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

Abstract

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.

Protein disulfide isomerase‐A1 regulates intraplatelet reactive oxygen species–thromboxane A2‐dependent pathway in human platelets

“…Gimenez et al 29 demonstrated that in vascular smooth muscle cell PDIA1 increased phosphorylation of p47phox subunit in Nox1 activation. Interestingly, in parallel to our work, the role of PDIA1 in the regulation of platelet activation has been addressed in paper published recently by Gaspar et al 50 These authors have proposed that Nox1 and PDIA1 cooperate with each other to regulate platelet activation; however, they control two distinct pathways downstream of GPVI receptor. Nox1 was suggested to be involved in the regulation of phosphoinositide 3‐kinase signaling pathway, whereas PDIA1 can regulate the activation of protein kinase C and mitogen‐activated protein kinases (MAPKs), which interact with p47phox to assemble the Nox1 complex.…”

“…Nox1 was suggested to be involved in the regulation of phosphoinositide 3‐kinase signaling pathway, whereas PDIA1 can regulate the activation of protein kinase C and mitogen‐activated protein kinases (MAPKs), which interact with p47phox to assemble the Nox1 complex. Superoxide generated by Nox1 may then activate protein kinase C and MAPKs in a positive feedback loop 50 . At the molecular level, the activation of TxA 2 synthesis by Nox1‐derived ROS in response to GPVI receptor stimulation is mediated by the activation of p38 MAPK because the inhibition of p38 MAPK with the selective inhibitor SB202190 reduced TxA 2 production following GPVI receptor stimulation 41 .…”

“…Superoxide generated by Nox1 may then activate protein kinase C and MAPKs in a positive feedback loop. 50 At the molecular level, the activation of TxA 2 synthesis by Nox1‐derived ROS in response to GPVI receptor stimulation is mediated by the activation of p38 MAPK because the inhibition of p38 MAPK with the selective inhibitor SB202190 reduced TxA 2 production following GPVI receptor stimulation. 41 These results and the findings reported by Gaspar et al, 50 showing that PDIA1 regulates the activation of MAPKs (ERK 1/2, p38) and subsequently Nox1 activity, support our concept that PDIA1 specifically controls GPVI receptor‐Nox1‐ROS and subsequent generation of TxA 2 demonstrated here for the first time.…”

“… 50 At the molecular level, the activation of TxA 2 synthesis by Nox1‐derived ROS in response to GPVI receptor stimulation is mediated by the activation of p38 MAPK because the inhibition of p38 MAPK with the selective inhibitor SB202190 reduced TxA 2 production following GPVI receptor stimulation. 41 These results and the findings reported by Gaspar et al, 50 showing that PDIA1 regulates the activation of MAPKs (ERK 1/2, p38) and subsequently Nox1 activity, support our concept that PDIA1 specifically controls GPVI receptor‐Nox1‐ROS and subsequent generation of TxA 2 demonstrated here for the first time. In addition, the reduced production of Nox1‐derived ROS caused by PDIA1 inhibition may impair various intraplatelet redox mechanisms, including peroxynitrite‐mediated “peroxide tone” 51 or have also a direct effect on intraplatelet inside‐out αIIβ3 integrin activation (see Masselli et al 40 for a recent review on intraplatelet redox regulations).…”

Thiol Isomerases Orchestrate Thrombosis and Hemostasis

“…This is of particular relevance to the platelet, given that others and we proposed the inhibition of the C-terminal active site of PDI as a new antithrombotic strategy 93,95 . Indeed, we have recently shown that PDI and Nox-1 translocate to a closer proximity in CRP-activated platelets and that the expression levels of these enzymes are increased in platelets of individuals presenting cardiometabolic risk factors, such as obesity and high blood pressure 96 .…”