Abstract
Platelets are specialized cellular elements of blood and play a central role in maintaining normal hemostasis, wound healing, and host defense but also are implicated in pathologic processes of thrombosis, inflammation, and tumor progression and dissemination. Transfusion of platelet concentrates is an important treatment for thrombocytopenia (low platelet count) due to disease or significant blood loss, with the goal being to prevent bleeding or to arrest active bleeding. In blood circulation, platelets are in a resting state; however, when triggered by a stimulus, such as blood vessel injury, become activated (also termed procoagulant). Platelet activation is the basis of their biological function to arrest active bleeding, comprising a complex interplay of morphological phenotype/shape change, adhesion, expression of signaling molecules, and release of bioactive factors, including extracellular vesicles/microparticles. Advances in high-throughput mRNA and protein profiling techniques have brought new understanding of platelet biological functions, including identification of novel platelet proteins and secreted molecules, analysis of functional changes between normal and pathologic states, and determining the effects of processing and storage on platelet concentrates for transfusion. However, because platelets are very easily activated, it is important to understand the different in vitro methods for platelet isolation commonly used and how they differ from the perspective for use as research samples in clinical chemistry. Two simple methods are described here for the preparation of research-scale platelet samples from human whole blood, and detailed notes are provided about the methods used for the preparation of platelet concentrates for transfusion.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Goto S, Hasebe T, Takagi S (2015) Platelets: small in size but essential in the regulation of vascular homeostasis – translation from basic science to clinical medicine. Circ J 79(9):1871–1881. https://doi.org/10.1253/circj.CJ-14-1434
Stevens H, McFadyen JD (2019) Platelets as central actors in thrombosis-reprising an old role and defining a new character. Semin Thromb Hemost 45(8):802–809. https://doi.org/10.1055/s-0039-1698829
Tokarz-Deptula B, Palma J, Baraniecki L, Stosik M, Kolacz R, Deptula W (2021) What function do platelets play in inflammation and bacterial and viral infections? Front Immunol 12:770436. https://doi.org/10.3389/fimmu.2021.770436
Sim X, Poncz M, Gadue P, French DL (2016) Understanding platelet generation from megakaryocytes: implications for in vitro-derived platelets. Blood 127(10):1227–1233. https://doi.org/10.1182/blood-2015-08-607929
Schubert S, Weyrich AS, Rowley JW (2014) A tour through the transcriptional landscape of platelets. Blood 124(4):493–502. https://doi.org/10.1182/blood-2014-04-512756
Garofano K, Park CS, Alarcon C, Avitia J, Barbour A, Diemert D, Fraser CM, Friedman PN, Horvath A, Rashid K, Shaazuddin M, Sidahmed A, O’Brien TJ, Perera MA, Lee NH (2021) Differences in the platelet mRNA landscape portend racial disparities in platelet function and suggest novel therapeutic targets. Clin Pharmacol Ther 110(3):702–713. https://doi.org/10.1002/cpt.2363
Shevchuk O, Begonja AJ, Gambaryan S, Totzeck M, Rassaf T, Huber TB, Greinacher A, Renne T, Sickmann A (2021) Proteomics: a tool to study platelet function. Int J Mol Sci 22(9). https://doi.org/10.3390/ijms22094776
Loosse C, Swieringa F, Heemskerk JWM, Sickmann A, Lorenz C (2018) Platelet proteomics: from discovery to diagnosis. Expert Rev Proteomics 15(6):467–476. https://doi.org/10.1080/14789450.2018.1480111
Greening DW, Glenister KM, Kapp EA, Moritz RL, Sparrow RL, Lynch GW, Simpson RJ (2008) Comparison of human platelet-membrane cytoskeletal proteins with the plasma proteome: towards understanding the platelet-plasma nexus. Proteomics Clin Appl 2:63–77
Rabani V, Davani S, Gambert-Nicot S, Meneveau N, Montange D (2016) Comparative lipidomics and proteomics analysis of platelet lipid rafts using different detergents. Platelets 27(7):634–641. https://doi.org/10.3109/09537104.2016.1174203
Pagel O, Walter E, Jurk K, Zahedi RP (2017) Taking the stock of granule cargo: platelet releasate proteomics. Platelets 28(2):119–128. https://doi.org/10.1080/09537104.2016.1254762
Schmidt GJ, Reumiller CM, Ercan H, Resch U, Butt E, Heber S, Liutkeviciute Z, Basilio J, Schmid JA, Assinger A, Jilma B, Zellner M (2019) Comparative proteomics reveals unexpected quantitative phosphorylation differences linked to platelet activation state. Sci Rep 9(1):19009. https://doi.org/10.1038/s41598-019-55391-5
Lemmens TP, Coenen DM, Swieringa F, Niessen ICL, Coort SLM, Koenen RR, Kutmon M, Cosemans J (2022) Finding the “switch” in platelet activation: prediction of key mediators involved in reversal of platelet activation using a novel network biology approach. J Proteome 261:104577. https://doi.org/10.1016/j.jprot.2022.104577
Milioli M, Ibanez-Vea M, Sidoli S, Palmisano G, Careri M, Larsen MR (2015) Quantitative proteomics analysis of platelet-derived microparticles reveals distinct protein signatures when stimulated by different physiological agonists. J Proteome 121:56–66. https://doi.org/10.1016/j.jprot.2015.03.013
Suades R, Padro T, Vilahur G, Badimon L (2022) Platelet-released extracellular vesicles: the effects of thrombin activation. Cell Mol Life Sci 79(3):190. https://doi.org/10.1007/s00018-022-04222-4
Maguire PB, Parsons ME, Szklanna PB, Zdanyte M, Munzer P, Chatterjee M, Wynne K, Rath D, Comer SP, Hayden M, Ni Ainle F, Gawaz M (2020) Comparative platelet releasate proteomic profiling of acute coronary syndrome versus stable coronary artery disease. Front Cardiovasc Med 7:101. https://doi.org/10.3389/fcvm.2020.00101
Parsons MEM, Szklanna PB, Guerrero JA, Wynne K, Dervin F, O’Connell K, Allen S, Egan K, Bennett C, McGuigan C, Gheveart C, Ni Ainle F, Maguire PB (2018) Platelet releasate proteome profiling reveals a core set of proteins with low variance between healthy adults. Proteomics 18(15):e1800219. https://doi.org/10.1002/pmic.201800219
Salunkhe V, De Cuyper IM, Papadopoulos P, van der Meer PF, Daal BB, Villa-Fajardo M, de Korte D, van den Berg TK, Gutierrez L (2019) A comprehensive proteomics study on platelet concentrates: platelet proteome, storage time and Mirasol pathogen reduction technology. Platelets 30(3):368–379. https://doi.org/10.1080/09537104.2018.1447658
Sonego G, Abonnenc M, Tissot JD, Prudent M, Lion N (2017) Redox proteomics and platelet activation: understanding the redox proteome to improve platelet quality for transfusion. Int J Mol Sci 18(2). https://doi.org/10.3390/ijms18020387
Wang S, Jiang T, Fan Y, Zhao S (2019) A proteomic approach reveals the variation in human platelet protein composition after storage at different temperatures. Platelets 30(3):403–412. https://doi.org/10.1080/09537104.2018.1453060
Grande R, Dovizio M, Marcone S, Szklanna PB, Bruno A, Ebhardt HA, Cassidy H, Ni Ainle F, Caprodossi A, Lanuti P, Marchisio M, Mingrone G, Maguire PB, Patrignani P (2019) Platelet-derived microparticles from obese individuals: characterization of number, size, proteomics, and crosstalk with cancer and endothelial cells. Front Pharmacol 10:7. https://doi.org/10.3389/fphar.2019.00007
Linge CP, Jern A, Tyden H, Gullstrand B, Yan H, Welinder C, Kahn R, Jonssen A, Semple JW, Bengtsson AA (2022) Enrichment of complement, immunoglobulins and autoantibody targets in the proteome of platelets from patients with Systemic Lupus Erythematosus (SLE). Thromb Haemost 122:1486. https://doi.org/10.1055/a-1825-2915
Gawrys K, Turek-Jakubowska A, Gawrys J, Jakubowski M, Debski J, Szahidewicz-Krupska E, Trocha M, Derkacz A, Doroszko A (2022) Platelet-derived drug targets and biomarkers of ischemic stroke-the first dynamic human LC-MS proteomic study. J Clin Med 11(5). https://doi.org/10.3390/jcm11051198
Murphy S (2005) Platelets from pooled buffy coats: an update. Transfusion 45(4):634–639
Vassallo RR, Murphy S (2006) A critical comparison of platelet preparation methods. Curr Opin Hematol 13(5):323–330
Greening DW, Glenister KM, Sparrow RL, Simpson RJ (2010) International blood collection and storage: clinical use of blood products. J Proteome 73(3):386–395
Greening DW, Simpson RJ, Sparrow RL (2017) Preparation of platelet concentrates for research and transfusion purposes. Methods Mol Biol 1619:31–42. https://doi.org/10.1007/978-1-4939-7057-5_3
Greening DW, Sparrow RL, Simpson RJ (2011) Preparation of platelet concentrates. Methods Mol Biol 728:267–278. https://doi.org/10.1007/978-1-61779-068-3_18
Murphy S, Gardner FH (1969) Effect of storage temperature on maintenance of platelet viability–deleterious effect of refrigerated storage. N Engl J Med 280(20):1094–1098
Getz TM (2019) Physiology of cold-stored platelets. Transfus Apher Sci 58(1):12–15. https://doi.org/10.1016/j.transci.2018.12.011
Greening DW, Glenister K, Sparrow R, Simpson RJ (2009) Enrichment of human platelet membrane-cytoskeletal proteins for proteomic analysis. In: Pierce M (ed) Proteomic analysis of membrane proteins: methods and protocols. Methods in molecular medicine series, vol 528. Humana Press, pp 245–258
Neufeld M, Nowak-Gottl U, Junker R (1999) Citrate-theophylline-adenine-dipyridamol buffer is preferable to citrate buffer as an anticoagulant for flow cytometric measurement of platelet activation. Clin Chem 45(11):2030–2033
Saris A, Kreuger AL, Ten Brinke A, Kerkhoffs JLH, Middelburg RA, Zwaginga JJ, van der Meer PF (2019) The quality of platelet concentrates related to corrected count increment: linking in vitro to in vivo. Transfusion 59(2):697–706. https://doi.org/10.1111/trf.14868
van der Meer PF, de Korte D (2018) Platelet additive solutions: a review of the latest developments and their clinical implications. Transfus Med Hemother 45(2):98–102. https://doi.org/10.1159/000487513
Thomas S (2016) Platelets: handle with care. Transfus Med 26(5):330–338. https://doi.org/10.1111/tme.12327
Smethurst PA (2016) Aging of platelets stored for transfusion. Platelets 27(6):526–534. https://doi.org/10.3109/09537104.2016.1171303
Bassuni WY, Blajchman MA, Al-Moshary MA (2008) Why implement universal leukoreduction? Hematol Oncol Stem Cell Ther 1(2):106–123
Godbey EA, Thibodeaux SR (2019) Ensuring safety of the blood supply in the United States: donor screening, testing, emerging pathogens, and pathogen inactivation. Semin Hematol 56(4):229–235. https://doi.org/10.1053/j.seminhematol.2019.11.004
Devine DV, Schubert P (2016) Pathogen inactivation technologies: the advent of pathogen-reduced blood components to reduce blood safety risk. Hematol Oncol Clin North Am 30(3):609–617. https://doi.org/10.1016/j.hoc.2016.01.005
Escolar G, Diaz-Ricart M, McCullough J (2022) Impact of different pathogen reduction technologies on the biochemistry, function, and clinical effectiveness of platelet concentrates: an updated view during a pandemic. Transfusion 62(1):227–246. https://doi.org/10.1111/trf.16747
Moroff G, Luban NL (1997) The irradiation of blood and blood components to prevent graft-versus-host disease: technical issues and guidelines. Transfus Med Rev 11(1):15–26
Kelley WE, Edelman BB, Drachenberg CB, Hess JR (2009) Washing platelets in neutral, calcium-free, Ringer’s acetate. Transfusion 49(9):1917–1923
Waters L, Cameron M, Padula MP, Marks DC, Johnson L (2018) Refrigeration, cryopreservation and pathogen inactivation: an updated perspective on platelet storage conditions. Vox Sang 113(4):317–328. https://doi.org/10.1111/vox.12640
Wood B, Padula MP, Marks DC, Johnson L (2021) Cryopreservation alters the immune characteristics of platelets. Transfusion 61(12):3432–3442. https://doi.org/10.1111/trf.16697
Albanyan AM, Harrison P, Murphy MF (2009) Markers of platelet activation and apoptosis during storage of apheresis- and buffy coat-derived platelet concentrates for 7 days. Transfusion 49(1):108–117
Cardigan R, Turner C, Harrison P (2005) Current methods of assessing platelet function: relevance to transfusion medicine. Vox Sang 88(3):153–163
Tynngard N (2009) Preparation, storage and quality control of platelet concentrates. Transfus Apher Sci 41(2):97–104
Author information
Authors and Affiliations
Corresponding authors
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2023 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Sparrow, R.L., Simpson, R.J., Greening, D.W. (2023). Protocols for the Isolation of Platelets for Research and Contrast to Production of Platelet Concentrates for Transfusion. In: Greening, D.W., Simpson, R.J. (eds) Serum/Plasma Proteomics. Methods in Molecular Biology, vol 2628. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2978-9_1
Download citation
DOI: https://doi.org/10.1007/978-1-0716-2978-9_1
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-2977-2
Online ISBN: 978-1-0716-2978-9
eBook Packages: Springer Protocols