Understanding the mechanisms of the regulation of platelet function by thiol isomerase enzymes

Abstract

Background: Human platelets contain a number of thiol isomerases that are released to the cell surface upon platelet activation, such as protein disulphide isomerase (PDI), and endoplasmic reticulum (ER) proteins (ERp5, ERp57, and ERp72). Previous findings indicate that these enzymes play a role in regulating integrin αIIbβ3, the adhesion receptor that mediates platelet aggregation and thrombus formation, and the similarity in their inhibitory effects suggests functional overlaps. However, thiol isomerases are believed to facilitate disulphide exchange among themselves in mammalian cells. This suggests a potential cooperative interaction between these enzymes to regulate platelet function. Aims: The main aims of this research were to elucidate the mechanisms underlying the actions of thiol isomerases during platelet activation and explore potential interactions among these enzymes. The investigation aimed to identify common and distinctive substrates associated with PDI, ERp5, ERp57, and ERp72, and detect functional interactions among these enzymes. To understand more completely the associations between these enzymes, the study aimed to identify and characterise additional thiol isomerases in platelets that interact with these enzymes in other human cell types. To understand how these interactions affect platelet function, the investigation aimed to test the effects of novel broad- spectrum inhibitors on both platelet function and thiol isomerase interactions with potential substrates. Results: Experimental investigations of thiol isomerase interactions revealed inter-thiol complexes during platelet stimulation. The characterisation of platelet thiol isomerases revealed ERp18, a novel family member. Interactions among PDI, ERp5, ERp57, and ERp72 were detected and were particularly seen during platelet activation. Proteomics experiments revealed potential substrates and/or binding partners of these enzymes, which included key players in integrin inside-out signalling, such as the target receptor integrin αIIbβ3, thioredoxin, and heat shock proteins. In experiments to explore the collective function effects of thiol isomerases in the control of platelet function, the effects of the newly identified pan-thiol isomerase inhibitor were explored. Benserazide was demonstrated to inhibit the enzymatic activity of PDI, ERp5, ERp57, and ERp72, along with its capacity to inhibit various platelet functions. In proteomics studies, benserazide was shown to block the interactions between thiol isomerases and their substrates. Conclusions: These findings provide evidence that thiol isomerase enzymes participate in common and distinct protein complexes to regulate platelet function. The broad inhibition observed, resembling single inhibition of these enzymes, further supports the notion that platelet thiol isomerases collaborate to elicit their function roles upon their translocation to the cell surface. Lastly, benserazide, a well-tolerated drug with established therapeutic uses, holds promise as a potential fast-track option for developing a new anti-thrombotic therapy.