Platelet aggregometry assay for evaluating the effects of platelet agonists and antiplatelet compounds on platelet function in vitro

Alexandros Tsoupras, Ioannis Zabetakis, Ronan Lordan, Alexandros Tsoupras, Ioannis Zabetakis, Ronan Lordan

Abstract

Platelet aggregometry assays are generally used for the analysis of platelet function but can also be adapted for further research and therapy focused applications. This method describes the procedures for the preparation of human platelet-rich plasma (PRP) and platelet-poor plasma (PPP) for the assessment of human platelet aggregation induced by agonists such as platelet-activating factor (PAF), thrombin, collagen, adenosine diphosphate (ADP), arachidonic acid, etc. •This method can be applied in vitro to evaluate the aggregatory effects of these agonists and to assess the antiaggregatory effects of several bioactive antiplatelet agents (compounds of natural or pharmacological origin) in human PRP.•This versatile method can be used in both basic and clinical research for the assessment of platelet aggregation (a major cardiovascular risk factor), platelet agonists, and inhibitors, in physiological or pathological conditions.•This method can be adapted to assess platelet activity in postprandial and intervention studies ex vivo.

Keywords: ADP, adenosine diphosphate; Agonist; Antiplatelet; Human platelet aggregometry assay; In vitro; Inhibitors; LTA, light transmission aggregometry; Light transmission aggregometry; PAF, platelet-activating factor; PPP, platelet-poor plasma; PRP, platelet-rich plasma; Platelet aggregation; Platelet-activating factor; Thrombin.

Figures

Graphical abstract
Graphical abstract
Fig. 1
Fig. 1
Application of LTA assay for evaluating and monitoring the effect of several agonists (i.e. PAF or thrombin) on platelet aggregation of PRP. (A): Detection of increased light transmission during platelet aggregation; baseline light transmission is acquired when platelets of PRP suspensions are in an aggregometer cuvette in the absence of an agonist. Increased light transmission is detected when platelets start to aggregate in the presence of an agonist in the aggregometer cuvette leading to the development of an aggregation curve. (B): Characteristic examples of an aggregation curve of a maximum irreversible platelet aggregation [B1: Trace 1 (blue)] and of an aggregation curve of a maximum-reversible platelet aggregation [B2: trace 2 (black)]. The latent aggregation curve (B2) of maximum-reversible platelet aggregation of hPRP induced by an agonist (PAF or thrombin) is determined as the 100% aggregation of platelets. (C): The addition of several concentrations of an agonist (PAF or thrombin) within a specific range can induce reversible platelet aggregation in a concentration dependent manner; i.e. the higher the concentration of the agonist (C5 > C4 > C3 > C2 > C1) the higher the platelet aggregation curve (E > D > C > B > A) that is obtained by the aggregometer detector of the LTA assay. (D): A linear relationship exists between the concentrations of an agonist (i.e. PAF or thrombin) within a specific range that induces platelet aggregation within the 20%–80% of the maximum-reversible platelet aggregation of hPRP. From this linear curve the concentration of the agonist needed to induce 50% of platelet aggregation can be calculated, which is known as the EC50 value of the specific agonist for platelet aggregation. The lower the EC50 value for an agonist the higher the potency of its platelet aggregation effect. All experiments must be performed in triplicate (n = 3), using a different donor’s blood sample for each replicate, to ensure reproducibility. Abbreviations: LTA: light transmission aggregometry; PAF: platelet-activating factor; hPRP: human platelet-rich plasma.

References

    1. Tsoupras A., Lordan R., Demuru M., Shiels K., Saha S.K., Nasopoulou C., Zabetakis I. Structural elucidation of Irish organic farmed salmon (Salmo salar) polar lipids with antithrombotic activities. Mar. Drugs. 2018;16(6):176.
    1. Tsoupras A., Lordan R., Zabetakis I. Inflammation, not cholesterol, is a cause of chronic disease. Nutrients. 2018;10(5):604.
    1. Tsoupras A.B., Iatrou C., Frangia C., Demopoulos C.A. The implication of platelet-activating factor in cancer growth and metastasis: potent beneficial role of PAF-inhibitors and antioxidants. Infect. Disord. Drug Targets. 2009;9(4):390–399.
    1. Lordan R., Tsoupras A., Zabetakis I. The potential role of dietary platelet-activating factor inhibitors in cancer prevention and treatment. Adv. Nutr. 2018 In Press.
    1. Lordan R., Tsoupras A., Zabetakis I. Phospholipids of animal and marine origin: structure, function, and anti-inflammatory properties. Molecules. 2017;22(11):1964.
    1. Lordan R., Zabetakis I. Invited review: the anti-inflammatory properties of dairy lipids. J. Dairy Sci. 2017;100(6):4197–4212.
    1. Turck D., Bresson J.-L., Burlingame B., Dean T., Fairweather-Tait S., Heinonen M., Hirsch-Ernst K.I., Mangelsdorf I., McArdle H.J., Naska A., Neuhäuser-Berthold M., Nowicka G., Pentieva K., Sanz Y., Sjödin A., Stern M., Tomé D., Van Loveren H., Vinceti M., Willatts P., Martin A., Strain J.J., Heng L., Valtueña Martínez S., Siani A. Guidance for the scientific requirements for health claims related to antioxidants, oxidative damage and cardiovascular health. EFSA J. 2018;16(1) e05136-n/a.
    1. A. European Food Safety Water-soluble tomato concentrate (WSTC I and II) and platelet aggregation. EFSA J. 2009;7(5) 1101-n/a.
    1. Born G.V.R. Aggregation of blood platelets by adenosine diphosphate and its reversal. Nature. 1962;194:927.
    1. O’Brien J.R. The adhesiveness of native platelets and its prevention. J. Clin. Pathol. 1961;14(2):140–149.
    1. Cattaneo M., Cerletti C., Harrison P., Hayward C., Kenny D., Nugent D., Nurden P., Rao A.K., Schmaier A., Watson S. Recommendations for the standardization of light transmission aggregometry: a consensus of the working party from the platelet physiology sub-committee of SSC/ISTH. J. Thromb. Haemost. 2013;11(6):1183–1189.
    1. Megalemou K., Sioriki E., Lordan R., Dermiki M., Nasopoulou C., Zabetakis I. Evaluation of sensory and in vitro anti-thrombotic properties of traditional Greek yogurts derived from different types of milk. Heliyon. 2017;3(1) Article e00227.
    1. Tsoupras A.B., Papakyriakou A., Demopoulos C.A., Philippopoulos A.I. Synthesis, biochemical evaluation and molecular modeling studies of novel rhodium complexes with nanomolar activity against platelet activating factor. J. Inorg. Biochem. 2013;120:63–73.
    1. Verouti S.N., Tsoupras A.B., Alevizopoulou F., Demopoulos C.A., Iatrou C. Paricalcitol effects on activities and metabolism of platelet activating factor and on inflammatory cytokines in hemodialysis patients. Int. J. Artif. Organs. 2013;36(2):87–96.
    1. Tsoupras A.B., Chini M., Tsogas N., Fragopoulou E., Nomikos T., Lioni A., Mangafas N., Demopoulos C.A., Antonopoulou S., Lazanas M.C. Anti-platelet-activating factor effects of highly active antiretroviral therapy (HAART): a new insight in the drug therapy of HIV infection? AIDS Res. Hum. Retroviruses. 2008;24(8):1079–1086.
    1. Tsoupras A.B., Roulia M., Ferentinos E., Stamatopoulos I., Demopoulos C.A., Kyritsis P. Structurally diverse metal coordination compounds, bearing imidodiphosphinate and diphosphinoamine ligands, as potential inhibitors of the platelet activating factor. Bioinorg. Chem. Appl. 2010;(2010)
    1. Tsoupras A.B., Chini M., Tsogas N., Lioni A., Tsekes G., Demopoulos C.A., Lazanas M.C. In vitro anti-inflammatory and anti-coagulant effects of antibiotics towards platelet activating factor and thrombin. J. Inflammation. 2011;8 17-17.
    1. Tsoupras A., Demopoulos C.A., Pappas K.M. Platelet‐activating factor detection, metabolism, and inhibitors in the ethanologenic bacterium Zymomonas mobilis. Eur. J. Lipid Sci. Technol. 2012;114(2):123–133.
    1. Sioriki E., Smith T.K., Demopoulos C.A., Zabetakis I. Structure and cardioprotective activities of polar lipids of olive pomace, olive pomace-enriched fish feed and olive pomace fed gilthead sea bream (Sparus aurata) Food Res. Int. 2016;83:143–151.
    1. Nasopoulou C., Tsoupras A.B., Karantonis H.C., Demopoulos C.A., Zabetakis I. Fish polar lipids retard atherosclerosis in rabbits by down-regulating PAF biosynthesis and up-regulating PAF catabolism. Lipids Health Dis. 2011;10(213):1–18.
    1. Poutzalis S., Lordan R., Nasopoulou C., Zabetakis I. Phospholipids of goat and sheep origin: structural and functional studies. Small Rumin. Res. 2018;167:39–47.
    1. Sioriki E., Lordan R., Nahra F., Van Hecke K., Zabetakis I., Nolan S.P. In vitro anti-atherogenic properties of N-heterocyclic carbene aurate(I) compounds. ChemMedChem. 2018;13:2484.
    1. Tsoupras A.B., Antonopoulou S., Baltas G., Samiotaki M., Panayotou G., Kotsifaki H., Mantzavinos Z., Demopoulos C.A. Isolation and identification of hydroxyl–platelet-activating factor from natural sources. Life Sci. 2006;79(19):1796–1803.
    1. Antonopoulou S., Tsoupras A., Baltas G., Kotsifaki H., Mantzavinos Z., Demopoulos C.A. Hydroxyl-platelet-activating factor exists in blood of healthy volunteers and periodontal patients. Mediators Inflamm. 2003;12(4):221–227.

Source: PubMed

Sponsorer och medarbetare

Medicinska tillstånd

Läkemedelsinterventioner

3
Prenumerera