REVIEW ARTICLE Ferrata Storti Foundation Haematologica 2021 Volume 106(12):3034-3045 Correspondence: PAOLO GRESELE paolo.gresele@unipg.it Received: May 24, 2021 Accepted: August 4, 2021. Pre-published: August 19, 2021. https://doi.org/10.3324/haematol.2021.279289 ©2021 Ferrata Storti Foundation Material published in Haematologica is covered by copyright. All rights are reserved to the Ferrata Storti Foundation. Use of published material is allowed under the following terms and conditions: https://creativecommons.org/licenses/by-nc/4.0/legalcode. Copies of published material are allowed for personal or inter- nal use. Sharing published material for non-commercial pur- poses is subject to the following conditions: https://creativecommons.org/licenses/by-nc/4.0/legalcode, sect. 3. Reproducing and sharing published material for com- mercial purposes is not allowed without permission in writing from the publisher. Interactions of adenoviruses with platelets and coagulation and the vaccine-induced immune thrombotic thrombocytopenia syndrome Paolo Gresele,1 Stefania Momi,1 Rossella Marcucci,2 Francesco Ramundo,3 Valerio De Stefano3 and Armando Tripodi4 1Department of Medicine and Surgery, Section of Internal and Cardiovascular Medicine, University of Perugia, Perugia; 2Department of Experimental and Clinical Medicine, University of Florence; Atherothrombosis Center, AOU Careggi, Florence; 3Section of Hematology, Department of Radiological and Hematological Sciences, Catholic University, Fondazione Policlinico A. Gemelli IRCCS, Rome and 4Fondazione IRCCS Ca' Granda, Ospedale Maggiore Policlinico, Angelo Bianchi Bonomi Hemophilia and Thrombosis Center, Milan, Italy ABSTRACT The COVID-19 pandemic has had a heavy impact on global health and economy and vaccination remains the primary way of con- trolling the infection. During the ongoing vaccination campaign some unexpected thrombotic events have emerged in subjects who had recently received the AstraZeneca (Vaxzevria) vaccine or the Johnson&Johnson (Janssen) vaccine, two adenovirus vector-based vac- cines. Epidemiological studies confirm that the observed/expected ratio of these unusual thromboses is abnormally increased, especially in women in fertile age. The characteristics of this complication, with venous thromboses at unusual sites, most frequently in the cerebral vein sinuses but also in splanchnic vessels, often with multiple associated thromboses, thrombocytopenia, and sometimes disseminated intravas- cular coagulation, are unique and the time course and tumultuous evolu- tion are suggestive of an acute immunological reaction. Indeed, platelet- activating anti-PF4 antibodies have been detected in a large proportion of the affected patients. Several data suggest that adenoviruses may interact with platelets, the endothelium and the blood coagulation system. Here we review interactions between adenoviral vectors and the hemostatic system that are of possible relevance in vaccine-associated thrombotic thrombocytopenia syndrome. We systematically analyze the clinical data on the reported thrombotic complications of adenovirus-based ther- apeutics and discuss all the current hypotheses on the mechanisms trig- gering this novel syndrome. Although, considering current evidence, the benefit of vaccination clearly outweighs the potential risks, it is of para- mount importance to fully unravel the mechanisms leading to vaccine- associated thrombotic thrombocytopenia syndrome and to identify prognostic factors through further research. Introduction The coronavirus disease 19 (COVID-19) pandemic has prompted an unprecedent- ed effort to develop highly effective vaccines to prevent further spreading of the infection, the associated mortality and the enormous strain on healthcare systems. Indeed, in a previously unimaginable short time, many vaccines have been devel- oped. Several of them underwent controlled randomized phase III clinical trials and, as of 22 June, 2021, 13 have been licensed globally for clinical use. By July 18, 2021 they had been administered to more than 1.9 billion subjects worldwide (923 mil- lion of whom are fully vaccinated; 3.66 billion doses have been administered glob- ally; 26.3% of the world’s population has received at least one dose of a COVID-19 vaccine). This represents the most massive vaccination campaign ever undertaken 3034 haematologica | 2021; 106(12) (https://www.who.int/emergencies/diseases/novel-coronavirus- 2019/covid-19-vaccines; https://ourworldindata.org/covid-vacci- nations). Although careful scrutiny of vaccine safety in con- trolled randomized phase III clinical trials did not high- light significant thrombotic risks, exceedingly rare events may have been missed and indeed during the vaccination campaign several cases of thrombosis, in particular thrombotic events at unusual sites associated with thrombocytopenia, were reported. Most events occurred in subjects who had received the ChAdOx1 (Vaxzevria) vaccine in the preceding weeks, but more recently several cases have also been reported following the Ad26-CoV2S Johnson&Johnson (Janssen) vaccine.1-9 Not only was the observed/expected ratio of these thromboses abnormally high in subjects receiving the Vaxzevria vaccine, but the clinical characteristics of the events were unique, associ- ating unusual site venous thromboses, mainly cerebral vein sinus thrombosis (CVST), with thrombocytopenia and sometimes disseminated intravascular coagulation (DIC). In contrast, no thromboses were reported in about 90 million subjects who had received the messenger RNA (mRNA)-based Pfizer BioNTech and only very few in those who had received the Moderna vaccine (Spikevax), although the latter had characteristics apparently dissim- ilar from those observed in Vaxzevria recipients, with one exception.10 These findings suggest that the reported thrombotic complications, which have variously been called vaccine- induced prothrombotic immune thrombocytopenia (VIPIT), vaccine-induced immune thrombotic thrombo- cytopenia (VITT), thrombotic thrombocytopenia syn- drome (TTS) and vaccine-associated thrombotic throm- bocytopenia syndrome (VATTS),2,11-13 are peculiar to aden- oviral (Ad) vector-based vaccines and have led to limita- tions and/or temporary suspensions of the use of such vaccines in several countries. From the most recently available UK pharmacovigilance data (July 7, 2021), CVST and other major thromboembol- ic events with concurrent thrombocytopenia had been reported in 147 (average age, 54 years) and 258 subjects (average age, 54 years), respectively, among an estimated 24.6 million recipients of a first dose and an estimated 22.3 million recipients of a second dose of the Vaxzevria vac- cine. Thus, the overall incidence after first or unknown doses was 14.8 cases per million doses in the UK (https://www.gov.uk/government/publications/coronavirus- covid-19-vaccine-adverse-reactions/coronavirus-vaccine-summa- ry-of-yellow-card-reporting). Concerning Europe, as of June 27, 2021, to EudraVigilance of 479 suspected cases, 100 of which had had a fatal outcome, among recipients of about 51.4 mil- lion doses of Vaxzevria, i.e. 19.3 cases per million doses (https://www.ema.europa.eu/en/documents/covid-19-vaccine- safety-update/covid-19-vaccine-safety-update-vaxzevria-previ- ously-covid-19-vaccine-astrazeneca-14-july-2021_en.pdf), and 21 cases of suspected TTS associated with the Janssen COVID-19 vaccine, four of which were fatal, among recipients of about 7 million doses of this vaccine, i.e. 3 cases per million doses (https://www.ema.europa.eu/en/docu- ments/covid-19-vaccine-safety-update/covid-19-vaccine-safety- update-covid-19-vaccine-janssen-14-july-2021_en.pdf). there were spontaneous reports This review aims to discuss the interactions between Ad vectors and Ad-based vaccines and the hemostatic system and the hypotheses on the mechanisms triggering VITT. Adenovirus vaccines and thrombosis Adenoviruses, platelets and the blood coagulation system Based on available data and given that VITT has been associated with Ad-vector-based vaccines, hypotheses on a direct role of the interaction between Ad and blood components can be made. Ad are non-enveloped DNA viruses with a nucleopro- tein core encapsulated by an icosahedral protein capsid from which proteinaceous fibers protrude. The C-termi- nal knob domain at the distal end of these fibers is responsible for virus binding to its primary cellular recep- tor, a 46-kDa transmembrane protein14-16 which also func- tions as a receptor for Coxsackie B virus and is, therefore, called coxsackie and Ad receptor (CAR).15-17 The high affinity binding of Ad to CAR starts receptor-mediated endocytosis.18 Moreover, Ad have evolved other mecha- nisms to facilitate cell entry via recognition of the argi- nine-glycine-aspartate (RGD) sequence on cell surface integrins. Molecules expressed on host cell surfaces involved in cell infection include the vitronectin-binding integrins αvb3 and αvb5,19 the fibronectin-binding integrin 20 and others, such as αVb1,21 all characterized by a com- α5b1 mon RGD peptide sequence which is recognized by the RGD ligand in the HI fiber knob loop of the Ad penton base protein. Although the CAR is expressed in almost all tissues, including the adult nervous system and cerebral vasculature,22,23 muscle,24 heart25 and the hematopoietic system,26 its presence in platelets is debated. Othman et al. identified CAR (by flow cytometry) and its mRNA (by reverse transcriptase polymerase chain reaction) in human platelets27 while Shimony et al. did not confirm the presence of the receptor and proposed that binding of Ad to platelets is mediated by an interaction between 28 (Figure 1). RGD-binding motifs of Ad and platelet αVb3 Indeed, human megakaryocytes either do not express mRNA for CAR or express it at extremely low levels (J. Rowley and A.S. Weyrich, University of Utah, personal communication). After intravenous inoculation in mice, Ad rapidly bind circulating platelets causing their activation and subsequent entrapment in liver sinusoids where virus-platelet aggregates are taken up by Küpffer cells and degraded. Platelet activation is followed by activation of blood coagulation, leading to DIC.29 Activated platelets also release cytokines promoting endothelial cell activa- tion with secretion of von Willebrand factor, binding of platelets to endothelial cells and the formation of platelet/leukocyte aggregates, eventually triggering the development of microthrombi in liver sinusoids.21,29 There is also a complex interplay between Ad and the coagula- tion system. In fact, the distribution and activity of Ad in blood is affected by interactions with plasma proteins, including complement and vitamin K-dependent coagula- tion factors, which act as opsonizing agents. Our knowl- edge of these interactions derives mainly from in vitro observations and it is unknown whether the interplay of Ad with coagulation proteins affects the activity of the latter. Vitamin K-dependent coagulation factors, including the anticoagulant protein C, interact with Ad-5, the most widely used Ad vector. Activated protein C is generated on endothelial cells via the interaction of protein C with the the thrombin-thrombomodulin complex and endothelial protein C receptor (EPCR). Activated protein C requires protein S to express anticoagulant activity.30 Protein S circulates either free or associated with C4BP, a haematologica | 2021; 106(12) 3035 P. Gresele et al. (Ad) Figure 1. Hypothesized interactions between platelets and adenovirus- es. Adenoviruses induce platelet activation either by binding to platelet coxsackie and adeno - virus receptor (CAR) or to platelet- surface integrins, such as αvb3 or α5b1. Moreover, circulating Ad-elicit- ed IgG or immune complexes may directly activate platelets through FcgRIIa. Gas6 exposed by cerebral vein endothelial cells may bind Ad and activate platelets acting on Tyro3, Axl and Mer (TAM) receptors. Ad may also bind CAR expressed by cerebral vein vessels in this way activating endothelial cells which in turn may elicit platelet activation. regulatory protein of the complement system.31 C4BP binds to activated platelets through mechanisms involv- ing chondroitin sulfate expressed on activated platelets32 and to membrane-associated protein S on platelets.33 Interestingly, the protein C anticoagulant pathway plays a peculiar pathophysiological role in CVST.34 Small but measurable amounts of EPCR are also found in plasma. Soluble EPCR binds both protein C and acti- vated protein C with an affinity similar to that of mem- brane-bound EPCR35 but, in contrast to the latter, it inhibits activated protein C anticoagulant activity thus limiting its ability to inactivate activated factor V, and also binds protein C impeding its activation by thrombin- thrombomodulin complexes.32,36 An increase in soluble EPCR was observed in CVST, possibly leading to a proco- agulant condition and enhanced risk of thrombosis.37 Finally, Gas6 (encoded by the growth arrest-specific 6 gene), a vitamin K-dependent protein with 44% sequence homology with protein S but devoid of anticoagulant activity, is widely expressed in the cerebral nervous sys- tem where it is found on resting endothelial cells. Gas6 potentiates platelet activation acting on Tyro3, Axl and Mer (TAM) receptors leading to thrombus formation38 and in vitro studies have shown that Gas6 binds to Ad enhancing their gene expression.39 The affinity of different Ad for coagulation factors is variable, with a considerable number of Ad types unable to bind them. Ad-5, Ad-2 and Ad-16 bind strongly to fac- tor X.40 Moreover, the ability of Ad to bind coagulation factors is species-specific, e.g., Ad-5 binds human and mouse factor X with similar affinity, but Ad-2 binds human factor X with 10-fold lower affinity than mouse factor X.41 Vitamin K-dependent coagulation factors VII, IX, X, and protein C mediate the binding of Ad to hepato- cytes.42-44 For instance, for Ad-5 hepatotropism is critically dependent on the ability of the Ad-5 hexon to bind factor X. In contrast, non-factor X-binding Ad, such as Ad-48 and Ad-26, do not show hepatocyte tropism.45 The pri- mary reason why factor X is required for Ad-5 transduc- tion to the liver is that it protects Ad-5 from attack by complement.46 It has been previously ascertained that components of intramuscularly-injected vaccines, including the Ad vec- tor, are disseminated in the circulation47 and it is thus con- ceivable that some of the above described activating interactions between Ad and platelets, endothelium and the blood clotting system can occur in recipients of Ad- vector-based vaccines. However, so far no experimental evidence that this may have a role in VITT is available and actually it seems unlikely that sufficiently high circu- lating levels of a non-replicating Ad vector may be reached to trigger platelet activation or blood coagulation changes. In fact, it should be considered that around 2,500 billion virions/kg are required to trigger this reac- tion in mice and non-human primates,29,48 and even if all the Vaxzevria viral content were to spill-over into the blood after intramuscular administration, a concentration of 0.7 billion/kg Ad viral vectors would be reached, which is probably insufficient to activate platelets/coagulation.49 Antibody-dependent enhancement and vaccine-associated adverse events (ADE) Antibody-dependent enhancement is an immunological form of a more general phenomenon called enhanced respiratory disease, leading to the clinical worsening of respiratory viral infections. ADE can occur either through an antibody-mediated increase of virus uptake by Fcg receptor IIa (FcgRIIa)-expressing phagocytic cells, thus facilitating viral infection and replication, or by boosting immune activation through excessive Fc-medi- ated immunological cell effector functions or immune 3036 haematologica | 2021; 106(12) Adenovirus vaccines and thrombosis complex formation with consequent increase of inflam- mation and immunopathology.50 Both ADE pathways can occur when non-neutralizing antibodies or antibodies at sub-neutralizing levels bind to viral antigens without blocking or clearing the infection. ADE has been reported for vaccines against both severe acute respiratory syn- drome corona virus (SARS-CoV) and Middle East respira- tory syndrome corona virus (MERS-CoV) in vitro and in animal models.50 The cytoplasmic tail of FcgRIIa activates the protein-tyrosine kinases Src 51,52 and Syk.53-55 Src- dependent signaling has been shown to be crucial for ADE triggered by Ebola virus, enhancing viral uptake into cells and thus worsening the infection.56 Circulating antibodies activating platelet IgG FcgRIIa may be key determinants of a host response leading to uncontrolled platelet aggregation and thrombosis. Studies in transgenic mice expressing human FcgRIIa on platelets showed that the administration of anti-CD9 antibodies caused thrombosis accompanied by platelet consump- tion, a response that was absent in mice lacking the receptor.57 The clinical relevance of this pathway for thrombotic disorders in humans is confirmed by the observation that FcgRIIa expression is higher in patients with stroke58 and that relatively common FcgRIIa poly- morphisms are associated with increased risk of throm- bosis in patients with heparin-induced thrombocytopenia (HIT).59 Immuno-complex formation, complement depo- sition and local immune activation are likely mechanisms triggered by SARS-CoV-2 vaccine. Furthermore, pre- existing antibodies to coronavirus strains endemic in humans could mediate ADE by facilitating cross-reactive recognition of SARS-CoV-2 in the absence of viral neu- tralization.60 Interestingly, compared to Ad-5 and Ad-6, chimpanzee adenoviruses (ChAd) are much less frequently neutral- ized by pre-existing antibodies present in humans. The prevalence of vector-neutralizing antibodies against Y25, now renamed ChAdOx1, the vector of the Vaxzevria vac- cine, in human sera from British and Gambian adults was found to be 0% (n=100) and 9% (n=57), respectively.61 The presence of these antibodies in rare patients in Europe might theoretically represent one potential mech- anism triggering ADE, and possibly VITT, in vaccine recipients but no data on this are available yet. Despite the above hypotheses, preliminary in vitro evi- dence suggests that serum from convalescent COVID-19 patients does not induce either enhancement of SARS- CoV-2 infection or innate immunity responses in human macrophages, indicating that ADE may not be involved in the immune-pathological processes associated with COVID-19 infection or immunization.62 Use of adenovirus vectors and thrombotic events Adenovirus vectors for gene therapy Ad vectors have been used therapeutically for their ability to transduce and deliver transgenes to different cell types. However, for these indications the clinical use of Ad vectors has been limited to a few tens of patients and the main concerns have been the development of humoral and cellular immunity occurring upon repeated administration and/or the possible neutralization of the vector by pre-existing immunity against the virus, while little attention had been paid to the possible interactions of Ad vectors with platelets and the blood clotting sys- tem. The first use of Ad vectors for gene therapy of inherited disorders or to treat neoplasia dates back to the 1990s. An analysis of the risks associated with the use of Ad-vec- tored gene therapies among 90 individuals who received 140 administrations for various diseases (cystic fibrosis, metastatic colorectal cancer, cardiovascular disease), showed that 13 deaths were recorded. The authors con- cluded that none was linked to the Ad vector.63 The reported hematologic abnormalities were decreased hemoglobin, leukocytosis, thrombocytopenia, and pro- longation of the activated partial thromboplastin time (aPTT), with no cases of DIC.63 It is, however, puzzling that a recently European Medicines Agency-licensed Ad-vectored gene therapy for spinal muscular atrophy received a warning about the possible risk of thrombotic microangiopathy based on the infants five cases (https://www.ema.europa.eu/en/medicines/human/EPAR/zol- gensma). reporting of treated in Adenovirus-vectored vaccines Beside SARS-CoV-2, Ad vectors have been used for the preparation of other vaccines, including the ChAdOx1- vectored vaccines for MERS-CoV and Chikungunya; with only a few hundred volunteers having received these vac- cines up to June 2020,64 no excess of thrombotic events had been noted.65 Even for the Ebola vaccination campaign, the largest previous example of large-scale vaccination using an Ad vector, a maximum of around 200,000 volunteers were treated, with only one vena cava thrombosis reported (Table 1). However, it may be extremely difficult to prove that adverse events following immunization are caused by the vaccine itself when their occurrence is extremely rare (https://www.nature.com/articles/d41586-021-00880-9. Accessed on April 9, 2021). Except for common mild/moderate reactome reactions, the most frequently recorded adverse events in clinical trials were hematologic (e.g., mild hemoglobin decrease, thrombocytopenia, leukopenia) the majority of which recovered a few days or weeks after vaccination. The extent and rate of hematologic adverse events associated with Ad-vectored vaccines are summarized in Table 1. Occasional abnormalities of coagulation were reported, with prolongation of the aPTT, possibly due to the devel- opment of transient antiphospholipid antibodies. Thrombotic events were rare both for human and non- human Ad-vectored vaccines. One case of phlebitis was observed among 114 volunteers who received a recombi- nant, replication-defective Ad-5-vectored vaccine expressing human immunodeficiency virus (HIV)-1 anti- genic proteins.66 Another case of deep vein thrombosis was observed among 58 volunteers after administration of a recombinant, replication-defective Ad-35-vectored vaccine expressing HIV-1 antigens.67 Both events were considered unrelated to the vaccine. A systematic review identified 200 clinical studies on active immunization against SARS-CoV-2. The second most used vaccine platform, after mRNA-based vaccines, was represented by Ad vectors (24%).68 Concerning chimpanzee Ad-vectored vaccines (ChAdOx1 nCoV-19), neutropenia was the most common hematologic abnor- mality (Table 1). Across all studies, vaccines had a good haematologica | 2021; 106(12) 3037 P. Gresele et al. Table 1. Studies with adenovirus-vectored vaccines reporting hematologic adverse effects. Adenoviral Pathogens Study N. of Thrombocytopenia Venous Coagulation Other Other systemic vector (type of) participants (n) thromboembolism disorders hematologic adverse events (n) complications Chimpanzee Adenovirus Influenza A Phase Ia, 15 NR NR NR Leukopenia Fatigue, malaise, dose-escalation (S1) headache MERS Phase I, dose-escalation, 24 NR NR NR Anemia, neutropenia, Fatigue, ChAdOx1 non-randomized, lymphopenia headache, myalgia uncontrolled (64) SARS-CoV-2 Phase I/II, 1077 NR NR NR Neutropenia Fatigue, headache single-blind, randomized, controlled (70) Ebola Phase I, dose-escalation, 20 NR NR aPTT prolongation Leukopenia Fever open-label (S2) (15%) Phase I/IIa, double-blind, 120 NR NR aPTT prolongation Anemia, Fatigue, malaise, placebo-controlled, (n=1) lymphopenia, headache dose-finding (S3) neutropenia Phase I, dose-escalation, 60 1 NR aPTT prolongation Leukopenia, Fatigue, headache, open-label (S4) (n=4) eosinophilia myalgia ChAd3 Phase II, randomized, 3030 7 Vena cava thrombosis NR Anemia Fever, headache observer-blind, (n=1) placebo-controlled (S5) Phase II, randomized, 600 5 NR NR Anemia Fever, headache observer-blind, placebo-controlled (S6) RSV Phase I, open-label, 42 2 NR NR Anemia Fatigue, headache, single-site, myalgia, nausea dose-escalation (S7) Human Adenovirus HIV Phase I, double-blind, 114 NR Phlebitis NR Leukopenia, Fatigue, malaise, randomized, (n=1) anemia headache placebo-controlled (66) Phase I, double-blinded, 36 NR NR NR Neutropenia Fever, malaise, placebo-controlled (S8) myalgia, chills Phase IIb, double-blind, 801 NR NR NR Neutropenia, Headache, malaise, randomized, controlled (S9) anemia myalgia Ad5 Ebola Phase I, randomized, 120 NR NR NR Anemia, leukopenia Fever double-blind, placebo-controlled (S10) Phase I, single-site, double-blind, 32 NR NR aPTT prolongation NR Malaise, myalgia, randomized, placebo-controlled, (n=2) headache, chills dose-escalation (S11) SARS-Cov2 Phase I, single-centre, 108 4 NR NR NR Fever, fatigue, dose-escalation, double-blind, headache, non-randomized (S12) muscle pain SARS-CoV-2 Phase III, randomized, 44325 NR DVT (n=6), PE (n=4), NR NR Fatigue, headache, double-blind, CSVT (n=1) myalgia, nausea placebo-controlled (71) Ad26 Ebola Phase II, randomized, 423 NR NR NR Anemia, Fatigue, headache, double-blind, neutropenia myalgia, chills placebo-controlled (S13) HIV Phase I, double-blind, 58 NR DVT (n=1) NR NR Malaise, myalgia, randomized, headache, chills placebo-controlled (67) Ad35 Plasmodium Phase Ib, randomized, 48 1 NR NR WBC count Myalgia, chills, Falciparum controlled, double-blind, abnormalities headache, fever, dosage-escalation (S14) vomiting Phase I, randomized, 72 NR NR NR Neutropenia, Fever, headache, placebo-controlled, eosinophilia malaise, myalgia, dose-escalation (S15) nausea Ad26-Ad5 SARS-CoV-2 Phase III, non-randomized, 21977 NR DVT (n=1) NR NR Fever, headache, single-center (72) fatigue, myalgia The reference numbers preceded by an S refer to references listed in the Online Supplementary Material. The adverse events reported are related only to the vaccinated groups and not to the placebo groups. MERS: Middle East respiratory syndrome virus; SARS-CoV-2: severe acute respiratory syndrome coronavirus 2; RSV: respiratory syncytial virus; HIV: human immunodeficiency virus; NR: not reported; DVT: deep vein thrombosis; PE: pulmonary embolism; CSVT: cerebral sinus venous thrombosis; aPTT: activated partial thromboplastin time; WBC: white blood cell. 3038 haematologica | 2021; 106(12) Adenovirus vaccines and thrombosis safety profile with no difference in severe reactions between study arms.69,70 In a phase III trial with a recom- binant, replication-incompetent human Ad-26 vector encoding the SARS-CoV-2 spike protein, with 43,783 par- ticipants, 11 venous thromboembolic events were observed in the vaccine group compared to three in the placebo group (Table 1); however, most subjects had underlying medical conditions that might have con- tributed to these events. In the vaccine group there were six cases of lower leg deep venous thrombosis and four cases of pulmonary embolism. Interestingly, however, a CVST, with cerebral hemorrhage and thrombocytopenia, occurred 21 days after vaccination in a 25-year-old male who had multiple predisposing factors, including pre- existing cerebral sigmoid sinus stenosis and infection from an unknown pathogen. Subsequent testing identi- fied anti-PF4 antibodies at the time of the event. The patient recovered.71 In a phase III controlled randomized clinical trial with a recombinant Ad-26-vectored and a recombinant Ad-5- vectored vaccine (Sputnik V) among 16,501 participants, ten vascular events (0.061%) were observed including: one deep vein thrombosis (0.006%), one transient ischemic attack (0.006%), one cerebral circulation failure (0.006%), one vascular encephalopathy (0.00659%) and two acute myocardial infarctions (0.012%) (4 additional events were non thrombotic)72 (Table 1). The vaccine-induced immune thrombotic thrombocytopenia syndrome When the anti-SARS-CoV-2 vaccination campaign was well underway worldwide a few cases of spontaneous, severe thromboembolic events in otherwise healthy sub- jects began to be reported, leading to a pause in the admin- istration of the Vaxzevria vaccine in several European countries (https://www.ema.europa.eu/en/news/emas-safety- committee-continues-investigation-covid-19-vaccine-astrazeneca- thromboembolic-events). Soon after several case reports were published, mainly concerning young females, with new ones continuing to accrue, although many were not subject to rigorous central review and with anti-PF4 antibodies measured using disparate methods, not allowing to con- clude that all were typical VITT cases Up to July 17, 2021, 105 such cases with two Ad-vec- tored vaccines had been published (Table 2) with some common clinical features characterizing a new syndrome, including thrombocytopenia, often severe, venous throm- bosis at unusual sites, in particular of the cerebral sinuses but also of the splanchnic veins, frequently associated with thromboses in multiple sites, both venous and arte- rial, and sometimes DIC combined with hemorrhage. A comparative evaluation of the clinical characteristics of the published ChAdOx1 or Ad26.CoV2.S VITT cases suggests that while clinical symptoms are comparable, Ad26.CoV2.S-associated cases show more thrombosis and intracerebral hemorrhage, lower D-dimer and less altered aPTT, but a similar mortality.9 In a recent, large nationwide healthcare register-based study in Denmark and Norway involving 281,264 ChAdOx1-S-vaccinated subjects aged 18-65 and as controls the entire age- matched populations of the two countries studied in the period 2016-2019, the standardized morbidity ratio for CVST was 20.25 (8.14-41.7), with an excess of 2.5 events per 100,000 vaccinations, particularly evident in women 18-44 years old,73 confirming the crucial relationship between Vaxzevria administration and occurrence of VITT. The catastrophic syndrome, burdened by a 20-50% mortality rate, has the time course and tumultuous evolu- tion of an acute immunological reaction and indeed three groups of investigators identified, in several of their patients, circulating antibodies to PF4/heparin complexes using an enzyme-linked immunosorbent assay (ELISA) and a heparin-induced platelet activation assay,2-4 and thus proposed that this disorder is a peculiar form of autoimmune HIT. The autoimmune heparin-induced thrombocytopenia hypothesis HIT is a rare immune-mediated adverse drug reaction that may occur after exposure to heparin. Circulating heparin binds to PF4, a positively charged platelet protein released in plasma upon activation. PF4 normally binds to negatively charged glycosaminoglycans on the endotheli- um, displacing antithrombin and thus activating coagula- tion. However, PF4 binds with greater affinity to heparin, forming heparin/PF4 complexes which become neoanti- gens inducing the formation of autoantibodies. Heparin- PF4-IgG immune complexes in turn bind to platelet FcgRIIA receptors causing activation, aggregation, and additional release of PF4, with ignition of a positive feed- back loop leading to further platelet activation and con- sumption. Moreover, these complexes also activate monocytes, which release tissue factor, thus promoting concomitant activation of coagulation. HIT is a potentially fatal condition, associated with the thrombosis.74 development of arterial or venous Thrombocytopenia occurs in more than 85% of HIT patients and is usually of moderate severity, with median platelet counts of approximately 50-60×109/L, although values <20×109/L can be found in approximately 10% of cases. Typically, the platelet count starts to decrease 5-10 days after initiation of heparin, but early-onset thrombo- cytopenia (rapid-onset), within 24 h of exposure, can develop in 25-30% of cases if patients have been treated the preceding 3 months.75 with heparin Thromboembolic complications occur in 35-75% of HIT patients and are usually severe. They can be venous (i.e., deep vein thrombosis and pulmonary embolism, but rarely also CVST or splanchnic thrombosis), arterial (ischemic stroke, myocardial infarction, acute occlusion of limb arteries) or microvascular (digital infarction).76 in Recently, another clinical picture not triggered by expo- sure to heparin has been recognized and defined as autoimmune HIT.77 The main characteristic of this condi- tion is the presence of circulating antibodies able to acti- vate platelets also in the absence of heparin. Polyanion molecules potentially involved in the development of autoimmune HIT are typically bacteria and virus compo- nents, hypersulfated chondroitin sulfate, DNA and RNA and polyphosphates.78 Patients with this syndrome show slightly different clinical features from those with classi- cal HIT, including severe thrombocytopenia (<20x109/L), sometimes in combination with DIC, microvascular thrombosis and CVST in up to 40% of cases.78 From a therapeutic standpoint, besides the indication for an alter- native anticoagulant, valid also for HIT, the intravenous haematologica | 2021; 106(12) 3039 P. Gresele et al. Table 2. Cases of vaccine-induced immune thrombotic thrombocytopenia reported in the literature as of July 17, 2021. Vaccine VITT Female, Age range, CVST, Platelet count Negative anti Heparin-treated Time form References cases, n (%) years n/total nadir-range PF4, with success, vaccination n x109/L n (total) n/treated range, days ChAdOx-1 ChAdOx-1 11 9(82%) 22-49 9/11 8-107 0/9 1/5 5-16 2 ChAdOx-1 5 4(80%) 32-54 4/5 10/70 0/5 2(3)/5 5-30 4 ChAdOx-1 23 14(61%) 21-77 13/22a 7/113 1/23 NRb 6-24 3 ChAdOx-1 2 0(0%) 25-32 2/2 7/17 0/1 0/1 6-9 S1 ChAdOx-1 1 1(100%) 55 0/1c 30 1/1 0/1 10 6 ChAdOx-1 1 1(100%) 51 0/1 37 0/0 1/1 11 5 ChAdOx-1 1 1(100%) 60 0/1 5 0/1 0/1 7 S2 ChAdOx-1 3 3(100%) 22-46 3/3 60-92 0/3 0/3 4-17 S3 ChAdOx-1 1 1(100%) 54 1/1 NR 0/0 3/3 12 S4 ChAdOx-1 5 5(100%) 41-67 1/5 12-105 0/5 0/1 5-11 S5 ChAdOx-1 1 0(0%) 63 0/1 36 0/1 (1) 20 S6 ChAdOx-1 1 1(100%) 69 1/1 18 0/1 0/1 11 S7 ChAdOx-1 1 0(0%) 50 1/1 15 0/0 0/0 11 S8 ChAdOx-1 2 1(50%) 37-50 1/2 7-9 0/2 0/2 10 S9 ChAdOx-1 7d 4(57%) 24-53 4/7 8/71 0/7 NR 6-20 S10 ChAdOx-1 2 0(0%) 25-32 2/2 19-30 1/1 0/1 6-9 S11 ChAdOx-1 1 1(100%) 62 0/1e 19 0/1 0/0 8 S12 ChAdOx-1 1 1(100%) 30 1/1 56 0/1 0/1 8 S13 ChAdOx-1 1 1(100%) 30 0/1 72 0/1 1/1 8 S14 ChAdOx-1 4f 3(75%) 29-50 1/4 24-110 0/4 0/1g 7-20 S15 ChAdOx-1 1 1(100%) 41 1/1 36 0/1 0/0 7 S16 ChAdOx-1 1 1(100%) 36 1/1 94 0/0 0/1 14 S17 ChAdOx-1 1 0/1(0%) 51 1/1 NRh 1/1 1/1 6 S18 ChAdOx-1 1 0/1(0%) 38 1/1 14 0/1 0/0 8 S19 ChAdOx-1 1 0/1(0%) 50 1/1 15 0/1 0/0 7 S20 ChAdOx-1 1 1(100%) 35 1/1 50 0/1 0/0 14 S21 ChAdOx-1 1 0(0%) 54 1/1 34 0/1 0/0 21 S22 ChAdOx-1 1 1(100%) 36 0/1 133 0/1 0/1g 17 S23 ChAdOx-1 1 0(0%) 44 0/1 6 0/1 0/0 8 S24 ChAdOx-1 1 0(0%) 27 1/1 68 0/1 0/0 2 S25 ChAdOx-1 1 0(0%) 63 0/1 36 0/1 0/0 20 S26 ChAdOx-1 1 0(100%) 30 1/1 37 0/1 0/0 7 S27 ChAdOx-1 2 2(100%) 24-39 2/2 29-36 0/2 0/2 8-12 S28 ChAdOx-1 3 2(66.6)% 53-61 2/3 21-25 0/3 0/0g 10-16 S29 Ad26.COV2.s Ad26.COV2.s 1 1(100%) 48 0/1 13 1/1 1/1 14 1 Ad26.COV2.s 12 12(100%) 18-<60l 7/12 9-127 0/11 NR/6 6-15 12 Ad26.COV2.s 1 1(100%) 40 1/1 20 0/1 0/0 5 S30 TOTAL 105 73(69.5%) 18-77 65/105 (62%) 5-133 5/92 10(4)/41 2-30 - The reference numbers preceded by an S refer to references listed in the Online Supplementary Material. aOne patient had only thrombocytopenia. bThe patient was reported to be thrombocytopenic. cBilateral superior ophthalmic vein thrombosis. dAn eighth case included in this series was reported in reference S10. eNo thromboses were detected (pos- sibly not a VITT). fOne of the cases did not have thrombosis. gOne case was initially treated with full dose low molecular weight heparin, with no apparent worsening, and was then changed to a non-heparin anticoagulant upon identification of positive anti PF4 antibodies. hThe platelet count was stated to be normal. iAges are reported as ranges, not individual. NR: not reported; PF4: platelet factor 4; VITT: vaccine-induced immune thrombotic thrombocytopenia. administration of high doses of IgG in combination with steroids has been proposed for autoimmune HIT.77 Very early after the first reports of unusual types of thrombosis associated with the Vaxzevria vaccine, a German group suggested a tentative pathogenic mecha- nism underlying these rare events, which they named VIPIT, based on findings in nine cases of previously vac- cinated subjects.11 Indeed, in sera from four of these sub- jects the investigators detected antibodies to PF4/heparin complexes using an enzyme-immunoassay; these anti- bodies were inhibited by the addition of high concentra- tions of heparin (i.e., 100 U/mL), and the sera were able to activate washed control platelets when either PF4 or the Vaxzevria vaccine was added in vitro to the samples.11 3040 haematologica | 2021; 106(12) Adenovirus vaccines and thrombosis Figure 2. The autoimmune heparin-induced thrombocytopenia hypothesis. Vaccine components leaking into the bloodstream from the vaccination site (facilitated by ethylenediaminetetraacetic acid present in the vaccine) activate platelets to release platelet factor 4 (PF4). Vaccine constituents, likely polyanions or viral DNA, form complexes with positively-charged PF4 which are recognized as neoantigens by B cells that then produce antibodies against these complexes. The resulting immune complexes activate platelets through FcgRIIa, triggering the release of additional PF4 and polyphosphates thereby initiating a positive feedback loop that leads to further platelet activation and consumption. Extracellular DNA in neutrophil extracellular traps binds PF4 and the resulting DNA/PF4 complexes further recruit anti-PF4 antibodies inducing massive Fcg receptor-dependent activation of neutrophils, platelets, monocytes and endothelial cells leading to massive activa- tion of coagulation and thrombosis. EDTA: ethylenediaminetetraacetic acid. The investigators showed that platelet activation was triggered by FcgRIIA stimulation, because an FcgRIIA- blocking antibody prevented this phenomenon. Given the absence of previous exposure to heparin, the authors suggested a condition resembling autoimmune HIT. More recently, in a preliminary report published in a non-peer- reviewed internet repository, the German group went on to suggest that the Ad vector and/or some protein com- ponents of the Vaxzevria vaccine activate platelets to release PF4 which then forms complexes with virus pro- teins and other anionic constituents of the vaccine, gener- ating neoantigens against which antibodies develop and induce strong platelet activation via FcgRIIa stimulating granulocyte activation with NETosis and ultimately cata- strophic thrombosis.47 The presence of EDTA in the vac- cine would favor vascular leakage at the inoculation site, facilitating dissemination of the vaccine components in blood (Figure 2). Very recently a study using alanine scanning mutagen- esis explored the binding sites on PF4 of antibodies isolat- ed from patients with VITT or with classical HIT. While the binding of VITT anti-PF4 antibodies was restricted to eight surface amino acids, all located within the heparin- binding site of PF4, HIT anti-PF4 antibodies bound amino acids corresponding to two different sites on PF4; more- over, VITT antibodies had a stronger binding response than HIT antibodies. The authors concluded that VITT antibodies mimic the effect of heparin by binding to a similar site on PF4, allowing PF4 tetramers to cluster with the formation of immuno-complexes which, in turn, cause FcγRIIa-dependent platelet activation.79 These peculiar characteristics explain why the identifi- cation of VITT requires different tests from those needed for the identification of classical HIT.80 In suspected VITT anti-PF4 antibodies can be identified by an ELISA, but not by other rapid immunological assays typically positive in HIT, such as the STic® Expert HIT kit, latex immunoas- says and chemiluminescence-based assays,4,80 and it is better characterized by a heparin-induced platelet aggre- gation (HIPA) or PF4-induced platelet activation (PIPA) test.2,4,81 It should be noted that no single ELISA method detected all possible/probable VITT cases.82 While the autoimmune HIT hypothesis is important and provides the basis for understanding this novel cata- strophic autoimmune thrombotic syndrome, several aspects do not fit completely with the clinical presenta- tion of VITT and various issues remain unclarified. First, among the reported VITT cases in which anti-PF4 antibodies were measured, in a few they were negative (Table 2).4,5 Second, it is expected that treatment with heparin would worsen the clinical evolution of these patients, and indeed it is generally cautiously recom- mended not to use this drug.2,4,13 However, in around one fourth of the heparin-treated cases, the use of this antico- agulant was successful (Table 2). Third, while VITT resembles autoimmune HIT in several respects, the latter is not as frequently associated with CVST and rarely with DIC, and the above summarized mechanistic hypothesis does not explain the preferential localization of the venous thrombotic events in the cerebral and splanchnic circulations. Other unclear aspects are its relatively precocious onset, as early as 4 days after vaccination, which seems too soon to generate high-titer, class-switched, high-affin- haematologica | 2021; 106(12) 3041 P. Gresele et al. Figure 3. A suggested clinical surveillance and diagnostic approach to suspected vaccine-induced immune thrombotic thrombocytopenia. Subjects receiving the Vaxzevria and the Janssen vaccines who develop new onset headache, especially if severe or with unprecedented characteristics, and/or associated with other clin- ical manifestations (blurred vision, gait disturbance, focal neurological symptoms and/or abdominal pain, vomiting, bloody stool, shortness of breath, petechiae or ecchymoses) should be referred for immediate laboratory evaluation (platelet count and D-dimer measurement). If thrombocytopenia is detected they should under- go anti-PF4 antibody testing, imaging and evaluation for cerebral vein sinus thrombosis, splanchnic vein thrombosis or pulmonary embolism. If confirmed, therapy for vaccine-induced immune thrombocytopenic thrombosis (VITT) should be immediately started according to the statement from the Italian Society for the Study of Haemostasis and Thrombosis (SISET)13 Although almost one quarter of the reported patients with VITT in whom unfractionated or low-molecular weight heparin was used apparently responded well to treatment (Table 2), subjects positive for anti-PF4 antibodies, as determined by a heparin-induced platelet aggregation (HIPA) test and/or enzyme-linked immunosorbent assay, or who have not been tested should, for prudence, be treated with alternative anticoagulants, until new information becomes available. In subjects in whom these anti-PF4 antibodies do not cross-react with heparin, as shown by a HIPA test in the presence of a low concentration of heparin, the use of heparin as anticoagulant may be allowed. ELISA: enzyme-linked immunosorbent assay; MRI: magnetic resonance imaging; CT: computed tomography; US: ultrasound. ity anti-PF4 antibodies. Furthermore, there is no evidence that the anti-PF4 antibodies isolated from patients with VITT cause thrombosis and thrombocytopenia in animal models.49,83 Finally, recent observations show that 1.2% to 8.0% of subjects receiving a first dose of Vaxzevria devel- op circulating anti-PF4 antibodies while the prevalence of VITT ranges from 0.0006% to 0.00125%.84,85 Other possible pathogenic mechanisms of vaccine-induced immune thrombotic thrombocytopenia Very recently a preliminary report, published in a non- peer-reviewed repository, provided an interesting alterna- tive potential pathogenic mechanism of VITT.86 COVID- 19 is caused by SARS-CoV-2 which is a single-strand RNA virus that is translated and replicates only in the cytosol of infected cells in the absence of processes which are necessary when nuclear-encoded genes are tran- scribed, and in particular of mRNA splicing. Nuclear encoded genes have intronic sequences, thus their tran- scripts require splice reactions at consensus RNA sequences to eliminate them. When an Ad-vectored viral RNA sequence is administered the vector infects host cells, adenoviral DNA enters the nucleus and is then tran- scribed by the host transcription machinery. However, the viral piece of DNA deriving from the SARS-CoV-2 virus is not optimized to be transcribed into the nucleus and its open reading frame may thus be disrupted by arbi- trary splice events. These splice events would produce shorter spike protein variants, including forms missing the C-terminal membrane anchor, thus leading to soluble circulating spike protein molecules. The soluble spike protein may cause a strong activation of endothelial cells expressing ACE2.87 Moreover, when the host immune system starts to produce antibodies against the spike pro- tein, endothelial cells binding soluble spike would also be decorated by these antibodies, triggering a strong inflam- matory reaction through antibody-dependent or comple- ment-dependent cytotoxicity, thus eliciting VITT. With this hypothesis, the preferential involvement of cerebral 3042 haematologica | 2021; 106(12) Adenovirus vaccines and thrombosis veins could be explained by the non-unidirectional blood flow in these vessels due to the lack of venous valves, with prolonged residence time of the soluble spike pro- tein in this district depending on body posture or when sleeping. The immunological part of this hypothesis is also in agreement with the apparent higher prevalence of VITT in young women, because they have stronger immune reactions than men and older people. To explain the rarity of VITT the authors hypothesized that only some individuals, due to specific major histocompatibili- ty complex combinations, are not able to produce neu- tralizing anti-spike antibodies which would instead pre- vent the binding of soluble spike to endothelial ACE2 and its ominous consequences in most vaccine recipients. This hypothesis was partly validated by the identification through in silico analysis of potential splice sites in the AstraZeneca and Johnson&Johnson codon-optimized spike opening frames and by in vitro studies with HeLa cells showing that vaccine-transduced cells generate tran- scripts smaller than the full spike protein. It would also explain why VITT has not been reported with mRNA vaccines, which release their cargo mRNA directly into the host cells’ cytosol where it is translated into spike protein without undergoing splicing reactions. Finally, it would account for why the incidence of VITT seems to be lower with the Johnson&Johnson vaccine than with the AstraZeneca one, given that the latter carries more splice donor sequences than the former.86 Additional hypotheses on the mechanisms triggering VITT include a genetically determined enhanced expres- sion of FcgRIIa in susceptible subjects, an altered glycosy- lation state of IgG produced in response to vaccination in some individuals making these antibodies more reactive to platelet FcgRIIa,49 the leakage of the Ad vector into the circulation and/or the prior presence of cross-reactive antibodies to other coronaviruses forming immune com- plexes activating platelets.88 However, the hypothesis that VITT develops in subjects with previous, not appar- ent SARS-CoV-2 infection with prior circulating IgG anti- bodies against the spike protein able to activate platelet FcgRIIa89 should be excluded by the observation that most VITT subjects tested for previous or recent COVID-19 infections were negative. Excessive transcription of the spike protein, which would then activate platelets bind- ing to ACE2,90 and vaccine-induced expression of the spike by megakaryocytes and platelets, leading to a thrombo-inflammatory storm,49,91 have also been pro- posed. Another hypothesis starts from the observation that both the ChAdOx1 and the Ad26.CoV2.S vaccines use polysorbate 80 as an excipient. Polysorbate 80 is a non-anionic surfactant that crosses the blood-brain barri- er and enhances microparticle uptake by endothelial cells. Therefore leakage of Ad vector and polysorbate into the circulation and the spike protein produced by vaccination could preferentially localize in brain vessels triggering endothelial activation.92 However, considering that VITT usually develops at least 1 week after vaccination, it is very unlikely that circulating Ad vector or vaccine excipi- ents would still be present in blood, making the alterna- tive explanations, and in particular an immunological reaction, more likely. Conclusive remarks At least two Ad vector-based vaccines against SARS- CoV-2 have been associated with an excess rate of a special form of catastrophic thrombotic syndrome associated with thrombocytopenia of likely autoimmune origin, not observed so far with mRNA-based vaccines, suggesting that the vectors may play a role in eliciting it. Several ele- ments of the Ad vectors and/or of vaccine composition may theoretically interact with platelets, the endothelium and the blood clotting system precipitating this rare com- plication. However, the exact sequence of events leading to the development of this syndrome and, most importantly, the reason why it evolves in only very few subjects with- out apparent predisposing factors remain to be clarified. It is clear that our understanding of the pathogenesis of VITT is far from complete and that more mechanistic studies are required to clarify it and, it is hoped, to iden- tify risk factors predictive of its development. What is quite likely is that the Ad vector-based vaccine triggers an immunological reaction which, for unknown reasons, in some rare subjects involves especially blood platelets, and possibly some peculiar vascular endothelial districts such as those of the cerebral and splanchnic veins, precipitat- ing the catastrophic vaccine-induced autoimmune throm- bocytopenia thrombosis syndrome. Awareness of this condition and the prompt identification/evaluation of affected patients may lead to successful treatment and recovery (Figure 3).13,84 clearly outweigh COVID-19 continues to be a serious global health prob- lem and vaccination against SARS-CoV-2 is the most effective way of limiting illness and death due to the pan- demic. Based on the current available information, and in light of the relative rarity of VITT, the benefits of vacci- nation risks (https://www.ema.europa.eu/en/documents/dhpc/direct-health- care-professional-communication-dhpc-zolgensma-onasemno- gene-abeparvovec-risk-thrombotic_en.pdf). However, once the global pandemic begins to retreat, the relative impor- tance of even small risks will increase,65 making it critical- ly important to understand the mechanisms leading to this ominous thrombotic syndrome, to identify the prog- nostic factors for its development and to define the best management strategies.13 potential the Disclosures No conflicts of interest to disclose. Contributions PG, VDS, AT, RM, SM and FR wrote the review article. Acknowledgments This work was supported in part by grants from Fondazione Cassa di Risparmio di Perugia (#19663 (2020.0508) and FISR 2020 (# 1049) to PG. haematologica | 2021; 106(12) 3043 P. Gresele et al. References 1. Muir KL, Kallam A, Koepsell SA, Gundabolu K. Thrombotic thrombocy- topenia after Ad26.COV2.S vaccination. N Engl J Med. 2021;384(20):1964-1965. 2. Greinacher A, Thiele T, Warkentin TE, Weisser K, Kyrle PA, Eichinger S. Thrombotic after ChAdOx1 nCov-19 vaccination. N Engl J Med. 2021;384(22):2092-2101. thrombocytopenia 3. Schultz NH, Sørvoll IH, Michelsen AE, et al. Thrombosis and thrombocytopenia after ChAdOx1 nCoV-19 vaccination. N Engl J Med. 2021;384(22):2124-2130. 4. Scully M, Singh D, Lown R, et al. Pathologic antibodies to platelet factor 4 after ChAdOx1 nCoV-19 vaccination. N Engl J Med. 2021,384(23):2202-2211. 5. Bayas A, Menacher M, Christ M, Behrens L, Rank A, Naumann M. Bilateral superior ophthalmic vein thrombosis, ischaemic stroke, and immune thrombocytopenia after ChAdOx1 nCoV-19 vaccination. Lancet. 2021;397(10285):e11. 6. Muster V, Gary T, Raggam RB, WÖlfler A, Brodmann M. Pulmonary embolism and thrombocytopenia following ChAdOx1 vaccination. Lancet. 2021;397(10287):1842. 7. Shay DK, Gee J, Su JR, et al. Safety moni- toring of the Janssen (Johnson & Johnson) COVID-19 vaccine - United States, March- April 2021. MMWR Morb Mortal Wkly Rep. 2021;70(18):680-684. 8. Rodeghiero F, Balduini CL. A new enemy is emerging in the fight against the SARS- CoV-2 pandemic. Haematologica. 2021;106 (8):2040-2041. 9. Hwang J, Lee SB, Lee SW. Comparison of events vaccine-induced and between ChAdOx1 Ad26.COV.2.S vaccines. J Autoimmun. 2021;122:102681. thrombotic nCoV-19 10. Sangli S, Virani A, Cheronis N. Thrombosis with thrombocytopenia after the messen- ger RNA-1273 vaccine. Ann Intern Med. 2021;L21-0244. 11. Greinacher A, Thiele T, Warkentin TE, Weisser K, Kyrle P, Eichinger S. A pro- thrombotic thrombocytopenic disorder resembling heparin-induced thrombocy- topenia following coronavirus vaccination. Blood Transfus. 2021; 19(4): 281-283 12. See I, Su JR, Lale A, Woo EJ, et al. US case reports of cerebral venous sinus thrombosis with after thrombocytopenia Ad26.COV2.S vaccination, March 2 to April 21, 2021. JAMA. 2021;325(24):2448- 2456. 13. Gresele P, Marietta M, Ageno W, et al. Management of cerebral and splanchnic vein thrombosis associated with thrombo- cytopenia in subjects previously vaccinated with Vaxzevria (AstraZeneca): a position statement from the Italian Society for the Study of Haemostasis and Thrombosis (SISET). Blood Transfus. 2021;19(4):281- 283. 14. Hsu KH, Lonberg-Holm K, Alstein B, Crowell RL. A monoclonal antibody specif- ic for the cellular receptor for the group B coxsackieviruses. J Virol. 1988;62(5):1647- 1652. 15. Bergelson JM, Cunningham JA, Droguett G, et al. Isolation of a common receptor for coxsackie B viruses and adenoviruses 2 and 5. Science. 1997;275(5304):1320-1323. 16. Bewley MC, Springer K, Zhang YB, Freimuth P, Flanagan JM. Structural analy- sis of the mechanism of adenovirus binding to its human cellular receptor CAR. Science. 1999;286(5444):1579-1583. 17. Roelvink PW, Mi Lee G, Einfeld DA, Kovesdi I, Wicham TJ. Identification of a conserved receptor-binding site on the fiber proteins of CAR-recognizing Adenoviridae. Science. 1999;286(5444):1568-1571. 18. Greber U, Willets M, Webster P, Helenius A. Stepwise dismantling of adenovirus 2 during entry into cells. Cell. 1993;75(3):477- 486. 19. Wickham TJ, Mathias P, Cheresh DA, Nemerow GR. Integrins alpha v beta 3 and alpha v beta 5 promote adenovirus internal- ization but not virus attachment. Cell. 1993;73(2):309-319. 20. Davison E, Diaz RM, Hart IR, Santis G, Marshall JF. Integrin a5b1-mediated aden- ovirus infection is enhanced by the inte- grin-activating antibody TS2/16. J Virol. 1997;71(8):6204-6207. 21. Zhang Y, Bergelson JM. AD receptors. J Virol. 2005;79(19):12125-12131. 22. Honda T, Saitoh H, Masuko M, et al. The coxsackievirus-adenovirus receptor protein as a cell adhesion molecule in the develop- ing mouse brain. Brain Res Mol Brain Res. 2000;77(1):19-28. 23. Patzke C, Max KEA, Behlke J, et al. The coxsackievirus-adenovirus receptor reveals complex homophilic and heterophilic inter- actions on neural cells. J Neurosci. 2010;30(8):2897-2910. 24. Nalbantoglu J, Pari G, Karpati G, et al. Expression of the primary coxsackie and adenovirus receptor is downregulated dur- ing skeletal muscle maturation and limits the efficacy of adenovirus-mediated gene delivery to muscle cells. Hum Gene Ther. 1999;10(6):1009-1019. 25. Noutsias M, Fechner H, de Jonge H, et al. Human coxsackie-adenovirus receptor is colocalized with integrins alpha(v)beta(3) and alpha(v)beta(5) on the cardiomyocyte sarcolemma and upregulated in dilated car- diomyopathy: implications for cardiotropic viral infections. Circulation. 2001;104(3): 275-280. 26. Rebel V, Hartnett S, Denham J, et al. Maturation and lineage-specific expression of the coxsackie and adenovirus receptor in hematopoietic cells. Stem Cells. 2000;18 (13):176-182. 27. Othman M, Labelle A, Mazzetti I, Elbatarny HS, Lillicrap D. Adenovirus- induced thrombocytopenia: the role of von Willebrand factor and P-selectin in mediat- ing accelerated platelet clearance. Blood. 2007;109(7):2832-2839. 28. Shimony N, Elkin G, Kolodkin-Gal D, Krasny L, Urieli-Shoval S, Haviv YS. Analysis of adenoviral attachment to human platelets. Virol J. 2009;6:25. 29. Stone D, Liu Y, Shayakhmetov D, Li ZY, Ni S, Lieber A. Adenovirus-platelet interaction in blood causes virus sequestration to the reticuloendothelial system of the liver. J Virol. 2007;81(9):4866-4871. 30. Griffin JH, Zlokovic BV, Mosnier LO. Activated protein C: biased for translation. Blood. 2015;125(19):2898-2907. 31. Esmon CT. Protein C. Prog Hemost Thromb. 1984;7:25-54. 32. Hamad OA, Nilsson PH, Lasaosa M, et al. Contribution of chondroitin sulfate A to the binding of complement proteins to acti- vated platelets. PLoS One. 2010;5(9): e12889. 33. Dahlbäck B, Wiedmer T, Sims PJ. Binding of anticoagulant vitamin K-dependent pro- tein S to platelet-derived microparticles. Biochemistry. 1992;31(51):12769-12777. F; 34. Ferro JM, Canhão P, Stam J, Bousser MG, Barinagarrementeria ISCVT Investigators. Prognosis of cerebral vein and dural sinus thrombosis: results of the International Study on Cerebral Vein and Dural Sinus Thrombosis (ISCVT). Stroke. 2004;35(3):664-670. 35. Kurosawa S, Stearns-Kurosawa DJ, Hidar N, Esmon CT. Identification of functional endothelial protein C receptor in human plasma. J Clin Invest. 1997;100(2):411-418. 36. Gandrille S. Endothelial cell protein C receptor and the risk of venous thrombosis. Haematologica. 2008;93(6):812-816. 37. Javanmard SH, Shahsavarzadeh T, Saadatnia M. Soluble thrombomodulin and endothelial cell protein C receptor levels in patients with cerebral venous and sinus thrombosis. Eur Neurol. 2013;70(3-4):156- 158. 38. Gould WR, Baxi SM, Schroeder R, et al. Gas6 receptors Axl, Sky and Mer enhance platelet activation and regulate thrombotic responses. Haemost. 2005;3(4):733-741. Thromb J 39. Nidetz NF, Gallagher TM, Wiethoff CM. Inhibition of type I interferon responses by adenovirus serotype-dependent Gas6 bind- ing. Virology. 2018;515:150-157. 40. Allen RJ, Byrnes AP. Interaction of aden- ovirus with antibodies, complement, and coagulation factors. FEBS Letters. 2019;593 (24):3449-3460. 41. Lenman A, Muller S, Nygren MI, Frangsmyr L, Stehle T, Arnberg N. Coagulation factor IX mediates serotype- specific binding of species A adenoviruses to host cells. J Virol. 2011;85(24):13420- 13431. 42. Parker AL, McVey JH, Doctor JH, et al. Influence of coagulation factor zymogens on the infectivity of adenoviruses pseudo- typed with fibers from subgroup D. J Virol. 2007;81(7):3627-3631. 43. Parker AL, Waddington SN, Nicol CG, et al. Multiple vitamin K-dependent coagulation zymogens promote adenovirus-mediated gene delivery to hepatocytes. Blood. 2006;108(8):2554-2561. 44. Shayakhmetov DM, Gaggar A, Ni S, Li, Lieber A. Adenovirus binding to blood fac- tors results in liver cell infection and hepa- totoxicity. J Virol. 2005;79(12):7478-7491. 45. Kalyuzhniy O, Di Paolo NC, Silvestry M, et al. Adenovirus serotype 5 hexon is critical for virus infection of hepatocytes in vivo. Proc Natl Acad Sci U S A. 2008;105(14): 5483-5488. 46. Duffy MR, Doszpoly A, Turner G, Nicklin SA, Baker AH. The relevance of coagula- tion factor X protection of adenoviruses in human sera. Gene Ther. 2016;23(7):592- 596. 47. Greinacher A, Selleng K, Wesche J, et al. Towards understanding ChAdOx1 nCov- 19 vaccine-induced immune thrombotic thrombocytopenia (VITT). Research Square. 2021 Apr 20. [Epub ahead of print] 48. Brunetti-Pierri N, Palmer DJ, Beaudet AL, Carey KD, Finegold M, Ng P. Acute toxicity injection of after high-dose systemic helper-dependent adenoviral vectors into nonhuman primates. Hum Gene Ther. 2004;15(1):35-46. K. Post-adenoviral-based COVID-19 vaccines thrombosis: a pro- posed mechanism. J Thromb Haemost. 2021;19(7):1831-1832. 49. Kadkhoda 50. Lee WS, Wheatley AK, Kent SJ, DeKosky BJ. Antibody-dependent enhancement and SARS-CoV-2 vaccines and therapies. Nat 3044 haematologica | 2021; 106(12) Microbiol. 2020;5(10):1185-1191. 51. Hamada F, Aoki M, Akiyama T, Toyoshima K. Association of immunoglobulin G Fc receptor II with Src like protein-tyrosine kinase Fgr in neutrophils. Proc Natl Acad Sci U S A. 1993;90(13):6305-6309. 52. Ghazizadeh S, Bolen JB, Fleit HB. Physical and functional association of Src-related protein tyrosine kinases with Fc gamma RII in monocytic THP-1 cells. J Biol Chem. 1994;269(12):8878-8884. 53. Kiener PA, Rankin BM, Burkhardt AL, et al. Cross-linking of Fc gamma receptor I (Fc gamma RI) and receptor II (Fc gamma RII) on monocytic cells activates a signal trans- duction pathway common to both Fc receptors that involves the stimulation of p72 Syk protein tyrosine kinase. J Biol Chem. 1993;268(32):24442-24448. 54. Nimmerjahn F, Ravetch JV. Fcγ receptors as regulators of immune responses. Nat Rev Immunol. 2008;8(1):34-47. 55. Joshi T, Butchar JP, Tridandapani S. Fcg receptor signaling in phagocytes. Int J Hematol. 2006;84(3):210-216. 56. Furuyama W, Marzi A, Carmody AB, et al. Fcg-receptor IIa-mediated Src signaling pathway is essential for the antibody- dependent enhancement of Ebola virus infection. PLoS Pathog. 2016;12(12): e1006139. shock 57. Taylor SM, Reilly MP, Schreiber AD, Chien P, Tuckosh JR, McKenzie SE. Thrombosis and activating antiplatelet antibodies in human Fc gamma RIIA transgenic mice: the interplay among antibody, spleen, and Fc receptor. Blood. 2000;96(13):4254-4260. induced by 58. Calverley DC, Brass E, Hacker MR, et al. Potential role of platelet FcgammaRIIA in col- lagen-mediated platelet activation associated with atherothrombosis. Atherosclerosis. 2002;164(2):261-267. 59. Pamela S, Anna Maria L, Elena D et al. Heparin-induced thrombocytopenia: the role of platelets genetic polymorphisms. Platelets. 2013;24(5):362-368. 60. Tetro JA. Is COVID-19 receiving ADE from other coronaviruses? Microbes Infect. 2020;22(2):72-73. 61. Collocca S, Barnes E, Folgori A, et al. Vaccine vectors derived from a large collec- tion of simian adenoviruses induce potent cellular immunity across multiple species. Sci Transl Med. 2012;4(115):115ra2. 62. Obdulio GN, V'kovski V, Zettl F, Zimmer G, Thiel V, Summerfield A. No evidence for human monocyte-derived macrophage infection and antibody-mediated enhance- ment of SARS-CoV-2 Infection. Front Cell Infect Microbiol. 2021;11:644574. 63. Crystal RG, Harvey BG, Wisnivesky JP, et al. Analysis of risk factors for local delivery of low- and intermediate-dose adenovirus gene transfer vectors to individuals with a spectrum of comorbid conditions. Hum Gene Ther. 2002;13(1):65-100. 64. Folegatti PM, Bittaye M, Flaxman A, et al. Safety and immunogenicity of a candidate Middle East respiratory syndrome coron- avirus viral-vectored vaccine: a dose-escala- tion, open-label, non-randomised, uncon- trolled, phase 1 trial. Lancet Infect Dis. 2020;20(7):816-826. 65. Kupferschmidt K, Vogel G. What’s the future of vaccines linked to rare clotting disorders? Science breaks down the latest. 2021 May 3. [Epub ahead of print] 66. Jaoko W, Karita E, Kayitenkore K, et al. Safety and immunogenicity study of multi- clade HIV-1 Adenoviral vector vaccine alone or as boost following a multiclade HIV-1 DNA vaccine in Africa. PLoS One. 2010;5(9):e12873. 67. Keefer MC, Gilmour J, Hayes P, Gill D, et al. A phase I double blind, placebo-con- trolled, randomized study of a multigenic HIV-1 adenovirus subtype 35 vector vac- cine in healthy uninfected adults. PLoS One. 2012;7(8):e41936. 68. Rego GNA, Nucci MP, Alves AH, et al. Current clinical trials protocols and the global effort for immunization against SARS-CoV-2. Vaccines (Basel). 2020;8(3): 474. in 69. Van Doremalen N, Lambe T, Spencer A, et al. ChAdOx1 nCoV-19 vaccination prevents SARS-CoV-2 rhesus pneumonia macaques. Nature. 2020;586(7830):578-582. 70. Folegatti PM, Ewer KJ, Aley PK, et al. Oxford COVID Vaccine Trial Group. Safety and the ChAdOx1 nCoV-19 vaccine against SARS- CoV-2: a preliminary report of a phase 1/2, single-blind, randomised controlled trial. Lancet. 2020;396(10249):467-478. immunogenicity of 71. Sandoff J, Gray G, Vandebosch A, et al. Safety and efficacy of single-dose Ad26.COV2.S vaccine against Covid-19. N Engl J Med. 2021;384(23):2187-2201. 72. Logunov DY, Dolzhikova IV, Shcheblyakov DV, et al. Safety and efficacy of an rAd26 and rAd5 vector-based heterologous prime- boost COVID-19 vaccine: an interim analy- sis of a randomized controlled phase 3 trial in Russia. Lancet. 2021;397(10275):671- 681. 73. Pottegård A, Lund LC, Karlstad Ø, et al. Arterial events, venous thromboembolism, thrombocytopenia, and bleeding after vac- cination with Oxford-AstraZeneca ChAdOx1-S in Denmark and Norway: population based cohort study. BMJ. 2021;May 5;373:n1114. 74. Marcucci R, Berteotti M, Gori AM, et al. Heparin induced thrombocytopenia: posi- tion paper from the Italian Society on Thrombosis and Haemostasis (SISET). Blood Transfus. 2021;19(1):14-23. 75. Cuker A, Arepally GM, Chong BH, et al. American Society of Hematology 2018 guidelines for management of venous thromboembolism: heparin-induced thrombocytopenia. Blood Adv. 2018;2(22): 3360-3392. 76. Greinacher A, Farner B, Kroll H, et al. Clinical features of heparin-induced throm- bocytopenia including risk factors for thrombosis. A retrospective of 408 patients. Thromb Haemost. 2005;94(1): 132-135. 77. Greinacher A, Selleng K, Warkentin TE. Autoimmune heparin-induced thrombocy- topenia. J Thromb Haemost. 2017;15(11): Adenovirus vaccines and thrombosis 2099-2114. 78. Warkentin TE, Greinacher A. Spontaneous HIT syndrome: knee replacement, infec- tion, and parallels with vaccine-induced immune thrombotic thrombocytopenia. Thromb Res. 2021;204:40-51. 79. Huynh A, Kelton JG, Arnold DM, Daka M, Nazy I. Antibody epitopes in vaccine- induced immune thrombotic thrombocy- topenia. Nature. 2021;596(7873):565-569. 80. Favaloro EJ. Laboratory testing for suspect- ed COVID-19 vaccine-induced (immune) thrombotic thrombocytopenia. Int J Lab Hematol. 2021;43(4): 559-570 81. Vayne C, Guery EA, Kizlik-Masson C, et al. Beneficial effect of exogenous platelet fac- tor 4 for detecting pathogenic heparin- induced thrombocytopenia antibodies. Br J Haematol. 2017;179(5):811-819. 82. Platton S, Bartlett A, MacCallum P, et al. Evaluation of laboratory assays for anti- platelet factor 4 antibodies after ChAdOx1 nCOV-19 vaccination. J Thromb Haemost. 2021;19(8):2007-2013. 83. Cines DB, Bussel JB. SARS-CoV-2 Vaccine- induced immune thrombotic thrombocy- topenia. N Engl J Med. 2021;384(23):2254- 2256. 84. Sørvoll IH, Horvei KD, Ernstsen SL, et al. An observational study to identify the prevalence of thrombocytopenia and anti- PF4/polyanion antibodies in Norwegian health care workers after COVID-19 vacci- nation. J Thromb Haemost. 2021;19(7): 1813-1818. 85. Thiele T, Ulm L, Holtfreter S, et al. Frequency of positive anti-PF4/polyanion antibody tests after COVID-19 vaccination with ChAdOx1 nCoV-19 and BNT162b2. Blood. 2021;138(4):299-303. 86. Kowarz E, Krutzke L, Reis J, Bracharz S, Kochanek S, Marschalek R. Vaccine- induced Covid-19 spike open reading frame results in spike protein variant that may cause thromboembolic events in patients immunized with vector-based vac- cine. Research Square. 2021 May 26. [Epub ahead of print] 87. Lei Y, Zhang J, Schiavon CR, et al. SARS- CoV-2 spike protein impairs endothelial function via downregulation of ACE 2. Circ Res. 2021;128(9):1323-1326. 88. Chakraborty S, Gonzalez J, Edwards K. Proinflammatory IgG Fc structures in patients with severe COVID-19. Nat Immunol. 2021;22(1):67-73. 89. Douxfils J, Dogné J, Favresse JM. Hypotheses behind the very rare cases of thrombosis with thrombocytopenia syn- drome after SARS-CoV-2 vaccination. Thromb Res. 2021;203:163-171. 90. Shen S, Zhang J, Fang Y, et al. SARS-CoV-2 interacts with platelets and megakary- ocytes via ACE2-independent mechanism. J Hematol Oncol. 2021;29;14(1):72. 91. Millington-Burgess SL, Harper MT. A dou- ble-edged sword: antibody-mediated pro- coagulant platelets in COVID-19. Platelets. 2021; 32(5):579-581. 92. Choi PHI. Thrombotic thrombocytopenia after ChAdOx1 nCoV-19 vaccination. N Engl J Med. 2021;385(3):e11. haematologica | 2021; 106(12) 3045