Keywords
thromboelastometry - fibrinogen/fibrin - thrombotic complications
Coronaviruses (CoVs) are enveloped, single-stranded ribonucleic acid viruses that
usually infect birds, mammals, and humans. Human CoVs may cause respiratory, enteric,
and neurological affections.[1] In late December 2019, a cluster of pneumonia cases of unknown cause was reported
in Wuhan, Hubei Provence, China. Thereafter, many countries reported similar cases,
leading to the discovery of a novel CoV. In February 2020, the World Health Organization
named SARS-CoV-2 as the cause of the Coronavirus Disease 2019 (COVID-19). Early reports
showed that very high D-dimer levels are common in COVID-19 pneumonia and correlate
with a worse prognosis.[2] To better characterize COVID-19-related coagulation changes, we investigated traditional
parameters and whole blood thromboelastometry profiles in all consecutive patients
admitted to the intensive care unit (ICU) of Padua University Hospital between March
7 and 19 2020 for acute respiratory distress syndrome due to COVID-19. Exclusion criteria
were: known preexisting congenital bleeding or thrombotic disorders and/or preexisting
acquired coagulopathies, active cancer and/or chemotherapy, pregnancy, and ongoing
anticoagulant therapy. Demographic characteristics, comorbidities, and Sequential
Organ Failure Assessment score were recorded. A group of 44 healthy, age-, sex-, and
body weight-matched subjects served as controls for laboratory data. Within 30 minutes
of ICU admission, venous blood samples were drawn from each enrolled patient into
two BD Vacutainer tubes (Becton–Dickinson, Franklin Lakes, New Jersey, United States)
containing sodium citrate 109 mmol/L (3.8%) and one BD Vacutainer tube containing
ethylenediaminetetraacetic acid 5.4 mg. In all enrolled patients, hemoglobin, platelet
count, prothrombin time/international normalized ratio, activated partial thromboplastin
time, fibrinogen, antithrombin, and D-dimer were measured. Whole blood thromboelastometry
profiles were obtained using a ROTEM delta apparatus (Instrumentation Laboratory –
Werfen, Barcelona, Spain), as previously described.[3] INTEM and EXTEM assays (evaluation of intrinsic and extrinsic coagulation pathways)
and FIBTEM test (evaluation of fibrinogen contribution to blood clot) were performed
in each enrolled patient. The following ROTEM parameters were analyzed: (1) clotting
time corresponding to the initiation phase of the clotting process; (2) clot formation
time (CFT) reflects the measure of the propagation phase of whole blood clot formation;
(3) maximum clot firmness (MCF) is the maximum amplitude in millimeters reached in
thromboelastogram; and (4) area under the curve (mm*100), defined as the area under
the velocity curve, that is the area under the first derivative curve ending at a
time point that corresponds to MCF.[4] The protocol was conducted in compliance with the Helsinki Declaration and notified
to the Institutional Ethical Committee of Padua University Hospital. Written informed
consent was obtained from each patient when possible or relatives and from each control.
Statistical analysis was performed using the PASW Statistics 17.0.2 (SPSS Inc.) for
Windows. Continuous variables were expressed as mean ± standard deviation and categorical
variables as number and fraction (%). The parametric t-Student's test or the nonparametric Mann–Whitney U was used to test for differences between variables, when appropriate. A p-value of < 0.05 was considered significant.
Among 30 eligible patients, 8 were excluded: active cancer (n = 4), anticoagulant therapy (n = 3), and pregnancy (n = 1). [Table 1] shows the main clinical and laboratory characteristics of the study population.
The vast majority of COVID-19 patients were male (91%), obese (86%), with a PaO2/FiO2 < 150 (91%) at admission, and mechanically ventilated (86%) (tidal volume 6 mL × ideal
body weight and driving pressure < 12 cm H2O). Fibrinogen and D-dimer plasma levels were significantly higher in COVID-19 patients
than controls (p < 0.0001 in both comparisons). Cases showed markedly hypercoagulable ROTEM profiles
characterized by significantly shorter CFT in INTEM (p = 0.0002) and EXTEM (p = 0.01) and by a higher MCF in INTEM, EXTEM, and FIBTEM (p < 0.001 in all comparisons). One patient died of multiorgan failure at day +1 after
enrolment and presented with the most hypercoagulable ROTEM profiles ([Fig. 1]).
Table 1
Patient characteristics and laboratory data
|
Cases
|
Controls
|
p-Value
|
|
Patients, n
|
22
|
44
|
–
|
|
Gender M/F, n
|
20/2
|
40/4
|
–
|
|
Age, y
|
67 ± 8
|
68 ± 7
|
0.63
|
|
BMI, kg/m2
|
30 ± 6
|
29 ± 4
|
0.49
|
|
SOFA score
|
4 ± 2
|
–
|
–
|
|
PT, %
|
93 ± 10
|
91 ± 10
|
0.46
|
|
INR
|
1.08 ± 0.06
|
1.09 ± 0.06
|
0.54
|
|
aPTT, s
|
26 ± 12
|
26 ± 2
|
0.95
|
|
Fibrinogen, mg/dL
|
517 ± 148
|
297 ± 78
|
< 0.0001
|
|
Antithrombin, %
|
96 ± 13
|
90 ± 14
|
0.1
|
|
D-dimer, ng/L
|
5,343 ± 2,099
|
225 ± 158
|
< 0.0001
|
|
Hb, g/L
|
121 ± 16
|
138 ± 15
|
0.0002
|
|
Htc, %
|
38 ± 4
|
41 ± 4
|
0.008
|
|
Plts, ×109/L
|
249 ± 119
|
218 ± 67
|
0.27
|
|
INTEM
|
|
|
|
|
CT, s
|
185 ± 49
|
174 ± 23
|
0.33
|
|
CFT, s
|
57 ± 13
|
70 ± 18
|
0.0002
|
|
MCF, mm
|
68 ± 6
|
62 ± 7
|
< 0.0001
|
|
ML, % (range)
|
1 ± 3
|
2 ± 3
|
0.22
|
|
AUC, U
|
6,808 ± 603
|
6,743 ± 563
|
0.68
|
|
EXTEM
|
|
|
|
|
CT, s
|
75 ± 16
|
72 ± 8
|
0.11
|
|
CFT, s
|
66 ± 20
|
78 ± 26
|
0.01
|
|
MCF, mm
|
69 ± 6
|
64 ± 5
|
0.0003
|
|
ML, % (range)
|
1 ± 3
|
2 ± 3
|
0.22
|
|
AUC, U
|
6,924 ± 591
|
6,882 ± 569
|
0.79
|
|
FIBTEM
|
|
|
|
|
MCF, mm
|
31 ± 9
|
18 ± 6
|
< 0.0001
|
|
AUC, U
|
3,101 ± 852
|
2,249 ± 1072
|
0.001
|
Abbreviations: aPTT, activated partial thromboplastin time; AUC, area under the curve;
BMI, body mass index; CFT, clot formation time; CT, clotting time; Hb, hemoglobin;
Htc, hematocrit; INR, international normalized ratio; MCF, maximum clot firmness;
ML, maximum lysis; Plts, platelet count; PT, prothrombin time; SOFA, Sequential Organ
Failure Assessment.
Note: p-Values marked in bold are statistically significant.
Fig. 1 Thromboelastometry profiles of the deceased patient. (A) INTEM test; (B) EXTEM test; and (C) FIBTEM test. ɑ, ɑ-angle; AUC, area under the curve; CFT, clot formation time; CT,
clotting time; MCF, maximum clot firmness.
Coagulation profiles observed in our study population reflect a severe hypercoagulability
rather than a consumptive coagulopathy (e.g., disseminated intravascular coagulation).
Such a laboratory pattern and association can be linked to both markedly increased
levels of fibrinogen and an excessive fibrin polymerization due to the infection.
SARS-CoV-2 is likely to promote massive fibrin formation and deposition which can
also account for the very high D-dimer levels found in these patients.[2] Fibrin deposition in alveolar and interstitial lung spaces, in addition to microcirculation
thrombosis,[5] may contribute to worsen respiratory failure resulting in prolonged mechanical ventilation,
poor prognosis, and death. Furthermore, other major venous thromboembolic events and
arterial complications (e.g., acute myocardial infarction) have been reported[6] and are likely to be largely underestimated. Notably, 5 (23%) of our patients developed
an in-hospital deep vein thrombosis despite anticoagulant prophylaxis. In this regard,
anticoagulant therapy may improve the prognosis in COVID-19 patients as reported by
Tang et al.[7] In light of the severe hypercoagulable state observed in these patients, effective
anticoagulant prophylaxis should be considered to reduce the risk of thrombotic complications.
Unfortunately, our data did not allow to assess the impact of adequate dosages of
anticoagulants on clotting parameters. Measuring antifactor Xa (anti-Xa) activity
in plasma was deemed as the most accurate way to monitor therapeutic dosing of low
molecular weight heparin (LMWH, the anticoagulant used in our study). However, we
were not able to evaluate anti-Xa activity in our study as it should have been measured
4 to 6 hours after the last injection of LMWH (peak level) whereas we collected blood
samples immediately after admission to the ICU and thus at varying times from the
last administration of heparin. It bears noting that if on the one hand thromboelastometry
has the advantage to provide a global assessment of whole blood's ability to clot,
on the other hand it is not able to evaluate the contribution to clot formation of
each element (e.g., endothelium, platelets, and clotting factors). In conclusion,
COVID-19 patients with acute respiratory failure present with severe hypercoagulability
due to hyperfibrinogenemia resulting in increased fibrin formation and polymerization
that may predispose to thrombosis. Larger studies are needed to define new therapeutic
strategies to limit hypercoagulability and improve outcomes.
