Protein Disulphide Isomerase 6 (PDIA6) Attenuates Platelet Endoplasmic Reticulum Stress and Secretion in a Mouse Model

Background: Platelet hyperreactivity involves increased secretion of their granule content which promotes platelet aggregation and thrombosis. Platelet hyperreactivity is observed in conditions such as diabetes mellitus and is associated with decreased cardioprotective effect from antiplatelet agents in this patient group. Diabetes is associated with increased endoplasmic reticulum (ER) stress from hyperglycemia and hyperlipidemia. Protein disulphide isomerase 6 (PDIA6) is an endoplasmic reticulum protein which folds nascent proteins by reduction and oxidation of their disulphide bonds. PDIA6 has been shown to inhibit downstream ER stress pathways by inhibiting the phosphorylation of IRE-1 in fibroblasts (Eletto, Mol Cell, 2014). We hypothesized that ER stress pathways are functional in platelets and that PDIA6 may inhibit ER stress pathways leading to platelet secretion. Methods: We generated conditional PDIA6 knockout mice (PF4Cre+ Pdia6 fl/fl) (CKO) in the megakaryocyte/platelet lineage by CRISPR-Cas9 technology (Fig.1A). Megakaryopoiesis and haemostasis was assessed by bone marrow histology, coagulation assays, platelet aggregation and tail bleeding studies. We induced ER stress of purified platelets by incubation with thapsigargin and tunicamycin. Activation of the PERK and IRE1 pathways was measured by Western blot. Thrombosis was assessed in vitro by microfluidic devices and in vivo by electrolytic injury of the carotid artery. Results: PDIA6 CKO mice displayed a mild macrothrombocytopenia: the mean (+/-SD) platelet count in Pf4Cre+/Pdia6fl/fl was 775 +/- 98 x10 3/ul compared with 874 +/- 55 x10 3/ul in Pdia6fl/fl (p<0.005). The median platelet volume was 6.3 fL in Pf4Cre+/Pdia6fl/fl compared with 5.7 fL in Pdia6fl/fl (p<0.005). Megakaryopoiesis was normal at baseline. However, PDIA6 CKO mice showed significant upregulation of intracellular platelet PDIs including PDIA1, PDIA3 and PDIA4. PDIA6 deficient platelets displayed significant increase of disulphide reductase activity and the generation of free thiols on the platelet surface. Activation of the PERK and IRE-1 pathway at baseline and after induction of ER stress was increased in PDIA6 deficient platelets (Figure 1B, C). There was striking hypersecretion of PDIA1 (Figure 1D) and α-granule proteins (Figure 1E, F) in response to shear and stimulation with thrombin. PDIA6 CKO mice displayed a prothrombotic phenotype with increased platelet adhesion to fibrinogen under shear (500 s-1) and decreased time to carotid artery occlusion (mean+/SD: 10.8 +/-3.2 min in Pf4Cre+/Pdia6fl/fl compared with 15.3 +/-5.2 min in Pdia6fl/fl, n=8-10, p<0.05). Conclusion: We have identified a role for platelet PDIA6 in attenuating platelet ER stress and secretion. This opens avenues for further study into the role of platelet PDIs in conditions with increased ER stress, such as obesity and diabetes. Figure 1: PDIA6 deficient platelets have increased endoplasmic reticulum (ER) stress and are hypersecretory. A. Western blot of PDIA6 protein in platelets from Pf4Cre+/Pdia6fl/fl mice and control mice (Pdia6fl/fl) showing efficient deletion of PDIA6 in platelets. B. PDIA6 deficient platelets have increased phosphorylation of pEIF2a (PERK phosphorylation pathway) at baseline and after induction of ER stress by thapsigargin, representative image. C. Normalized band intensity (peIF2a/beta actin) in platelets treated with DMSO control or thapsigargin. D. Increased secretion of thiol isomerase PDIA1. E. alpha granule proteins: platelet factor 4 (PF4) and F. von Willebrand factor (vWF) from PDIA6 deficient platelets compared with controls after stimulation with thrombin 0.5 U/ml. n=3-5 Pf4Cre+/Pdia6fl/fl (red boxes) and n=3-5 Pdia6fl/fl mice (grey boxes). Columns are presented as mean+/-SD, *p<0.05, ** p<0.001 by Mann Whitney. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Protein disulphide isomerase (PDI) is protective against amyotrophic lateral sclerosis (ALS)-related mutant Fused in Sarcoma (FUS) in in vitro models

Mutations in Fused in Sarcoma (FUS) are present in familial and sporadic cases of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). FUS is localised in the nucleus where it has important functions in DNA repair. However, in ALS/FTD, mutant FUS mislocalises from the nucleus to the cytoplasm where it forms inclusions, a key pathological hallmark of neurodegeneration. Mutant FUS also inhibits protein import into the nucleus, resulting in defects in nucleocytoplasmic transport. Fragmentation of the neuronal Golgi apparatus, induction of endoplasmic reticulum (ER) stress, and inhibition of ER-Golgi trafficking are also associated with mutant FUS misfolding in ALS. Protein disulphide isomerase (PDI) is an ER chaperone previously shown to be protective against misfolding associated with mutant superoxide dismutase 1 (SOD1) and TAR DNA-binding protein-43 (TDP-43) in cellular and zebrafish models. However, a protective role against mutant FUS in ALS has not been previously described. In this study, we demonstrate that PDI is protective against mutant FUS. In neuronal cell line and primary cultures, PDI restores defects in nuclear import, prevents the formation of mutant FUS inclusions, inhibits Golgi fragmentation, ER stress, ER-Golgi transport defects, and apoptosis. These findings imply that PDI is a new therapeutic target in FUS-associated ALS.

Protein disulphide isomerase (PDI) is protective against several types of DNA damage, including that induced by amyotrophic lateral sclerosis-associated mutant TDP-43 in neuronal cells/ in vitro models

Protein disulphide isomerase (PDI) is a chaperone that catalyses the formation of thiol-disulphide bonds during protein folding. Whilst up-regulation of PDI is a protective mechanism to regulate protein folding, an increasingly wide range of cellular functions have been ascribed to PDI. Originally identified in the endoplasmic reticulum (ER), PDI has now been detected in many cellular locations, including the nucleus. However, its role in this cellular compartment remains undefined. PDI is implicated in multiple diseases, including amyotrophic lateral sclerosis (ALS), a fatal and rapidly progressing neurodegenerative condition affecting motor neurons. Loss of essential proteins from the nucleus is an important feature of ALS. This includes TAR DNA-binding protein-43 (TDP-43), a DNA/RNA binding protein present in a pathological form in the cytoplasm in almost all (97%) ALS cases, that is also mutated in a proportion of familial cases. PDI is protective against disease-relevant phenotypes associated with dysregulation of protein homeostasis (proteostasis) in ALS. DNA damage is also increasingly linked to ALS, which is induced by pathological forms of TDP-43 by impairment of its normal function in the non-homologous end-joining (NHEJ) mechanism of DNA repair. However, it remains unclear whether PDI is protective against DNA damage in ALS. In this study we demonstrate that PDI was protective against several types of DNA damage, induced by either etoposide, hydrogen peroxide (H2O2), or ALS-associated mutant TDP-43M337V in neuronal cells. This was demonstrated using widely used DNA damage markers, phosphorylated H2AX and 53BP1, which is specific for NHEJ. Moreover, we also show that PDI translocates into the nucleus following DNA damage. Here PDI is recruited directly to sites of DNA damage, implying that it has a direct role in DNA repair. This study therefore identifies a novel role of PDI in the nucleus in preventing DNA damage.

Protein Disulphide Isomerase and NADPH Oxidase 1 Cooperate to Control Platelet Function and Are Associated with Cardiometabolic Disease Risk Factors

Background: Protein disulphide isomerase (PDI) and NADPH oxidase 1 (Nox-1) regulate platelet function and reactive oxygen species (ROS) generation, suggesting potentially interdependent roles. Increased platelet reactivity and ROS production have been correlated with cardiometabolic disease risk factors. Objectives: To establish whether PDI and Nox-1 cooperate to control platelet function. Methods: Immunofluorescence microscopy was utilised to determine expression and localisation of PDI and Nox-1. Platelet aggregation, fibrinogen binding, P-selectin exposure, spreading and calcium mobilization were measured as markers of platelet function. A cross-sectional population study (n = 136) was conducted to assess the relationship between platelet PDI and Nox-1 levels and cardiometabolic risk factors. Results: PDI and Nox-1 co-localized upon activation induced by the collagen receptor GPVI. Co-inhibition of PDI and Nox-1 led to additive inhibition of GPVI-mediated platelet aggregation, activation and calcium flux. This was confirmed in murine Nox-1−/− platelets treated with PDI inhibitor bepristat, without affecting bleeding. PDI and Nox-1 together contributed to GPVI signalling that involved the phosphorylation of p38 MAPK, p47phox, PKC and Akt. Platelet PDI and Nox-1 levels were upregulated in obesity, with platelet Nox-1 also elevated in hypertensive individuals. Conclusions: We show that PDI and Nox-1 cooperate to control platelet function and are associated with cardiometabolic risk factors.