Disulfide bond status links the open and closed conformations of prothrombin

Emerging evidence indicates that structural changes in proteins can influence autoimmune recognition. Antibodies targeting the central blood coagulation protein, prothrombin, are frequently detected in patients with the autoimmune disorder, antiphospholipid syndrome. Prothrombin exists as a dynamic equilibrium between closed and open conformations, and the open conformation is more antigenic. Using differential cysteine alkylation and mass spectrometry, we demonstrate that prothrombin circulates as an array of partially disulfide-bonded protein states. Immunoprecipitation of prothrombin from pooled plasma with an antibody that recognizes the open conformation resulted in enrichment of prothrombin molecules containing unformed disulfide bonds. Genetic ablation of disulfides revealed that individual bonds modulate the equilibrium between prothrombin's open and closed conformations. Unformed disulfide bonds in the kringle-1 (C65-C143 and C114-C138), kringle-2 (C191-C231), and protease domain (C293-C439, C493-C507, and C521-C551) were associated with a shift in the protein's structure toward a more open conformation, as assessed by enhanced chymotrypsin accessibility and increased binding to the open conformation antibody. These findings indicate that the disulfide bond status of prothrombin contributes to the open and closed conformational equilibrium.