Vascular thiol isomerases in thrombosis: The yin and yang

Abstract

There has recently been considerable progress of the field of extracellular protein disulfide isomerases with vascular thiol isomerases in the forefront. Four members of protein disulfide isomerase (PDI) family of enzymes, PDI, ERp57, ERp72, and ERp5, have been shown to be secreted from activated platelets and endothelial cells at the site of vascular injury. Each isomerase individually supports platelet accumulation and coagulation, as indicated by multiple levels of evidence, including inhibitory antibodies, targeted knockout mice, and mutant isomerases. The transmembrane PDI family member TMX1 was recently shown to inhibit platelet function and thrombosis, demonstrating that the PDIs can have opposing functions in thrombosis. These observations provide a new concept that thiol isomerases can both positively and negatively regulate hemostasis, constituting off-on redox switches controlling activation of hemostatic factors. This redox network serves to maintain vascular homeostasis. Integrins such as the αIIbβ3 fibrinogen receptor on platelets appear to be major substrates, with the platelet receptor for von Willebrand factor, glycoprotein Ibα, as another substrate. S-nitrosylation of the prothrombotic PDIs may additionally negatively regulate platelets and thrombosis. Thiol isomerases also regulate coagulation in mouse models, and a clinical trial with the oral PDI inhibitor isoquercetin substantially decreased markers of coagulation in patients at risk for thrombosis. This review updates recent findings in the field and addresses emerging evidence that thiol/disulfide-based reactions mediated by the prothrombotic secreted PDIs are balanced by the transmembrane member of this family, TMX1.

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

FIGURE 1

The CXYC-containing thiol isomerases of the protein disulfide isomerase family

FIGURE 2

Thiol (SH) and disulfide bond (S-S)-based reactions catalyzed by thiol isomerases. A, Disulfide bond cleavage. The mixed disulfide formed between protein disulfide isomerase (PDI) and the substrate decomposes releasing the oxidized PDI and a reduced disulfide bond in the substrate. B, Disulfide isomerization can occur in two ways: rearrangement of a four cysteine system containing two disulfide bonds based on the transient formation of an intermolecular mixed disulfide between the PDI and the substrate. Alternatively, intramolecular rearrangement of a three-cysteine system containing one disulfide bond. For intramolecular thiol-disulfide exchange, the nucleophilic sulphur can come from the protein itself, which attacks a redox sensitive disulfide bond resulting in disulfide bond cleavage. It is possible that mechanical forces and conformational changes in a protein can lead to intramolecular thiol-disulfide exchange. C, The disulfide form of the CXYC active site inserts disulfides into protein substrates, resulting in the pairing of two substrate cysteines into disulfides and the reduction of the PDI active site

FIGURE 3

Schematic of protein disulfide isomerase (PDI) fragments and mutants used in binding studies with platelet αIIbβ3. The PDI fragments containing an intact b′ domain bind to platelets and purified αIIbβ3. The a and a′ domains cooperate with the b′ domain in PDI binding. The abb′ x fragment lacking the C-terminal active site inhibits platelet aggregation and platelet accumulation in vivo

FIGURE 4

The protein disulfide isomerase (PDI) inhibitor isoquercetin improves markers of coagulation in advanced cancer. Shown is the PDI molecule with a, b, b′, x, and a′ domains. The hydrophobic pocket in the b′ domain is highlighted in green with small molecule inhibitors like isoquercetin and bepristat targeting this pocket (black balls). 1000 mg of oral isoquercetin per day inhibits plasma PDI activity, and platelet dependent thrombin generation; and decreases d-dimer, and soluble P-selectin levels in patients

FIGURE 5

Model of αIIbβ3 activation by thiol isomerases (yin-yang). TMX1 maintains αIIbβ3 an inactive or quiescent state by keeping thiols in an oxidized state (the yin). Upon platelet activation with inside-out signaling in αIIbβ3, disulfide bonds in this integrin are cleaved generating thiols. This allows a protein disulfide isomerase (PDI)-catalyzed thiol disulfide exchange reaction that results in the high affinity fibrinogen binding confirmation (the yang). This working model is likely oversimplified: TMX1 may act at later stages in integrin activation to counterbalance the prothrombotic PDIs (TMX1?), and ERp57, ERp5, and ERp72 may also act at earlier steps (PDIs?)