Therapeutic implications of protein disulfide isomerase inhibition in thrombotic disease

Abstract

The study of thrombus formation has increasingly applied in vivo tools such as genetically modified mice and intravital microscopy to the evaluation of molecular and cellular mechanisms of thrombosis. Among several unexpected findings of this approach was the discovery that protein disulfide isomerase serves an essential role in thrombus formation at sites of vascular injury. The observation that the commonly ingested quercetin flavonoid, quercetin-3-rutinoside, inhibits protein disulfide isomerase and blocks thrombus formation in preclinical studies has set the stage for clinical trials using protein disulfide isomerase antagonists as antithrombotics. Although the mechanisms by which protein disulfide isomerase facilitates platelet activation and fibrin formation have yet to be elucidated, protein disulfide isomerase antagonists are currently being developed as antithrombotics. This review will consider what is known about the role of protein disulfide isomerase in platelet accumulation and fibrin generation with a focus on pharmacological strategies for blocking protein disulfide isomerase activity in the context of thrombus formation. Potential indications and clinical trial design for testing the efficacy of protein disulfide isomerase inhibition to reduce the incidence of thrombosis will be considered.

Keywords: blood platelet; platelet inhibitors; thrombosis.

Figure 1. Structure and function of protein disulfide isomerase

A, The structure of protein disulfide isomerase (PDI) as determined by x-ray crystallography. The a, b, b', x, and a' domains are indicated. Arrows denote the location of the CGHC catalytic motifs (adapted from Wang et al., Antioxid. Redox Signal., 2013). B, The primary function of the CGHC motifs is to catalyze the oxidation and reduction of disulfide bonds to facilitate proper folding of proteins as they are synthesized in the endoplasmic reticulum. However, PDI can also be secreted from vascular cells and extracellular PDI is essential for thrombus formation.

Figure 2. Model of potential roles of PDI in thrombus formation

Little is known about the mechanisms by which PDI functions in thrombus formation. This model illustrates several hypotheses that have been offered. In platelets, PDI localizes to T-granules and is released upon platelet activation. Extracellular PDI is thought to act as an isomerase for platelet receptors, such as α_IIbβ₃, converting them to an activated conformation. However, the influence of PDI on α_IIbβ₃ conformation and the importance of PDI in activating α_IIbβ₃ during thrombus formation are currently unknown. In endothelial cells, PDI localizes to secondary granules (that do not contain von Willebrand factor) and is release with cell activation. One theory is that PDI facilitates the conversion of resting tissue factor to activated tissue factor by facilitating disulfide bond formation between Cys186 and Cys209, but this remains to be proven. The action of PDI on the components of thrombus formation remains to be determined.

Figure 3. Structure of ML359

Figure 4. Randomized, phase II/III clinical trial to evaluate the efficacy of oral isoquercetin to prevent thrombosis in advanced cancer patients

Phase II studies will be completed as two sequential dosing cohorts A and B. Based on evidence of safety in cohort A, patients will then enroll into cohort B using a higher dose of daily isoquercetin. D-dimer will be measured before and following two months of daily isoquercetin administration. Based on safety, efficacy in reducing D-dimer levels as well as preliminary data on venous thromboembolic events, the dose will be selected to proceed to phase III. The phase III trial will involve randomization between placebo and isoquercetin with a primary endpoint of venous thromboemoblic events.