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Novel antiplatelet role for a protein disulfide isomerase‐targeted peptide: Evidence of covalent binding to C‐terminal CGHC redox motif

2017; Wiley; Volume: 31; Issue: S1 Linguagem: Inglês

10.1096/fasebj.31.1_supplement.674.21

1530-6860

Renato Simões Gaspar, Hiran Reis Sousa, Elyjany Sena, Samira Abdalla da Silva, João Lucas de Lima Fontelles, Thais Araujo, Diana M. Fries, Ana Paula Silva de Azevedo dos Santos, Francisco Rafael Martins Laurindo, Andrés Trostchansky, Antonio Marcus de Andrade Paes,

Calcium signaling and nucleotide metabolism

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 PDI redox motif (CGHC) that inhibited both PDI reductase activity and PDI‐modulated superoxide generation by neutrophil Nox2. Thus, we hypothesized this peptide would also inhibit platelet aggregation by association to surface PDI. 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. We showed that 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. In thrombin‐activated platelets, surface expression of activated αIIbβ3 integrin, P‐selectin and granulophysin were analyzed by flow cytometry. CxxC decreased αIIbβ3 activation, but had no effect on the other markers. In addition, CxxC was the only peptide able to decrease cell‐surface PDI pull down, with no detectable unspecific binding to total free thiols. Finally, the capacity of CxxC to covalently bind to PDI active site was analyzed by ESI LC‐MS/MS. We detected the addition of one CxxC molecule to PDI through binding to Cys 400 , an important site for PDI prothrombotic activity. In summary, our data support an antiplatelet activity for CxxC through binding to Cys 400 in PDI a ′ domain, which can be further exploited as a model for site‐driven antithrombotic agents development. Support or Funding Information Foundation for Research and Scientific Development of Maranhão ‐ FAPEMA, Nacional Counsel for Scientific Development and Technology ‐ CNPq, Coordination of Improvement of Higher Level Personnel ‐ CAPES and CSIC grupos I+D 2014.