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 5

SEC61B overexpression in HEK cells does not induce activation of IRE1 but is associated with decreased protein synthesis.

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SEC61B overexpression in HEK cells does not induce activation of IRE1 bu...
(A) SEC61B (red) and tubulin (green) immunostaining of lentiviral vector-transfected control or SEC61B-OE HEK293 cells. Nuclei are stained with Hoechst 33258 (blue). Representative images are shown. Scale bar: 20 μm. Normalized SEC1B intensity/cell in control (gray) and OE (red) cells. n = 20–30 cell clusters from n = 3 independent experiments (Welch’s test). (B) Representative Western blots of SEC61B, anti-Myc, and GAPDH of control and OE cells. SEC61B in HEK293 OE cells runs as 2 bands: native SEC61B (~10 kDa) and Myc-tagged SEC61B (~15 kDa). (C) Band intensity ratio of SEC61B to GAPDH in lysate of control (gray) versus OE cells (red). n = 7 independent experiments per group (Welch’s test). (D) Representative Western blots of SEC61A and GAPDH of control and OE cells. Band intensity ratio of SEC61A to GAPDH in lysate of control (gray) versus OE cells (red). n = 8 independent experiments (Welch’s test). (E) Representative Western blots of p-IRE1, IRE1, and GAPDH of control and OE cells. Band intensity ratio of p-IRE1 to IRE1 in HEK293 lysate of control (gray) versus OE cells (red). n = 10 independent experiments (Welch’s test). (F) Representative images of L-AHA fluorescence intensity (green) as a measure of protein synthesis in control and SEC61B-OE cells. Scale bar: 20 μm. L-AHA fluorescence intensity per cell area measured in n = 30 cell clusters per group from n = 3 independent experiments (Mann Whitney test). (G) Representative images of L-AHA fluorescence intensity (green) in platelets from normoglycemic (non-DM) and streptozotocin-induced hyperglycemic (DM) mice. Scale bar: 5 μm. L-AHA fluorescence intensity per platelet in non-DM (gray) and DM (red) mice. 20 platelets analyzed per mice, n = 7–8 mice per group (Welch’s t test). SEC61B OE, SEC61B overexpressing cells; L-AHA, L-azidohomoalanine.