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

Abstract

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 Aα > Bβ > γ. 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 (α polymer, α-chains, γ-γ dimer and β-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 μg, whereas S. grantii and W. tinctoria latex extracts were non-toxic and did not induce any hemorrhagic effect at the tested dose (> 200 μg). 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.

Key words

Calotropis gigantea - Asclepiadaceae - Synadenium grantii - Euphorbiaceae - Wrightia tinctoria - Apocynaceae - hemostasis - wound healing - folk medicine

References

• 1 Silva J RA, Amaral A CF, Siani A C, Rezende C M, Felcman J, Pinto A C. Contribution to the study of Himatanthus sucuuba: latex macromolecule, microelements and carbohydrates.  Acia Amazonica. 2003;  33 105-10.
• 2 Yagami T, Sato M, Nakamura A, Komiyama T, Kitagawa K, Akasawa A. et al . Plant defense-related enzymes as latex antigens.  Allergy Clin Immunol. 1998;  101 379-85.
• 3 Dhanukar K A, Kulakarni R A, Rege N N. Pharmacology of medicinal plants and national products.  Indian J Pharmacol. 2000;  32 S81-118.
• 4 Pankaj O. Doomar or Gular (Ficus glomerata) as medicinal herbs in Chattishgarh, India. Research Note. Available at botanical.com/site/column_poudhia/127_doomar.html Accessed in 2003.
• 5 Bhuyan D K. Herbal drugs used by the tribal people of Lohit district of Arunachal Pradesh for abortion and easy delivery a report.  Adv Plant Sci. 1994;  7 197-202.
• 6 Ashwani K. Ayurvedic medicines: some potential plants for medicine from India. Meeting of the International Forum on Traditional Medicines Toyama; 1999.
• 7 Thankamma L. Hevea latex as wound healer and pain killer.  Curr Sci. 2003;  84 971-2.
• 8 Richter G, Hans P S, Friedrich D, Peter L. Activation and inactivation of human factor X by proteases derived from Ficus carica .  Br J Haematol. 2002;  119 1042-51.
• 9 Rajesh R, Raghavendra Gowda C D, Nataraju A, Dhananjaya B L, Kemparaju K, Vishwanath B S. Procoagulant activity of Calotropis gigantia latex associated with fibrin (ogen) olytic activity.  Toxicon. 2005;  46 84-92.
• 10 Lowry O H, Rosenbrough N J, Farr A, Randall R J. Protein measurement with Folin phenol reagent.  J Biol Chem. 1951;  193 265-75.
• 11 Davis B J. Disk electrophoresis. II. Method and application to human serum proteins.  Ann NY Acad Sci. 1964;  12 404-27.
• 12 LaemmLi U K. Cleavage of structural proteins during the assembly of head of bacteriophage T4.  Nature. 1970;  227 680-5.
• 13 Murata J, Satake M, Suzuki T. Studies on snake venom. XII. Distribution of proteinase activities among Japanese and Formosan snake venoms.  J Biochem. 1963;  53 431-43.
• 14 Ouyang C, Teng C M. Fibrinogenolytic enzymes of Trimeresurus mucrosquamatus venom.  Biochim Biophys Acta. 1976;  420 298-308.
• 15 Condrea E, Yang C C, Rosenberg P. Anticoagulant activity and plasma phophatidylserine hydrolysis by snake venom phospholipase A2.  Thromb Haemost. 1983;  49 151.
• 16 Denson K WE. Coagulant and anticoagulant actions of snake venoms.  Toxicon. 1969;  7 5-11.
• 17 Kondo H, Kondo S, Ikezawa H, Murata R, Ohsaka A. Studies on the quantitative method for determination of haemorrhagic activity of Habu snake venom.  Jpn J Med Sci Biol. 1960;  13 43-51.
• 18 Mrinalini M, Vithayathil P J, Raju S M, Ramadoss C S. Isolation and characterization of proteolytic enzymes from the latex of Synadenium grantii Hook, ‘f’.  Plant Sci. 2002;  163 131-9.
• 19 Civello D J, Moran J B, Green C R. Substrate specificity of a haemorrhagic proteinase from timber rattlesnake venom.  Biochemistry. 1983;  22 755-62.
• 20 Komori Y, Sugihara H, Tu A T. Specificity of haemorrhagic proteinase from Crotalus atrox (Western diamond back rattle snake) venom.  Biochim Biophys Acta. 1985;  829 127-30.
• 21 Siigur J, Aaspollu A, Tonismagi K, Trummal K, Samel M, Vija H. et al . Proteases from Vipera labetina venom affecting coagulation and fibrinolysis.  Haemostasis. 2001;  31 123-32.
• 22 Lijnen H R. Matrix metalloproteinases and cellular fibrinolytic activity.  Biochemistry (Moscow). 2002;  67 92-8.
• 23 Davie E W, Fujikawa K, Kurachi K, Kisiel W. The role of serine proteases in the blood coagulation cascade.  Adv Enzymol Relat Areas Mol Biol. 1979;  48 277-318.
• 24 Burke D E, Lewis S D, Shafer J A. A two-step procedure for purification of papain from extract of papaya latex.  Arch Biochem Biophys. 1974;  164 30-6.
• 25 Sengupta A, Bhattacharya D, Pal G, Sinha N K. Comparative studies on calotropins DI and DII from the latex of Calotropis gigantea .  Arch Biochem Biophys. 1984;  232 17-25.
• 26 Rajesh R, Nataraju A, Gowda C D, Frey B M, Frey F J, Vishwanath B S. Purification and characterization of a 34-kDa, heat stable glycoprotein from Synadenium grantii latex: action on human fibrinogen and fibrin clot.  Biochimie. 2006;  88 1313-22.
• 27 Szecsi P B. The aspartic proteases.  Scand J Clin Lab Invest Suppl. 1992;  210 5-22.
• 28 Stoka V, Turk B, Turk V. Lysosomal cysteine proteases: structural features and their role in apoptosis.  IUBMB Life. 2005;  57 347-53.
• 29 Swenson S, Markland FS J r. Snake venom fibrin(ogen)olytic enzymes.  Toxicon. 2005;  45 1021-39.