Approach for analysis of intracellular markers in phosphatidylserine-positive platelets

Cover Page

Cite item

Full Text

Open Access Open Access
Restricted Access Access granted
Restricted Access Subscription Access

Abstract

Phosphatidylserine (PS)-positive platelets play an important role in thrombosis and hemostasis. They have high procoagulant activity, the ability to vesiculate, and can aggregate with activated PS-negative platelets. They are found in growing thrombus in vitro, but there remain a number of mysteries associated with them. In particular, intracellular signaling and cytoskeletal reorganization in these platelets is very poorly studied, because they are destroyed upon permeabilization, which is necessary for antibodies to intracellular markers to penetrate the cell. In this work, we propose an approach that allows the analysis of intracellular signaling in calcium ionophore A23187-induced PS-positive platelets using flow cytometry or confocal microscopy. We used the mildest permeabilization of fixed PS-positive platelets using saponin and showed that such permeabilization allows us to preserve PS-positive platelets. As an example, we analyzed the state of the polymerized form of actin in PS-positive platelets and showed that, despite the significant rearrangement of the cytoskeleton that occurs upon activation in such platelets, actin in them is partially presented in a polymerized form.

Full Text

Restricted Access

About the authors

E. O. Artemenko

Center for Theoretical Problems of Physicochemical Pharmacology; National Scientific and Practical Center of Pediatric Hematology, Oncology and Immunology

Author for correspondence.
Email: lartemenko@yandex.ru
Russian Federation, Moscow, 109029; Moscow, 117997

S. I. Obydennyi

Center for Theoretical Problems of Physicochemical Pharmacology; National Scientific and Practical Center of Pediatric Hematology, Oncology and Immunology

Email: lartemenko@yandex.ru
Russian Federation, Moscow, 109029; Moscow, 117997

M. A. Panteleev

Center for Theoretical Problems of Physicochemical Pharmacology; National Scientific and Practical Center of Pediatric Hematology, Oncology and Immunology; Moscow Lomonosov State University

Email: lartemenko@yandex.ru

Moscow Lomonosov State University, Faculty of Physics

Russian Federation, Moscow, 109029; Moscow, 117997; Moscow, 119991

References

  1. Bevers E.M., Tilly R.H., Senden J.M., Comfurius P., Zwaal R.F. 1989. Exposure of endogenous phosphatidylserine at the outer surface of stimulated platelets is reversed by restoration of aminophospholipid translocase activity. Biochemistry. 28 (6), 2382–2387. doi: 10.1021/bi00432a007
  2. Kotova Y.N., Ataullakhanov F.I., Panteleev M.A. 2008. Formation of coated platelets is regulated by the dense granule secretion of adenosine 5'diphosphate acting via the P2Y12 receptor. J. Thromb. Haemost. 6 (9), 1603–1605. doi: 10.1111/j.1538-7836.2008.03052.x
  3. Podoplelova N.A., Nechipurenko D.Y., Ignatova A.A., Sveshnikova A.N., Panteleev M.A. 2021. Procoagulant platelets: Mechanisms of generation and action. Hamostaseologie. 41 (2), 146–153. doi: 10.1055/a-1401-2706
  4. Dale G.L. 2005. Coated-platelets: An emerging component of the procoagulant response. J. Thromb. Haemost. 3 (10), 2185–2192. doi: 10.1111/j.1538-7836.2005.01274.x
  5. Sinauridze E.I., Kireev D.A., Popenko N.Y., Pichugin A.V., Panteleev M.A., Krymskaya O.V., Ataullakhanov F.I. 2007. Platelet microparticle membranes have 50- to 100-fold higher specific procoagulant activity than activated platelets. Thromb. Haemost. 97 (3), 425–434.
  6. Yakimenko A.O., Verholomova F.Y., Kotova Y.N., Ataullakhanov F.I., Panteleev M.A. 2012. Identification of different proaggregatory abilities of activated platelet subpopulations. Biophys. J. 102 (10), 2261–2269. doi: 10.1016/j.bpj.2012.04.004.
  7. Nechipurenko D.Y., Receveur N., Yakimenko A.O., Shepelyuk T.O., Yakusheva A.A., Kerimov R.R., Obydennyy S.I., Eckly A., Léon C., Gachet C., Grishchuk E.L., Ataullakhanov F.I., Mangin P.H., Panteleev M.A. 2019. Clot contraction drives the translocation of procoagulant platelets to thrombus surface. Arterioscler. Thromb. Vasc. Biol. 39 (1), 37–47. doi: 10.1161/ATVBAHA.118.311390
  8. Gaffet P., Bettache N., Bienvenüe A. 1995. Phosphatidylserine exposure on the platelet plasma membrane during A23187-induced activation is independent of cytoskeleton reorganization. Eur. J. Cell Biol. 67 (4), 336–345.
  9. Verhallen P.F., Bevers E.M., Comfurius P., Zwaal R.F. 1987. Correlation between calpain-mediated cytoskeletal degradation and expression of platelet procoagulant activity. A role for the platelet membrane-skeleton in the regulation of membrane lipid asymmetry? Biochim. Biophys. Acta. 903 (1), 206–217. doi: 10.1016/0005-2736(87)90170-2
  10. Artemenko E.O., Yakimenko A.O., Pichugin A.V., Ataullakhanov F.I., Panteleev M.A. 2016. Calpain-controlled detachment of major glycoproteins from the cytoskeleton regulates adhesive properties of activated phosphatidylserine-positive platelets. Biochem. J. 473 (4), 435–448. doi: 10.1042/BJ20150779
  11. Dale G.L., Friese P., Batar P., Hamilton S.F., Reed G.L., Jackson K.W., Clemetson K.J., Alberio L. 2002. Stimulated platelets use serotonin to enhance their retention of procoagulant proteins on the cell surface. Nature. 415 (6868), 175–179. doi: 10.1038/415175a.
  12. Pasquet J.M., Dachary-Prigent J., Nurden A.T. 1998. Microvesicle release is associated with extensive protein tyrosine dephosphorylation in platelets stimulated by A23187 or a mixture of thrombin and collagen. Biochem. J. 333 (Pt 3)(Pt 3), 591–599. doi: 10.1042/bj3330591
  13. Rochat S., Alberio L. 2015. Formaldehyde-fixation of platelets for flow cytometric measurement of phosphatidylserine exposure is feasible. Cytometry A. 87 (1), 32–36. doi: 10.1002/cyto.a.22567
  14. Jamur M.C., Oliver C. 2010. Permeabilization of cell membranes. Methods Mol. Biol. 588, 63–66. doi: 10.1007/978-1-59745-324-0_9

Supplementary files

Supplementary Files
Action
1. JATS XML
Download
2. Fig. 1. Flow cytometry of non-activated and activated A23187 platelets (n/a – non-activated, A23187 – activated FS-positive). a–c – Dot plots of typical experiments (n = 3). The first column shows FSC – forward light scatter, the second column FL4-A – CD61-Alexa Fluor 647 fluorescence, the third column FL1-A – phalloidin-FITC fluorescence; SSC – side light scatter. d – Averaged data for three donors are shown, the number of platelets without treatment with detergent is taken as 1. d – Relative fluorescence values ​​of phalloidin-FITC staining of fixed platelets after their permeabilization are shown (n = 3).

Download (2MB)
Indexing metadata
3. Fig. 2. Study of the structure of PS-positive platelets obtained by activation with the ionophore A23187 using confocal microscopy. a – Fixed PS-positive platelets permeabilized with saponin, stained with CD61-Alexa Fluor 647 and phalloidin-FITC. b – Fixed PS-positive platelets, non-permeabilized, stained only with CD61-Alexa Fluor 647. c – Fixed PS-positive platelets, permeabilized with saponin, stained only with CD61-Alexa Fluor 647. DIC – differential interference contrast. Scale bar is 10 μm.

Download (261KB)
Indexing metadata

Copyright (c) 2025 The Russian Academy of Sciences