The b' domain of protein disulfide isomerase cooperates with the a and a' domains to functionally interact with platelets.

Background Protein disulfide isomerase (PDI) is an oxidoreductase consisting of four domains arranged in the order a-b-b '-a ' with an x-linker between the b ' and a ' domains. PDI binds to alpha(IIb)beta(3) integrin on activated platelets, and potentiates activation of this integrin through the C-terminal CGHC active-site motif. How PDI binds to platelet alpha(IIb)beta(3) is unknown. Objective and methods We used PDI domain fragments and full-length PDI containing point mutations to study inhibition of Alexa 488-labeled PDI binding to thrombin-activated platelets. The effect of the PDI variants on platelet aggregation was tested. Results Only PDI fragments containing the b ' domain bound to activated platelets. A double mutant of the b ' domain had decreased binding, confirming the essential role of the b ' domain. Addition of mutations in the a and a ' domains further decreased binding, suggesting that these domains contribute to the interaction of PDI with platelets. The ability of the b ' domain to interact directly with alpha(IIb)beta(3) was demonstrated with surface plasmon resonance, with contributions from the a and a ' domains. The abb ' x PDI fragment that binds to platelets but lacks the critical C-terminal active site inhibited platelet aggregation and in vivo thrombosis. Moreover, site mutations in the a, b ' and a ' domains that resulted in partial binding to platelets partially recovered aggregation of PDI-null platelets. PDI mutants that did not bind showed no recovery. Conclusion PDI functionally interacts with alpha(IIb)beta(3) on platelets through the substrate-binding b ' domain, with the a and a ' domains contributing to efficient binding.