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

Renato Simões Gaspar,Giordano Pula,Tanya Sage,Neline Kriek,Gemma Little,Jonathan M Gibbins

doi:10.3390/antiox10030497

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.

Highlights

Cardiometabolic risk factors, such as central obesity, hyperglycaemia, insulin resistance, hypertension and dyslipidaemia are associated with increased production of reactive oxygen species (ROS) [1,2]
Protein disulphide isomerase (PDI) and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 1 (Nox-1) Cellular Localization in Resting and Activated Platelets. Since both PDI and Nox-1 are able to regulate ROS generation [12], we would anticipate them to be present in a similar location, and close to their site of activity
Platelets, PDI and p47phox were localized primarily in the intracellular space, while Nox-1 staining has been reported to be able to associate with p47phox, which is a cytosolic subunit regulates the activation of Nox-1 [12]

Summary

Introduction

Cardiometabolic risk factors, such as central obesity, hyperglycaemia, insulin resistance, hypertension and dyslipidaemia are associated with increased production of reactive oxygen species (ROS) [1,2]. There is a higher risk of developing thrombotic events partly due to platelet hyperactivation, which is pivotal to thrombus formation [3]. Upon vascular injury, these anucleated cells become activated through adhesion receptors and secondary mediator responses to form thrombi and reduce blood loss. These anucleated cells become activated through adhesion receptors and secondary mediator responses to form thrombi and reduce blood loss Of note, both ROS and enzymes that regulate ROS generation are key to platelet activation in health and disease [4]. The study of enzymes that regulate ROS production in platelets may be exploited to decrease thrombotic events associated with cardiometabolic risk factors. PDI itself is released upon platelet activation [8] and was found to bind to integrin β3 during thrombus

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