title: Investigation of thrombin activity with PAR 1-based fluorogenic peptides.
abstract: Thrombin, a highly specific protease of blood coagulation, has two exosites that modulate its specificity. We designed two sets of synthetic substrate FRET peptides with 25- or 11- amino acids (aa) each, based on the PAR 1 sequence, to characterize the effect of exosite 1 engagement on substrate catalysis and preference. The 25-aa set encompassed a sequence binding to exosite 1, and structural modeling showed that binding to thrombin did not differ significantly from that of PAR 1 peptide. Modification at the P3´position of the 25 or 11-aa peptides resulted in small effect on kinetic parameters. Ionic strength higher than physiologic depressed thrombin action on the 25-aa peptides. Addition of ligands of the exosite 1 negatively modulated the catalysis of 25-aa substrates. In conclusion, we succeeded to mimic and study in real time, using these synthetic peptides, the influence of ligand binding to exosite 1 on thrombin activity.
authors.affiliation: Instituto de Bioquímica Médica, UFRJ, Brasil.
authors.forename: Saulo Martins
authors.initials: SM
authors.lastname: Vieira
authors.forename: Flávia Garcia
authors.initials: FG
authors.lastname: dos Reis
authors.forename: Reinaldo
authors.initials: R
authors.lastname: Geraldo
authors.forename: Denis Luis da Silva
authors.initials: DL
authors.lastname: Dutra
authors.forename: Luiz
authors.initials: L
authors.lastname: Juliano
authors.forename: Maria Aparecida
authors.initials: MA
authors.lastname: Julianod
authors.forename: Julio Alberto
authors.initials: JA
authors.lastname: Mignaco
authors.forename: Russolina Benedeta
authors.initials: RB
authors.lastname: Zingali
mesh_terms: D000595:Amino Acid Sequence; D057075:Enzyme Assays; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D062105:Molecular Docking Simulation; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000378:metabolism*; D044463:Receptor, PAR-1 / Q000737:chemistry / Q000378:metabolism*; D013379:Substrate Specificity; D013917:Thrombin / Q000737:chemistry / Q000378:metabolism*
chemical_list: D005456:Fluorescent Dyes; D010455:Peptides; D044463:Receptor, PAR-1; D013917:Thrombin