Thiol isomerases in thrombus formation

Abstract

Protein disulfide isomerase (PDI), ERp5, and ERp57, among perhaps other thiol isomerases, are important for the initiation of thrombus formation. Using the laser injury thrombosis model in mice to induce in vivo arterial thrombus formation, it was shown that thrombus formation is associated with PDI secretion by platelets, that inhibition of PDI blocked platelet thrombus formation and fibrin generation, and that endothelial cell activation leads to PDI secretion. Similar results using this and other thrombosis models in mice have demonstrated the importance of ERp5 and ERp57 in the initiation of thrombus formation. The integrins, αIIbβ3 and αVβ3, play a key role in this process and interact directly with PDI, ERp5, and ERp57. The mechanism by which thiol isomerases participate in thrombus generation is being evaluated using trapping mutant forms to identify substrates of thiol isomerases that participate in the network pathways linking thiol isomerases, platelet receptor activation, and fibrin generation. PDI as an antithrombotic target is being explored using isoquercetin and quercetin 3-rutinoside, inhibitors of PDI identified by high throughput screening. Regulation of thiol isomerase expression, analysis of the storage, and secretion of thiol isomerases and determination of the electron transfer pathway are key issues to understanding this newly discovered mechanism of regulation of the initiation of thrombus formation.

Keywords: antiplatelet agents; antithrombotic agents; blood platelets; platelet aggregation inhibitor; thrombosis.

Figure 1. The thiol isomerase family

Protein disulfide isomerase, the prototypic thiol isomerase, has four domains: a, b, b’, a’. Catalytically active thioredoxin-like domains containing the CGHC motif are colored yellow. Catalytically active thioredoxin-like domains with a motif other than CGHC are colored orange. Catalytically inactive thioredoxin-like domains are colored dark blue and light blue. Thiol isomerases found or secreted from platelets are highlighted with a yellow background. Modified from .

Figure 2. PDI is released during and is required for thrombus formation

Intravital microscopy images of thrombus formation in a live mouse. A. PDI antigen (green) is secreted from platelets and endothelium during thrombus formation. B. Blocking anti-PDI antibody inhibits platelet thrombus formation (red) and fibrin (green) generation. Left, 0 μg/g mouse; right, 3.0 μg/g mouse. Time of images: 60 seconds post-injury.

Figure 3. Co-localization of PDI with TLR9 in T-granules

Electron microscopy demonstrates (A) TLR9, (B) PDI, or (C and D) co-localization of both TLR9 and PDI to electron-dense membrane-encapsulated regions adjacent to the plasma membrane of platelets.

Figure 4. Inhibition of PDI by quercetin flavonoids with a 3-O-glycosidic linkage

High throughput screening identified quercetin-3-rutinoside as an inhibitor of PDI. Subsequent studies evaluating structure activity relationships demonstrated that a sugar at 3’ position in the C ring of quercetin-3-rutinoside is critical for its ability to inhibit PDI. All analogs tested with a sugar in this position inhibited PDI (Active), while analogs lacking this sugar failed to demonstrate inhibition (Inactive).