Novel Pheretima guillelmi-derived antithrombotic protein DPf3: Identification, characterization, in vitro evaluation and antithrombotic mechanisms investigation.

In this study, the antithrombotic protein, named DPf3, was purified from Pheretima guillelmi by ion-exchange chromatography. The protein pattern of DPf3 was mainly at 26-34 kDa; its two main proteins, DPf3 ID NO.1 and NO.2, were detected to be 36,121.745 Da and 24,485.004 Da consisting of 329 and 241 amino acids, respectively; the full covered protein sequences were consistent with Ac44553_g1_i1_1 and Dc43026_g1_i1_2 in our previous constructed P. guillelmi local database. The secondary structure of DPf3 is the mixture of α-helix (0.19), β-sheet (0.30) and random coil (0.51). DPf3 was predicted to possess a direct effect on fibrin, fibrinogen and plasminogen by protein-protein docking analysis, which was further confirmed by in vitro and ex vivo study. DPf3 was determined to possess antithrombotic ability by showing outstanding direct-hydrolysis ability on fibrin, fibrinogen and blood clot, and slight plasminogen activation activity. DPf3 could significantly prolong APTT and decrease fibrinogen content, indicating that DPf3 exerted antithrombotic activity via the intrinsic and/or common pathway, and the third coagulation phase. By this approach, the functional protein DPf3 was fully revealed and found to confer excellent anticoagulant and thrombolytic activity, and could be developed into a promising antithrombotic agent.