Novel antiplatelet role for a protein disulfide isomerase-targeted peptide: evidence of covalent binding to the C-terminal CGHC redox motif
Novel antiplatelet role for a protein disulfide isomerase-targeted peptide: evidence of covalent binding to the C-terminal CGHC redox motif
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DOI：
10.1111/jth.13633
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发表时间：
2017-04-01
影响因子：
10.4
通讯作者：
Paes, A. M.
中科院分区：
文献类型：
--
作者：
Sousa, H. R.;Gaspar, R. S.;Paes, A. M.
背景：蛋白二硫键异构酶(PDI)在血小板聚集中起重要作用，其抑制剂已成为新型抗血栓药物。在之前的工作中，我们设计了一种基于PDI氧化还原基序(CGHC)的肽，它既能抑制PDI还原酶的活性，又能抑制PDI调节的中性粒细胞NOX2产生的超氧化物。因此，我们假设这种多肽也会通过与表面PDI结合来抑制血小板聚集。方法：使用三种多肽：CxxC，含有PDI氧化还原基序；SCR，呈现相同残基的扰乱序列；以及AxxA，用丙氨酸取代半胱氨酸。用血小板聚集和流式细胞仪检测这些多肽，以确定它们可能的抗血小板活性。我们标记了膜游离硫醇和电喷雾电离液相色谱串联质谱来测试相互作用。结果：CxxC以剂量依赖的方式抑制血小板聚集，且在较低浓度激动剂时作用更强，而AxxA和Scr多肽均无此作用。CxxC降低aIIbb3活性，但对其他标志物无影响。CxxC还可降低细胞表面PDI下拉力，但不影响细胞内总硫醇蛋白含量。最后，我们通过质谱学检测到一个CxxC分子通过与Cys400结合而添加到还原的PDI中。有趣的是，CxxC不与氧化的PDI反应。讨论：CxxC一直显示出其在PRP和洗涤血小板中的抗血小板作用，aIIbb3活性的降低证实了这一点。可能的作用机制是通过与PDI的Cys400的混合二硫键，这已被证明是PDI的作用所必需的。结论：综上所述，我们的数据支持CxxC通过与PDIa0结构域上的Cys400结合来发挥抗血小板活性，这可以进一步作为现场驱动抗血栓药物开发的模型。
Background: Protein disulfide isomerase (PDI) plays a major role in platelet aggregation, and its inhibitors have emerged as novel antithrombotic drugs. In previous work, we designed a peptide based on a PDI redox motif (CGHC) that inhibited both PDI reductase activity and PDI-modulated superoxide generation by neutrophil Nox2. Thus, we hypothesized that this peptide would also inhibit platelet aggregation by association with surface PDI. Methods: Three peptides were used: CxxC, containing the PDI redox motif; Scr, presenting a scrambled sequence of the same residues and AxxA, with cysteines replaced by alanine. These peptides were tested under platelet aggregation and flow cytometry protocols to identify their possible antiplatelet activity. We labeled membrane free thiol and electrospray ionization liquid chromatography tandem mass spectrometry to test for an interaction. Results: CxxC decreased platelet aggregation in a dose-dependent manner, being more potent at lower agonist concentrations, whereas neither AxxA nor Scr peptides exerted any effect. CxxC decreased aIIbb3 activation, but had no effect on the other markers. CxxC also decreased cell surface PDI pulldown without interfering with the total thiol protein content. Finally, we detected the addition of one CxxC molecule to reduced PDI through binding to Cys400 through mass spectrometry. Interestingly, CxxC did not react with oxidized PDI. Discussion: CxxC has consistently shown its antiplatelet effects, both in PRP and washed platelets, corroborated by decreased aIIbb3 activation. The probable mechanism of action is through a mixed dissulphide bond with Cys400 of PDI, which has been shown to be essential for PDI's actions. Conclusion: In summary, our data support antiplatelet activity for CxxC through binding to Cys400 in the PDI a0 domain, which can be further exploited as a model for sitedriven antithrombotic agent development.