Background: Reactive oxygen species (ROS) levels are often elevated in cardiovascular disease and thought to be both cause and consequence of damage induced by thrombosis. NADPH oxidase 1 (Nox-1) and protein disulphide isomerase (PDI) are key regulators of ROS production in cardiovascular cells. Both of these proteins contribute to platelet activation independently of one another. Understanding how platelets produce ROS and ultimately if this involves Nox-1 and PDI can provide better tools to treat and prevent thrombotic events.

Aims: To assess if and how PDI and Nox-1 regulate platelet function.

Methods: Nox-1 and PDI were visualized in resting and activated human washed platelets (WP) through immunofluorescence. WP were co-incubated with Nox-1 inhibitor ML171 and/or different PDI inhibitors and assays carried to assess ROS production, reductase activity, as well as platelet function through aggregometry, fibrinogen binding and P-selectin exposure. Similar experiments were performed in Nox-1 -/- mice in the presence or absence of PDI inhibitor. Phosphorylation of key proteins was investigated through immunoblot analysis in both human and mouse platelets.

Results: PDI and Nox-1 colocalization increased after activation with CRP. Likewise, ROS production was decreased by both PDI and Nox-1 inhibitors, whereas platelet membrane reductase activity increased in the presence of the Nox-1 inhibitor. These data indicate PDI may co-localize and modulate Nox-1 function and vice-versa. Co-incubation of PDI and Nox-1 inhibitors synergized to decrease platelet function only in collagen or CRP-activated platelets. A similar synergistic effect was seen using Nox-1 -/- platelets incubated with PDI inhibitor. Immunoblots of human and mouse platelets suggest Nox-1 and PDI co-inhibition induce a steep decrease in p47phox phosphorylation, whereas upstream molecules were not affected.

Conclusions: This is the first report of a synergistic effect amongst redox proteins in platelets, and has implications for the development of more effective ways to prevent thrombotic events.

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ISTH Congress Abstracts - https://abstracts.isth.org/abstract/nadph-oxidase-1-and-protein-disulphide-isomerase-synergize-to-modulate-platelet-function/