The constructed networks presented in Fig. 2 provide noteworthy information on how diverse terms are closely interlinked within the context of thrombocytopenia induced by SARS-CoV-2 infection or through vaccination. The term thrombocytopenia appears with a rather high frequency in the COVID-19/VITT network (Fig. 2A). Similarly, the term VITT is included in the COVID-19/thrombocytopenia network (Fig. 2B). COVID-19 and VITT share several comorbidities implicating vascular and epithelial dysfunction and thrombocytopenia. The nodes represent the top 50 words with a cosine similarity score of each word vector >0.9.

Subsequently, two interactomes were constructed: The first one involving 119 nodes is described in Table I and illustrated in Fig. 3. Collectively, 119 nodes are involved in COVID-19, while 57 are implicated in thrombocytopenia [the latter profits from an unpublished work of ours (unpublished data). Of these, 14 nodes were common in both entities (Figs. 3 and 4), namely AIM2, IFI16, NOD2, CD8A, IL-1B, 1L-6, JAK2, NCAM1, HLA-DRB1, SERPINE1, TGFB1, TLR2, TNF and VWF. The major hubs detected are displayed in the center of the constructed circular network, while the less connected nodes are shown at the periphery of the circle (Fig. 3). The thrombocytopenia-related nodes are represented in square bullets, and the COVID-19-related ones are presented in circles, whilst the common nodes are depicted in rhomboids. The calculated average node degree of the entire interactome was extremely high (11.9).

The second one including 61 molecules, is described in Table I and illustrated in Fig. 5. Of these, 47 are common with thrombocytopenia (indicated by a polygon), and 16 with COVID-19 (represented by circles). The VITT-related molecules are denoted with triangles.

Venn diagrams were further created to illustrate the nodes that are common between thrombocytopenia and COVID-19 or VITT (Fig. 4A and B, respectively), between COVID-19 and VITT (Fig. 4C), and amid the three morbid entities (Fig. 4D). The common nodes are listed in each diagram in detail.

All included molecules herein are listed in Table I. The figure (network) in which each molecule is implicated is also noted in a separate column in Table I.

Ariadne's thread appears to be the angiotensin I converting enzyme 2 (ACE2), which clearly plays a crucial role. SARS-CoV-2, via its spike S protein, a surface glycoprotein that surrounds the spherical virus, is attached to ACE2 and this is followed by entry into cells of the host (22-27). ACE2 is expressed in cells of a number of human organs (including the skin, nasal and oral mucosa, lung, nasopharynx, brain, lymph nodes, thymus, stomach, small intestine, colon, bone marrow, spleen, liver and kidneys). Additionally, its expression in lung alveoli (type 2 pneumonocytes) and small intestine endothelium, as well as in the arterial and other tissue smooth muscle epithelium (28), may trigger the release of anaphylatoxin (29). There is clinical evidence to confirm the aforementioned knowledge of COVID-19 (29).

In the generated network illustrated in Fig. 2, ACE2 interacts with CYP11B2 and with IL-6. The latter is the greatest hub in this vastly interconnected network, with 63 interactions, confirming that the progress of SARS-CoV-2-induced infection would profit from therapeutic blockade of IL-6. As noted by Mazzoni et al (24), blocking this mechanism would 'suppress noxious systemic inflammation but also restore the protective antiviral potential'. It has been established that innate immunity via natural killer (NK) cells exerts the frontline defense, with CD8⁺ T-lymphocytes being important for the long-term surveillance against viruses, while adaptive immune responses play a key role in the control of viral infections (28). Both responses are mediated either via cytotoxicity or by IF-γ, IL-12 and IL-18. Virus-induced cytotoxicity is primarily moderated by perforin and granzymes. Increased severity in viral infections may lead to dysregulated immunity and tissue/organ damage (30). Clinical evidence in SARS-CoV-2 infection has demonstrated that high IL-6 levels in patients in intensive care units, are inversely associated with the concentration of NK cells (24,31).

The network included dense interactions illustrating clearly that SARS-CoV-2-specific T-cells are critical for the extended damage caused by the 'cytokine storm' (or 'cytokine release syndrome') (30,32) (Fig. 3). This excessive inflammatory response may be lethal for some patients (29,33). Although the phenomenon may manifest in other inflammatory conditions, including bacterial sepsis, pneumonia, sterile inflammation, etc., the extent in the secretion of several specific cytokines is different in COVID-19-related storm (29). Of note, COVID-19 infection has been associated with changes in the blood coagulation mechanisms, with differing manifestations in different patients, in distinct phases of the disease, and independently of disease severity.

Autoimmune destruction of platelets, cytokine release and high consumption of coagulation factors and platelets have been observed in patients with SARS-CoV-2 infection (Geronikolou et al, unpublished data) and initial hypercoagulability (34). Thromboembolic events increase by 31% in patients with COVID-19 admitted in intensive care units (35,36); the phenomenon may be interpreted by the 'two way activation theory' (20,37), i.e., thrombogenesis via inflammation-relevant pathways, with parallel occurrence of release of VWF large polymers. The coagulation and platelet profiles of patients with COVID-19 are then rather normal, unlike in patients with sepsis where platelets are activated and consumed, with the occurrence of thrombocytopenia (38). Only a few patients may then survive, particularly of those with extensive disseminated intravascular coagulation (38). Thrombosis has been observed in situ in the lungs, as well as in a systemic manner, in a similar fashion with classic sepsis and acute respiratory distress syndrome. Reported thromboembolic complications include mostly venous pulmonary embolism (38), aortic graft thrombosis, and mesenteric ischemia; coronary and cerebral thrombosis cases have been reported, although these are rare. The so-called 'COVID toe' is a sign of thrombosis accompanied by arterial and venous clots, urgent oxygen demand and multiple organ dysfunction (20,36,39).

COVID-19 and thrombocytopenia interactomes share only 14 nodes (AIM2, IFI16, TLR2, NOD2, NKAM1, IL-6, TNF, JAK2, IL-1B, SERPINE1, HLA-DRB1, TGFB1, CD8A, and VWF) (Fig. 3), most of which serve as major hubs (IL-6, TNF, JAK2, IL-1B, SERPINE1, TGFB1, CD8A and VWF) in the herein presented interactome (Figs. 1 and 2).

Cytokines, such as IL-1B, 1L-6 and TNF contribute to the so-called cytokine storm, as aforementioned. Moreover, JAK2 is a kinase suspected to be implicated in thrombocytopenia via reduced levels of thrombopoietin or via decreased expression levels of their cognate receptors (cMpl receptors). JAK2 mutations (V617F) that are present in the majority of patients with myeloproliferative disease, may increase hematopoietic cell sensitivity to erythropoietin and thrombopoietin. NKAM1 or CD56 is a homophilic binding glycoprotein expressed on the surface of neurons, glia cells and skeletal muscles. NKAM1 is a prototypic marker of NK cells, also present in CD8⁺ T-cells. These cell types exhibit diminished antiviral ability and cytotoxic impairment during COVID-19 infection (24). CD8A1 is a cytotoxic marker for T-cell populations. SERPINE1 or plasminogen activator inhibitor-1 is a protein encoded by the SERPINE1 gene, which participates in both thrombosis and atherogenesis (40).

TGFB1 is a multifunctional peptide, with diverse activities, including the control of cell growth, proliferation, differentiation, and apoptosis. It can also down-regulate the activity of immune cells via decreasing the expression levels of cytokine receptors, such as that of IL-2. Several types of T-cells secrete TGFB1, so as to inhibit cytotoxicity and the secretion of certain cytokines, such as interferon-γ, TNF-α and various interleukins, such as IL-6. This makes this molecule a potential target of therapeutic value. On the other hand, the hemostatic VWF is detected in blood plasma, endothelium and megakaryocytes, as well as in subendothelial connective tissue. This factor appears to be also increased and implicated in autoimmune diseases, such as thrombotic thrombocytopenic purpura, as well as in stroke and atrial fibrillation, due to the platelet clots that are potentially formed when its levels are elevated.

Recent literature has further revealed that an HLA class I and II molecule, that is, HLA-DRB1, which is common in COVID-19 and in thrombocytopenia networks (Fig. 2), may play a role in the observed COVID-19 individual and ethnic diversity in clinical severity and/or response to therapy or vaccination (41-44). Of note, HLA-DRB1 is interconnected with the lymphocyte function markers CD3D, CD3E, CD3G, CD4, lymphocyte regulation positive FCGR1A, FCGR1B, HLA class I and II molecules, such as HLA-A, HLA-B, similar to the NCAM1, PTPN1, SHC1 and VCAM1 molecules that have been implicated in thrombosis and atherosclerosis. NCAM1 is involved in cell-cell adhesion in neural-muscle cells in the embryonic phase and later, and more notably, in the responsiveness to viral infections (rabies virus and papilloma virus) (45). PTPN1 is a potential therapeutic target of obesity and type 2 diabetes mellitus as well (46); SHC1 is implicated in reactive oxygen species regulation, thus, in the oxidative stress response (47), while VCAM1 is directly involved in thrombosis and atherogenesis and acute respiratory syndrome (48-51).

Various coagulation mechanisms have been implicated in VITT: High levels of D-dimers and low levels of fibrinogen have been observed in patients (2,52,53). On the other hand, early reports of VITT described a higher incidence of the syndrome in young women, exhibiting both age-dependence and sexual dimorphism. VITT, though very rare, is of utmost importance. Yet, in March, 2021, the European Medicines Agency (EMA) issued a statement noting that the thromboembolic events of VITT in vaccinated populations were not higher than in general population (54). Subsequently, the 'risk vs. benefit' equilibrium was weighed by the World Health Organization (WHO), promoting the benefit of the vaccination vs. the extremely low risk of thromboembolic risk of VITT in the general population (55).

VITT is currently termed 'thrombosis with thrombocytopenia syndrome (TTS)' by the Centers for Disease Control and Prevention (CDC) and the US Food and Drug Administration (FDA) (56), and is characterized by arterial and venous thrombosis at unexpected sites (i.e., cerebral venous sinuses, splanchnic vessels of variant severity and/or positive platelet factor (PF) 4-heparin ELISA ('HIT' ELISA) syndrome (52), exhibiting both age dependence and sexual dimorphism (more frequent in individuals <50 years old and of the female sex) (2). The laboratory and clinical features of this syndrome are similar to those of the heparin-induced thrombocytopenia (HIT) syndrome and/or the HIT-like autoimmune thrombosis with thrombocytopenia syndrome (2,52,53), both of which have already been reported following surgery, the uptake of certain pharmaceuticals, or during some infections in patients that are not being treated with heparin. The therapeutic suggestions of this recently coined syndrome include early initiation of non-heparin anticoagulation, high-dose IVIG, and/or prednisolone (57).

The genetic basis of the VITT syndrome appears to be closely intertwined with that of the COVID-19 disease and, as such, they share 16 nodes: CASP1, CXCL8, FGF7, HLA-A, HLA-B, IL1B, IL6, MS4A1, NFATC1, NFKB1, NLP3, P2RX7, PYCARD, TNF, TFP1, VWF (Figs. 3-5). The purpose of the vaccine is to inhibit pathways that mediate this condition (52,58). More importantly, the relevant research is ongoing with the extremely rare cases of this syndrome, as VITT incidence is ~0.74-1 cases per 100,000 vaccinated subjects (52). Of note, the anti-COVID-19 vaccines do not cause illness and the two morbid entities (COVID-19 and VITT) are by no means identical, with the etiopathology of the latter being actually autoimmune, with auto-antibodies against platelet factor 4. More explicitly, COVID-19 network shares 14 nodes with thrombocytopenia (AIM2, CD8A, HLA-DRB1, IFI16, IL1B, IL6, JAK2, NCAM1, NOD2, SERPINE1, TGFB1, TLR2, TNF and VWF), while VITT (which is a type of thrombocytopenia) shares 46 nodes with thrombocytopenia (Figs. 3-5). Notably, SHC1, STXBP2, CDC20 and ADAM10 are silenced in VITT, while AURKA, CD46, CD19 are uniquely expressed following vaccination (apparently not expressed in common thrombocytopenia or in COVID-19) (Figs. 3-5). These molecules were not previously identified as VITT-related and are, thus, a novel finding, at least to the best of our knowledge.

It is known that the NLP3 inflammasome is implicated in both COVID-19 and VITT, apart from its participation in other inflammatory reactions (59). It has also been previously demonstrated that acute thrombotic events may manifest during hypoxia, as shown in COVID-19, due to an early proinflammatory state in the venous milieu, mediated by a HIF-induced NLP3 inflammasome complex (60,61). In the network constructed in the present study, NLP3 connects with CASP1, IL-IB, IL17A, CXCL8, IL-6, MYD88, NFKB1, P2RX7, PYCARD and TNF.

P2RX7 exhibits sexually dimorphic and contrasting roles in the pathogenesis of thrombosis, depending on the pathogen type, the severity of infection, the cell type infected and the level of tissue activation (62). In the thrombocytopenia/ COVID-19/VITT cases, the viral load, the cell-type infected and the infecting virus strain or certain vaccine types have been associated with NLP3 hyperactivation, which in the presence of comorbidities, such as liver, renal, gut or respiratory tract illnesses, diabetes mellitus, previous infections, exposure to pollutants, and/or lifestyle factors, such as smoking and obesity, may upend the roles of P2RX7 and PYCARD to those of tissue-damaging, or even lethal factors (62,63). More importantly, the persistent neurological effects ('long-COVID-19') observed in a large percentage of patients with COVID-19 may be explained via the activation of these pathways. Thus, P2RX7 antagonists may be promising therapeutics in the management of both VITT and 'long-COVID-19' (62,64), as P2RX7 receptor stimulation has been implicated in lung damage, psychiatric disorders and pathological inflammation (65,66). In the COVID-19 interactome, P2RX7 directly interacts with NLP3, CASP1 and P2RX1. On the contrary, in the VITT network, P2RX7 directly interacts only with NLP3, IL1B and CASP1. Accordingly, PYCARD interacts with NLP3, CASP1, IL1B, IL18 and IKBKG in COVID-19, and with NLP3, CASP1 and IL1B in the VITT syndrome (Table II). The common node in all possible combinations, as shown in Table II, is CASP1, a downstream event of the NLP3 inflammasome; CASP1 activation promotes IL1B production, which may be prevented by a pan-caspase inhibitor or by glyburide treatment (67).

To this end, the present study investigated the aforementioned issues through the construction of molecular networks and the detection of at least one known COVID/VITT/thrombocytopenia molecule that confirmed that endothelial dysfunction and blood thrombosis are the key players of both COVID-19 and VITT morbid entities. One limitation of the present study is that it included only wild-type genes and their products. To the best of our knowledge, however, this is the first effort made at providing a comprehensive network map of the molecules involved in the underlying mechanisms of COVID-19, long COVID-19 and/or VITT pathophysiology.

In conclusion, the interactomes presented herein revealed therapeutic and vaccination modification targets (i.e., SHC1, NCAM1, HLAs, CD8A, PTPN1, VWF and TBP1). It was also demonstrated that: i) NCAM1 is involved in SARS-CoV-2 infection responsiveness, apart from papilloma and rabies virus infections, and may be responsible for relevant vaccination side effects; ii) NLP3, P2RX7 and PYCARD contribution may help explain (partly or mostly) VITT and/or 'long COVID-19 side-effects'; iii) furthermore, the antagonism of these latter nodes should focus on potential pharmacological targets in the context of SARS-CoV-2 infection and/or vaccine immunization responsiveness. In conclusion, network construction is a powerful tool, which may be used to elucidate the physiology and pathophysiology of different states in clinical investigation. The highly interconnected network presented herein highlights the complexity of COVID-19/VITT pathophysiology, mapping the key role of cytokines, enzymes and immune response markers (lymphocyte regulators and human leucocyte antigens) that may be potential drug or vaccine targets. It was constructed using wild-type genes and gene products, revealing the body's predisposition to COVID-19 infection or VITT. Of note, the COVID-19 and thrombocytopenia common nodes appear to be key players in the natural history of the illness.