Morphofunctional state of platelets in their concentrates depending on storage time

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Abstract

Background. Evaluation of the functional state of platelets in the composition of concentrates is necessary to improve the methods of their production, optimize storage conditions and terms, increase therapeutic efficacy, and reduce the risk of transfusion complications.

Aim. Comprehensive study of the morphofunctional state of platelets during the storage of their concentrates.

Material and methods. 202 samples of human platelet apheresis concentrates were studied for storage periods of up to 1–7 days at a temperature of 22–24 °C. The functional state of platelets was assessed using flow cytometry for the expression of P-selectin, active αIIbβ3 integrin, and phosphatidylserines before and after biochemical stimulation. In addition, the mitochondrial potential of platelets, the concentration of intracellular adenosine triphosphate, contractile properties, induced aggregation, and morphological characteristics were studied according to scanning electron microscopy. Statistical analysis was performed by analysis of variance (ANOVA or Kruskal–Wallis).

Results. In non-activated platelets during storage, the spontaneous expression of P-selectin increased by an average of 7 times, and phosphatidylserines — by 3 times. The induced expression of P-selectin, integrin αIIbβ3 and phosphatidylserines was the highest on the day of receiving the concentrate and gradually decreased during storage to 1/2 of the initial level. Platelet aggregation activity in response to collagen stimulation progressively decreased by an average of 100 times, in response to the TRAP peptide — by 1.5 times. In contrast to the expression of activation markers, the ability of platelets to compress plasma clots in most samples changed slightly, within a few percent; in single samples, the contractile function dropped to zero by 4–7 days of storage, which was combined with platelet hyperactivation and depletion. The mitochondrial potential of platelets and the content of adenosine triphosphate were relatively constant. Morphologically, discoid platelets predominated, however, starting from the 1st day of storage, spindle-shaped platelets, and from the 3rd day — spherical forms and microaggregates of platelets accumulated.

Conclusion. Platelets in the composition of concentrates initially have a high functional potential, which gradually decreases due to progressive spontaneous activation with a simultaneous decrease in reactivity and structural changes.

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About the authors

Alina I. Khabirova

Institute of Fundamental Medicine and Biology

Author for correspondence.
Email: alina.urussu.95@gmail.com
ORCID iD: 0000-0002-7243-8832
ResearcherId: AAO-3282-2021

Junior Researcher, Research Laboratory “Protein-Cell Interactions”, Institute of Fundamental Medicine and Biology

Russian Federation, Kazan, Russia

Luciya S. Fatkhullina

Interregional Clinical Diagnostic Center

Email: Lusik65@rambler.ru
ORCID iD: 0000-0001-8303-7455

M.D., Head, Depart. of Procurement of Blood and Its Components

Russian Federation, Kazan, Russia

Izabella A. Andrianova

Institute of Fundamental Medicine and Biology

Email: izabella2d@gmail.com
ORCID iD: 0000-0003-3973-3183

Cand. Sci. (Biol.), Senior Researcher, Research Laboratory “Protein-Cell Interactions”, Institute of Fundamental Medicine and Biology

Russian Federation, Kazan, Russia

Natalia G. Evtugina

Institute of Fundamental Medicine and Biology

Email: natalja.evtugyna@gmail.com
ORCID iD: 0000-0002-4950-3691

PhD Stud., Depart. of Biochemistry, Biotechnology and Pharmacology, Institute of Fundamental Medicine and Biology

Russian Federation, Kazan, Russia

Rustem I. Litvinov

Institute of Fundamental Medicine and Biology

Email: rustempa@gmail.com
ORCID iD: 0000-0003-0643-1496

M.D., D. Sci. (Med.), Prof., Chief Researcher, Research Laboratory “Protein-Cell Interactions”, Institute of Fundamental Medicine and Biology

Russian Federation, Kazan, Russia

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Supplementary files

Supplementary Files
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1. JATS XML
2. Рис. 1. Спонтанная (A) и индуцированная (Б) экспрессия маркёров активации в тромбоцитах на разных сроках хранения концентратов тромбоцитов. Число образцов в дни исследования составило для P-селектина n=33–49; для активного интегрина αIIbβ3 n=23–36; для фосфатидилсерина n=25–37. Результаты представлены как медиана и интер­квартильный интервал; ***p <0,0001, **p <0,001; *p <0,05; тест Краскела–Уоллиса с последующим тестом Данна для парных сравнений

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3. Рис. 2. Степень агрегации тромбоцитов, индуцированной коллагеном (А) или пептидом TRAP-6 (Б), на разных сроках хранения концентратов тромбоцитов. При агрегации, вызванной коллагеном, число образцов в дни исследования составило n=19–29; при агрегации, вызванной TRAP, n=26–36. Результаты представлены как медиана и интерквартильный интервал; ***p <0,0001, **p <0,001, *p <0,05; тест Краскела–Уоллиса с последующим тестом Данна для парных сравнений

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4. Рис. 3. Сократительная функция тромбоцитов на разных сроках хранения концентратов тромбоцитов, определённая по способности вызывать сокращение (контракцию) сгустков в плазме крови. Число образцов в дни исследования составило n=12–36. Результаты представлены как медиана и интерквартильный интервал

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5. Рис. 4. А. Митохондриальный потенциал неактивированных тромбоцитов на разных сроках хранения концентратов тромбоцитов. Число образцов в дни исследования составило n=31–41. Б. Содержание внутриклеточного аденозинтрифосфата (АТФ) в концентратах тромбоцитов на разных сроках хранения. Концентрация АТФ нормализована на единицу массы белка в тромбоцитарных лизатах. Число образцов в дни исследования составило n=9–20. Результаты представлены в виде среднего значения и стандартного отклонения (А) и как медиана и интерквартильный интервал (Б)

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6. Рис. 5. Различные морфологические варианты тромбоцитов в составе концентратов тромбоцитов по данным сканирующей электронной микроскопии: А — дисковидные; Б — овальные; В — недеформированные или слегка деформированные с 1–3 филоподиями; Г — сильно деформированные с множественными филоподиями; Д — сферические с шероховатой поверхностью; Е — веретенообразные. Линейки = 1 мкм

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