Protein disulfide isomerase-A1 regulates intraplatelet reactive oxygen species-thromboxane A 2 -dependent pathway in human platelets

Background Platelet-derived protein disulfide isomerase 1 (PDIA1) regulates thrombus formation, but its role in the regulation of platelet function is not fully understood. Aims The aim of this study was to characterize the role of PDIA1 in human platelets. Methods Proteomic analysis of PDI isoforms in platelets was performed using LC-MS/MS, and the expression of PDIs on platelets in response to collagen, TRAP-14, or ADP was measured with flow cytometry. The effects of bepristat, a selective PDIA1 inhibitor, on platelet aggregation, expression of platelet surface activation markers, thromboxane A2 (TxA2 ) and reactive oxygen species (ROS) generation were evaluated by optical aggregometry, flow cytometry, enzyme-linked immunosorbent assay and dihydrodichlorofluorescein diacetate-based fluorescent assay, respectively. Results PDIA1 was less abundant compared to PDIA3 in resting platelets and platelets stimulated with TRAP-14, collagen or ADP. Collagen, but not ADP, induced a significant increase in PDIA1 expression. Bepristat potently inhibited the aggregation of washed platelets induced by collagen or convulxin, but only weakly inhibited platelet aggregation induced by TRAP-14 or thrombin, and had the negligible effect on platelet aggregation induced by arachidonic acid. Inhibition of PDIA1 by bepristat resulted in the reduction of thromboxane A2 and ROS production in collagen- or thrombin-stimulated platelets. Furthermore, bepristat reduced the activation of αIIbβ3 integrin and expression of P-selectin. Conclusions PDIA1 acts as an intraplatelet regulator of the ROS-TxA2 pathway in collagen-GP VI receptor-mediated platelet activation that is a mechanistically distinct pathway from extracellular regulation of αIIbβ3 integrin by PDIA3.