Thrombosis in COVID-19 is influenced by several complex pathophysiological mechanisms, which require further investigation. The analysis and discovery of complex genetic networks is highly effective when gene co-expression patterns are examined. As part of our research, RNAseq data derived from platelets obtained from eight patients with COVID-19 and ten healthy controls with matched sex and age were used for gene co-expression analysis. We identified DEGs from GSE176480 using the R package 'DESeq2' and WGCNA, then overlapped these genes and performed enrichment analysis. Finally, we identified 16 hub genes using multi-calculations based on five topological algorithms.
Enrichment analysis indicated that the overlapped genes were primarily associated with platelet activation, signaling and aggregation. Indeed, other studies showed the COVID-19 disease to be associated with platelet activation and increased platelet alpha granule secretion, which are critical in developing thrombosis in those patients 11,12. Previous research found enhanced p-selectin and CD36 expression, markers of platelet alpha and dense granule secretion respectively, in COVID-19 patients compared to healthy controls, and this phenomenon was more evident in patients admitted to ICU versus common patients 13. In accordance with this, Zaid et al. reported enhanced platelet activation in both severe and non-severe patients 14.
It is noteworthy that platelet activation is important not only for thrombus formation but also in inflammation 15. In our study, we found that these overlapped genes were involved in neutrophil degranulation and immune system. A number of granules are associated with bactericidal activity in neutrophils, including aspergillus granules, specific granules, and tertiary granules 16. The asplenophilic granules, specific to neutrophils, contain a large amount of myeloperoxidase, neutrophil elastase, proteinase E, and other bactericidal substances. By degranulating their contents, neutrophils kill pathogenic bacteria 17. In addition to the degranulation pathway, the interaction between platelets and leucocytes may also generate neutrophil extracellular traps (NETs), which consist of DNA and histones and are released when pattern recognition receptors or chemokines stimulate neutrophils 18. There was evidence that leukocyte-platelet interactions in patients with COVID-19 resulted in migration, secretion, degranulation, and formation of NETs, which up-regulated tissue factors, induced thrombosis, and altered thrombin receptor 1 to cause coagulation dysfunction and inflammatory responses 19,20. The autopsy of patients with COVID-19 also revealed the presence of platelets, neutrophils, and NETs in the lung and structures consistent with blood vessels, suggesting that the interaction between platelets and neutrophils could result in the formation of NETs and ultimately thrombosis 21.
Enrichment analysis showed that the VEGFA-VEGFR2 signaling pathway was important in the pathophysiology of thrombosis in COVID-19 patients, and these pathways were related to endothelial dysfunction. A growing body of evidence indicated that endothelial cells played an essential role in the transmission of severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)22. Previous studies collected postmortem data from nineteen COVID-19 patients and eleven age- and sex-matched autopsy controls without underlying lung diseases. They found up-regulation of VEGFR-2 expression in lung tissue and plasma samples of COVID-19 patients, which might be explained by compensatory angiogenesis 23. Our study is consistent with previous studies.
Bioinformatics analysis enables us to understand the molecular mechanisms of disease initiation and progression, providing a new and effective method for identifying potential gene biomarkers for assessing COVID-19 thrombosis. In this study, nine co-upregulated genes, RPLP0, RPS4X, RPL13A, RPL13, RPL10, TPT1, PSMA7, ATP5D and AKT1, and seven co-downregulated genes, HIST1H2AC, HIST1H2BH, H3F3B, KDM6A, GATA3, ITGAM and RBMX were identified from COVID-19 and thrombosis datasets. Among these hub genes, RPLP0, RPS4X, RPL13A, RPL13 and RPL10 belonged to the ribosomal protein family. It had been reported that ribosomes were present in circulating platelets, the cellular effectors of hemostasis and thrombosis 24–26. For example, the ribosomal protein RPS6 had been found in circulating platelets 27. The platelets promoted neutrophil extracellular trap formation, which led to deep vein thrombosis propagation by supporting the accumulation of innate immune cells 28. Another research conducted on a Saudi population with thrombosis showed that all patients detected mutations of RPL5 or RPL9. The mutations were represented by a novel variant of the ribosomal protein gene, which was correlated with thrombosis 29. Currently, fewer data exist regarding the relationship between these ribosomal protein family genes and thrombosis, so in view of their association with platelets, we suggest that DEGs of RPLP0, RPS4X, RPL13A, RPL13 and RPL10 may also be closely linked to thrombosis in COVID-19 patients.
Our study reported several hub genes related to thrombocytopenia, including HIST1H2AC, HIST1H2BH. HIST1H2AC and HIST1H2BH belong to the histone family. Histones are essential proteins in the chromatin and prokaryotic cells of eukaryotic organisms, which act as spools for DNA entanglement, form nucleosome structures together with DNA and regulate chromatin structure in eukaryotic cells. Histones have translation modifications, including methylation, acetylation, acylation, ubiquitination, phosphorylation, et al 30,31. In addition to changing chromosome structures, histone modification also affects gene expression. HIST1H2AC and HIST1H2BH have been studied primarily in tumors, with HIST1H2BH being used to predict cervical cancer survival outcomes 32. HIST1H2BH was also highly expressed in squamous cell carcinoma of the head and neck and strongly associated with patient survival 33. The relationship between these genes and thrombosis has been less studied. However, a recent bioinformatics study in patients with idiopathic thrombocytopenia before and after eltrombopag treatment found that HIST1H2BH and HIST1H4H genes were up-regulated in idiopathic thrombocytopenic purpura (ITP) patients after treatment 34. It may be suggested that the histone family played a role in platelet regeneration in COVID-19, but further studies are needed to determine the exact mechanisms.
Akt1 was the hub gene in our study, and Akt1 is one of the serine/threonine protein kinases called Akt kinase (Akt1, Akt2 and Akt3). The over-expressed Akt1 can significantly enhance virus protein synthesis and promote viral release 35. Recently, a study revealed crosstalk between Akt/mTOR/HIF-1 signaling pathway and SARS-CoV-2. Inhibition of the mTOR pathway using an Akt inhibitor showed a significant reduction in viral production. Furthermore, The Akt protein is also essential to the differentiation, proliferation, and migration of immune cells, which are involved in the development of systemic and local inflammation 36. Lucas et al. found increased phosphorylation of the kinase Akt and hyperactivation of the metabolic checkpoint kinase mTOR in T cells from patients who had a substantial deficiency in naive T cells and increased senescent effector T cells. Inhibition of mTOR activity partially improved T cell defects 37. In COVID-19 patients, there was also reduction and exhaustion of T cells. We suspected inhibition of the Akt pathway might improve immune response and prognosis during COVID-19. Our study also found Akt1 had a higher inference score with cardiovascular diseases especially acute myocardial infarction (AMI). It is well known that thrombosis contributes significantly to the development of AMI, as disruption of atherosclerotic plaques exposes flowing blood to substances such as subendothelial collagen, tissue factor, and other pro-coagulant molecules, which trigger platelet activation and fibrin synthesis within the vessel lumen 38. As a result of the above evidence, we conclude that the AKT1 gene may play a role in the development of thrombosis, and we need to explore this mechanism further.
There are some limitations to the present study. The first is that the analysis was limited to only one dataset as limited access to platelet gene expression data collected from COVID-19 patients was available. In order to validate our findings, additional experimental studies should be conducted. Furthermore, the number of samples was extremely small, which could lead to a degree of heterogeneity in the study.