Endothelial activation and dysfunction are associated with COVID-19 severity.
Epidemiological studies suggest that severe cases or deaths due to COVID-19 frequently
present with underlying comorbidities, such as advanced age, hypertension, diabetes, and
cardiovascular diseases. Endothelial dysfunction, with the subsequent use of the
complement, the hypercoagulable state and production of thrombin appears to be the common
denominator of varieties of clinical signs and symptoms of COVID-19, which are directly
associated with thromboembolic events. Necropsy findings in patients with COVID-19 have
shown catastrophic microvascular injury, with pulmonary endotheliitis, thrombosis and
angiogenesis being distinct pathophysiological features of the lungs in patients with
COVID-19 compared with H1N1 patients and uninfected controls.

Venous thromboembolic disease, which can manifest as Deep Vein Thrombosis (DVT) or
Pulmonary Embolism (PE), often occurs in patients with severe COVID-19, despite
prophylactic anticoagulation. PE has been found in almost 20% of patients, despite having
received anticoagulant treatment. In another study, necropsy was performed in 12 patients
with COVID-19, of whom were receiving anticoagulants. DVT was detected in 7 (58%)
patients, although none of them had a clinical suspicion. PE was the leading cause of
death in 4 patients. Venous thrombosis occurs in particularly high rates (14-81%) in
critically ill COVID-19 patients admitted to Intensive Care Units (ICU). In fact, PE has
been recorded as the most common thrombotic complication in these patients with a
frequency of up to 81%. In hospitalized patients with COVID-19, venous thrombosis occurs
less frequently (3-21%), while for outpatients there are insufficient data. In addition
to DVT, cases of acute ischemia of the upper or lower extremity with a potential need for
surgery have also been reported as a less common arterial thrombotic complication in
COVID-19.

In addition, acute myocardial ischemia is the most commonly reported cardiovascular
complication of COVID-19. The prevalence of myocardial ischemia varies from 7% to 28%.
Studies have correlated the increase in troponin in hospitalized patients with a more
severe clinical course and worse prognosis.

Ischemic stroke is another common extrapulmonary thromboembolic complication of COVID-19.
There are reports of ischemic stroke as the first symptom of infection (appearing
especially as a middle cerebral artery thrombosis, even in young patients) and as a
complication during hospitalization. In retrospective observational studies, the
prevalence of ischemic stroke in this population ranges from 1.3% to 46%. The most likely
mechanism of strokes during COVID-19 is considered to be subsequent hypercoagulability
and vascular endothelial dysfunction through activation of inflammatory cytokines.

Human serum albumin is a peptide, which consists of 585 amino acids bonded in a specific
sequence in humans. The ischemia modified albumin (IMA) molecule was identified in early
2000s, when it was observed that hypoxic-induced ischemia resulted in a modification in
the circulating albumin molecule. Under ischemic conditions, the N-terminus of albumin is
differentiated, possibly as a result of hypoxia, oxidation, free radicals and
energy-dependent changes in membranes. This modification at the N-terminus of albumin can
be indirectly evaluated. When cobalt (in vitro) is added to an ischemic sample, normal
albumin molecules will bind to it, leaving a small percentage free, while IMA cannot bind
to the added cobalt due to its modification at its binding site. Elevated IMA percentages
result in more unbound cobalt, which can be detected by the addition of a chromatographic
agent, such as dithiothreitol, and evaluated photospectrometrically. The increase of IMA
is inversely related to the amount of cobalt, which causes an increase in the colour
product. This is the basis of the Albumin Cobalt - Binding test, ACB test.

IMA has been studied as an indicator of cardiac ischemia but has also been found
increased in patients with endothelial dysfunction, infections and sepsis. It also
increases in patients with cerebral ischemia, pulmonary embolism, deep vein thrombosis,
heart failure, intestinal ischemia etc. IMA values have a normal distribution in the
control population and are not related to age and gender. IMA, which is the only ischemia
biomarker that has reached the level of clinical evaluation, increases within a few
minutes of the onset of ischemia regardless of the affected organ-tissue.

SuPAR (Soluble urokinase Plasminogen Activating Receptor) is the soluble form of uPAR, a
protein that is primarily expressed in cells of the immune system, including neutrophils,
activated T-lymphocytes, and macrophages. The uPAR binds to the cell membrane with a
glycosylphosphatidyl-inositol (GPI). When uPAR is released from the cell membrane, it
becomes soluble (SuPAR) and can be measured as a stable protein in various biological
fluids, such as plasma, urine and cerebrospinal fluid. SuPAR levels in healthy people are
quite stable in the blood and urine. Elevated levels of SuPAR reflect the activation of
the immune system and have been evaluated as an indicator of inflammation and organ
damage in a few diseases, including COVID-19. Studies on the concentration of SuPAR in 57
patients with COVID-19 pneumonia, showed that SuPAR concentrations were higher in
patients who eventually developed severe respiratory failure and required mechanical
ventilation, compared to patients who did not develop severe respiratory failure. More
specifically, concentrations greater than or equal to 6 ng/ml had a sensitivity of 85.7%
to predict the adverse outcome even 12 days before happening. SuPAR appears to be an
important risk stratification tool in patients with COVID-19, as it can early identify
patients who will develop severe respiratory failure or that need mechanical ventilation.

Since this is the first study of IMA in patients with COVID-19, the primary aim of the
study is to investigate whether IMA levels at admission or at day 10 from symptoms onset
(peak of symptoms) will be higher in patients with COVID-19 pneumonia compared to healthy
individuals. The secondary aim is to assess whether IMA can predict severe respiratory
failure and deterioration risk and whether combination with SuPAR will increase its
accuracy.

Participants: 2 study groups will be included:

i) 128 patients with confirmed COVID-19 disease (study group, group 1) ii) 64 healthy
volunteers without any of the exclusion criteria (control group, group 2) Patients from
the study group will be divided into two subgroups, those with severe respiratory failure
(PaO2/FiO2 <150mmHg) and those without severe respiratory failure (PaO2/FiO2 >150mmHg)
Furthermore, they will be divided into those with or without deterioration risk defined
as need for high flow nasal cannula, mechanical ventilation, ICU admission or death.

Type of study: This will be a prospective observational study that will be carried out at
the Pulmonology/Covid-19 Department of the University General Hospital of Larissa.

Power analysis: For a statistical power of 90% with a probability of α-error of 0.05 and
Group 1/Group 2 ratio 2:1, it was estimated that 64 people are needed in the control
group (group 2) and 128 in the study group (group 1).

Interventions: Blood samples will be collected at admission for measurement of IMA and
SuPAR levels and at day 10 from symptom onset (peak of symptoms) for IMA level.

Blood sample collection: Samples will be collected in blood collection tubes containing
K2EDTA as anticoagulant and serum separation gel tubes for SuPAR and IMA assays
respectively. The samples will be centrifuged (at 3000 x g for 10 minutes) within the
first 3 hours of sampling. Then, both plasma and serum samples will be placed in
Eppendorf tubes and stored at -80oC.

IMA and SuPAR measurements: IMA levels will be determined according to the principles of
the ACB assay, using the commercially available "Ischemia Modified Albumin Assay Kit"
method (Abbexa LTD, Cambridge, UK), on the Architect c8000 automatic analytical system
(Abbott, USA). SuPAR levels will be determined by the suPARnostic TurbiLatex quantitative
turbidimetric immunoassay (ViroGates A/S, Denmark), also on the Architect c8000 automatic
analytical system.

Monitoring/Recording: Demographics (age, sex, nationality), date of admission,
comorbidities, medication, symptoms, vital signs (heart rate, temperature, blood
pressure, respiratory rate, arterial blood oxygen saturation and PaO2/FiO2 ratio),
arterial blood gases (PaO2, PaCO2, pH, HCO3), lactic acid, viral load from RT-PCR for
SARS-COV2 and the findings from the imaging and laboratory test (hemoglobin, hematocrit,
white blood cells, neutrophils, lymphocytes, platelets, PT, INR, aPTT, fibrinogen,
d-dimers, CRP, ferritin, LDH, troponin, urea, creatinine, AST, ALT, ALP, CPK, potassium,
sodium, calcium, total protein, albumin, total bilirubin) will be recorded on admission.

Vital signs (heart rate, temperature, blood pressure, respiratory rate, arterial blood
oxygen saturation and PaO2/FiO2 ratio), laboratory findings (as mentioned above) and the
presence of any complications (pneumonia, ARDS, pulmonary embolism, stroke, acute renal
failure, multiorgan failure, arrhythmias, shock, pseudomembranous colitis, etc.) will be
recorded daily.

Expected results: i) IMA levels are expected to be higher in patients with COVID-19
compared to healthy volunteers ii) IMA levels at admission are expected to be higher in
patients that will develop severe respiratory failure or with deterioration risk iii) IMA
levels at day 10 from symptom onset are expected to be higher in patients with severe
respiratory failure and predict deterioration risk with a greater accuracy iv) SuPAR
levels are expected to be higher in patients with COVID-19 compared to healthy volunteers
v) SuPAR levels at admission are expected to be higher in patients that will develop
severe respiratory failure or with deterioration risk vi) Combination of IMA and SuPAR,
is expected to increase the predictive accuracy of SuPAR

To our knowledge, this will be the first study of Ischemia Modified Albumin in patients
with COVID-19. Prognosis is a key driver of clinical decision-making. If the correlation
between IMA levels and poor prognosis is proved, it will become a useful tool for the
risk stratification of COVID-19 patients. Furthermore, the combination of an ischemia
biomarker, such as IMA, with an inflammatory biomarker, like SuPAR might be the key to
this approach.