Transmembrane thiol isomerase TMX1 counterbalances the effect of ERp46 to inhibit platelet activation and integrin αIIbβ3 function

Background: Previous studies have shown that thiol isomerases such as ERp46 positively regulate platelet function by reducing integrin αIIbβ3 disulfides, and the transmembrane thiol isomerase TMX1 negatively regulates integrin αIIbβ3 activation. However, whether and how the positive and negative thiol isomerases interact with each other and their interactions participate in platelet activation remain unknown. Objectives: To investigate whether and how TMX1 regulates the effect of ERp46 on platelet function. Methods: Using ERp46- and TMX1-deficient platelets, anti-TMX1 antibody, and wild-type TMX1 (TMX1-CPAC, TMX1-SS) and inactive TMX1 (TMX1-SPAS, TMX1-OO) proteins, we studied the antagonistic effect of TMX1 on ERp46 in platelet aggregation, clot retraction, and integrin αIIbβ3 signaling. The underlying mechanisms were further determined using thiol labeling, reductase activity, and other assays. Results: Anti-TMX1 antibody and TMX1-OO reversed the decreased aggregation of ERp46-deficient platelets induced by thrombin, convulxin, and U46619. Anti-TMX1 antibody reversed the attenuated integrin αIIbβ3 function of ERp46-deficient platelets. TMX1 inhibited ERp46 reductase activity in a concentration-dependent manner. TMX1 oxidized thiols of ERp46 and those of integrin αIIbβ3 generated by ERp46. Moreover, TMX1 deficiency increased free thiols of ERp46 in platelets, which was reversed by the addition of wild-type TMX1 protein. Besides, anti-TMX1 antibody increased free thiols of ERp46 in wild-type activated platelets. Conclusion: TMX1 not only oxidizes integrin αIIbβ3 disulfides that are reduced by ERp46 but also directly oxidizes ERp46 to suppress its reduction of integrin αIIbβ3. Thus, TMX1 is critical for maintaining platelets in a quiescent state and counterbalancing the effect of ERp46 to prevent platelet overactivation.