title: Novel antiplatelet role for a protein disulfide isomerase-targeted peptide: evidence of covalent binding to the C-terminal CGHC redox motif.
abstract: Essentials Inhibitors of protein disulfide isomerase (PDI) have been considered a new antithrombotic class. CxxC is a PDI-targeted peptide that has been previously shown to inhibit its reductase activity. CxxC binds to surface PDI and inhibits ADP- and thrombin-evoked platelet activation and aggregation. CxxC binds to Cys400on CGHC redox motif of PDI a' domain, a site for PDI prothrombotic activity.
  
  
  
  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.
authors.affiliation: Laboratory of Experimental Physiology, Department of Physiological Sciences, Federal University of Maranhão, São Luís, MA, Brazil.
authors.forename: H R
authors.initials: HR
authors.lastname: Sousa
authors.affiliation: Laboratory of Experimental Physiology, Department of Physiological Sciences, Federal University of Maranhão, São Luís, MA, Brazil.
authors.forename: R S
authors.initials: RS
authors.lastname: Gaspar
authors.affiliation: Laboratory of Experimental Physiology, Department of Physiological Sciences, Federal University of Maranhão, São Luís, MA, Brazil.
authors.forename: E M L
authors.initials: EM
authors.lastname: Sena
authors.affiliation: Laboratory of Experimental Physiology, Department of Physiological Sciences, Federal University of Maranhão, São Luís, MA, Brazil.
authors.forename: S A
authors.initials: SA
authors.lastname: da Silva
authors.affiliation: Laboratory of Experimental Physiology, Department of Physiological Sciences, Federal University of Maranhão, São Luís, MA, Brazil.
authors.forename: J L
authors.initials: JL
authors.lastname: Fontelles
authors.affiliation: Laboratory of Vascular Biology, Heart Institute, School of Medicine of the University of São Paulo, São Paulo, SP, Brazil.
authors.forename: T L S
authors.initials: TL
authors.lastname: AraUjo
authors.affiliation: Departamento de Bioquímica and Center for Free Radical and Biomedical Research, Facultad de Medicina, Universidad de la República, Montevideo, Uruguay.
authors.forename: M
authors.initials: M
authors.lastname: Mastrogiovanni
authors.affiliation: Laboratory of Vascular Biology, Heart Institute, School of Medicine of the University of São Paulo, São Paulo, SP, Brazil.
authors.forename: D M
authors.initials: DM
authors.lastname: Fries
authors.affiliation: Laboratory of Immunophysiology, Department of Pathology, Federal University of Maranhão, São Luís, MA, Brazil.
authors.forename: A P S
authors.initials: AP
authors.lastname: Azevedo-Santos
authors.affiliation: Laboratory of Vascular Biology, Heart Institute, School of Medicine of the University of São Paulo, São Paulo, SP, Brazil.
authors.forename: F R M
authors.initials: FR
authors.lastname: Laurindo
authors.affiliation: Departamento de Bioquímica and Center for Free Radical and Biomedical Research, Facultad de Medicina, Universidad de la República, Montevideo, Uruguay.
authors.forename: A
authors.initials: A
authors.lastname: Trostchansky
authors.affiliation: Laboratory of Experimental Physiology, Department of Physiological Sciences, Federal University of Maranhão, São Luís, MA, Brazil.
authors.forename: A M
authors.initials: AM
authors.lastname: Paes
mesh_terms: D000409:Alanine / Q000737:chemistry; D020816:Amino Acid Motifs; D001792:Blood Platelets / Q000378:metabolism; D020134:Catalytic Domain; D003545:Cysteine / Q000737:chemistry; D004220:Disulfides; D006801:Humans; D010084:Oxidation-Reduction; D010455:Peptides / Q000737:chemistry; D015539:Platelet Activation; D010974:Platelet Aggregation; D010975:Platelet Aggregation Inhibitors / Q000737:chemistry*; D011393:Procollagen-Proline Dioxygenase / Q000737:chemistry*; D011485:Protein Binding; D019704:Protein Disulfide-Isomerases / Q000737:chemistry*; D000072417:Protein Domains; D017510:Protein Folding
chemical_list: D004220:Disulfides; D010455:Peptides; D010975:Platelet Aggregation Inhibitors; D011393:Procollagen-Proline Dioxygenase; C526497:P4HB protein, human; D019704:Protein Disulfide-Isomerases; D003545:Cysteine; D000409:Alanine
keywords: antithrombotic agents; oxidation-reduction; peptides; platelet aggregation; protein disulfide isomerase