Background/Purpose: Antiphospholipid syndrome (APS) is characterized by thrombosis and/or pregnancy morbidity and the presence of antiphospholipid antibodies (aPL). Phosphatidylserine-dependent antiprothrombin antibodies (aPS/PT) recognize the phosphatidylserine/prothrombin (PS/PT) complex, and are highly associated with APS. Recently, it has been reported that misfolded beta2-glycoprotein I (beta2-GPI) are transported to the cell surface by human leukocyte antigen (HLA) class II molecules and are targeted by autoantibodies in patients with APS [1, 2]. APS patients with anti-beta2-GPI are highly likely to share the thrombophilic pathophysiology with those with aPS/PT, therefore we hypothesized that misfolded prothrombin (PT), likewise beta2-GPI, are transported to the cell surface by HLA class II molecules in procoagulant cells, consequently being targeted by aPS/PT.

Methods: 1) The interaction of PT with HLA-DR was analyzed by flow cytometry (FCM) using PT / HLA-DR overexpressed HEK293T cells. 2) PT synthesis from monocyte was investigated in phorbol-12-myristate-13-acetate (PMA) treated THP-1 cells by western blotting (WB) and FCM. 3) Cell surface transportation of synthesized PT with HLA-DR was evaluated by FCM in PMA-treated THP-1 cells.

Results: 1) PT protein in the presence of transcripted HLA-DR was detected on the cell surface and PT/HLA-DR complex was recognized by a mouse monoclonal aPS/PT (231D). 2) PMA treated THP-1 cells synthetized PT which showed stronger binding to 231D than to control monoclonal anti-PT antibody, the latter recognizes PT in the absence of phosphatidylserine (Fig). 3) 231D binding to PT/HLA-DR complex was confirmed in THP-1 cells co-stimulated with PMA and interferon gamma. No binding was observed between control monoclonal anti-PT antibody and PT/HLA-DR complex.

Conclusion: Structurally altered PT is transported to the cell surface by HLA class II molecules in monocyte after PMA stimulation, indicating that PT/HLA-DR complexes may be targets for aPS/PT.

References: [1] Tanimura K, et al. Beta2-Glycoprotein I/HLA class II complexes are novel autoantigens in antiphospholipid syndrome. Blood 2015. [2] Arase N, et al. Novel autoantibody against the beta2-glycoprotein I/human leucocyte antigen-DR complex in patients with refractory cutaneous ulcers. Br J Dermatol 2018

 Figure. Prothrombin expression in PMA treated THP-1 cells