Antigenic assessment for the β2-glycoprotein I/Platelet factor 4 complex in thrombotic patients with antiphospholipid syndrome

Objective: Antiphospholipid syndrome (APS) is an autoimmune condition characterized by recurrent thrombosis, pregnancy-related complications and circulating antiphospholipid antibodies, including anti-β2-glycoprotein I (β2-GPI). Platelet factor 4 (PF4) is a pro-coagulant protein expressed by activated platelets. It is considered a potential platelet ligand for oxidized β2-GPI, and this interaction may play a role in the thrombotic manifestations of APS. This study aims to assess β2-GPI/PF4 complex autoantibodies in sera of thrombotic patients with APS and their potential functional role in platelet activation. Methods: We analysed sera from 73 patients with thrombotic APS, 20 with obstetric APS, 20 with systemic lupus erythematosus (SLE), 20 with non-APS thrombosis, 3 with vaccine-induced immune thrombotic thrombocytopenia (VITT), 20 with COVID-19 and 45 healthy donors (HDs). These samples were tested by ELISA for antibodies to the β2-GPI/PF4 complex after in vitro induction of spontaneous β2-GPI protein oxidation. Results: Anti-β2-GPI/PF4 were detected in 34.24% of thrombotic APS patients and 20% of obstetric APS. All VITT and none of the SLE, non-APS thrombosis, COVID-19 patients and HDs were positive for anti-β2-GPI/PF4. In thrombotic APS, a significant association was found between anti-β2-GPI/PF4 positivity and antibody titer with venous thrombotic complications (p = 0.032, p = 0.01) as well as between anti-β2-GPI/PF4 and triple positivity to conventional aPLs (p = 0.028). Notably, antibody titer correlated with the young age both at diagnosis (p<0.001) and at the current evaluation (p=0.001). Moreover, HDs’ platelets, in vitro treated with Ig fractions from APS patients, exhibited a significant increase in phospho-ERK and phospho-p38 expression, leading to NF-κB activation and TF expression. Conclusion: This study demonstrated the presence of anti-β2-GPI/PF4 in patients with APS, where it may be involved in the mechanism underlying the hypercoagulable state and correlated with a greater risk of developing thrombosis, especially in the young population.