Covid-19 Patient Cohorts
A total of 222 Covid-19 patients were enrolled from five prospective registries from university medical centers in Munich (CORKUM, WHO trial ID DRKS00021225), Freiburg (WHO trial ID DRKS00021206), Tuebingen (approval by the local ethics committee 240/2018BO2), Greifswald (DRKS-ID: DRKS00023770) and Brescia, Italy (approval by the local ethics committee, ID Number: NP 4463). Patients between the age of 4 months to 88 years with available serum and positive PCR testing of SARS-CoV-2 in nasopharyngeal swabs were enrolled. Registries began recruiting patients at varying start dates ranging from February 2020 until October 2020. Patient characteristics are summarized in Table 1; registries are described in detail in the Supplementary Material 1.
Sera from 6 VITT patients presenting with thrombocytopenia and thromboembolic events approximately 5-20 days after COVID-19 Vaccine AstraZeneca vaccination were available.9
Identification of immunogenic epitopes and homologies of human PF4 and SARS-CoV-2 spike protein and the comparative analysis of their 3D structures
The protein sequence for human PF4/CXCL4 was retrieved from the ENSEMBL gene data base (ENSG00000163737).15 Similarly, the protein sequence of the SARS-CoV-2 spike protein (1273 amino acids) was retrieved from publicly available data bases (NCBI: Gene ID 43740568).16 Using the online prediction tool of the University of Madrid, Spain (http://imed.med.ucm.es/Tools/antigenic.pl),17 we identified potential immunogenic peptide sequences (epitopes) in both protein sequences. In the SIM Alignment online Tool (https://web.expasy.org/sim/),18 the following default setting parameters were applied (comparison matrix BLOSUM62, gap opening penalty=12 and gap extension penalty=4). For 3D analysis, the MacMYPOL program (https://pymol.org/2/)19 was used together with the files 6vxx.pbd and 4r9w.pbd available for the PBD database (http://www.rcsb.org)20 to compare the epitopes on the published structures of the proteins.
Cloning and expression of SARS-CoV-2 spike protein
The SARS-CoV2 spike ectodomain amino acids 17-1213 and the RBD-SD1 domain aa 319‑519 (based on QHD43416)21 were cloned and expressed in the human cell line Expi293 (Thermo Fisher Scientific, Germany) (details Supplementary Material 2).
Testing for PF4/heparin-reactive and platelet-activating immunoglobulin G antibodies
For screening of all sera of the Covid-19 cohorts and the patients with VITT, we used an IgG-specific anti-PF4/heparin ELISA, with antibody binding measured using a secondary antihuman IgG antibody, as described.22 Optical density (OD) results <0.5 units were considered negative, ≥0.5<1.0 weak-positive, and OD≥1.0 strong-positive.
We performed platelet activation assays using purified, washed platelets from healthy volunteers, as described,9 using patient sera, or the respective purified anti-PF4/heparin IgG fractions with and without addition of PF4 (10 µg/mL) (Chromatec, Greifswald, Germany). Unfractionated heparin (100 IU/mL, final) was added to evaluate inhibition of antibody- and PF4-dependent platelet activation. Platelet activation was judged positive if at least two of 3 donor cells aggregated within 30 minutes.23,24
Affinity purification of PF4 and PF4/heparin IgG antibodies
Biotinylated PF4 (biotin-PF4) (Chromatec, Greifswald, Germany) and biotin-PF4/heparin complexes were coupled to streptavidin-conjugated paramagnetic microbeads (Dynabeads MyOne Streptavidin T1, Invitrogen). Beads were incubated with the serum, unbound antibodies and plasma removed by washing, and the IgG fractions were eluted (details in Supplementary Material 2).
Binding studies of affinity purified anti-PF4 and anti-PF4/heparin IgG to SARS-CoV-2 S-1 domain, receptor-binding domain, full-length spike protein, PF4 and PF4/heparin complexes
We identified sera testing positive for anti-PF4/heparin antibodies from two patient groups, (a) patients with Covid-19 disease (only a minority tested positive), and (b) patients with VITT (all tested positive). These sera were assessed for anti-spike protein antibodies using the SARS-CoV-2 full-length spike protein, the receptor-binding domain (RDB) using in-house ELISAs, and a commercially-available CoV-2 ELISA (recombinant S1-domain; EI 2606-9620 G; EUROIMMUN Medizinische Labordiagnostika AG, Lübeck, Germany). Anti-PF4 and anti-PF4/heparin affinity-purified IgG fractions of two VITT patients with documented thromboembolic events were used in a 1:20 dilution (detailed description in Supplementary Material 2).