Novel Anticoagulant Peptide from Lactoferrin Binding Thrombin at the Active Site and Exosite-I.

Thrombin is currently one of the important targets for the treatment and prevention of thrombosis. At present, there are few reports on the application of lactoferrin peptides in anticoagulation. In this study, a peptide with the amino acid sequence of LRPVAAEIY (LF-LR) derived from lactoferrin was shown to possess antithrombotic activity. LF-LR (5 mM) significantly prolonged activated partial thromboplastin time, prothrombin time, and thrombin time for 13.4, 1.7, and 5.1 s, respectively. It prolonged the coagulation time of fibrinogen from 15.3 ± 0.4 to 20.2 ± 0.5 s by affecting the conformation of thrombin. Using circular dichroism analysis, LF-LR can increase the α-helix content of thrombin from 25.6 to 56.7% and made the β-sheet disappear. In addition, LF-LR also quenched fluorescence of thrombin at about 346 nm (λEx = 280 nm). By means of molecular docking, it was found that LF-LR could bind to both the active site and the exosite-I of thrombin, and the combined LYS60F, TRP60D, ASP189, LYS36, and ARG77A are typical amino acids in the two domains, respectively.