Advances in vascular thiol isomerase function

Abstract

Purpose of review: The present review will provide an overview of several recent advances in the field of vascular thiol isomerase function.

Recent findings: The initial observation that protein disulfide isomerase (PDI) functions in thrombus formation occurred approximately a decade ago. At the time, there was little understanding regarding how PDI or other vascular thiol isomerases contribute to thrombosis. Although this problem is far from solved, the past few years have seen substantial progress in several areas that will be reviewed in this article. The relationship between PDI structure and its function has been investigated and applied to identify domains of PDI that are critical for thrombus formation. The mechanisms that direct thiol isomerase storage and release from platelets and endothelium have been studied. New techniques including kinetic-based trapping have identified substrates that vascular thiol isomerases modify during thrombus formation. Novel inhibitors of thiol isomerases have been developed that are useful both as tools to interrogate PDI function and as potential therapeutics. Human studies have been conducted to measure circulating PDI in disease states and evaluate the effect of oral administration of a PDI inhibitor on ex-vivo thrombin generation.

Summary: Current findings indicate that thiol isomerase-mediated disulfide bond modification in receptors and plasma proteins is an important layer of control of thrombosis and vascular function more generally.

Figure 1. The allosteric switch mechanism

In its unligated state, the x-linker interacts with the hydrophobic binding pocket on the b' domain (capped conformation). In this conformation, the active site motifs on the a and a' domains are prone to disulfide bonding. The binding of a substrate to the hydrophobic pocket results in displacement of the x-linker. This displacement causes a conformational change resulting in a more compact structure and a propensity towards a free dithiol state for the catalytic cysteines in the active site motif.

Figure 2. Kinetic mechanism-based trapping of thiol isomerase substrates

A. Reduced thiol isomerase cleaves disulfide bonds in substrates by nucleophilic attack in which the active site sulfur ion nucleophile of the thiol isomerase attacks the adjacent sulfur atoms of the disulfide bond in the substrate. This nucleophilic substitution results in a transient mixed disulfide. The mixed disulfide then decomposes with a disulfide bond forming in the thiol isomerase. B. A trapping mutant with a CGHA active site motif has a free thiol that is capable of nucleophilic attack of a disulfide bond on a substrate. However, the mixed disulfide bond that forms fails to resolve into a disulfide on the thiol isomerase since it lacks a second cysteine. Instead, the mixed disulfide is stable, enabling isolation of the complex and identification of the substrate.

Figure 3. Mechanism of action of PDI antagonists

Several PDI inhibitors have been identified over the past few years. Evaluation of their mechanism of action indicates that most act either at the catalytic cysteines within the active site motifs or at the hydrophobic binding pocking on the b' domain. In general, compounds that act at the active site motif tend to be irreversible inhibitors while those that act at the hydrophobic binding pocket tend to be reversible inhibitors.