Methodological approaches for assessment of platelet functional activity under the action of antiplatelet drugs: 616.15:612.115

Abstract

Summary. Background. Adequate control of platelet functional activity, including exposure to antiplatelet drugs, is an unresolved problem in laboratory diagnostics. Optical aggregation and flow cytometry are the most commonly used methods for determining of platelet functional activity. Objective: to assess changes in platelet functional activity under the influence of antiplatelet drugs using optical aggregation and flow cytometry methods. Patients/Methods. The prospective study involved 22 healthy women of fertile age (26.0 ± 4.7 years). Blood was taken from the cubital vein into a vacutainer with an anticoagulant — sodium citrate in ratio blood: anticoagulant = 9:1. Acetylsalicylic acid (ASA) and eptifibatide were used as antiplatelet agents. The degree and rate of platelet aggregation (РA) were accessed by optical aggregation. Flow cytometry was used to assess the expression of P-selectin (CD62P), glycoprotein GPIIb/IIIa (CD41a) and the formation of platelet-leukocyte complexes. Results. Determination of the functional activity of ADP-induced platelets under ASA action revealed a decrease in the rate and degree of РA activity and a decrease in the level of CD41a and CD62P expression. Platelet processing with eptifibatide caused an increase of CD62P and CD41a expression and a pronounced suppression of РA rate and degree. ASA addition had no significant effect on ADP-induced formation of platelet- leukocyte complexes. On the contrary, more intense РA with leukocytes was observed in the presence of eptifibatide. Conclusions. Assessment of platelet functional activity should take into account all stages of platelet activation and their participation in inter- cellular interactions. Expansion of approaches for assessing of antiplatelet therapy effectiveness by laboratory methods is of great importance for searching safe and effective methods of treating patients with thrombophilic conditions at various stages of their development.

For citation: Chepanov S.V., Pavlov O.V., Oparina T.I., Kudryavtseva T.A., Orlova E.S., Milyutina Yu.P., Kakhiani M.I., Selkov S.A. Methodological approaches for assessment of platelet functional activity under the action of antiplatelet drugs. Tromboz, gemostaz i reologiya. 2022;(4):75–84. (In Russ.).

References

1. Mazurov A.V. Physiology and pathology of platelets. Moscow: Litterra, 2011. 480 pp. (In Russ.).
2. Dolgov V.V., Svirin P.V. Laboratory diagnosis of hemostasis disorders. Moscow-Tver’: OOO “Triada”, 2005. 227 рр. (In Russ.).
3. Sirotkina O.V., Laskovets A.B., Andoskin P.A. et al. Increase in GP IIB-IIIA and P2Y12 receptors in activated platelets as the possible indicator of de novo protein synthesis. Molekulyarnaya biologiya. 2016;50(1):128–35. (In Russ.). DOI: 10.7868/S0026898416010183.
4. Shitikova A.S. Thrombocytopathies, congenital and acquired. Eds. L.N. Papayan, O.G. Golovina. SPb.: OOO “Bastion”, 2008. 320 pp. (In Russ.).
5. SharmaP.,SachdevaM.S.,KumarN.etal.Acomparativestudy between light transmission aggregometry and flow cytometric platelet aggregation test for the identification of platelet function defects in patients with bleeding. Blood Res. 2021;56(2):109– 18. DOI: 10.5045/br.2021.2020232.
6. Althaus K., Zieger B., Bakchoul T., Jurk K. Standardization of light transmission aggregometry for diagnosis of platelet disorders: An inter-laboratory external quality assessment. Thromb Haemost. 2019;119(7):1154–61. DOI: 10.1055/s-0039–1688791.
7. FrelingerA.L., Rivera J., David E. et al. Consensus recommendations on flow cytometry for the assessment of inherited and acquired disorders of platelet number and function: Communication from the ISTH SSC Subcommittee on Platelet Physiology. J Thromb Haemost. 2021;19(12):3193–202. DOI: 10.1111/jth.15526.
8. Selyutin A.V., Chepanov S.V., Pavlov O.V. et al. The role of peripheral blood platelet-monocyte aggeregates in reproductive processes and their study methods. Akusherstvo i ginekologiya. 2021;(8):50– 9. (In Russ.). DOI: 10.18565/aig.2021.8.50–58.
9. Shabalina A.A., Roitman E.V., Tanashyan M.M. The comparison between two methods of laboratory evaluation for platelet aggregation and for resistance to antiplatelet therapy. Tromboz, gemostaz i reologiya. 2019;(3):64–71. (In Russ.). DOI: 10.25555/ THR.2019.3.0892.
10. Ermakova A.I. Gaikovaya L.B., Vavilova T.V., Sirotkina O.V. Reference intervals of P-selectin expression on platelets by flow cytometry. Profilakticheskaya i klinicheskaya medicina. 2020;(4):87–92. (In Russ.). DOI: 10.47843/2074–9120_2020_4_87.
11. Granja T., Schad J., Schüssel P. et al. Using six-colour flow cytometry to analyse the activation and interaction of platelets and leukocytes — A new assay suitable for bench and bedside conditions. Thromb Res. 2015;136(4):786–96. DOI: 10.1016/j. thromres.2015.07.009 0049–3848.
12. Panchenko E.P. Modern concepts of the place of acetylsalicylic acid in the treatment of patients with various manifestations of atherothrombosis. Aterotromboz. 2021;(1):89–105. (In Russ.). DOI: 10.21518/2307–1109–2021–11–1–89–105.
13. Tereshchenko A.S., Merkulov E.V., Samko A.N. Glycoprotein IIb/IIIa receptor inhibitors in patients with ST-segment elevation myocardial infarction and primary percutaneous coronary intervention. Racional’naya farmakoterapiya v kardiologii. 2019;15(6):918–27. (In Russ.). DOI: 10.20996/1819–6446–2019–15– 6–918–927.
14. Scholz T., Zhao L., Temmler U. et al. The GPIIb/IIIa antagonist eptifibatide markedly potentiates platelet-leukocyte interaction and tissue factor expression following platelet activation in whole blood in vitro. Platelets. 2002;13(7):401–6. DOI:10.1080/0953710021000024367.
15. Zhao L., Bath P., May J. et al. P-selectin, tissue factor and CD40 ligand expression on platelet-leucocyte conjugates in the presence of a GPIIb/IIIa antagonist. Platelets. 2003;14(7–8):473–80. DOI: 10.1080/09537100310001638562.
16. Simon D.I., Chen Z., Xu H. et al. Platelet glycoprotein Ibα is a counterreceptor for the leukocyte integrin Mac-1 (CD11b/CD18). J Exp Med. 2000;192(2):193–204. DOI: 10.1084/jem.192.2.193.
17. Gils J.M., Zwaginga J.J., Hordijk P.L. Molecular and functional interactions among monocytes, platelets, and endothelial cells and their relevance for cardiovascular diseases. J Leukoc Biol. 2009;85(2):195–204. DOI: 10.1189/jlb.0708400.
18. Haselmayer P., Grosse-Hovest L., von Landenberg P. et al. TREM-1 ligand expression on platelets enhances neutrophil activation. Blood. 2007;110(3):1029–35.
19. Hur J., Jang J.H., Oh I.Y. et al. Human podoplanin-positive monocytes and platelets enhance lymphangiogenesis through the activation of the podoplanin/CLEC-2 axis. Mol Ther. 2014;22(8):1518– 529. DOI: 10.1038/mt.2014.61.