Luminespib and AZ5104 are effective antithrombotic drugs via targeting the platelet Ero1α-PDI pathway.

The platelet-surface Ero1α-protein disulfide isomerase (PDI) system is essential for integrin αIIbβ3 activation and platelet aggregation. Targeting the functional interplay between Ero1α and PDI emerges as a promising antithrombotic strategy. Using a tiered high-throughput screen, we identified two clinical-stage compounds, Luminespib and AZ5104, as selective inhibitors of the Ero1α-PDI interaction. They bind a hydrophobic pocket in the PDI b' domain, inhibiting the pathway in biochemical and cellular assays without affecting other PDI family members. The antiplatelet effects of Luminespib and AZ5104 were abolished in megakaryocyte-specific Pdi or Ero1a knockout mice, confirming on-target specificity. Mechanistically, they concurrently inhibit Ero1α-PDI-driven extracellular integrin activation and intracellular Ca2+ signaling. Both compounds potently reduced arterial thrombosis in mice without prolonging bleeding time. Our work establishes Luminespib and AZ5104 as clinical-stage antithrombotic agents that target the platelet Ero1α-PDI system, offering an effective strategy to achieve potent antithrombosis without compromising hemostasis.