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Flaxseed Cysteine Protease Exhibits Strong Anticoagulant, Antiplatelet, and Clot-Dissolving Properties

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Abstract

In this study, we purified and characterized flaxseed cysteine protease (FSCP) with strong anticoagulant, antiplatelet, and clot-dissolving properties. The enzyme was purified to homogeneity by a combination of gel permeation and ion-exchange column chromatography techniques. The purity of the enzyme was evaluated by SDS-PAGE, RP-HPLC, and MALDI-TOF. FSCP was observed as a single band of approximately 160 kDa in SDS-PAGE under reducing and non-reducing conditions. The exact molecular mass of FSCP was found to be 168 kDa by MALDI-TOF spectrometry. The CD spectra of FSCP revealed the presence of 25.6% helices, 25.8% turns, and 48% random coils with no beta-sheet 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 optimum for the proteolytic activity of FSCP was pH 6.0; the temperature optimum was 30°C. FSCP exhibited strong anticoagulant effect in both platelet-rich plasma (PRP) and platelet-poor 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 fibrinogen degradation by thrombin and FSCP were different. Furthermore, FSCP inhibited aggregation of washed platelets triggered 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.

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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:

phenylmethylsulfonyl fluoride

PPP:

platelet-poor plasma

PRP:

platelet-rich plasma

PT:

prothrombin time

RP-HPLC:

reversed-phase high-performance liquid chromatography

SDS-PAGE:

sodium dodecyl sulphate polyacrylamide gel electrophoresis

TCA:

trichloroacetic acid

TFA:

trifluoroacetic acid

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Acknowledgements

D. S. and S. K. thank the Department of Science and Technology, Government of India, New Delhi and Vision Group on Science and Technology, Government of Karnataka, Bangalore for financial assistance.

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Authors and Affiliations

  1. Department of Studies and Research in Biochemistry and Centre for Bioscience and Innovation, Tumkur University, 572103, Tumkur, India

    S. K. M. Nandish, J. Kengaiah, Ch. Ramachandraiah,  Chandramma, A. Shivaiah & D. Sannaningaiah

  2. Department of Medicinal Biochemistry and Microbiology (IMBM), Uppsala Biomedical Centre, 75237, Uppsala, Sweden

    S. M. Santhosh

  3. Department of Biochemistry and Molecular Biology, School of Life Sciences, Pondicherry University, 605014, Pondicherry, Tamil Nadu, India

    Thirunavukkarasu

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  1. S. K. M. Nandish
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  2. J. Kengaiah
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Correspondence to D. Sannaningaiah.

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Nandish, S.K.M., Kengaiah, J., Ramachandraiah, C. et al. Flaxseed Cysteine Protease Exhibits Strong Anticoagulant, Antiplatelet, and Clot-Dissolving Properties. Biochemistry Moscow 85, 1113–1126 (2020). https://doi.org/10.1134/S0006297920090102

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