Lu Wang1,2, Junsong Zhou1,3, Lei Wang2,4, Chih-Chen Wang2,4, David W Essex1. 1. Sol Sherry Thrombosis Research Center, Division of Hematology, Department of Medicine, Temple University School of Medicine, Philadelphia, PA, USA. 2. National Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China. 3. The Cyrus Tang Hematology Center, Jiangsu Institute of Hematology, Collaborative Innovation Center of Hematology, Soochow University, Suzhou, China. 4. College of Life Sciences, University of Chinese Academy of Sciences, Beijing, China.
Abstract
Essentials Protein disulfide isomerase (PDI) interacts with the αIIbβ3 integrin on platelets We generated PDI domain fragments and full-length PDI containing point mutations PDI interacts with αIIbβ3 through the b' domain, with the a and a' domains contributing This is the first report demonstrating PDI binding to a native protein on intact cells SUMMARY: 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 αII b β3 integrin on activated platelets, and potentiates activation of this integrin through the C-terminal CGHC active-site motif. How PDI binds to platelet αII b β3 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 αII b β3 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 αII b β3 on platelets through the substrate-binding b' domain, with the a and a' domains contributing to efficient binding.
Essentials Protein disulfide isomerase (n class="Gene">PDI) interacts with the αIIbβ3 integrin on platelets We generated PDI domain fragments and full-length PDI containing point mutations PDI interacts with αIIbβ3 through the b' domain, with the a and a' domains contributing This is the first report demonstrating PDI binding to a native protein on intact cells SUMMARY: 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 αII b β3 integrin on activated platelets, and potentiates activation of this integrin through the C-terminal CGHC active-site motif. How PDI binds to platelet αII b β3 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 αII b β3 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 αII b β3 on platelets through the substrate-binding b' domain, with the a and a' domains contributing to efficient binding.
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