Molecular mechanism for platelet hyperreactivity in diabetes mellitus

It is known that hyperreactivity of platelets increases the risk of thrombosis and cardiovascular diseases in subjects with diabetes mellitus1. Also, platelet hyperactivity attenuates the effects of antiplatelet drug therapy. Various factors are involved in platelet hyperreactivity in subjects with diabetes mellitus. One possible pathway is that hyperglycemia increases platelet reactivity by nonenzymatic glycation of platelet membrane glycoproteins and by producing glycated low-density lipoprotein (LDL) that provokes oxidative stress. Another possible pathway is that endoplasmic reticulum (ER) stress is involved in platelet hypersensitivity in diabetes mellitus2. It has also been thought that one of the key features of diabetic platelets is the higher cytosolic calcium, but the mechanism responsible remained unknown. Understanding the mechanisms for the increase of platelet calcium response in diabetes mellitus would exert favorable effects on the development of drugs against platelet hyperreactivity (Figure 1). Cytosolic calcium must remain at submicromolar levels so that platelets remain in a resting state, while calcium is at high submillimolar levels in the ER and millimolar levels in the extracellular space3. Calcium is pumped into the ER by sarco/ER Ca2+-ATPase (SERCA). When platelets are stimulated with some stimuli, inositol-1,4,5-trisphosphate (IP3) is produced, which stimulates IP3 receptor channels to release calcium from the ER. Consequently, calcium enters the cells through calcium channels, leading to the activation of the platelets. Calcium leaks out of the ER through calcium leak channels, and this process is counteracted by SERCA. The SEC61 translocon is the main channel of calcium leak from the ER. The SEC61 translocon functions as a transporter of newly synthesized polypeptides into the ER. When not engaged in protein transport, calcium leaks through SEC61 into the cytosol. Blocking SERCA function leads to a leak from the ER through the translocon and an increase in cytosolic calcium. Although there are various reports about SERCA function in platelets, the role of SEC61 in platelets has not been investigated. To investigate the potential protein alteration in platelets in diabetes mellitus, the authors employed a high-sensitivity proteomic platform that they developed previously4. They compared platelets obtained from subjects with diabetes mellitus, those with coronary artery disease, and those with similar risk factors without diabetes mellitus. Previously, they showed that SEC61B expression was upregulated in platelets of hyperglycemic mice and that SEC61B overexpression was associated with increased calcium leak from the ER into the cytosol, which was inhibited with the SEC61 inhibitor. Thereby, the authors have identified SEC61 as a calcium leak channel in platelets and described SEC61-mediated calcium leak as a mechanism of dysregulated calcium flux and platelet hyperreactivity under diabetic conditions. In this study, the authors detected many intracellular proteins and identified proteins that were differentially released by platelets in type 2 diabetes5. They showed that SEC61 translocon subunit β (SEC61B) was increased in platelets from humans and mice with diabetes mellitus. SEC61 is known to act as an ER calcium leak channel. They showed that SEC61B overexpression increased calcium flux into the cytosol and decreased protein synthesis and that platelets in hyperglycemic mice mobilized more calcium and decreased protein synthesis. Platelets in both humans and mice with hyperglycemia had increased ER stress, which led to the increase of platelet SEC61B expression and increased cytosolic calcium. Conversely, inhibition of SEC61 with anisomycin decreased platelet calcium flux and inhibited platelet aggregation. The data in this study clearly demonstrate that ER stress upregulates SEC61B expression in platelets, which leads to the increase of cytosolic calcium and platelet hyperreactivity under diabetic conditions. The increase of platelet reactivity is a critical issue under diabetic conditions, because in subjects with diabetes mellitus, the effects of antiplatelet therapy are weakened, which leads to cardiovascular risk1. Therefore, it would lead to provide possible novel therapy for cardiovascular diseases to identify the mechanism for platelet hyperreactivity. To identify this under diabetic conditions, the authors used a high-sensitivity proteomic platform which they had used for another purpose4. Although there was a large limitation in prior platelet proteomic studies, the method in this study enabled them to provide the largest proteomic analysis of platelet proteins in diabetes mellitus with coronary artery disease. The most important finding in this study was that SEC61B expression in platelets is increased under diabetic conditions. SEC61B expression is upregulated in a separate human diabetic cohort and in diabetic mice, and it seems that SEC61B upregulation is involved in hyperglycemia-related phenomena. They showed that increased SEC61B was associated with increased cytosolic calcium, while SEC61 inhibition decreased calcium leak and platelet aggregation. These findings clearly demonstrate that SEC61B plays crucial roles in platelet hyperreactivity which is observed in diabetes mellitus. Platelets from subjects with diabetes mellitus show elevated basal cytosolic calcium probably due to impaired SERCA. In consideration of the role of SEC61 as a calcium leak channel, they propose that SEC61B is involved in calcium dysregulation under diabetic conditions. It is likely that SEC61B upregulation is due to platelet ER stress under diabetic conditions because SEC61B is upregulated by chemical induction of ER stress. It has been shown that SEC61B is closely associated with the ER, which is likely involved in the rapid protein synthesis after platelet ER stress. In addition, it is known that the spliced form of XPB1, a transcription factor downstream of ER stress activation, binds the SEC61B promoter region. Taken all these data together, the authors' working hypotheses are based upon many firm results and thus are very reasonable and convincing. Increased expression of SEC61B provides a positive feedback loop to further increase platelet ER stress due to the decrease of calcium in the ER. One interesting point is how the β subunit of SEC61 regulates calcium leak through the SEC61 channel because it is peripheral to the pore of the channel. Although calcium movement through the translocon occurs through the SEC61A pore, it has been proposed that SEC61B and SEC61G play major roles. At present, there is no information about the role of SEC61B in calcium flux, and further examination of channel subunits would be necessary to unveil the precise function of the channel. It is also possible that the increased calcium leak by SEC61B overexpression is due to decreased peptide transfer. Decreased peptide transfer would enable calcium to leak through the SEC61 channel into the cytosol. Overexpression of SEC61B reduced protein synthesis, further indicating that the β subunit is crucial in optimal SEC61 function. Furthermore, their analysis highlighted the enrichment of oxidation pathways in platelets of subjects with diabetes mellitus. Indeed, diabetic platelets had increased expression levels of redox proteins such as glutathione peroxidase, which can alter oxidative stress and/or ER stress. It is thought that such redox imbalance leads to activation of platelets together with CD36. Other danger signals such as oxidized LDL also act together with CD36. Taken all data into consideration, it is likely that oxidative stress and/or ER stress are also involved in platelet activation under diabetic conditions. There is a limitation in human sample variability. To increase the precision, however, they carefully adhered to the method of platelet isolation and measurements and validated their findings of SEC61B upregulation in a separate cohort of subjects. They also tried to eliminate confounders by matching diabetes and non-diabetes subjects at various points. Although some confounders were not excluded in this study, it is likely that they performed very well in order to demonstrate their working hypotheses. In this study, the authors clearly demonstrate that SEC61 translocon plays a crucial role in platelet biology as a calcium leak channel. SEC61B functions to regulate calcium influx in platelets under diabetic conditions. It is likely that the upregulation in diabetes leads to platelet hyperreactivity. Furthermore, the data in this study is very important in that they show some potential biomarker or therapeutic target in cardiovascular disease. Data sharing not applicable to this article as no datasets were generated or analysed during the current study.