Alterations in Platelet Activity and Elastic Modulus of Healthy Subjects, Carriers of G20210A Polymorphism in the Prothrombin Gene

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Summary

Platelet activation is a complex process in which platelet reorganization takes place associated with changes in the cell shape, topology, membrane elasticity and microparticle production. The aim of this study was to investigate the changes/aberrations in the platelet activity, elasticity and morphology in healthy subjects, carriers of A allele of prothrombin G20210A polymorphism. Blood samples from 18 healthy subjects were used for platelet analysis by force-mode atomic force microscopy. Restriction analysis was used to investigate the carriage of G20210A polymorphism in the prothrombin gene. Flow- cytometry was applied to evaluate platelet activation. Young’s modulus of the plasma membranes of platelets derived from healthy subjects, carriers of variant A allele of prothrombin 20210G>A polymorphism (407±69 kPa) is two times higher than the one determined for non­carriers (195.4±48.7 kPa; p<0.05). The background activity of platelets measured as an interrelation of Cd41/Cd61 and CD62 by flow cytometry was also higher in carriers of variant A allele of prothrombin 20210G>A polymorphism (5.0%) than in non-carriers (1.3%). Platelets isolated from healthy carriers of variant A allele of prothrombin 20210G>A polymorphism exhibited a higher level of activity and a higher degree of stiffness at the stage of spreading as compared to platelets from non­carriers.

1. Hartwig J. Mechanism of actin rearrangements mediating platelet activation. J Cell Biol. 1992;118:1421-42.

2. Rheinlaender J, Vogel S, Seifert J, Schachtele M, Borst O, Lang F, et al. Imaging the elastic modulus of human platelets during thrombininduced activation using scanning ion conductance microscopy. J Thromb Haemost. 2015; 113(2):305-11.

3. Parise LV, Smyth SS, Coller BS. Platelet morphology, biochemistry, and function. In: Lichtman MA, Beutler E, Kaushansky K, Kipps TJ, Seligsohn U, Prchal J, editors. Williams Hematology. 7th ed. New York: McGraw-Hill Professional; 2005. p. 1357-408.

4. Pompe T, Glorius S, Bischoff T, Uhlmann 1, Kaufmann M, Brenner S, et al. Dissecting the impact of matrix anchorage and elasticity in cell adhesion. Biophys J. 2009;97(8):2154-63.

5. Engler AJ, Sen S, Sweeney HL, Discher DE. Matrix elasticity directs stem cell lineage specification. Cell. 2006.126:677-89.

6. McBeath R, Pirone DM, Nelson CM, Bhadriraju K, Chen CS. Cell shape, cytoskeletal tension, and RhoA regulate stem cell lineage commitment. Dev Cell. 2004;6:483-95.

7. Offermanns S. Activation of platelet function through G protein-coupled receptors. Circ Res. 2006.99(12): 1293-304.

8. Jackson SP, Nesbitt WS, Westein E. Dynamics of platelet thrombus formation. J Thromb Haemost. 2009;7 Suppl 1:17-20. doi:

9. Poort SR, Rosendaal FR, Reitsma PH, Bertina RM. A common genetic variation in the 3'-untranslated region of the prothrombin gene is associated with elevated plasma prothrombin levels and an increase in venous thrombosis. Blood. 1996;88(10):3698-703. PubMed PMID: 8916933.

10. Martinelli I, Bucciarelli P, MannucciPM. Thrombotic risk factors: basic pathophysiology. Crit Care Med. 2010;38 (2 Suppl):S3-9. doi:

11. Evaluation of Genomic Applications in Practice and Prevention (EGAPP) Working Group. Recommendations from the EGAPP Working Group: routine testing for Factor V Leiden (R506Q) and prothrombin (20210G>A) mutations in adults with a history of idiopathic venous thromboembolism and their adult family members. Genet Med. 2011 ;13(l):67-76. doi:

12. Franz CM, Puech P-H. Atomic Force Microscopy: A versatile tool for studying cell morphology, adhesion and mechanics. Cell Mol Bioeng. 2008;l(4):289-300. doi:

13. Lee IS, Marchant RE. Force measurements on platelet surfaces with high spatial resolution under physiological conditions. Colloids Surf B Biointerfaces. 2000;19(4):357-65.

14. Ortega JE, Barron-Gonzalez MP, Gracia-Pinilla MA, Perez E, Lopez-Aldrete A, Menchaca J-L. Biological samples observed in vitro by Atomic Force Microscopy: Morphology and Elastic Properties. In: Mendez-Vilas A, editor. Microscopy: advances in scientific research and education. Badajoz: Formatex Research Center; 2014.

15. Komsa-Penkova R, Kovacheva-Kotseva K, Angelova S, Savov A, Simeonova M. [Selected methods of DNA analysis and clinical applications], Pleven: VMI-Pleven; 2004. p. lb-28. Bulgarian.

16. Kwok S, Higuchi R. Avoiding false-positives with PCR. Nature. 1989;339:237-8.

17. Dhurat R, Sukesh MS. Principles and methods of preparation of platelet-rich plasma: A review and author's perspective. J Cutan Aesthet Surg. 20 14;7(4): 1 89-97. do i: 1 0.410 3/0974-2077.150734.

18. Radmacher M, Tillamnn RW, Fritz M, Gaub HE. From molecules to cells: imaging soft samples with the atomic force microscope. Science. 1992 ;257:1900-5. doi:

19. Kuznetsova TG, Starodubtseva MN, Yegorenkov NI, Chizhik SA, Zhdanov RI. Atomic force microscopy probing of cell elasticity. Micron. 2007;38(8):824-33.

20. Digital Instruments/Veeco Metrology Group. NanoScope Command Reference Manual Version 5.12 Revision B. Digital Instruments/Veeco Metrology Group Inc.; 2001.

21. Domke J, Radmacher M. Measuring the elastic properties of thin polymer films with the Atomic Force Microscope. Langmuir. 1998;14:3320-25.

22. Starodubtseva M, Chizhik S, Yegorenkov N, Nikitina I, Drozd E. Study of the mechanical properties of single cells as biocomposites by atomic force microscopy. In: Mendez-Vilas A, Diaz J, editors. Microscopy: science, technology, applications and education. Badajoz: Formatex; 2010.

23. Du Plooy JN, Buys A, Duim W, Pretorius E. Comparison of platelet ultrastructure and elastic properties in thrombo-embolic ischemic stroke and smoking using atomic force and scanning electron microscopy. PLoS One. 2013;8(7):e69774.

24. van Rooy M-J, Duim W, Ehlers R, Buys AV, Pretorius E. Platelet hyperactivity and fibrin clot structure in transient ischemic attack individuals in the presence of metabolic syndrome: a microscopy and thromboelastography study. Cardiovasc Diabetol. 2015; 14:86. doi:

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