Protein disulfide isomerase regulation by nitric oxide maintains vascular quiescence and controls thrombus formation

Abstract

Essentials Nitric oxide synthesis controls protein disulfide isomerase (PDI) function. Nitric oxide (NO) modulation of PDI controls endothelial thrombogenicity. S-nitrosylated PDI inhibits platelet function and thrombosis. Nitric oxide maintains vascular quiescence in part through inhibition of PDI. SUMMARY: Background Protein disulfide isomerase (PDI) plays an essential role in thrombus formation, and PDI inhibition is being evaluated clinically as a novel anticoagulant strategy. However, little is known about the regulation of PDI in the vasculature. Thiols within the catalytic motif of PDI are essential for its role in thrombosis. These same thiols bind nitric oxide (NO), which is a potent regulator of vessel function. To determine whether regulation of PDI represents a mechanism by which NO controls vascular quiescence, we evaluated the effect of NO on PDI function in endothelial cells and platelets, and thrombus formation in vivo. Aim To assess the effect of S-nitrosylation on the regulation of PDI and other thiol isomerases in the vasculature. Methods and results The role of endogenous NO in PDI activity was evaluated by incubating endothelium with an NO scavenger, which resulted in exposure of free thiols, increased thiol isomerase activity, and enhanced thrombin generation on the cell membrane. Conversely, exposure of endothelium to NO carriers or elevation of endogenous NO levels by induction of NO synthesis resulted in S-nitrosylation of PDI and decreased surface thiol reductase activity. S-nitrosylation of platelet PDI inhibited its reductase activity, and S-nitrosylated PDI interfered with platelet aggregation, α-granule release, and thrombin generation on platelets. S-nitrosylated PDI also blocked laser-induced thrombus formation when infused into mice. S-nitrosylated ERp5 and ERp57 were found to have similar inhibitory activity. Conclusions These studies identify NO as a critical regulator of vascular PDI, and show that regulation of PDI function is an important mechanism by which NO maintains vascular quiescence.

A review on possible mechanistic insights of Nitazoxanide for repurposing in COVID-19

“…Nitazoxanide block this oxidoreductase mechanism ( Fig. 2 A) by interacting with cysteine residues of surface-bound PDI through S-nitrosylation ( Bekendam et al, 2018 ; Flaumenhaft et al, 2015 ; Müller et al, 2008a ; Piacentini et al, 2018 ). Further, inhibition of furin ( Bonacci et al, 2011 ; Denault et al, 2002 ; McCarthy et al, 2008 ) by nitazoxanide can facilitate blocking of other protease signalling pathways, including Wnt/β-catenin ( Miner et al, 2019b ; Qu et al, 2018 ), MAPK/ERK ( Shou et al, 2019 ), and PI3K/Akt/mTOR ( Senkowski et al, 2015 ; Shou et al, 2020 ).…”

Vascular thiol isomerases in thrombosis: The yin and yang

“…PDI inhibitors inhibit endothelial cell and platelet-dependent thrombin generation in plasma, and factor Xa generation on endothelium. 59 PDI directly modulates activation of tissue factor, factor XI, and platelet factor V, [60][61][62] and regulates platelet and endothelial celldependent coagulant activity. 39,63 However, PDI has been found to have both positive and negative regulatory roles on tissue factor and procoagulant activity, 39,60,63 .…”

Microvascular thrombosis: experimental and clinical implications

“…78,89,90 Both methods are used in contemporary studies by various authors to define the kinetics of microvascular thrombosis in both venules and arterioles in vivo. [91][92][93][94][95][96] Although admittedly photochemical and laser stimulation represent artificial methods of microvascular injury, a significant Schematic of experimental models of microvascular thrombosis induced by focal stimulation of individual microvessels, adapted from a schematic from reference. 78 advantage of both approaches is the ability to standardize the degree of microvascular injury yielding highly reproducible results.…”