Investigation of Thrombin Activity with PAR 1-based Fluorogenic Peptides

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 P-3' 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.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)INBEBUniv Fed Rio de Janeiro, Inst Bioquim Med, Rio de Janeiro, BrazilUniv Fed Rio de Janeiro, Inst Biofis Carlos Chagas Filho, BR-21941 Rio De Janeiro, BrazilUniv Fed Rio de Janeiro, Inst Nacl Biol Estrut & Bioimagem, Rio de Janeiro, BrazilUniv Fed Sao Paulo, Escola Paulista Med, Dept Biofis, Sao Paulo, BrazilUniv Fed Sao Paulo, Escola Paulista Med, Dept Biofis, Sao Paulo, BrazilWeb of Scienc