European Heart Journal (2021) 42, 4064–4072
doi:10.1093/eurheartj/ehab506

CLINICAL RESEARCH
Thrombosis and antithrombotic treatment

Immune complexes, innate immunity, and
NETosis in ChAdOx1 vaccine-induced
thrombocytopenia
Sverre Holm1†, Hassen Kared2,3,4†, Annika E. Michelsen
1,5, Nina H. Schultz6,7, Tuula A. Nyman
Tuva B. Dahl
Ingebjørg Seljeflot
Guro Løvik Goll12, Lise Sofie Nissen-Meyer3, Pa˚l Aukrust1,4,13, Karolina Skagen14,
Ida Gregersen1, Mona Skjelland
and Bente Halvorsen

4,9, Thor Ueland1,4,10, Maria Stensland3,4, Siri Mjaaland11,

1,4†, Xiang Yi Kong
3,4, Cathrine Fladeby8,

4,14, Pa˚l A. Holme1,4,6*‡, Ludvig A. Munthe

1,4‡

1,

2,3,4‡,

1Research Institute of Internal Medicine, Oslo University Hospital, Postbox 4950, 0424 Oslo, Norway; 2KG Jebsen Centre for B Cell Malignancies, University of Oslo, Postbox
4950, 0424 Oslo, Norway; 3Department of Immunology, Oslo University Hospital, Postbox 4950, 0424 Oslo, Norway; 4Institute of Clinical Medicine, University of Oslo,
Postbox 1171, Blindern 0318 Oslo, Norway; 5Division of Emergencies and Critical Care, Department of Research and Development, Oslo University Hospital, Postbox 4950,
N-0424 Oslo, Norway; 6Department of Haematology, Oslo University Hospital, Postbox 4950, N-0424 Oslo, Norway; 7Department of Haematology, Akershus University
Hospital, Postbox 1000, 1478 Lørenskog, Norway; 8Department of Microbiology, Oslo University Hospital, Postbox 4950, N-0424 Oslo, Norway; 9Department of Cardiology,
Oslo University Hospital, Postbox 4950, N-0424 Oslo, Norway; 10Faculty of Health Sciences, K.G. Jebsen TREC, University of Tromsø, Postbox 6050, Langnes 9037 Tromsø,
Norway; 11Division of Infection Control and Environmental Health, Norwegian Institute of Public Health, Postbox 222, Skøyen, 0213 Oslo, Norway; 12Division of
Rheumatology and Research, Diakonhjemmet Hospital, Postbox 23 Vindern, 0319 Oslo, Norway; 13Section of Clinical Immunology and Infectious Diseases, Oslo University
Hospital, Postbox 4950, N-0424 Oslo, Norway; and 14Department of Neurology, Oslo University Hospital, Postbox 4950, N-0424 Oslo, Norway

Received 9 June 2021; revised 25 June 2021; editorial decision 13 July 2021; accepted 6 August 2021; online publish-ahead-of-print 18 August 2021

See page 4073 for the editorial comment for this article ‘Thromboinflammatory findings and clinical predictors of mortality in vaccine-
induced immune thrombotic thrombocytopenia’, by J.M. Connors, https://doi.org/10.1093/eurheartj/ehab585.

Aims

We recently reported five cases of vaccine-induced immune thrombotic thrombocytopenia (VITT) 7–10 days after
receiving the first dose of the ChAdOx1 nCoV-19 adenoviral vector vaccine against corona virus disease 2019
(COVID-19). We aimed to investigate the pathogenic immunological responses operating in these patients.

...................................................................................................................................................................................................
Methods and
We assessed circulating inflammatory markers by immune assays and immune cell phenotyping by flow cytometry
results
analyses and performed immunoprecipitation with anti-platelet factor (PF)4 antibody in plasma samples followed by
mass spectrometry from all five patients. A thrombus was retrieved from the sinus sagittal superior of one patient
and analysed by immunohistochemistry and flow cytometry. Precipitated immune complexes revealed multiple in-
nate immune pathway triggers for platelet and leucocyte activation. Plasma contained increased levels of innate im-
mune response cytokines and markers of systemic inflammation, extensive degranulation of neutrophils, and tissue
and endothelial damage. Blood analyses showed activation of neutrophils and increased levels of circulating H3Cit,
dsDNA, and myeloperoxidase–DNA complex. The thrombus had extensive infiltration of neutrophils, formation of
neutrophil extracellular traps (NETs), and IgG deposits.

...................................................................................................................................................................................................
Conclusions
The results show that anti-PF4/polyanion IgG-mediated thrombus formation in VITT patients is accompanied by a
massive innate immune activation and particularly the fulminant activation of neutrophils including NETosis. These
results provide novel data on the immune response in this rare adenoviral vector-induced VITT.

(cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2) (cid:2)

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* Corresponding author. Tel: þ47 23070460, Email: pholme@ous-hf.no
† These authors shared first authorship.
‡ These authors shared senior authorship.
VC The Author(s) 2021. Published by Oxford University Press on behalf of the European Society of Cardiology.
This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (http://creativecommons.org/licenses/by-nc/4.0/),
which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact
journals.permissions@oup.com

ICs, innate immunity, and NETosis

4065

Graphical Abstract

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Keywords

Vaccine-induced immune thrombotic thrombocytopenia (cid:129) Thrombus (cid:129) Immune activation (cid:129) Neutrophils

Introduction

On March 11, after 132 686 individuals in Norway had received the
first dose of the ChAdOx1 nCoV-19 adenoviral vector vaccine
against COVID-19,1 this vaccination was stopped following reports
from Denmark of a possible connection between the vaccine and
fatal cases of thrombosis.2 Within 10 days after vaccination with
ChAdOx1 nCov-19, five healthcare workers from 32 to 54 years of
age were admitted to Oslo University Hospital with severe cases of
thrombosis,
thrombocytopenia, remarkably high titres of anti-
platelet factor (PF)4/polyanion IgG, and atypical, hyperactive platelet
aggregation assays. Three of the five patients died, of what has been
termed as vaccine-induced immune thrombotic thrombocytopenia
(VITT) in two previous reports.3,4 More recently, Scully et al.5
reported 23 similar cases with severe thrombotic episodes 6–24 days
following ChAdOx1 nCoV-19 vaccination. Based on reported cases
of VITT from nine countries with moderate-to-high data quality, the
estimated risk of developing VITT after the first dose of ChAdOx1
nCoV-19 ranges from 1 case per 26 500 to 1 case per 148 200.6 All

...........................................................

these studies have identified a pathogenic anti-PF4/polyanion-de-
pendent syndrome, unrelated to the use of heparin therapy.
However, the molecular triggers are not fully elucidated, and detailed
examination of the thrombus has been lacking. Here, we describe im-
mune complexes (ICs) that incorporated multiple triggers of innate
immunity, and inflammatory and thrombogenic profiles in five VITT
patients admitted to Oslo University Hospital. This includes the com-
position of sinus venous thrombi retrieved from one of the patients
with fatal outcome.

Methods

Patients
In brief, the patients were five healthcare workers from 32 to 54 years
old: one man and four women. They were admitted to Oslo University
Hospital with thrombosis and thrombocytopenia 7–10 days after vaccin-
ation with ChAdOx1 nCoV-19. Four of the patients had major cerebral
haemorrhage and three of the patients died. Clinical data are summarized
in Supplementary material online, Table S1 and have previously been

4066

S. Holm et al.

described.3 Blood samples from health careworkers of both sexes, in the
same age group as the patients, were used as controls for plasma studies
(unvaccinated n = 11, ChAdOx1 nCov-19 vaccinated n = 8). Controls for
flow cytometry analysis of blood cells included samples from non-
vaccinated blood bank donors (n = 11) and vaccinated healthcare work-
ers (n = 8).

Ethical considerations
Written informed consent for publication was obtained from all patients
and donors. The study and all control and reference material were
approved by Regional Committees for Medical and Health Research
(2014/2078; 2020/135924, 2020/175588, 2021/235424). In the event that
the patient was not able to provide consent, family guardians provided
written informed consent.

Further description of methodology is found in the Supplementary ma-

terial online.

Results

Analysis of immune complexes
The spontaneous aggregation of platelets by anti-PF4/polyanion IgG
patient samples3 demonstrated that circulating IC could activate pla-
telets directly in the absence of added heparin. We precipitated IC
with anti-PF4 antibodies and analysed for protein content. Analysis
revealed the expected antibodies and PF4, and principal component
analysis of the mass spectrometry data showed that the patient group
clearly separated from the healthy vaccinated or non-vaccinated con-
trols (Figure 1A). In addition, the IC contained multiple innate immune
pathway activators (Figure 1B) that included complement pathway
(classical pathways: C1s, C2, lectin pathway; MBL: MASP2), the acute
phase C-reactive protein (CRP) that also can activate the classical
complement pathway, nucleosomes/chromatin [histones and toll-like
receptor
cathelicidin antimicrobial peptide
(CAMP)], lipopolysaccharide-binding protein (LBP), a co-activator of
CD14, and the platelet activator von Willebrand factor (VWF) that is
released upon endothelial cell activation. The IC thus included ligands
for innate immune response pathways including FccR, PF4 receptors,
complement receptors, CD14, and platelet thrombosis—glycopro-
tein Ib (GPIb). Proteoglycan 4 contains glycosaminoglycan (GAG)
chondroitin sulphate and keratan sulphate polyanions and may po-
tentially serve as a PF4 ligand. Leucine-rich alpha-2-glycoprotein is
expressed mainly in granula of neutrophils. Kinesin-like protein
(KIF25) is a cytosolic microtubule-dependent motor protein that
would require release via necrosis or potentially also neutrophil
extracellular trap (NET)osis.

(TLR)9 cofactor

We also found serine-protease inhibitors (serpin family A mem-
bers) 10, 3, and 1 that may have anti-coagulative effects (see
Discussion). The acute phase proteins, serum amyloid A-1 and A-2
proteins (SAA1, SAA2), bleomycin hydrolase, cysteine proteinase
inhibitors (CST1 or 4), inter-alpha-trypsin inhibitor heavy chain H3 (a
hyaluronan binder), cholesteryl ester transfer protein, and the apoli-
poprotein, apolipoprotein A-V (APOA5), have unclear consequence
and/or function in terms of the IC. No peptides from the viral vector
(chimpanzee adenovirus serotype Y25) and no SARS-CoV-2 spike
peptide sequences were detected.

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Figure 1 Analysis of immune complexes using immunoprecipita-
tion and mass spectrometry. (A) Principal component analysis of the
protein identification and quantification. Patient samples are marked
with red, healthy vaccinated controls are marked with blue, and
non-vaccinated controls are marked with green. (B) Heatmap of
the proteins that were more abundant (P < 0.05) in patient samples
compared to vaccinated controls.

............................................

Inflammatory markers
To reveal the potential effects of the IC, we analysed inflamma-
tory markers in all plasma samples. Patients had significantly ele-
vated levels of proinflammatory cytokines [interleukin (IL)-6, IL-
18], signs of monocyte activation (release of sCD163, sCD14),
neutrophil degranulation [myeloperoxidase (MPO)] or both
fpentraxin-related protein (PTX3), S100A8/A9, YKL-40 [chiti-
nase-3-like protein 1 (CHI3L1)]g, endothelial cell activation
(P-selectin), markers of tissue damage [growth differentiation
factor (GDF)-15 and cfDNA], and systemic inflammation (LBP)
compared to healthy vaccinated or non-vaccinated controls.
Three markers of NETosis, citrullinated histone H3 (H3Cit),
MPO–DNA complexes, and circulating dsDNA were also signifi-
cantly increased (Figure 2A).

ICs, innate immunity, and NETosis

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Figure 2 Characteristics of immune mediators in plasma from vaccine-induced immune thrombotic thrombocytopenia patients. Biomarkers in
plasma from patients (P, n = 5), unvaccinated controls (HC, n = 11), and healthy vaccinated controls (HV, n = 8). (A) Violin plots of biomarkers, dashed
lines indicate median and interquartile range. P-values indicate comparisons between HC and P. (B) Correlogram showing Pearson’s correlation be-
tween all plasma molecules detected in non-vaccinated and healthy/non-healthy vaccinated donors. A heat scale where red colour shows positive lin-
ear correlation coefficients, and blue colour shows negative linear correlation. The size of the dot corresponds to the r correlation value and the
significant correlations are indicated with stars (*P < 0.05, **P < 0.01, P-values were adjusted for multiple testing using the Bonferroni method).

4068

S. Holm et al.

Multi-parameter analysis was performed to find significant correla-
tions. Pearson’s correlation analysis showed that the alarmins
(S100A8/A9) significantly correlated with markers of platelet de-
granulation (PF4, platelet-derived growth factor-DD), endothelial
damage (P-selectin) and NETosis (dsDNA and H3Cit; Figure 2B). We
combined these parameters in a Volcano plot, which establish a hier-
archy among our markers and highlighted the importance of the alar-
mins S100A8/A9, P-selectin, MPO (neutrophil marker), and PTX3
(potentiation of complement) for VITT (Supplementary material on-
line, Figure S1). The same markers together with GDF-15 (associated
with tissue damage) correlated significantly with fatal outcome among
the hospitalized individuals (Supplementary material online, Figure
S1).

Activation of blood leucocytes
Flow cytometry analysis of whole blood from the patients demon-
strated an increased frequency of activated (HLA-DRþ and CD38þ)
and immature neutrophil subpopulations that had decreased expres-
sion of CD15, CD16, CD66b, CD101, and CD107,8 (Figure 3A and
Supplementary material online, Figure S2). This expansion of imma-
ture neutrophil was not observed in healthy vaccinated donors. In
two of the patients, intravenous immunoglobulin treatment was asso-
ciated with normalized cell count, neutrophil/lymphocyte ratio, and
neutrophil phenotype, as illustrated by the follow-up time points
showing a similar profile as the healthy controls (Figure 3B and
Supplementary material online, Figure S3). In contrast, the flow analy-
ses did not reveal activation of T cells or isotype class switching for B
cells in VITT patients compared to controls.

........................................................................................

Analysis of cerebral sinus sagittal
superior thrombus
We next analysed the retrieved thrombus from one of the patients
who died (Patient 5) by immunohistochemistry (IHC) and flow
cytometry. Microscopic and flow analyses revealed unusual thrombi
with very high cellularity, massive polymorphonuclear leucocytes
(PMN) infiltrates (Figure 4A), platelets and neutrophils, IgG deposits
(Figure 4B), PMN degranulation (S100A8 and S100A9, Figure 4C), and
extensive neutrophil activation characterized by decondensated
chromatin (NETosis) and NET formation, as shown by the positive
staining of citrullinated histone H3 (CitH3), and elastase (Figure 4D).
No reliable level of spike protein was detected by IHC.

Flow cytometry analysis revealed that nearly 75% of infiltrating
cells were PMN (in comparison to 10% in blood from the same pa-
tient). Among non-granulocytes, the main population detected in the
clot was antigen-presenting cells (HLA-DRþCD3-). B cells were
barely detectable in the clot, which was thus essentially composed of
classical monocytes (CD14þCD16þ). The relative proportion of T
cells was decreased due to the increased frequency of APC in non-
granulocytes derived from the clot, but the proportion of CD4 and
CD8 or the activation, differentiation, and exhaustion status were
not altered (Figure 4E and Supplementary material online, Figure S4).

Discussion

We here describe the composition of PF4-containing IC in patients
with severe VITT, and demonstrate multiple ligands for innate im-
mune receptors as illustrated in the Graphical abstract. This was linked

Figure 3 Immune phenotype of vaccine-induced immune thrombotic thrombocytopenia patients. Analysis of 5 cases and 12 vaccinated healthy
control blood samples at three time points. (A) Peripheral blood of healthy donors and cryo-preserved PBMC from vaccine-induced immune throm-
botic thrombocytopenia patients were surface stained to characterize low-density neutrophils. After the removal of debris and dead cells based on
FSC-A and SSC-A, singlets were gated for haematopoietic cells. Granulocytes were defined as CD45þSSCHiCD66bþCCR3-CD123- (see also
Supplementary material online, Figure S2 for gating). (B) Longitudinal follow-up of neutrophils in surviving vaccine-induced immune thrombotic
thrombocytopenia patients. The expression of maturation markers on neutrophils was compared between healthy donors (HD, top panel) and fatal
vaccine-induced immune thrombotic thrombocytopenia (Patients 2 and 5, top) or in longitudinal samples for non-fatal cases (Cases 3 and 4 at Day 7,
Day 14 or 20 for Time-points 2 and 3, respectively). The intensity of each marker was normalized in the overlaid histograms. See also Supplementary
material online, Figure S4 for hierarchical clustering of all time points.

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ICs, innate immunity, and NETosis

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Figure 4 Thrombus from vaccine-induced immune thrombotic thrombocytopenia patients is rich in neutrophils and stain positive for IgG and neu-
trophil extracellular traps. (A) Haematoxylin and eosin staining of vaccine-induced immune thrombotic thrombocytopenia and control thrombus at
100(cid:2) and 400(cid:2) magnifications. (B) Immunofluorescent staining of IgG (red) in vaccine-induced immune thrombotic thrombocytopenia—control
venous thrombus and control cerebral arterial thrombus. (C) Double immunofluorescent staining of S100A8 (red) and S100A9 (green) in vaccine-
induced immune thrombotic thrombocytopenia thrombus and control venous thrombus; the lower panels are magnifications of selected parts of the
original image as indicated, showing the presence of the S100A8/A9 complex outside of cytosol in vaccine-induced immune thrombotic thrombo-
cytopenia thrombus. (D) Double immunofluorescent staining of elastase (green) and citrullinated histone H3 (red) in vaccine-induced immune
thrombotic thrombocytopenia thrombus; the image to the right is a magnification of the original image as indicated. DAPI serves as a nuclear DNA
counterstain (blue). Scale bars indicate 100 mm. (E) Single-cell suspension was made from the blood clot and analysed by flow cytometry, presented
as UMAP plots showing the clustering of all haematopoietic immune cells by tissue origin. Cells composing the clot occupy the upper right region of
the two-dimensional map. Clustering is based on the expression of all phenotypic markers assessed. Granulocytes can be identified as SSC-AHI
CD45low cells. Automatic clustering was generated by Phenograph. (F) Cluster quantification in samples derived from the fatal Case 5. A heat map
represents the frequency of each cluster. See also UMAP phonograph, Supplementary material online, Figure S4.

4070

S. Holm et al.

to a proinflammatory immune profile in all patients analysed, includ-
ing cytokines that trigger innate immunity with massive activation of
neutrophils in the circulation. We also found extensive neutrophil ac-
tivation and NET formation in venous sinus sagittal superior thrombi
from one of the patients with fatal outcome.

The present study highlights the role of innate immune responses
in VITT that includes an unusual fulminant focal neutrophil activation
in cell-rich thrombi as well as systemic activation of leucocytes and
circulating cytokines, free nucleic acids, and acute phase reactants.
Increased levels of alarmins S100A8 and S100A9 were observed
both in circulation and in the sinus thrombus. These constitute 45%
of cytosolar proteins in neutrophils9 and after secretion are potent
triggers of innate inflammatory cytokines such as IL-6 that we found
to be increased in the plasma of VITT patients.

An important finding in this study was the massive NET formation at
the site of thrombus formation in the one patient who died. All five
patients also have indirect signs of NET formation in peripheral blood
(H3Cit, dsDNA, MPO–DNA complex) as opposed to healthy con-
trols and vaccinated healthcare workers without signs of thrombus
formation. Neutrophil extracellular traps in interaction with platelets
are involved in thrombus formation during severe COVID-19 disease
and have also been seen during atherothrombus formation10,11 and
have been detailed in heparin-induced thrombocytopenia patients.12,13
However, the massive immune infiltration including high numbers of
neutrophils and related inflammatory molecules, as observed in these
patients, is unusual.

Mass spectrometry analysis of

immunoprecipitated PF4 from
plasma identified PF4 in complex with proteins such as histones, com-
plement pathway factors, GAG-binding lectins, and antibodies, sug-
gesting several inflammatory pathways involved. These are ligands for
a number of pathways that can explain the observations of marked in-
flammatory responses in these patients.

Although the mechanisms are not yet clear, adenovirus can acti-
vate platelets in a VWF and P-selectin14 and alternative complement
pathway dependent manner.15,16 It is thus plausible that the adeno-
viral vector itself activated platelets and provided a first trigger for
PF4 secretion. PF4/polyanion is a chemokine and can in itself activate
leucocytes via the CXCR3-B splice variant of CXCR3 and CCR1.17–
19 Platelets express CCR1 that can trigger aggregation,20 but the ef-
fect of PF4/GAG on platelet CCR1 is unclear. PF4 is secreted from a-
granules of platelets as a tetrameric complex bound to serglycin
GAG chains.21 The GAG serve as polyanion ligands for cationic
amine acid side chains of PF4. The complex includes four PF4 and
two GAGs, these may be chondroitin sulphate or dermatan sulphate.
After secretion, PF4 may bind other ligands with higher affinity, such
as endothelial-derived perlecan heparan sulphate side chains,22 or
other polyanions.

It is thought that the polyanions allow conformational changes in
PF4 exposing antigenic determinants for anti-PF4 IgG antibodies.23
Our patients all had high titres of anti-PF4/polyanion IgG and platelet
aggregation could not be abolished by high dose heparin in the major-
ity of patients.3 This indicated an unidentified non-heparin polyanion
in the patients. The anti-PF4 IgG constitutes a second trigger as plate-
lets express FccRIIA (CD32a). FccRIIA is a low-affinity Fc receptor
for the constant region of IgG that recognizes ICs with high avidity
and causes platelet aggregation and dense granule exposed release,24
exposure of phosphatidylserine, complement factor deposition, and

.............................................................................................................................................................................

accelerated prothrombinase activity.25 The IC included classical com-
plement pathway initiator C1qrs, CRP and the lectin pathway activa-
tors MBL–MASP1–MASP2 that typically responds to lectins similar to
the polyanion GAGs associated with PF4. MASP1 and MASP2 cleave
prothrombin, convert fibrinogen to fibrin monomers, activate factor
XIII, and promote fibrin cross-linking.26 von Willebrand factor was
also found in the IC and provides the ligand for GPIb on platelets, a
receptor that triggers platelet aggregation and degranulation.

Neutrophils express FccRIIA and the complement receptor
C5AR1 (for C5a) that activates degranulation and NETosis.27 The IC
also included histones, i.e. nucleosomes as well as CAMP, the cofac-
tor for human DNA/chromatin binding of TLR928 required for
inflammasome activation in myeloid cells. It should be noted that PF4
can bind and aggregate DNA (as a polyanion) and amplify TLR9 sig-
nalling by organizing fragmented DNA into liquid-crystalline ICs with
inter-DNA spacings optimal for TLR9 amplification.29 This suggests a
positive feedback loop that would be further stabilized by anti-PF4
IgG. Thus, it is likely that the FccRIIA, C5AR1, and TLR9 signalling
converged in a massive activation of neutrophils in the patients.

NET and thrombus formation could potentially be induced by
spike protein in the vaccine, but we could not detect any spike pro-
tein in plasma from the VITT patients or within the thrombus by IHC
or IP of spike followed by targeted mass spectrometry.

Apart from reduced fibrinogen levels, the patients did not display
convincing signs of disseminated intravascular coagulation. This may
seem strange given the range of pro-coagulant activators described
Immune complex, however, also included three serine-
above.
protease inhibitors (serpin family A members). SerpinA10 (protein Z-
dependent protease inhibitor) binds heparin and together with protein
Z inhibits coagulation protease factor Xa and Xia,30,31 serpinA3 (alpha-
1-antichymotrypsin) inhibits neutrophil cathepsin G (and thereby
inhibits conversion of angiotensin-I to the active angiotensin-II).
SerpinA1 (alpha-1-antitrypsin) has been described in NETs,32 inhibits
neutrophil elastase, and can also inhibit thrombin.33

In summary, we suggest that VITT is caused by a preliminary direct
activation of platelets by the ChAdOx1 nCoV-19 adenoviral vector vac-
cine,14–16 degranulation and PF4/GAG secretion, exchange of GAG
with an unknown polyanion, generation of IgG against PF4/polyanion,3–5
IgG-PF4/polyanion IC formation, secondary activation of platelets via IC
IgG/FccRIIA24, addition of complement factors and VWF to IC, addition
of DNA/nucleosomes/CAMP to IC, activation of neutrophils via mul-
tiple pathways27–29, NETosis, and further massive platelet activation.25,26
However, the permissive factors that allow these events and why
this is only seen in a minority of ChAdOx1 nCoV-19-vaccinated indi-
viduals are still not clear.

Supplementary material

Supplementary material is available at European Heart Journal online.

Acknowledgements
The authors thank Ellen Lund Sagen and Katrine Persga˚rd Lund for
technical assistance. The graphical abstract was made using modified
elements from Servier Medical Art images, www.smart.servier.com

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ICs, innate immunity, and NETosis

4071

Funding
This study was supported by the South-Eastern Norway Regional Health
Authority (2021071 and 29286) and the Core Facilities program;
National network of Advanced Proteomics Infrastructure (NAPI)—the
Research Council of Norway (RCN)
(295910) and RCN Covid
(312693); a KG Jebsen Foundation (grant 19); and the Coalition for
Epidemic Preparedness Innovations (CEPI); and the University of Oslo
and Oslo University Hospital.

Conflict of interest: N.H.S. has received honoraria from Pfizer, BMS,
and Bayer for lectures about management of anticoagulation treatment
and bleeding, and for participation in an advisory board on the same
topic. B.H. has a licenced patent (sCD36 as biomarker of atherosclerosis
and metabolic disorder) and a pending patent application (Cetoleic
acids—health effects). P.A. and M.S. have a licenced patent (sCD36 as
biomarker of atherosclerosis and metabolic disorder). M.S. has received
meeting honoraria paid from Bayer in 2019 (not relevant for this manu-
script). K.S. has received a personal fee from Bayer. L.A.M. has received
honoraria from pharma companies for two lectures last 3 years and has
no financial
interests. H.K. was a shareholder and employee of
ImmunoScape Pte Ltd in 2019–2020. G.L.G. has received honoraria from
AbbVie, Biogen, Eli Lilly, Novartis, Pfizer, Roche, Sandoz, Orion Pharma,
Celltrion, and Boehringer Ingelheim for lectures and advisory committees
the last 3 years and has no financial interests. A.E.M. has stock ownership
in Pfizer. P.A.H. has received research grants from Bayer, Pfizer, SOBI,
Roche within area of bleeding disorders to institution (not personal), lec-
ture honoraria, advisory boards in the area of bleeding disorders from
Takeda, SOBI, Bayer, Pfizer, Roche, Octapharma, NovoNordisk, CSL,
BMS, support for attending meetings from Takeda, Bayer, Roche, Pfizer,
Octapharma, NovoNordisk, CSL, SOBI and is a member of executive
committee of the ADVANCE group and ACHIEVE group, Bayer. All
other authors declared no conflict of interest.

Data availability
The data underlying this article will be shared on reasonable request
to the corresponding author.

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..................

Translational perspective
Recently, Norway and Denmark stopped the ChAdOx1 nCoV-19 vaccination after several reported cases of vaccine-induced syndrome
of severe thrombosis and thrombocytopenia with fatal outcome. Samples from vaccine-induced immune thrombotic thrombocytopenia
(VITT) patients allowed us to investigate mechanisms in this severe syndrome and we report immune complexes (ICs) with multi-path-
way triggers, innate immune response cytokines, activation of neutrophils in the blood, and extensive formation of neutrophil extracel-
lular traps (NETs) surrounded by IgG in a thrombus ectomized from the sagittal sinus vein. Our results shed light on the underlying
mechanisms in this rare adenoviral vector vaccine-induced syndrome of severe thrombosis and thrombocytopenia and suggest that anti-
body-mediated thrombus formation in VITT patients is accompanied by a massive innate immune activation with particular activation of
neutrophils, at least partly induced by IC-mediated mechanisms with NET formation as a major pathogenic event.

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