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Review
. 2018 May;11(5):437-448.
doi: 10.1080/17474086.2018.1452612. Epub 2018 Mar 28.

Regulatory Role of Thiol Isomerases in Thrombus Formation

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Free PMC article
Review

Regulatory Role of Thiol Isomerases in Thrombus Formation

Anish Sharda et al. Expert Rev Hematol. .
Free PMC article

Abstract

The protein disulfide isomerase (PDI) family of thiol isomerases are intracellular enzymes known to catalyze the oxidation, reduction and isomerization of disulfide bonds during protein synthesis in the endoplasmic reticulum. PDI and related members of the thiol isomerase family are known to localize extracellularly where they possess various functions. Among these, the role of PDI in the initiation of thrombus formation is best characterized. PDI is secreted within seconds from activated platelets and endothelial cells at the site of vascular injury and accumulates in the developing platelet-fibrin thrombus. Inhibition of PDI by antibodies or small molecule inhibitors blocks thrombus formation. Efforts are underway to identify extracellular substrates of PDI that participate in the network pathways linking thiol isomerases to thrombus formation. ERp57, ERp5 and ERp72 also play a role in initiation of thrombus formation but their specific extracellular substrates are unknown. Areas covered: The following review gives an overview of biochemistry of vascular thiol isomerases followed by a detailed description of their role in thrombosis and its clinical implications. Expert commentary: The thiol isomerase system, by controlling the initiation of thrombus formation, provides the regulatory switch by which the normal vasculature is protected under physiologic conditions from thrombi generation.

Keywords: Anti-thrombotic drugs; disulfide; endothelium; isomerase; oxidase; platelet; protein disulfide; reductase; thiol isomerases; thrombus formation.

Figures

Figure 1.
Figure 1.. PDI family of thiol isomerases.
PDI is the archetypal member of the thiol isomerase family and contains four domains: a, b, b’, a’. The a and a’ domains are catalytically active thioredoxin-like domains containing the CGHC motif (yellow), whereas b and b’ are catalytically inactive thioredoxin-like domains (blue and light blue, respectively). Other vascular thiol isomerases are highlighted in yellow. PDI, ERp57, ERp5 and ERp72 are implicated in thrombus formation. Adapted from [88].
Figure 2.
Figure 2.. X-ray crystal structure of PDI.
A.The structure of thioredoxin-like domain [106] B. The tertiary structure of PDI is U-shaped, composed of four thioredoxin-like domains: a, b, b’ and a’ [107]. The active site motifs CGHC are found in the a and a’ domains. b and b’ are catalytically inactive substrate binding domains with b’ thought to be important for substrate specificity. The b’ and a’ domains are connected via a short 19-amino acid peptide x-linker. Adapted from [88].
Figure 3.
Figure 3.. Mechanism-based trapping of PDI substrates.
Reduction of a substrate disulfide by PDI occurs through transient formation of a mixed disulfide between the N-terminal Cys in the PDI CGHC motif and a Cys in the substrate, as depicted in the top panel. Resolution of the mixed disulfide requires the C-terminal Cys of the CGHC motif. Mutation of the C-terminal Cys to alanine (Ala) in the PDI active site (CGHC to CGHA) makes this mixed PDI-substrate disulfide stable, as depicted in the lower panel. These PDI-substrate complexes can then be isolated and analyzed. The Western blot on the right shows one such reaction where no substrate is isolated with the use of wild-type PDI (CCCC) or inactive PDI (AAAA), but only with PDI trapping mutants with C-terminal Cys modified to Ala in one (CACC and CCCA) or both (CACA) catalytically active domains.
Figure 4.
Figure 4.. Vitronectin is a substrate of PDI.
A.Vitronectin, a major plasma protein, does not bind to its receptors, endothelial ∝vβ3 and platelet ∝IIbβ3, under normal physiologic conditions. B. Vascular injury results in endothelial activation and release of PDI at the site of injury. Extracellular PDI reduces one or two disulfide bonds in vitronectin resulting in a structural change. C and D. ‘Activated’ vitronectin can now engage with its receptors ∝vβ3 and ∝IIbβ3 to support platelet aggregation and fibrin generation. The graphs show impaired platelet accumulation and fibrin generation at the site of vessel wall injury in the laser injury model of thrombus formation in vitronectin−/− mice. The downstream substrates of vitronectin in thrombus formation remain elusive.

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