Unfolded Protein Response Differentially Modulates The Platelet Phenotype

Abstract

Background:

Unfolded protein response (UPR) is a multifaceted signaling cascade that alleviates protein misfolding. Although well studied in nucleated cells, UPR in absence of transcriptional regulation has not been described. Intricately associated with cardiovascular diseases, platelets, despite being anucleate, respond rapidly to stressors in blood. We investigate the UPR in anucleate platelets and explore its role, if any, on platelet physiology and function.

Methods:

Human and mouse platelets were studied using a combination of ex vivo and in vivo experiments. Platelet lineage specific knockout mice were generated independently for each of the three UPR pathways, protein kinase RNA (PKR)-like ER kinase (PERK), X-binding protein (XBP1) and activating transcription factor 6 (ATF6). Diabetes mellitus (DM) patients were prospectively recruited and platelets were evaluated for activation of UPR under chronic pathophysiological disease conditions.

Results:

Tunicamycin induced the IRE1α-XBP1 pathway in human and mouse platelets, while oxidative stress predominantly activated the PERK pathway. PERK deletion significantly increased platelet aggregation and apoptosis, and phosphorylation of PLCγ2, PLCβ3, and p38 MAPK. Deficiency of XBP1 increased platelet aggregation, with higher PLCβ3 and PKCδ activation. ATF6 deletion mediated a relatively modest effect on platelet phenotype with increased PKA. Platelets from DM patients exhibited a positive correlation between disease severity, platelet activation and protein aggregation, with only IRE1α-XBP1 activation. Moreover, IRE1α inhibition increased platelet aggregation, while clinically approved chemical chaperone, 4-PBA reduced the platelet hyperactivation.

Conclusion:

We show for the first time, that UPR activation occurs in platelets and can be independent of genomic regulation, with selective induction being specific to the source and severity of stress. Each UPR pathway plays a key role and can differentially modulate the platelet activation pathways and phenotype. Targeting the specific arms of UPR may provide a new anti-platelet strategy to mitigate thrombotic risk in DM and other cardiovascular diseases.

Graphical Abstract

Introduction

Several stressors, including hypoxia, oxidative stress and nutrient stress, can affect the protein folding capacity of the endoplasmic reticulum (ER), leading to the accumulation of misfolded proteins (ER stress) ¹. To alleviate the misfolded protein load and ensure protein folding fidelity, the unfolded protein response (UPR) is activated, which includes multifaceted signaling pathways to reprogram gene transcription, mRNA translation, and protein expression. Glucose regulated protein (GRP78), an ER chaperone, forms the master regulator of the UPR and regulates activation of transmembrane ER stress sensors protein kinase RNA (PKR)-like ER kinase (PERK), inositol-requiring enzyme-1alpha (IRE1α) and activating transcription factor 6 (ATF6), which work in a concerted manner in nucleated cells to restore protein folding homeostasis ².

Platelets, while anucleate, contain a variety of cellular organelles (including the dense tubular system, DTS), granules, and mitochondria. Platelets, short lived and small (2–5 μm), result from the fractionation of megakaryocytes, and ‘inherit’ the cytoplasm ³. Although platelets lack genomic DNA, they contain a heterogenous array of functional coding and non-coding RNAs, spliceosome components and translational machinery ^{4, 5}. They are thus capable of highly efficient de novo protein synthesis ⁴ and upon activation, can rapidly synthesize proteins and respond efficiently to perturbations through activation of diverse signaling cascades. Indeed, platelets possess the machinery for several stress induced signaling pathways including apoptosis, autophagy, ubiquitin proteasome system and mitophagy ^6–9; the UPR however remains largely unexplored in these anucleate cells.

Beyond hemostasis and thrombosis ¹⁰, platelets are involved in pathophysiological conditions including cardiovascular diseases (CVD), neurodegenerative diseases and cancer ^{11, 12}. Diabetes mellitus Type II (DM) affects millions globally and CVD forms the leading cause of morbidity and mortality in DM patients. Such patients present a higher risk for thrombotic events and several factors including hyperglycemia, impaired insulin signaling, and oxidative stress, contribute to the platelet hyperreactivity ^{7, 13}. Here, we set out to explore (1) whether the multifaceted UPR can be launched in platelets despite the lack of transcriptional regulation, (2) whether the UPR plays a role in platelet function in normal physiology (3) whether chronic pathophysiological (diabetes) stress affects the platelet UPR and 4) its therapeutic relevance in platelets (if any).

Methods

Data Availability.

The data supporting the findings of the study are available upon reasonable request from the corresponding authors.

A detailed description of materials and methods is available in the Online Supplemental Material.

Results

UPR induction by tunicamycin selectively activates IRE1α in platelets

The UPR has not been previously described or characterized in platelets. A robust expression of GRP78, IRE1α and spliced XBP1 (Fig S1A) was observed in resting platelets; PERK and ATF6 were minimally detected (Fig S1B). To investigate whether UPR plays a role in platelets, we treated platelets with a classic inducer of ER stress, tunicamycin (Tm), an N-linked glycosylation inhibitor. Platelet ER (DTS) exhibits rapid ultrastructural changes in response to platelet agonists and is involved in platelet activation ¹⁹. Following Tm treatment, the ER was visibly distended into a more elongated vesicular form (Figure 1A). Similar ultrastructural changes, following ER stress, have been shown in yeast, possibly due to the need for an increased ER surface area for translational reprogramming in response to misfolded proteins ²⁰. Next, Proteostat^® dye, a fluorescent molecular rotor dye, was used to estimate the relative percentage of protein aggregation in human platelets. Significantly higher protein aggregation was observed in Tm treated platelets as compared to control human (HC) platelets (Figure 1B). Activation of platelets with thrombin or ADP did not increase protein aggregation (Figure S1C). Tm increased ER chaperone expression including GRP78 (Figure 1C–D, Figure S1D), GRP94 and PDI. Incubation with tunicamycin increased surface P-selectin levels (released by the alpha granules upon platelet activation) (Figure S1 E). Immunostaining, as visualized by confocal microscopy, further confirmed the increase in GRP78 and P-selectin (Figure 1E). Surprisingly, Tm selectively induced the IRE1α pathway; PERK and ATF6α remained unaffected (Figure 1F–G). This was consistent, even with higher Tm concentrations or longer treatments with Tm (Figure S1 F–G). Activation of IRE1α causes the cytoplasmic mRNA splicing of X-binding protein (XBP1) and higher XBP1s protein expression (Figure 1G–H). We thus find, that, unlike most in vitro and in vivo studies in nucleated cells, which demonstrate the induction of ER stress within days of Tm treatment ²¹, anucleate platelets appear to quickly mount the ER stress response (within hours); thus demonstrating extreme sensitivity to changes in proteostasis.

Figure 1: Tunicamycin selectively activates IRE1α in platelets.

A-H. Freshly isolated human platelets were treated with Tm (5 μg/ml) or vehicle (saline) for 3 h at 37°C. A. Transmission electron microscopy (TEM) images of vehicle and Tm treated platelets, acquired on FEI Tecnai Biotwin (LaB6, 80 kV). Arrows show distended ER within treated platelets. Scale bar, 500nm. n=3 independent experiments for Veh and Tm. B. Percentage of protein aggregates in platelets measured using a rotor dye, ProteoStat^® in n=8/group. Values calculated based on standard curve generated using lysozyme IgG standards and controls provided by manufacturer and expressed as percentage. C-D. Representative immunoblots and respective quantitation for expression of ER chaperones following Tm treatment (3 × 10⁸ platelets/ml). n=6/group. E. Confocal microscopy images showing localization of GRP78 (red) and P-selectin (blue) on platelets stained with CD41 (green). Scale bar, 10 μm. Images acquired on Perkin Elmer 5-Laser Spinning Disk Confocal Microscope (equipped with Nikon Ti-E Eclipse inverted microscope), objective 100X (oil). Platelet counts adjusted between samples prior to staining. n=3/group. F-G. Immunoblotting and respective quantitation for key UPR proteins in platelets. For phosphorylated proteins, quantification represents ratio of phosphorylated to total protein, normalized to the loading control, GAPDH. H. Levels of spliced and unspliced XBP1 mRNA measured by qPCR in platelet RNA. For Fig I-H, n=6/group. See also Supplemental Figure S1. I-N. Wild type (WT) mice injected with 1 μg/ml Tm, i.p or vehicle (150 mM dextrose), n=15 per group, 8 males/7 females. Platelets isolated 24 h post injection. I. Soluble P-selectin measured in plasma from WT and Tm injected mice (n=14/group). J. TMRE assay for mitochondrial apoptosis in mice platelets (n=5/group), with or without addition of calcium. K. Protein aggregate levels in mice platelets (n=15/ group). L. Intraplatelet (green, CD41) expression of GRP78 (red) as seen by confocal microscopy in mice platelets. Scale bar, 10 μm. Images acquired on Perkin Elmer 5-Laser Spinning Disk Confocal Microscope with 100× oil immersion lens, n=3/group. M-N. Representative images for UPR protein expression by immunoblotting and respective quantification from n=4/group. Values expressed as mean ± SEM and the p value corresponds to Student t test (B, I, K) or Mann-Whitney test (D, G, H, N). See also Supplemental Figure S1–S2.

Examining the response in vivo, we injected mice with Tm ²¹ and observed higher surface and soluble sP-selectin levels (Figure S2A–B, Figure 1I), loss of membrane mitochondrial potential (Figure 1J, Figure S2C), and higher apoptosis (caspase-3 and Bax) (Figure S2D–E). Platelets from Tm-injected mice had higher intraplatelet protein aggregates (Figure 1K), along with increased GRP78 expression (Figure 1L–N, Figure S2F). Concurrent with human platelets, we observed a selective increase in only IRE1α and spliced XBP1 (Figure 1M–N). In contrast, heart and liver tissues isolated from same mice exhibited simultaneous activation of all three arms of the UPR (Figure S2G).

Oxidative stress selectively activates only PERK while DTT stress-induced protein misfolding activates all three UPR arms in platelets

We next investigated whether other stressors could selectively activate different arms of UPR. Oxidative stress (OS), through the disruption of ER redox homeostasis, inhibits protein folding; thereby inducing ER stress and UPR ²². We applied hydrogen peroxide, H₂O₂ (oxidative stressor) onto freshly isolated human platelets ¹⁴. Accompanied with higher platelet apoptosis (Figure S3A), transmission electron microscopy (TEM) revealed altered platelet morphology, with presence of protein aggregate like fibrils and degranulation (Figure 2A). OS mediated a significant increase in protein carbonylation (Figure S3B) and intraplatelet protein aggregation (Figure 2B). Surprisingly, while GRP78 expression increased (Figure 2C–D, Figure S3C,E), IRE1α levels and spliced XBP1 remained unaffected, at protein (Figure 2C, Figure S3C) and mRNA (Figure S3D) levels. Interestingly, OS mediated a distinct increase in PERK expression (Figure 2E–F) along with increase in downstream effectors including phosphorylated eIF2α and ATF4 (Figure 2E–F) and was consistent even with higher H₂O₂ concentrations and longer incubation times (Figure S3F–G). Expression of cleaved (activated) ATF6α also increased (Figure 2E–F). This provided further evidence of pathway selectivity of the platelet UPR. Although more extensive studies are needed to detail the roles of ATF4 and ATF6 in absence of a nucleus, the rapid selective induction of UPR components within platelets is likely an effective strategy when dealing with specific stressors.

Figure 2: Acute oxidative stress activates only PERK pathway while stress-induced protein misfolding activates all three arms of the UPR in platelets.

A-G. Freshly isolated human platelets were treated with H₂O₂ (50 μM) or vehicle (saline) for 30 min at 37°C. A. TEM images of vehicle and H₂O₂ treated platelets acquired on FEI Tecnai Biotwin (LaB6, 80 kV). Arrows show protein aggregate fibrils and microparticle release. Scale bar, 1 μm. B. Protein aggregation in platelets. n=5/group. C. Representative immunoblots for expression of GRP78, IRE1α and sXBP1 following H₂O₂ treatment. D. GRP78 (red) levels in individual platelets (green) as seen by microscopy. Scale bar, 10 μm. Images acquired on Perkin Elmer 5-Laser Spinning Disk Confocal Microscope (equipped with Nikon Ti-E Eclipse inverted microscope), 100× oil immersion lens. Platelet counts adjusted between samples prior to staining. TEM and immunostaining micrographs representative of n=3 independent experiments. E-F. Expression and quantification of PERK pathway proteins and ATF6. n=6/group. See also Supplemental Figure S3. G-J. Freshly isolated human platelets were treated with DTT (2 mM) or vehicle (saline) for 30 min at 37°C. G. TEM images of the vehicle and DTT treated platelets acquired on FEI Tecnai T12 (LaB6, 120 kV). Arrows indicate presence of protein aggregates. Zoom in shows magnified view of protein fibril aggregates. Scale bar, 1 μm. Higher magnification of HC platelets shown in Online Data Supplemental Figure S3. TEM micrographs representative of n=3/group. H. Protein aggregation in DTT-treated platelets. n=6/control group and n=9/DTT treated group. I. Representative immunoblots for expression of UPR proteins following DTT treatment; n= 6/group. J. Confocal Microscopy images showing co-localization of IRE1α (magenta) and PERK (cyan) on the platelets stained with CD41 (green). Scale bar, 10 μm. Zoom in depicts levels of IRE1α and PERK in individual platelets. n= 3 independent experiments/group. For phosphorylated proteins, quantification represents ratio of phosphorylated to total protein, normalized to GAPDH. Values expressed as mean ± SEM and precise p value corresponds to Mann-Whitney test.

Treatment of human platelets with dithiothreitol (DTT; disulfide bond disruption and protein misfolding) led to a significant increase in protein aggregates/fibril like structures, by TEM (Figure 2G, higher magnification in Figure S3H) and biochemically (Figure 2H), along with elevated mitochondrial depolarization and apoptosis (Figure S3I–J). Such visible direct evidence of protein aggregates in platelets has not been reported previously. Protein misfolding led to simultaneous upregulation of IRE1α (Figure 2I, FigureS3 K), XBP1 splicing (Figure 2I, Figure S3K–L), PERK and ATF6α (Figure 2I, Figure S3K), confirmed further by the distinct colocalization of IRE1α and PERK (Figure 2J, Figure S3M). Our results, for the first time, demonstrate the presence of a robust UPR in the anucleate platelet; this selective UPR likely equips circulating platelets with a finely tuned, rapid, highly sensitive response to stress

Platelet-specific deletion of UPR genes differentially modulates platelet phenotype

Next, we wanted to investigate the pathophysiological significance of UPR in platelets in vivo. Since global knockout mice for most UPR genes are embryonically lethal ²³, we utilized the platelet factor 4-Cre recombinase (Pf4-Cre) megakaryocyte lineage specific transgenic mice¹⁸, to generate platelet specific knockout mice (Figure 3A).

Figure 3: Platelet-specific deletion of UPR genes differentially modulates platelet physiology.

A. Platelet-specific knockout mice were generated by crossing respective floxed mice with Pf4-iCre transgenic mice, as depicted in schematic. Freshly drawn blood and isolated platelets from each KO mice strain and their respective littermate controls (n=16 mice per group per strain, 8 males and 8 females) were used for experiments. Results from genotyping are shown in Supplemental Figure S4. Panel B-D represents results from the PERK conditional KO mice. Panel E-G depicts results from XBP1 cKO mice. Panel H-J shows results from ATF6 cKO mice. B, E, H. Representative platelet aggregation tracings in response to ADP (20μM) for each strain and quantitation shown taking 20 ohms (maximal impedance) as the 100% response. Aggregation response to collagen and CLEC-2 stimulatory antibody shown in Supplemental Figure S5. Aggregation curves reflect results from n=6 WT_P and n=6 PERK cKO, n=4 WT_X and n, =6 XBP1 cKO, n=5 WT_A and n=6 ATF6 cKO mice respectively. C, F, I. Levels of soluble P-selectin in platelet-specific KO mice. Results shown are from n=9 WT_P and n=16 PERK cKO, n=6 WT_X and n=10 XBP1 cKO, n=6 WT_A and n=9 ATF6 cKO mice respectively. D, G, J. Quantification of TMRE assay in the platelets following deletion of specific UPR genes. Graphs reflect results from n=7 WT_P and n=9 PERK cKO, n=7 WT_X and n=9 XBP1 cKO, n=6 WT_A and n=9 ATF6 cKO mice respectively. Representative flow cytometry scatter plots provided in Supplemental Figure S6. K. Intraplatelet protein aggregation levels in platelet-specific KO mice. Results from n=8 WT_P and n=12 PERK cKO, n=8 WT_X and n=12 XBP1 cKO, n=8 WT_A and n=12 ATF6 cKO mice respectively. Values expressed as Mean ± SEM. Exact p values correspond to Mann Whitney test (B-C, E-F, I-J), Student t test (D, G) or one way ANOVA, with Tukey multiple comparisons (K). See also Supplemental Figure S4–S8.

Mice with the platelet-specific PERK deletion (PERK cKO) demonstrated normal litter frequencies and survival (Figure S4A); both male and female PERK cKO mice had lower body weights (Figure S4B) and platelet counts (Figure S4C), compared to WT littermate (WT_P) controls. Agonist-induced platelet aggregation in whole blood forms the standard assay for measurement of prothrombotic tendency. PERK deficient platelets had significantly higher platelet aggregation in response to agonists like ADP (Figure 3B), collagen (Figure S5 A, C) and CLEC-2 (C-type lectin-like receptor 2) stimulatory antibody (Figure S5B, D). A marked elevation was also observed in P-selectin (Figure 3C) and mitochondrial depolarization (Figure 3D, Figure S6A). Additionally, a modest reduction occurred in the percentage of reticulated platelets (immature platelets) (Figure S6B). Platelets lacking PERK had elevated protein aggregates (Figure 3K) and a higher expression of ER chaperones, GRP78 and GRP94 (Figure S7 A–C).

PERK cKO platelets also displayed higher IRE1α and spliced XBP1 expression (Figure S7 A, D–E), even at basal conditions, likely as a compensatory mechanism to cope with physiological stressors. Levels of phosphorylated eIF2α, which occurs downstream of PERK, were significantly lowered (Figure S7 A,G). Interestingly, the UPR-mediated increase in PERK expression also regulates mitochondrial integrity and function in other cells ²⁴. This likely explains the marked increase in mitochondrial dysfunction, and subsequent platelet activation and aggregation observed in PERK cKO mice. This is consistent with our results from the ex vivo studies showing the selective increase in PERK and not IRE1α, following acute oxidative stress. These results further highlight the presence of a highly sensitive yet selective stress response in platelets, despite the lack of transcriptional machinery.

We next targeted the IRE1α-XBP1 pathway to identify its effect on platelet function. Mice with platelet-specific deletion of XBP1 (XBP1 cKO, Figure S4D), had normal survival, body weight, and platelet counts (Figure S4E–F). ADP and collagen induced platelet aggregation was higher in the XBP1 cKO than the WT_X (Figure 3E, collagen, Figure S5 A, C), along with increased platelet activation (Figure 3F). In contrast to PERK cKO, the XBP1 deficient platelets had a modest increase in apoptosis (Figure 3G, Figure S6C). The percentage of reticulated platelets was significantly higher (Figure S6D), suggesting a potential role for XBP1 in the regulation of platelet production from megakaryocytes or platelet lifespan in circulation. Although not significant, the expression of GRP78, GRP94 and PERK was elevated in the XBP1 cKO mice platelets (Figure S7), further supporting a distinct role for XBP1 as compared to PERK in platelets.

Finally, to identify the role of ATF6, we generated platelet-specific knockouts (ATF6 cKO, Figure S4G). Body weight and platelet counts in the ATF6 cKO mice did not differ significantly from littermate controls (WT_A) (Figure S4H–I). Unlike the PERK and XBP1 cKO mice, ATF6 cKO mice showed higher platelet aggregation in response to only ADP (Figure 3H) but not collagen and CLEC-2 stimulatory antibody (Figure S5) and platelet activation (Figure 3I) was distinctly lower. No significant difference was observed in apoptosis markers (Figure 3J, Figure S6E) and reticulated platelet levels in the ATF6 cKO (Figure S6F). Albeit still significant, the increase in protein aggregates was relatively lower in ATF6 cKO mice (Figure 3K). Concurrent with the overall relatively lower consequence of ATF6 on platelet phenotype, there were no significant changes in the expression of the ER chaperones and UPR pathway proteins (PERK, IRE1α, XBP1s) (Figure S7). Consistent with the differential induction of UPR observed in human ex vivo studies, platelet-specific ablation of specific UPR components causes distinct changes in the platelet phenotype in vivo. Even at normal physiological conditions, PERK and XBP1 deficient platelets displayed a higher prothrombotic tendency than the ATF6 cKO and WT mice.

We next wanted to investigate whether UPR pathways play a selective and differential role in thrombus formation in vivo. We used the ferric chloride-induced carotid artery injury model which employed the topical application of FeCl₃ to induce oxidative stress arterial damage, and subsequent thrombus formation. Post injury, carotid arteries from cKO mice were stained to visualize the thrombus composition. Different components of the thrombus display different colors in Carstairs staining (bright red for fibrin, gray-blue to navy blue for platelets, and bright blue for collagen). Platelet-fibrin rich thrombus area was highest in the PERK cKO and XBP1 cKO mice as compared to both wild type littermate controls and ATF6 cKO mice (Figure S8 A–B). This in vivo evidence is consistent with the distinct platelet aggregation response, and increase in activation biomarkers observed in platelets from the cKO mice ex vivo as well. Indeed, platelets from PERK cKO mice, which display the most pronounced increase in aggregation in response to all three agonists, also show the maximal increase in platelet rich thrombus formation following injury. Taken together, it is evident that each arm of the UPR pathway plays a key protective role in platelets; indeed, deletion of the UPR pathways promotes platelet hyperactivation and prothrombotic phenotype in mice.

Platelet-specific deletion of UPR genes selectively affects activation signaling

Based on increased platelet aggregation and thrombus formation associated with the PERK cKO and XBP1 cKO, we now needed to address how the key UPR response pathways intersect with the platelet activation signaling cascades. We also wanted to explore whether the distinct differences in the role of PERK, XBP1 or ATF6 on the observed platelet phenotype, arises from their differential regulation of the complex network of platelet activation responses. Using our platelet specific KO mice, we thus set out to investigate the effect of deletion of these UPR components on some of the known signaling pathways involved in platelet activation.

Early events of receptor mediated signaling, followed by the convergence and amplification of the signaling networks, leads to intraplatelet cytoskeletal re-arrangements, eventually causing platelet aggregation. There are two major classes of activation receptors in platelets – G-protein coupled receptors (GPCRs) and immunoreceptor tyrosine-based activation motif (ITAM) receptors ²⁵. Phospholipase C (PLC) in platelets plays a key role in transducing surface receptor activation stimuli into a intracellular signaling cascade events. The GPCR pathway primarily activates PLC-β2/3 ²⁶, while ITAM pathway has been known to activate PLCγ2 ²⁷. Both PLCγ2 and PLCβ2/3 are involved in earlier events of α_IIbβ₃ inside -out signaling, forming a major determinant of platelet activation ^{25, 28, 29}. PLCγ2 phosphorylation increased significantly in PERK cKO but not XBP1 cKO and the ATF6 cKO platelets (Figure 4A). PLCβ2/3 expression and phosphorylation increased significantly in the platelets from PERK cKO and XBP1 cKO mice (Figure 4B).

Figure 4: Platelet-specific deletion of UPR genes selectively affects specific platelet activation pathways.

Platelets (3 × 10⁸ per mL) from each of the mouse strains were lysed using RIPA buffer, resolved on SDS PAGE and immunoblotted for respective proteins. A total of n=10 mice (n=4 males, n=6 females) per strain and respective littermate controls used for platelet activation signaling responses. Immunoblotting and respective quantitation for A. phospho- PLCγ2 and total PLCγ2. B. phospho PLCβ3 and Total PLCβ3. C. phospho-Akt1 and Total Akt. D. phospho-p38 and Total p-38. E. phospho-PKCδ and Total PKCδ. F. phospho-PKA Cα and total PKA Cα. Quantification of immunoblotting reflects the results from n=6 WT_P and n=6 PERK cKO, n=6 WT_X and n=6 XBP1 cKO, n=6 WT_A and n=6 ATF6 cKO mice respectively. Data presented as mean ± SEM. Precise p value corresponds to Kruskal-Wallis test with Dunn’s multiple comparisons.

Platelets are known to express all isoforms of Akt, which are involved in signal amplification of initial receptor signals, leading to robust platelet activation responses ^{30, 31}. Deletion of UPR genes led to upregulation of Akt1 in all our three cKO mice (Figure 4C). This is not surprising, as the precise mechanism by which Akt modulates platelet activation response (positive or negative) is multifactorial and is still not completely understood. Mitogen Activated Protein Kinases (MAPKs) are a family of serine/threonine kinases present in all nucleated cells and p38 MAPK regulates platelet aggregation and apoptosis ³². Mice deficient in PERK show higher phospho-p38 levels, as compared to the other UPR cKO mice (Figure 4D). While p38 phosphorylation in nucleated cells can be modulated by the PERK/ATF4/e-IF2α pathway ^{33, 34}, the p38-RTN4-Bcl-xl pathway was recently shown to regulate ER physiology in platelet activation ³⁵. We find, that, consistent with the higher aggregation in response to ADP, collagen as well as CLEC-2, PERK cKO mouse platelets show the most pronounced increase in phosphorylation and activation of downstream signaling effectors. Our results provide new insights into PERK as an integral regulator of platelet physiological responses via modulation of PLC and p38 activation pathways.

Another signaling mediator, Protein Kinase C δ (PKCδ) regulates platelet activation response via calcium independent activation and aggregation ^{36, 37} and plays a complex role with opposing signaling functions³⁶. Phospho-PKCδ expression increased significantly in the XBP1 cKO and ATF6 cKO platelets, but not in PERK cKO mice (Figure 4E). PKCδ has been linked with ER stress induced apoptosis, primarily via the IRE1α/JNK pathway and has been associated with atherosclerotic plaque stability ³⁸. Our results highlight a possible mechanism for the elevated platelet aggregation and activation observed in the XBP1 cKO mice. Cyclic AMP (cAMP) and cAMP-dependent protein kinase (PKA) form the negative regulators of platelet activation ³⁹. Platelets deficient in PERK and XBP1 displayed no significant PKA activation; a marked increase in phospho-PKA was observed in ATF6 cKO platelets (Figure 4F). PKA serves as regulator of apoptosis and determines platelet life span and survival ⁴⁰. PKA activation likely mediates the least increase in platelet aggregation and no changes in apoptosis, as observed with the ATF6 ckO platelets.

These results are consistent with our observations on the modulation of platelet aggregation phenotype in response to ADP, Collagen and CLEC-2. ADP signals through the GPCRs (Gq), while collagen and CLEC-2 mediate their effects through the ITAM (GpVI) pathway^{25, 41}. These synergistic platelet activation pathways and their complex downstream events, lead to cytoskeletal rearrangements within the platelets, ultimately causing aggregation. Platelets, following deletion of the individual UPR components, particularly PERK and XBP1, appear to be primed for enhanced hyperreactivity, as evident in effects on key signaling mediators of GPCR and ITAM pathways. ATF6 cKO platelets display no change in aggregation due to collagen and CLEC-2; this may be mediated by the higher PKA expression, which acts via a negative feedback mechanism. Thus, platelet specific ablation of individual UPR components, PERK and XBP1 differentially increases platelet aggregation (in response to agonists), activation and promotes a pro thrombotic phenotype in vivo, likely via enhanced activation of selective platelet signaling pathways.

Platelet-specific deletion of UPR genes differentially affects the response to ex vivo stressors

We next wanted to explore whether ablation of platelet UPR genes modifies the stress response in platelets using oxidative stress as an acute stressor ¹⁴. Deficiency of PERK and XBP1 leads to increased sensitivity to oxidative stress ^{42, 43}. This was evident with maximum increase in protein aggregation levels in PERK and XBP1 deficient platelets (Figure 5A). Elevated mitochondrial depolarization following H₂O₂ treatment was most pronounced in PERK cKO platelets (Figure 5B–C). OS induced apoptosis was highest in the PERK cKO platelets, followed closely by the XBP1 cKO (Figure 5D). Interestingly, PERK expression increased in both XBP1 and ATF6 deficient platelets; PERK deficient platelets displayed higher IRE1α and XBP1, likely as a compensatory mechanism in absence of PERK (Figure 5E, Figure S9). Our results demonstrate that both PERK and XBP1 form a key component of the pro-survival mechanism in platelets as well as their ability to respond to external stress stimuli.

Figure 5: Platelet-specific deletion of UPR genes differentially affects the response to ex vivo stressors.

A-F. Freshly isolated platelets (3 × 10⁸ platelets per mL, n=3 samples pooled to obtain the appropriate platelet counts) from each KO strain and littermate controls, incubated for 30 min with 50 μM H₂O₂ or vehicle. Blood drawn from a total of n=12 mice/ strain. For PERK and XBP1 strain, n=7 male/ n=5 female mice were used. For ATF6, n=6 male/ n=6 female mice were used. A. Protein aggregation in platelets following acute stress (n=5 pooled samples). B-C. Representative plots and quantification for TMRE assay each mouse strain (n=4 pooled samples). D. Intraplatelet Caspase-3 activity following H₂O₂ treatment (n=4 pooled samples). E. Representative immunoblotting for UPR protein expression in KO mice platelets. Quantitation shown in Supplemental Figure S9. Data presented as mean ± SEM. The precise p value corresponds to Mann Whitney test for comparison between vehicle and H₂O₂ treated groups; or Kruskal-Wallis test with Dunn’s multiple comparisons between treated strains. See also Supplemental Figure S9.

Diabetes mellitus activates IRE1α in platelets

Hyperglycemia and hyperlipidemia, observed in diabetes mellitus (DM), leads to ER stress and UPR^{44, 45}. Previously, our lab has shown the role of stress-induced signaling in platelets from DM patients ^{7, 46}. We now explored whether the chronic stress, associated with Type II DM, activates the UPR in platelets (Supplementary Table 1). Consistent with our previous study ⁷, DM platelets displayed higher activation and apoptosis (Figure 6A, S10A). TEM revealed a more pronounced and clearly distended ER in the DM platelets compared to HC (Figure 6B, Figure S10B). Consistent with previous studies in yeast ²⁰, increased ER surface area and membrane expansion likely allows for more efficient clearance and resolution of misfolded protein accumulation and subsequent stress under the hyperglycemic conditions ⁴⁵. Presence of intraplatelet protein aggregation was significantly increased in DM platelets (Figure 6C). DM platelets showed a marked upregulation of IRE1α and spliced XBP1 protein (Figure 6D–E); however, PERK (and downstream effectors) and ATF6α expression did not change remarkably (Figure 6D–E). Platelet gene expression array for UPR, confirmed upregulation of genes associated predominantly with the IRE1α pathway (Figure 6F), the increase being more pronounced in patients with more severe DM (hemoglobin A1c (HbA1c) > 8.5, a prognostic marker for diabetes). This is likely due to higher persistent hyperglycemic stress faced by not only the platelets, but also megakaryocytes. Levels of selected genes were validated by qPCR experiments (Figure S11 A–F). Colocalization immunostaining studies further demonstrated a higher number of DM platelets, positive for both GRP78 and IRE1α vs HC platelets (Figure 6G, Fig S11G). Interestingly, levels of protein aggregates correlated strongly with the platelet activation marker (sP-selectin), (r=0.690, p=1.1 × 10⁻¹¹; Figure 6H) and HbA1c levels (r=0.710, p=1.3 × 10⁻¹²; Figure 6I). These results from our prospectively recruited DM patients highlight the clinical and translational relevance of platelet UPR.

Figure 6: Diabetes mellitus selectively activates IRE1α in platelets.

Freshly isolated platelets from DM patients (n=102) were compared to healthy age matched controls(HC), processed simultaneously. A. Levels of soluble P-selectin in DM (n=75) and controls (n=38). B. Representative TEM from DM and HC platelets, acquired on FEI Tecnai Biotwin (LaB6, 80 kV). Arrows highlight presence of protein aggregates and ER expansion within DM platelets. Scale bar, 500 nm. Data representative of n=3 independent experiments. Lower magnification shown in Supplemental Figure S10. C. Protein aggregation in DM (n=102) and HC platelets (n=40) measured using the Proteostat Dye. D-E. Platelets (3 × 10⁸ per mL) were used for immunoblotting. Representative immunoblots and quantification for expression of UPR pathway proteins in HC (n=10) and DM (n=18) samples. F. Heat map shows differential expression of key ER stress/UPR genes measured and quantified using the RT² Profiler Human Unfolded Protein Response PCR array. Heat map represents fold change in patients with less (HbA1c<8.5) and more (HbA1c>8.5) severe diabetes. Results normalized to housekeeping genes and expressed over HC. n=6 each, for HC and DM platelets. Validation by qPCR for UPR genes shown in Supplemental Figure S11. G. Confocal microscopy images from DM and control platelets (green, CD41) stained for IRE1α (blue) with GRP78 (red). Zoom in as shown by dotted line shows expression in individual platelets. Images acquired on Perkin Elmer 5-Laser Spinning Disk Confocal Microscope (equipped with Nikon Ti-E Eclipse inverted microscope), 100X (oil) objective. Microscopy images representative of n=3 independent experiments. Platelet counts adjusted between samples prior to staining. Scale bar, 10 μm H. Pearson’s correlation between protein aggregates and P-selectin. I. Pearson’s Correlation between protein aggregates and HbA1c (%), an indicator of the severity of diabetes. Values expressed as mean ± SEM. Precise p value corresponds to Mann Whitney test (A) and Student’s t test (C, E). See also Supplemental Data Table 1 and Supplemental Figure S10–12

IRE1α-XBP1 pathway has been shown to regulate glucose biosynthesis and mitochondrial respiration ^47–50 and we hypothesize that its selective activation in platelets is mediated by glucose levels and overall metabolic homeostasis in diabetes. Ex vivo hyperglycemia (Figure S12A–C) led to a trend (not significant) towards increase in protein aggregates in human platelets (Figure S12A) along with significantly higher IRE1α and XBP1s expression; PERK remained unchanged (Figure S12B–C). To further exclude effects of comorbidities and medications, we used the recognized streptozotocin (STZ) mouse model of diabetes. STZ platelets had elevated protein aggregation and consistent with human studies, only IRE1α and XBP1s but not PERK was increased (Figure S12E–F). In contrast, all three UPR pathways were activated in heart tissues from the same mice (Figure S12G). Taken together, the disruption of glucose homeostasis in human and mouse platelets, both acute (ex vivo) and chronic (DM), selectively induces the IRE1α-XBP1 pathway.

Chemical chaperone 4-PBA and IRE1α inhibition differentially modulates platelet reactivity in diabetes mellitus.

Growing recognition of UPR in human diseases has led to an emerging interest in therapeutic targeting of UPR components. Pharmacological chaperones are low molecular mass compounds that stabilize protein conformation and buffer the misfolded protein aggregates ⁵¹. Sodium 4-phenylbutyrate (PBA) is the most well studied chemical chaperone and is clinically approved for urea cycle disorders. Previously, PBA has been shown to improve insulin sensitivity, glucose homeostasis and leptin resistance in diabetes mellitus ^{52, 53}. With the significant increase in protein misfolding observed in our patient cohort, we were interested in investigating the effect of PBA on platelet phenotype. Ex vivo administration of PBA reduced platelet aggregation in both HC and DM platelets, with the decline being significant in DM platelets (Figure 7A–B). Both ADP and Collagen induced aggregation was substantially decreased following treatment with PBA in the control samples (Figure S13A). Further, PBA considerably reduced platelet apoptosis (Figure 7C) and protein aggregates levels (Figure 7D) in both the HC and DM platelet samples, ex vivo. These results demonstrate the ability of PBA to potentially mitigate platelet hyperactivation in diabetes mellitus.

Figure 7: Chemical chaperone 4-PBA and IRE1α inhibition differentially modulates platelet activation in diabetes mellitus.

Healthy control (HC) and diabetes mellitus (DM) patients were randomly selected for ex vivo interventions to study effect of UPR modulation on platelet phenotype. A-D. Freshly isolated platelet rich plasma (PRP) from either HC or DM patients (n=8 per group) incubated with 4- phenylbutyric acid (PBA, 5mM) or vehicle (PBS) for 3 hr at 37°C. A-B. Representative platelet aggregation tracings using ADP (2.5μM) as agonist. Quantitation shows percentage light transmittance. n=5 samples per group used for aggregation studies. p-value shown between DM veh vs DM + PBA corresponds to Wilcoxon test between pre- and post PBA treated samples. C. Caspase-3 activity (n=6 per group). D. Protein aggregation levels in HC and DM samples (n=8 per group) treated with Veh or PBA were measured in the lysates. E-H. Freshly isolated PRP from HC and DM patients (n=8 per group) was incubated with STF-083010 (STF, 30μM) or vehicle. E-F. Representative platelet aggregation tracings from HC and DM platelet rich plasma (n=5 per group) treated with STF and respective quantitation. Exact p-value shown between HC veh vs HC + STF corresponds to Wilcoxon test between pre- and post STF treated samples. G. Caspase -3 activity following incubation of HC and DM samples (n=6/group) with STF. H. Intraplatelet protein aggregates in HC and DM samples (n=8 per group). Exact p value corresponds to Wilcoxon test between pre- and post-treatment groups (B, F), Kruskal-Wallis test with Dunn’s multiple comparisons (C, G) or one way ANOVA with Tukey multiple comparisons (D-H).

With the observed selective activation of the IRE1α-XBP1 pathway in DM platelets, we next investigated whether pharmacological targeting of this pathway affects the prothrombotic phenotype observed in DM. STF-080310 is a novel IRE1α inhibitor which specifically inhibits the RNase activity of IRE1α and thus can block XBP1 splicing and decrease XBP1s levels ⁵⁴. Ex vivo treatment with STF led to higher platelet aggregation (Figure 7E–F, Figure S13B), caspase-3 activity (Figure 7G) and protein aggregates (Figure 7H) in both HC and DM platelets. In DM platelets, this selective upregulation of the IRE1α-XBP1 likely plays a protective role and limits platelet hyperactivation. Consistent with XBP1 cKO mice results, this further confirms the role and translational significance of XBP1 pathway in platelet pathophysiology. Although more in depth studies are needed to completely elucidate the effect of PBA in ameliorating platelet hyper-reactivity, our results, statistically significant even in a relatively small cohort, suggest its therapeutic value as an anti-platelet agent. As shown comprehensively using a combination of ex vivo, in vivo and human studies, the understanding of the finely tuned UPR in platelets could pave the way for the development of an attractive new milieu for developing safer anti-platelet therapies.

Discussion

The UPR is a ubiquitous response to alleviate the load of misfolded proteins in nucleated cells; however, whether it can occur in absence of nucleus and transcriptional regulation has not been described. Here, we report, for the first time, that platelets contain a functional highly selective UPR machinery; the presence of mRNA transcripts indicates that all components for this complex process are prepackaged in the platelet. Our platelet specific knockout models for specific UPR genes, further exemplify the differential role played by each pathway in the functional platelet phenotype. This is particularly important in DM wherein the platelet mediated atherothrombosis forms a major cause of morbidity and mortality. Results from our DM cohort (and the STZ mice) reveal a significant, yet selective, activation of IRE1α following a robust increase in the intraplatelet protein aggregate accumulation. Targeting the protein misfolding, using the chemical chaperone PBA, was beneficial in diminishing platelet hyperactivation in DM platelets, and could be developed as a potential new therapeutic strategy.

ER stress and UPR has been identified in the platelet precursor cells, the megakaryocytes and the role of ER stress induced apoptosis described in platelet formation and thrombopoiesis^{55, 56}. Inhibition of eIF2α phosphorylation, inhibits pro-platelet formation from Meg-01 cell lines, in vitro⁵⁶. Although, a few limited studies have suggested the presence of UPR proteins (mainly ER chaperones) ⁵⁷, the key signaling components of this multifaceted response are not yet described in platelets. In our human (ex vivo and translational) and mouse studies, inhibition of N-linked glycosylation (Tm) and hyperglycemia both led to an increase in the IRE1α-XBP1 but not the PERK pathway. XBP1 modulates the N-linked glycome ⁵⁸ and with N-linked glycosylation forming a crucial posttranslational modification for several platelet proteins ⁵⁹, the role of XBP1 in platelets likely extends beyond the UPR. Indeed, selective activation of XBP1 is involved in anti-tumor immunity and host defense ^{48, 60, 61} and more studies are needed to elucidate its role in platelets. Metabolic dysregulation associated with diabetes has been linked to vascular dysfunction and DM platelets display exaggerated aggregation and prothrombotic risk ⁶². The selective activation of the IRE1α-XBP1 in platelets from diabetic patients is likely mediated by glucose levels and metabolic homeostasis. While the XBP1-deficient mice platelets displayed increased activation and apoptosis and a higher population of immature platelets, the inhibition of IRE1α-XBP1 by STF worsened platelet apoptosis in both HC and DM platelets. Thus, along with upregulating the translation of cytoprotective proteins, the selective activation of the IRE1α-XBP1 pathway likely contributes to the maintenance of platelet integrity, function, and lifespan.

PBA is an clinically approved orally bioavailable chemical chaperone, shown to improve glucose homeostasis ^{52, 53}. In patients with insulin resistance, treatment with PBA improved glucose tolerance ⁶³. We demonstrate the effect of PBA on platelets for the first time in reducing platelet aggregation, thereby decreasing the associated prothrombotic risk. With its low toxicity, stability and ease of oral supplementation, PBA is under consideration for use in several diseases including thalassemia, neurodegenerative diseases and cancers ⁶⁴. Although more extensive studies are needed to investigate the effect of other chemical chaperones on platelets, we show that PBA is beneficial in alleviating platelet hyperactivation and could be potentially developed as a clinical therapy in vascular diseases.

PERK pathway plays a key role in the hypoxia tolerance of tumor cells ^65–67 with an increase in hypoxia-induced cell death in PERK mutant cell lines ⁶⁵. Induction of OS preferentially activated PERK pathway in human platelets; this could be an acute stress response providing the signal for upregulation of antioxidant proteins and reduction of intraplatelet ROS levels. PERK deficient platelets showed a distinct procoagulant phenotype and enhanced sensitivity to oxidative stress. Since ROS is a signaling molecule in platelets and promotes platelet pro-coagulant phenotype ⁶⁸, higher PERK and eIF2α phosphorylation could drive both pro-survival and apoptotic decisions. Platelet mitochondria regulate the pro-thrombotic function via redox signaling and initiation of apoptosis ⁶⁹ and PERK can modulate mitochondrial function ²⁴. PERK, thus, may be involved in the protection of mitochondria against stress, regulation of platelet function and translation of selective proteins.

Genetic manipulation of UPR in murine models provide insights into its role in normal physiology and disease conditions ²³. Although few individual studies have investigated the tissue-specific deletion of all three UPR sensors, Gass et al demonstrated that the deletion of XBP1, but not PERK or ATF6, impaired B cell differentiation ⁷⁰. Our mouse studies establish, for the first time, that ablation of these genes differentially affects platelet aggregation, activation and apoptosis responses as well as thrombus formation, in vivo. Mechanistically, enhanced activation of PLCγ2 and p38 MAPKs in PERK cKO mice, likely forms the basis of increased aggregation in absence of PERK; suggesting the role of PERK in regulating platelet prothrombotic propensity. XBP1 deletion led to higher aggregation and activation likely via increase in PKC δ. Conversely, deletion of ATF6 cKO led to higher phosphorylation of PKA, the negative regulator of platelet activation. Our results, thus, highlight a critical role for UPR proteins in modulating these intricately linked complex pathways in platelets; further, it would be interesting to explore the regulation of these ubiquitous signaling pathways in nucleated cells by UPR components. Platelet functional responses encompasses complex mechanisms to maintain platelets in their resting state, modulate the activation pathways by platelet agonists and thrombus formation as well as negative feedback mechanisms ²⁵. As we observed the regulation of selective platelet activation pathways by individual UPR molecules, it would be interesting to examine the effect on UPR when studying platelet activation pathways. Our study, provides novel directions and forms the basis of future more detailed and focused studies for each key signaling molecule and how it may be regulated by activation of a selective yet robust UPR. Although the deletion of PERK and XBP1 increases the propensity for platelet rich thrombus formation in the semi-occlusive carotid artery injury model used, our study did not measure the occlusion time. Future studies involving other murine thrombosis models such as the photochemical injury model, mechanical injury model ⁷¹ could provide more detailed insights into the role of platelet UPR during in vivo thrombus formation.

Previously, using siRNA mediated knockdown or chemical inhibitors of specific UPR arms, it has been shown that inhibition of one UPR branch did not necessarily lead to compensatory activation of other branches of UPR^{72, 73}. In platelets from our UPR cKO mice, however, both under physiological baseline conditions and OS, we observed a compensatory increase in the arms of UPR. With UPR being an integral stress response, it is likely that platelets deficient in one of the UPR branches, would modulate the compensatory upregulation of other UPR arms. Moreover, since UPR is critical for ER stress induced apoptosis and subsequent platelet formation from megakaryocytes^{55, 56}, the compensatory role of each of the arms is highly relevant. This is also reflected in the selective stress-specific regulation of individual UPR arms in platelets, which has been observed in few studies with nucleated cells⁷⁴. More detailed studies using megakaryocytes from cKO mice would provide new insights into the compensatory mechanisms within the platelet UPR.

While we show the effect of PBA and STF on platelet function in DM, it would be important to study the impact, if any, of other modulators of UPR pathways, including pharmacological chaperones on platelet phenotype, especially in atherothrombosis. IRE1α, upon activation, can also induce degradation of mRNAs through a process termed as RIID (Regulated IRE1α-Dependent Decay)⁷⁵. While RIDD and its mechanism is still being explored in other nucleated cells, platelet RNA decay is a growing field of interest⁷⁶ and it would be exciting to use IRE1α cKO mice to investigate RIDD in platelets. With XBP1 being crucial for platelet production and lifespan, it would be interesting to evaluate its role in thrombocytopenia and bleeding disorders. The apoptosis-like phase, which, forms a key step in megakaryocyte maturation and platelet formation, has been previously shown to be induced by ER stress⁵⁶. It is likely that lineage specific deletion of UPR genes could affect thrombopoiesis and megakaryocyte maturation, as is also evident by the decreased platelet counts in the PERK cKO mice. Further, UPR could potentially affect the downstream signaling and consequences in the bone marrow niche, especially under physiological conditions and hypoxia.

Thus, the diversity of the signaling responses observed, demonstrates for the first time, a non-redundant, specific role for the UPR sensors in platelets. Our results emphasize the distinct role of platelet UPR under various stimuli and in different microenvironments and how these may independently or in a concerted manner modulate the platelet activation signaling. Indeed, this specificity could be closely linked to the ability of platelets to sense and respond rapidly to multiple kinds of blood stressors. More in-depth studies are needed to investigate the relevance of selective UPR in context of other diseases including cancers, cardiovascular and neurodegenerative diseases, with a known platelet etiology. A highly specific platelet UPR highlights the significance of therapeutic targeting of selective arms and provides novel avenues for development of anti-platelet therapy. Beyond platelet function and physiology, our results also have significance in understanding the transcription independent regulation of UPR and reveal a new dimension of UPR in absence of a nucleus.

NOVELTY AND SIGNIFICANCE.

What is known?

• The unfolded protein response (UPR) is well described in nucleated cells under diverse conditions of stress.

• Platelet hyper-reactivity is associated with increased thrombotic events and cardiovascular diseases including diabetes mellitus (DM)

• ER stress induced apoptosis has been shown to be involved in megakaryocyte maturation and thrombopoiesis

What new information does this article contribute?

• Circulating platelets contain the UPR pathway components, each of which can be activated selectively in absence of a nucleus.

• Platelet UPR components PERK, XBP1 and ATF6 regulate platelet function via distinct underlying platelet activation signalling pathways.

• Protein aggregate levels in platelets from Type II DM patients correlate significantly with the disease severity, and the IRE1a-XBP-1 UPR axis is selectively activated in platelets from these DM patients.

• UPR modulators including chemical chaperones could be developed as potential targeted novel anti-platelet therapies in cardiovascular diseases.

The diversity of the platelet responses, demonstrate for the first time, a non-redundant, yet defined role for the UPR sensors in platelets. Our results emphasize the distinct role of platelet UPR under various stimuli and in different microenvironments and how these may, independently or in a concerted manner, modulate the platelet activation signaling. Diabetes Mellitus Type II (DM) affects millions globally and potentiates a risk for thrombotic events as well as cardiovascular diseases. Platelet hyperreactivity is a known phenomenon in DM. Understanding platelet UPR pathways and its effect on platelet reactivity highlights the significance of therapeutic targeting of selective UPR arms, especially in DM. Our results thus also provide novel avenues for development of anti-platelet therapy. Beyond platelet function and physiology, our results are relevant in understanding the transcription independent regulation of UPR and thus reveal a new dimension of UPR in the absence of a nucleus.

Non-standard Abbreviations and Acronyms

ADP

Adenosine diphosphate

ATF6

Activating Transcription Factor 6

cKO

conditional knockout

CLEC-2

C-type lectin-like receptor 2

CVD

Cardiovascular Diseases

DM

Diabetes Mellitus

DTS

Dense Tubular System

DTT

Dithiothreitol

eIF2

eukaryotic Initiation Factor 2

ER

Endoplasmic Reticulum

GPCR

G-protein coupled receptors

GRP78

Glucose Regulated Protein 78

GRP94

Glucose Regulated Protein 94

HC

Healthy Control

IRE1α

Inositol-Requiring Enzyme-1alpha

ITAM

immunoreceptor tyrosine-based activation motif

OS

Oxidative Stress

PBA

Sodium 4-phenylbutyrate

PDI

Protein Disulfide Isomerase

PERK

Protein kinase RNA (PKR)-like ER kinase

Pf4

Platelet factor 4

PKA

Protein Kinase A

PKC

Protein Kinase C

PLC

Phospholipase C

RIID

Regulated IRE1-Dependent Decay

ROS

Reactive Oxygen Species

RTN4

Reticulon-4

SEM

Standard Error of Mean

STZ

Streptozotocin

TEM

Transmission Electron Microscopy

Tm

Tunicamycin

UPR

Unfolded Protein Response

XBP1

X Binding Protein