Regulation of thiol isomerases in thrombus formation

Garnier, C.L. (Céline) (2016) Regulation of thiol isomerases in thrombus formation. thesis, Medicine.

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Abstract

Protein Disulfide Isomerase (PDI) is the archetypal member of the thiol isomerase family, a group of enzymes that catalyzes posttranslational disulfide bond formation in the endoplasmic reticulum. In certain pathological conditions, PDI is released onto membrane surfaces. Platelets and endothelial cells, for example, secrete PDI following vascular injury. Inhibition of extracellular PDI blocks platelet accumulation as well as fibrin formation during thrombus formation and therefore has potential as a therapeutic intervention in venous thromboembolic disease. PDI contains two catalytically active domains, termed a and a', that possess a Cysteine-Glycine-Histidine-Cysteine (CGHC) motif. It also has two substrate binding domains: b and b’. These are arranged in an a-b-b’-x-a’ conformation, with the x-linker being a small flexible peptide that connects b’ and a’. Two other thiol isomerases important for thrombus formation are ERp5 and ERp57, which also possess CGHC motifs in their catalytically active domains but have distinct substrate binding domains. Nitric oxide has an inhibitory effect on platelet aggregation and is shown to inhibit PDI reductase activity. The effect of nitric oxide on ERp5 and ERp57 is not known. This report is divided into two parts. In part 1, we demonstrate that PDI, ERp5 and ERp57 reductase activities are all inhibited by S-nitrosylation following exposure to nitric oxide donors. This part of the report provides evidence that S-nitrosylation is an important post-translational modification that equally affects PDI, ERp5 and ERp57 activities. In part 2, we describe the mechanism of action of three novel PDI inhibitors that block platelet aggregation. These compounds reversibly and selectively inhibit PDI in an insulin reductase assay, but paradoxically stimulate PDI activity in a di-eosin-GSSG-based reductase assay. These compounds elicit augmentation of PDImediated reduction of GSSG through an allosteric switch mechanism that involves binding to the b’x domain of PDI and displacement of the x-linker. Mutational analysis of amino acids adjacent to the hydrophobic pocket of the b' domain identified specific amino acids that mediate this allosteric switch mechanism. These studies demonstrate a molecular mechanism whereby substrate binding elicits the activation of remote catalytic domains.

Item Type:	Thesis (Thesis)
Supervisor name:	Faculty supervisor: and Lisman, Ton and Section of Hepatobiliary Surgery and Liver Transplantation and Groningen, University Medical Center Groningen
Supervisor name:	Local supervisor: and Flaumenhaft, Robert and Division of Hemostasis and Thrombosis, Department of Medicin and Center, Harvard Medical School and Section of Hepatobiliary Surgery and Liver Transplantation
Faculty:	Medical Sciences
Date Deposited:	25 Jun 2020 10:44
Last Modified:	25 Jun 2020 10:44
URI:	https://umcg.studenttheses.ub.rug.nl/id/eprint/568

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