Platelet Surface PDI Controls the Ligand-Binding Function of Glycoprotein Ibalpha and Platelet-Neutrophil Interactions Under Thromboinflammatory Conditions

We previously showed that the isomerase activity of platelet surface protein disulfide isomerase (PDI), a prototypic thiol isomerase, regulates the full activation of αIIbβ3 integrin and is important for platelet accumulation, but not hemostasis, following vascular injury (Kim et al. Blood 2013). However, it remains unclear whether platelet PDI regulates the function of other platelet surface receptors. Since it was reported that platelet PDI and glycoprotein Ibα (GPIbα) were physically close on the platelet surface (Burgess et al. JBC 2000), we investigated whether platelet PDI regulates GPIbα function using megakaryocyte-specific PDI conditional knockout (CKO) mice developed by us. We found that PDI-null platelets showed a significant defect in GPIbα-mediated agglutination and von Willebrand factor (vWF) binding. Those defects were completely rescued by exogenously-added recombinant wild-type PDI (wtPDI), but not mutant PDI (dmPDI) lacking the isomerase activity. Consistently, inhibition of platelet PDI with blocking antibodies impaired GPIbα-mediated agglutination and vWF binding in human and mouse platelets, suggesting that the isomerase activity of platelet surface PDI controls the ligand-binding function of GPIbα. Studies using surface plasmon resonance revealed that wtPDI and dmPDI bound to immobilized GPIbα with a dissociation constant of 0.9 and 1.2 µM, respectively. Mass spectrometric analysis indicated that the Cys4-Cys17 disulfide bond in GPIbα, which has an allosteric –RHStaple configuration, was preferentially reduced by wtPDI, but not dmPDI. Consistent with previous studies showing that platelet GPIbα is important for platelet-neutrophil interactions, we observed that deletion of platelet PDI significantly reduced platelet-neutrophil interactions under shear conditions. Importantly, using real-time fluorescence intravital microscopy with megakaryocyte-specific PDI CKO mice, we demonstrated that platelet-specific PDI deletion did not influence neutrophil adhesion but markedly reduced platelet adhesion and accumulation on the adherent neutrophils on TNF-α-inflamed cremaster muscle venules. Infusion of wtPDI, but not dmPDI, restored reduced platelet-neutrophil interactions in the PDI CKO mice, implying the importance of platelet surface PDI activity in regulating GPIbα-mediated platelet-neutrophil interactions. Using a thromboinflammation model of hepatic ischemia/reperfusion injury, we found that platelet-specific PDI CKO mice exhibited a significant reduction in the infarct size and the serum levels of aspartate aminotransferase, a marker of liver damage. Our results suggest that the isomerase activity of platelet surface PDI regulates the redox state of the Cys4-Cys17 disulfide bond in GPIbα and is required for the ligand-binding function of GPIbα, thereby playing a critical role during platelet-neutrophil interactions under thromboinflammatory conditions.