Ab0127 antigenic assessment for the β2gpiox-pf4 complex in a monocentric cohort of patients with aps, thrombosis during sars-cov-2 infection and vitt

Background Platelet factor 4 (PF4) is a protein with a pro-clotting function expressed by activated platelets with a high affinity for anionic glycosaminoglycans present on the platelet surface. It has been shown that the positively charged surface of PF4 tetramer interacts with the negatively charged regions of β2glycoprotein I (β2GPI) domains, stabilising the link between the antigen and the phospholipid surface, thus increasing the possibility of binding with the respective antibodies. In particular, a tetramer of PF4 selectively binds two molecules of β2GPI, favouring the dimerization of the same, which is crucial in platelet activation and therefore in thrombotic manifestations of antiphospholipid syndrome (aPS). PF4 may be a common denominator in syndromes such as aPS and heparin-induced thrombocytopenia (HIT), which share similar clinical manifestations as thrombocytopenia and thrombosis. Other syndromes, which share the same clinical and laboratory features of HIT despite not having previously received heparin, appear to be associated with the presence of anti-PF4 antibodies. Such pathologies could only be explained by HIT antibodies with heparin-independent platelet-activating properties. One of these could be vaccine-induced Immune thrombotic thrombocytopenia (VITT) post-somministration of ChAdOx1 nCoV-19 vaccine. Recent studies have shown structural similarities between heparin and β2GPI, which may be responsible for thrombotic events in those infected with SARS-Cov-2 and VITT, who never had heparin. In particular, oxidised-β2GPI (β2GPIox) may mimic heparin by structural analogy and link to PF4. Considering the structural similarities between heparin and β2GPIox, and demonstrating the immunogenicity of the hypothesised complex in APS, the alternative molecule could be represented by β2GPIox itself, thus explaining the thrombotic events following vaccination in subjects who have never received heparin. Objectives The aim of the study is to test the potential immunogenicity of the β2GPIox-PF4 complex and the presence of antibodies against this complex in patients with aPS, thrombosis during infection with SARS-CoV-2 or VITT. Methods 34 patients with proven diagnosis of APS, 17 patients with thrombosis related to infection SARS CoV-2 and 3 patients with VITT were enrolled. Only one aPS patient received heparin prior to testing. Antibodies to the β2GPIox-PF4 complex were evaluated by home made ELISA immunoenzyme testing. Competitive inhibition (homologous) experiments of the bond of PF4 with β2GPIox in the fluid phase were conducted in order to verify the binding specificity. Results Anti-β2GPIox-PF4 antibodies were detected in 11 of 34 aPS patients (32%) and all VITT patients (100%), while none of covid-19 patients tested positive. In addition, Anti-β2GPIox-PF4 antibodies positivity significantly correlated with the presence of anti-β2GPI antibodies (p=0.0259) and with a triple positive antibody profile (p=0.0017) (Figure 1). There was no significant association between anti-β2GPIox-PF4 antibodies’ title and clinical features of aPS patients. Conclusion The results of this study show a new antibody positivity in aPS and VITT. In particular we have identified a high prevalence of anti-β2GPIox-PF4 antibodies which may be involved in the pathophysiology of both aPS and VITT. In addition the absence of anti-β2GPIox-PF4 antibodies in covid-19 patients suggest that the thrombotic mechanism that underlies VITT is different from covid-19 and, on the contrary, could resemble aPS. Figure 1. References [1]Alessandri C et al. New autoantigens in the antiphospholipidsyndrome. Autoimmun Rev. 2011 [2]Sikara MP et al. {beta}2 Glycoprotein I ({beta}2GPI) binds platelet factor 4 (PF4): implications for the pathogenesis of antiphospholipid syndrome. Blood. 2010 Jan 21 [3] Favaloro EJ et al. Antibodies against Platelet Factor 4 and Their Associated Pathologies: From HIT/HITT to Spontaneous HIT-Like Syndrome, to COVID-19, to VITT/TTS. Antibodies (Basel). 2022 Jan 21 Acknowledgements: NIL. Disclosure of Interests None Declared.