Recent advances in vascular thiol isomerases and redox systems in platelet function and thrombosis

Abstract

There have been substantial advances in vascular protein disulfide isomerases (PDIs) in platelet function and thrombosis in recent years. There are 4 known prothrombotic thiol isomerases; PDI, endoplasmic reticulum protein (ERp)57, ERp72, and ERp46, and 1 antithrombotic PDI; transmembrane protein 1. A sixth PDI, ERp5, may exhibit either prothrombotic or antithrombotic properties in platelets. Studies on ERp46 in platelet function and thrombosis provide insight into the mechanisms by which these enzymes function. ERp46-catalyzed disulfide cleavage in the α_IIbβ₃ platelet integrin occurs prior to PDI-catalyzed events to maximally support platelet aggregation. The transmembrane PDI transmembrane protein 1 counterbalances the effect of ERp46 by inhibiting activation of α_IIbβ₃. Recent work on the prototypic PDI found that oxidized PDI supports platelet aggregation. The a' domain of PDI is constitutively oxidized, possibly by endoplasmic reticulum oxidoreductase-1α. However, the a domain is normally reduced but becomes oxidized under conditions of oxidative stress. In contrast to the role of oxidized PDI in platelet function, reduced PDI downregulates activation of the neutrophil integrin αMβ₂. Intracellular platelet PDI cooperates with Nox1 and contributes to thromboxane A2 production to support platelet function. Finally, α_IIb and von Willebrand factor contain free thiols, which alter the functions of these proteins, although the extent to which the PDIs regulate these functions is unclear. We are beginning to understand the substrates and functions of vascular thiol isomerases and the redox network they form that supports hemostasis and thrombosis. Moreover, the disulfide bonds these enzymes target are being defined. The clinical implications of the knowledge gained are wide-ranging.

Keywords: disulfide; integrin; platelet; protein disulfide isomerase; sulfhydryl; thrombosis.

Figure 1.

Thiol isomerases mediate integrin activation in thrombosis and inflammation. PDI family members potentiate activation of the αIIbβ3 and α2β1 platelet integrins and the αMβ2 neutrophil integrin. The PDIs are secreted from platelets, endothelial cells, and neutrophils at the site of vascular injury or inflammation. They directly interact with these integrins facilitating conversion of each integrin to the high affinity state. The ligand for αIIbβ3 is fibrinogen, for α2β1 is collagen, and for αMβ2 is ICAM-1. The PDIs thus support the processes of platelet adhesion and aggregation and neutrophil rolling, crawling and adhesion. ERp46 and PDI sequentially mediate activation of αIIbβ3, PDI mediates activation of α2β1, and ERp72 mediates activation of αMβ2. PDI also regulates coagulation resulting in fibrin formation.

Figure 2.

The CXYC-containing thiol isomerases of the PDI family involved in platelet function and thrombosis. The x-linker between the b′ and a′ domains of PDI facilitates domain flexibility. Five secreted and one transmembrane member of the PDI family are depicted.

Figure 3.

Structures of ERp46 and PDI. ERp46 is composed of three redox-active a°, a and a′ catalytic domains that form an open V-shaped structure. The domains are linked by unusually long, flexible, extended loops. Redox-active sites are shown as red spheres. Solvent-exposed active sites allow easy access to clients in all three catalytic domains. PDI is horseshoe shaped with the actives sites facing inwards. The crystal structures for each catalytic domain of human ERp46 and for the PDI structure were obtained from the Research Collaboratory for Structural Bioinformatics Protein Data Bank (RCSB PDB) and prepared with the PyMOL Molecular Graphics System, Version 2.0 Schrödinger, LLC.

Figure 4.

Disulfide bonds cleaved by ERp46 in the β3 subunit of integrin αIIbβ3. Shown in red are disulfide bonds targeted by ERp46 in which at least one of the two cysteine residues in the β3 subunit had at a > 2-fold increase in thiol labeling in the presence of ERp46 (ref. [1]). Additional disulfide bonds are shown in yellow. Results are from experiments repeated 2 to 3 times. The model of integrin β3 (PDB code 3ije) is in an extended conformation. To open the structure, the peptide bonds at residues 475–476 in chain B was broken to simulate the opening of the hinge between EGF-1 and EGF-2 (‘β knee’).

Figure 5.

Working model of αIIbβ3 activation by thiol isomerases. TMX1 maintains αIIbβ3 in an inactive state by maintaining cysteines in the oxidized state. Upon platelet activation with inside-out signaling in αIIbβ3, disulfide bonds in this integrin are reduced by ERp46 resulting in the generation of thiols. This event occurs before fibrinogen binding. A PDI-catalyzed event then leads to the high affinity fibrinogen binding confirmation. ERp57 and ERp72 may also act after the ERp46 catalyzed reaction. This model is likely oversimplified as TMX1 may also act at later stages in integrin activation.

Figure 6.

Disulfide bonds in the neutrophil integrin αMβ2 cleaved by PDI and ERp72. ERp72 cleaves Cys654-Cys711 in the thigh region of αM mediating activation of αMβ2 and binding of αMβ2 to its endothelial cell ligand ICAM-1. PDI cleaves two disulfide bonds in the βI domain of integrin αMβ2. Cleavage of the Cys224-Cys264 disulfide bond shifts the conformation of integrin αMβ2 from a high to low affinity form.