Research Article

Evaluation of Antiplatelet Activity of Phenolic Compounds by Flow Cytometry

Konstantinos D. Kyriakidis
Colchester Hospital University Foundation Trust, UK
* Corresponding author
Eyrysthenis G. Vartholomatos
University Hospital of Ioannina, Greece
Georgios S. Markopoulos
University Hospital of Ioannina, Greece
DOI icon 10.24018/ejmed.2021.3.1.703

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Submitted 2021-02-02
Published 2021-02-18

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Article Main Content

Platelets play a pivotal role in coagulation, or clot formation, resulting in haemostasis, after endothelium injury. Disturbance of platelet activation may lead to pathologic thrombosis. Platelet activation and aggregation are common factors in atherothrombotic events, critical in the atherothrombotic process, and cardiovascular diseases. Several drugs are being used for antiplatelet therapy to prevent and/or treat atherosclerosis and cardiovascular diseases. Synthetic antiplatelet drugs hold possible undesired health consequences (cardiovascular diseases, carcinogenicity, etc.), advocating their replacement with natural, effective, and non-toxic compounds. Many phenolic compounds are created as secondary metabolites of plants, are found in many fruits and vegetables, and constitute a wide family of high-added-value molecules. Their biological activities include antioxidant, anti-platelet, and anti-inflammatory action. Based on the above, we examined five phenolic compounds (ellagic acid, ferulic acid, gallic acid, quercetin, and kaempferol) for their effect on platelet reactivity in whole blood samples using flow cytometry.

Quantification of activated platelet marker CD62-P by flow cytometry showed that all five compounds inhibited platelet activation in vitro, induced by adenosine diphosphate (ADP) and collagen. Interestingly, based on the IC50 values obtained for expression of CD62-P, among ellagic, ferulic, and gallic acid, gallic acid showed significantly higher inhibition than the other two. Kaempferol found to be a more potent inhibitor than quercetin, following previously reported results from aggregometry. Results obtained from our flow cytometry screening indicate antiplatelet activity from novel phenolic compounds and their potential use as drugs for thrombosis and cardiovascular diseases.

Keywords: phenolic acids, flavonols, platelet biomarkers, P-selectin, flow cytometry

References

  1. L.K. Jennings, “Mechanisms of platelet activation: Need for new strategies to protect against platelet-mediated atherothrombosis,” Thromb Haemostas, vol.103, pp. 248-257, 2009.
    DOI  |   Google Scholar
  2. B. Jr Nagy, I.B. Debreceni, J. Kappelmayer, “Flow cytometric investigation of classical and alternative platelet activation markers,” J of Intern Fed Clin. Chem & Lab. medic, vol. 23, pp.11124-1134, 2012.
     Google Scholar
  3. J.K. Cha, M.H. Jeong, J.Y. Jang, H.R. Bae, Y.J. Lim, J.S. Kim, S.H. Kim, and J.W Kim, “Serial measurement of surface expressions of CD63, P-selectin, and CD40 ligand on platelets in atherosclerotic ischemic stroke. A possible role of CD40 ligand on platelets in atherosclerotic ischemic stroke,” Cerebrovasc. Dis, vol.16, pp. 376-382, 2003.
    DOI  |   Google Scholar
  4. N.W. Tsai, W.N, Cheng, C.F. Shaw, C.R. Jan, H.W. Chang, C.R. Huang, S.A.D. Chen, Y.C. Chuang, T.H. Lee, and C.H. Lu “Levels and value of platelet activation markers in different subtypes of acute non-cardio-embolic ischemic stroke,” Thromb Res, vol. 124, pp. 213-218, 2009.
     Google Scholar
  5. C.L. Campbelli, S. Smyth, G. Montalescot, and S.R. Steinhubl, “Aspirin dose for the prevention of cardiovascular disease: a systematic review,” The J of the Am Med Assoc, vol. 298(18), pp. 2018-2024, 2007.
    DOI  |   Google Scholar
  6. V.J. Marder, M.H. Rosove, and M. Minning, “Foundation and sites of action of antithrombotic agents, Best Practice and Research,” Clin Haematol, vol. 17, pp. 3-22, 2004.
    DOI  |   Google Scholar
  7. A. Battaglia, E. Stragliotto, F. Heiman, “Indobufen in the prevention of cerebral ischemic attack (TIA): a prospective randomized, controlled study,” Thromb and Haemost, vol. 69, pp.1349-1393,1993.
     Google Scholar
  8. P. Libby, “Mechanisms of acute coronary syndromes and their implications for therapy,” The New Eng. J of Medicine, vol. 368 (21), pp. 2004-2013, 2013.
    DOI  |   Google Scholar
  9. B.E. Nignpense, K.A. Chinkwo, L. Christopher, C.L. Blanchard, and A.B. Santhakumarm, “Polyphenols: Modulators of Platelet Function and Platelet Microparticle Generation?” Int. J. Mol. Sci, vol. 21, pp. 146-153, 2020. https://doi.org/10.3390/ijms21010146
    DOI  |   Google Scholar
  10. A. Moschona A, K.D. Kyriakidis, A.D. Kleontas, and M. Liakopoulou-Kyriakides, “Comparative study of natural phenolic acids and flavonols as antiplatelet and anti-inflammatory agents,” The Grant Medical J, vol. 2, pp. 57-66, 2017.
     Google Scholar
  11. A. Moschona, K. Rouptsiou, S. Theodoridou, and M. Liakopoulou-Kyriakides, “Studies on polyphenols from sea buckthorn berries and pomegranate peels extracts. Recovery, bioactivities and encapsulation into polymers,” Int. J Pharm & Biol Sci, vol. 10, pp.158-171, 2020.
     Google Scholar
  12. D. Menssen, K. Melber, N. Brandt, and E. Thiel, “The Use of Hirudin as Universal Anticoagulant in Haematology. Clinical Chemistry and Blood Grouping,” Clin Chem & Lab Medicine, vol. 39(12), pp. 1267-1277, 2002.
    DOI  |   Google Scholar
  13. N.H. Wallén, M. Ladjevardi, J. Albert, and A. Bröijersén, “Influence of Different Anticoagulants on Platelet Aggregation in Whole Blood; A Comparison Between Citrate, Low Molecular Mass Heparin and Hirudin,” Thromb Res, vol. 87, pp.151-157, 1997.
    DOI  |   Google Scholar
  14. M.L. Kalb, L. Potura, G. Scharbert, and S.A. Kozek-Langenecker, “The effect of ex vivo anticoagulants on whole blood platelet aggregation,” Platelets, vol. 20, pp. 7-11, 2009. doi: 10.1080/09537100802364076 PMID:19172515.
    DOI  |   Google Scholar
  15. J. Kappelmayer, B. Jr Nagy, K. Miszti-Blasius, Z. Havessy, and H. Sertidi, “The emerging value of P-selectin as a disease marker,” Clin Chem Lab Med, vol. 42, pp. 475-486, 2004.
    DOI  |   Google Scholar
  16. W. Russel, G. Duthle, “Plant secondary metabolites and gut health: the case for phenolic acids,” The proceedings of the Nutrition Society, vol. 10(2), pp. 389-396, 2011.
    DOI  |   Google Scholar
  17. E. Barone, V. Calabrese, and C. Mancuso, “Ferulic acid its therapeutic potential as a hormetin for age-related diseases,” Biotechnol, vol. 10, pp. 97-108, 2009.
    DOI  |   Google Scholar
  18. H.X. Zhang, H. Lin, C. Qu, YP. Tang, N.G. Li, J. Kai, G. Shang, and B. Li, “Design, synthesis, and in vitro antiplatelet aggregation Activities of Ferulic acid derivatives,” J of Chem, hindawi publishing corporation, article id 376527, 2015.
    DOI  |   Google Scholar
  19. M. Modi, T. Goel, T. Das, S. Malik, S. Suri, A.K. Rawat, et al, “Ellagic acid & gallic acid from Lagerstroemia speciosa L inhibit HIV-1 infection through inhibition of HIV-1 protease & reverse transcriptase activity,” Indian J Med Res, vol. 137, pp. 540-548, 2013.
     Google Scholar
  20. D. Lee, S. Eom, Y. Kim, H. Kim, M. Yim, S. Lee, et al, “Antibacterial and synergic effects of gallic acid-grafted-chitosan with beta-lactams against methicillin-resistant Staphylococcus aureus (MRSA),” Can J Microbiol, vol. 60, pp. 629-638, 2014.
    DOI  |   Google Scholar
  21. C. Liao, K. Lai, A. Huang, J. Yang, J. Lin, S. Wu, et al, “Gallic acid inhibits migration and invasion in human osteosarcoma U-2 OS cells through suppressing the matrix metalloproteinase-2/-9, protein kinase B (PKB) and PKC signaling pathways,” Food Chem Toxicol, vol. 50, pp. 1734-1740, 2012.
    DOI  |   Google Scholar
  22. A. Borges, C. Ferreira, M. Saavedra, and M. Simoes, “Antibacterial activity and mode of action of ferulic and gallic acids against pathogenic bacteria,” Microb Drug Resist, vol. 19, pp. 256–265, 2013.
    DOI  |   Google Scholar
  23. A. Sahebkar, M. Reza Zirak, and A. Sahebkar, “Ellagic Acid: A Logical Lead for Drug Development?” Cur Pharmac Design, vol. 24(2), pp. 106-122, 2018.
    DOI  |   Google Scholar
  24. C. Guo, S. Liu, Y. Guo, Y. Yin, J. Lin, X. Chen, and MA. Sun, “Comparative function-structural analysis of antiplatet and antiradical activities of flavonoid phytochemicals,” J Anim Plant Sci, vol. 4, pp. 926-935, 2014.
     Google Scholar
  25. M. Bijak, R. Ziewiecki, J. Saluk, M. Ponczek, I. Pawlaczyk, H. Krotkiewski, P. Wachowicz, and P. Nowak, “Thrombin inhibitory activity of some polyphenolic compounds,” Med. Chem. Res, vol. 23, pp. 2324-2337, 2014.
    DOI  |   Google Scholar
  26. G.P. Hubbard, S. Wolffram, J.A. Lovegrove, and J.M. Gibbins, “Ingestion of quercetin inhibits platelet aggregation and essential components of the collagen-stimulated platelet activation pathway in humans,” J Thromb Haemost, vol. 2(12), pp. 2138-2145, 2004.
    DOI  |   Google Scholar
  27. K-H. Lee, E. Park, H-J. Lee, et al, “Effects of daily quercetin-rich supplementation on cardiometabolic risks in male smokers,” Nutr Res Pract, vol. 5(01), pp. 28–33, 2011.
    DOI  |   Google Scholar
  28. A.R. Stainer, P. Sasikumar, A.P. Bye, A.J. Unswort, L.M. Holbrook, M. Tindall, J.A. Lovegrove, and J.M. Gibbins, “The Metabolites of the Dietary Flavonoid Quercetin Possess Potent Antithrombotic Activity, and Interact with Aspirin to Enhance Antiplatelet Effects,” TH Open, vol. 3(3), pp. e244–e258, 2019.
    DOI  |   Google Scholar
  29. S.Cid-Ortega, and J.A. Monroy-Rivera, “Extraction of Kaempferol and Its Glycosides Using Supercritical Fluids from Plant Sources: A Review,” Food Technol Biotechnol, vol. 56(4), pp. 480-493, 2018.
    DOI  |   Google Scholar
  30. A.Y. Chen, and Y.C. Chen, “A review of the dietary flavonoid, kaempferol on human health and cancer chemoprevention,” Food Chem, vol. 138(4), pp. 2099-2107, 2013. https://doi.org/10.1016/j.foodchem.2012.11.13.
    DOI  |   Google Scholar
  31. Y. Cui, H. Morgenstern, S. Greenland, D.P. Tashkin, J.T. Mao, L. Cai, et al, “Dietary flavonoid intake and lung cancer -A population-based case-control study,” Cancer, vol. 112 (10), 2241-2248, 2008. https://doi.org/10.1002/cncr.2339831.
    DOI  |   Google Scholar
  32. J.M. Calderon-Montano, E. Burgos-Morón, C. Pérez-Guerrero, and M. López-Lázaro, “A review on the dietary flavonoid kaempferol,” Mini Rev Med Chem, vol. 11(4), pp. 298-344, 2011. https://doi.org/10.2174/138955711795305335.
    DOI  |   Google Scholar
  33. N.W. Tsai, W.N. Chang, C.F. Shaw, C.R. Jan, C.R. Chang, S.D. Chem, Y.C. Chuang, T.H. Lee, H.C. Wang, and C.H. Lu, “Levels and value of platelet activation markers in different sub types of acutenon-cardio-embopolic ischaemic stroke,” Thromb Res, vol. 124, pp. 213-218, 2009.
    DOI  |   Google Scholar
  34. D.J. McCabe, P. Harrison, I.J. Macklie, PS. Sidhu, G. Purdy, A.S. Lawrie, H. Watt, M.M. Brown, and S.J. Machin, “Platelet degranulation and monocyte-platelet complex formation are increased in the acute and convalescent phases after ischaemic stroke or transient ischaemic attack,” Br J Haematol, vol. 125, pp. 777-787, 2004.
    DOI  |   Google Scholar
  35. R.A. Preston, J.O. Coffey, B.J. Materson, M. Ledfordand, and A.B. Alonso, “Elevated platelet P-selectin expression and platelet activation in high risk patients with uncontrolled severe hypertension,” Atherosclerosis, vol. 192, pp. 148-154, 2007.
    DOI  |   Google Scholar
  36. M. Lukasik, G. Dworacki, J. Kufel-Grabowska, C. Watala, W. Kozubski, “Chronic hyper-reactivity of platelets resulting in enhanced monocyte recruitment in patients after ischaemic stroke,” Platelets, vol. 20, pp. 235-241, 2009.
     Google Scholar
  37. S. Fateh-Moghadam, P. Htum, B. Tomandi, D. Sander, K. Stellios, et al, “Hyper responsiveness of platelets in ischaemic stroke,” Thromb Haemost, vol. 97, pp. 974-978, 2007.
    DOI  |   Google Scholar
  38. E. Csongrady, B. Jr Nagy, T. Fulop, Z. Varga, Z. Karanyl, M.T. Magyar, L. Olah, et al, “Increased levels of platelet activation markers are positively associated with carotid wall thickness and other atherosclerotic risk factors in obese patients,” Thromb Haemost, vol. 106, pp. 683-692, 2011.
    DOI  |   Google Scholar
  39. F. Zeigel, S. Stephan, G. Hoheisel, D. Pfeiffer, C. Riehlmann, and M. Koksch, “P-selectin expression, platelet aggregation, and platelet-derived microparticle formation are increased in peripheral arterial disease,” Blood Coagul Fibrinol, vol. 11, pp.723-728, 2000.
    DOI  |   Google Scholar
  40. T. Gremmel, R. Koppensteiner, and S. Panze, “Comparison of Aggregometry with Flow Cytometry for the Assessment of Agonists-Induced Platelet Reactivity in Patients on Dual Antiplatelet Therapy,” PLoS ONE 10:e0129666, 2015.
    DOI  |   Google Scholar