Pomab, a Novel Monoclonal Type-I Antiphospholipid Antibody, Reduces Thrombin Generation By Selectively Binding to the Open Form of Prothrombin

Background. Anti-prothrombin (anti-PT) antibodies are a type of antiphospholipid antibodies frequently found in antiphospholipid syndrome (APS) patients. Previously, we identified two types of anti-PT antibodies, one binding to epitopes in kringle-1 that are hidden in the closed form but available in the open form (Type-I), and another one in which the epitopes are located in fragment-1 but accessible in both forms (Type-II). Our understanding of their functional characteristics remains limited. Goal. To identify Type-I monoclonal antibodies for mechanistic studies. Methods. Type-I anti-prothrombin antibodies were generated by immunizing mice with fragment-1 and producing monoclonal antibodies using hybridoma technology. Binding mechanisms and epitope recognition for one representative Type-I antibody, POmAb, were elucidated using surface plasmon resonance (SPR), single-molecule FRET (smFRET), and cryo-electron microscopy (cryo-EM). Functional evaluations were performed in human plasma and with purified components. In vivo hemostatic activity was assessed in a laser injury model of the cremasteric arteriole followed by intravital microscopy. Results. Type-I antibodies recognizing kringle-1 were obtained from immunization experiments. One of them, POmAb (Prothrombin Open monoclonal Antibody), was selected for in-depth mechanistic and structural studies. SPR studies showed that POmAb binds to the open form of prothrombin with low nanomolar affinity, and binding was impaired by the mutations S91A and Y93A in kringle-1. smFRET provided evidence of conformational selection, where POmAb preferentially selects the open conformation of prothrombin, thereby shifting the equilibrium towards the open form. Cryo-EM studies of the POmAb-prothrombin complex revealed an extended binding interface in kringle-1 which overlaps with the region occupied by the serine protease domain in the closed form, explaining why POmAb binds to the open form. In human plasma, POmAb prolonged the activated partial thromboplastin time and Russel's viper venom time, though the effect was modest. Further investigations using purified proteins and antibody fragments confirmed that POmAb reduces thrombin generation, although it does not completely abolish it, as the inhibition of thrombin generation occurs through a non-competitive mechanism with prothrombinase. In vivo studies corroborated in vitro findings, further proving reduced fibrin accumulation at the site of injury. Conclusions. We discovered POmAb, a Type-I antiphospholipid antibody. We demonstrated that it selectively binds to the open form of prothrombin, resulting in reduced thrombin generation. POmAb represents a novel tool for studying the role of Type-I antibodies in APS and advances the provocative new idea of conformational trapping of prothrombin as a novel class of anticoagulants.