Nasopharyngeal swab samples and serological tests are being routinely used in clinics to detect SARS-CoV-2 infection diagnosis; however, biomarkers for prognosis of the disease before it could lead to fatal symptoms are yet to be found. Understanding the host response towards the viral infection might provide important clues on the progression of the disease from non-severe to severity. A proteomics approach was applied for an in-depth understanding of the disease mechanism. Few studies have already reported differences in the level of blood-based proteins such as lactate dehydrogenase (LDH), d-dimers, and inflammatory markers such as C-reactive protein (CRP), ferritin, and fibrinogen in COVID-19 patients.19,20 One specific forte of our study is the in-depth profiling of plasma proteome from a cohort of COVID-19 patients (n = 73), facilitating the robust and statistically significant evaluation of differential expression between non-severe and severe disease groups. The Indian subcontinent being reasonably unscathed by the severity of the pandemic, reporting a few of the lowest case fatality rates per million, 21 our study holds importance in understanding the biology associated with the same.
Deep proteome comparison between COVID-19 positive and COVID-19 negative patients who exhibited symptoms similar to COVID-19 revealed a subset of proteins that differentiates COVID-19 from other febrile respiratory maladies. Of importance was the upregulation of the von Willebrand factor (VWF). Animal studies have shown that increased VWF might be due to hypoxic conditions in the lung endothelial cells22; however, this induces a risk of arterial or venous thrombosis since it directly promotes the thrombotic process during inflammation.23 The increase in Haptoglobin-related protein (HPR) is also found in cases of idiopathic pulmonary fibrosis24 and as a factor of non-bacterial pneumonia 25 thus may act as a biomarker of lung trauma. Carboxypeptidase B2 has anti-inflammatory and anti-fibrinolytic effects. Its increase in this cohort indicates the natural response to systemic inflammation brought about by COVID-19.26 Another protein Profilin-1 (PFN1) overexpression implicated in the vascular hyperpermeability, vascular Hypertrophy can perhaps explain the aberrant physiology of COVID-19 patients. These apart acute phase response proteins were like SAA-4 and S100A8 were also upregulated in response to COVID-19.
Interestingly, however, the protein Lymphatic vessel endothelial hyaluronic acid receptor 1 (LYVE1) was downregulated and might indicate liver injury 27. At the same time, attenuated Histidine-rich glycoprotein (HRG) expression might explain the altered hemostasis in the patients.28 However there lies a caveat, most of these patients were under medications the results might also be due to the ongoing therapies than the disease itself.
In our plasma deep proteome study of non-severe vs. severe patients, we observed that only 38 were differentially expressed proteins such as FGG, S100A8, VWF, SAA4, SERPIND1, and SERPINA6 to be upregulated in the COVID-19 positive patients while interestingly, the mitochondrial 60 kDa heat shock protein, HSPD1 was only expressed in the severe patients. It has been already reported that high levels of circulatory HSPD1 are associated with cardiac failures.29 Therefore increased HSPD1in severe patients can act as a clinical biomarker of cardiac malfunction in the severe group. Also, our results indicated an increase in plasma Cholinesterase (BCHE) in the severe group, which is upregulated in patients suffering from mild ischemic stroke.30 These findings thus implicate severe COVID-19 associated risk of cardiac and CNS injury that has been already reported by clinicians, and these biomarkers could help prognosis of the threat.
The plasma levels of carbonic anhydrase 1 (CA1) was found to be substantially elevated in the severe group. Increased carbonic anhydrase has been found to mediate hemorrhagic retinal and cerebral vascular permeability.31 The ramifications of increased CA1 are also substantiated by earlier reports on a cohort study of sepsis secondary to pneumonia32, where it was found to be upregulated during sepsis. Moreover, the role of increased CA1 in the worsening of ischemic diabetic cardiomyopathy also paints a rather gloomy picture of the cardiac sequelae of COVID-19, especially in diabetic patients33 and might also contribute to the increased fatality of diabetic patients.34
The protein Fibrinogen (FGG) was also found to be upregulated in severe patients when compared to non-severe. FGG is an oligomeric glycoprotein produced in the liver and secreted in the blood. The increased fibrin formation and breakdown correlated with the high level of D-dimers observed in the COVID-19 patients with the worst outcomes.35 The increasing level of FGG in severe might be due to liver injury, impairing hepatic fibrinogen secretion with acquired fibrinogen storage disease.36 The protein S100A8 (calgranulin A/myeloid-related protein 8) belongs to the group of alarmins or damage-associated molecular patterns (DAMPs), which are released in response to stress against the microbial infection that leads to exacerbating the inflammatory response. Chen et al. and his co-workers reported that the level of S100A8 positively correlated with the Ct value and oxygen demand, indicating the severity of the acute respiratory distress (ARDs) in COVID-19 patients.37 A recent study showed that severe COVID-19 patients release massive amounts of S100A8, which is accompanied by changes in monocytes and neutrophil subsets.38 The protein AGT was found to be significantly upregulated in severe patients as compared to the non-severe. Angiotensinogen (AGT) is a component of the renin-angiotensin system (RAS), a substrate of renin that regulates blood pressure and fluid balance. The dysregulation of AGT and RAS might lead to acute lung injury and acute respiratory distress leading to a severe prognosis.39 Apolipoprotein B-100 (APOB) is involved in lipid transport and low-density lipoprotein (LDL) catabolism. The high levels of APOB in the plasma might be an indicator of significant cardiovascular manifestation seen in COVID-19 infected severe patients.40These results are consistent with previous findings41 on COVID-19 patient sera, which had identified dysregulation of multiple apolipoproteins. Several serine protease inhibitors (SERPINs) such as SERPING1 and SERPINA3 were also identified to be upregulated in severe patients. The increasing level of SERPINs, an acute-phase protein, positively correlates and associates with a high level of IL-6 seen in severe patients.42 The validation study by MRM based assay could specifically detect AGT, FGG, APOB, SERPING1, and SERPINA3 host peptides in COVID-19 patients using. The mass spectrometry-based detection of host peptides used in our study can be used in the clinics for the prognosis of disease severity.
A subset of proteins was also downregulated in the severe group. The protein peptidase inhibitor 16 (PI16) was severely downregulated (FC= -2.45, P <0.005). It concurs well with the previous studies wherein it has been shown that while PI16 protective role against atherosclerosis, PI16 inhibition by circulating inflammatory cytokines that act through the NF-κB signaling pathway.43 These results demonstrate the pathogenesis of cardiac maladies in severe COVID-19 patients.44 Patients with severe COVID-19 often report lower platelet count.45 Our studies have demonstrated that a crucial factor in platelet biogenesis TPM4 46 is inhibited (FC-1.94, P < 0.001) in severe cases, thereby providing novel biological insight into COVID-19 severity. Another ubiquitously present protein βII spectrin was found to be downregulated in severe COVID-19, given that inadequate βII spectrin might precipitate into arrhythmia, heart failure, or even neurodegeneration 47 the findings hold much importance. Two proteins, namely APOM, which is known to protect the lungs and kidneys from injuries 48 and APOA2, were also downregulated; similar observations were previously reported.41
Functional enrichment analysis of the 38 differentially expressed proteins in severe vs. non-severe cohort revealed that these proteins are enriched in pathways related to blood coagulation, fibrin clot formation, complement system, leukocyte activation, regulation of peptidase activity, regulated exocytosis, and extracellular structure organization, among others. Proteins like A2M, SERPINA4, SERPINA3, SERPING1, and FGG that are involved in regulated exocytosis of platelets were upregulated in the severe cohort suggesting an increased consumption of platelets. This could be a possible reason for the lower platelet count (clinically called thrombocytopenia) commonly reported in many severe cases of COVID-19,49, which is also associated with coagulation abnormalities, disease severity, and mortality.50–52 There is enough evidence to suggest that platelets have potent immune and inflammatory effector functions aside from their role in hemostasis. Interaction between viruses and platelets has been known to stimulate platelet degranulation leading to the release of a variety of cytokines and chemokines.53,54 They also directly interact with leucocytes and endothelial cells to trigger and modulate inflammatory reactions and immune responses.54 Thus, platelet hyperactivity due to the upregulation of these proteins correlates with the over-exuberant host inflammatory response as COVID-19 progresses from non-severe to severe.
Many of the peptidase activity regulator proteins, including SERPINA4, SERPING1, SERPINA3, SERPIND1, and A2M, are involved in blood coagulation and inflammation pathways. SERPINA4 is an inhibitor of the kallikrein-kinin system involved in coagulation and inflammation.55,56 SERPING1 is an inhibitor of the classical pathway of the complement system as well as of several proteins involved in blood coagulation. 57 SERPIN A3 is a significant inhibitor of cathepsin G, a key proteolytic enzyme and inflammatory effector released by neutrophils. 58 A2M is an inhibitor of a variety of proteases involved in blood coagulation and inflammation including thrombin, kallikrein, plasmin, and cathepsin G;59 SERPIND1 regulates blood clot formation by inhibiting thrombin.60 Moreover, FGG or fibrinogen gamma chain is a component of the clotting factor fibrinogen, promoting tissue repair. High fibrinogen levels are associated with bleeding and thrombosis and correlate with the increased erythrocyte sedimentation rate (ESR) observed in severe cases.61,62 COVID-19 associated coagulopathy is common in severe patients, 63 while overt disseminated intravascular coagulopathy, a critical condition characterized by abnormal blood clotting and bleeding, is observed in most the critically ill patients who do not survive. 64,65 These thrombotic complications can be characterized by dysregulation of proteins involved in blood coagulation, fibrin clot formation, and platelet exocytosis. Conversely, these proteins can be associated with disease severity and mortality risk and can serve as biomarkers for a better prognosis.
Consistent with previous studies, multiple acute phase proteins (APPs) like APCS, C4B, A2M, SERPING1, SERPINA3, and FGG were upregulated in severe patients.41,66 APPs are manifested as the body's innate response to any kind of stress. Tissue damage caused by injury or infection instigates a local inflammatory response that leads to the release of pro-inflammatory cytokines. APPs are synthesized and released mainly by liver hepatocytes in response to these cytokines.67 Severe COVID-19 patients tend to have higher levels of pro-inflammatory cytokines68–70, which explains the elevated APP levels and the acute inflammatory state correlating with disease severity. The complement system is a significant contributor to the acute phase response against infection. C4B is a proteolytic product of complement factor C4 and is involved in the propagation of all the three complement pathways; 71APCS or serum amyloid P component (SAP) is an activator of the classical pathway of the complement system.72 Other proteins involved in the complement system that were downregulated in the severe cohort include CFI, C8A, CFD, CFP. Complement factor I (CFI) down-regulates the complement system by inhibiting complement C3b and C4b 73, while complement factor D (CFD) and properdin (CFP) play an essential role in the initiation and propagation of the alternate pathway in complement activation.74,75 Complement activation is the first line of defense against invading pathogens. However, unrestrained and prolonged complement activation can lead to fatal consequences associated with most of the severe COVID-19 cases.76–78This disrupted fine-tune between the regulatory complement proteins is consistent with the prolonged systemic complement activation observed in severe patients.
Multiple antiviral drug therapies, as well as clinical drug trials, are going on to come up with a definitive solution for this life-threatening viral infection. Current approaches of treating various stages of COVID-19 patients with commercially available drugs include two major categories; treatment with antiviral drugs and immune modulators. HIV protease inhibitors are quite famous as they belong to the former category, but still, no definitive studies have proven those drugs to be potent inhibitors; hence the quest has to be continued.79 In our study, we performed in silico drug re-purposing analysis with 9 proteins from our proteomic analysis against a library of 58 small molecules. The chosen drugs are previously found to target the protein-protein interactions happening between SARS-CoV-2 and human proteins in a cell line model.80 Two FDA-approved drugs, Selinexor and Ponatinib, were found to inhibit most of the proteins belonging to two different cohorts; COVID-19 positive vs. COVID-19 negative and non-severe Vs. Severe patients' plasma. Previously, Food and drug administration (FDA) had approved Selinexor for the treatment of multiple myeloma in combination with Dexamethasone.81 The drug is a first-class exportin-1 (XPO1) inhibitor that brings apoptosis in cancer cells by blocking nucleocytoplasmic transport of tumor suppressor proteins.82Although developed originally as anticancer-drugs, Exportin inhibitors can act as antiviral drugs as they have the potential to block the intracellular replication of viral particles by inhibiting the transport of viral replication proteins into the cytoplasm.83 Hence, the drug is currently under phase 2 clinical trial for COVID-19 infection (ClinicalTrials.gov Identifier: NCT04349098). Five plasma proteins from our study, including Thyroxine-binding globulin (SERPIN A7) and Heparin cofactor 2 (SERPIND1) belonging to the family of serine protease inhibitors (SERPINs) shown to interact tightly with Selinexor, suggesting these SERPINs could be a target for the drug. Both proteins are also seen to interact with another FDA-approved drug called ponatinib. Originally a tyrosine kinase inhibitor, Ponatinib is used to treat patients with chronic myeloid leukemia (CML).84It is currently under no clinical trial for COVID-19 patients but, recent studies in mice models showed it could suppress the cytokine storms from viral infections like influenza.85 Hence ponatinib, as an immune modulator, appears to be a suitable drug for making therapeutic cocktails against COVID-19 infection in the future.
A stitch in time saves nine. Perhaps no proverb holds true as this when it comes to managing the case fatality in COVID-19. While the COVID-19 pandemic has unleashed an unprecedented crisis on most lives and livelihood, it is the health workers who have borne the brunt of the pandemic. With limited availability of hospital infrastructure and overworked staff, it was pertinent to uncover factors leading to the severity of symptoms associated with COVID-19 to reduce the caseload in hospitals and manage severe cases at an accelerated pace. Further, the validation of these biomarkers can help clinicians in faster disease prognosis and better survival rates, especially in the vulnerable population, while selecting drugs based on the severity of infection would help better manage symptoms.