Identification of Platelet Subpopulations in Cryopreserved Platelet Components Using Multi-colour Imaging Flow Cytometry

Overview

Journal Sci Rep

Specialty Science

Date 2023 Jan 21

PMID 36681723

Authors

Lacey Johnson

Pearl Lei

Lauren Waters

Matthew P Padula

Denese C Marks

Affiliations

Soon will be listed here.

Abstract

Cryopreservation of platelets, at - 80 °C with 5-6% DMSO, results in externalisation of phosphatidylserine and the formation of extracellular vesicles (EVs), which may mediate their procoagulant function. The phenotypic features of procoagulant platelets overlap with other platelet subpopulations. The aim of this study was to define the phenotype of in vitro generated platelet subpopulations, and subsequently identify the subpopulations present in cryopreserved components. Fresh platelet components (n = 6 in each group) were either unstimulated as a source of resting platelets; or stimulated with thrombin and collagen to generate a mixture of aggregatory and procoagulant platelets; calcium ionophore (A23187) to generate procoagulant platelets; or ABT-737 to generate apoptotic platelets. Platelet components (n = 6) were cryopreserved with DMSO, thawed and resuspended in a unit of thawed plasma. Multi-colour panels of fluorescent antibodies and dyes were used to identify the features of subpopulations by imaging flow cytometry. A combination of annexin-V (AnnV), CD42b, and either PAC1 or CD62P was able to distinguish the four subpopulations. Cryopreserved platelets contained procoagulant platelets (AnnV/PAC1/CD42b/CD62P) and a novel population (AnnV/PAC1/CD42b/CD62P) that did not align with the phenotype of aggregatory (AnnV/PAC1/CD42b/CD62P) or apoptotic (AnnV/PAC1/CD42b/CD62P) subpopulations. These data suggests that the enhanced haemostatic potential of cryopreserved platelets may be due to the cryo-induced development of procoagulant platelets, and that additional subpopulations may exist.

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References

Agbani E, Bosch M, Brown E, Williams C, Mattheij N, Cosemans J . Coordinated Membrane Ballooning and Procoagulant Spreading in Human Platelets. Circulation. 2015; 132(15):1414-24. DOI: 10.1161/CIRCULATIONAHA.114.015036. View

Obydennyy S, Sveshnikova A, Ataullakhanov F, Panteleev M . Dynamics of calcium spiking, mitochondrial collapse and phosphatidylserine exposure in platelet subpopulations during activation. J Thromb Haemost. 2016; 14(9):1867-81. DOI: 10.1111/jth.13395. View

Sodergren A, Ramstrom S . Platelet subpopulations remain despite strong dual agonist stimulation and can be characterised using a novel six-colour flow cytometry protocol. Sci Rep. 2018; 8(1):1441. PMC: 5780418. DOI: 10.1038/s41598-017-19126-8. View

Kemble S, Dalby A, Lowe G, Nicolson P, Watson S, Senis Y . Analysis of preplatelets and their barbell platelet derivatives by imaging flow cytometry. Blood Adv. 2022; 6(9):2932-2946. PMC: 9092408. DOI: 10.1182/bloodadvances.2021006073. View

Heemskerk J, Mattheij N, Cosemans J . Platelet-based coagulation: different populations, different functions. J Thromb Haemost. 2012; 11(1):2-16. DOI: 10.1111/jth.12045. View

Hindle M, Spurgeon B, Cheah L, Webb B, Naseem K . Multidimensional flow cytometry reveals novel platelet subpopulations in response to prostacyclin. J Thromb Haemost. 2021; 19(7):1800-1812. DOI: 10.1111/jth.15330. View

Remenyi G, Szasz R, Friese P, Dale G . Role of mitochondrial permeability transition pore in coated-platelet formation. Arterioscler Thromb Vasc Biol. 2004; 25(2):467-71. DOI: 10.1161/01.ATV.0000152726.49229.bf. View

Schoenwaelder S, Jarman K, Gardiner E, Hua M, Qiao J, White M . Bcl-xL-inhibitory BH3 mimetics can induce a transient thrombocytopathy that undermines the hemostatic function of platelets. Blood. 2011; 118(6):1663-74. DOI: 10.1182/blood-2011-04-347849. View

Handtke S, Steil L, Greinacher A, Thiele T . Toward the Relevance of Platelet Subpopulations for Transfusion Medicine. Front Med (Lausanne). 2018; 5:17. PMC: 5807390. DOI: 10.3389/fmed.2018.00017. View

10.

Johnson L, Vekariya S, Tan S, Padula M, Marks D . Extended storage of thawed platelets: Refrigeration supports postthaw quality for 10 days. Transfusion. 2020; 60(12):2969-2981. DOI: 10.1111/trf.16127. View

11.

Holley A, Marks D, Johnson L, Reade M, Badloe J, Noorman F . Frozen blood products: clinically effective and potentially ideal for remote Australia. Anaesth Intensive Care. 2013; 41(1):10-9. DOI: 10.1177/0310057X1304100104. View

12.

Marks D, Johnson L . Assays for phenotypic and functional characterization of cryopreserved platelets. Platelets. 2018; 30(1):48-55. DOI: 10.1080/09537104.2018.1514108. View

13.

Dale G . Coated-platelets: an emerging component of the procoagulant response. J Thromb Haemost. 2005; 3(10):2185-92. DOI: 10.1111/j.1538-7836.2005.01274.x. View

14.

Nevzorova T, Mordakhanova E, Daminova A, Ponomareva A, Andrianova I, Le Minh G . Platelet factor 4-containing immune complexes induce platelet activation followed by calpain-dependent platelet death. Cell Death Discov. 2019; 5:106. PMC: 6591288. DOI: 10.1038/s41420-019-0188-0. View

15.

Donati A, Gupta S, Reviakine I . Subpopulations in purified platelets adhering on glass. Biointerphases. 2016; 11(2):029811. DOI: 10.1116/1.4953866. View

16.

Michelson A, Barnard M, Hechtman H, MacGregor H, Connolly R, Loscalzo J . In vivo tracking of platelets: circulating degranulated platelets rapidly lose surface P-selectin but continue to circulate and function. Proc Natl Acad Sci U S A. 1996; 93(21):11877-82. PMC: 38152. DOI: 10.1073/pnas.93.21.11877. View

17.

Johnson L, Winter K, Reid S, Hartkopf-Theis T, Marks D . Cryopreservation of buffy-coat-derived platelet concentrates in dimethyl sulfoxide and platelet additive solution. Cryobiology. 2011; 62(2):100-6. DOI: 10.1016/j.cryobiol.2011.01.003. View

18.

Tynngard N, Alshamari A, Mansson F, Ramstrom S . Variation in activation marker expression within the platelet population - a new parameter for evaluation of platelet flow cytometry data. Platelets. 2022; 33(8):1113-1118. DOI: 10.1080/09537104.2022.2078490. View

19.

Brambilla M, Rossetti L, Zara C, Canzano P, Giesen P, Tremoli E . Do methodological differences account for the current controversy on tissue factor expression in platelets?. Platelets. 2017; 29(4):406-414. DOI: 10.1080/09537104.2017.1327653. View

20.

Bohonek M, Kutac D, Landova L, Koranova M, Sladkova E, Staskova E . The use of cryopreserved platelets in the treatment of polytraumatic patients and patients with massive bleeding. Transfusion. 2019; 59(S2):1474-1478. DOI: 10.1111/trf.15177. View