Purification and characterization of a novel thermostable anticoagulant protein from medicinal leech Whitmania pigra Whitman
Protein purification study published in Journal of Ethnopharmacology (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.
Resumen
Novel WP-77 anticoagulant protein (20.8 kDa) from non-suck-blood Whitmania pigra was thermostable from 20 to 100°C and pH 2-8, prolonged APTT and TT, and inhibited thrombus formation in carrageenan-induced thrombosis mice — supports development of a heat-resistant anticoagulant drug candidate.
Por qué esto importa para la hirudoterapia
Este estudio purificó y caracterizó una nueva proteína anticoagulante termoestable, WP-77 (~20.8 kDa), a partir de la sanguijuela medicinal no hematófaga Whitmania pigra mediante decocción acuosa y cromatografía. WP-77 resistió amplios rangos de temperatura (20–100 °C) y pH (2–8), prolongó significativamente el APTT y el TT in vitro, protegió a las células endoteliales EA.hy926 lesionadas por trombina, e inhibió la formación de trombos en un modelo murino de trombosis inducida por carragenina tras su administración intragástrica. Para el dominio de la ASH, este trabajo amplía el conocimiento sobre anticoagulantes bioactivos termoestables de una especie de sanguijuela medicinal utilizada tradicionalmente en la medicina china, complementando a agentes conocidos derivados de sanguijuelas como la hirudina. Advertencia honesta: los datos in vivo provienen de un único modelo murino de trombosis y los hallazgos son preclínicos; no se presentan datos de eficacia ni seguridad en humanos, y las afirmaciones sobre desarrollo clínico son especulativas.
Citación
Purification and characterization of a novel thermostable anticoagulant protein from medicinal leech Whitmania pigra Whitman.
Zhang Y et al. · Journal of Ethnopharmacology, 2022
Contexto clínico relacionado
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Añadido a la biblioteca ASH: May 27, 2026 · Última actualización del sitio: 18 de junio de 2026