Flaxseed Cysteine Protease Exhibits Strong Anticoagulant, Antiplatelet, and Clot-Dissolving Properties
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Flaxseed Cysteine Protease Exhibits Strong Anticoagulant, Antiplatelet, and ClotDissolving Properties S. K. M. Nandish1, J. Kengaiah1, Ch. Ramachandraiah1, Chandramma1, A. Shivaiah1, S. M. Santhosh2, Thirunavukkarasu3, and D. Sannaningaiah1,a* 1
Department of Studies and Research in Biochemistry and Centre for Bioscience and Innovation, Tumkur University, 572103 Tumkur, India 2 Department of Medicinal Biochemistry and Microbiology (IMBM), Uppsala Biomedical Centre, 75237 Uppsala, Sweden 3 Department of Biochemistry and Molecular Biology, School of Life Sciences, Pondicherry University, 605014 Pondicherry, Tamil Nadu, India a email: [email protected] Received March 21, 2020 Revised May 26, 2020 Accepted May 29, 2020 Abstract—In this study, we purified and characterized flaxseed cysteine protease (FSCP) with strong anticoagulant, antiplatelet, and clotdissolving properties. The enzyme was purified to homogeneity by a combination of gel permeation and ionexchange column chromatography techniques. The purity of the enzyme was evaluated by SDSPAGE, RPHPLC, and MALDITOF. FSCP was observed as a single band of approximately 160 kDa in SDSPAGE under reducing and non reducing conditions. The exact molecular mass of FSCP was found to be 168 kDa by MALDITOF spectrometry. The CD spectra of FSCP revealed the presence of 25.6% helices, 25.8% turns, and 48% random coils with no betasheet structures. FSCP hydrolyzed both casein and gelatin with a specific activity of 3.5 and 4.2 unit/mg min respectively. The proteolytic activity of FSCP was completely abolished by iodoacetic acid (IAA), suggesting FSCP is a cysteine protease. The pH opti mum for the proteolytic activity of FSCP was pH 6.0; the temperature optimum was 30°C. FSCP exhibited strong antico agulant effect in both plateletrich plasma (PRP) and plateletpoor plasma (PPP) by extending the clotting time from 222 to 1100 s and from 256 to 1210 s, respectively. FSCP degraded human fibrinogen and fibrin clots. The products of fibrino gen degradation by thrombin and FSCP were different. Furthermore, FSCP inhibited aggregation of washed platelets trig gered by ADP, epinephrine, thrombin, collagen, arachidonic acid, and platelet activating factor (PAF). FSCP was found to be nontoxic as it did not damage the membrane of red blood cells (RBCs) and did not induce hemorrhage and edema in experimental mice. DOI: 10.1134/S0006297920090102 Keywords: cysteine protease, anticoagulant, antiplatelet, nontoxic
INTRODUCTION Flaxseeds contain a plethora of phytoconstituents including enzymes, nonenzymatic proteins, lipids, car Abbreviations: ADP, adenosine diphosphate; APTT, activated partial thromboplastin time; CD, circular dichroism; DEAE, diethyl amino ethyl; EDTA, ethylenediaminetetraacetic acid; FSCP, flaxseed cysteine protease; IAA, iodoacetic acid; OD, optical density; PAF, platelet activating factor; PMSF, phenyl methylsulfonyl fluoride; PPP, plateletpoor plasma; PRP, plateletrich plasma; PT, prothrombin time; RPHPLC, reversedphase highperformance liquid ch
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