Differential Sensitivity of Various Markers of Platelet Activation with Adenosine Diphosphate


A number of techniques have been available to assess platelet activation, but their relative sensitivity is unknown and their usage is variable and not based on any rational criteria. Here, we compared the ability of several techniques based on morphological and biochemical markers to detect the first signs of ADP-induced platelet activation. Scanning electron microscopy of platelets was performed in parallel with flow cytometry to quantify the surface expression of P-selectin (marked by labeled anti-CD62P antibodies), active αIIbβ3-intergrin (assessed by the binding of labeled fibrinogen), and phosphatidylserine (assessed by the binding of labeled Annexin V). When expressed as a fraction of activated platelets, shape changes were the most sensitive to a low ADP concentration compared to the biochemical markers in the following order of sensitivity: morphological changes>fibrinogen binding capacity>P-selectin expression> phosphatidylserine exposure. These results suggest the greater sensitivity of platelet microscopy and the importance of its combination with flow cytometry used to detect surface expression of the molecular markers of platelet activation.

This is a preview of subscription content, log in to check access.

Fig. 1
Fig. 2


  1. 1.

    Wendelboe, A. M., & Raskob, G. E. (2016). Global burden of thrombosis: epidemiologic aspects. Circulation Research, 118(9), 1340–1347. https://doi.org/10.1161/CIRCRESAHA.115.306841.

    Article  Google Scholar 

  2. 2.

    Peshkova, A. D., Malyasyov, D. V., Bredikhin, R. A., Le Minh, G., Andrianova, I. A., Tutwiler, V., Nagaswami, C., Weisel, J. W., & Litvinov, R. I. (2018). Reduced contraction of blood clots in venous thromboembolism is a potential thrombogenic and embologenic mechanism. TH Open, 2(1), e104–e115. https://doi.org/10.1055/s-0038-1635572.

    Article  Google Scholar 

  3. 3.

    Tutwiler, V., Peshkova, A. D., Andrianova, I. A., Khasanova, D. R., Weisel, J. W., & Litvinov, R. I. (2016). Contraction of blood clots is impaired in acute ischemic stroke. Arteriosclerosis, Thrombosis, and Vascular Biology, 37(2), 271–279. https://doi.org/10.1161/ATVBAHA.116.308622.

    Article  Google Scholar 

  4. 4.

    Le Minh, G., Peshkova, A. D., Andrianova, I. A., Sibgatullin, T. B., Maksudova, A. N., Weisel, J. W., & Litvinov, R. I. (2018). Impaired contraction of blood clots as a novel prothrombotic mechanism in systemic lupus erythematosus. Clinical Science, 132(2), 243–254. https://doi.org/10.1042/CS20171510.

    Article  Google Scholar 

  5. 5.

    Jennings, L. K. (2009). Mechanisms of platelet activation: need for new strategies to protect against platelet-mediated atherothrombosis. Thrombosis and Haemostasis, 101(2), 248–257. https://doi.org/10.1160/TH09-03-0192.

    MathSciNet  Article  Google Scholar 

  6. 6.

    Daniel, J. L., Dangelmaier, C., Jin, J., Ashby, B., Smith, J. B., & Kunapuli, S. P. (1998). Molecular basis for ADP-induced platelet activation I. evidence for three distinct ADP receptors on human platelets. Journal of Biological Chemistry, 273(4), 2024–2029. https://doi.org/10.1074/jbc.273.4.2024.

    Article  Google Scholar 

  7. 7.

    Kamath, S., Blann, A. D., & Lip, G. Y. (2001). Platelet activation: assessment and quantification. European Heart Journal, 22(17), 1561–1571. https://doi.org/10.1053/euhj.2000.2515.

    Article  Google Scholar 

  8. 8.

    Tutwiler, V., Litvinov, R. I., Lozhkin, A. P., Peshkova, A. D., Lebedeva, T., Ataullakhanov, F. I., Spiller, K. L., Cines, D. B., & Weisel, J. W. (2015). Kinetics and mechanics of clot contraction are governed by the molecular and cellular composition of the blood. Blood, 127(1), 149–159. https://doi.org/10.1182/blood-2015-05-647560.

    Article  Google Scholar 

  9. 9.

    Harrison, P. (2000). Progress in the assessment of platelet function. British Journal of Haematology, 111(3), 733–744. https://doi.org/10.1111/j.1365-2141.2000.02269.x.

    Google Scholar 

  10. 10.

    Fijnheer, R., Frijns, C. J., Korteweg, J., Rommes, H., Peters, J. H., Sixma, J. J., & Nieuwenhuis, H. K. (1997). The origin of P-selectin as a circulating plasma protein. Thrombosis and Haemostasis, 77(6), 1081–1085.

    Article  Google Scholar 

  11. 11.

    van Velzen, J. F., Laros-van Gorkom, B. A., Pop, G. A., & van Heerde, W. L. (2012). Multicolor flow cytometry for evaluation of platelet surface antigens and activation markers. Thrombosis Research, 130(1), 92–98. https://doi.org/10.1016/j.thromres.2012.02.041.

    Article  Google Scholar 

  12. 12.

    Ramström, S., Rånby, M., & Lindahl, T. (2003). Platelet phosphatidylserine exposure and procoagulant activity in clotting whole blood: different effects of collagen, TRAP and calcium ionophore A23187. Thrombosis and Haemostasis, 89(1), 132–141. https://doi.org/10.1055/s-0037-1613552.

    Article  Google Scholar 

  13. 13.

    Merten, M., & Thiagarajan, P. (2000). P-selectin expression on platelets determines size and stability of platelet aggregates. Circulation, 102(16), 1931–1936. https://doi.org/10.1161/01.CIR.102.16.1931.

    Article  Google Scholar 

  14. 14.

    Ferroni, P., Speziale, G., Ruvolo, G., Giovannelli, A., Pulcinelli, F. M., Lenti, L., Pignatelli, P., Criniti, A., Tonelli, E., Marino, B., & Gazzaniga, P. P. (1998). Platelet activation and cytokine production during hypothermic cardiopulmonary bypass–a possible correlation? Thrombosis and Haemostasis, 80(1), 58–64. https://doi.org/10.1055/s-0037-1615139.

    Article  Google Scholar 

  15. 15.

    Ferroni, P., Riondino, S., Vazzana, N., Santoro, N., Guadagni, F., & Davì, G. (2012). Biomarkers of platelet activation in acute coronary syndromes. Thrombosis and Haemostasis, 108(6), 1109–1123. https://doi.org/10.1160/TH12-08-0550.

    Article  Google Scholar 

  16. 16.

    Michelson, A. D., Barnard, M. R., Hechtman, H. B., MacGregor, H., Connolly, R. J., Loscalzo, J., & Valeri, C. R. (1996). In vivo tracking of platelets: circulating degranulated platelets rapidly lose surface P-selectin but continue to circulate and function. Proceedings of the National Academy of Sciences of the United States of America, 93(21), 11877–11882. https://doi.org/10.1073/pnas.93.21.11877.

    Article  Google Scholar 

  17. 17.

    Born, G. V. (1962). Aggregation of blood platelets by adenosine diphosphate and its reversal. Nature, 194, 927–929. https://doi.org/10.1038/194927b0.

    Article  Google Scholar 

  18. 18.

    Paul, B. Z., Daniel, J. L., & Kunapuli, S. P. (1999). Platelet shape change is mediated by both calcium-dependent and-independent signaling pathways role of p160 Rho-associated coiled-coil-containing protein kinase in platelet shape change. Journal of Biological Chemistry, 274(40), 28293–28300. https://doi.org/10.1074/jbc.274.40.28293.

    Article  Google Scholar 

  19. 19.

    Bearer, E. L. (1995). Cytoskeletal domains in the activated platelet. Cytoskeleton, 30(1), 50–66. https://doi.org/10.1002/cm.970300107.

    Article  Google Scholar 

  20. 20.

    Woronowicz, K., Dilks, J. R., Rozenvayn, N., Dowal, L., Blair, P. S., Peters, C. G., Woronowicz, L., & Flaumenhaft, R. (2010). The platelet actin cytoskeleton associates with SNAREs and participates in α-granule secretion. Biochemistry, 49(21), 4533–4542. https://doi.org/10.1021/bi100541t.

    Article  Google Scholar 

  21. 21.

    Aslan, J. E. (2017). Platelet shape change. In P. Gresele, N. Kleiman, J. Lopez, & C. Page (Eds.), Platelets in thrombotic and non-thrombotic disorders (pp. 321–336). Cham: Springer. https://doi.org/10.1007/978-3-319-47462-5_24.

    Google Scholar 

  22. 22.

    Baker-Groberg, S. M., Phillips, K. G., & McCarty, O. J. (2013). Quantification of volume, mass, and density of thrombus formation using brightfield and differential interference contrast microscopy. Journal of Biomedical Optics, 18(1), 016014. https://doi.org/10.1117/1.JBO.18.1.016014.

    Article  Google Scholar 

  23. 23.

    Bennett, J. S. (2005). Structure and function of the platelet integrin αIIbβ3. The Journal of Clinical Investigation, 115(12), 3363–3369. https://doi.org/10.1172/JCI26989.

    Article  Google Scholar 

  24. 24.

    Mehrbod, M., Trisno, S., & Mofrad, M. R. (2013). On the activation of integrin αIIbβ3: outside-in and inside-out pathways. Biophysical Journal, 105(6), 1304–1315. https://doi.org/10.1016/j.bpj.2013.07.055.

    Article  Google Scholar 

  25. 25.

    Harrison, P., & Cramer, E. M. (1993). Platelet α-granules. Blood Reviews, 7(1), 52–62. https://doi.org/10.1016/0268-960X(93)90024-X.

    Article  Google Scholar 

  26. 26.

    Thiagarajan, P., & Tait, J. F. (1990). Binding of annexin V/placental anticoagulant protein I to platelets. Evidence for phosphatidylserine exposure in the procoagulant response of activated platelets. Journal of Biological Chemistry, 265(29), 17420–17423.

    Google Scholar 

  27. 27.

    Tonon, G., Luo, X., Greco, N. J., Chen, W., Shi, Y., & Jamieson, G. A. (2002). Weak platelet agonists and U46619 induce apoptosis-like events in platelets, in the absence of phosphatidylserine exposure. Thrombosis Research, 107(6), 345–350. https://doi.org/10.1016/S0049-3848(02)00338-9.

    Article  Google Scholar 

  28. 28.

    Beguin, S., Kumar, R., Keularts, I. M., Seligsohn, U., Coller, B. S., & Hemker, H. C. (1999). Fibrin-dependent platelet procoagulant activity requires GPIb receptors and von Willebrand factor. Blood, 93(2), 564–570.

    Google Scholar 

  29. 29.

    Heemskerk, J. W., Bevers, E. M., & Lindhout, T. (2002). Platelet activation and blood coagulation. Thrombosis and Haemostasis, 88(2), 186–193. https://doi.org/10.1055/s-0037-1613209.

    Google Scholar 

Download references


The work was supported by the NIH grant UO1HL116330 and HL090774, National Science Foundation grant DMR150566, the Program for Competitive Growth at Kazan Federal University, and the Russian Foundation for Basic Research/Republic of Tatarstan grant 18-415-16004.

Author information



Corresponding author

Correspondence to Rustem I. Litvinov.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Le Minh, G., Peshkova, A.D., Andrianova, I.A. et al. Differential Sensitivity of Various Markers of Platelet Activation with Adenosine Diphosphate. BioNanoSci. 9, 53–58 (2019). https://doi.org/10.1007/s12668-018-0586-4

Download citation


  • Platelet activation
  • Scanning electron microscopy
  • Flow cytometry