Cryogenic electron microscopy (cryo-EM) resolves the structures of proteins and their complexes under near-native conditions that are often inaccessible to other structural techniques. Cryo-EM has recently contributed significant advances in basic knowledge to the field of coagulation. New paradigms regarding individual domain role in protein-protein interactions have emerged, casting the main reactions of the coagulation cascade in a much-needed structural context. This review focuses on recent advances in the study of coagulation factors using cryo-EM.

Disorders of the blood coagulation cascade continue to pose a major clinical challenge, necessitating the development of new therapeutic agents capable of modulating this process. Several oligonucleotide aptamers targeting coagulation factors have been developed, and some are undergoing preclinical or clinical evaluation. Among them, anti-thrombin anticoagulant aptamers are promising dual-targeting agents in that, in addition to inhibiting enzyme activity, they may limit thrombin generation by binding to its precursor, prothrombin. In the present study, combined calorimetric and spectroscopic analyses reveal that these aptamers recognize proexosite I of prothrombin and exosite I of thrombin through broadly similar thermodynamic binding mechanisms. Integration of structural SAXS studies and limited proteolysis shows that aptamer binding to proexosite I alters prothrombin structure, shifting the equilibrium from its more abundant closed form to the open conformation. Taken together, these results support the classification of these aptamers as dual-targeting agents capable of recognizing both thrombin and prothrombin and provide guidance for their continued development as anticoagulant therapeutics.

The concomitant presence of 4 antiphospholipid antibody types, namely lupus anticoagulant (LA), anticardiolipin antibodies, anti-β2-glycoprotein I, and antiprothrombin antibodies (tetra-positive), constitutes a high-risk antiphospholipid antibody profile and predisposes to thrombosis and catastrophic antiphospholipid syndrome (CAPS). However, tetra-positivity is also seen in asymptomatic carriers with LA-hypoprothrombinemia syndrome (LAHS).

Anti-β2glycoprotein I (β2GPI) antibodies are the hallmark of the antiphospholipid syndrome (APS). β2GPI consists of five domains, DI-DV. While DI is the primary target, the clinical relevance of antibodies against other domains remains uncertain. We analyzed two large anti-β2GPI IgG positive cohorts, to investigate whether different domain binding affects anti-β2GPI IgG assay testing and APS diagnosis.

[This corrects the article DOI: 10.3389/fimmu.2026.1809192.].