Cryo-EM structure and functional basis of prothrombin recognition by a Type-I anti-prothrombin antiphospholipid antibody.

Anti-prothrombin (anti-PT) antibodies are found in antiphospholipid patients, but how they interact with prothrombin remains elusive. Prothrombin adopts closed and open forms. We recently discovered Type-I and Type-II antibodies and proposed that Type-I recognize the open form. In this study, we report the discovery, structural and functional characterization in human plasma of a Type-I antibody, POmAb. Using surface plasmon resonance and single-molecule spectroscopy, we show that POmAb interacts with kringle-1 of prothrombin, shifting the equilibrium towards the open form. Using single-particle cryogenic electron microscopy (cryo-EM), we establish that the epitope targeted by POmAb is in kringle-1, comprising an extended binding interface centered at residues R90-Y93. The 3.2Å cryo-EM structure of the complex reveals that the epitope overlaps with the position occupied by the protease domain of prothrombin in the closed state, explaining the exclusive binding of POmAb to the open form. In human plasma, POmAb prolongs phospholipid-initiated and diluted Russel Viper Venom clotting time, which could be partly rescued by excess phospholipids, indicating POmAb is an anticoagulant but exerts a weak lupus anticoagulant effect. These studies reveal the structural basis of prothrombin recognition by a Type-I antiphospholipid antibody and uncover an exciting new strategy to achieve anticoagulation in human plasma.