The Immunobiology of Vaccine-induced Immune Thrombotic Thrombocytopenia

ABSTRACT Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a complication of adenoviral (AdV)-based COVID-19 vaccination. Antibodies (Abs) against the 70-aa chemokine, platelet (Plt) factor 4 (PF4), have been noted in both VITT and in the related immune, prothrombotic disorder, heparin-induced thrombocytopenia (HIT), but target different sites on the human (h) PF4 tetramer. The site on hPF4 targeted in VITT is conserved in the related chemokine, 94-aa -granule, Plt-basic protein (PBP) and its N-terminal-truncated isoforms (PBPi), including 70-aa neutrophil-activating peptide 2 (NAP2) that uniquely activates neutrophils (PMNs) via CXCR2. We hypothesize that PF4 and/or PBP can initiate VITT and contribute to its prothrombotic state. We show that VITT Abs bind hPBPi and activate Plts. Mice injected with VITT plasma develop a prothrombotic state even in the absence of PF4, and unlike in murine HIT, PMNs become incorporated into arterial as well as venular thrombi. We also show by dynamic light scattering (DLS) that both chemokines bind directly to AdVs. In a murine VITT model, Abs develop to either PF4 or PBPi. We will pursue our observation and test the above hypothesis as follows: Aim 1: Characterize the range of antigenic targets of VITT Abs. We will confirm that VITT Abs, VITT monoclonal (mo) Abs, and murine Abs generated in Aim 3 bind to hPBPi. We will also determine where VITT Abs bind on hPBPi using select amino acid substitutions of hPBP. Aim 2: Examine the importance of hPF4 and hPBPi in VITT in vitro and in vivo. We will test the relative contribution of hPF4 vs. hPBPi to thrombus formation in vitro using an injured-endothelium microfluidic model. Transgenic FcRIIA+ mice that express either no PF4 or PBP, or have the murine or human versions, will be injected with VITT Abs to recreate the prothrombotic state to validate the importance of PF4 vs. PBP in the development of thrombi in vivo. Aim 3: Examine the mechanistic basis for the onset of VITT in a murine model. We have developed a novel murine VITT model that suggests that the prothrombotic state occurs after AdV vaccination independent of expressing the COVID-19 spike protein. Both anti-PF4 and anti-PBPi Abs were noted. These studies were supported by DLS studies which showed that both chemokines complex with AdV, but that some AdV bind better to hPF4 and others to hPBPi. The mechanistic basis of VITT will be further pursued, including defining the site(s) by which these chemokines interact with AdV, and the characterization of the formed Abs. Taken together, Project 3 should provide important, novel conceptual insights into VITT. A number of new VITT-like pro-thrombotic disorders are being recognized, and our studies should also be applicable to these disorders. Finally, AdV-based vaccines have been developed for other disorders, and our models in Aim 3 may guide AdV modifications to enhance vaccine safety. These advances will greatly benefit from synergy between Dr. Poncz and the expertise provided by shared Core B at Versiti's Blood Research Institute, as well as with the other Project Leaders in our Program - each studying thematically related, clinically important immune thrombocytopenic disorders.