Abstract

Platelet surface Protein Disulphide-Isomerase (psPDI) is indispensable for thrombus formation at vascular injury sites, primarily through its role in αIIbβ3 integrin activation and is influenced by oxidative and nitrosative stress. Given limitations of traditional assays, a sensitive fluorometric method employing the fluorescence-quenched pseudo-substrate Di-E-GSSG was utilised to determine psPDI reductase activity. The Di-E-GSSG probe was synthesised and validated, exhibiting a greater than 6-fold fluorescence increase with Di-Thio-Threitol (DTT), confirming its suitability. Experiments demonstrated the probe’s resistance to low DTT concentrations, with 5μM DTT deemed appropriate. The assay successfully monitored both bovine liver PDI and psPDI-dependent disulphide reduction in a concentration-dependent manner, confirming psPDI’s contribution to reductase activity and the assay’s efficacy as a simple, single-step tool. Furthermore, psPDI activity was consistently inhibited by known PDI inhibitors: DTNB (100%), bacitracin (80%), PAO (55%) and rutin (14%), serving to confirm the presence of psPDI-contributed surface reductase activity. Most significantly, exposure to oxidative and nitrosative stress agents resulted in a dose-dependent disturbance of psPDI activity. At the highest concentrations, oxidative stress, specifically using hydrogen peroxide and hypochlorous acid, led to complete (100%) ablation of psPDI activity, whereas the nitric oxide donors, nitrosoglutathione and diethylamine nitric oxide, resulted in 97% and 86% inhibition, respectively, thereby retaining 3% and 14% activity with equimolar concentrations. This highlights that oxidative stress exerts a more potent inhibitory effect on psPDI than nitrosative stress. In conclusion, this study demonstrates that the reductive function of psPDI is markedly inhibited within oxidative/nitrosative stress environments, offering insights into its regulation.

Keywords:Blood platelets; Protein disulphide-isomerase; Cell surface; Redox potential; Fluorescent probes