Remember Me
Or use your Academic/Social account:


Or use your Academic/Social account:


You have just completed your registration at OpenAire.

Before you can login to the site, you will need to activate your account. An e-mail will be sent to you with the proper instructions.


Please note that this site is currently undergoing Beta testing.
Any new content you create is not guaranteed to be present to the final version of the site upon release.

Thank you for your patience,
OpenAire Dev Team.

Close This Message


Verify Password:
Verify E-mail:
*All Fields Are Required.
Please Verify You Are Human:
fbtwitterlinkedinvimeoflicker grey 14rssslideshare1
Tseday Z. Tegegn; Silvia H. De Paoli; Martina Orecna; Oumsalama K. Elhelu; Samuel A. Woodle; Ivan D. Tarandovskiy; Mikhail V. Ovanesov; Jan Simak (2016)
Publisher: Taylor & Francis Group
Journal: Journal of Extracellular Vesicles
Languages: English
Types: Article
Subjects: Cytology, blood products, extracellular vesicles, thrombin, nanoparticle tracking analysis, microparticles, Extracellular vesicles; Microparticles; Platelet physiology; Blood products; Thrombin; Transfusion medicine; Nanoparticle tracking analysis; Flow cytometry; Atomic force microscopy; Electron microscopy, QH573-671, atomic force microscopy, platelet physiology, Original Research Article, transfusion medicine, electron microscopy, flow cytometry
Background: Freezing is promising for extended platelet (PLT) storage for transfusion. 6% DMSO cryopreserved PLTs (CPPs) are currently in clinical development. CPPs contain significant amount of platelet membrane vesicles (PMVs). PLT-membrane changes and PMV release in CPP are poorly understood, and haemostatic effects of CPP PMVs are not fully elucidated. This study aims to investigate PLT-membrane alterations in CPPs and provide comprehensive characterization of CPP PMVs, and their contribution to procoagulant activity (PCA) of CPPs.Methods: CPPs and corresponding liquid-stored PLTs (LSPs) were characterized by flow cytometry (FC), fluorescence polarization (FP), nanoparticle tracking analysis (NTA), electron microscopy (SEM, TEM), atomic force microscopy (AFM) and thrombin-generation (TG) test.Results: SEM and TEM revealed disintegration and vesiculation of the PLT-plasma membrane and loss of intracellular organization in 60% PLTs in CPPs. FP demonstrated that 6% DMSO alone and with freezing–thawing caused marked increase in PLT-membrane fluidity. The FC counts of annexin V-binding PMVs and CD41a+ PMVs were 68- and 56-folds higher, respectively, in CPPs than in LSPs. The AFM and NTA size distribution of PMVs in CPPs indicated a peak diameter of 100 nm, corresponding to exosome-size vesicles. TG-based PCA of CPPs was 2- and 9-folds higher per PLT and per volume, respectively, compared to LSPs. Differential centrifugation showed that CPP supernatant contributed 26% to CPP TG-PCA, mostly by the exosome-size PMVs and their TG-PCA was phosphatidylserine dependent.Conclusions: Major portion of CPPs does not show activation phenotype but exhibits grape-like membrane disintegration with significant increase of membrane fluidity induced by 6% DMSO alone and further aggravated by freezing–thawing process. DMSO cryopreservation of PLTs is associated with the release of PMVs and marked increase of TG-PCA, as compared to LSPs. Exosome-size PMVs have significant contribution to PCA of CPPs.Keywords: extracellular vesicles; microparticles; platelet physiology; blood products; thrombin; transfusion medicine; nanoparticle tracking analysis; flow cytometry; atomic force microscopy; electron microscopy(Published: 4 May 2016)Citation: Journal of Extracellular Vesicles 2016, 5: 30422 - http://dx.doi.org/10.3402/jev.v5.30422
  • The results below are discovered through our pilot algorithms. Let us know how we are doing!

    • 1. Dumont LJ, Slichter SJ, Reade MC. Cryopreserved platelets: frozen in a logjam? Transfusion. 2014;54:1907 10.
    • 2. Valeri CR. Hemostatic effectiveness of liquid-preserved and previously frozen human platelets. N Engl J Med. 1974;290: 353 8.
    • 3. Valeri CR, Feingold H, Marchionni LD. A simple method for freezing human platelets using 6 per cent dimethylsulfoxide and storage at 80 degrees C. Blood. 1974;43:131 6.
    • 4. Valeri CR, Ragno G, Khuri S. Freezing human platelets with 6 percent dimethyl sulfoxide with removal of the supernatant solution before freezing and storage at 80 degrees C without postthaw processing. Transfusion. 2005;45:1890 8.
    • 5. Dumont LJ, Cancelas JA, Dumont DF, Siegel AH, Szczepiorkowski ZM, Rugg N, et al. A randomized controlled trial evaluating recovery and survival of 6% dimethyl sulfoxidefrozen autologous platelets in healthy volunteers. Transfusion. 2013;53:128 37.
    • 6. Santos NC, Figueira-Coelho J, Martins-Silva J, Saldanha C. Multidisciplinary utilization of dimethyl sulfoxide: pharmacological, cellular, and molecular aspects. Biochem Pharmacol. 2003;65:1035 41.
    • 7. Jacob SW, Herschler R. Pharmacology of DMSO. Cryobiology. 1986;23:14 27.
    • 8. Fahy GM. The relevance of cryoprotectant ''toxicity'' to cryobiology. Cryobiology. 1986;23:1 13.
    • 9. Oh JE, Karlmark Raja K, Shin JH, Pollak A, Hengstschla¨ger M, Lubec G. Cytoskeleton changes following differentiation of N1E-115 neuroblastoma cell line. Amino Acids. 2006;31: 289 98.
    • 10. Jiang G, Bi K, Tang T, Wang J, Zhang Y, Zhang W, et al. Down-regulation of TRRAP-dependent hTERT and TRRAPindependent CAD activation by Myc/Max contributes to the differentiation of HL60 cells after exposure to DMSO. Int Immunopharmacol. 2006;6:1204 13.
    • 11. Cox MA, Kastrup J, Hrubisko M. Historical perspectives and the future of adverse reactions associated with haemopoietic stem cells cryopreserved with dimethyl sulfoxide. Cell Tissue Bank. 2012;13:203 15.
    • 12. Hornsey VS, McMillan L, Morrison A, Drummond O, Macgregor IR, Prowse CV. Freezing of buffy coat-derived, leukoreduced platelet concentrates in 6 percent dimethyl sulfoxide. Transfusion. 2008;48:2508 14.
    • 13. Valeri CR, Macgregor H, Ragno G. Correlation between in vitro aggregation and thromboxane A2 production in fresh, liquid-preserved, and cryopreserved human platelets: effect of agonists, pH, and plasma and saline resuspension. Transfusion. 2005;45:596 603.
    • 14. Khuri SF, Healey N, MacGregor H, Barnard MR, Szymanski IO, Birjiniuk V, et al. Comparison of the effects of transfusions of cryopreserved and liquid-preserved platelets on hemostasis and blood loss after cardiopulmonary bypass. J Thorac Cardiovasc Surg. 1999;117:172 83;discussion 83 4.
    • 15. Klein E, Toch R, Farber S, Freeman G, Fiorentino R. Hemostasis in thrombocytopenic bleeding following infusion of stored, frozen platelets. Blood. 1956;11:693 9.
    • 16. Slichter SJ, Jones M, Ransom J, Gettinger I, Jones MK, Christoffel T, et al. Review of in vivo studies of dimethyl sulfoxide cryopreserved platelets. Transfus Med Rev. 2014;28: 212 25.
    • 17. Schiffer CA, Aisner J, Wiernik PH. Clinical experience with transfusion of cryopreserved platelets. Br J Haematol. 1976;34: 377 85.
    • 18. Lelkens CC, Koning JG, de Kort B, Floot IB, Noorman F. Experiences with frozen blood products in the Netherlands military. Transfus Apher Sci. 2006;34:289 98.
    • 19. Neuhaus SJ, Wishaw K, Lelkens C. Australian experience with frozen blood products on military operations. Med J Aust. 2010;192:203 5.
    • 20. Johnson L, Coorey CP, Marks DC. The hemostatic activity of cryopreserved platelets is mediated by phosphatidylserineexpressing platelets and platelet microparticles. Transfusion. 2014;54:1917 26.
    • 21. Shibeko AM, Woodle SA, Lee TK, Ovanesov MV. Unifying the mechanism of recombinant FVIIa action: dose dependence is regulated differently by tissue factor and phospholipids. Blood. 2012;120:891 9.
    • 22. Woodle SA, Shibeko AM, Lee TK, Ovanesov MV. Determining the impact of instrument variation and automated software algorithms on the TGT in hemophilia and normalized plasma. Thromb Res. 2013;132:374 80.
    • 23. Simak J, Holada K, Janota J, Strana´k Z. Surface expression of major membrane glycoproteins on resting and TRAPactivated neonatal platelets. Pediatr Res. 1999;46:445 9.
    • 24. Simak J, Gelderman MP. Cell membrane microparticles in blood and blood products: potentially pathogenic agents and diagnostic markers. Transfus Med Rev. 2006;20:1 26.
    • 25. Orecna M, De Paoli SH, Janouskova O, Tegegn TZ, Filipova M, Bonevich JE. Toxicity of carboxylated carbon nanotubes in endothelial cells is attenuated by stimulation of the autophagic flux with the release of nanomaterial in autophagic vesicles. Nanomedicine. 2014;10:939 48.
    • 26. Semberova J, De Paoli Lacerda SH, Simakova O, Holada K, Gelderman MP, Simak J. Carbon nanotubes activate blood platelets by inducing extracellular Ca2+ influx sensitive to calcium entry inhibitors. Nano Lett. 2009;9:3312 7.
    • 27. Lacerda SH, Semberova J, Holada K, Simakova O, Hudson SD, Simak J. Carbon nanotubes activate store-operated calcium entry in human blood platelets. ACS Nano. 2011;5: 5808 13.
    • 28. Dobrovolskaia MA, Patri AK, Simak J, Hall JB, Semberova J, De Paoli Lacerda SH, et al. Nanoparticle size and surface charge determine effects of PAMAM dendrimers on human platelets in vitro. Mol Pharm. 2012;9:382 93.
    • 29. De Paoli SH, Diduch LL, Tegegn TZ, Orecna M, Strader MB, Karnaukhova E, et al. The effect of protein corona composition on the interaction of carbon nanotubes with human blood platelets. Biomaterials. 2014;35:6182 94.
    • 30. Shinitzky M, Barenholz Y. Fluidity parameters of lipid regions determined by fluorescence polarization. Biochim Biophys Acta. 1978;515:367 94.
    • 31. Rooney TA, Hager R, Stubbs CD, Thomas AP. Halothane regulates G-protein-dependent phospholipase C activity in turkey erythrocyte membranes. J Biol Chem. 1993;268:15550 6.
    • 32. Popov VM, Vladareanu AM, Bumbea H, Kovacs E, Moisescu MG, Onisai M, et al. Assessment of changes in membrane properties of platelets from patients with chronic myeloid leukaemia in different stages of the disease. Blood Coagul Fibrinolysis. 2014;25:142 50.
    • 33. Watala C, Golanski J, Boncler MA, Pietrucha T, Gwozdzinski K. Membrane lipid fluidity of blood platelets: a common denominator that underlies the opposing actions of various agents that affect platelet activation in whole blood. Platelets. 1998;9:315 27.
    • 34. Spector JI, Flor WJ, Valeri CR. Ultrastructural alterations and phagocytic function of cryopreserved platelets. Transfusion. 1979;19:307 12.
    • 35. Spector JI, Skrabut EM, Valeri CR. Oxygen consumption, platelet aggregation and release reactions in platelets freezepreserved with dimethylsulfoxide. Transfusion. 1977;17:99 109.
    • 36. Odink J, Brank A., Platelet preservation V. Survival, serotonin uptake velocity, and response to hypotonic stress of fresh and cryopreserved human platelets. Transfusion. 1977;17:203 9.
    • 37. Holtz GC, Davis RB. Inhibition of human platelet aggregation by dimethylsulfoxide, dimethylacetamide, and sodium glycerophosphate. Proc Soc Exp Biol Med. 1972;141:244 8.
    • 38. Kim BK, Baldini MG. Biochemistry, function, and hemostatic effectiveness of frozen human platelets. Proc Soc Exp Biol Med. 1974;145:830 5.
    • 39. Owens M, Cimino C, Donnelly J. Cryopreserved platelets have decreased adhesive capacity. Transfusion. 1991;31:160 3.
    • 40. Crowley JP, Rene A, Valeri CR. Changes in platelet shape and structure after freeze preservation. Blood. 1974;44:599 603.
    • 41. Baythoon H, Tuddenham EG, Hutton RA. Morphological and functional disturbances of platelets induced by cryopreservation. J Clin Pathol. 1982;35:870 4.
    • 42. Murphy S, Sayar SN, Abdou NL, Gardner FH. Platelet preservation by freezing. Use of dimethylsulfoxide as cryoprotective agent. Transfusion. 1974;14:139 44.
    • 43. Valeri CR. Cryopreservation of human platelets and bone marrow and peripheral blood totipotential mononuclear stem cells. Ann N Y Acad Sci. 1985;459:353 66.
    • 44. van der Pol E, Coumans F, Varga Z, Krumrey M, Nieuwland R. Innovation in detection of microparticles and exosomes. J Thromb Haemost. 2013;11(Suppl 1):36 45.
    • 45. Dragovic RA, Collett GP, Hole P, Ferguson DJ, Redman CW, Sargent IL, et al. Isolation of syncytiotrophoblast microvesicles and exosomes and their characterisation by multicolour flow cytometry and fluorescence Nanoparticle Tracking Analysis. Methods. 2015;87:64 74.
    • 46. Dragovic RA, Southcombe JH, Tannetta DS, Redman CW, Sargent IL. Multicolor flow cytometry and nanoparticle tracking analysis of extracellular vesicles in the plasma of normal pregnant and pre-eclamptic women. Biol Reprod. 2013;89:151.
    • 47. Gardiner C, Ferreira YJ, Dragovic RA, Redman CW, Sargent IL. Extracellular vesicle sizing and enumeration by nanoparticle tracking analysis. J Extracell Vesicles. 2013;2:19671, doi: http://dx.doi.org/10.3402/jev.v2i0.19671
    • 48. Raynel S, Padula MP, Marks DC, Johnson L. Cryopreservation alters the membrane and cytoskeletal protein profile of platelet microparticles. Transfusion. 2015;55:2422 32.
    • 49. Filella M, Zhang J, Newman ME, Buffle J. Analytical applications of photon correlation spectroscopy for size distribution measurements of natural colloidal suspensions: capabilities and limitations. Colloids Surfaces A. 1997;120:27 46.
    • 50. Gardiner C, Harrison P, Belting M, B o¨ing A, Campello E, Carter BS, et al. Extracellular vesicles, tissue factor, cancer and thrombosis discussion themes of the ISEV 2014 Educational Day. J Extracell Vesicles. 2015;4:26901, doi: http://dx.doi.org/ 10.3402/jev.v4.26901
  • No related research data.
  • No similar publications.

Share - Bookmark

Cite this article