In vivo murine studies demonstrate that neutrophil activation by anti-NAP2 antibodies contributes to vaccine-induced immune thrombocytopenia and thrombosis (VITT)

ABSTRACT

Background: VITT involves thrombocytopenia and thrombosis post-initial anti-SARS-CoV-2 adenoviral vaccination. Most patients are found to have platelet-activating antibodies to the chemokine, platelet factor 4 (PF4) in the absence of heparin. VITT antibodies differ from those in heparin-induced thrombocytopenia (HIT), in which PF4 is bound to heparin. Distinct epitope sites on PF4 for VITT and HIT antibodies were defined (PMID34233346). We noted that the VITT antigenic site is conserved in mouse (m) PF4, and in the platelet-specific chemokine neutrophil-activating peptide 2 (NAP2), both human and mouse. We observed that VITT antibodies bind strongly to NAP2. In an active patient with VITT, we found that VITT antibodies circulate as immune complexes containing either PF4 or NAP2. Importantly, VITT antibodies plus NAP2 activates platelets. Aim(s) We tested in a passive-immunization murine model with VITT antibodies the ability to induce neutrophil-endothelial activation as an indicator of a prothrombotic state and identify the chemokines involved. Method(s) We studied two systems A femoral vein and a cremaster venule model, using confocal intravital imaging and labeled neutrophils. VITT antibodies were infused into mice transgenic for FcgammaRIIA and lacking PF4 (FcgammaRIIA+/mPF4-/-). Result(s) This led to an immediately reduced neutrophil rolling by ~80% (14-3 m/sec) (Figure 1A,B). Subsequent infusion of PF4 slowed neutrophil rolling by another ~80% (3-0.6 m/sec). In contrast, VITT antibodies did not slow neutrophil speed in FcgammaRIIA+/ mPF4-/-/ mNAP2-/-mice (Figure 1C). Conclusion(s) These data suggest that both NAP2 and PF4 contribute to thrombosis in VITT and may explain the pathogenesis of VITT in patients with no detectable anti-PF4 antibodies. VITT may be prothrombotic because it involves co-activation of neutrophils via NAP2 by way of CXCR2 and FcgammaRIIA. Targeting NAP2 pathobiology may enhance understanding of the pathogenesis of VITT and lead to new therapeutics.