Plasma markers of COVID-19 severity: a pilot study

The coronavirus disease of 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Most infected people experience mild to moderate respiratory symptoms and recover without requiring special care. However, some people get seriously ill or die. In general, older people and people with other medical conditions like obesity, cardiovascular disease, diabetes, chronic respiratory disease, or cancer are more likely to develop serious illness. The wide spectrum of clinical presentations of SARS-CoV-2 infected patients implies the involvement of many biological pathways like immunological, inflammatory, and coagulative, and to those pathways related biomarkers. Accumulated data shows that in response to the SARS-CoV-2 infection global changes occur in protein synthesis, processing and posttranslational modifications, like phosphorylation and glycosylation [1]. Some of these changes might reflect the severity of COVID-19. Hitherto, changes in concentrations of D-dimer, cardiac troponin I, renal biomarkers, such as serum urea, creatinine and markers of glomerular filtration rate, C-reactive protein (CRP), ferritin, and certain cytokines/chemokines may have a diagnostic value [2‐4]. Several studies reported that circulating levels of damage associated molecular patterns, such as cytoskeletal keratin-18 (CK18) fragment M30/M65 ratio (an indicator of cell apoptosis in relation to the total cell death), HMGB-1 or mitochondrial DNA may also reflect the severity of COVID-19 patients [5‐7]. The activity of ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type 1 motif 13), a regulator of von Willebrand factor (VWF) multimer disruption, typically decline with the increasing extent of inflammatory responses [8]. Recent data show that VWF antigen (VWF:Ag) to ADAMTS13 activity ratio is strongly associated with SARS-CoV-2 severity [9].

High levels of pro-inflammatory cytokines also linked to severity and poor outcomes in SARS-CoV-2 infection [10, 11]. For example, the levels of IL-6 rise sharply in severe manifestations of COVID-19. One meta-analysis reviewing six studies reported that mean IL-6 concentrations are 2.9-fold higher in patients with severe compared to those with non-severe COVID-19 disease [12]. As a matter of fact, IL-6 is the only cytokine that affects liver synthesis and secretion of the full spectrum of acute phase proteins (APPs) [13]. Other inflammatory cytokines, like IL-1β and TNFα, modulate synthesis of specific APPs and can have additive, or inhibitory effects on IL-6-induced APPs expressions [14]. In turn, the synthesis of APPs may be influenced by insulin, dexamethasone, and glucagon [15].

As mentioned above, C-reactive protein (CRP) can be used as a diagnostic marker reflecting the severity and prognosis of COVID-19 [16, 17], and the levels of CRP are helpful to differentiate between viral and bacterial infections [18, 19]. Decrease in serum albumin, which is a negative APP, also suggested as a marker of the severity of SARS-CoV-2 infection [20].

Some studies reveal that the host’s protein glycosylation is altered upon infection with SARS-CoV-2 as well. For example, critically sick patients develop high concentrations of IgG antibodies where the conserved N-glycan in the Fc domain is not fucosylated thereby causing an amplified cytokine storm [21]. The glycosylation pattern of AAT is also modified in severe cases of COVID-19, which affects anti-inflammatory properties of AAT protein [22].

Alterations in human plasma protein levels and/or their molecular forms indicate pathophysiological changes caused by various diseases, including viral infections. Therefore, host proteins might not only provide valuable insights for the pathogenesis of diseases, including COVID-19, but also might be useful as clinical biomarkers, either individually or in combinations, to monitor and evaluate the disease development. To explore the clinical value of plasma inflammatory proteins, we enrolled twenty COVID-19 patients, with moderate or severe symptoms and ten non-COVID-19 controls. We performed whole plasma protein N-glycosylation analysis, and measured plasma levels of M30 and M65, as cell death markers, ADAMTS13, as a marker limiting platelet-rich thrombus formation, and various APPs, including albumin (ALB), α1-acid glycoprotein (AGP), α1-antitrypsin (AAT), ceruloplasmin (CP), haptoglobin (HP), high-sensitivity C-reactive protein (hs-CRP), α2-macroglobulin (A2MG), α1-antichymotrypsin (ACT) and serum amyloid A (SAA). We hoped that in addition to previous reports, results from this pilot study could confirm and/or uncover plasma marker candidates associated with COVID-19 symptom severity.

For unknown reasons some individuals infected with SARS-CoV-2 develop moderate symptoms and others become severely ill. Furthermore, patients differ in their response to therapies [30, 31]. These variable clinical manifestations reflect the host`s genetics, immunity, and comorbidities [32, 33]. Therefore, the discovery of biomarker signatures differentiating COVID-19 patients with different severities remains of importance. Since plasma/serum can be collected noninvasively, analyses of circulating protein levels and posttranslational modifications can give much information about the host`s proteome response to COVID-19 [34]. We conducted a pilot study in a small cohort of COVID-19 patients exhibiting severe or moderate symptoms to determine plasma levels of M30/M65, ADAMST13, various APPs and whole protein N-glycosylation profile. Although other studies have already investigated many proteins in COVID-19 patients, our findings may still contribute to the better understanding of the proteome responses to the infection. We provide results from a parallel measurement of nine plasma APPs concentrations, six of which were significantly higher in COVID-19 patients than in healthy subjects. Among these latter, median levels above reference values were found for AGP: 167.0 mg/dl (ref. 51–117 mg/dl), HP: 271.5 mg/dl (ref. 36–195 mg/dl), AAT: 235.5 mg/dl (ref. 88–174 mg/dl) and hs-CRP: 9.68 mg/dl (ref. less than 0.744 mg/dl). Albeit within the reference range, the levels of ALB were significantly lower, but CP were significantly higher in COVID-19 patients as compared to healthy controls.

Though changes in APP levels reflect general inflammatory response, altered magnitudes and profiles of these proteins might mirror distinct processes involved in the SARS-CoV-2 infection and disease progression. For instance, repeated studies show that levels of CRP are useful as a biomarker to follow COVID-19 progression [35], and patient management [36‐38]. AAT is also acknowledged as one of the key immunomodulatory proteins during the SARS-CoV-2 infection [22, 39] beneficial for COVID-19 patients [40, 41]. AAT is a broad inhibitor of proteases and interacts with different pro-inflammatory substances [42]. Moreover, AAT inhibits the metalloproteinase domain 17 [43] and the type II transmembrane serine protease [39], two host proteases facilitating SARS-CoV-2 viral entry, replication and the pathogenesis of viral infections. AGP, which also was found to be higher in severe versus moderate COVID-19 patients, belongs to the lipocalin protein family. Functions of AGP remain incompletely understood, although typically associated with anti-inflammatory, immune modulatory, and sphingolipid metabolism [44, 45]. On the contrary, levels of A2MG and, especially ALB, decreased in association with COVID-19 severity. A2MG is one of the major blood proteins binding a wide range of substances, such as TGF-β1, TNF-α, IL-1β, and hormones, and inhibiting proteases, like trypsin, chymotrypsin, elastase, metalloproteinases as well as parasite-derived proteinases, and it is involved in blood coagulation and fibrinolysis [46]. Moreover, A2MG binds blood iron, zinc, and copper ions stronger than ALB, and acts as a serum copper transporter. The sequestering of metal ions by A2MG or ALB is one of the host defense strategies against infections [47]. Previous experimental studies have shown that the administration of A2MG prolongs graft survival and protects against sepsis [48, 49]. The impaired fibrinolysis together with lower levels of A2MG suggested as a risk of asthma exacerbations [50]. COVID-19 disease has not yet been associated with low plasma levels of A2MG, which inspires further investigations. However, low serum levels of ALB have already been associated with poor COVID-19 patient prognosis [51, 52]. Researchers also demonstrated a negative relationship between serum ALB levels and a risk of developing thromboembolism [53]. In some patients, however, increased hemolysis and altered levels of hemoglobin and heme-scavenging proteins (i.e., hemopexin, ALB or HP) were described [54]. Thus, triggered induction of AAT, AGP, CRP or other APPs as well as the reduction of A2MG and ALB during the SARS-CoV-2 infection may mirror specific pathological processes. However, it remains unknown which concentrations of these proteins are required to be protective and/or become detrimental.

N-glycosylation is a highly dynamic posttranslational modification strongly affected by viral infections and inflammation [55]. APPs represent a fraction of mostly glycosylated plasma proteins, and therefore, we anticipated changes in N-glycan levels upon SARS-CoV-2 infection. Our N-glycan analysis of whole plasma proteins revealed an increase in the di-antennary di-sialylated glycan A2G2S2(6,6) in COVID-19 patients, which is the most abundant N-glycan on AAT [56]. Increased relative levels of A2G2S2(6,6) could be explained by the observed higher plasma levels of AAT in COVID-19 patients. Also, the di-antennary mono-sialylated glycan A2G2S1(6) is highly elevated in the serum of COVID-19 patients whereas the corresponding non-sialylated di-antennary N-glycan A2G2 was even significantly decreased in COVID-19 patients. These findings hint towards an elevated sialylation of serum protein N-glycans upon SARS-CoV-2 infection. Accordingly, scientists reported that SARS-CoV-2 not only induces a production but also increases the sialylation of AAT [22]. This latter was linked to enhance anti-inflammatory properties of AAT [57]. Furthermore, we detected increased levels of oligomannose N-glycans (M5, M9) from serum proteins of COVID-19 patients. It is well known that glycoproteins with terminal mannoses are more rapidly cleared from the circulation than their complex glycosylated counterparts [58] which might therefore exert broad effects on serum levels of diverse glycoproteins including immunoglobulins [59].

Finally, there are increasing reports regarding cell damage and thromboembolism in COVID-19 cases. As COVID-19 is associated with hypercytokinaemia, cytokines, like IL-1, IL-6 and TNFα, may induce cell death. Therefore, we sought to quantify circulating levels of full length (M65) and caspase-cleaved (M30) cytokeratin 18 (CK-18), as markers of apoptotic and necrotic cell death. Indeed, M65 (total cell death) and M30 (apoptosis) were significantly elevated in COVID-19 patients compared with healthy controls. It is noteworthy that M30/M65 ratio did not differ between COVID-19 patients with different severities but was significantly lower than in controls. This latter suggests that cytotoxic or ischemic necrosis might be the predominant pathway of cell death in these patients. In line, other studies suggested that increased cell death in COVID-19 patients relates to defective organ perfusion, particularly in those who develop microvascular thrombosis [60]. We also found significantly lower levels of ADAMTS13 in COVID-19 patients relative to controls. The reciprocal relationship between VWF and ADAMTS13 in thrombosis is widely studied [61], and several groups reported that COVID-19 causes a significant increase in formation of large VWF multimers, and decrease in activity and/or levels of ADAMTS13 [62‐64].

The number of patients and controls analyzed in this study is too small to allow for controlling potential confounding factors such as age, sex, smoking status, comorbidities, and therapies. The correlations between identified protein levels and changes in glycosylation need further investigations in larger cohorts. Nevertheless, our findings support a notion that inflammatory markers of different pathways when monitored simultaneously and combined with clinical data may help to improve prognosis and outcomes for COVID-19 patients.