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 1

Unbiased, high-sensitivity proteomics of human platelets identifies increased SEC61B in type 2 diabetes.

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Unbiased, high-sensitivity proteomics of human platelets identifies incr...
(A) Workflow demonstrating collection of clinicolaboratory and coronary angiogram data from patients without (non-DM) and with diabetes (DM); quality check of platelets by flow cytometry; platelet aggregation; and separation of resting and low-dose thrombin-activated platelet intracellular fraction “lysate” and released fraction “releasate” from patients without and with DM. (B) Levels of serum fructosamine in patients without (n = 33) versus with DM (n = 42) (Mann-Whitney test). (C) Median intensity of K310 glycated albumin peptides detected by mass spectrometry in the plasma of patients without (n = 29) and with DM (n = 39) (Welch’s t test). (D) Heatmap of 100 highest fold change differences between DM and non-DM platelet lysate proteomes in the resting state. 2,467 proteins were consistently detected in >50% of samples. Enrichment in proteins involved in response to oxidative stress (Z-scores shown). (E) Correlation of the top 100 upregulated platelet lysate proteins, in response to low-dose thrombin 0.025 U/mL, with serum fructosamine. SEC61B was the only platelet protein significantly correlated with serum fructosamine (red circle) (Spearman’s rho = 0.33, P = 0.029). ARMT1, CPNE2, and COMMD3 were negatively correlated with serum fructosamine (blue circles). (F) SEC61B, SEC61A, and SEC61G levels determined by mass spectrometry in platelets grouped in normal fructosamine (200–290 μmol/L) (n = 28) (gray) and high fructosamine (>290 μmol/L) (n = 15) (red) (Mann-Whitney test). Data are shown as the mean ± SD. GPX7, glutathione peroxidase 7; AMBP, α-1-microglobulin/bikunin precursor; MGMT, O-6-methylguanine-DNA methyltransferase; RWDD1, RWD domain containing 1; ARG1, arginase 1; SEPP1, selenoprotein P; HP, haptoglobin; APOA4, apolipoprotein A4; APOD, apolipoprotein D; APOE, apolipoprotein E; ARMT1, acidic residue methyltransferase 1; COMMD3, COMM domain containing 3; CPNE2, calcium-dependent phospholipid-binding protein; SEC61B, SEC61 translocon subunit β; SEC61A, SEC61 translocon subunit α; SEC61G, SEC61 translocon subunit γ.