Kenaf Seed Cysteine Protease (KSCP) Inhibits the Intrinsic Pathway of the Blood Coagulation Cascade and Platelet Aggregation


如何引用文章

全文:

详细

Background:Thrombosis is the key event that obstructs the flow of blood throughout the circulatory system, leading to stroke, myocardial infarction and severe cardiovascular complications. Currently, available antithrombotic drugs trigger several life-threatening side effects.

Introduction:Antithrombotic agents from natural sources devoid of adverse effects are grabbing high attention. In our previous study, we reported the antioxidant, anticoagulant and antiplatelet properties of kenaf seed protein extract. Therefore, in the current study, purification and characterization of cysteine protease from kenaf seed protein extract responsible for potential antithrombotic activity was undertaken.

Methods:Purification of KSCP (Kenaf Seed Cysteine Protease) was carried out using gel permeation and ion exchange column chromatography. The purity of the enzyme was evaluated by SDS PAGE (Sodium Dodecyl-Sulfate Polyacrylamide Gel Electrophoresis). RP-HPLC (Reverse Phase High-Performance Liquid Chromatography), MALDI-TOF (Matrix-Assisted Laser Desorption Ionization Time-Of-Flight) and CD (Circular Dichroism techniques) were employed for its characterization. Proteolytic, fibrinolytic and kinetic study was done using spectroscopy. Plasma recalcification time, Prothrombin Time (PT), Thrombin clotting time (TCT), Activated Partial Thromboplastin Time (APTT), bleeding time and platelet aggregation studies were carried out for antithrombotic activity of KSCP.

Result:A single sharp band of KSCP was observed under both reduced and non-reduced conditions, having a molecular mass of 24.1667kDa. KSCP was found to contain 30.3 % helix turns and 69.7 % random coils without a beta-pleated sheet. KSCP digested casein and fibrin, and its activity was inhibited by iodoacetic acid (IAA). KSCP was optimally active at pH 6.0 at the temperature of 40°C. KSCP exhibited anticoagulant properties by interfering in the intrinsic pathway of the blood coagulation cascade. Furthermore, KSCP dissolved both whole blood and plasma clots and platelet aggregation.

Conclusion:KSCP purified from kenaf seed extract showed antithrombotic potential. Hence, it could be a better candidate for the management of thrombotic complications.

作者简介

Sujatha Hanumegowda

Department of Biochemistry Jnansahydri, Kuvempu University

Email: info@benthamscience.net

Chandramma Srinivasa

Department of Studies and Research in Biochemistry, Tumkur University

Email: info@benthamscience.net

Ashwini Shivaiah

Department of Studies and Research in Biochemistry, Tumkur University

Email: info@benthamscience.net

Manjula Venkatappa

Department of Biochemistry Jnansahydri, Kuvempu University

Email: info@benthamscience.net

Rohith Shankar

Department of Seribiotechnology, Yuvaraja’s College, University of Mysore

Email: info@benthamscience.net

Ramesh Lakshmaiah

Department of Food Science, Maharani College for Women, University of Mysore

Email: info@benthamscience.net

Sathisha Gonchigar

Department of Biochemistry Jnansahydri, Kuvempu University

Email: info@benthamscience.net

Devaraja Sannaningaiah

Department of Studies and Research in Biochemistry, Tumkur University

编辑信件的主要联系方式.
Email: info@benthamscience.net

参考

  1. Segarra, M.; Aburto, M.R.; Acker-Palmer, A. Blood–brain barrier dynamics to maintain brain homeostasis. Trends Neurosci., 2021, 44(5), 393-405. doi: 10.1016/j.tins.2020.12.002 PMID: 33423792
  2. Fathima, S. N. Acute Myocardial Infarction; StatPearls Publishing: Treasure Island (FL), 2021.
  3. Periayah, M.H.; Halim, A.S.; Mat Saad, A.Z. Mechanism action of platelets and crucial blood coagulation pathways in hemostasis. Int. J. Hematol. Oncol. Stem Cell Res., 2017, 11(4), 319-327. PMID: 29340130
  4. Induruwa, I.; Moroi, M.; Bonna, A.; Malcor, J.D.; Howes, J.M.; Warburton, E.A.; Farndale, R.W.; Jung, S.M. Platelet collagen receptor Glycoprotein VI-dimer recognizes fibrinogen and fibrin through their D-domains, contributing to platelet adhesion and activation during thrombus formation. J. Thromb. Haemost., 2018, 16(2), 389-404. doi: 10.1111/jth.13919 PMID: 29210180
  5. Neubauer, K.; Zieger, B. Endothelial cells and coagulation. Cell Tissue Res., 2022, 387(3), 391-398. doi: 10.1007/s00441-021-03471-2 PMID: 34014399
  6. Ashwini, S.A. Evidences for the presence of proteolytic enzyme in finger millet. AJST, 2018, 2018, 9122-9130.
  7. Maas, C.; Renné, T. Coagulation factor XII in thrombosis and inflammation. Blood, 2018, 131(17), 1903-1909. doi: 10.1182/blood-2017-04-569111 PMID: 29483100
  8. Naudin, C.; Burillo, E.; Blankenberg, S.; Butler, L.; Renné, T. Factor XII contact activation. Semin. Thromb. Hemost., 2017, 43(8), 814-826. doi: 10.1055/s-0036-1598003 PMID: 28346966
  9. Kamikubo, Y.; Mendolicchio, G.L.; Zampolli, A.; Marchese, P.; Rothmeier, A.S.; Orje, J.N.; Gale, A.J.; Krishnaswamy, S.; Gruber, A.; Østergaard, H.; Petersen, L.C.; Ruf, W.; Ruggeri, Z.M. Selective factor VIII activation by the tissue factor–factor VIIa–factor Xa complex. Blood, 2017, 130(14), 1661-1670. doi: 10.1182/blood-2017-02-767079 PMID: 28729433
  10. Hillegass, E. Essentials of Cardiopulmonary Physical Therapy, 5th ed; Elsevier, Inc: Philadelphia, 2021.
  11. Malik, A.; Rehman, F.U.; Shah, K.U.; Naz, S.S.; Qaisar, S. Hemostatic strategies for uncontrolled bleeding: A comprehensive update. J. Biomed. Mater. Res. B Appl. Biomater., 2021, 109(10), 1465-1477. doi: 10.1002/jbm.b.34806 PMID: 33511753
  12. Kohli, S.; Shahzad, K.; Jouppila, A.; Holthöfer, H.; Isermann, B.; Lassila, R. Thrombosis and inflammation—a dynamic interplay and the role of glycosaminoglycans and activated protein C. Front. Cardiovasc. Med., 2022, 9, 866751. doi: 10.3389/fcvm.2022.866751 PMID: 35433860
  13. Manjappa, B.; Gangaraju, S.; Girish, K.S.; Kemparaju, K.; Gonchigar, S.J.; Shankar, R.L.; Shinde, M.; Sannaningaiah, D. Momordica charantia seed extract exhibits strong anticoagulant effect by specifically interfering in intrinsic pathway of blood coagulation and dissolves fibrin clot. Blood Coagul. Fibrinolysis, 2015, 26(2), 191-199. doi: 10.1097/MBC.0000000000000191 PMID: 25192240
  14. Mehta, L.; Sharma, G.; Douglas, P.; Rzeszut, A. Discrimination and harrassment in cardiology: Insights from the 2019 American College of cardiology global professional life survey. J. Am. Coll. Cardiol., 2020, 75(11), 3631. doi: 10.1016/S0735-1097(20)34258-3
  15. Tafur, A.; Douketis, J. Perioperative management of anticoagulant and antiplatelet therapy. Heart, 2018, 104(17), 1461-1467. doi: 10.1136/heartjnl-2016-310581 PMID: 29217632
  16. Hassanpour, S.; Kim, H.J.; Saadati, A.; Tebon, P.; Xue, C.; van den Dolder, F.W.; Thakor, J.; Baradaran, B.; Mosafer, J.; Baghbanzadeh, A.; de Barros, N.R.; Hashemzaei, M.; Lee, K.J.; Lee, J.; Zhang, S.; Sun, W.; Cho, H.J.; Ahadian, S.; Ashammakhi, N.; Dokmeci, M.R.; Mokhtarzadeh, A.; Khademhosseini, A. Thrombolytic agents: Nanocarriers in controlled release. Small, 2020, 16(40), 2001647. doi: 10.1002/smll.202001647 PMID: 32790000
  17. MacDougall, C.; Canonica, T.; Keh, C.; P Phan, B.A.; Louie, J. Systematic review of drug-drug interactions between rifamycins and anticoagulant and antiplatelet agents and considerations for management. Pharmacotherapy, 2022, 42(4), 343-361. doi: 10.1002/phar.2672 PMID: 35152432
  18. Łubek-Nguyen, A.; Ziemichód, W.; Olech, M. Application of enzyme-assisted extraction for the recovery of natural bioactive compounds for nutraceutical and pharmaceutical applications. Appl. Sci., 2022, 12(7), 3232. doi: 10.3390/app12073232
  19. Norhisham, D.A.; Saad, N.M.; Ahmad Usuldin, S.R.; Vayabari, D.A.G.; Ilham, Z.; Ibrahim, M.F.; Wan-Mohtar, W.A.A.Q.I. Bioactivities of kenaf biomass extracts: A review. Processes, 2023, 11(4), 1178. doi: 10.3390/pr11041178
  20. Ayadi, R.; Hanana, M.; Mzid, R.; Hamrouni, L.; Khouja, M.; Salhi Hanachi, A. Hibiscus cannabinus L. – Kenaf : A review paper. J. Nat. Fibers, 2016, 14(4), 466-484. doi: 10.1080/15440478.2016.1240639
  21. Gonchigar, S.J.; Sannaningaiah, D.; Hanumegowda, S.M.; Srinivasa, C.; Shivaiah, A.; Venkatappa, M.M.; Hanumanthappa, R.; Rangappa, R.; Laxmaiah, R.K. Protein extract of kenaf seed exhibits anticoagulant, antiplatelet and antioxidant activities. Asian Pac. J. Trop. Biomed., 2022, 12(2), 47. doi: 10.4103/2221-1691.335693
  22. Bradford, M.M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem., 1976, 72(1-2), 248-254. doi: 10.1016/0003-2697(76)90527-3 PMID: 942051
  23. Laemmli, U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 1970, 227(5259), 680-685. doi: 10.1038/227680a0 PMID: 5432063
  24. Leach, B.S.; Collawn, J.F., Jr; Fish, W.W. Behavior of glycopolypeptides with empirical molecular weight estimation methods. 1. In sodium dodecyl sulfate. Biochemistry, 1980, 19(25), 5734-5741. doi: 10.1021/bi00566a011 PMID: 7459341
  25. Saran, S.; Isar, J.; Saxena, R.K. A modified method for the detection of microbial proteases on agar plates using tannic acid. J. Biochem. Biophys. Methods, 2007, 70(4), 697-699. doi: 10.1016/j.jbbm.2007.03.005 PMID: 17434593
  26. Satake, M.; Murata, Y.; Suzuki, T. Studies on snake venom. XIII. Chromatographic separation and properties of three proteinases from Agkistrodon halys blomhoffii venom. J. Biochem., 1963, 53(6), 438-447. doi: 10.1093/oxfordjournals.jbchem.a127720 PMID: 13986686
  27. Quick, A.J.; Stanley-Brown, M.; Bancroft, F.W. A study of the coagulation defect in hemophilia and in jaundice. Am. J. Med. Sci., 1935, 190(4), 501-510. doi: 10.1097/00000441-193510000-00009
  28. Denis, C.; Methia, N.; Frenette, P.S.; Rayburn, H.; Ullman-Culleré, M.; Hynes, R.O.; Wagner, D.D. A mouse model of severe von Willebrand disease: Defects in hemostasis and thrombosis. Proc. Natl. Acad. Sci., 1998, 95(16), 9524-9529. doi: 10.1073/pnas.95.16.9524 PMID: 9689113
  29. Neofotistos, D.; Oropeza, M.; Ts’ao, C.H. Stability of plasma for add-on PT and APTT tests. Am. J. Clin. Pathol., 1998, 109(6), 758-763. doi: 10.1093/ajcp/109.6.758 PMID: 9620036
  30. Evans, H.J. Cleavage of the Aα-chain of fibrinogen and the α-polymer of fibrin by the venom of spitting cobra (Naja nigricollis). Biochimica et Biophysica Acta (BBA) - Enzymology, 1981, 660(2), 219-226. doi: 10.1016/0005-2744(81)90163-7 PMID: 6793073
  31. Prasad, S.; Kashyap, R.S.; Deopujari, J.Y.; Purohit, H.J.; Taori, G.M.; Daginawala, H.F. Development of an in vitro model to study clot lysis activity of thrombolytic drugs. Thromb. J., 2006, 4(1), 14. doi: 10.1186/1477-9560-4-14 PMID: 16968529
  32. Matsubara, K.; Hori, K.; Matsuura, Y.; Miyazawa, K. Purification and characterization of a fibrinolytic enzyme and identification of fibrinogen clotting enzyme in a marine green alga, Codium divaricatum. Comp. Biochem. Physiol. B Biochem. Mol. Biol., 2000, 125(1), 137-143. doi: 10.1016/S0305-0491(99)00161-3 PMID: 10840649
  33. Rajesh, R.; Raghavendra Gowda, C.D.; Nataraju, A.; Dhananjaya, B.L.; Kemparaju, K.; Vishwanath, B.S. Procoagulant activity of Calotropis gigantea latex associated with fibrin(ogen)olytic activity. Toxicon, 2005, 46(1), 84-92. doi: 10.1016/j.toxicon.2005.03.012 PMID: 15922393
  34. Ardlie, N.G.; Han, P. Enzymatic basis for platelet aggregation and release: the significance of the ‘platelet atmosphere’ and the relationship between platelet function and blood coagulation. Br. J. Haematol., 1974, 26(3), 331-356. doi: 10.1111/j.1365-2141.1974.tb00477.x PMID: 4851871
  35. Born, G.V.R.; Cross, M.J. Effects of inorganic ions and of plasma proteins on the aggregation of blood platelets by adenosine diphosphate. J. Physiol., 1964, 170(2), 397-414. doi: 10.1113/jphysiol.1964.sp007340 PMID: 14165175
  36. Stoykova, S.; Goranova, Y.; Pantcheva, I.; Atanasov, V.; Danchev, D.; Petrova, S. Hemolytic activity and platelet aggregation inhibitory effect of vipoxin’s basic sPLA2 subunit. Interdiscip. Toxicol., 2013, 6(3), 136-140. doi: 10.2478/intox-2013-0021 PMID: 24678250
  37. Kondo, H.; Kondo, S.; Ikezawa, H.; Murata, R.; Ohsaka, A. Studies on the quantitative method for determination of hemorrhagic activity of Habu snake venom. Jpn. J. Med. Sci. Biol., 1960, 13(1-2), 43-51. doi: 10.7883/yoken1952.13.43 PMID: 13853435
  38. Sannanaik Vishwanath, B.; Manjunatha Kini, R.; Veerabasappa Gowda, T. Characterization of three edema-inducing phospholipase A2 enzymes from habu (Trimeresurus flavoviridis) venom and their interaction with the alkaloid aristolochic acid. Toxicon, 1987, 25(5), 501-515. doi: 10.1016/0041-0101(87)90286-8 PMID: 3617087
  39. Marathe, K.R.; Patil, R.H.; Vishwakarma, K.S.; Chaudhari, A.B.; Maheshwari, V.L. Protease inhibitors and their applications: An overview. In: Studies in Natural Products Chemistry; Elsevier, 2019; 62, pp. 211-242.
  40. Silva-López, R.E.; Gonçalves, R.N. Therapeutic proteases from plants: biopharmaceuticals with multiple applications. J. Appl. Biol. Biotechnol. Bioeng., 2019, 6(2), 101-109. doi: 10.15406/jabb.2019.06.00180
  41. Mnif, I.H.; Siala, R.; Nasri, R.; Mhamdi, S.; Nasri, M.; Kamoun, A.S. A cysteine protease isolated from the latex of Ficus microcarpa: purification and biochemical characterization. Appl. Biochem. Biotechnol., 2015, 175(3), 1732-1744. doi: 10.1007/s12010-014-1376-2 PMID: 25424283
  42. Joshi, B.N.; Sainani, M.N.; Bastawade, K.B.; Gupta, V.S.; Ranjekar, P.K. Cysteine protease inhibitor from pearl millet: A new class of antifungal protein. Biochem. Biophys. Res. Commun., 1998, 246(2), 382-387. doi: 10.1006/bbrc.1998.8625 PMID: 9610368
  43. Nandish, S.K.M.; Kengaiah, J.; Ramachandraiah, C.; Chandramma; Shivaiah, A.; Santhosh, S.M.; Thirunavukkarasu; Sannaningaiah, D. Flaxseed cysteine protease exhibits strong anticoagulant, antiplatelet, and clot-dissolving properties. Biochemistry, 2020, 85(9), 1113-1126. doi: 10.1134/S0006297920090102 PMID: 33050855
  44. Sundd, M.; Kundu, S.; Pal, G.P.; Medicherla, J.V. Purification and characterization of a highly stable cysteine protease from the latex of Ervatamia coronaria. Biosci. Biotechnol. Biochem., 1998, 62(10), 1947-1955. doi: 10.1271/bbb.62.1947 PMID: 9836431
  45. Liggieri, C.; Arribére, M.C.; Trejo, S.A.; Canals, F.; Avilés, F.X.; Priolo, N.S.; Avilés, F.X.; Priolo, N.S. Purification and biochemical characterization of asclepain c I from the latex of Asclepias curassavica L. Protein J., 2004, 23(6), 403-411. doi: 10.1023/B:JOPC.0000039554.18157.69 PMID: 15517987
  46. Barco, S.; Mahmoudpour, S.H.; Valerio, L.; Klok, F.A.; Münzel, T.; Middeldorp, S.; Ageno, W.; Cohen, A.T.; Hunt, B.J.; Konstantinides, S.V. Trends in mortality related to pulmonary embolism in the European Region, 2000–15: Analysis of vital registration data from the WHO Mortality Database. Lancet Respir. Med., 2020, 8(3), 277-287. doi: 10.1016/S2213-2600(19)30354-6 PMID: 31615719
  47. Nimjee, S.M.; Povsic, T.J.; Sullenger, B.A.; Becker, R.C. Translation and clinical development of antithrombotic aptamers. Nucleic Acid Ther., 2016, 26(3), 147-155. doi: 10.1089/nat.2015.0581 PMID: 26882082
  48. Salekeen, R.; Haider, A.N.; Akhter, F.; Billah, M.M.; Islam, M.E.; Islam, D.K.M. Lipid oxidation in pathophysiology of atherosclerosis: Current understanding and therapeutic strategies. IJCCRP, 2022, 14, 200143. doi: 10.1016/j.ijcrp.2022.200143 PMID: 36060286
  49. Castellucci, L.A.; Cameron, C.; Le Gal, G.; Rodger, M.A.; Coyle, D.; Wells, P.S.; Clifford, T.; Gandara, E.; Wells, G.; Carrier, M. Efficacy and safety outcomes of oral anticoagulants and antiplatelet drugs in the secondary prevention of venous thromboembolism: Systematic review and network meta-analysis. BMJ, 2013, 347(1), f5133-f5133. doi: 10.1136/bmj.f5133 PMID: 23996149
  50. Angiolillo, D.J.; Guzman, L.A.; Bass, T.A. Current antiplatelet therapies: Benefits and limitations. Am. Heart J., 2008, 156(S2), 3S-9S. doi: 10.1016/j.ahj.2008.06.003 PMID: 18657680
  51. Santangeli, R.; Montozzi, G.; Gamba, L.; Salvucci, S.; Manfredi, L.; Moroncini, G. Ab1514 unusual association between thrombotic thrombocytopenic purpura and primary antiphospholipid syndrome. Ann. Rheum. Dis., 2022, 81(S1), 1859.2-1860. doi: 10.1136/annrheumdis-2022-eular.1830
  52. Shivaprasad, H.V.; Riyaz, M.; Venkatesh Kumar, R.; Dharmappa, K.K.; Tarannum, S.; Siddesha, J.M.; Rajesh, R.; Vishwanath, B.S. Cysteine proteases from the Asclepiadaceae plants latex exhibited thrombin and plasmin like activities. J. Thromb. Thrombolysis, 2009, 28(3), 304-308. doi: 10.1007/s11239-008-0290-2 PMID: 18979066
  53. Sannaningaiah, D.; Shivaiah, A.; Kengaiah, J.; Srinivasa, C.; Nandish, S.K.M.; Ramachandraiah, C.; Hanumegowda, S.; Manjappa, B.; Martin, S.S.; Laxmaiah, R.K.; Shinde, M. Sorghum protein extract protects RBC from sodium nitrite-induced oxidative stress and exhibits anticoagulant and antiplatelet activity. Folia Med, 2021, 63(6), 884-894.
  54. Nandish, S.K.M.; Kengaiah, J.; Ramachandraiah, C.; Chandramma; Shivaiah, A.; Thirunavukkarasu; Shankar, R.L.; Sannaningaiah, D. Purification and characterization of non-enzymatic glycoprotein (NEGp) from flax seed buffer extract that exhibits anticoagulant and antiplatelet activity. Int. J. Biol. Macromol., 2020, 163, 317-326. doi: 10.1016/j.ijbiomac.2020.06.270 PMID: 32629053
  55. Altaf, F.; Wu, S.; Kasim, V. Role of fibrinolytic enzymes in anti-thrombosis therapy. Front. Mol. Biosci., 2021, 8, 680397. doi: 10.3389/fmolb.2021.680397 PMID: 34124160
  56. Barzkar, N.; Jahromi, S.T.; Vianello, F. Marine microbial fibrinolytic enzymes: An overview of source, production, biochemical properties and thrombolytic activity. Mar. Drugs, 2022, 20(1), 46. doi: 10.3390/md20010046 PMID: 35049901
  57. Shivaprasad, H.V.; Rajaiah, R.; Frey, B.M.; Frey, F.J.; Vishwanath, B.S. ‘Pergularain e I’ – a plant cysteine protease with thrombin-like activity from Pergularia extensa latex. Thromb. Res., 2010, 125(3), e100-e105. doi: 10.1016/j.thromres.2009.10.002 PMID: 19853890
  58. Ramachandraiah, C.; Nandish, S.K.M.; Kengaiah, J.; Srinivas, C.; Shivaiah, A.; Martin, S.S.; Shinde, M.; Sannaningaiah, D. Macrotyloma uniflorum seed aqueous extract exhibits anticoagulant, antiplatelet and clot dissolving properties. Asian J. Pharm. Pharmacol., 2019, 5(3), 589-603. doi: 10.31024/ajpp.2019.5.3.23
  59. Lordan, R.; Tsoupras, A.; Zabetakis, I. Platelet activation and prothrombotic mediators at the nexus of inflammation and atherosclerosis: Potential role of antiplatelet agents. Blood Rev., 2021, 45, 100694. doi: 10.1016/j.blre.2020.100694 PMID: 32340775

补充文件

附件文件
动作
1. JATS XML
下载

版权所有 © Bentham Science Publishers, 2024