Protein disulfide isomerase 1 (PDIA1) regulates platelet-derived extracellular vesicle release

Abstract

Background

Protein disulfide isomerase 1 (PDIA1) and 3 (PDIA3) regulate platelet activation and thrombus formation. However, their role in the formation of platelet-derived extracellular vesicles (pEVs) remains unknown.

Aim

To characterise the effects of PDIA1 and PDIA3 inhibition on pEV formation in washed murine platelets in response to platelet glycoprotein VI (GPVI) receptor or intracellular calcium signal activation.

Methods

Washed platelets were isolated from C57BL/6 mice and activated using convulxin or the calcium ionophore A23187. Then, the resulting pEVs were analysed using nano flow cytometry (FC), platelet aggregation was measured by FC and a 96-well plate-based assay, and intracellular free calcium concentration ([Ca²⁺]i) was measured by indicator fluorescence. Platelet PDIs were blocked by a classic selective PDIA1 inhibitor (bepristat 2a) and sulphonamides of aziridine-2-carboxylic acid derivatives, novel PDI inhibitors relatively selective for PDIA1 or PDIA3 (C-3389 and C-3399, respectively). Clinically relevant antiplatelet drugs were used for comparison.

Results

Convulxin and A23187 concentration-dependently induced pEV formation. However, unlike convulxin, platelet activation by A23187 did not stimulate their aggregation. Bepristat 2a, C-3389 and C-3399 inhibited convulxin-induced pEV release accompanied by the reduction of [Ca²⁺]i. In contrast, only bepristat 2a inhibited A23187-induced pEV release, but without effect on [Ca²⁺]i. Cangrelor and tirofiban, but not acetylsalicylic acid (ASA), inhibited convulxin-induced pEV release, but neither of them inhibited A23187-induced pEV release.

Conclusion

The inhibition of PDIA1 represents a novel approach to inhibit pEV formation by a mechanism independent of platelet aggregation and calcium signaling.