LAUSR

Vaccination against severe acute respiratory syndrome coronavirus 2 (SARSCoV2) is one of the most important measures to fight the coronavirus 2019 (COVID19) pandemic. One of the vaccines, ChAdOx1S (AstraZeneca), is a recombinant, replicationdeficient chimpanzee adenovirus encoding the SARSCoV2 spike glycoprotein. The vector is propagated in TREx293 human embryonic kidney (HEK) cells, a genetically modified derivative of the HEK 293 cell line.1 Hundreds of cases of unusual thrombotic events in combination with thrombocytopenia after vaccination with ChAdOx1S have been observed.2– 4 This socalled vaccineinduced immune thrombotic thrombocytopenia (VITT; also known as thrombotic thrombocytopenia syndrome [TTS]) resembles a subtype of autoimmune heparininduced thrombocytopenia (aHIT) known as “spontaneous HIT syndrome.”5 Its pathogenesis remains unclear and many potential mechanisms have been discussed.6 It is unequivocally accepted that VITT is associated with high titers of IgG class antibodies directed against the cationic platelet chemokine, platelet factor 4 (PF4; CXCL4).7 These antibodies potently activate platelets via platelet FcγIIa receptors with platelet activation greatly enhanced by PF4.3,8 PF4 is a positively charged, compact homotetrameric globular protein, which binds to a wide array of polyanions, especially sulfated glycosaminoglycans (GAG) like heparins, heparan sulfates, and chondroitin sulfates, and other polyanions like polyphosphates and nucleic acids.9– 12 Recently, we have shown that constituents of the vaccine form complexes with PF4.7 These complexes contain adenoviral hexon proteins as shown by staining with a monoclonal antihexon antibody. However, it is unresolved whether additional molecules are involved in PF4 complex formation. Potential binding partners are sulfated GAG13 from the TREx293 HEK cells. It is technically challenging to detect low concentrations of GAGs in a complex matrix such as a vaccine, which contains many proteins. We report the experimental approach by which we excluded relevant amounts of contaminating GAG in the vaccine. 2 | METHODS