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 6

Eeyarestatin I, which stabilizes SEC61 in its “open” — calcium permeable — conformation, increases ER calcium leak in HEK293 cells overexpressing SEC61B and in platelets.

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Eeyarestatin I, which stabilizes SEC61 in its “open” — calcium permeable...
(A) Schematic for 2-step induction of SEC6 calcium leak. In step 1, eeyarestatin I (ES1) “opens” the SEC61 channel allowing calcium to leak from the ER into the cytosol. In step 2, TG inhibits SERCA from pumping calcium back into the ER to measure maximal SEC61-mediated ER calcium leak. (B) Cytosolic calcium was quantified over time in Cal-520–loaded control or SEC61B-overexpressing (SEC61B-OE) HEK293 cells using a fluorescent plate reader. HEK293 control and OE cells were treated with vehicle (gray) or ES1 (red) for 1 minute, followed by addition of TG, in the presence of EGTA (solid line indicates mean; shaded region indicates SEM). (C) Basal and (D) peak fluorescence intensity of Cal-520 in the presence of vehicle or ES1 in control (gray) and OE cells (red). n = 10 independent experiments (1-way ANOVA with Dunn’s multiple comparisons test). (E) Cytosolic calcium was quantified over time in Fura2-loaded human platelets in the presence of vehicle or ES1 50 μM for 10 minutes, followed by addition of TG. (F) Basal and peak fluorescence intensity of Fura2 10 minutes after incubation with vehicle or ES1 (basal) and after addition of TG (peak). Mean ± SD, n = 5 healthy donors (Mann-Whitney test). (G and H) Cytosolic calcium was quantified over time in Cal-520–loaded platelets from (G) normoglycemic (non-DM) mice and (H) streptozotocin-induced hyperglycemic (DM) mice. Platelets were treated with ES1 for 5 minutes, followed by TG, in the presence of EGTA (solid line indicates mean; shaded region indicates SEM). (I and J) Basal and peak fluorescence intensity in Cal-520–loaded platelets from vehicle-treated (Veh, gray) versus ES1-treated (red) platelets from (I) non-DM mice and (J) DM mice. n = 7 mice, per group (Welch’s t test). SERCA 2b, sarco/endoplasmic reticulum calcium ATPase; ES1, eeyarestatin I; TG, thapsigargin.