SEC61B regulates calcium flux and platelet hyperreactivity in diabetes
Yvonne X. Kong, Rajan Rehan, Cesar L. Moreno, Søren Madsen, Yunwei Zhang, Huiwen Zhao, Miao Qi, Callum B. Houlahan, Siân P. Cartland, Declan Robertshaw, Vincent Trang, Frederick Jun Liang Ong, Michael Liu, Edward Cheng, Imala Alwis, Alexander Dupuy, Michelle Cielesh, Kristen C. Cooke, Meg Potter, Jacqueline Stöckli, Grant Morahan, Maggie L. Kalev-Zylinska, Matthew T. Rondina, Sol Schulman, Jean Y. H. Yang, G. Gregory Neely, Simone M. Schoenwaelder, Shaun P. Jackson, David E. James, Mary M. Kavurma, Samantha L. Hocking, Stephen M. Twigg, James C. Weaver, Mark Larance, Freda H. Passam
Yvonne X. Kong, Rajan Rehan, Cesar L. Moreno, Søren Madsen, Yunwei Zhang, Huiwen Zhao, Miao Qi, Callum B. Houlahan, Siân P. Cartland, Declan Robertshaw, Vincent Trang, Frederick Jun Liang Ong, Michael Liu, Edward Cheng, Imala Alwis, Alexander Dupuy, Michelle Cielesh, Kristen C. Cooke, Meg Potter, Jacqueline Stöckli, Grant Morahan, Maggie L. Kalev-Zylinska, Matthew T. Rondina, Sol Schulman, Jean Y. H. Yang, G. Gregory Neely, Simone M. Schoenwaelder, Shaun P. Jackson, David E. James, Mary M. Kavurma, Samantha L. Hocking, Stephen M. Twigg, James C. Weaver, Mark Larance, Freda H. Passam
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Research Article Cardiology Cell biology Hematology

SEC61B regulates calcium flux and platelet hyperreactivity in diabetes

Abstract

Platelet hyperreactivity increases the risk of cardiovascular thrombosis in diabetes and failure of antiplatelet drug therapies. Elevated basal and agonist-induced calcium flux is a fundamental cause of platelet hyperreactivity in diabetes; however, the mechanisms responsible for this remain largely unknown. Using a high-sensitivity, unbiased proteomic platform, we consistently detected over 2,400 intracellular proteins and identified proteins that were differentially released by platelets in type 2 diabetes. We identified that SEC61 translocon subunit β (SEC61B) was increased in platelets from humans and mice with hyperglycemia and in megakaryocytes from mice with hyperglycemia. SEC61 is known to act as an endoplasmic reticulum (ER) calcium leak channel in nucleated cells. Using HEK293 cells, we showed that SEC61B overexpression increased calcium flux into the cytosol and decreased protein synthesis. Concordantly, platelets in hyperglycemic mice mobilized more calcium and had decreased protein synthesis. Platelets in both humans and mice with hyperglycemia had increased ER stress. ER stress induced the expression of platelet SEC61B and increased cytosolic calcium. Inhibition of SEC61 with anisomycin decreased platelet calcium flux and inhibited platelet aggregation in vitro and in vivo. These studies demonstrate the existence of a mechanism whereby ER stress–induced upregulation of platelet SEC61B leads to increased cytosolic calcium, potentially contributing to platelet hyperreactivity in diabetes.

Authors

Yvonne X. Kong, Rajan Rehan, Cesar L. Moreno, Søren Madsen, Yunwei Zhang, Huiwen Zhao, Miao Qi, Callum B. Houlahan, Siân P. Cartland, Declan Robertshaw, Vincent Trang, Frederick Jun Liang Ong, Michael Liu, Edward Cheng, Imala Alwis, Alexander Dupuy, Michelle Cielesh, Kristen C. Cooke, Meg Potter, Jacqueline Stöckli, Grant Morahan, Maggie L. Kalev-Zylinska, Matthew T. Rondina, Sol Schulman, Jean Y. H. Yang, G. Gregory Neely, Simone M. Schoenwaelder, Shaun P. Jackson, David E. James, Mary M. Kavurma, Samantha L. Hocking, Stephen M. Twigg, James C. Weaver, Mark Larance, Freda H. Passam

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Figure 7

Anisomycin, which promotes a “sealed” SEC61 conformation, decreases ER calcium leak in HEK293 cells and platelets and inhibits platelet thrombus formation in vivo. (A)

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Anisomycin, which promotes a “sealed” SEC61 conformation, decreases ER c...
Schematic of inhibition of SEC61 by anisomycin (ANX). ANX inhibits ribosomal elongation of the peptide, locking the peptide within the pore, thus preventing calcium leak when SERCA2 is inhibited with TG. (B) Cytosolic calcium was quantified with Fura2-loaded human platelets in response to TG, after pretreatment with vehicle or ANX (200 μM for 2 hours). Solid line indicates mean; shaded region indicates SEM. (C) Peak Fura2 in platelets treated with vehicle (gray) or ANX (blue) after TG addition. n = 6 healthy donors (Welch’s t test). (D) Platelet aggregation over time in response to TG, after pretreatment of platelet-rich plasma (PRP) with vehicle or ANX 200 μM for 2 hours. Maximal percentage aggregation of PRP, and time to initiation of platelet aggregation, in response to TG, after pretreatment with vehicle (gray) or ANX (blue). n = 5 healthy donors (Welch’s t test). (E) Cytosolic calcium in Cal520-loaded non-DM platelets after treatment with vehicle (gray) or ANX (blue) (100 μM for 1 hour), prior to TG addition. Peak Cal520 in non-DM platelets treated with vehicle (gray) or ANX (blue) followed by TG. n = 6 mice per group (Welch’s t test). (F) Cytosolic calcium in Cal520-loaded DM platelets, as described before. n = 6 mice per group (Welch’s t test). (G) Differential interference contrast images depicting thrombi (dotted line) in mouse mesenteric venules forming 4 minutes after needle insertion in non-DM and DM mice. Mice were pretreated with i.v. vehicle control (Veh) or ANX (20 mg/kg, 1.5 hours prior to surgery). The scale bar is 50 μm.(H) Surface area of thrombi generated in non-DM and (I) in DM mice was quantified at the indicated time points after needle insertion. Results are expressed as mean ± SD. n = 3–5 mice per group, 6–8 thrombi per mouse (2-way ANOVA).