Pathogenesis of vaccine-induced immune thrombotic thrombocytopenia (VITT)

Abstract

Vaccine-induced immune thrombotic thrombocytopenia (VITT; synonym, thrombosis with thrombocytopenia syndrome, is associated with high-titer immunoglobulin G antibodies directed against platelet factor 4 (PF4). These antibodies activate platelets via platelet FcγIIa receptors, with platelet activation greatly enhanced by PF4. Here we summarize the current concepts in the pathogenesis of VITT. We first address parallels between heparin-induced thrombocytopenia and VITT, and provide recent findings on binding of PF4 to adenovirus particles and non-assembled adenovirus proteins in the 2 adenovirus vector-based COVID-19 vaccines, ChAdOx1 nCoV-19 and Ad26.COV2.S. Further, we discuss the potential role of vaccine constituents such as glycosaminoglycans, EDTA, polysorbate 80, human cell-line proteins and nucleotides as potential binding partners of PF4. The immune response towards PF4 in VITT is likely triggered by a proinflammatory milieu. Human cell-line proteins, non-assembled virus proteins, and potentially EDTA may contribute to the proinflammatory state. The transient nature of the immune response towards PF4 in VITT makes it likely that-as in heparin-induced thrombocytopenia -marginal zone B cells are key for antibody production. Once high-titer anti-PF4 antibodies have been formed 5 to 20 days after vaccination, they activate platelets and granulocytes. Activated granulocytes undergo NETosis and the released DNA also forms complexes with PF4, which fuels the Fcγ receptor-dependent cell activation process, ultimately leading to massive thrombin generation. Finally, we summarize our initial observations indicating that VITT-like antibodies might also be present in rare patients with recurrent venous and arterial thrombotic complications, independent of vaccination.

Keywords: COVID-19; ChAdOx1 nCoV-19; Platelet factor 4; TTS; VITT; Vaccination.

Fig. 1

PF4 binds to adenovirus. (A) The ChAdOx1 nCoV-19 vaccine preparation binds PF4. Brownian dynamics simulations show frequent interactions (red spots) between the PF4 tetramer and the ChAdOx1 surface (grey). (B) this interaction is inhibited in the presence of the polyanion fondaparinux. Taken from .

Fig. 2

Super-resolution imaging of PF4 vaccine complexes with VITT anti-PF4 antibodies. 3D structured illumination microscopy (3D-SIM) shows binding of ChAdOx1 nCoV-19 components (hexon polypeptide in magenta) to complexes of VITT IgG (cyan) bound to PF4 (green). Arrowheads indicate localization of VITT IgG on PF4-hexon complexes. The scale bar indicates 200 nm.

Fig. 3

Binding of human antibodies to ChAdOx1 nCoV-19 vaccine components. Binding of IgG antibodies to the vaccine was determined with a LI-COR fluorescence detection system after Western blotting using sera from non-vaccinated and vaccinated healthy individuals (each n = 7) and VITT patients (n = 7) at a serum dilution of 1:40. PageRuler Prestained Protein Ladder was used (Invitrogen/Thermo Fisher) as a molecular mass standard and for the negative control lane no serum was used to assess unspecific binding of the secondary antibody. The binding of antibodies to vaccine components was determined by immunoblotting using a LI-COR Odyssey CLx imaging system (LI-COR, Lincoln, NE, USA) for quantitation of signal intensities according to the manufacturers protocol.

Fig. 4

¹H-NMR spectrum of ChAdOx1 nCoV-19 vaccine. Analysis of ChAdOx1 nCoV-19 vaccine confirmed the solution composition and showed the NMR signals of sucrose, ethanol, and histidine. EDTA was detected (approx. 0.1 mM). TRIS was not found in the vaccine. X-axis: NMR chemical shift signals in ppm relative to internal standard TSP; y-axis: relative signal intensity.

Fig. 5

Summary of pathogenesis of VITT. The initial mechanisms underlying B-cell activation are still hypothetical and are derived from the known mechanisms in HIT. For a more detailed description please see the “summary section” of the manuscript. Figure is a modified version of a figure on pathogenesis of VITT in .

Fig. 6

Confocal immunofluorescence microscopy images of platelets from blood smears (A) COVID-19 vaccinated healthy individual and from (B) VITT patient labeled for spike protein (green) and platelet myosin (magenta). The inset graphs show the fluorescence intensities (in grayscale) of both spike protein and myosin from line profiles (dotted line). The arrowhead (B) points at filopodia-like projection, a typically activated platelet morphology in acute VITT patients.