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Adenovirus vaccines and thrombosis
Figure 2. The autoimmune heparin-induced thrombocytopenia hypothesis. Vaccine components leaking into the bloodstream from the vaccination site (facilitated by ethylenediaminetetraacetic acid present in the vaccine) activate platelets to release platelet factor 4 (PF4). Vaccine constituents, likely polyanions or viral DNA, form complexes with positively-charged PF4 which are recognized as neoantigens by B cells that then produce antibodies against these complexes. The resulting immune complexes activate platelets through FcgRIIa, triggering the release of additional PF4 and polyphosphates thereby initiating a positive feedback loop that leads to further platelet activation and consumption. Extracellular DNA in neutrophil extracellular traps binds PF4 and the resulting DNA/PF4 complexes further recruit anti-PF4 antibodies inducing massive Fcg receptor-dependent activation of neutrophils, platelets, monocytes and endothelial cells leading to massive activa- tion of coagulation and thrombosis. EDTA: ethylenediaminetetraacetic acid.
The investigators showed that platelet activation was triggered by FcgRIIA stimulation, because an FcgRIIA- blocking antibody prevented this phenomenon. Given the absence of previous exposure to heparin, the authors suggested a condition resembling autoimmune HIT. More recently, in a preliminary report published in a non-peer- reviewed internet repository, the German group went on to suggest that the Ad vector and/or some protein com- ponents of the Vaxzevria vaccine activate platelets to release PF4 which then forms complexes with virus pro- teins and other anionic constituents of the vaccine, gener- ating neoantigens against which antibodies develop and induce strong platelet activation via FcgRIIa stimulating granulocyte activation with NETosis and ultimately cata- strophic thrombosis.47 The presence of EDTA in the vac- cine would favor vascular leakage at the inoculation site, facilitating dissemination of the vaccine components in blood (Figure 2).
Very recently a study using alanine scanning mutagen- esis explored the binding sites on PF4 of antibodies isolat- ed from patients with VITT or with classical HIT. While the binding of VITT anti-PF4 antibodies was restricted to eight surface amino acids, all located within the heparin- binding site of PF4, HIT anti-PF4 antibodies bound amino acids corresponding to two different sites on PF4; more- over, VITT antibodies had a stronger binding response than HIT antibodies. The authors concluded that VITT antibodies mimic the effect of heparin by binding to a similar site on PF4, allowing PF4 tetramers to cluster with the formation of immuno-complexes which, in turn, cause FcγRIIa-dependent platelet activation.79
These peculiar characteristics explain why the identifi-
cation of VITT requires different tests from those needed for the identification of classical HIT.80 In suspected VITT anti-PF4 antibodies can be identified by an ELISA, but not by other rapid immunological assays typically positive in HIT, such as the STic® Expert HIT kit, latex immunoas- says and chemiluminescence-based assays,4,80 and it is better characterized by a heparin-induced platelet aggre- gation (HIPA) or PF4-induced platelet activation (PIPA) test.2,4,81 It should be noted that no single ELISA method detected all possible/probable VITT cases.82
While the autoimmune HIT hypothesis is important and provides the basis for understanding this novel cata- strophic autoimmune thrombotic syndrome, several aspects do not fit completely with the clinical presenta- tion of VITT and various issues remain unclarified.
First, among the reported VITT cases in which anti-PF4 antibodies were measured, in a few they were negative (Table 2).4,5 Second, it is expected that treatment with heparin would worsen the clinical evolution of these patients, and indeed it is generally cautiously recom- mended not to use this drug.2,4,13 However, in around one fourth of the heparin-treated cases, the use of this antico- agulant was successful (Table 2). Third, while VITT resembles autoimmune HIT in several respects, the latter is not as frequently associated with CVST and rarely with DIC, and the above summarized mechanistic hypothesis does not explain the preferential localization of the venous thrombotic events in the cerebral and splanchnic circulations.
Other unclear aspects are its relatively precocious onset, as early as 4 days after vaccination, which seems too soon to generate high-titer, class-switched, high-affin-
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