Purification and identification of thrombolytic peptides from enzymatic hydrolysate of Pheretima vulgaris

Pheretima vulgaris has been prescribed for the treatment of cardiovascular diseases in China for several hundred years in the form of dried powder in the clinic. However, the peptides with the potential antithrombotic activity of this source have never been reported. The total active proteins from Pheretima vulgaris were hydrolyzed by eight different commercial proteases and the alcalase hydrolysate showed the strongest thrombolytic activity. Four original thrombolytic peptides were isolated and characterized using bioactivity-directed fractionation of the active hydrolysate. The amino acid sequences were identified as HEPLPEP (m/z 818.40076), EYPLPEP (m/z 844.39648), LGEPSVP (m/z 698.39648), and LLAPP (m/z 510.28043) by nanoLC-ESI-Orbitrap mass spectrometry with PEAKS software. HEPLPEP and EYPLPEP, containing the common -PLPEP residue, showed superior thrombolytic activity in plasmin assay and fibrinogen-thrombin time assay. This research confirmed that Pheretima vulgaris was a potential source of active peptides with thrombolytic activities and provided novel candidates for the thrombolytic agents. PRACTICAL APPLICATIONS: Thrombosis has become the leading cause of mortality as it was the common underlying pathology of cardiovascular diseases, such as ischemic heart disease, and stroke. The demand for thrombolytics has increased gradually as the incidence trends of thrombosis-related diseases raise with the aging of the population. Four novel thrombolytic peptides were characterized from Pheretima vulgaris proteins hydrolysates, among which HEPLPEP and EYPLPEP could prevent the formation of thrombus and degrade existing thrombus in vitro. These peptides are promising to be meritorious templates for developing thrombolytic agents. The structure-function relationship of peptides resulting from the presence of specific residues in these sequences may contribute to extending the knowledge about their thrombolytic activity, which may be useful in designing novel thrombolytic agents. The present research based on a bioactivity-directed isolation strategy could also be applied to other animal-derived traditional Chinese medicines with proteins or peptides as their function basis.