Thromboses belong to the most serious complications in COVID-19. The trigger for rarely occurring thromboses associated with thrombocytopenia upon adenoviral vaccination against COVID-19 is unknown. As the spike protein could represent a pathogenetic link between the two conditions, we investigated the fate of spike protein, its processing to S1 and S2 subunits and its localization upon de novo expression from plasmids, approved COVID-19 vaccines and wildtype SARS-CoV-2.

Independent from the source, a significant proportion of the spike protein was found being not cell-associated. Rather, the spike protein was abundantly present in the medium. Depending on, whether or not the spike protein was proteolytically processed by furin, we found it either (i) anchored as full-length spike protein in extracellular vesicles (EVs), (ii) anchored as S2 subunit in EVs not associated with S1, or (iii) as free S1 subunit in the medium not associated with S2 or EVs.

We show that extracellular full-length spike protein and the S1 subunit attached to ACE2-expressing cells and mediated the binding of human plasma-derived spike protein-specific IgGs to the cell surface.

Similar results were obtained with another viral transmembrane protein, the glycoprotein of Vesicular Stomatitis Indiana Virus (VSV-G), suggesting a general applicable phenomenon for de novo expressed transmembrane proteins.

The extracellular presence of considerable amounts of functional spike protein might provide a possible explanation for reported pathological effects observed during COVID-19 or following genetic vaccination