Plant Latex Proteases Research Articles

Biotechnological application prospects of latex proteases from Apocynaceae family

Plant latex proteases from different species find applications in diverse industries ranging from food to medicine. Family Apocynaceae consists of species producing large quantities of milky latex. Latex is known to be a rich source of proteolytic enzymes or proteases. Proteases, mainly of the cysteine and occasionally serine type, have been characterized from the latices of different species of Apocynaceae. Many of these proteases have also demonstrated stability under a wide range of parameters including temperature, pH and presence of metal ions. The current review is an attempt to elucidate on latex proteases belonging to Apocynaceae family, highlighting those properties that offer potential for their large-scale applications in different industrial sectors.

Drupin, a thrombin-like protease prompts platelet activation and aggregation through protease-activated receptors.

Hemostasis is a proteolytically regulated process that requires activation of platelets and the blood coagulation cascade upon vascular injury. Activated platelets create a thrombogenic environment and amplify the coagulation process. Plant latex proteases (PLPs) have been used as therapeutic components to treat various ailments by folk healers. One of the main applications of plant latices is to stop bleeding from minor injuries and to enhance wound healing activity. Although many studies have reported the pro-coagulant activities of PLPs, an in-depth investigation is required to understand the mechanism of action of PLPs on platelets. Here, the effect of PLPs on platelet aggregation was studied systematically to validate the observed pharmacological effect by folk healers. Among 29 latices from theFicus genus tested, Ficus drupacea exhibited potent pro-coagulant and thrombin-like activity. Drupin, a thrombin-like cysteine protease responsible for platelet aggregation was purified from F. drupacea latex. Drupin exhibits pro-coagulant activity and reduces the bleeding time in mice tail. It induces platelet aggregation by activating mitogen-activated protein kinases andthe nuclear factor-κB and PI3K/Akt signalling cascade, which, in turn, phosphorylats, cytosolic phospholipase A2 leading to the release of thromboxane A2 from the granules to activate the nearby platelets to aggregate. Furthermore, we investigated the involvement of protease-activated receptors in drupin-induced platelet aggregation using specific protease activated receptor 1 (PAR1) and PAR4 receptor antagonists. The results confirmed that the drupin-induced platelet aggregation was mediated by both PAR1 and PAR4, synergistically. Overall, drupin reduces the bleeding time by exerting pro-coagulant activity and induces platelet aggregation by activating the intracellular signalling cascade.

Purification, characterization and fibrino(geno)lytic activity of cysteine protease from Tabernaemontana divaricata latex.

Protease was isolated and purified from Tabernaemontana divaricata latex and its hemostatic potential was analyzed. Crude latex enzyme was purified through ion exchange and gel filtration chromatography. Purified protease was characterized and its thrombin-like (coagulant assay, fibrinogen polymerizing, and fibrinogenolytic activity) and plasmin-like (blood and plasma clot lysis) activities were assessed accordingly. The homogeneous nature of protease was confirmed with the identification of a single band approximately at 25-kDa molecular weight position. The purified enzyme showed an enhancement of 77.32% in clot inducing ability and 89.86% improvement in blood clot lysis in comparison to that by the crude enzyme. All three subunits (Aα, Bβ and γ chains) of human fibrinogen were hydrolyzed by the purified enzyme. PAGE results of the fibrinolytic activity and blood clot lytic effect by the purified enzyme indicated the plasmin-like activity. The study lays a foundation for the development of enzyme-based approaches for pharmaceutical innovations, in which plant latex proteases can be utilized as a potential natural agent for wound healing applications.

Thrombin-like serine protease, antiquorin from Euphorbia antiquorum latex induces platelet aggregation via PAR1-Akt/p38 signaling axis

Plant latex proteases (PLPs) are pharmacologically essential and are integral components of traditional medicine in the management of bleeding wounds. PLPs are known to promote blood coagulation and stop bleeding by interfering at various stages of hemostasis. There are a handful of scientific reports on thrombin-like enzymes characterized from plant latices. However, the role of plant latex thrombin-like enzymes in platelet aggregation is not well known. In the present study, we attempted to purify and characterize thrombin-like protease responsible for platelet aggregation. Among tested plant latices, Euphorbia genus latex protease fractions (LPFs) induced platelet aggregation. In Euphorbia genus, E. antiquorum LPF (EaLPF) strongly induced platelet aggregation and attenuated bleeding in mice. The purified thrombin-like serine protease, antiquorin (Aqn) is a glycoprotein with platelet aggregating activities that interfere in intrinsic and common pathways of blood coagulation cascade and alleviates bleeding and enhanced excision wound healing in mice. In continuation, the pharmacological inhibitor of PAR1 inhibited Aqn-induced phosphorylation of cPLA2, Akt, and P38 in human platelets. Moreover, Aqn-induced platelet aggregation was inhibited by pharmacological inhibitors of PAR1, PI3K, and P38. These data indicate that PAR1-Akt/P38 signaling pathways are involved in Aqn-induced platelet aggregation. The findings of the present study may open up a new avenue for exploiting Aqn in the treatment of bleeding wounds.

Drupin, a cysteine protease from Ficus drupacea latex accelerates excision wound healing in mice

Wound healing is a tightly regulated physiological process that restores tissue integrity after injury. Plant latex proteases (PLPs) are considered an integral part in herbal wound care as it interferes at different phases of the wound healing process. Although many studies have reported the involvement of PLPs in healing process, an in-depth investigation is required to understand the molecular mechanism. Hence, the effect of PLPs with fibrinolytic activity on wound healing was investigated systematically using mouse excision wound model. Among 29 latices from Ficus genus tested, Ficus drupacea exhibited potent fibrinolytic activity. Cysteine protease responsible for fibrinolysis was purified from the F. drupacea latex named it as drupin, tested for its wound healing efficacy. The accelerated wound healing was mediated by downregulation of matrix metalloprotease (MMP)-9 without altering MMP-8 expression. Besides, drupin enhanced the rate of collagen synthesis at the wound site by increasing arginase 1 activity. And also, drupin increased the expression of arginase 1 in macrophages and involved in cell proliferation, and migration via MAP kinase and PI3K/Akt pathways. Overall, the present study highlights the interference of drupin in wound healing by increased arginase 1 activity and collagen synthesis, and cell proliferation and migration.

Plant latex thrombin-like cysteine proteases alleviates bleeding by bypassing factor VIII in murine model.

Hemostasis is a tightly regulated process which maintains a fluid state of blood within the vasculature and provides thrombotic response upon tissue injury. Various scientific studies have implicated the role of plant latex proteases in hemostasis using in vitro experiments. However, in vivo models substantiate their role in hemostasis. Therefore, in the present study, the effect of plant latex thrombin-like proteases (PTLPs) on hemostasis was investigated systematically using mice tail bleeding as a preclinical model. In this direction, latex protease fractions (LPFs), which showed potent thrombin-like activity, were selected as they act directly on fibrinogen to form clot and quickly stop bleeding. Thrombin-like activity was exhibited mainly by cysteine proteases. Calotropis gigantea, Carica papaya, Jatropha curcas, Oxystelma esculentum, Tabernaemontana divaricata, and Vallaris solanacea LPFs and papain from C. papaya latex significantly reduced bleeding on a topical application in normal and aspirin administered mice. In addition, PTLPs accelerated the clotting of factor VIII deficient plasma, while, papain brought back the clotting time to normal levels acting like a bypassing agent. Further, papain failed to show activity in the presence of specific cysteine protease inhibitor iodoacetic acid; confirming protease role in all the activities exhibited. At the tested dose, PTLPs except C. gigantea did not show toxicity. Further, structural and sequence comparison between PTLPs and human thrombin revealed structural and sequence dissimilarity indicating their unique nature. The findings of the present study may open up a new avenue for considering PTLPs including papain in the treatment of bleeding wounds.

Investigation of plant latices of Asteraceae and Campanulaceae regarding proteolytic activity

Occurrence of plant latices is widespread, there are more than 40 families of plants characterized to establish lactiferous structures. The appearance of hydrolytic active proteins, incorporated in latices is already characterized, and hydrolytic active proteins are considerable, and for several plant families, the occurrence of hydrolytic active proteins is already specified e.g. Apocynaceae Juss., Caricaceae Dumort, Euphorbiaceae Juss., Moraceae Gaudich and Papaveraceae Juss. In our investigation, focused on latex bearing plants of order Asterales, Asteraceae and Campanulaceae in particular. The present outcomes represent a comprehensive study, relating to the occurrence of proteolytic active enzymes of order Asterales for the first time. 131 different species of Asteraceae and Campanulaceae were tested, and the appearance of plant latex proteases were determined in different quantities. Proteolytic activity was investigated by inhibitory studies and determination of residual activity in the following, enable us to characterize the proteases. Most of the considered species exhibit a serine protease activity and a multiplicity of species exhibited two or more subclasses of proteases.

Detecting of fibrino(geno)lytic activity of plant latex proteases by a specific fluorogenic substrate and fibrin(ogen)-zymography

Plant latex is repeatedly examined in terms of blood clotting and fibrinolytic activity, due to the proteases often contained. Some examples are the latex of Synadenium grantii Hook (Euphorbiaceae) [1], Pergularia extensa (Jacq.) N.E. Br. (Apocynaceae) [2] or Artocarpus heterophyllus Lam. (Moraceae) [3]. Previously we described the in vitro influence of a protease from the latex of Euphorbia mauritanica L. (Euphorbiaceae) on some steps of the coagulation and fibrinolysis process, by using different methods such as a fibrin plate assay, turbidimetry or in-gel-digestion [4]. We examined over hundred species of the family Euphorbiaceae for fibrinolytic activity with the specific fluorogenic substrate Boc-Val-Leu-Lys-AMC. We have combined the results for the most active plant latices with fibrin(ogen) zymography to confirm the results of the screening and to get information whether the active proteases are the same in the different latices. We also wanted to confirm the results of the above methods for the mentioned isolated protease. The zymograms confirm the activity for all latices tested. However, different proteases seem to be responsible for the activity in each latex.

Plant latex proteases with potential influence on blood coagulation and fibrinolysis

Proteases are widespread in latices of different plant families (e.g. Apocynaceae, Asteraceae, Caricacea, Convolvulaceae, Euphorbiaceae and Moraceae) [1]. Their function has not been completely clarified yet; involvement in wound healing after injury and protection from predators appear logical. Potential use of plant proteases are in the food industry (e.g. cheese production), washing industry or in the pharmaceutical industry. For the latter one possible application is the blood coagulation and fibrinolysis process. Some latex-containing plants are already under investigation in this aspect, like for example Synadenium grantii Hook (Euphorbiaceae), Wrightia tinctoria R.Br. (Apocynaceae), Ficus carica L. (Moraceae) and Euphorbia hirta L. (Euphorbiaceae) [2, 3]. We analysed the in vitro influence of the protease-containing latex of Euphorbia mauritanica L. (Euphorbiaceae) on some parts of the coagulation and fibrinolysis procedure, by using different methods like a fibrin plate assay, specific substrates or In-Gel-Digestion. It shows thombin-like activity, since it leads to an opacity in the plate assay and a moderate conversion of two thrombin-specific fluorogenic substrates. However it also shows plasmin-like activity, since it is able to dissolve small fibrin clots and coverts two plasmin-specific fluorogenic substrates. In-Gel-Digestion of fibrin and fibrinogen chains confirmed the activity, but the cleavage sites appear to be different.

74. Plant Latex Proteases: An Insight into their Procoagulant Activity

74. Plant Latex Proteases: An Insight into their Procoagulant Activity

Comparative Study on Plant Latex Proteases and their Involvement in Hemostasis: A Special Emphasis on Clot Inducing and Dissolving Properties

In the present study we compared the clot inducing and dissolving properties of Calotropis gigantea R. Br. (Asclepiadaceae), Synadenium grantii Hook. f. (Euphorbiaceae) and Wrightia tinctoria R. Br. (Apocynaceae) latex extracts. All the three latex extracts hydrolyzed casein, fibrinogen and crude fibrin dose-dependently. The proteolytic action on fibrinogen subunity was in the order of Aalpha > Bbeta > gamma. All extracts exhibited procoagulant activity as assayed by re-calcification time. However, thrombin like activity is restricted to C. gigantea. In addition, the extracts dose-dependently hydrolyzed blood and plasma clots. Furthermore, the hydrolyzing pattern of fibrin in the plasma clot was substantiated by SDS-PAGE. The extracts hydrolyzed all the subunits (alpha polymer, alpha-chains, gamma-gamma dimer and beta-chain) of fibrin efficiently. Both fibrinogenolytic and fibrinolytic activity potency of the extracts were in the order of C. gigantea > S. grantii > W. tinctoria. Among the three latices, C. gigantea is toxic with a minimum hemorrhagic dose (MHD) of > 75 microg, whereas S. grantii and W. tinctoria latex extracts were non-toxic and did not induce any hemorrhagic effect at the tested dose (> 200 microg). The proteolytic activity of C. gigantea latex extract on different substrates was inhibited by IAA. On the other hand, the proteolytic activities of S. grantii and W. tinctoria were inhibited by PMSF. Thus, this study provides the basis for the probable action of plant latex proteases to stop bleeding and effect wound healing as exploited in folk medicine.