CoV-2 infection was based on positive reverse-transcriptase-polymerasechain-reaction (RT-PCR) assay [18].

Exclusion criteria included pregnancy, previous known coagulopathy, currently use of systemic anticoagulants or anti-platelet therapy or vitamin K antagonists, moribund patients and patients who presented cardiac arrest.

Participants were recruited between March 29, 2020 through May 13, 2020 and they could represent the majority of severe patients infected by COVID-19, once there were few exclusion criteria and the participants were recruited with waiver of informed consent once it was an observational study without any intervention and consecutive patients admitted in the Intensive Care Unit were recruited following the inclusion and exclusion criteria until we completed 30 patients included in the study.

Laboratory analysis

Laboratory tests were performed at the time of study inclusion (baseline), and at days 1, 3, 7 and 14 after enrollment unless the patient had died or was discharged from the hospital.

Conventional coagulation tests

Conventional coagulation tests included: platelet count (XE 2100, Sysmex, São Paulo, Brazil), plasma fibrinogen concentration [Clauss method (Hemosil QFA thrombin (bovine), IL Instrumentation Laboratory Company, Bedford MA, USA], aPTT (Hemosil Synthasil, IL Instrumentation Laboratory Company, Bedford MA, USA), PT and INR (Hemosil PT-Fibrinogen HS Plus, IL Instrumentation Laboratory Company, Bedford MA, USA) and ionic calcium (ABL 800 FLEX, Radiometer Medical ApS, Brønshøj, Denmark).

Rotational thromboelastometry

Rotational thromboelastometry analyses were performed with EXTEM (extrinsic coagulation pathway assessment), INTEM (intrinsic coagulation pathway assessment) and FIBTEM (extrinsic coagulation pathway assessment with additional platelet inhibition using cytochalasin D) tests according to the manufacturer’s instructions [16]. The following parameters were recorded during ROTEM analysis: clotting time [CT; seconds (sec)], which represents the beginning of the test until clot firmness of 2 mm; clot formation time (CFT; sec), which represents time between detection of a clot firmness of 2 and 20 mm and maximum clot firmness (MCF; mm), which represents the greatest amplitude of thromboelastometric trace and reflects clot “strength” [19, 20]. ROTEM tests were performed by laboratory technicians. Blood samples of approximately 3 ml were collected by venipuncture into a tube with citrate (3.2%; Sarsted1, Wedel, Germany). Blood samples were immediately processed for ROTEM analysis. The analyses were performed by pipetting 340 μl of citrated whole blood and 20 μl of 0.2 M calcium chloride with specific activators into a cup. There was no change in methodology for test performance nor test controls (Rotrol N and Rotrol P) throughout the study period [15, 20].

Normal coagulation profile on the ROTEM was defined according to reference values for CT, CFT and MCF (INTEM CT: 100–240 sec, INTEM CFT: 30–110 sec, INTEM MCF: 50–72 mm; EXTEM CT: 38–79 sec, EXTEM CFT: 34–159 sec, EXTEM MCF: 50–72 mm; FIBTEM MCF: 9–25 mm) [15, 19, 20]. Hypocoagulability in ROTEM was defined as prolongation of CT (INTEM CT >240 sec or EXTEM CT >79 sec) and/or CFT (INTEM CFT >110 sec or EXTEM CFT >159 sec) and/or MCF reduction (MCF INTEM or EXTEM MCF <50 mm or FIBTEM MCF <9 mm) [15, 19]. Hypercoagulability in ROTEM was defined as a reduction in clotting time (INTEM CT <100 sec or EXTEM CT <38 sec), or clot formation time (INTEM CFT <30 sec or EXTEM CFT <34 sec) and/or an increase in MCF (MCF INTEM or EXTEM MCF >72 mm or FIBTEM MCF >25 mm) [15, 19].

Platelet function test

Platelet function test was assessed in whole blood samples using impedance aggregometry (The ROTEM®‐Platelet; TEM Innovations GmbH, Munich Germany) [21]. Platelets were activated with arachidonic acid (ARATEM test) and adenosine diphosphate (ADPTEM) [21].

Fibrinolysis and endogenous anticoagulation system

D-dimer (Hemosil D-dimer HS 500 and hemosil D-dimer HS 500 controls, L Instrumentation Laboratory Company, Bedford MA, USA), serum plasminogen (Plasmin Inhibitor, IL Instrumentation Laboratory Company, Bedford MA, USA), alpha-2 antiplasmin (Plasmin Inhibitor, IL Instrumentation Laboratory Company, Bedford MA, USA), antithrombin (IL Instrumentation Laboratory Company, Bedford MA, USA), protein C (IL Instrumentation Laboratory Company, Bedford MA, USA) and free protein S (IL Instrumentation Laboratory Company, Bedford MA, USA) were measured.

Data collection

All study data were retrieved from Epimed Monitor System (Epimed Solutions, Rio de Janeiro, Brazil), which is an electronic structured case report form where patients data are prospectively entered by trained ICU case managers [22].

Collected clinical variables included demographics, comorbidities, Simplified Acute Physiology score (SAPS 3 score) at ICU admission [23], Sequential Organ Failure Assessment score (SOFA score) [24] at ICU admission, and at days 1, 3, 7 and 14 after enrollment unless the patient had died or was discharged from the ICU, total maximum SOFA score (from the time of study inclusion (baseline) up to 14 after enrollment unless the patient had died or was discharged from the ICU) [25], body-mass index, treatment measures (i.e, hydroxychloroquine, macrolides, corticosteroids, interleukin-6 receptor antagonist, convalescent plasma and lopinavir-ritonavir), supportive therapy [use of vasopressors, mechanical ventilation, noninvasive mechanical ventilation and renal replacement therapy (RRT)] during ICU stay, hospital length of stay (LOS) prior to ICU admission, ICU and hospital LOS, and ICU mortality.

Blood component transfusion [platelet concentrate, fresh frozen plasma (FFP) and cryoprecipitate] and hemostatic agents [fibrinogen concentrate, prothrombin complex concentrate (PCC) and tranexamic acid] were collected. The presence of thrombotic or hemorrhagic events and the use of prophylactic or therapeutic doses of low-molecular-weight heparin (LMWH) or unfractionated heparin (UFH) during ICU stay were recorded.

Statistical analysis

Based on median total maximum SOFA score, patients were divided in two groups: group SOFA ≤ 10 and group SOFA > 10. Categorical variables were presented as n/n total (%). Continuous variables were presented as median with interquartile range (IQR). Categorical variables were compared between groups with Fisher’s exact test, and continuous variables were compared using independent t test or Mann-Whitney U test in case of non-normal distribution, tested by the Kolmogorov-Smirnov test.

To account for longitudinal (repeated measurements) and correlated response continuous variables, between-group differences and within-group differences over time were assessed using generalized estimating equations (GEE), with group (SOFA ≤ 10 and group SOFA > 10) and study time points (time) as predictors. P values for group effect, time effect, and time-group interaction were presented. When a time effect was detected in pooled patients, each time point (Day 1, 3, 7 and 14) was compared against Day 0. When a group effect or a time-group interaction were detected, between group comparisons (group SOFA>10 vs. group SOFA ≤10) were performed at each time point. The Bonferroni method was used to account for multiple comparisons.

Two-tailed tests were used and when p<0.05, the test was considered statistically significant. No adjustment was made for missing data. The SPSS™ (IBM™ Statistical Package for the Social Science version 26.0) was used for statistical analyses, and GraphPad Prism version 8.0.0 (GraphPad Software, San Diego, California, USA) was used for graph plotting.

Results

Baseline characteristics of patients

From March 29, 2020 through May 13, 2020, thirty patients were included in this study. The median (IQR) age of pooled patients was 61 (52–83) years, 50.0% were man and median (IQR) SAPS III of 49 (41–61). Of the 30 patients, 24 (80.0%) had one or more coexisting medical conditions. Obesity [12/29 (41.4%)], systemic hypertension [12/30 (40.0%)] and diabetes mellitus [11/30 (36.7%)] were the most common coexisting conditions. Most patients received invasive mechanical ventilation [27/30 (90.0%)] and/or vasopressors [27/30 (90.0%)] during the ICU stay. Clinical characteristics of patients are shown in Table 1.

Table 1. Characteristics of study participants.

Characteristics	All patients (n = 30)	SOFA ≤10 (n = 16)	SOFA >10 (n = 14)	P value
Age, years (median, IQR)	61 (52–83)	53 (45–64)	78 (60–85)	0.002^a
Men, n (%)	15/30 (50.0)	7/16 (43.8)	8/14 (57.1)	0.715^b
SAPS III score, points (median, IQR)^§	49 (41–61)	50 (43–64)	47 (41–55)	0.307^a
SOFA score D0, points (median, IQR)^#	6 (4–8)	5 (3–6)	8 (6–9)	0.002^a
Maximum SOFA score, points (median, IQR)^#	10 (7–12)	7 (6–9)	13 (11–14)	<0.001^a
Number of coexisting conditions, (median, IQR)	2 (1–3)	1 (0–2)	3 (2–3)	<0.001^a
Coexisting conditions, n (%)
Obesity	12/29 (41.4)	7/16 (43.8)	5/13 (38.5)	1.000^b
Systemic hypertension	12/30 (40.0)	5/16 (31.3)	7/14 (50.0)	0.457^b
Diabetes mellitus	11/30 (36.7)	2/16 (12.5)	9/14 (64.3)	0.007^b
Malignancy	4/30 (13.3)	2/16 (12.5)	2/14 (14.3)	1.000^b
Congestive heart failure	3/30 (10.0)	0/16 (0.0)	3/14 (21.4)	0.090^b
COPD / Asthma	3/30 (10.0)	1/16 (6.3)	2/14 (14.3)	0.586^b
Chronic kidney disease	4/30 (13.3)	1/16 (6.3)	3/14 (21.4)	0.315^b
Coronary artery disease	2/30 (6.7)	0/16 (0.0)	2/14 (14.3)	0.209^b
Body-mass index	29.3 (24.4–32.2)	29.7 (24.2–32.5)	27.2 (24.4–31.4)	0.793^a
Treatment, n (%)
Macrolides	28/30 (93.3)	16/16 (100.0)	12/14 (85.7)	0.209^b
Glucocorticoids	25/30 (83.3)	12/16 (75.0)	13/14 (92.9)	0.336^b
Hydroxychloroquine	24/30 (80.0)	13/16 (81.3)	11/14 (78.6)	1.000^b
Convalescent plasma	10/30 (33.3)	6/16 (37.5)	4/14 (28.6)	0.709^b
Interleukin-6 receptor antagonist	3/30 (10.0)	2/16 (12.5)	1/14 (7.1)	1.000^b
Lopinavir-ritonavir	2/30 (6.7)	1/16 (6.3)	1/14 (7.1)	1.000^b
Support during ICU stay, n (%)
Vasopressors	27/30 (90.0)	13/16 (81.3)	14/14 (100.0)	0.228^b
Mechanical ventilation	27/30 (90.0)	13/16 (81.3)	14/14 (100.0)	0.228^b
Noninvasive ventilation	6/30 (20.0)	3/16 (18.8)	3/14 (21.4)	1.000^b
Renal replacement therapy	10/30 (33.3)	0/16 (100.0)	10/14 (71.4)	<0.001^b
Time from symptom onset to study inclusion, days (median, IQR)	9 (6–14)	9 (6–15)	8 (6–13)	0.951^c
Hospital LOS prior ICU admission, days (median, IQR)	1 (0–3)	2 (1–3)	1 (0–2)	0.400^c

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Values represent median (IQR) or n (%). ICU: intensive care unit, SAPS III: simplified acute physiology score III

§: scores on SAPS III range from 0 to 217, with higher scores indicating more severe illness and higher risk of death, SOFA: sequential organ failure assessment score

#: SOFA score ranges from 0 to 24, with higher scores indicating more severe organ dysfunction, COPD: chronic obstructive pulmonary disease, LOS: length of stay. P values were calculated with the use of (a) Independent t-test, (b) Fisher's exact test or (c) Mann-Whitney U test.

Compared with patients in group SOFA ≤ 10 [16/30 (53.3%) patients], patients in group SOFA > 10 [14/30 (46.7%)] were older [median (IQR), 78 (60–85) vs. 53 (45–64) years, p = 0.002], had a higher SOFA score at study inclusion [median (IQR), 8 (6–9) vs. 5 (3–6) points, p = 0.002], a higher number of coexisting conditions [median (IQR), 3 (2–3) vs. 1 (0–2), p<0.001], had diabetes mellitus more frequently [9/14 (64.3) vs. 2/16 (12.5), p = 0.007] and received renal replacement therapy more frequently [10/14 (71.4) vs. 0/16 (0.0), p<0.001] (Table 1).

Anticoagulants, blood transfusion and clinical outcomes

During the study period, 22/30 (73.3%) patients received anticoagulants as deep vein thrombosis (DVT) prophylaxis and 7/30 (23.3%) patients received systemic anticoagulation (Table 2). The proportion of patients receiving anticoagulants as DVT prophylaxis and as systemic anticoagulation did not differ between the groups (p = 0.830) (Table 2). Unfractionated heparin was more commonly used as DVT in group SOFA > 10 compared to group SOFA ≤ 10 [7/10 (70.0%) vs. 2/12 (16.7%), p = 0.027] (Table 2).

Table 2. Administered treatments and clinical outcomes.

Characteristics	All patients (n = 30)	SOFA ≤10 (n = 16)	SOFA >10 (n = 14)	P value
Anticoagulants, n (%)				0.830^a
DVT prophylaxis	22/30 (73.3)	12/16 (75.0)	10/14 (71.4)
UFH	9/22 (40.9)	2/12 (16.7)	7/10 (70.0)	0.027^a
LMWH	13/22 (59.1)	10/12 (83.3)	3/10 (30.0)
Systemic anticoagulation	7/30 (23.3)	3/16 (18.8)	4/14 (28.6)
UFH	5/7 (71.4)	1/3 (33.3)	4/4 (100.0)	0.143^a
LMWH	2/7 (28.6)	2/3 (66.7)	0/4 (0.0)
Transfused blood product, n (%)
Red blood cells	4/30 (13.3)	0 (0.0)	4/14 (28.6)	0.037^a
Platelet concentrate	0/30 (0.0)	0 (0.0)	0 (0.0)
Fresh frozen plasma	0/30 (0.0)	0 (0.0)	0 (0.0)
Cryoprecipitate	0/30 (0.0)	0 (0.0)	0 (0.0)
Thrombotic events, n (%)	6/30 (20.0)	3/16 (18.8)	3/14 (21.4)	1.000^a
DVT	4/6 (66.7)	2/3 (66.7)	2/3 (66.7)
Pulmonary embolism	2/6 (33.3)	1/3 (33.3)	1/3 (33.3)
Hemorrhagic events, n (%)	3/30 (10.0)	2/16 (12.5)	1/14 (7.1)	1.000^a
Outcomes^#
Died in ICU	4/30 (13.3)	0/16 (0.0)	4/14 (28.6)	0.014^a
Discharged from ICU	25/30 (83.3)	16/16 (100.0)	9/14 (64.3)
Still in ICU as of 06/10/2020	1/30 (3.3)	0/16 (0.0)	1/14 (7.1)
ICU LOS (days), median (IQR)^#	15 (7–22)	7 (6–15)	22 (16–35)	<0.001^b
Hospital LOS (days), median (IQR)^#	26 (15–38)	17 (13–30)	38 (25–51)	0.012^b

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Values represent median (IQR) or n (%). ICU: intensive care unit, DVT: deep vein thrombosis, HFH: unfractionated heparin, LMWH: low-molecular-weight heparin, LOS: length of stay.

#: the number of patients who died, were discharged, and were still admitted in the ICU as of June 10, 2020 were recorded, and ICU and hospital length of stay also were determined. P values were calculated with the use of (a) Fisher's exact test and (b) Mann-Whitney U test.

Four patients (13.3%) received red blood cells transfusion, all of them in group SOFA > 10 (Table 2). No patient received platelet concentrate, FFP, cryoprecipitate, fibrinogen concentrate, PCC or tranexamic acid during the study period. Thrombotic events occurred in 6/30 (20.0%) patients and hemorrhagic events were observed in 3/30 (10.0%) patients. The incidence of thrombotic and hemorrhagic events did not differ between the groups (Table 2).

As of June 10, 2020, one patient [1/30 (3.3%)] was still hospitalized in the ICU while 25/30 (83.3%) of patients were discharged alive from the ICU. Four [4/30 (13.3%)] patients died at the ICU (Table 2). Compared with patients in group SOFA ≤ 10, patients in group SOFA > 10 had a higher ICU mortality [4/14 (28.6%) vs. 0/16 (0.0%), p = 0.014], exhibited a higher ICU [median (IQR), 22 (16–35) vs. 7 (6–15), p<0.001], and hospital [38 (25–51) vs. 17 (13–30), p = 0.012] LOS (Table 2).

Laboratory analysis

Arterial pH increased in both groups from Day 0 to Day 14 (p<0.001 for time effect), while ionized calcium increased from Day 0 to Day 14 in group SOFA ≤ 10, and decreased in group SOFA > 10 (p = 0.038 for time-group interaction) (Table 3). Peripheral temperature remained stable during study period (Table 3).

Table 3. Arterial pH, ionized calcium, peripheral temperature, conventional coagulation tests and hemoglobin.

Parameters	Reference range	Day 0	Day 1	Day 3	Day 7	Day 14	P value
Arterial pH	7.35–7.45
All patients		7.40 (7.35–7.41)	7.38 (7.31–7.40)	7.40 (7.34–7.43)	7.42 (7.38–7.45)	7.46 (7.43–7.50)^*	<0.001^a
SOFA≤10		7.40 (7.36–7.41)	7.39 (7.35–7.42)	7.43 (7.40–7.45)	7.43 (7.42–7.47)	7.45 (7.42–7.48)	<0.001^b
SOFA >10		7.39 (7.31–7.40)	7.32 (7.27–7.39)^#	7.34 (7.30–7.39)^#	7.41 (7.32–7.45)^#	7.47 (7.44–7.50)	0.001^c
Ionized calcium (mmol/L)	1.14–1.31
All patients		1.12 (1.09–1.15)	1.15 (1.12–1.20)	1.13 (1.10–1.16)	1.17 (1.15–1.21)	1.15 (1.06–1.24)	0.030^a
SOFA≤10		1.14 (1.13–1.21)	1.15 (1.12–1.19)	1.13 (1.10–1.14)	1.16 (1.15–1.19)	1.24 (1.17–1.25)	0.151^b
SOFA >10		1.12 (1.09–1.12)^#	1.14 (1.10–1.20)	1.13 (1.09–1.21)	1.19 (1.16–1.26)	1.09 (1.06–1.17)	0.038^c
Temperature (°C)
All patients		36.4 (36.0–37.1)	36.6 (36.2–37.0)	36.5 (36.2–37.1)	36.2 (36.0–36.7)	36.1 (36.0–36.6)	0.149^a
SOFA≤10		36.2 (36.0–36.8)	36.6 (36.4–37.2)	36.7 (36.2–37.2)	36.3 (36.2–36.7)	36.1 (36.0–36.6)	0.351^b
SOFA >10		36.5 (35.9–37.5)	36.5 (36.0–37.0)	36.3 (35.3–36.7)	36.1 (35.9–36.8)	36.1 (35.7–36.6)	0.324^c
Platelets (x10⁹/L)	150–450
All patients		226 (176–261)	236 (182–268)	272 (230–314)^*	349 (242–444)^*	306 (229–480)^*	<0.001^a
SOFA≤10		243 (185–274)	236 (210–307)	271 (252–310)	419 (307–462)	469 (232–679)	0.009^b
SOFA >10		197 (141–237)^#	220 (160–264)	277 (197–314)	282 (175–349)^#	286 (212–383)	0.213^c
Fibrinogen (g/dL)	200–400
All patients		600 (480–680)	642 (470–722)	625 (513–782)	532 (348–592)^*	397 (303–537)^*	<0.001
SOFA≤10		633 (503–690)	642 (524–722)	592 (513–680)	482 (348–592)	372 (298–439)	0.365^b
SOFA >10		552 (480–680)	610 (470–851)	700 (513–822)	564 (473–646)	470 (303–550)	0.048^c
Prothrombin time (sec)	70–100
All patients		82 (76–89)	81 (69–89)	78 (70–88)	85 (73–88)	80 (67–89)	0.323^a
SOFA≤10		82 (78–93)	78 (70–86)	78 (70–88)	85 (78–87)	83 (67–97)	0.226^b
SOFA >10		83 (67–86)	86 (69–89)	75 (67–86)	76 (70–89)	77 (63–87)	0.463^c
INR	0.96–1.30
All patients		1.13 (1.07–1.18)	1.14 (1.07–1.25)	1.17 (1.10–1.25)	1.11 (1.07–1.21)	1.17 (1.07–1.34)	0.503^a
SOFA≤10		1.13 (1.05–1.17)	1.16 (1.09–1.25)	1.16 (1.07–1.24)	1.10 (1.09–1.16)	1.12 (1.02–1.33)	0.303^b
SOFA >10		1.13 (1.09–1.28)	1.10 (1.07–1.26)	1.19 (1.10–1.29)	1.18 (1.07–1.27)	1.21 (1.10–1.39)	0.143^c
aPTT (sec)	25.6–35.5
All patients		28.8 (27.2–32.6)	27.8 (25.6–32.8)	28.6 (25.9–33.2)	27.9 (26.4–31.9)	28.1 (25.6–35.7)	0.079^a
SOFA≤10		28.0 (27.0–31.1)	27.2 (25.6–31.2)	26.7 (25.6–31.0)	27.4 (27.0–30.8)	28.1 (23.7–31.9)	0.497^b
SOFA >10		30.0 (28.3–33.0)	28.7 (25.6–33.4)	30.8 (27.0–38.0)	28.4 (26.0–33.3)	28.3 (26.6–36.7)	0.512^c
Hemoglobin (g/dL)	13.5–17.5
All patients		12.1 (11.2–12.9)	11.4 (10.2–12.2)^*	10.5 (9.6–11.8)^*	10.2 (9.3–11.1)^*	9.2 (8.7–10.6)^*	<0.001^a
SOFA≤10		12.2 (11.3–12.9)	11.4 (10.7–12.1)	10.6 (9.7–11.8)	10.7 (10.1–11.1)	9.2 (9.1–11.7)	0.483^b
SOFA >10		11.7 (11.2–13.4)	11.3 (10.1–13.2)	10.5 (9.0–11.8)	9.8 (8.3–10.3)	9.3 (8.1–10.2)	0.618^c

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Values represent median (IQR). INR: international normalized ratio, aPTT: activated partial thromboplastin time. P values were calculated with the use of generalized estimating equations (GEE): (a): time effect, (b): group effect and (c): time-group interaction. Pairwise comparisons significant at the 0.05 level:

(*): time effect—pooled patients: each time point vs. Day 0.

(#): between group comparisons (group SOFA>10 vs. group SOFA ≤10) at each time point.

Conventional coagulation tests

Platelet count increased from baseline to Day 14 in both groups (p<0.001 for time effect), although lower platelet count was observed over the time in group SOFA > 10 compared to group SOFA ≤ 10 (p = 0.009 for group effect) (Table 3). Fibrinogen levels were increased in both groups at baseline, with the highest values observed at day 1 in group SOFA ≤ 10 and at day 3 in group SOFA > 10 (p<0.001 for time effect). Patients in group SOFA ≤ 10 exhibited a more pronounced decrease in plasma fibrinogen levels at study end than patients in group SOFA > 10 (p = 0.048 for time-group interaction) (Table 3). Prothrombin time, INR, aPTT, and hemoglobin remained unchanged during the study period (Table 3).

Rotational thromboelastometry

The majority of patients in both groups exhibited a hypercoagulability state based on ROTEM (Fig 1). ROTEM (INTEM and EXTEM) maximum clot firmness slightly increased in both study groups from Day 0 to Day 14 (p<0.001 for time effect) (Table 4). ROTEM–FIBTEM maximum clot firmness was high in both groups during the study period, with a slight decrease from Day 0 to Day 14 in group SOFA ≤ 10 and a slight increase during the same period in group SOFA > 10 (p = 0.050 for time-group interaction) (Table 4).

Table 4. Rotational thromboelastometry and platelet function test.

Parameters	Reference range	Day 0	Day 1	Day 3	Day 7	Day 14	P value
ROTEM—INTEM
Clotting time (sec)	100–240
All patients		164 (155–184)	160 (156–191)	173 (159–196)	171 (157–193)	163 (153–190)	0.300^a
SOFA≤10		165 (141–180)	157 (153–168)	161 (142–180)	172 (162–193)	171 (148–188)	0.035^b
SOFA >10		159 (157–184)	176 (156–194)	184 (172–214⁾^#	169 (152–185)	157 (153–194)	0.378^c
Clot formation time (sec)	30–110
All patients		48 (46–59)	47 (42–57)	44 (39–55)	42 (39–48)	41 (34–47)	0.096^a
SOFA≤10		48 (45–55)	46 (41–52)	40 (35–47)	42 (37–47)	34 (29–49)	0.026^b
SOFA >10		53 (47–62)	51 (43–63)^#	52 (45–62)^#	43 (40–61)	43 (39–46)	0.567^c
Maximum clot firmness (mm)	50–72
All patients		70 (67–72)	71 (69–73)^*	73 (70–76)	75 (70–78)^*	76 (69–79)^*	<0.001^a
SOFA≤10		70 (68–72)	72 (70–74)	74 (72–76)	75 (70–79)	78 (70–80)	0.312^b
SOFA >10		70 (66–73)	71 (67–73)	72 (70–76)	75 (71–77)	75 (69–78)	0.914^c
Maximum lysis (%)	£15
All patients		10 (6–12)	8 (5–10)^*	6 (3–9)^*	3 (2–6)^*	4 (2–6)^*	<0.001^a
SOFA≤10		11 (6–13)	8 (4–10)	7 (6–9)	5 (2–6)	5 (2–6)	0.223^b
SOFA >10		9 (6–12)	8 (5–11)	3 (2–7)	2 (1–3)^#	3 (2–7)	0.004^c
ROTEM—EXTEM
Clotting time (sec)	38–79
All patients		72 (66–79)	73 (66–88)	78 (70–84)^*	73 (62–88)	72 (61–80)	0.013^a
SOFA≤10		76 (71–81)	75 (67–94)	76 (68–78)	74 (68–81)	78 (61–83)	0.486^b
SOFA >10		68 (66–71)	72 (64–75)	85 (77–94)^#	68 (61–89)	67 (65–80)	0.001^c
Clot formation time (sec)	34–159
All patients		54 (44–61)	56 (45–64)	48 (41–63)	49 (41–57)	47 (41–56)	0.123^a
SOFA≤10		53 (44–61)	52 (43–62)	45 (40–64)	46 (37–56)	38 (31–59)	0.124^b
SOFA >10		54 (50–64)	60 (45–73)	56 (47–63)	53 (45–62)	51 (44–55)	0.839^c
Maximum clot firmness (mm)	50–72
All patients		73 (69–74)	73 (70–75)	75 (71–77)	74 (70–79)^*	74 (69–79)	<0.001^a
SOFA≤10		73 (70–75)	74 (71–76)	76 (71–77)	75 (70–79)	75 (69–80)	0.344^b
SOFA >10		71 (68–73)	72 (68–73)	73 (71–80)	74 (72–78)	74 (69–78)	0.415^c
Maximum lysis (%)	£15
All patients		10 (7–12)	9 (6–13)	8 (5–10)^*	8 (3–11)	7 (5–11)	0.006^a
SOFA≤10		10 (8–12)	9 (6–11)	8 (6–9)	8 (4–12)	7 (4–11)	0.863^b
SOFA >10		9 (6–15)	10 (7–13)	8 (4–11)	4 (3–10)	8 (5–11)	0.617^c
ROTEM—FIBTEM
Maximum clot firmness (mm)	9–25
All patients		36 (32–38)	37 (30–40)	41(30–44)	37 (31–45)	35 (28–47)	0.080^a
SOFA≤10		37 (30–41)	37 (30–41)	40 (28–43)	42 (31–46)	32 (27–49)	0.836^b
SOFA >10		36 (33–38)	36 (31–40)	42 (36–46)	37 (32–42)	38 (31–43)	0.050^c
PLATELET function test
ARATEM test (sec)	70–153
All patients		79 (54–110)	87 (58–113)	81 (62–112)	114 (89–124)^*	110 (83–154)^*	0.014^a
SOFA≤10		79 (53–108)	88 (65–112)	85 (71–110)	121 (115–154)	140 (85–182)	0.068^b
SOFA >10		79 (54–122)	76 (36–113)	62 (43–112)	95 (65–110)^#	109 (83–116)	0.161^c
ADPTEM test (sec)	56–139
All patients		96 (65–111)	105 (72–128)	92 (76–117)	127 (86–138)	112 (67–134)	0.004^a
SOFA≤10		105 (83–126)	105 (89–127)	92 (78–116)	135 (115–148)	126 (73–159)	0.024^b
SOFA >10		85 (56–107)	106 (51–135)	91 (61–118)	82 (63–129)^#	96 (67–116)	0.121^c

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Values represent median (IQR). SOFA: sequential organ failure assessment score. P values were calculated with the use of generalized estimating equations (GEE): (a): time effect, (b): group effect and (c): time-group interaction. Pairwise comparisons significant at the 0.05 level:

(*): time effect—pooled patients: each time point vs. Day 0.

(#): between group comparisons (group SOFA>10 vs. group SOFA ≤10) at each time point.

Platelet function test

Median (IQR) values of ARATEM and ADPTEM tests remained within the normal range during the study period, although both ARATEM (p = 0.014 for time effect) and ADPTEM (p = 0.004 for time effect) slightly increased over time in both groups (Table 4).

Fibrinolysis and endogenous inhibitors of coagulation

Fibrinolysis (INTEM and EXTEM maximum lysis) was low and decreased over time in all groups, with the most pronounced decrease observed in INTEM maximum lysis in group SOFA > 10 (p = 0.004 for time-group interaction) (Table 4). D-dimer plasma levels were 2 to 3-fold higher than normal reference range in both groups (Fig 2; S1 Table). Plasminogen median values remained within the normal range during the study period, although with a slight increase over time in both groups (p<0.001 for time effect). Alpha-2 antiplasmin remained unchanged during the study period (Fig 2; S1 Table).

Antithrombin slightly increased over time in group SOFA ≤ 10 while it remained stable in group SOFA > 10 (p = 0.021 for time-group interaction) (Fig 2; S1 Table). Protein C plasma levels increased over time in both groups, although patients in group SOFA > 10 exhibited lower values in comparison to patients in group SOFA ≤ 10 (p = 0.015 for time-group interaction) (Fig 2; S1 Table). Free Protein S plasma levels were low in both groups at baseline and increased over time with no between-group differences (p<0.001 for time effect) (Fig 2; S1 Table).

Discussion

In this prospective longitudinal single center study, we demonstrated that patients admitted to the ICU with severe SARS-CoV-2 infection exhibited a pronounced hypercoagulability state, characterized by increased plasma fibrinogen levels, decreased free protein S plasma levels, and decreased fibrinolysis. The severity of coagulation derangements seems to correlate with the intensity of organ dysfunction according to the SOFA score. Finally, the hypercoagulability state of severe COVID-19 patients was detected by ROTEM and modifications on some coagulation tests related to the fibrinolytic and endogenous anticoagulation system, while the more common conventional coagulation tests, as aPTT (sec) INR and platelets, remained unchanged.

Approximately 5% of patients infected with SARS-CoV-2 become critically ill, developing organ dysfunction and failure [26]. Coagulopathy is frequently observed in COVID-19 patients [5–11] and has been associated with worse outcomes [3, 11, 27, 28]. For instance, Tang and cols. showed that abnormal conventional coagulation tests, especially markedly elevated D-dimer and FDP levels during hospitalization, were associated with poor prognosis in COVID-19 patients [11]. Moreover, disseminated intravascular coagulation (DIC) was more frequently observed in non-survivals (71.4% vs. 0.6%, respectively) compared to survivors [11].

The hemostatic disorders observed in critically ill COVID-19 patients are complex and multifactorial. The combination of varying degrees of hypoxemia, immune-mediated endothelial damage/dysfunction, and systemic inflammation have been implicated in the physiopathology of hypercoagulability state observed in COVID-19 patients [29]. Therefore, several authors have demonstrated that COVID-19 infection is associated with a high rate of venous and arterial thrombotic manifestations, such as DVT, pulmonary embolism, catheter-related thrombosis and ischemic stroke [5, 12, 30, 31]. Therefore, caution for thromboembolic events should be advised especially in the most severe patients, and the strategy for DVT prophylaxis / anticoagulation individualized.

Panigada and cols. recently demonstrated that patients admitted to the ICU infected with COVID-19 exhibited hypercoagulability by thromboelastography [8]. Similarly, we found that most patients admitted to the ICU with severe SARS-CoV-2 infection exhibited a pronounced hypercoagulability state, characterized by increased ROTEM INTEM, EXTEM and FIBTEM maximum clot firmness. Nevertheless, we did not observe consumption coagulopathy and laboratory findings suggesting DIC as observed by Tang and cols [11]. Our results are in line with other studies that did not show a DIC state in severe critical ill COVID-19 patients [5, 8, 30].

Hypercoagulability may be related to augmented levels of pro-coagulant factors, reduced levels of the naturally occurring anti-coagulant factors or both. Fibrinogen levels, which were initially high in both groups at ICU admission, decreased during the ICU stay. The decrease was more pronounced in patients with lower maximum SOFA score (group SOFA ≤ 10) and after day 7. Also, the naturally occurring anti-coagulant factors, such as protein C and antithrombin were lower in the sicker patients (SOFA >10). The differences observed over time between the groups in our study may reflect a higher pro-coagulant tendency in the higher SOFA group and may explain the worst clinical outcomes observed in the group of patients with SOFA > 10.

Protein C has anti-coagulant, profibrinolytic, and anti-inflammatory effects [33]. It has been shown that septic patients with low plasma protein C concentrations have a higher incidence of organ dysfunction and worse outcomes [33–35]. Furthermore, the occurrence of fibrinolysis suppression in patients with septic shock was associated with disease severity and lower survival [36]. Additionally, it has been shown that patients with acute respiratory distress syndrome (ARDS) have higher levels of plasminogen activator inhibitor-1 (PAI-1), which contribute to fibrin deposition in lung parenchyma [37]. This abnormality has been shown also during the SARS-CoV-1 pandemic [38].

There are some pathophysiological changes secondary to COVID-19 infection that may contribute to a greater chance of patients infected with COVID-19 to develop thrombotic complications as an increased angiotensin II expression secondary to angiotensin-converting enzyme 2 receptor binding and consequently augmented plasminogen activator inhibitor C-1 expression with a reduced fibrinolysis in the anticoagulation system [39].

Patients in group SOFA > 10 had lower plasminogen levels than patients with SOFA <10, which may represent less fibrinolysis activity during a state of hypercoagulability and, consequently, a greater probability of microthrombi formation in the microcirculation of different organs. This might explain the higher rate of organ dysfunction in group SOFA > 10. Nevertheless, our data do not allow us to confirm or refute this hypothesis. We did not evidence differences in the fibrinolytic behavior in both groups accordingly to ROTEM. One possible explanation for this fact is the high values of fibrinogen present in both groups, which compromise detection of increased thrombotic activity by ROTEM maximum lysis. The hypothesis that the fibrinolytic system is overwhelmed during COVID-19 infection has been commented in a recent paper discussing the association of COVD-19 and the fibrinolytic pathway [7].

We believe that despite several studies addressing the alterations in the coagulation system of COVID-19 patients, [41–46] our study continues to bring important news because it was one of the few that evaluated the behavior of the coagulation system, of critically critical patients infected with COVID-19, during two weeks of hospitalization. Also, our study was one of the few where we tried to assess the impact of the degree of change in the coagulation system and its impact on the evolution of organ dysfunctions presented by patients during the two weeks of study.

Several mechanics can explain the relationship between viral infection and our findings, as endothelial cell disruption, tissue factor expression, and activation of the coagulation cascade by cytokines released during viral infections are other possible mechanisms of thrombosis. This pro-inflammatory state can promote microthrombosis in the vascular lung system and consequently promoting more hypoxia with local impact creating a deleterious positive thrombo-inflammatory feedback loop [39, 47, 48].

Our study has some limitations. First, it is a single-center study with a relatively small sample size. However, to the best of our knowledge, it was the first study to perform a comprehensive and longitudinal assessment of coagulation profile in critically ill COVID-19 patients during the first two weeks of ICU stay. Secondly, during the study period, all the ICU beds available in our department were destinated to COVID-19 patients. Thus, inclusion of a control group without severe SARS-CoV-2 infection was not possible. Nevertheless, the coagulation profile of patients admitted to the ICU in our center has been recently addressed [15]. Also, a recent study has already demonstrated a higher incidence of thrombotic complications were diagnosed in COVID-19 ARDS patients than in patients with non-COVID-19 ARDS [5]. Third, all patients were already receiving anticoagulants as DVT prophylaxis or systemic anticoagulation and these could change the ROTEM results. Fourth, we did not evaluate all factors involved in fibrinolysis, such as PAI-1 and plasmin, which preclude us to fully understand the role of fibrinolytic system on COVID-19 induced coagulopathy and also other laboratory tests as prothrombin fragment 1+2, thrombin-anti-thrombin complexes and endogenous thrombin potential assays were not done to better understand the hypercoagulability state of such patients. Finally, we not measured any marker for endothelial dysfunction which probably contribute to the modifications on the coagulation system of the severe patients infected with SARS-CoV-2. Nevertheless, our study was the first to demonstrate some aspects of coagulation disorders that can occur in critical patients infected by COVID-19, especially the deficiency of naturally anti-coagulant factors.

Conclusion

Patients admitted to the ICU with COVID-19 have a pronounced hypercoagulability state, characterized by impaired endogenous anticoagulation system and decreased fibrinolysis. Moreover, the magnitude of coagulation abnormalities seems to correlate with the severity of organ dysfunction. The hypercoagulability state of patients infected with SARS-CoV-2 was detected by ROTEM and other coagulation tests but not with usual coagulation tests. Our findings highlight the role of rotational thromboelastometry when monitoring the coagulation system in ICU patients with COVID-19 and also demonstrated that the mechanisms to explain the hypercoagulability state of patients infected with SARS-CoV-2 is very complex and need more studies.

Supporting information

S1 Table. Fibrinolysis and endogenous inhibitors of coagulation.

(DOCX)

Click here for additional data file.^{(18KB, docx)}

Acknowledgments

We thank Helena Spalic for proofreading this manuscript.

List of abbreviations

ARDS: acute respiratory distress syndrome
aPTT: activated partial thromboplastin time
CFT: clot formation time
CCT: conventional coagulation tests
COPD: chronic obstructive pulmonary disease
COVID-19: coronavirus disease 2019
CT: clotting time
DVT: deep vein thrombosis
HFH: unfractionated heparin
ICU: intensive care unit
INR: international normalized ratio
LMWH: low-molecular-weight heparin
LOS: length of stay
MCF: maximum clot firmness
PAI-1: plasminogen activator inhibitor-1
PCC: prothrombin complex concentrate
PT: prothrombin time
RRT: renal replacement therapy
ROTEM: rotational thromboelastometry
RT-PCR: reverse-transcriptase-polymerasechain-reaction
SAPS III: simplified acute physiology score III
SARS-CoV-2: severe acute respiratory syndrome coronavirus 2
SOFA: sequential organ failure assessment score
TT: thrombin time
UFH: unfractionated heparin

Data Availability

All relevant data are available from Dryad (doi:10.5061/dryad.pg4f4qrn3).

Funding Statement

The author(s) received no specific funding for this work.

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PLoS One. doi: 10.1371/journal.pone.0243604.r001

Decision Letter 0

Pablo Garcia de Frutos

17 Sep 2020

PONE-D-20-25608

COAGULATION PROFILE OF COVID-19 PATIENTS ADMITTED TO THE ICU: AN EXPLORATORY STUDY

PLOS ONE

Dear Dr. Cordioli,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

The study has been revised by two experts in the field. They note several concerns that should be adequatedly considered. The study does not have a group of ICU patients to look for specific factors altered in COVID-19. The authors should provide convincing published evidence in order to understand in which way the altered parameters are specific of COVID-19. Furhter, heparin could affect ROTEM measurements. Finally, protein S values are reported as free protein S values. This should be indicated in the table and description of the results. The total values of protein S would be essential to understand the type of deficiency associated with COVID19. If possible protein S activity values would be interesting to know. The authors should try to update their discussion with the latest research on this very active field of research.

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We look forward to receiving your revised manuscript.

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Reviewer #1: The authors have measured coagulation tests serially in the intensive care unit in 30 COVID19 patients. The authors found hypercoagulability. A main problem is that there is no control group with non-COVID ICU patients. We will therefor not be able to know whether ICU treated COVID19 patients have higher coagulation activation than other ICU patients. Still of course the longitudinal measurement is of interest.

Comments

1. No non-COVID19 ICU control group

2. I number of patients have been treated with UFH ie heparin. This may affect the ROTEM test.

3. It would have been nice to have a figures where the individuals patients values are plotted over time for the most important analysis at least.

4. In table 3 and 4 it is unclear what the p-values actually is testing.

5. There is a number of papers already published on this topic. What is the novelty with the present paper? For instance: a) Hardy M, Douxfils J, Bareille M, Lessire S, Gouin-Thibault I, Fontana P, Lecompte T, Mullier F. Studies on hemostasis in COVID-19 deserve careful reporting of the laboratory methods, their significance and their limitations. J Thromb Haemost. 2020 Aug 13:10.1111/jth.15061. b) Collett LW, Gluck S, Strickland RM, Reddi BJ. Evaluation of coagulation tatus using viscoelastic testing in intensive care patients with coronavirus disease 2019 (COVID-19): An observational point prevalence cohort study. Aust Crit Care. 2020 Jul 21:S1036-7314(20)30254-X. d) Creel-Bulos C, Auld SC, Caridi-Scheible M, Barker N, Friend S, Gaddh M, Kempton CL, Maier C, Nahab F, Sniecinski R. Fibrinolysis Shutdown and Thrombosis in A COVID-19 ICU. Shock. 2020 Aug 4. C) Ibañez C, Perdomo J, Calvo A, Ferrando C, Reverter JC, Tassies D, Blasi A. High D dimers and low global fibrinolysis coexist in COVID19 patients: what is going on in there? J Thromb Thrombolysis. 2020 Jul 15:1–5. D) Nougier C, Benoit R, Simon M, Desmurs-Clavel H, Marcotte G, Argaud L, David JS, Bonnet A, Negrier C, Dargaud Y. Hypofibrinolytic state and high thrombin generation may play a major role in SARS-COV2 associated thrombosis. J Thromb Haemost. 2020 Jul 15:10.1111/jth.15016. doi: 10.1111/jth.15016. E)Pavoni V, Gianesello L, Pazzi M, Stera C, Meconi T, Frigieri FC. Evaluation of coagulation function by rotation thromboelastometry in critically ill patients with severe COVID-19 pneumonia. J Thromb Thrombolysis. 2020

Reviewer #2: This is an intriguing study focused on an important topic linked to COVID-19.

The relevance of coagulation abnormalities in COVID-19 patients is clearly underscored by the relationship between degree of severity of disease and indicators of abnormal clotting.

It would be interesting to more clearly postulate in the discussion the mechanism by which the viral infection may lead to the reported findings.

Furthermore, the role of Vitamin K abnormalities in patients with defective ROTEM should be discussed.

Future investigations should attempt to validate the current observation in independent patients’ cohorts. Finally, conducting a long-term follow up on patients with ROTEM severe abnormalities may provide further insights into the long-term vascular complications of the disease.

**********

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Reviewer #2: No

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PLoS One. 2020 Dec 15;15(12):e0243604. doi: 10.1371/journal.pone.0243604.r002

Author response to Decision Letter 0

26 Oct 2020

Dear Editor and Reviewers

Thank you very much for the interesting and important inputs to our study

PONE-D-20-25608 - COAGULATION PROFILE OF COVID-19 PATIENTS ADMITTED TO THE ICU: AN EXPLORATORY STUDY

Responses to the Editor

The study does not have a group of ICU patients to look for specific factors altered in COVID-19.

We didn’t do a control group at the time of the study, as the study planning and even data collection occurred at the beginning of the pandemic in Brazil and the Hospital Israelita Albert Einstein, where the study was done, was reorganized to treat, almost exclusively, patients infected with COVID-19 as it happened around the world. We addressed such limitation in our text “Secondly, during the study period, all the ICU beds available in our department were designated to COVID-19 patients. Thus, inclusion of a control group without severe SARS-CoV-2 infection was not possible. Nevertheless, the coagulation profile of patients admitted to the ICU in our center has been recently addressed [15]” The reference cited above (#15) evaluated 531 critical ill patients and showed normal ROTEM profille in the majority of ICU, non COVID-19, patients.

Also, to make it clearer, we included this sentence following the previous paragraph “Also, a recent study have already demonstrated a higher incidence of thrombotic complications were diagnosed in COVID-19 ARDS patients than in patients with non-COVID-19 ARDS”. (included reference number 5 already cited in our study)

So, it was not possible to include a control group during the study period, only on July the pandemic started to be more controlled in our city and we returned to have non-COVID-19 patients in our ICU. By the reasons discussed, it would have taken a long time to have had a control group and then delay the manuscript submission.

The authors should provide convincing published evidence in order to understand in which way the altered parameters are specific of COVID-19

We agree that there are indirect effects of any infection, such as through severe illness with subsequently inflammatory response that may predispose patients to thrombotic events and this is not specific for COVID-19 patients as well as hypoxia can contribute to thrombotic events and again it is not specific for hypoxemic COVID-19 patients. However, COVID-19 patients demonstrated a higher incidence of thrombotic events that highlighted the importance of better understand its specific changes in the coagulation system and it is the main objective of our study. The following epidemiological studies highlighted the greater incidence of thrombosis events in patients hospitalized with COVID-19 specially in ICU patients

1. Klok FA, Kruip M, van der Meer NJM, Arbous MS, Gommers D, Kant KM, et al. Incidence of thrombotic complications in critically ill ICU patients with COVID-19. Thrombosis research. 2020;191:145-7.

2. Helms J, Tacquard C, Severac F, Leonard-Lorant I, Ohana M, Delabranche X, et al. High risk of thrombosis in patients with severe SARS-CoV-2 infection: a multicenter prospective cohort study. Intensive care medicine. 2020;46(6):1089-98.

3. Lodigiani C, Iapichino G, Carenzo L, Cecconi M, Ferrazzi P, Sebastian T, et al. Venous and arterial thromboembolic complications in COVID-19 patients admitted to an academic hospital in Milan, Italy. Thrombosis research. 2020;191:9-14.

4. Bikdeli B, Madhavan MV, Jimenez D, Chuich T, Dreyfus I, Driggin E, et al. COVID-19 and Thrombotic or Thromboembolic Disease: Implications for Prevention, Antithrombotic Therapy, and Follow-Up: JACC State-of-the-Art Review. Journal of the American College of Cardiology. 2020;75(23):2950-73.

5. Klok FA, et al. .Confirmation of the high cumulative incidence of thrombotic complications in critically ill ICU patients with COVID-19: An updated analysis. Thromb Res. 2020. PMID: 32381264

We included the further sentence, on the Discussion section, to address the correlation of COVID-19 and thrombosis profile of patients with COVID-19: “There are some pathophysiological changes secondary to COVID-19 infection that may contribute to a greater chance of patients infected with COVID-19 to develop thrombotic complications as an increased angiotensin II expression secondary to angiotensin-converting enzyme 2 receptor binding and consequently augmented plasminogen activator inhibitor C-1 expression with a reduced fibrinolysis in the anticoagulation system. (reference included: Dolhnikoff M, Duarte-Neto AN, de Almeida Monteiro RA, et al. Pathological evidence of pulmonary thrombotic phenomena in severe COVID-19. Thromb Haemost 2020 Jun;18(6):1517-1519.)

Further, angiotensin II–mediated pulmonary vasoconstriction can predispose to stasis and hypercoagulability, as can COVID-19 induction of antiphospholipid antibodies and complement during cytokine storms, causing vasculitis and microthromboses. (reference included: Medcalf RL, Keragala CB, Myles PS. Fibrinolysis and COVID-19: a plasmin paradox. J Thromb Haemost. 2020.)

Heparin could affect ROTEM measurements

We agree with this statement and it is one of the reasons that we changed in the Discussion section where it was included on the limitation paragraph: Third, all patients were already receiving anticoagulants as DVT prophylaxis or systemic anticoagulation and these could change the ROTEM results.

Concerning the comments regarding protein S:

We changed on Material and Methods section at the Laboratory analysis subsection “Fibrinolysis and endogenous anticoagulation system” that we measured the free protein S and in the Table and when it was cited in the text we changed in the manuscript and always wrote “free protein S” to be more exact.

We understand your comment about the importance of total protein S but protein S is a vitamin K-dependent glycoprotein, which acts as a cofactor for Activated Protein C, increasing its anticoagulant and profibrinolytic effects. Protein S is present in plasma in two forms: free protein S (40%) and protein S bound to the transport protein of the complement C4b fraction (60%). The two forms are in dynamic equilibrium and only free Protein S has biological activity. These are the reasons that we decided to include free protein S since its biological activity.

References to our decision:

1. Suzuki K, Nishioka J. Plasma Protein S Activity measured using Protac, a Snake Venom Derived Activator of Protein C, Thromb. Res. 1988; 49: 214-251.

2. Faioni EM, Valsecchi C, Palla A, Taioli E, Razzari C, Mannucci PM. Free protein S deficiency is a Risk Factor for Venous Thrombosis. Thromb Haemost 1997; 78: 1343-1346

Response to Journal Requirements:

When submitting your revision, we need you to address these additional requirements. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming.

We did the changed above according to PLOS ONE's style requirements

In your Methods section, please provide additional information about the participant recruitment method and the demographic details of your participants. Please ensure you have provided sufficient details to replicate the analyses such as:

a) the recruitment date range (month and year),

b) a statement as to whether your sample can be considered representative of a larger population, and

c) a description of how participants were recruited.

We included the paragraph on the Methods Section: “Participants were recruited between March 29, 2020 through May 13, 2020 and they could represent the majority of severe patients infected by COVID-19, once there were few exclusion criteria and the participants were recruited with waiver of informed consent once it was an observational study without any intervention and consecutive patients admitted in the Intensive Care Unit were recruited following the inclusion and exclusion criteria until we completed 30 patients included in the study.”

Response to the comments of the reviewers

Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: No.

We provided now all our dataset, in the datadryad.org following the Plos One policy after your comment.

Please, find bellow the message from datadryad: “Your dataset has been assigned a unique identifier, called a DOI (doi:10.5061/dryad.pg4f4qrn3). If your dataset is associated with a manuscript submission, you may provide this DOI to the journal, although it will not be live until the dataset is published. For private access prior to publication, you may share your dataset using this temporary link: https://datadryad.org/stash/share/iz0FlU7yXkYg4_Yh2PM_2y0cPIPTyiD6_lry0NMfJKM.”

Review Comments to the Author

Comments

No non-COVID19 ICU control group

We agree that it should be interest to have a control group of ICU non-COVID-19 patients, but we didn’t do a control group at the time of the study, as the study planning and even data collection occurred at the beginning of the pandemic in Brazil and the Hospital Israelita Albert Einstein, where the study was done, was reorganized to treat, almost exclusively, patients infected with COVID-19 as it happened around the world. We addressed such limitation in our text “Secondly, during the study period, all the ICU beds available in our department were destinated to COVID-19 patients. Thus, inclusion of a control group without severe SARS-CoV-2 infection was not possible. Nevertheless, the coagulation profile of patients admitted to the ICU in our center has been recently addressed [15]”

The reference cited above (#15) evaluated 531 critical ill patients and showed normal ROTEM profilte in the majority of ICU, non COVID-19, patients.

I number of patients have been treated with UFH ie heparin. This may affect the ROTEM test.

Before the pandemic started, the ICU of Hospital Israelita Albert Einstein have already a DVT prophylaxis algorithm and a culture in the institution to introduce drug prophylaxis for DVT in critically ill patients, unless contraindicated, and even more so in patients with COVID-19, due to reports of initial studies already demonstrating a significant incidence of thrombotic events in such patients, then it would take too much time to include patients with COVID-19, whose prophylaxis for DVT was contraindicated, and then the study would take much time to be finished.

It would have been nice to have a figures where the individuals patients values are plotted over time for the most important analysis at least.

We included a figure

Figure 2. Fibrinolysis and endogenous inhibitors of coagulation.

Values represent median (IQR). Red lines represent group SOFA >10 (n=14), blue lines represent group SOFA ≤10 (n=16) and black dotted lines represent all patients (n=30). P values were calculated with the use of generalized estimating equations (GEE): (a): time effect, (b): group effect and (c): time-group interaction. Pairwise comparisons significant at the 0.05 level: (*): time effect - pooled patients: each time point vs. Day 0. (#): between group comparisons (group SOFA>10 vs. group SOFA ≤10) at each time point.

And we put the Table 5 as a supplemental material

In table 3 and 4 it is unclear what the p-values actually is testing.

We included the sentence in the Statistical analysis Section to clarify what p-values actually tested: “To account for longitudinal (repeated measurements) and correlated response continuous variables, between-group differences and within-group differences over time were assessed using generalized estimating equations (GEE), with group (SOFA ≤ 10 and group SOFA > 10) and study time points (time) as predictors. P values for group effect, time effect, and time-group interaction were presented. When a time effect was detected in pooled patients, each time point (Day 1, 3, 7 and 14) was compared against Day 0. When a group effect or a time-group interaction were detected, between group comparisons (group SOFA>10 vs. group SOFA ≤10) were performed at each time point. The Bonferroni method was used to account for multiple comparisons”

Also, we did some modifications on Table 3 to make it clearer and we changed the legend to the following phrase: “Values represent median (IQR). INR: international normalized ratio, aPTT: activated partial thromboplastin time. P values were calculated with the use of generalized estimating equations (GEE): (a): time effect, (b): group effect and (c): time-group interaction. Pairwise comparisons significant at the 0.05 level: (*): time effect - pooled patients: each time point vs. Day 0. (#): between group comparisons (group SOFA>10 vs. group SOFA ≤10) at each time point.”

We also changed the Table 4 as well as its legend to: “Values represent median (IQR). SOFA: sequential organ failure assessment score. P values were calculated with the use of generalized estimating equations (GEE): (a): time effect, (b): group effect and (c): time-group interaction. Pairwise comparisons significant at the 0.05 level: (*): time effect - pooled patients: each time point vs. Day 0. (#): between group comparisons (group SOFA>10 vs. group SOFA ≤10) at each time point.”

We also changed the Table 5, to a supplemental table as well as its legend to: “Values represent median (IQR). SOFA: sequential organ failure assessment score. P values were calculated with the use of generalized estimating equations (GEE): (a): time effect, (b): group effect and (c): time-group interaction. Pairwise comparisons significant at the 0.05 level: (*): time effect - pooled patients: each time point vs. Day 0. (#): between group comparisons (group SOFA>10 vs. group SOFA ≤10) at each time point.”

There is a number of papers already published on this topic. What is the novelty with the present paper?

For instance: a) Hardy M, Douxfils J, Bareille M, Lessire S, Gouin-Thibault I, Fontana P, Lecompte T, Mullier F. Studies on hemostasis in COVID-19 deserve careful reporting of the laboratory methods, their significance and their limitations. J Thromb Haemost. 2020 Aug 13:10.1111/jth.15061.

This is an interesting letter that mainly discussed limits of another study (Nougier C, Benoit R, Simon M, Desmurs-Clavel H, Marcotte G, Argaud L, et al. Hypofibrinolytic state and high thrombin generation may play a major role in sars-cov2 associated thrombosis. J Thromb Haemost. 2020. Online ahead of print. DOI: 10.1111/jth.15016.) showing the complexity in studying the coagulation profile of COVID-19 patients, but our study, different from this letter and also from the study cited above, analyzed evaluated different coagulation parameters on COVID-19 patient and for a longer period.

b) Collett LW, Gluck S, Strickland RM, Reddi BJ. Evaluation of coagulation tatus using viscoelastic testing in intensive care patients with coronavirus disease 2019 (COVID-19): An observational point prevalence cohort study. Aust Crit Care. 2020 Jul 21:S1036-7314(20)30254-X.

This is an interesting paper, but with a small sample (6 patients) and it evaluated mainly the ROTEM ® profile and also only in a single measurement, our study, on the other hand, evaluated more coagulation examens and also during 14 days

c) Creel-Bulos C, Auld SC, Caridi-Scheible M, Barker N, Friend S, Gaddh M, Kempton CL, Maier C, Nahab F, Sniecinski R. Fibrinolysis Shutdown and Thrombosis in A COVID-19 ICU. Shock. 2020 Aug 4.

Similar to our study, this study also evaluated 21 patients, and it was demonstrated a fibrinolysis shutdown, but this study assessed mainly the ROTEM® profile and also only in a single day and unlike our study that evaluated different coagulation parameters over time and also compared different clinical groups (SOFA < or > 10).

d) Ibañez C, Perdomo J, Calvo A, Ferrando C, Reverter JC, Tassies D, Blasi A. High D dimers and low global fibrinolysis coexist in COVID19 patients: what is going on in there? J Thromb Thrombolysis. 2020 Jul 15:1–5.

Similar to our study, this study also evaluated 19 patients, and it was also demonstrated a hypofibrinolytic pattern on the viscoelastic examen and elevated D-dimers value as we demonstrated, however this study above did not evaluated other coagulation parameters as we did and we demonstrated a time effect on ROTEM® profile that was not demonstrated in the study above.

e) Nougier C, Benoit R, Simon M, Desmurs-Clavel H, Marcotte G, Argaud L, David JS, Bonnet A, Negrier C, Dargaud Y. Hypofibrinolytic state and high thrombin generation may play a major role in SARS-COV2 associated thrombosis. J Thromb Haemost. 2020 Jul 15:10.1111/jth.15016. doi: 10.1111/jth.15016.

It is a very interesting paper which the authors investigated several different coagulation parameters, however the patients included were less sicker with a lower mean SOFA score, lower rate of mechanical ventilation and also it was only done discussed one single day measurement of the coagulation profile, so it was not studied the coagulation behavior along two weeks as we done and our study the patients included were general more sicker.

f)Pavoni V, Gianesello L, Pazzi M, Stera C, Meconi T, Frigieri FC. Evaluation of coagulation function by rotation thromboelastometry in critically ill patients with severe COVID-19 pneumonia. J Thromb Thrombolysis. 2020

This study is very interesting, since as our study, the authors investigated the coagulation profile over 10 days (we investigated during two weeks) but the study above include less severe patients, where only 10% of the patients need mechanical ventilation, the mean SOFA was less than the less critical group form our study (group SOFA < 10), and there are not two groups as our study to evaluate the impact and relationship of the coagulation system disorders and the organ dysfunction in ICU COVID-19 patients, one issue that our study has addressed.

Thank you for sharing these important and interesting references.

We included in our Discussion section the following sentence: “We believe that despite several studies addressing the alterations in the coagulation system of COVID-19 patients, (included all the references above) our study continues to bring important news because it was one of the few that evaluated the behavior of the coagulation system, of critically critical patients infected with COVID-19, during two weeks of hospitalization. Also, our study was one of the few where we tried to assess the impact of the degree of change in the coagulation system and its impact on the evolution of organ dysfunctions presented by patients during the two weeks of study”.

Reviewer #2: This is an intriguing study focused on an important topic linked to COVID-19.

The relevance of coagulation abnormalities in COVID-19 patients is clearly underscored by the relationship between degree of severity of disease and indicators of abnormal clotting.

It would be interesting to more clearly postulate in the discussion the mechanism by which the viral infection may lead to the reported findings.

We included this sentence on the Discussion section to address this relationship: “Several mechanics can explain the relationship between viral infection and our findings, as endothelial cell disruption, tissue factor expression, and activation of the coagulation cascade by cytokines released during viral infections are other possible mechanisms of thrombosis. This pro-inflammatory state can promote microthrombosis in the vascular lung system and consequently promoting more hypoxia with local impact creating a deleterious positive thromboinflammatory feedback loop (reference included: Dolhnikoff M, Duarte-Neto AN, de Almeida Monteiro RA, et al. Pathological evidence of pulmonary thrombotic phenomena in severe COVID-19. Thromb Haemost 2020 Jun;18(6):1517-1519. AND Wool GD, Miller JL. The Impact of COVID-19 Disease on Platelets and Coagulation Pathobiology. 2020 Oct 13:1-13. doi: 10.1159/000512007. AND Escher R, Breakey N, Lämmle B. Severe COVID-19 infection associated with endothelial activation. Thromb Res. 2020 Jun;190:62.)

Furthermore, the role of Vitamin K abnormalities in patients with defective ROTEM should be discussed.

We agree with the sentence above and is one of the reason that our exclusion criteria were, as cited in the manuscript: “Exclusion criteria included pregnancy, previous known coagulopathy, currently use of systemic anticoagulants or anti-platelet therapy or vitamin K antagonists, moribund patients and patients who presented cardiac arrests.

Future investigations should attempt to validate the current observation in independent patients’ cohorts.

We completely agree that it should be very interesting and important to validate the current observation in a ICU non COVID-19 population, however as explained in sentences above it would have taken a long time to have this population and we did not know when the peak of the pandemic in Brazil would be better allowing to return to a more stable situation where critical ill patients without COVID-19 infection would be hospitalized and we would be able to evaluate this population regarding deeply the coagulation system as we did in our study with severe ill COVID-19 patients. We intend to do this in a future research

Finally, conducting a long-term follow up on patients with ROTEM severe abnormalities may provide further insights into the long-term vascular complications of the disease.

We completely agree with this idea, and maybe we are going to evaluate for a longer period the coagulation system of critical ill ICU COVID-19 patients that stay more than 01 month in the ICU and/or hospital.

Thank you for your important inputs

Sincerely,

Ricardo Luiz Cordioli

26TH, October, 2020

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PLoS One. doi: 10.1371/journal.pone.0243604.r003

Decision Letter 1

Pablo Garcia de Frutos

18 Nov 2020

PONE-D-20-25608R1

COAGULATION PROFILE OF COVID-19 PATIENTS ADMITTED TO THE ICU: AN EXPLORATORY STUDY

PLOS ONE

Dear Dr. Cordioli,

The reviewers consider that their initial concerns have been addressed. One reviewers suggests some minor changes that should be considered by the authors, eitherr by changing their text or by providing a reasoned answer.

Please submit your revised manuscript by Jan 02 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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Academic Editor

PLOS ONE

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Reviewers' comments:

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Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

Reviewer #2: Yes

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: (No Response)

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4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

Reviewer #2: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

Reviewer #2: Yes

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6. Review Comments to the Author

Reviewer #1: Thea authors have changed the paper according to reviewer suggestions. I have only a few minor but important comments.

1. The authors do not present in the abstract the increased d-dimer levels nor the transient decreased free protein S levels and plasminogen levels.

2. I do not agree with this sentence in the abstract ¨The hypercoagulability state of COVID-19 patients was only detected by ROTEM¨ or the sentences in discussion or conclusions:¨ Finally, the hypercoagulability state of severe COVID-19 patients was detected by ROTEM, while conventional coagulation tests remained unchanged.¨ ¨The hypercoagulability state of patients infected with SARS-CoV-2 was detected by ROTEM but not with conventional coagulation tests.¨ Ordinary coagulation analysis showed increased fibrinogen, increased d-dimer, slightly increased antiplasmin, transient decreased free protein S. and slightly transient decreased plasminogen. Thus, not only ROTEM could identify a hypercoagulable state in COVID infection.

3. A limitation is that the authors have not measured markers of hypercoagulability such as F1+2, TAT and endogenous thrombin potential. Nor have the authors measured any marker for endothelial dysfunction. I think this should be stated among limitations in the discussion. This has not only relevance for the authors conclusion that the hypercoagulability of COVID19 may be detected only by ROTEM and not conventional coagulation tests but also regarding the cause of hypercoagulability.

Reviewer #2: No further comments .................................................................................................................................

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PLoS One. 2020 Dec 15;15(12):e0243604. doi: 10.1371/journal.pone.0243604.r004

Author response to Decision Letter 1

19 Nov 2020

Dear Editor and Reviewers

Thank you very much for the interesting and important inputs to our study

PONE-D-20-25608 - COAGULATION PROFILE OF COVID-19 PATIENTS ADMITTED TO THE ICU: AN EXPLORATORY STUDY

Response to the comments of the reviewers

1. The authors do not present in the abstract the increased d-dimer levels nor the transient decreased free protein S levels and plasminogen levels.

We changed the result paragraph on the Abstract Section:

“Thirty patients were studied. Some conventional coagulation tests, as aPTT, PT and INR remained unchanged during the study period, while alterations on others coagulation laboratory tests were detected. Fibrinogen levels were increased in both groups. ROTEM maximum clot firmness increased in both groups from Day 0 to Day 14. Moreover, ROTEM – FIBTEM maximum clot firmness was high in both groups, with a slight decrease from day 0 to day 14 in group SOFA ≤ 10 and a slight increase during the same period in group SOFA > 10. Fibrinolysis was low and decreased over time in all groups, with the most pronounced decrease observed in INTEM maximum lysis in group SOFA > 10. Also, D-dimer plasma levels were higher than normal reference range in both groups and free protein S plasma levels were low in both groups at baseline and increased over time, Finally, patients in group SOFA > 10 had lower plasminogen levels and Protein C than patients with SOFA <10, which may represent less fibrinolysis activity during a state of hypercoagulability.”

2. I do not agree with this sentence in the abstract ¨The hypercoagulability state of COVID-19 patients was only detected by ROTEM¨ or the sentences in discussion or conclusions:¨Finally, the hypercoagulability state of severe COVID-19 patients was detected by ROTEM, while conventional coagulation tests remained unchanged.¨ ¨The hypercoagulability state of patients infected with SARS-CoV-2 was detected by ROTEM but not with conventional coagulation tests.¨ Ordinary coagulation analysis showed increased fibrinogen, increased d-dimer, slightly increased antiplasmin, transient decreased free protein S. and slightly transient decreased plasminogen. Thus, not only ROTEM could identify a hypercoagulable state in COVID infection.

We changed the conclusion paragraph on the Abstract Section:

“The hypercoagulability state of COVID-19 patients was not only detected by ROTEM but it much more complex, where changes were observed on the fibrinolytic and endogenous anticoagulation system.”

We changed the paragraph on the Discussion Section:

“Finally, the hypercoagulability state of severe COVID-19 patients was detected by ROTEM and modifications on some coagulation tests related to the fibrinolytic and endogenous anticoagulation system, while the more common conventional coagulation tests, as aPTT (sec) INR and platelets, remained unchanged.”

We changed the paragraph on the Conclusion Section:

“The hypercoagulability state of patients infected with SARS-CoV-2 was detected by ROTEM and other coagulation tests but not with usual coagulation tests. Our findings highlight the role of rotational thromboelastometry when monitoring the coagulation system in ICU patients with COVID-19 and also demonstrated that the mechanisms to explain the hypercoagulability state of patients infected with SARS-CoV-2 is very complex and need more studies”.

We agree with your important comment and we included in the limitation paragraph:

“Fourth, we did not evaluate all factors involved in fibrinolysis, such as PAI-1 and plasmin, which preclude us to fully understand the role of fibrinolytic system on COVID-19 induced coagulopathy and also other laboratory tests as prothrombin fragment 1+2, thrombin-anti-thrombin complexes and endogenous thrombin potential assays were not done to better understand the hypercoagulability state of such patients. Finally, we not measured any marker for endothelial dysfunction which probably contribute to the modifications on the coagulation system of the severe patients infected with SARS-CoV-2. Nevertheless, our study was the first to demonstrate some aspects of coagulation disorders that can occur in critical patients infected by COVID-19, especially the deficiency of naturally anti-coagulant factors.”

Thank you for your important inputs

Sincerely,

Ricardo Luiz Cordioli

19TH, November, 2020

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PLoS One. doi: 10.1371/journal.pone.0243604.r005

Decision Letter 2

Pablo Garcia de Frutos

25 Nov 2020

COAGULATION PROFILE OF COVID-19 PATIENTS ADMITTED TO THE ICU: AN EXPLORATORY STUDY

PONE-D-20-25608R2

Dear Dr. Cordioli,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Pablo Garcia de Frutos

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

PLoS One. doi: 10.1371/journal.pone.0243604.r006

Acceptance letter

Pablo Garcia de Frutos

1 Dec 2020

PONE-D-20-25608R2

COAGULATION PROFILE OF COVID-19 PATIENTS ADMITTED TO THE ICU: AN EXPLORATORY STUDY

Dear Dr. Cordioli:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

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on behalf of

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PLOS ONE

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Table. Fibrinolysis and endogenous inhibitors of coagulation.

(DOCX)

Click here for additional data file.^{(18KB, docx)}

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Data Availability Statement

All relevant data are available from Dryad (doi:10.5061/dryad.pg4f4qrn3).

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PERMALINK

Coagulation profile of COVID-19 patients admitted to the ICU: An exploratory study

Thiago Domingos Corrêa

Ricardo Luiz Cordioli

João Carlos Campos Guerra

Bruno Caldin da Silva

Roseny dos Reis Rodrigues

Guilherme Martins de Souza

Thais Dias Midega

Niklas Söderberg Campos

Bárbara Vieira Carneiro

Flávia Nunes Dias Campos

Hélio Penna Guimarães

Gustavo Faissol Janot de Matos

Valdir Fernandes de Aranda

Leonardo José Rolim Ferraz

Roles

Abstract

Background

Objective

Methods

Results

Conclusion

Introduction

Materials and methods

Study design and setting

Study participants

Laboratory analysis

Conventional coagulation tests

Rotational thromboelastometry

Platelet function test

Fibrinolysis and endogenous anticoagulation system

Data collection

Statistical analysis

Results

Baseline characteristics of patients

Table 1. Characteristics of study participants.

Anticoagulants, blood transfusion and clinical outcomes

Table 2. Administered treatments and clinical outcomes.

Laboratory analysis

Table 3. Arterial pH, ionized calcium, peripheral temperature, conventional coagulation tests and hemoglobin.

Conventional coagulation tests

Rotational thromboelastometry

Fig 1. Coagulation profile accordingly to rotational thromboelastometry (ROTEM).

Table 4. Rotational thromboelastometry and platelet function test.

Platelet function test

Fibrinolysis and endogenous inhibitors of coagulation

Fig 2. Fibrinolysis and endogenous inhibitors of coagulation.

Discussion

Conclusion

Supporting information

Acknowledgments

List of abbreviations

Data Availability

Funding Statement

References

Decision Letter 0

Pablo Garcia de Frutos

Roles

Author response to Decision Letter 0

Decision Letter 1

Pablo Garcia de Frutos

Roles

Author response to Decision Letter 1

Decision Letter 2

Pablo Garcia de Frutos

Roles

Acceptance letter

Pablo Garcia de Frutos

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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