Journal of Ginseng Research

The Korean Society of Ginseng (고려인삼학회)
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Anti-thrombotic effects of ginsenoside Rk3 by regulating cAMP and PI3K/MAPK pathway on human platelets

  • Hyuk-Woo Kwon (Department of Biomedical Laboratory Science, Far East University) ;
  • Jung-Hae Shin (Department of Veterinary Medicine, College of Veterinary Medicine, Kyungpook National University) ;
  • Man Hee Rhee (Department of Veterinary Medicine, College of Veterinary Medicine, Kyungpook National University) ;
  • Chang-Eun Park (Department of Biomedical Laboratory Science, Namseoul University) ;
  • Dong-Ha Lee (Department of Biomedical Laboratory Science, Namseoul University)
  • Received : 2023.01.23
  • Accepted : 2023.04.20
  • Published : 2023.11.01
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Abstract

Background and objective: The ability to inhibit aggregation has been demonstrated with synthetically derived ginsenoside compounds G-Rp (1, 3, and 4) and ginsenosides naturally found in Panax ginseng 20(S)-Rg3, Rg6, F4, and Ro. Among these compounds, Rk3 (G-Rk3) from Panax ginseng needs to be further explored in order to reveal the mechanisms of action during inhibition. Methodology: Our study focused to investigate the action of G-Rk3 on agonist-stimulated human platelet aggregation, inhibition of platelet signaling molecules such as fibrinogen binding with integrin αIIbβ3 using flow cytometry, intracellular calcium mobilization, dense granule secretion, and thromboxane B2 secretion. In addition, we checked the regulation of phosphorylation on PI3K/MAPK pathway, and thrombin-induced clot retraction was also observed in platelets rich plasma. Key Results: G-Rk3 significantly increased amounts of cyclic adenosine monophosphate (cAMP) and led to significant phosphorylation of cAMP-dependent kinase substrates vasodilator-stimulated phosphoprotein (VASP) and inositol 1,4,5-trisphosphate receptor (IP3R). In the presence of G-Rk3, dense tubular system Ca2+ was inhibited, and platelet activity was lowered by inactivating the integrin αIIb/β3 and reducing the binding of fibrinogen. Furthermore, the effect of G-Rk3 extended to the inhibition of MAPK and PI3K/Akt phosphorylation resulting in the reduced secretion of intracellular granules and reduced production of TXA2. Lastly, G-Rk3 inhibited platelet aggregation and thrombus formation via fibrin clot. Conclusions and implications: These results suggest that when dealing with cardiovascular diseases brought upon by faulty aggregation among platelets or through the formation of a thrombus, the G-Rk3 compound can play a role as an effective prophylactic or therapeutic agent.

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References

  1. Mozaffarian D, Benjamin EJ, Go AS, Arnett DK, Blaha MJ, Cushman M, Das SR, de Ferranti S, Despres JP, Fullerton HJ, et al. Heart disease and stroke statistics-2016 update a report from the American Heart Association. Circulation 2016;133:e38-48.  https://doi.org/10.1161/CIR.0000000000000366
  2. Andrews RK, Berndt MC. Platelet physiology and thrombosis. Thromb Res 2004;114:447-53.  https://doi.org/10.1016/j.thromres.2004.07.020
  3. Barrett NE, Holbrook L, Jones S, Kaiser WJ, Moraes LA, Rana R, Wright B, et al. Future innovations in anti-platelet therapies. Br J Pharmacol 2008;154:918-39.  https://doi.org/10.1038/bjp.2008.151
  4. Badimon L, Vilahur G, Padro T. Nutraceuticals and atherosclerosis: human trials. Cardiovasc Ther 2010;28:202-15.  https://doi.org/10.1111/j.1755-5922.2010.00189.x
  5. Estruch R, Ros E, Salas-Salvado J, et al. Primary prevention of cardiovascular disease with a Mediterranean diet. N Engl J Med 2013;368:1279-90.  https://doi.org/10.1056/NEJMoa1200303
  6. Irfan M, Kwon TH, Yun BS, Park NH, Rhee MH. Eisenia bicyclis (brown alga) modulates platelet function and inhibits thrombus formation via impaired P2Y12 receptor signaling pathway. Phytomedicine 2018;40:79-87.  https://doi.org/10.1016/j.phymed.2018.01.003
  7. Rastogi S, Pandey MM, Rawat A. Traditional herbs: a remedy for cardiovascular disorders. Phytomedicine 2016;23:1082-9.  https://doi.org/10.1016/j.phymed.2015.10.012
  8. Schwarz UR, Walter U, Eigenthaler M. Taming platelets with cyclic nucleotides. Biochem Pharmacol 2001;62:1153-61.  https://doi.org/10.1016/S0006-2952(01)00760-2
  9. Cavallini L, Coassin M, Borean A, Alexandre A. Prostacyclin and sodium nitroprusside inhibit the activity of the platelet inositol 1,4,5-trisphosphate receptor and promote its phosphorylation. J Biol Chem 1996;271:5545-51.  https://doi.org/10.1074/jbc.271.10.5545
  10. Laurent V, Loisel TP, Harbeck B, Wehman A, Grobe L, Jockusch BM, et al. Carlier M.F. Role of proteins of the Ena/VASP family in actin-based motility of Listeria monocytogenes. J Cell Biol 1999;144:1245-58.  https://doi.org/10.1083/jcb.144.6.1245
  11. Sudo T, Ito H, Kimura Y. Phosphorylation of the vasodilator-stimulated phosphoprotein (VASP) by the anti-platelet drug, cilostazol, in platelets. Platelets 2003;14:381-90.  https://doi.org/10.1080/09537100310001598819
  12. Irfan M, Jeong D, Kwon HW, Shin JH, Park SJ, Kwak D, et al. Rhee M.H.Ginsenoside-Rp3 inhibits platelet activation and thrombus formation by regulating MAPK and cyclic nucleotide signaling. Vascul Pharmacol 2018;109:45-55.  https://doi.org/10.1016/j.vph.2018.06.002
  13. Adam F, Kauskot A, Rosa JP, Bryckaert M. Mitogen activated protein kinases in hemostasis and thrombosis. J Thromb Haemost 2008;6:2007-16.  https://doi.org/10.1111/j.1538-7836.2008.03169.x
  14. Bugaud F, Nadal-Wollbold F, Levy-Toledano S, Rosa JP, Bryckaert M. Regulation of c-jun-NH2 terminal kinase and extracellular-signal regulated kinase in human platelets. Blood 1990;94:3800-5.  https://doi.org/10.1182/blood.V94.11.3800.423k25_3800_3805
  15. Kramer RM, Roberts EF, Strifler BA, Johnstone EM. Thrombin induces activation of p38 MAP kinase in human platelets. J Biol Chem 1995;270:27395-8.  https://doi.org/10.1074/jbc.270.46.27395
  16. Nadal-Wollbold F, Pawlowski M, Levy-Toledano S, Berrou E, Rosa JP, Bryckaert M. Platelet ERK2 activation by thrombin is dependent on calcium and conventional protein kinases C but not Raf-1 or B-Raf. FEBS Lett 2002;531:475-82.  https://doi.org/10.1016/S0014-5793(02)03587-1
  17. Michelson AD. Antiplatelet therapies for the treatment of cardiovascular disease. Nat Rev Drug Discov 2010;9:154-69.  https://doi.org/10.1038/nrd2957
  18. Flevaris P, Li Z, Zhang G, Zheng Y, Liu J, Du X. Two distinct roles of mitogenactivated protein kinases in platelets and a novel Rac1-MAPK-dependent integrin outside-in retractile signaling pathway. Blood 2009;113:893-901.  https://doi.org/10.1182/blood-2008-05-155978
  19. Kramer RM, Roberts EF, Um SL, Borsch-Haubold AG, Watson SP, Fisher MJ. Jakubowski J.A.p38 mitogenactivated protein kinase phosphorylates cytosolic phospholipase A2 (cPLA2) in thrombin-stimulated platelets. Evidence that proline-directed phosphorylation is not required for mobilization of arachidonic acid by cPLA2. J Biol Chem 1996;271:27723-9.  https://doi.org/10.1074/jbc.271.44.27723
  20. McNicol A, Shibou TS. Translocation and phosphorylation of cytosolic phospholipase A2 in activated platelets. Thromb Res 1998;92:19-26.  https://doi.org/10.1016/S0049-3848(98)00097-8
  21. Chuang WY, Kung PH, Kuo CY, Wu CC. Sulforaphane prevents human platelet aggregation through inhibiting the phosphatidylinositol 3-kinase/Akt pathway. Thromb Haemost 2013;109:1120-30.  https://doi.org/10.1160/TH12-09-0636
  22. Nam KY. The comparative understanding between red ginseng and white ginsengs, processed ginsengs (Panax ginseng CA Meyer). J Ginseng Res 2005;29:1-18.  https://doi.org/10.5142/JGR.2005.29.1.001
  23. Ha YW, Lim SS, Ha IJ, Na YC, Seo JJ, Shin H, Son SH, Kim YS. Preparative isolation of four ginsenosides from Korean red ginseng (steam-treated Panax ginseng CA Meyer), by high-speed counter-current chromatography coupled with evaporative light scattering detection. J Chromatogr A 2007;1151:37-44.  https://doi.org/10.1016/j.chroma.2007.01.038
  24. Duan Z, Deng J, Dong Y, Zhu C, Li W, Fan D. Anticancer effects of ginsenoside Rk3 on non-small cell lung cancer cells: in vitro and in vivo. Food Funct 2017;8:3723-36.  https://doi.org/10.1039/C7FO00385D
  25. Qu L, Zhu Y, Liu Y, Yang H, Zhu C, Ma P, Qu L, Deng J, Fan D. Protective effects of ginsenoside Rk3 against chronic alcohol-induced liver injury in mice through inhibition of inflammation, oxidative stress, and apoptosis. Food Chem Toxicol 2019;126:277-84.  https://doi.org/10.1016/j.fct.2019.02.032
  26. Baek SH, Shin BK, Kim NJ, Chang SY, Park JH. Protective effect of ginsenosides Rk3 and Rh4 on cisplatin-induced acute kidney injury in vitro and in vivo. J Ginseng Res 2017;41:233-9.  https://doi.org/10.1016/j.jgr.2016.03.008
  27. Lee DH. Inhibitory effects of isoscopoletin on thrombus formation via regulation of cyclic nucleotides in collagen-induced platelets. J Appl Biol Chem 2020;63:235-41.  https://doi.org/10.3839/jabc.2020.032
  28. Grynkiewicz G, Poenie M, Tsien RY. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem 1985;260:3440-50.  https://doi.org/10.1016/S0021-9258(19)83641-4
  29. Berridge MJ, Irvine RF. Inositol phosphates and cell signalling. Nature 1989;341:197-205.  https://doi.org/10.1038/341197a0
  30. Nishikawa M, Tanaka T, Hidaka H. Ca2+ -calmodulin-dependent phosphorylation and platelet secretion. Nature 1980;287:863-5.  https://doi.org/10.1038/287863a0
  31. Kuo JF, Andersson RG, Wise BC, Mackerlova L, Salomonsson I, Brackett NL, Katoh N, Shoji M, Wrenn RW. Calcium-dependent protein kinase: widespread occurrence in various tissues and phyla of the animal kingdom and comparison of effects of phospholipid, calmodulin, and trifluoperazine. Proc Natl Acad Sci 1980;77:7039-43.  https://doi.org/10.1073/pnas.77.12.7039
  32. Wentworth JK, Pula G, Poole AW. Vasodilator-stimulated phosphoprotein (VASP) is phosphorylated on Ser157 by protein kinase C-dependent and-independent mechanisms in thrombin-stimulated human platelets. Biochem J 2006;393:555-64.  https://doi.org/10.1042/BJ20050796
  33. Napenas J, Oost FC, DeGroot A, Loven B, Hong CH, Brennan MT, Lockhart PB, van. Diermen D.E.Review of postoperative bleeding risk in dental patients on antiplatelet therapy. Oral Surg Oral Med Oral Pathol Oral Radiol 2013;115:491-9.  https://doi.org/10.1016/j.oooo.2012.11.001
  34. Gao J, Tao J, Liang W, Zhao M, Du X, Cui S, Duan H, Kan B, Su X, Jiang Z. Identification and characterization of phosphodiesterases that specifically degrade 3'3'-cyclic GMP-AMP. Cell Res 2015;25:539-50.  https://doi.org/10.1038/cr.2015.40
  35. Haslam RJ, Dickinson NT, Jang EK. Cyclic nucleotides and phosphodiesterases in platelets. Thromb Haemost 1999;82:412-23.  https://doi.org/10.1055/s-0037-1615861
  36. Quinton TM, Dean WL. Cyclic AMP-dependent phosphorylation of the inositol-1,4,5-trisphosphate receptor inhibits Ca2+ release from platelet membranes. Biochemical and Biochem Biophys Res Commun 1992;184:893-9.  https://doi.org/10.1016/0006-291X(92)90675-B
  37. Menshikov MYU, Ivanova K, Schaefer M, Drummer C, Gerzer R. Influence of the cGMP analog 8-PCPT-cGMP on agonist-induced increases in cytosolic ionized Ca2+ and on aggregation of human platelets. Eur J Pharmacol 1993;245:281-4.  https://doi.org/10.1016/0922-4106(93)90108-L
  38. Cipollone F, Patrignani P, Greco A, Panara MR, Padovano R, Cuccurullo F, Patrono C, Rebuzzi AG, Liuzzo G, Quaranta G, et al. Differential suppression of thromboxane biosynthesis by indobufen and aspirin in patients with unstable angina. Circulation 1997;96:1109-16.  https://doi.org/10.1161/01.CIR.96.4.1109
  39. Calderwood DA. Integrin activation. J. Cell Sci 2004;117:657-66.  https://doi.org/10.1242/jcs.01014
  40. Shin JH, Kwon HW, Irfan M, Rhee MH, Lee DH. Ginsenoside Rk1 suppresses platelet mediated thrombus formation by downregulation of granule release and aIIbb3 activation. J Ginseng Res 2021;45:490-7. https://doi.org/10.1016/j.jgr.2020.11.001