key: cord-0653946-wgkmoyqg
authors: Kircheis, Ralf
title: Coagulapathies after vaccination against SARS-CoV-2 may be derived from a combination effect of SARS-CoV-2 spike protein and adenovirus vector-triggered signaling pathways
date: 2021-08-31
journal: nan
DOI: nan
sha: 7fc061a487f1abef61b3d794693966bd699e8582
doc_id: 653946
cord_uid: wgkmoyqg

The novel coronavirus SARS-CoV-2 has resulted in a global pandemic with worldwide 6-digital infection rates and thousands death tolls daily. Enormeous effords are undertaken to achieve high coverage of immunization in order to reach herd immunity to stop spreading of SARS-CoV-2 infection. Several SARS-CoV-2 vaccines, based either on mRNA, viral vectors, or inactivated SARS-CoV-2 virus have been approved and are being applied worldwide. However, recently increased numbers of normally very rare types of thromboses associated with thrombocytopenia have been reported in particular in the context of the adenoviral vector vaccine ChAdOx1 nCoV-19 from Astra Zeneca. While statistical prevalence of these side effects seem to correlate with this particular vaccine type, i.e. adenonoviral vector based vaccines, the exact molecular mechanisms are still not clear. The present review summarizes current data and hypotheses for molecular and cellular mechanisms into one integrated hypothesis indicating that coagulopathies, including thromboses, thrombocytopenia and other related side effects are correlated to an interplay of the two components in the vaccine, i.e. the spike antigen and the adenoviral vector, with the innate and immune system which under certain circumstances can imitate the picture of a limited COVID-19 pathological picture.

The novel coronavi rus SARS-CoV-2 (severe acute respi ratory syndrome coronavi rus 2) fi rst reported in Wuhan, China, at the end of 2019 has developed into the heaviest global pandemic since the Spanish flu from 1918-1920, wi th worldwide more than 212 million infected persons and more than 4,4 million deaths by August 23rd, 2021, and 6-digi t infection rates daily. show good safety profiles. A panel of typical side effects, including pain at the injection si te, fever, chills, fatique, and muscle pain have been reported, but no signi ficant number of severe side effects has been reported from clinical studies (1) (2) (3) (4) (5) .

However, recently reports of various types of venous thrombosis, and in particular of normally very rare cerebral venous sinus thrombosis (CVST) in a timely correlation to vaccination against SARS-CoV -2 wi th ChAdOx1 nCoV-19 have raised safety concerns. A variety of vaccine-associated thrombotic events including cerebral venous thrombosis, splanchnic-vein thrombosis, pulmonary embolism, and other thromboses, as well as disseminated intravascular coagulation have been reported in a time frame between a few days up to three weeks days after ChAdOx1 nCoV-19 vaccination against SARS-CoV-2 (6) (7) (8) .

Among the various vaccine-associated thrombotic events special at tension has been focussed on the normally very rare cerebral venous sinus thrombosis (CVST) in combination wi th pronounced thrombocytopenia, wi th most of these patients showing high levels of antibodies to platelet factor 4-polyanion complexes; wi thout previous exposure to heparin pointing to a rare vaccine-related variant of spontaneous heparin-induced thrombocytopenia refered to as vaccine-induced immune thrombotic thrombocytopenia (VITT) (6) (7) (8) .

A recent ret rospective survey which estimated the incidence of CVST and other cerebrovascular events in temporal relation to COVID-19 vaccination wi th BNT162b2, ChAdOx1 nCoV-19, and mRNA-1273 in Germany showed an at least 10-fold higher CVST incidence rate in patients who received a fi rst ChAdOx1 nCoV-19 vaccine shot compared wi th the highest estimate of CVST incidence rate from empi rical data. Furthermore, an almost 10 fold higher risk for CVST following vaccination wi th ChAdOx1 nCov-19 compared to mRNA-based vaccines (9) together wi th recent reports on individuals who developed CVST wi th a severe thrombopenia wi thin two weeks after immunization wi th Ad26.COV2.S (10, 11) suggest that VITT-associated thrombotic events may be associated wi th adenovi rus vector-based vaccines coding for the SARS-CoV-2 spike protein indicating that the underlying mechanism relies on -or at least includes -the adenovi ral vectors used in this vaccine type.

The underlying mechanism of action of these thrombotic events after adenovi ral vector-based SARS-CoV-2 vaccines is still unknown. The present review summarizes the published data related to thrombotic events and di fferent hypotheses for the VITT associated thrombotic events, and presents an integrated model indicating that both, SARS-CoV-2 spike protein and adenovi rus vector can t rigger signaling pathways which individually -and at a higher probabili ty in combination -may t rigger thromboses and thrombocytopenia following vaccination. SARS-CoV-2 and COVID-19the virus and the disease SARS-CoV-2 belongs to enveloped posi tive-sense, single-st randed RNA vi ruses, similar to the two other highly pathogenic coronavi ruses, SARS-CoV and Middle East Respiratory Syndrome (MERS-CoV) (12) . SARS-CoV-2 binds to the angiotensin-converting enzyme-related carboxypeptidase-2 (ACE-2) receptor on the target cells by i ts spike (S) protein. The spike (S) protein is composed of the S1 subuni t containing the highly conserved receptor binding domain (RBD) and the S2 subuni t, which mediates fusion between the vi ral and host cell membranes after cleavage by the cellular serine protease TMPRSS2 (13) . This furin-like cleavage si te is unique to the S protein of SARS-CoV-2 and may together wi th the particularly high binding affinity to the target receptor and the peculiari ty of a long symptom-f ree but nevertheless highly infectious time period between infection and appearance of fi rst symptoms or asymptomatic t ransmission be responsible for the particularly efficient spread of SARS-CoV-2 compared to previous pathogenic hCoVs (14) . The ACE-2 receptor is widely expressed in pulmonary and cardiovascular tissues and hematopoietic cells, including monocytes and macrophages, which may explain the broad range of pulmonary and extrapulmonary effects of SARS-CoV-2 infection, including cardiac, gast rointestinal organs, and kidney affection (13) (14) (15) .

The majori ty of individuals infected wi th SARS-CoV-2 show mild-to moderate symptoms, and up to 20% of infections may be asymptomatic. Symptomatic patients show a wide spec trum of clinical mani festations ranging from mild febrile illness and cough up to acute respi ratory dist ress syndrome (ARDS), mul tiple organ failure, and death. Thus, the clinical picture of severe cases is very similar to that seen in SARS-CoV-and MERS-CoV-infected patients (12) . While younger individuals show predominantly mild-to-moderate clinical symptoms, elderly individuals frequently exhibi t severe clinical mani festations (17) (18) (19) (20) (21) (22) (23) . Pre-existing comorbidi ties, including diabetes, respi ratory and cardiovascular diseases, renal failure and sepsis, higher age and male sex seem to be associated wi th more severe disease and higher mortali ty (20) (21) (22) (23) (24) . Postmortem analysis of fatal COVID-19 showed di ffuse alveolar disease wi th capillary congestion, cell necrosis, intersti tial edema, platelet-fibrin thrombi, and infil t rates of macrophages and lymphocytes (25) . Furthermore, induction of endotheliitis in various organs (including lungs, heart, kidney and intestine) by SARS-CoV-2 infection as a di rect consequence of vi ral involvement and of the host inflammatory response has been demonst rated (15, 16) .

The molecular mechanisms for the morbidi ty and mortali ty of SARS-CoV-2 are still incompletely understood. Vi rus-induced cytopathic effects and vi ral evasion of the host immune response, in particular the inhibi tion of the host IFN type I response by the SARS-CoV-2 (26), seem to play a role in disease severi ty. Furthermore, clinical data from patients, in particular those wi th severe clinical mani festations, indicate that highly dysregulated exuberant inflammatory and immune responses correlate wi th the severi ty of disease and lethali ty (15, 16, 18, (27) (28) (29) . Signi ficantly elevated cytokine and chemokine levels, also termed "cytokine storm", are assumed to play a cent ral role in severi ty and lethali ty in SARS-CoV-2 infections . Elevated plasma levels of IL-1b, IL-7, IL-8, IL-9, IL-10, G-CSF,   GM-CSF, IFNg, IP-10, MCP-1, MIP-1a, MIP-1b, PDGF, TNFa, and VEGF have been reported in both ICU (intensive care uni t) patients and non-ICU patients. Notably, signi ficantly higher plasma levels of IL-2, IL-7, IL-10, G-CSF, IP-10, MCP-1, MIP-1a, and TNFa were found in patients wi th severe pneumonia developing ARDS and requiring ICU admission and oxygen therapy compared to non-ICU patients showing pneumonia wi thout ADRS (18) . Various studies have shown that highly stimulated epi thelialimmune cell interactions lead to exuberant dysregulated inflammatory responses wi th signi ficantly (topically and systemically) elevated cytokine and chemokine release (30, 31) .

Regarding the underlying signaling pathways, recent data suggest that the NF-kB pathway is one of the cent ral signaling pathways for the SARS-CoV-2 infection-induced proinflammatory cytokine/ chemokine response, playing a cent ral role in the severi ty and lethali ty of COVID-19 (32) (33) (34) (35) (36) (37) (38) . This NF-kB-t riggered proinflammatory response in acute COVID-19 is shared wi th other acute respi ratory vi ral infections caused by highly pathogenic influenza A vi rus of H1N1 (e.g. Spanish flu) and H5N1 (avian flu) origin, SARS-CoV and MERS-CoV (38) .

Excessive activation of exuberant inflammatory responses wi th involvement of endothelial cells, epi thelial cells and immune cells are assumed to lead to further disturbances in a variety of other integrated systems, including the complement system, coagulation, and bradikinine systems, leading to increased coagulopathies and feeding back into posi tive signaling feedback loops accelerating COVID-19-associated inflammatory processes (39) (40) (41) (42) (43) (44) . In particular, vascular occlusion by neutrophil extracellular t raps (NET) and disturbances of coagulation wi th various types of thromboses and mul tiple micro thromboses seem to be another hallmark of COVID-19 disease and development of coagulopathies is one of the key and persistent features associated wi th poor outcome. In particular elevated D-dimer levels, prolonged prothrombin time, and thrombocytopenia, together wi th low fibrinogen (indicating fibrinogen consumption) have been found as prognostic indicators for poor outcome (45) (46) (47) (48) (49) . Lung histopathology often reveals fibrin-based blockages in the small blood vessels of patients who succumb to COVID-19 (25) .

Furthermore, various types of antiphospholipid (aPL) antibodies targeting phospholipids and phospholipid-binding proteins including anti-cardiolipin IgG, IgM, and IgA; anti-β2 glycoprotein I IgG, IgM, and IgA; and anti-phosphatidylserine/prothrombin (aPS/PT) IgG and IgM were found in 52% of serum samples from 172 patients hospitalized wi th COVID-19. Higher titers of aPL antibodies were associated wi th neutrophil hyperactivi ty, including the release of neutrophil extracellular t raps (NETs), higher platelet counts, more severe respi ratory disease, and lower clinical estimated glomerular fil tration rate. Similar to IgG from patients wi th anti-phospholipid syndrome, IgG fractions isolated from patients wi th COVID-19 promoted NET release from neutrophils isolated from heal thy individuals. Furthermore, injection of IgG puri fied from COVID-19 patient serum into mice accelerated venous thrombosis in two mouse models. These findings suggest that hal f of patients hospitalized wi th COVID-19 become at least t ransiently posi tive for aPL antibodies and that these auto-antibodies are potentially pathogenic (50) . High rates of thrombosis and thrombotic-related complications have been reported in adul t patients wi th severe COVID-19 as well as in children developing COVID-19 or mul tisystem inflammatory syndrome (MIS-C). Studies in adul ts have invoked thrombotic micro angiopathy (TMA) as a potential cause for severe mani festations of COVID-19 (51) (52) (53) . TMA resul ts from endothelial cell damage to small blood vessels, leading to hemolytic anemia, thrombocytopenia, and, in some cases, organ damage (54) (55) (56) (57) (58) . TMA has been reported in postmortem studies of adul t patients wi th COVID-19 (59) . Regarding therapeutic intervension, a ret rospective analysis examined the association of in-hospi tal anticoagulation procedures wi th mortali ty, intubation, and major bleeding. In-hospital anticoagulation was associated wi th lower mortali ty and intubation among hospitalized COVID-19 patients (60).

Frequently decribed side effects after vaccination against SARS-CoV-2 Before certi fication by the regulatory authori ties, i.e. FDA and EMA respectively, the vaccines had been tested in thousands of volunteers in large clinical t rials (1) (2) (3) (4) (5) 61) . A panel of typical side effects have been reported, such as short-term, mild-to-moderate pain, redness, and swelling at the injection si te and systemic flu-like symptoms including fatigue, headache, muscle pain, chills, joint pains, and fever shown at varying degrees for all vaccines. There were no major di fferences between the mRNA vaccine and the adenovi ral vector vaccines, wi th one exception, i.e. that the mRNA vaccine (i.e. BNT162b2 and mRNA-1273) showed more pronounced side effects after the second immunization and whereas the adenovi ral vector vaccines showed more pronounced side effects after the fi rst immunization, wi th lower intensity/prevalence of side effects after the second vaccination for ChAdOx1 nCoV-19 vaccine (AZD1222). Furthermore, younger individuals (<55 hears) showed genereally a higher incidence and intensity of side effects compared to aged persons (>55 years) reported for both types of vaccines, mRNA and adenovi ral vector-based.

Importantly, no signi ficant increase in prevalence of thrombotic events has been reported during the clinical studies, wi th large Phase 3 t rials tested in more 30.000 -40.000 volunteers the various vaccines (1-6, 61).

Following mass vaccination after market approval recent reports of cerebral venous sinus thrombosis (CVST) and a variety of other thrombotic events after ChAdOx1 vaccination against SARS-CoV-2 have raised safety concerns. vaccine and 1.3 per 100,000 person-for BNT162b2. Before the COVID-19 pandemic, the incidence rate of CVST has been estimated between 0.22 -1.75 per 100,000 person-years in four European count ries, Australia, Iran and Hong Kong. Accordingly, a 10 to 90 fold higher CVST incidence rate in patients who received a fi rst ChAdOx1 nCov-19 vaccine shot compared wi th the highest or lowest estimate of CVT incidence rate from empi rical data, respectively. The incidence rate of a CVST event after fi rst dose COVID-19 vaccination was also statistically signi ficantly increased for ChAdOx1 nCov-19 compared to mRNA-based vaccines (9.68, 3.46 to 34.98) and for females compared to nonfemales(3.14, 1.22 to 10.65) (9). The 10 fold higher risk for CVST following vaccination wi th ChAdOx1 nCov-19 compared to mRNA-based vaccines together wi th recent reports on individuals who developed CVST wi th a severe thrombopenia wi thin two weeks after immunization wi th Ad26.COV2.S (10, 11, 62) suggests that VITT-associated thrombotic events may be associated wi th adenovi rus vector-based vaccines coding for the SARS-CoV-2 spike protein indicating that the mechanism of action relies or at least includes the adenovi ral vector used in this vaccine. The underlying mechanism of action of these thrombotic events after adenovi ral vector-based SARS-CoC-2 vaccines is still not completely known, al though various data and hypotheses have been raised (see below).

Beside vaccine-associated thrombotic events several addi tional serious condi tions have been reported in association wi th vaccination against SARS-CoV-2, including single cases of capillary leakage syndrome and of coronary myocardi tis after immunization wi th the ChAdOx1 nCov-19 (AZD1222) (Ast raZeneca) and BNT162b2 (BioNTech/Pfizer), respectively.

In order to elucidate the underlying mechanisms of various rare side effects following anti SARS-CoV-2 vaccination, one particular disease pattern appearing in a timely context to SARS-CoV-2 pandemics may be of interest. During the COVID-19 pandemic a new deadly disease in children named mul tisystem inflammatory syndrome in children (MIS-C) has got much at tension, which rapidly progresses to hyperinflammation, shock and can lead to mul tiple organ failure in a high percentage of affected children. MIS-C has been found temporally associated wi th COVID-19 pandemic wi th a few weeks delay following peaks in SARS-CoV-2 infection incidence and was found often associated wi th SARS-CoV-2 exposure or presence of SARS-CoV-2 reactive antibodies in affected children. After initial reports in the UK, an increasing number of cases has been reported in Europe and New York, a few weeks after epidemic peaks, respectively. MIS-C mani fests as persistent high fever, hyperinflammation wi th mul tiorgan system involvement including cardiac, gast rointestinal, renal, hematologic, dermatologic, and neurologic symptoms. The overall clinical picture of MIS-C, however, is often similar in many aspec ts to the late, severe CODIV-19 phase in adul ts, characterized by cytokine storm, hyperinflammtion, and mul tiorgan damage, severe myocardi tis and acute kidney injury (63) . A causal link between SARS-CoV-2 infection and MIS-C has not yet been fi rmly established; however, many patients wi th MIS-C were reportedly exposed to someone known or suspected to have COVID-19. Furthermore, only around a thi rd of patients wi th MIS-C were tested posi tive for SARS-CoV-2 by PCR, but a large majori ty of PCR-negative were posi tive serologically for SARS-CoV-2 antibodies and/or had a history of mild COVID-19 infection or exposure several weeks before presentation. Such timing suggests that MIS-C is a postinfectious disease or an immune or autoimmune disease t riggered by SARS-CoV-2 infection. Although initially MIS-C was seen to resemble Kawasaki diseases (KD) clinical, and laboratory characteristics indicate that MIS-C is rather reminiscent to the toxic shock syndrome (TSS) usually found in severe cases after sepsis with Grampositive bacteria such as Staphylococcus aureus or Streptococcus pyogenes as indicated by typical gast rointenstinal involvement, myocardial disfunction and cardiovascular shock, pronounced lymphopenia and thrombocytopenia, and high coagulation parameters, such as D-dimersfound in MIS-C and TSS, but typically not in KD (63) . Notably, TSS is known to be caused by di fferent types of superantigens (SAgs) including bacterial and vi ral, wi th the bacterial SAgs being broadly studied. They include proteins secreted by Staphylococcus aureus and Streptococcus pyogenes that stimulate massive production of inflammatory cytokines and toxic shock. Typical examples are toxic shock syndrome (TSS) toxin 1, and staphylococcal enterotoxins B (SEB) and H (SEH). They are highly potent T cell activators that can bind to major histocompatibili ty complex (MHC) class II (MHCII) molecules and/or di rectly to T cell receptors (TCRs) of both CD4+ and CD8+ T cells. The abili ty of SAgs to bypass the antigen specifici ty of the TCRs resul ts in broad activation of T cells and a cytokine storm, leading to toxic shock. Notably, SAgs do not bind the major (antigenic) peptide-binding groove of MHCII, but instead bind other regions or the αβTCRs, di rectly (63) .

Importantly, using st ructure-based computational models, Cheng et al demonstrated that the SARS-CoV-2 spike (S) glycoprotein exhibi ts a high-affini ty moti f for binding TCRs, and may form a ternary complex wi th MHCII. The binding epi tope on the spike protein harbors a sequence moti f unique to SARS-CoV-2 (which is not present in other SARS-related coronavi ruses), which is highly similar in both sequence and st ructure to the bacterial superantigen staphylococcal enterotoxin B. Furthermore, the interfacial region includes selected residues from an intercellular adhesion molecule (ICAM)-like moti f shared between the SARS vi ruses from the 2003 and 2019 pandemics. A neurotoxin-like sequence moti f on the receptor-binding domain also exhibi ts a high tendency to bind TCRs. Analysis of the TCR repertoi re in adul t COVID-19 patients demonst rated that those wi th severe hyperinflammatory disease exhibi t TCR skewing consistent wi th superantigen activation (64) . A blood test that determines the presence of specific TCR variable gene segments for identi fication of patients at risk for severe MIS-C has been developed (65) . These data suggest that SARS-CoV-2 Spike protein i tsel f may act as a superantigen to t rigger the development of MIS-C as well as cytokine storm in adul t COVID-19 patients (66) . Interestingly, fi rst cases of MIS following SARS-CoV-2 infection in adul ty have been reported (67) .

In the context of the superantigen hypothesis i t is noteworthy that during severe sepsis activation of blood coagulation plays a cri tical pathophysiological role resul ting in septic shock, microthrombi and mul ti organ dysfunction. During severe sepsis and septic shock, a massive release of cytokines and activation of the coagulation system resul t in disseminated intravascular coagulation (DIC) and mul tiorgan dysfunction syndrome (68) (69) (70) . The procoagulant activi ty and tissue factor induction by various superantigens from Staphylococcus aureus, including enterotoxin A (EA), enterotoxin B (EB), and toxic shock syndrome toxin (TSST)-1, were tested for their abili ty to induce procoagulant activi ty and tissue factor (TF) expressiona major initiator of blood coagulation expressend predominantly on monocytic cells and endothelial cells -in human whole blood and in peripheral blood mononuclear cells. Determination of clotting time showed that all, enterotoxin A, B and toxic shock syndrome toxin 1 from S. aureus induced procoagulant activi ty in whole blood and in mononuclear cells. The procoagulant activi ty was dependent on the expression of TF in monocytes. In the supernatants from staphylococcal toxin-stimulated mononuclear cells, interleukin (IL)-1 beta was detected by ELISA. The increased procoagulant activi ty and TF expression in monocytes induced by the staphylococcal toxins were inhibi ted in the presence of IL-1 receptor antagonist, a natural inhibi tor of IL-1 beta. The study demonst rated that superantigens from S. aureus activate the extrinsic coagulation pathway by inducing expression of TF in monocytes, and that the expression is mainly t riggered by superantigen-induced IL-1 beta release (69, 70) .

Furthermore, is has been demonst rated that toxic shock syndrome toxin-1 (TSST-1) and staphylococcal enterotoxins A and B induce the activation of NF-B, that acts as a t ranscriptional enhancer by binding to sequences found in both the IL-1 beta and TNF-alpha promoters. Induction of both NF-B DNA-binding proteins and NF-B enhancer func tion was down-regulated by inhibi tors of protein kinase C and protein tyrosine kinase, indicating a role for these protein kinases in the induction of NF-B by MHC class II ligands. Using neutralizing antibodies, i t was demonst rated that after the stimulation of cells wi th TSST-1, TNF-alpha acted to up-regulate binding of NF-B to DNA and the activation of the NF-B -promoter CAT const ruct indicating that induction of NF-B by superantigens is up-regulated in part by an autocrine loop involving TNF (71) .

The cent ral role of the NF-B pathway in superantigen mediated T-cell activation has also been demonst rated in studies showing that proteasome inhibi tion reduced superantigen-mediated T cell activation. PS-519 as a potent and selective proteasome inhibi tor was shown to inhibi t NF-κB activation by blocking the degradation of i ts inhibi tory protein IκB and to reduce superantigenmediated T cell-activation in vit ro and in vivo. Proli feration was inhibi ted along wi th the expression of very early (CD69), early (CD25), and late T cell (HLA-DR) activation molecules. Moreover, expression of E-selectin ligands relevant to dermal T cell homing was reduced, as was E-selectin binding in vit ro (72) . Furthermore, inhibi tion of NF-B pathway by two anti-oxidants, N-acetylcysteine (NAC) and pyrrolidine di thiocarbamate (PDTC) was shown to dose-dependently inhibi t SEstimulated T-cell proli feration (by 98%), production of cytokines and chemokines by PBMC and expression of SE-induced cell surface activation markers. The potency of both NAC and PDTC corresponded to their abili ty to inhibi t NF-κB activation (73) . Beside the MHCII dependent activation of T-cells, also a MHC/II independent di rect stimulation of TCR Vb by Staphylococcus aureus enterotoxin via PKCtheta/ NF-B and IL2R/STAT signaling pathways has been shown (74) .

In this context i t has to be mensioned that normal or physiological antigen stimulation of TCR signaling to NF-κB is required for T cell proli feration and di fferentiation of effector cells. Engagement of the TCR by an MHC-antigen complex initiates a whole chain of downst ream events, desc ribed in detail by Paul et al., which ul timately t rigger calcium release and PKC activation, respectively.

Activation of a specific PKC isoform, PKCθ, connects TCR proximal signaling events to distal events that ul timately lead to NF-κB activation. Importantly, PKCθ activation is also driven by engagement of the T cell costimulatory receptor CD28 by B7 ligands on antigen presenting cells and via intermediate steps leads to the activation of IKKβ. IKKβ then phosphorylates IκBα, t riggering i ts proteasomal degradation, enabling nuclear t ranslocation of canonical NF-κB heterodimers comprised of p65 (RELA) and p50 proteins. Once in the nucleus, NF-κB governs the t ranscription of numerous genes involved in T cell survival, proli feration, and effector func tions (75) .

Importantly, the pathogenesis of COVID-19 has been shown to involve over-activation of NF-B pathway (37, 38) . In several studies i t has been studied which part(s) of the SARS-CoV-2 is responsible for the massive NF-B pathway activation. Khan et al investigated di rect inflammatory func tions of major st ructural proteins of SARS-CoV-2 and showed that spike (S) protein potently induces inflammatory cytokines and chemokines including IL-6, IL-1ß, TNF, CXCL1, CXCL2, and CCL2, but not IFNs in human and mouse macrophages. No such inflammatory response was observed in response to membrane (M), envelope (E), and nucleocapsid (N) proteins. When stimulated wi th extracellular S protein, human lung epi thelial cells A549 also produced inflammatory cytokines and chemokines. Interestingly, epi thelial cells expressing S protein intracellularly are non-inflammatory, but elici ted an inflammatory response in macrophages when co-cultured. Biochemical studies revealed that S protein t riggers inflammation via activation of the NF-κB pathway in a MyD88dependent manner. Further, such an activation of the NF-κB pathway was abrogated in TLR2deficient macrophages. Consistently, administration of S protein induced IL-6, TNF, and IL-1 ß in wild-type, but not TLR2-deficient mice. In this study, both S1 and S2 were demonst rated to show high NF-B activation, wi th S2 showing the higher potency on an equimolar basis (76) . In a second study the spike protein was demonst rated to promote an angiotensin II type 1 receptor (AT1) mediated signaling cascade, induced the t ranscriptional regulatory molecules NF-κB and AP-1/c-Fos via MAPK activation, and increased IL-6 release (77) . A third study has demonst rated that SARS-CoV-2 spike protein subuni t 1 (CoV2-S1) induces high levels of NF-κB activations, production of pro-inflammatory cytokines and mild epi thelial damage, in human bronchial epi thelial cells. CoV2-S1induced NF-κB activation requires S1 interaction wi th human ACE2 receptor and early activation of endoplasmic reticulum (ER) st ress, and associated unfolded protein response (UPR), and MAP kinase signaling pathways. The FDA-approved ER st ress inhibi tor, 4-phenylburic acid (4-PBA), and MAP kinase inhibi tors, t rametinib and ulixertinib, ameliorated CoV2-S1-induced inflammation and epi thelial damage (78) . In a fourth study, the effects of a recombinant SARS-CoV-2 spike glycoprotein S1 was investigated in human peripheral blood mononuclear cells (PBMCs). Stimulation of PBMCs wi th spike glycoprotein S1 (100 ng/mL) resul ted in signi ficant increase in TNFα, IL-6, IL-1β and IL-8. Pre-treatment wi th dexamethasone (100 nM) caused signi ficant reduction in the release of these cytokines. Further experiments revealed that S1 stimulation of PBMCs increased phosphorylation of NF-B p65 and IBα, and IBα degradation. DNA binding of NF-B p65 was also signi ficantly increased following stimulation wi th spike glycoprotein S1. Treatment of PBMCs wi th dexamethasone (100 nM) or the specific NF-B inhibi tor BAY11-7082 (1 μM) resul ted in inhibi tion of spike glycoprotein S1-induced NF-B activation. Activation of p38 MAPK by S1 was blocked in the presence of dexamethasone and SKF 86002. CRID3, but not dexamethasone pre-treatment produced signi ficant inhibi tion of S1-induced activation of NLRP3/caspase-1. Further experiments revealed that S1-induced increase in the production of TNFα, IL-6, IL-1β and IL-8 was reduced in the presence of BAY11-7082 and SKF 86002, while CRID3 pre-treatment resul ted in the reduction of IL-1β production.

These resul ts suggest that SARS-CoV-2 spike glycoprotein S1 stimulated PBMCs to release proinflammatory cytokines through mechanisms involving activation of NF-B, p38 MAPK and NLRP3 inflammasome (79) .

Furthermore, an interaction between SARS-CoV-2 spike (S) protein and LPS was shown to lead to aggravated inflammation in vit ro and in vivo. Native gel elect rophoresis demonst rated that SARS-CoV -2 S protein binds to LPS. Microscale thermophoresis yielded a KD of ∼47 nM for the interaction. showing st rong similari ty to data recorded for SARS-CoV S protein. Similar to SARS-CoV-2 also the clinical picture of severe acute respi ratory syndrome (SARS) is characterized by an over-exuberant immune response wi th lung lymphomononuclear cells infil teration and proli feration that may account for tissue damage more than the di rect effect of vi ral replication. Puri fied recombinant S protein was studied for stimulating murine macrophages (RAW264.7) to produce proinflammatory cytokines (IL-6 and TNF) and chemokine IL-8. The authors found that a di rect induction of IL-6 and TNF-release in the supernatant in a dose-, time-dependent manner and highly spike protein-speci fic.

IL-6 and TNF-production were dependent on NF-B, which was activated through IB degradation facili tates platelet aggregation to form a thrombus. However, PF4 has addi tional activi ties beyond simply promoting blood coagulation, and the regulation of PF4 is very complex. PF4 expression is elevated following t rauma wi th the physiological role to prevent blood loss from injury. Notably, a signi ficant amount of PF4 is also released by activated platelets in response to infection (82) .

After exposure of patients to heparin, i t binds to PF4 and promotes PF4 aggregation, so that they was found, indicating frequent pre-immunization to modified PF4. Thus, PF4 may have a role in bacterial defense, and HIT is probably a misdi rected antibacterial host defense mechanism (85).

We hypotized that cationic PF4 may charge-dependently associate not only wi th bacterial surface st ructures, but also wi th vi ruses harboring a negative surface charge such as adenovi rus. Due to the highly negatively charged hexone proteinwhich represents the major capsid protein -adenovi rus expose a highly negative surface charge (86, 87) . In this context, complexation wi th polycations has been used to enhance t ransfection efficacy of adenovi ral vectors into target cells. It has been demonst rated that that a cationic component can charge-associate wi th adenovi rus particles, which carry a net negative surface charge, and will facili tate at tachment to the negatively charged cell membranean approach employed for increasing gene t ransfer efficacy of adenovi ral vectors (88) .

Similiarly adsorption of Ad2 beta gal2 in the presence of di fferent polycations, such as polybrene, protamine, DEAE-dext ran, or poly-L-lysine signi ficantly increased t ransfection efficacy into various cell types. The polyanion heparin completely abrogated the effects of polycations (89) . Finally, a recent bioRxiv preprint has revealed the st ructure of the ChAdOx1/AZD-1222. ChAdOx1 shares the archetypal icosahedral, T=25, capsid common to adenovi ruses. Binding of fiber knob to the coxsackie and adenovi rus receptor (CAR) was shown to be the primary mechanism ChAdOx1 uses to at tach to cells. Further, the work revealed that the surface of the ChAdOx1 vi ral capsid has a st rong elect ronegative potential. The ChAdOx1 hexon hypervariable rgions (HVRs) are di fferent to other adenovi ruses, in terms of apical elect rostatic surface potential, calculated on equilibrated hexon st ructures, ChAdOx1 has the most elect ronegative surface potential which can be expec ted to influence the st rength of incidental charge-based interactions wi th other molecules. Molecular simulations have suggested that this charge, together wi th shape complementari ty, are a mechanism by which an opposi tely charged protein, e.g. platelet factor 4 (PF4) may bind the vector surface (90) . They showed further that adenovi rus interferes wi th adhesion of platelets to a fibronectin-coated surface and flow cytomet ry revealed the presence of the Coxsackie adenovi rus receptor on the platelet surface. They conclude that VWF and P-selec tin are cri tically involved in a complex plateletleukocyte-endothelial interplay, resul ting in platelet activation and accelerated platelet clearance following adenovi rus administration (98) . Furthermore, replication-deficient adenovi ruses are known to induce acute injury and inflammation of infected tissues, thus limi ting their use for human gene therapy. The chemokine expression was evaluated in DBA/2 mice following the intravenous administration of various adenovi ral vectors.

Administ ration of adCMVbeta gal, adCMV-GFP, or FG140 intravenously rapidly induced a consistent pattern of C-X-C and C-C chemokine expression in mouse liver in a dose-dependent fashion. One hour following infection wi th 10(10) PFU of adCMVbeta gal, hepatic levels of MIP-2 mRNA were increased >60-fold over baseline. MCP-1 and IP-10 mRNA levels were also increased immediately following infection wi th various adenovi ral vectors, peaking at 6 hr wi th >25-and >100-fold expression, respectively. Early induction of RANTES and MIP-1beta mRNA by adenovi ral vectors also (100) . Furthermore, i t was investigated whether the early phase of vi rus-cell interaction is sufficient to stimulate ICAM-1 upregulation. A549 cells were exposed to wild-type Ad5 (Ad5), to Ad.CFTR, and to Ad5 inactivated by incubation at 56 degrees C (Ad5/56 degrees C). All, Ad5, Ad.CFTR, and Ad5/56 degrees C activated NF -kappaB and increased ICAM-1 mRNA levels wi thin 4 h after exposure. The role of the mi togenactivated protein kinases (MAPKs) on the ICAM-1 mRNA induction was studied. ICAM-1 mRNA upregulation was inhibi ted upon incubation wi th several chemicals, auch as ERK1/2 inhibi tors PD98059 and AG1288 (by 98 and 67%, respectively), of the p38/MAPK pathway SB203580 (by 50%), of the JNK pathway dimethylaminopurine (by 83%), and of the NF-kappaB parthenolide (by 96%).

Ad5 and Ad5/56 degrees C stimulated ERK1/2, p38/MAPK, and JNK1 starting 10 min and peaking 20- In another study, the vaccinal adjuvant role of rAd independently of i ts vector func tion was evaluated. BALB/c mice received one subcutaneous injection of a mixture of six lipopeptides (LP6) used as a model immunogen, along wi th AdE1 degrees (10(9) particles), a fi rst-generation rAd empty vector. Although coinjected wi th a suboptimal dose of lipopeptides, AdE1 degrees signi ficantly improved the effectiveness of the vaccination, even in the absence of booster immunization. In contrast to mice that received LP6 alone or LP6 plus a mock adjuvant, mice injected wi th AdE1 degrees plus LP6 developed both a polyspeci fic T-helper type 1 response and an effector CD8 T-cell response specific to at least two class I-restricted epi topes. The helper response was still observed when immunization was performed using LP6 plus a mixture of soluble capsid components released from detergent-disrupted vi rions. When mice were immunized wi th LP6 and each individual capsid component, i.e., hexon, penton base, or fiber, the resul ts obtained suggested that hexon protein was responsible for the adjuvant effect exerted by disrupted Ad particles on the helper response to the immunogen (103).

Recombinant adenovi rus (rAd) infection is one of the most effective and frequently employed methods to t ransduce dendri tic cells (DC). Cont radictory resul ts have been reported concerning the influence of rAd on the di fferentiation and activation of DC. In one report i t was shown, as a resul t of rAd infection, mouse bone marrow-derived immature DC upregulate expression of major histocompatibili ty complex class I and II antigens, costimulatory molecules (CD40, CD80, and CD86), and the adhesion molecule CD54 (ICAM-1). rAd-t ransduced DC exhibi ted increased allostimulatory capaci ty and levels of interleukin-6 (IL-6), IL-12p40, IL-15, gamma interferon, and tumor necrosis factor alpha mRNAs. These effects were not related to specific t ransgenic sequences or to rAd genome t ranscription. The rAd effect correlated wi th a rapid increase (1 h) in the NF-kappaB-DNA binding activi ty detected by elect rophoretic mobili ty shi ft assays. rAd-induced DC maturation was blocked by the proteasome inhibi tor Nalpha-p-tosyl-L-lysine chloromethyl ketone (TLCK) or by infection wi th rAd-IkappaB, an rAd-encoding the dominant-negative form of IkappaB. In vivo studies showed that after intravenous administration, rAds were rapidly ent rapped in the spleen by marginal zone DC that mobilized to T-cell areas, a phenomenon suggesting that rAd also induced DC di fferentiation in vivo. These findings add a further pathway for the immunogenici ty of rAd (104) . 

The replication-incompetent adenovi rus (Ad) vector is one of the most promising vectors for gene therapy, however, systemic administration of Ad vectors resul ts in severe hepatotoxici ties, partly due to the leaky expression of Ad genes in the liver. In one study i t has been shown that NF-κB mediates the leaky expression of Ad genes from the Ad vector genome, and that the inhibi tion of NF-κB leads to the suppression of Ad gene expression and hepatotoxici ties following t ransduction wi th Ad vectors. Activation of NF-κB by recombinant TNFα signi ficantly enhanced the leaky expression of Ad genes. More than 50% suppression of the Ad gene expression was found by inhibi tors of NF-κB signaling and siRNA-mediated knockdown of NF-κB. Similar resul ts were found when cells were infected wi th wild-type Ad. Compared wi th a conventional Ad vector, an Ad vector expressing a dominant-negative IκBα (Adv-CADNIκBα), which is a negative regulator of NF-κB, mediated approximately 70% suppression of the leaky expression of Ad genes in the liver. Adv-CADNIκBα did not induce apparent hepatotoxici ties. These resul ts indicate that inhibi tion of NF-κB leads to suppression of Ad vector-mediated tissue damages via not only suppression of inflammatory responses but also reduction in the leaky expression of Ad genes (106) .

Overall, these studies refered to in the present and previous chapters demonst rate that both, SARS-CoV-2 spike protein AND adenovi ral vectors activate NF-κB pathway together wi th several addi tional signal t ransduction pathways.

The effects of the NF-κB pathway activation on coagulopathies as desc ribed in a various studies desc ribed below may be relevant in VITT.

Plasminogen activator inhibi tor-1 (PAI-1) is the major inhibi tor of plasminogen activation and likely plays important roles in coronary thrombosis and arteriosclerosis. Tumor necrosis factor-alpha (TNF) is one of many recognized physiological regulators of PAI-1 expression and may contribute to elevated plasma PAI-1 levels in sepsis and obesi ty. Although TNF is a potent inducer of PAI-1 expression in vi t ro and in vivo, the precise location of the TNF response si te in the PAI-1 promoter was not determined. Transient t ransfection studies using luciferase reporter const ructs containing PAI-1 promoter sequence up to 6.4 kb failed to detect a response to TNF. Moreover, TNF failed to induce expression of enhanced green fluorescent protein under the control of a 2.9-kb human PAI-1 promoter in t ransgenic mice. In contrast, a 5' distal TNF-responsive enhancer of the PAI-1 gene located 15 kb upstream of the t ranscription start si te containing a conserved NF-B-binding si te that mediates the response to TNF. This newly recognized si te was fully capable of binding NF-B subuni ts p50 and p65, whereas overexpression of the NF-B inhibi tor IkappaB prevents TNFinduced activation of this enhancer element (107) .

In another study the development of procoagulant activi ty and monocyte activation in heparinized whole blood during extracorporeal ci rculation was studied. Anaphylatoxins, C3a and C5a appeared in the blood wi thin 30 minutes of ci rculation. Ci rculated blood developed a marked potential for coagulation that reached maximal activi ty by 4 hours of ci rculation. This procoagulant activi ty was neutralized by anti-tissue factor antibody. Isolation of monocytes from ci rculated blood revealed that tissue factor expression is upregulated on the cell surface. Furthermore, they observed NF-B nuclear t ranslocation in monocytes from blood passing through the ci rcuit, suggesting that tissue factor expression was due to monocyte stimulation and t ranscriptional activation of the tissue factor gene. Tissue factor expression resul ted in an approximately 30-fold increase in thrombin generation.

Monocyte NF-B activation, monocyte tissue factor expression, thrombin generation, and the procoagulant activi ty of blood in extracorporeal ci rculation were all blocked by the proteasome inhibi tor MG132 indicating that intravascular tissue factor expression during extracorporeal ci rculation of blood is due to NF-B-mediated activation of monocytes (possibly by complement) (108) .

In further study i t was investigated whether complement components C3a and C5a regulate plasminogen activator inhibi tor (PAI-1) in human macrophages. C5a increased PAI-1 up to 11-fold in human monocyte-derived macrophages (MDM) and up to 2.7-fold in human plaque macrophages.

These resul ts were confirmed at the mRNA level. Pertussis toxin or anti-C5aR/CD88 antibody completely abolished the effect of recombinant human C5a on PAI-1 production, suggesting a role of the C5a receptor. Experiments wi th anti TNF antibodies and tiron showed that the effect of C5a was not mediated by TNF or oxidative burst. Furthermore, C5a induced NF-B binding to the cis element in human macrophages and the C5a-induced increase in PAI-1 was completely abolished by an NF-B inhibi tor indicating that C5a upregulates PAI-1 in macrophages via NF-B activation (109) .

Finally, platelets are megakaryocyte-derived fragments lacking nuclei and prepped to maintain primary hemostasis by initiating blood clots on injured vascular endothelia. Pathologically, platelets undergo the same physiological processes of activation, secretion, and aggregation yet wi th such pronouncedness that they orchest rate and make headway the progression of atherothrombotic diseases not only through clot formation but also via forcing a pro-inflammatory state. Indeed, NF-B has been implicated in platelet survival and func tion (110) Process-related impurities in the ChAdOx1 nCov-19 vaccine ChAdOx1 nCov-19 vaccine lots have been analyzed by biochemical and proteomic methods. A recent preprint has shown that the vaccine, in addi tion to the adenovi rus vector, contained substantial amounts of both human and non-structural vi ral proteins. The authors analyzed 3 di fferent lots of the ChAdOx1 nCov-19 vaccine by SDS polyacrylamide gel elect rophoresis (SDS-PAGE) followed by silver staining and compared the staining pattern of the separated proteins wi th those of HAdV-C5-EGFP, an adenovi rus vector puri fied by CsCl ul tracent ri fugation followed by mass spec tromet ry.

Based on intensity comparisons of LC/MS signals, they estimated that in one of three lots about 2/3 of the detected protein amounts were of human and 1/3 of vi rus origin, while the two other lots consisted of rather equal amounts of human and vi ral proteins. Beside the expec ted vi ral proteins that form the vi rion (hexon, penton base, IIIa, fiber, V, VI, VII,VIII, IX and others), also several nonst ructural vi ral proteins were detected at high abundancy al though they are not part of the mature vi ral particle. Furthermore, peptides from more than 1000 di fferent human proteins were detected being derived from the human vector production cell line. The detected proteins were derived from di fferent cellular compartments including cytoplasm, nucleus, endoplasmic reticulum, Golgi apparatus and others. Intriguingly, from the human proteins found in the vaccine and beside several cytoskeletal proteins including Vimentin, Tubulin, Actin and Actinin, the group of heat shock proteins (HSPs) and chaperones stood out in abundancy. Among the top abundant proteins (including vi ral proteins) HSP 90-beta and HSP-90-alpha as cytosolic HSPs (wi th 9.5 % and 4.3 % of the total protein) and 3 chaperones of the endoplasmic reticulum (t ransitional endoplasmic reticulum ATPase, Endoplasmin and Cal reticulin) were present. Ext racellular HSPs are known to modulate innate and adaptive immune-responses, they can exacerbate pre-existing inflammatory condi tion, have been associated wi th autoimmuni ty and can even become target auf auto-immune responses themselves.

They very efficiently initiate specific immune responses by receptor-mediated uptake of HSP-peptide complexes in antigen-presenting cells (APCs), mainly via CD91 and scavenger receptors. Furthermore, among the vi ral proteins detected, the adenovi ral penton base is another candidate for inducing early toxici ty via an RGD motive, present in a solvent-exposed loop of penton base, by interacting wi th integrins on cell membranes including of platelets (111) .

Discussion of an integrated mechanistic model for VITT following vaccination with adenovirus vector-based vaccines SARS-CoV-2 Spike protein is known to bind to the ACE2 receptor followed by endocytosis (13) , whereas the primary mode of adenovi rus (including replication defective E1/E3 deletion variants) is the high affinity binding of the fiber-knob to the CAR (coxsackie and adenovi rus receptor), followed by interaction between the arginine-glycine-aspartate (RGD) moti f wi thin the vi ral penton base wi th the integrins on the cell surface, which facili tates then vi ral internalization (96) . Beside the primary high affinity binding si tes for cellular uptake both, SARS-CoV-2 and adenovi rus have been desc ribed to bind and activate various Toll-like receptors (TLR) (76, 96, 112, 113) . All these binding events of both, SARS-CoV-2 S-protein and adenovi ral vector, have been desc ribed to activateamong various otherthe NF-B signaling pathway by activating IB kinase (IKK) complexes, release of p50/p65 heterocomplex from the inhibi tor complex wi th IBa by proteasomal degradation of IB, and t ranslocation of the released and phosphorylated p50/p65 heterocomplex to the nucleus where the t ranscription of a huge panel of pro-inflammatory genes, including those for cytokines, such as TNF, IL-1, IL-6, chemokines, such as MCP-1, MCP-3, adhesion molecule, such as ICAM-1 and VCAM-1, complement components and coagulation factors, such as PAI-1 (107) are induced. These processes are t riggered in a broad variety of cell types, which express the respective receptors, including epi thelial cells (e.g. in the lung), macrophages (including alveolar macrophages), and endothelial cells, found in all organs (76) (77) (78) (79) .

Expression of pro-inflammatory cytokines such as TNF, IL-1, and IL-6, but also increased integrin expression (114, 115) can resul t in auto-ampli fying posi tive feedback loops which affect also integrated systems such as complement and coagulation (39) (40) (41) (42) (43) (44) . The release of various chemokines, in particularly MCP-1 and IL-8 will stimulates migration and accumulation of various inflammatory and immune cells to the si te. In particular neutrophils are at tracted, activated by the proinflammatory cytokine/chemokine melieu and stimulated to interact wi th endothelial cells and platelets. Release of DNA chromatine nets, i.e. NETs have been desc ribed, which are considered one major mechanism for thromboses and occlusion of capillaries in COVID-19 (45) (46) (47) (48) .

While these events are known to accelerate and ampli fy in the case of massive vi ral infection by SARS-CoV-2 leading to COVID-19, the localized expression of the spike protein in case of vaccination is not expec ted to resul ts in levels leading to a major systemic symptomatic picture. However, in the case of adenovi ral vector vaccines, here comes the addi tive, and possibly in some cases, synergistic effects of the adenovi ral vector i tsel f. Beside addi tional activation of the NF-B pathwaywhich may be considered a vaccine adjuvant effectadenovi ruses have the abili ty to bind and activate platelets and endothelial cells (97, 98) . Adenoviral vectors can bind to ci rculating platelets which causes their activation and aggregation and subsequent ent rapment in liver sinusoids. Moreover, activation of platelets is known to resul t in increased release of the PF4a tetramer wi th high posi tive surface charge, which on the one hand side can bind to the negatively charged glycoseaminoglycane st ructures on endothelial cells. As adenovi ruses are known to activate platelets, i t is plausible that the replication-deficient adenovi ral vector could be di rectly responsible for the release of platelet-derived PF4. However, this hypothesis implies that signi ficant amounts of vaccine particles would reach the bloodst ream after intramuscular injection, which seems less likely. An al ternative scenario would involve endothelial cells. Indeed, endothelial cells are efficiently t ransduced upon intramuscular injection. Transduced endothelial cells might be di rectly damaged by the spike protein that they synthesize. Platelets might then be recrui ted and activated by the spike protein expressed by endothelial cells. PF4 released by activated platelets may combine wi th anionic proteoglycans located on the surface of endothelial cells or are shed from endothelial cells. In such a scenario, both the adenovi ral vector and the spike protein would contribute to the formation of immunogenic PF4 following vaccination wi th adenovi ral vector-based COVID-19 vaccines.

One important cent ral points of the VITT are the anti-PF4 antibodies found in the majori ty of patients wi th venous thromboses following SARS-CoV-2 adenovi ral vector-based vaccines. The VITT induced antibodies are most probably induced by elect rostatic binding of the posi tively charges PF4 tetramers to the negative surface of the adenovi ral vector hexones. In this complex conformational changes occursimilar as reported for heparin induced HITwhich makes them highly immunogenic t riggering the formation of anti-PF4 di rected antibodies. Binding of these induced antibodies to the PF4 at tached adenovi ral surface will facili tate uptake of these complexes via FcgammaR mediated process (90) . While macropages, monocytes, NK cells and DC will uptake antibody-PF4 (adenovi rus) complexes via their di fferent FCgamma receptors leading to activation of the cells, FcgammaRinduced complement activation, and degradation and sequestion of adenovi ral vectors in reticulendothelial cells, also platelets are able to bind such complexes via their FcgammRIIA receptor leading to activation, release of PF4, aggregation of platelets, and resul ting thromboses (116) .

There is an addi tional mechanism that might accelerate the induction of anti-PF4 antibodies that is the supposed superantigen features of the Spike protein. Di fferent from antigen specific activation of single T-cell clones in the case of conventional antigens, wi th the antigen presented in the MHC-TCR groove, superantigens have been decribed to bind outside this groove to MHC and /or TCR leading to the polyclonal activation (63) (64) (65) . This, together wi th the anyway highly immunogenic PF4-adenovi rus complexes, may facili tate the induction of PF4 specific antibodies. Notably, both, normal cell activation as well as superantigen-induced polyclonal T cell activation are dependent of NFB pathway signaling (71) (72) (73) (74) (75) .

Although originally being locally applied, a limi ted local dissemination of adenovi ral vaccines e.g. via binding to endothelial cells (117) (118) (119) together wi th the desc ribed NF-B-t riggered leaky expression of adenovius genes in originally replication incompetent adenovi ral vectors (120) may lead in rare cases to sel f-ampli fying cascades leading to activated or damaged endothelial cells, activated and aggregated platelets, and activation of the coagulation systeme also at si tes distant to the application si te, i.e. systemic prothrombotic procoagulation events together wi th a corresponding (consumption) thrombocytopenia as observed in rare cases of adenovi rus vector based SARS-CoV-2 vaccines (121) (122) (123) (124) (125) (126) .

Addi tional activation of the desc ribed molecular and cellular pathways may occur by impuri ties such as the signi ficant amounts of human heat shock proteins found in several ADT1222 vaccine preparations (111) .

Together, these molecular and cellular mechanisms desc ribed in the previous sections may explain the higher prevalence of rare thromboses and thrombocytopenia following adenovi ral vector-based anti SARS-CoV-2 vaccines compared to mRNA based anti SARS-CoV-2 vaccines.

The data supporting the conclusions of this article will be made available by the authors, wi thout undue reservation.

RK contributed projec t idea, discussion of data, and writing of manuscript, li terature search, and review of manuscript.

No conflict of interest to be reported. SARS-CoV-2 Spike protein (expressed and released by t ranfected cells following vaccination wi th ei ther mRNA or adenovi ral vector coding for Spike protein) binds to the ACE2 receptor followed by endocytosis. In contrast, high affinity binding of the adenovi rus (including replication defective E1/E3 deletion variants) occurs via binding of the fiber-knob to the CAR (coxsackie and adenovi rus receptor), followed by interaction between the arginine-glycineaspartate (RGD) moti f wi thin the vi ral penton base wi th the integrins on the cell surface, which facili tates then vi ral internalization. Beside the primary high affinity binding si tes for cellular uptake both, SARS-CoV-2 and adenovi rus may bind and activate various Toll-like receptors (TLR) leading to activationamong various othersof the NFB signaling pathway including activation of IKK complexes, release of p50/p65 complexs from the inhibi tor complex wi th IBa by proteasomal degradation of IB, and t ranslocation of the released and phosphorylated p50/p65 heterocomplex to the nucleus where the t ranscription of a huge variety of pro-inflammatory genes, including those for cytokines, such as TNF, IL-1, IL-6, chemokines, such as MCP-1, MCP-3, adhesion molecule, such as ICAM-1 and VCAM-1, complement components and coagulation factors, such as PAI-1 are induced. These processes are t riggered in a broad variety of cell types, which express the respective receptors, including epi thelial cells (e.g. in the lung), macrophages (including alveolar macrophages), and endothelial cells, found in all organs.

Expression of pro-inflammatory cytokines such as TNF, IL-1, and IL-6, and increased integrin expression can resul t in autoampli fying posi tive feedback loops, which may extend to other intergrated systems such as complement and coagulation. The release of chemokines, such as MCP-1 and IL-8 will at tract migration and accumulation of inflammatory and immune cells to the si te. In particular neutrophils are at tracted, and activated by the pro-inflammatory cytokine/chemokine melieu and stimulated to interact wi th endothelial cells and platelets.

Release of DNA chromatine nets, i.e. NETs are considered one major mechanism for thromboses and occlusion of capillaries.

While these events are known to accelerate and ampli fy in the case of massive vi ral infection by SARS-CoV-2 resul ting in COVID-19, the localized expression of the spike protein in case of vaccination is not expec ted to resul ts in levels inducing a major systemic symptomatic picture. However, in the case of adenovi ral vector vaccines, addi tive and synergistic effects are possible. Beside an addi tional activation of the NFB pathway adenovi ruses have the abili ty di rectly to bind and activate platelets and endothelial cells. Adenovirus rapidly bind to ci rculating platelets, which causes their activation/ aggregation. Activation of platelets resul ts in increased release of the PF4a tetramer wi th high posi tive surface charge, which on the one hand side can bind to the negartively charged glycoseaminoglycane on endothelial cells. Addi tionally, endothelial cells can be t ransduced upon intramuscular injection to express spike protein leading to further activation and damage of the endothelial cells.

One hallmark of the VITT are anti-PF4 antibodies detected in the majori ty of patients wi th venous thromboses and thrombocytopenia following SARS-CoV-2 adenovi ral vector based vaccines. The VITT induced antibodies are most probably induced by elect rostatic binding of the posi tively charges PF4 tetramers to the negative surface of the adenovi ral vector hexones. In this complex conformational changes occursimilar as reported for heparin induced HITwhich makes them highly immunogenic resul ting in formation of anti-PF4 di rected antibodies. Binding of these induced antibodies to the PF4 at tached adenovi ral surface will in turn facili tate uptake of these complexes via FcgammaR mediated process.

Macropages, monocytes, NK cells, and DC take up antibody-PF4 (adenovi rus) complexes via di fferent types of FCgamma-receptors leading to cellular activation, co-induced complement activation, and sequestion and degradation of adenovi ral vectors in reticulendothelial cells.

Also platelets are able to bind such complexes via their FcgammR2a receptor leading to activation, release of PF4, aggregation of platelets and resul ting thromboses.

The supposed superantigen features of the Spike protein may present an addi tional mechanism to accelerate the induction of anti-PF4 antibodies. Di fferent from antigen specific activation of single T-cell clones in the case of conventional antigens, wi th the antigen presented in the MHC-TCR groove, superantigens bind outside this groove to MHC and /or TCR leading to the polyclonal activation. This, together wi th the highly immunogenic PF4adenovi rus complexes, may further facili tate the induction of PF4 specific antibodies.

Notably, both, normal cell activation as well as superantigen-induced polyclonal T cell activation are dependent of NFB pathway signaling.

Although originally being locally applied, a starting local dissemination of adenovi ral vaccines via binding to endothelial cells and platelets together wi th the desc ribed NFB-promoted leaky expression of adenovius genes in originally replication-incompetent adenovi ral vectors can lead in rare cases to sel f-ampli fying cascades leading to activated damaged endothelial cells, activated and aggregated platelets, and activation of the coagulation systeme even at si tes distant to the application si te, resul ting in systemic pro-thrombotic procoagulation events going along wi th corresponding (consumption) thrombocytopenia as observed in rare cases of adenovi rus vector based SARS-CoV-2 vaccines. Fig. 1 

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The Balance of Neutrophil Ext racellular Trap Formation and Nuclease Degradation: an Unknown Role of Bacterial Coinfections in COVID-19 Patients? mBio

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A new storm on the horizon in COVID-19: Bradykinin-induced vascular complications