PB1100 - Disulfide-bonded Forms of Prothrombin are Differentially Targeted by Anti-Prothrombin Antiphospholipid Antibodies

Background: Anti-prothrombin (anti-PT) antibodies are a subset of autoantibodies commonly detected in individuals with antiphospholipid syndrome (APS). Previous findings showed that structural changes in prothrombin can modulate autoantibody binding. However, our understanding of antibody development and binding characteristics remain limited. Using a differential cysteine labelling and mass spectrometry approach we have recently determined that six of prothrombin’s twelve disulfide bonds are unformed in ~1 in 10 to ~1 in 5 prothrombin molecules. These findings demonstrate that prothrombin circulates in plasma in different disulfide-bonded states, suggesting potential significant structural variations.

Aims: Determine the functional implications of the different prothrombin disulfide-bonded states in coagulation and anti-PT antibody binding.

Methods: Prothrombin variants comprising a single ablated disulfide bond by replacing both Cys with Ala were produced in HEK293 cells. Functional assays were performed in a purified system. Autoantibody binding assays were performed using an in-house ELISA with APS patient plasma.

Results: Recombinant prothrombin from HEK293 cells displayed an identical redox state compared to plasma-isolated prothrombin. Removal of a single disulfide bond did not significantly impact the redox state of other disulfide bonds. Ablation of the kringle 1 disulfide bond C114,C138 or kringle 2 disulfide bond C191,C231 markedly decreased the rate of prothrombin activation, whereas other mutants remained unaffected. Among the mutants with a disulfide bond ablation in the serine protease domain, two out of four displayed approximately a threefold reduction in fibrinogen conversion. Examination of APS patient-derived anti-PT antibody binding to prothrombin mutants revealed a four- to fivefold reduced binding to prothrombin C114A,C138A and C191A,C231A relative to wild-type prothrombin.

Conclusion(s): Collectively, our results indicate that distinct disulfide-bonded states of prothrombin exhibit different functional characteristics. Interestingly, several prothrombin states exhibit significantly reduced binding to anti-PT antibodies from individuals with APS. These findings have the potential to provide novel insights into our understanding of autoantibody development and binding mechanisms.