Purification and characterization of a novel thermostable anticoagulant protein from medicinal leech Whitmania pigra
Biochemistry/pharmacology published in J Ethnopharmacol (2022)
Abstract
ETHNOPHARMACOLOGICAL RELEVANCE: The prevalence of cardiovascular disease (CVD) is increasing worldwide. Despite significant improvements in novel targeted treatment agents, natural products purified from medicinal animals with minimal side effects have attracted much attention. Several native proteins explored from suck-blood leeches, such as non-thermostable hirudin and its variants, revealed potent anticoagulant activity. Traditional Chinese medicine clinics have proved that non-suck-blood leech Whitmania pigra Whitman (W. pigra) also played notable roles in CVD treatments even after decoction. However, only a few natural proteins and peptides have been identified from the fresh material of this medicinal species. AIM OF THE STUDY: We aimed to purify and characterize thermostable anticoagulant proteins from W. pigra for further development of a therapeutic agent for thrombosis. MATERIALS AND METHODS: W. pigra crude extract was prepared by decoction in water. Anticoagulant proteins were purified by DEAE cellulose DE-52, Sephadex G-75, and reversed-phase liquid chromatography sequentially and analyzed by SDS-PAGE and LC-MS/MS for structural information. In addition, we conducted in vitro anticoagulant experiments, including plasma recalcification time (PRT) assay, fibrinolytic assay, activated partial thromboplastin time (APTT), prothrombin time (PT), thrombin time (TT), fibrinogen (Fib) assay, and cell viability assays. Furthermore, a carrageenan-induced chronic thromboembolism model was employed in ICR mice, and four coagulation factors (APTT, PT, TT, and Fib) activities were determined after intragastric administration. RESULTS: The anticoagulant protein WP-77 has a relative molecular weight of ca. 20.8 kDa. It was effective over a broad temperature range from 20 °C to 100 °C and a pH 2-8 condition. The anticoagulant activity of WP-77 was retained after incubation with pepsin but was greatly inhibited by trypsin (P < 0.01). It significantly prolonged APTT and TT (P < 0.05) but had little effect on PT and Fib in vitro. Furthermore, WP-77 of a low concentration resulted in the recovery of injured EA.hy926 by thrombin. The protein also significantly prolonged APTT and TT (P < 0.01) and inhibited thrombus formation in carrageenan-induced thrombosis mice, demonstrating its antithrombotic effect in vivo. CONCLUSION: Our results suggest that WP-77 from W. pigra plays a distinct role in treating thrombotic diseases, and it is an essential substance of anticoagulant activity of non-suck-blood medicinal leeches. This thermostable anticoagulant protein could be a promising candidate for the development of clinical antithrombosis medicines.
Abstract sourced from PubMed (NCBI) for the cited record. See the original publication for the authoritative version.
Summary
WP-77, a 20.8 kDa thermostable anticoagulant protein from Whitmania pigra, retains activity from 20–100 °C and pH 2–8. In carrageenan-induced thrombosis mice, WP-77 significantly prolonged APTT and TT and reduced thrombus burden after oral administration.
Why This Matters for Hirudotherapy
This study isolated and characterized a novel thermostable anticoagulant protein, WP-77, from the non-suck-blood leech Whitmania pigra. The research demonstrated that WP-77 prolonged coagulation times in vitro and inhibited thrombus formation in a mouse model, while also protecting endothelial cells. This work is relevant to the leech secretome domain by expanding the understanding of anticoagulant compounds derived from leeches and their potential therapeutic mechanisms. However, the study is strictly preclinical, relying on in vitro assays and an animal disease model, with no data on live hirudotherapy or human clinical application.
Citation
Purification and characterization of a novel thermostable anticoagulant protein from medicinal leech Whitmania pigra Whitman.
Zhang Y et al. · Journal of ethnopharmacology, 2022
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