Thrombin- and plasmin-like and platelet-aggregation-inducing activities of Plumeria alba L. latex: Action of cysteine protease

Abstract

Ethnopharmacological relevance: In folk medicine, parts of Plumeria alba L. are used for the treatment of many diseases, with its latex being used for curing skin diseases and promoting wound healing. Aim of the study: This study aimed to study the role of P. alba L. latex in hemostasis and platelet aggregation. Materials and methods: The latex of P. alba L. was processed to remove waxes and enrich protein content, and the final extract was named Plumeria alba L. natant latex (PaNL). PaNL was analyzed for protease activity against casein. The type of protease in PaNL was identified by using protease inhibitors such as E−64, phenylmethylsulfonyl fluoride, ethylenediaminetetraacetic acid, and pepstatin A. Human fibrinogen, fibrin, and collagen types I and IV were subjected to hydrolysis with different concentrations of PaNL. The thrombin-like activity of PaNL was determined by analyzing its fibrinogen-clotting and procoagulant activities. The role of PaNL in platelet aggregation was also investigated. Its hemorrhagic and edema-inducing activities were evaluated in a mouse model. Phytochemical compounds were identified by gas chromatography–mass spectroscopy. Results: The findings of casein/gelatin zymography confirmed that PaNL possesses protease activity. The results of the protease inhibition study indicated the presence of a cysteine-type protease(s) in PaNL. PaNL hydrolyzed the subunits of fibrinogen, fibrin, and collagen types I and IV. Its fibrin-degradation activity indicated that PaNL possesses plasmin-like activity. PaNL induced clotting of citrated human plasma within 3 min of incubation in the absence of CaCl2, indicating the presence of thrombin-like activity, which was further confirmed by the results of the fibrinogen-clotting assay. PaNL induced platelet aggregation in the absence of agonists. There was no hemolytic activity. Mice injected with PaNL did not show edema/ hemorrhagic activity. Conclusion: PaNL possesses procoagulant, fibrino(geno)lytic, thrombin- and plasmin-like activities and induces platelet aggregation, which could explain its usage for wound treatment in folk medicine. © 2021 Elsevier B.V