■先前的研究表明,硫醇异构酶如ERp46通过减少整合素αIIbβ3二硫化物来正向调节血小板功能,跨膜硫醇异构酶TMX1负调控整合素αIIbβ3的活化。然而,正负硫醇异构酶是否以及如何相互作用以及它们之间的相互作用参与血小板活化仍然未知.
■研究TMX1是否以及如何调节ERp46对血小板功能的影响。
■使用ERp46和TMX1缺陷型血小板,抗TMX1抗体,和野生型TMX1(TMX1-CPAC,TMX1-SS)和非活动TMX1(TMX1-SPAS,TMX1-OO)蛋白质,我们研究了TMX1在血小板聚集中对ERp46的拮抗作用,凝块回缩,和整合素αIIbβ3信号传导。使用硫醇标记进一步确定了潜在的机制,还原酶活性,和其他化验。
■抗TMX1抗体和TMX1-OO逆转了凝血酶诱导的ERp46缺陷型血小板聚集减少,惊厥素,U46619抗TMX1抗体逆转ERp46缺陷型血小板减弱的整合素αIIbβ3功能。TMX1以浓度依赖性方式抑制ERp46还原酶活性。ERp46的TMX1氧化硫醇和ERp46产生的整联蛋白αIIbβ3的硫醇。此外,TMX1缺乏增加了血小板中ERp46的游离硫醇,通过添加野生型TMX1蛋白而逆转。此外,抗TMX1抗体增加了野生型活化血小板中ERp46的游离硫醇。
■TMX1不仅氧化被ERp46还原的整联蛋白αIIbβ3二硫化物,而且直接氧化ERp46以抑制其整联蛋白αIIbβ3的还原。因此,TMX1对于维持血小板处于静止状态和抵消ERp46的作用以防止血小板过度活化是至关重要的。
UNASSIGNED: 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.
UNASSIGNED: To investigate whether and how TMX1 regulates the effect of ERp46 on platelet function.
UNASSIGNED: 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.
UNASSIGNED: 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.
UNASSIGNED: 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.