Identified in the 1990s as the primary target of antiphospholipid antibodies (aPL) in antiphospholipid syndrome (APS), β2-glycoprotein I (β2GPI) remains a central focus in hematology and immunology. Anti-β2GPI antibodies are important not only for diagnosing APS but also play a key role in causing thrombosis and pregnancy complications in these patients. Elucidating the molecular basis of antibody-β2GPI interactions is therefore critical for advancing APS research and has broad implications for understanding related thrombotic autoimmune disorders. In this review, we summarize recent progress on the structural biology of β2GPI, discuss mechanisms of autoantibody recognition, and provide an update on genetic polymorphisms. By resolving longstanding controversies and uncovering new regulatory principles, structural insights are paving the way for targeted approaches aimed at selectively neutralizing pathogenic autoantibodies without broadly impairing coagulation or immune function, offering promising paths toward transformative APS therapies.

Allosteric modulation is central to enzyme function and an attractive strategy for drug development. Protein Disulfide Isomerase (PDI), the prototypical thiol-isomerase, exemplifies this potential through its structural flexibility and involvement in neurodegeneration, cancer, and thromboinflammatory disorders such as sepsis, stroke, cancer-associated thrombosis, and antiphospholipid syndrome. PDI consists of four thioredoxin-like domains (a-b-b’-a’), with catalytic CGHC motifs in a and a’ domains and a ligand-binding pocket in the b’ domain. We previously reported that the b’-ligand bepristat 2a (Bep2a) inhibits PDI activity toward large macromolecular substrates while allosterically enhancing activity toward smaller physiological substrates such as GSSG and l-cystine. Here, we define the molecular, thermodynamic, and structural basis of this dual function. Bep2a features an indole ring with five substituents (R1-R5). Using mutagenesis and HDX-MS, we mapped the complex topology, identified five residues (F249, H256, I301, F304, I318) involved in binding, and uncovered a ligand-induced rearrangement of the left helix that acts as a dynamic gate controlling pocket accessibility, a previously unrecognized regulatory mechanism. AI-informed modeling, SAR analysis, and smFRET revealed that Bep2a’s indole core binds perpendicularly in the pocket, with the R1 hydroxyl forming a critical hydrogen bond with H256, which is essential for binding but not for allosteric activation. Conversely, the R4 amine projects outward, serving as a key allosteric site that engages the catalytic domains and promotes PDI compaction. These findings uncover fundamental principles of PDI allosteric regulation and provide a blueprint for optimizing existing ligands and designing new ones with defined functional outcomes.

Protein disulfide isomerase (PDI) functions in thrombus formation in vivo and represents a viable target for antithrombotic therapy. PDI is a redox sensor that can either reduce or oxidize substrates depending on the redox environment. Yet whether PDI functions primarily as a reductase or an oxidase in the context of thrombus formation is unknown. We have used pharmacological approaches and PDI mutants to determine how the redox state of PDI affects thrombus formation. LOC14, which inhibits PDI reductase activity and induces PDI oxidation, promoted thrombus formation in arteries exposed to FeCl3 and enhanced injury-induced platelet accumulation and fibrin formation in cremaster arterioles. Substitution of a single sulfur atom with oxygen in LOC14 reversed these prothrombotic effects. Blocking antibodies targeting PDI also reversed the effect of LOC14. Evaluation of sulfenylation-mediated PDI oxidation using C53A, C56A, R120D and T101A PDI mutants showed that the sulfenylation mechanism of PDI resembles that of H2O2 reduction by peroxiredoxins. These studies identified PDI mutants that failed to undergo H2O2-mediated oxidation, but showed normal reductase activity. When tested in vivo, either wild-type PDI or the R120D mutant fully restored normal thrombus formation following morphilino-induced knockdown of PDI. In contrast, the R120D mutant PDI was unable to fully restore thrombus formation in the setting of oxidative stress induced in mice with genetic deletion of glutathione peroxidase 3 null (GPx3-/-). These studies show that PDI-catalyzed oxidization drives thrombus formation in vivo and demonstrate a mechanism of peroxide-mediated oxidation of PDI that contributes to the prothrombotic response of oxidative stress.

Antiphospholipid antibodies targeting prothrombin are frequently found in antiphospholipid syndrome (APS), yet their impact on thrombin generation remains unclear. Prothrombin exists in equilibrium between closed and open conformations, influencing its activation to thrombin. We recently identified Prothrombin Open monoclonal Antibody (POmAb), a monoclonal antibody that binds the open form and reduces thrombin generation in plasma. However, the in vivo effects of POmAb on coagulation remain unknown.

Antiphospholipid antibodies targeting β2-glycoprotein I (β2GPI) are a hallmark of antiphospholipid syndrome (APS), associated with an increased risk of thrombosis and pregnancy morbidity. Among these, antibodies targeting domain I (DI) are common in individuals at higher risk; however, their epitopes and prevalence among APS phenotypes remain unclear. Here, we use a large collection of 29 structurally and functionally validated β2GPI variants to provide new insights into the molecular mechanisms of autoantibody recognition in APS. Using the prototypic human-derived monoclonal anti-DI antibody MBB2, we identified positively charged residue R39 as the key driver of MBB2 binding, followed by residues R43, N56, and T57. Structural analyses revealed that although R39 is solvent exposed, R43 is not, because it is caged by residues N56 and T57. The narrow epitope footprint explains why MBB2 exhibits a modest affinity for soluble β2GPI. The cage structure accounts for the epitope being conformational rather than linear. Mutational analyses of immunoglobulin G anti-β2GPI antibodies from 52 patients with triple-positive APS, 37 with a history of thrombosis and 15 nonvascular obstetric patients, confirmed significant reactivity against DI and showed signatures of 2 conformational epitopes: one similar to MBB2 (epitope I), in which the presence of R39 is essential, and another that does not require R39 (epitope II). Although less frequent than epitope II in our cohort, epitope I reactivity was notably enriched in patients with vascular-obstetric APS. Varying epitope specificities for DI may therefore aid in identifying different APS phenotypes and predicting clinical outcomes.