key: cord-0990030-sk9zai29
authors: Bayrakci, Nergiz; Ozkan, Gulsum; Mutlu, Levent Cem; Erdem, lknur; Yildirim, Ilker; Gulen, Dumrul; Celikkol, Aliye
title: Relationship between serum soluble endothelial protein C receptor level and COVID-19 findings
date: 2021-07-27
journal: Blood Coagul Fibrinolysis
DOI: 10.1097/mbc.0000000000001070
sha: e806cdd693455890e055e504c46ad6dbdac4b438
doc_id: 990030
cord_uid: sk9zai29

Coronavirus-related disease-2019 (COVID-19)-associated coagulopathy presents predominantly with thrombosis and leads to complications in close association with inflammatory process. Soluble endothelial protein C receptor (sEPCR), which is the soluble form of EPCR, reduces the anticoagulant and anti-inflammatory activity of activated protein C. The purpose of this study is to investigate the relationship between sEPCR and the laboratory parameters and thorax computed tomography (CT) findings in the course of COVID-19. Twenty-five laboratory-confirmed [reverse transcription-quantitative polimerase chain reaction (RT-qPCR) positive] and 24 clinically diagnosed (RT-qPCR negative) COVID-19 patients were enrolled in the study. Blood specimens were collected for sEPCR and haematological and biochemical parameter measurement. Thorax CT was performed to detect COVID-19 findings. These parameters from RT-qPCR positive and negative patients were then compared. Although there was no difference between the groups in terms of symptoms, the time between the onset of symptoms and the admission time was shorter in RT-qPCR positive group (P = 0.000). sEPCR levels were significantly higher in the RT-qPCR positive group (P = 0.011). Patients with ground-glass opacity and bilateral involvement on thorax CT have higher serum sEPCR levels (P = 0.012 and 0.043, respectively). This study has shown for the first time that serum sEPCR levels, which is a member of coagulation cascade and has also been reported to be associated with inflammation, is higher in patients with positive RT-qPCR test and patients with GGO or bilateral involvement on thorax CT regardless of the PCR result.

Coronavirus-related disease-2019 (COVID- 19) , which caused by severe acute respiratory distress syndrome coronavirus 2 (SARS-CoV-2), is a systemic infectious disease with a wide spectrum of symptoms and signs. The first case was reported in December 2019 and has become a pandemic as of March 2020 [1] . The most widespread and only direct method for determining SARS-CoV-2 for diagnosis of COVID-19 is reverse transcription-quantitative polimerase chain reaction (RT-qPCR) performed mostly on nasopharyngeal swab, has a reported sensitivity of 63-78%. RT-qPCR test might give false-negative result because of testing too early (due to low viral load) or too late (due to viral load displacement), inappropriate or insufficient sample and improper transportation and storage of the sample [2] [3] [4] . Therefore, in symptomatic patients with negative RT-qPCR test, thorax computed tomography (CT) findings guide the diagnosis and may prevent delay in diagnosis and treatment of COVID-19 [5] .

The host receptor that mediates the entry of SARS-CoV-2 into the cell is angiotensin-converting enzyme 2'dir (ACE2) as in SARS-CoV. SARS-CoV-2 binds to ACE2 through the receptor-binding gene region of its spike protein [6, 7] . A rapid cytokine response occurs following the entry of the virus into the cell followed by e series of complications such as cytokine release syndrome, thromboembolism and acute respiratory distress syndrome (ARDS). It is known that thromboembolic complications can occur at any age and without any risk factor [8] [9] [10] [11] [12] . This process, also called 'COVID-19 associated coagulopathy', has not been clearly identified. The main laboratory findings are, normal or slightly prolonged prothrombin time (PT) and activated partial thromboplastin time (aPTT); high d-Dimer and fibrinogen levels and variable platelet level, which are consistent with infection-induced inflammatory changes [13] [14] [15] .

Protein C system is an important member of primary anticoagulant system, wherein FVIIa and FVa activity regulated thorough activated protein C (APC). Thrombin-thrombomodulin complex activates protein C to produce APC. Subsequently, endothelial protein C receptor (EPCR), a transmembrane protein localized endothelial cell, accelerates this activation. In addition to its anticoagulant effects, APC has anti-inflammatory and cytoprotective effects. The effects of APC on leukocyte and the other cell functions involved in inflammatory response take place via EPCR [16] [17] [18] . Soluble EPCR (sEPCR), resulting from proteolysis of ERCP, inhibits APC-phospholipid bindings and causes APC dysfunction. This process is strictly regulated by inflammatory and coagulant factors [19] . There are several studies reporting high sEPCR levels in some disease accompanied by coagulopathies such as systemic lupus erythematosus (SLE), preeclampsia, malaria and that this is associated with poor prognosis, severe pneumonia and ARDS [20] [21] [22] [23] [24] .

The purpose of this study is to investigate the relationship between sEPCR level and COVID-19 findings, which is frequently accompanied thromboinflammatory complications.

Twenty-five laboratory-confirmed (RT-qPCR positive) and 24 clinically diagnosed [RT-qPCR negative) based on chest CT and clinical findings] COVID-19 patients over the age of 18 years admitted to Tekirdag Namik Kemal University Hospital, were included. Exclusion criteria were pregnancy, using anticoagulant or antiaggregant, hepatic disease and systemic infection. Patients were enrolled once the local ethical committee had confirmed that the study protocol was compatible with the second Declaration of Helsinki. Patients underwent detailed physical examinations, and their demographic data and vital findings were recorded. Nasopharyngeal swab specimens were collected for the COVID-19 RT-qPCR test. Repeat RT-qPCR specimens were collected after 48 h from patients with negative swab results. Chest CT was performed for the diagnosis of COVID-19 pneumonia. Patients were subsequently divided into laboratory-confirmed (RT-qPCR positive) and clinically diagnosed (RT-qPCR negative, thoracic CT and clinical findings positive) depending on their nasal swab specimen results. Blood specimens (10 ml) were collected from all patients immediately on hospitalization from a large vein in the antecubital region between 08.00 and 10.00 a.m. after 12-h fasting. Serum biochemistry, complete blood count and coagulation parameters measurements were performed on the same day. Blood specimens obtained for sEPCR measurement were immediately centrifuged for 10 min at 2500 x g for serum collection. Serum specimens were then stored at -808C until the day of study.

Human sEPCR levels were measured using a Bioassay Technology Laboratory (Shanghai Korain Biotech Co. Ltd. Shanghai, China) commercial ELISA kit (catalogue no. E1245Hu, sensitivity 0.25 ng/ml, intra-assay variation coefficient < 8%, inter-assay CV < 10%).

RT-qPCR tests involving oropharyngeal and nasopharyngeal swab specimens from patients presenting to the hospital on suspicion of COVID-19 infection were performed at the Medical Microbiology Laboratory. The swab specimens were placed into tubes containing 2-3 ml vNAT buffer. Viral RNAs were extracted in vNAT buffer with no additional extraction procedure. Amplification of open reading frame 1ab (ORF1ab) and nucleocapsid protein (N) target genes was performed using appropriate SARS-CoV-2 Double Gene RT-qPCR kits (Bioeksen R&D Technologies Ltd., Istanbul, Turkey) in line with the manufacturer's instructions. Reaction mixtures were prepared to a total volume of 20 ml with the addition of 10 ml 2X Prime Script Mix, 5 ml CVD Di Oligo Mix and 5 ml template nucleic acid. RT-qPCR tests were performed with reverse transcriptional reaction for 5 min at 528C, predenaturation at 958C for 10 s, 40-cycle denaturation at 958C for 1 s and 40 cycle extension at 558C for 30s, and fluorescence signal collection. Nonsigmoidal curves were defined as negative, while cycle threshold values (Cq) less than 38 were regarded as positive test results. Repeat specimens were requested for samples with Cq at least 38, and the RT-qPCR tests were repeated.

Thorax computed tomography findings Thorax CT findings classified according to lesion type [ground glass opacity (GGO), consolidation, tree-in-bud, nodule, reticular density, lymphadenopathy, pleural effusion] and lesion localization (bilateral, unilateral).

Compatibility with normal distribution was evaluated using the Kolmogorov-Smirnov test. The t-test was applied in the comparison of normally distributed data, and the Mann-Whitney U test for nonnormally distributed data. The Chi-square test was employed in the evaluation of demographic variables, and Pearson correlation analysis for determining correlations. P values less than 0.05 were regarded as statistically significant.

Patients diagnosed with COVID-19 were compared in two groups: 24 patients in RT-qPCR negative group and 25 patients in RT-qPCR positive group. There was no difference in terms of age and sex between the groups. History of contact with an infected person was more frequent in RT-qPCR positive group (P ¼ 0.002). Although there was no difference between the groups in terms of symptoms, the time between the onset of symptoms and the admission time was shorter in sEPCR and COVID-19 Bayrakci et al. 551

RT-qPCR positive group (P ¼ 0.000). Procalcitonin, Creactive protein (CRP), leukocyte and international normalization ratio (INR) levels were higher in RT-qPCR negative group (P ¼ 0.001; 0.003; 0.001; 0.022, respectively) ( Table 1) . All patients had CT findings in favour of COVID-19. The two most common signs were GGO and consolidation (77.6 and 40.8%, respectively). Bilateral involvement was detected in 66.7% of the patients.

Soluble endothelial protein C receptor levels sEPCR levels were 9.35 (5.68-14.11) ng/ml in the RT-qPCR negative group and 10.35 (7.0368) ng/ml, 552 Blood Coagulation and Fibrinolysis 2021, Vol 32 No 8 Serum soluble endothelial protein C receptor levels in RT-qPCR positive and negative patients. sEPCR, soluble endothelial protein C receptor; RT-qPCR, reverse transcription-quantitative polimerase chain reaction. P value ¼ 0.011.

significantly higher in the RT-qPCR positive group (P ¼ 0.011) ( Table 1 ) (Fig. 1) .

There was no correlation between sEPCR and other laboratory parameters.

When the study group was evaluated in terms of CT findings, serum sEPCR levels were higher in patients with GGO and bilateral involvement (P ¼ 0.012; 0.043, respectively) ( Table 2 ) (Fig. 2 ).

EPCR plays a key role in initiation and maintenance of the anticoagulant and anti-inflammatory effects of APC, which produced from protein C through thrombin-thrombomodulin complex. sEPCR, which is the soluble form of EPCR and also found in healthy population, inhibits the activation of APC. High serum sEPCR levels have been reported in some disease such as SLE, cancer, stroke where inflammation and thrombosis are common [20] [21] [22] [23] [24] .

The pathogenesis of SARS-Cov-2-associated hypercoagulability is not clearly understood. Major laboratory findings in this process, called thromboinflammation or COVID-19-associated coagulopathy, are normal or slightly prolonged PT and aPTT, variable levels of platelets, high levels of fibrinogen and d-Dimer. In addition, there are some studies reporting increased factor VIII activity, significantly elevated von-Willebrand factor level, slightly decreased antithrombin and protein S levels, slightly elevated protein C level [25] . It has been suggested that the direct effect of the virus disrupting endothelial integrity plays a key role in COVID-19 associated coagulopathy [15] . SARS-CoV-2 enters into the cell ACE2-mediated endocytosis. Intracellular changes following the endocytosis of the virus result in decreased nitric oxide production, aggravated vasoconstriction, increased leukocyte and platelet adhesion and finally increased thrombogenicity [26] . In this process, transmembrane protease serine 2 (TMPRSS-2) and tumour necrosis factor alpha converting enzyme (TACE), also called as A disintegrin and metalloprotease 17 (ADAM17) interact with ACE2 [7, 27, 28] . ADAM17 is a sheddase and is responsible for the extracellular breakdown of many transmembrane proteins and the release of their soluble forms into the circulation. ACE2, EPCR and tumour necrosis factor alpha (TNF alpha) are among the target proteins of ADAM17, which also takes place in sEPCR production. [29] . It has been reported that TNF alpha and interleukin 1-beta (IL-1beta) suppress serum EPCR level and simultaneously high sEPCR level was detected [30, 31] . TNF alpha and IL-1beta are known to sEPCR and COVID-19 Bayrakci et al. 553 Serum soluble endothelial protein C receptor levels based on computed tomography findings. GGO, ground glass opacity; sEPCR, soluble endothelial protein C receptor.

be among the major cytokines in the cytokine storm seen in COVID-19 course [32] . Steven et al. [33] have suggested that considering the role of protein C system in inflammation and coagulation, APC may be involved in the treatment of disease based on the close relationship between inflammation and coagulation cascade in the course of COVID-19.

In our study, sEPCR levels were higher in RT-qPCR positive group. These patients were diagnosed earlier.

The time between the onset of symptoms and admission time was shorter in this group. It is possible that sEPCR may increase suddenly with the onset of endothelial damage following virus entry in early stages of the disease. On the other side, major laboratory findings such as INR, leucocyte count, procalcitonin, CRP known to be consistent with disease progression and severity were higher and sEPCR levels were lower in RT-qPCR negative group. These patients were diagnosed later and they are more likely to have been detected at a later stage of the disease. Therefore, prominent findings may have varied. Considering all these issues it can be argued that sEPCR level increases in the early phase of the disease and then gradually decreases as some other parameters increase with the dynamic inflammatory response in the later stages of the disease.

Reported sensitivity and specificity for thorax CT findings in the diagnosis of COVID-19 are quite variable (60 -98% and 25 -53%, respectively). It is recommended in clinically suspicious but RT-qPCR negative patients [34] . In a study including 1014 patients, 308 of 403 RT-qPCR negative and clinically suspicious patients had CT findings in favour of COVID-19 (%75) [35] . Earliest and most common reported CT finding associated with COVID-19 is GGO and is usually bilaterally and peripherally located in lower zone [36] . Thorax CT was performed in all patients included in our study and COVID-19 associated findings were detected in all of them. Similar to the literature, the most common finding was GGO and bilateral involvement was found in most of the patients (77.6 and 66.7%, respectively). Regardless of the PCR results, patients with GGO and bilateral involvement had higher sEPCR levels than those without GGO and unilateral involvement. In a study including patients diagnosed with melioidosis-associated pneumonia, plasma sEPCR levels were found to be significantly higher and associated with high mortality. Authors suggested that high levels of sEPCR in these group could be secondary to TNF-alpha associated EPCR dysfunction [24] . Similar findings were reported in two different studies evaluating septic shock patients secondary to pneumococcal pneumonia and experimental malaria-associated ARDS model [21, 22] . These findings support the view that there is an intersection between coagulation and inflammation cascade in systemic infectious diseases with severe lung involvement and that protein C system located at this junction. Therefore, our findings can be interpreted according to this implication.

In conclusion, COVID-19-associated coagulopathy is a complex process whose pathophysiology is not yet understood. Different mechanisms and mediators are involved in different stages of the disease. Close relationship between endothelial damage, coagulopathy and inflammation has been reported in many studies. This study is the first study investigating the relationship of sEPCR with thromboinflammatory process and pulmonary findings in the course of COVID-19. We believe that more detailed investigations of protein C and EPCR-related pathways may provide important information for the prevention and treatment of COVID-19-associated thromboembolic complications.

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This study has not been presented or published previously.The authors have no funding of report.

There are no conflicts of interest.