Mechanism and inhibition kinetics of peptide P13 as thrombin inhibitor

Excessive coagulation can easily lead to arterial and venous thrombosis, which is the main reason for the evolution of myocardial infarction and cerebrovascular accidents. As a key coagulation factor for the coagulation pathway, thrombin has become a remarkable target for the control of thrombosis. The synthesized peptide P13 with amino acid sequence of N-RGDAGFAGDDAPR was expected to be an inhibitor with higher antithrombotic activity. The results showed that the IC₅₀ (50% inhibition of thrombin activity) of the peptide P13 was determined by colorimetric method to be 115 µM. And enzyme kinetic experiments showed that P13 was a competitive inhibitor of thrombin with K_i = 106 µM. Fluorescence spectra and three-dimensional fluorescence showed that P13 could alter the secondary structure of thrombin and the microenvironment of certain chromogenic amino acids. P13 can spontaneously bind with thrombin exosite 1 in the form of 1:1 mainly through hydrogen bonding and van der Waals force. And the optimal docking mode of P13 and thrombin was revealed by molecular docking with "-CDOCKER_Energy" of 178.679 kcal mol^-1. This study revealed P13 may become a potential anticoagulant drug widely used after further studies in preclinical and clinical trials.