Pediatric Hematology/Oncology and ImmunopathologyPediatric Hematology/Oncology and ImmunopathologyВопросы гематологии/онкологии и иммунопатологии в педиатрии1726-17082414-9314Fund Doctors, Innovations, Science for Children51110.24287/1726-1708-2021-20-2-65-73ORIGINAL ARTICLESОРИГИНАЛЬНЫЕ СТАТЬИPlatelet phenotype in children with ANKRD26-related thrombocytopeniaОсобенности фенотипа тромбоцитов у детей с ANKRD26-ассоциированной тромбоцитопениейhttps://orcid.org/0000-0001-6905-2878PolokhovD. M.ПолоховД. М.
Russian Federation

Dmitry М. Polokhov, pathologist, laboratory of clinical hemostasis

 1 Samory Mashela St., Moscow 117997

Полохов Дмитрий Михайлович, врач клинической лабораторной диагностики лаборатории клинического гемостаза

117997, Москва, ул. Саморы Машела, 1

dmitrii.polokhov@gmail.comhttps://orcid.org/0000-0003-4567-1871FedorovaD. V.ФедороваД. В.
Russian Federation

 1 Samory Mashela St., Moscow 117997

117997, Москва, ул. Саморы Машела, 1

https://orcid.org/0000-0002-2057-2036PshonkinA. V.ПшонкинА. В.
Russian Federation

 1 Samory Mashela St., Moscow 117997

117997, Москва, ул. Саморы Машела, 1

https://orcid.org/0000-0002-5217-3937IgnatovaA. A.ИгнатоваА. А.
Russian Federation

 1 Samory Mashela St., Moscow 117997

117997, Москва, ул. Саморы Машела, 1

https://orcid.org/0000-0002-8697-7570PonomarenkoE. A.ПономаренкоЕ. А.
Russian Federation

 1 Samory Mashela St., Moscow 117997

117997, Москва, ул. Саморы Машела, 1

https://orcid.org/0000-0002-2521-5353AleksenkoM. Yu.АлексенкоМ. Ю.
Russian Federation

 1 Samory Mashela St., Moscow 117997

117997, Москва, ул. Саморы Машела, 1

https://orcid.org/0000-0003-0471-2956MersiyanovaI. V.МерсияноваИ. В.
Russian Federation

 1 Samory Mashela St., Moscow 117997

117997, Москва, ул. Саморы Машела, 1

https://orcid.org/0000-0002-7534-3863SereginaE. A.СерегинаЕ. А.
Russian Federation

 1 Samory Mashela St., Moscow 117997

117997, Москва, ул. Саморы Машела, 1

VoroninK. A.ВоронинК. А.
Russian Federation

 1 Samory Mashela St., Moscow 117997

117997, Москва, ул. Саморы Машела, 1

https://orcid.org/0000-0001-5209-2099PoletaevA. V.ПолетаевА. В.
Russian Federation

 1 Samory Mashela St., Moscow 117997

117997, Москва, ул. Саморы Машела, 1

https://orcid.org/0000-0002-7634-2053RaykinaE. V.РайкинаЕ. В.
Russian Federation

 1 Samory Mashela St., Moscow 117997

117997, Москва, ул. Саморы Машела, 1

https://orcid.org/0000-0002-8128-7757PanteleevM. A.ПантелеевМ. А.
Russian Federation

 1 Samory Mashela St., Moscow 117997

117997, Москва, ул. Саморы Машела, 1

https://orcid.org/0000-0003-4384-6754ZharkovP. A.ЖарковП. А.
Russian Federation

 1 Samory Mashela St., Moscow 117997

117997, Москва, ул. Саморы Машела, 1

Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology and Immunology, Ministry of Healthcare of the Russian FederationФГБУ «Национальный медицинский исследовательский центр детской гематологии, онкологии и иммунологии им. Дмитрия Рогачева» Минздрава РоссииCenter for Theoretical Problems of Physicochemical PharmacologyФГБУН «Центр теоретических проблем физико-химической фармакологии» РАНLomonosov Moscow State UniversityФГБОУ ВО «Московский государственный университет им. М.В. Ломоносова»Moscow Institute of Physics and TechnologyФГАОУ ВО «Московский физико-технический институт (национальный исследовательский университет)»2205202120265732105202121052021Copyright ©; 2021, «D. Rogachev NMRCPHOI»Copyright ©; 2021, ФГБУ «НМИЦ ДГОИ им. Дмитрия Рогачева» Минздрава России2021«D. Rogachev NMRCPHOI»ФГБУ «НМИЦ ДГОИ им. Дмитрия Рогачева» Минздрава Россииhttps://creativecommons.org/licenses/by/4.0https://hemoncim.com/jour/article/view/511

The mechanisms of hemorrhagic manifestations in patients with ANKRD26associated thrombocytopenia (ANKRD26-AT) are poorly understood. The aim of this work is to detect possible morpho-functional disorders of platelets in patients with mutations in the ANKRD26gene by flow cytometry with activation. The study was approved by the Independent Ethics Committee of the Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology and Immunology. 8 children aged from 1.5 to 15 years were examined. The platelet count ranged from 29 to 172 thousand/μl, with a median of 60 thousand/μl. The severity of hemorrhagic manifestations was assessed on a standardized scale (Pediatric Bleeding Questionnaire, PBQ) and it ranged from 0 to 5 points, with a median of 3.5 points. Platelet activation was performed with a CRP + TRAP mixture. Comparison was carried out with the results of examination of 26 apparently healthy children (control group, CG) aged 2 to 15 years. When compared with CG, patients showed an increase in platelet size (FSC; p= 0.018) and granularity (SSC; p< 0.001) after activation. In contrast to the CG, the correlation between FSC and SSC of platelets in patients was not significant (cor. = 0.55; p= 0.15). Patients showed a high, significant relationship between the number and FSC of platelets (cor. = –0.93; p< 0.001), as well as an increased density of CD42b (p < 0.001) and a decrease in the proportion of procoagulant platelets (p= 0.01) after activation. The revealed changes indicate violations of the mechanisms of activation and shape changes of platelets in patients with ANKRD26-AT.

Механизмы геморрагических проявлений у пациентов с ANKRD26-ассоциированной тромбоцитопенией (ANKRD26-AT) плохо изучены. Цель работы: провести поиск возможных морфофункциональных нарушений тромбоцитов у пациентов с мутациями в гене ANKRD26методом проточной цитометрии с активацией. Данное исследование одобрено локальным этическим комитетом и утверждено решением ученого совета НМИЦ ДГОИ им. Дмитрия Рогачева. Обследованы 8 детей в возрасте от 1,5 до 15 лет. Количество тромбоцитов варьировало от 29 до 172 тыс/мкл, медиана 60 тыс/мкл. Тяжесть геморрагических проявлений оценивали по стандартизированной шкале Pediatric Bleeding Questionnaire (PBQ) и она варьировала от 0 до 5 баллов, медиана 3,5 балла. Активация тромбоцитов производилась смесью CRP + TRAP. Сравнение проводили с результатами обследования 26 условно здоровых детей (контрольная группа, КГ) в возрасте от 2 до 15 лет. При сравнении с КГ у пациентов отмечалось увеличение размера (FSC; р= 0,018) и гранулярности (SSC;p< 0,001) тромбоцитов после активации. В отличие от КГ корреляционная связь между FSC и SSC тромбоцитов пациентов была недостоверной (корреляция 0,55; p= 0,15). У пациентов наблюдалась высокая достоверная связь между количеством и FSC тромбоцитов (корреляция –0,93; p< 0,001), а также повышенная плотность CD42b (p< 0,001) и снижение доли прокоагулянтных тромбоцитов (p= 0,01) после активации. Выявленные изменения указывают на нарушения механизмов активации и изменения формы тромбоцитов у пациентов с ANKRD26-AT.

hereditary thrombocytopeniachildrenANKRD26flow cytometryhemorrhagic syndromeнаследственная тромбоцитопениядетиANKRD26проточная цитометриягеморрагический синдром1. Botero J.P., Stefanie N.D., Matthew W.A. ANKRD26-Related Thrombocytopenia 2018 Jun 21. GeneReviews 2019; 1–11.Botero J.P., Stefanie N.D., Matthew W.A. ANKRD26-Related Thrombocytopenia 2018 Jun 21. GeneReviews 2019; 1–11.2. Noris P., Perrotta S., Seri M., Pecci A., Gnan C., Loffredo G., et al. Mutations in ANKRD26are responsible for a frequent form of inherited thrombocytopenia: Analysis of 78 patients from 21 families. Blood 2011; 117 (24): 6673–80.Noris P., Perrotta S., Seri M., Pecci A., Gnan C., Loffredo G., et al. Mutations in ANKRD26are responsible for a frequent form of inherited thrombocytopenia: Analysis of 78 patients from 21 families. Blood 2011; 117 (24): 6673–80.3. Balduini C.L., Melazzini F., Pecci A. Inherited thrombocytopenias – recent advances in clinical and molecular aspects. Platelets 2017; 28: 3–13.Balduini C.L., Melazzini F., Pecci A. Inherited thrombocytopenias – recent advances in clinical and molecular aspects. Platelets 2017; 28: 3–13.4. Bluteau D., Balduini A., Balayn N., Currao M., Nurden P., Deswarte C., et al. Thrombocytopenia-associated mutations in the ANKRD26regulatory region induce MAPK hyperactivation. J Clin Invest 2014; 124: 580–91.Bluteau D., Balduini A., Balayn N., Currao M., Nurden P., Deswarte C., et al. Thrombocytopenia-associated mutations in the ANKRD26regulatory region induce MAPK hyperactivation. J Clin Invest 2014; 124: 580–91.5. Balduini C., Pecci A., Noris P. Diagnosis and management of inherited thrombocytopenias. Semin Thromb Hemost 2013; 39: 161–71.Balduini C., Pecci A., Noris P. Diagnosis and management of inherited thrombocytopenias. Semin Thromb Hemost 2013; 39: 161–71.6. Boutroux H., Petit A., Auvrignon A., Lapillonne H., Ballerini P., Favier R., Leverger G. Childhood diagnosis of genetic thrombocytopenia with mutation in the ankyrine repeat domain 26 gene. Eur J Pediatr 2015; 174: 1399–403.Boutroux H., Petit A., Auvrignon A., Lapillonne H., Ballerini P., Favier R., Leverger G. Childhood diagnosis of genetic thrombocytopenia with mutation in the ankyrine repeat domain 26 gene. Eur J Pediatr 2015; 174: 1399–403.7. Rabbolini D., Connor D., Morel-Kopp M.С., Donikian D., Kondo M., Chen W., et al. An integrated approach to inherited platelet disorders: results from a research collaborative, the Sydney Platelet Group. Pathology 2020; 52: 243–55.Rabbolini D., Connor D., Morel-Kopp M.С., Donikian D., Kondo M., Chen W., et al. An integrated approach to inherited platelet disorders: results from a research collaborative, the Sydney Platelet Group. Pathology 2020; 52: 243–55.8. Botero J.P., Chen D., He R., Viswanatha D.S., Majerus J.A., Coon L.M., et al. Clinical and laboratory characteristics in congenital ANKRD26mutation-associated thrombocytopenia: A detailed phenotypic study of a family. Platelets 2016; 27: 712–5.Botero J.P., Chen D., He R., Viswanatha D.S., Majerus J.A., Coon L.M., et al. Clinical and laboratory characteristics in congenital ANKRD26mutation-associated thrombocytopenia: A detailed phenotypic study of a family. Platelets 2016; 27: 712–5.9. Ignatova A.A., Ponomarenko E.A., Polokhov D.M., Suntsova E.V., Zharkov P.A., Fedorova D.V., et al. Flow cytometry for pediatric platelets. Platelets 2019, 30 (4): 428–37.Ignatova A.A., Ponomarenko E.A., Polokhov D.M., Suntsova E.V., Zharkov P.A., Fedorova D.V., et al. Flow cytometry for pediatric platelets. Platelets 2019, 30 (4): 428–37.10. Полохов Д.М., Пшонкин А.В., Игнатова А.А., Пономаренко Е.А., Федо-рова Д.В., Алексенко М.Ю. и др. Особенности пула хранения и морфологии тромбоцитов у детей с неуточненным геморрагическим синдромом. Вопросы гематологии/онкологии и иммунопатологии в педиатрии 2021; 20 (1): 58–67.Полохов Д.М., Пшонкин А.В., Игнатова А.А., Пономаренко Е.А., Федо-рова Д.В., Алексенко М.Ю. и др. Особенности пула хранения и морфологии тромбоцитов у детей с неуточненным геморрагическим синдромом. Вопросы гематологии/онкологии и иммунопатологии в педиатрии 2021; 20 (1): 58–67.11. Suntsova E.V., Demina I.M., Ignatova A.A., Ershov N.M., Trubina N.M., Dobrynina J., et al. Bleeding tendency and platelet function during treatment with romiplostim in children with severe immune thrombocytopenic purpura. Int J Hematol 2017, 105: 841–8.Suntsova E.V., Demina I.M., Ignatova A.A., Ershov N.M., Trubina N.M., Dobrynina J., et al. Bleeding tendency and platelet function during treatment with romiplostim in children with severe immune thrombocytopenic purpura. Int J Hematol 2017, 105: 841–8.12. Mittal N., Naridze R., James P., Shott S., Valentino L.A. Utility of a Paediatric Bleeding Questionnaire as a screening tool for von Willebrand disease in apparently healthy children. Haemophilia 2015; 21: 806–11.Mittal N., Naridze R., James P., Shott S., Valentino L.A. Utility of a Paediatric Bleeding Questionnaire as a screening tool for von Willebrand disease in apparently healthy children. Haemophilia 2015; 21: 806–11.13. Рыжкова О.П., Кардымон О.Л., Прохорчук Е.Б., Коновалов Ф.А., Масленников А.Б., Степанов В.А. и др. Руководство по интерпретации данных последовательности ДНК человека, полученных методами массового параллельного секвенирования (MPS) (редакция 2018, версия 2). Медицинская генетика 2019; 8: 3–23.Рыжкова О.П., Кардымон О.Л., Прохорчук Е.Б., Коновалов Ф.А., Масленников А.Б., Степанов В.А. и др. Руководство по интерпретации данных последовательности ДНК человека, полученных методами массового параллельного секвенирования (MPS) (редакция 2018, версия 2). Медицинская генетика 2019; 8: 3–23.14. Carubbi C., Masselli E., Vitale M. Flow Cytometry. In: Gresele P., Kleiman N.S.,Lopez J.A., Page C.P. (eds.). Platelets in Thrombotic and Non-Thrombotic Disorders. Pathophysiology, Pharmacology and Therapeutics: an Update. Springer International Publishing, Cham; 2017. Рp. 589–617.Carubbi C., Masselli E., Vitale M. Flow Cytometry. In: Gresele P., Kleiman N.S.,Lopez J.A., Page C.P. (eds.). Platelets in Thrombotic and Non-Thrombotic Disorders. Pathophysiology, Pharmacology and Therapeutics: an Update. Springer International Publishing, Cham; 2017. Рp. 589–617.15. Ignatova A.A., Karpova O.V., Trakhtman P.E., Rumiantsev S.A., Panteleev M.A. Functional characteristics and clinical effectiveness of platelet concentrates treated with riboflavin and ultraviolet light in plasma and in platelet additive solution. Vox Sang 2016; 110: 244–52.Ignatova A.A., Karpova O.V., Trakhtman P.E., Rumiantsev S.A., Panteleev M.A. Functional characteristics and clinical effectiveness of platelet concentrates treated with riboflavin and ultraviolet light in plasma and in platelet additive solution. Vox Sang 2016; 110: 244–52.16. López J.A. The Platelet Glycoprotein Ib-IX-V Complex. In: Gresele P., Kleiman N.S., Lopez J.A., Page C.P. (eds.). Platelets in Thrombotic and Non-Thrombotic Disorders. Pathophysiology, Pharmacology and Therapeutics: an Update. Springer International Publishing, Cham; 2017. Рp. 85–97.López J.A. The Platelet Glycoprotein Ib-IX-V Complex. In: Gresele P., Kleiman N.S., Lopez J.A., Page C.P. (eds.). Platelets in Thrombotic and Non-Thrombotic Disorders. Pathophysiology, Pharmacology and Therapeutics: an Update. Springer International Publishing, Cham; 2017. Рp. 85–97.17. Bennett J.S. GPIIb/IIIa Structure and Function. In: Gresele P., Kleiman N.S., Lopez J.A., Page C.P. (eds.). Platelets in Thrombotic and Non-Thrombotic Disorders. Pathophysiology, Pharmacology and Therapeutics: an Update. Springer International Publishing, Cham; 2017. Рp 99–112.Bennett J.S. GPIIb/IIIa Structure and Function. In: Gresele P., Kleiman N.S., Lopez J.A., Page C.P. (eds.). Platelets in Thrombotic and Non-Thrombotic Disorders. Pathophysiology, Pharmacology and Therapeutics: an Update. Springer International Publishing, Cham; 2017. Рp 99–112.18. Flaumenhaft R. Platelet Secretion. In: Gresele P., Kleiman N.S., Lopez J.A., Page C.P. (eds.). Platelets in Thrombotic and Non-Thrombotic Disorders. Pathophysiology, Pharmacology and Therapeutics: an Update. Springer International Publishing, Cham; 2017. Рp. 353–366.Flaumenhaft R. Platelet Secretion. In: Gresele P., Kleiman N.S., Lopez J.A., Page C.P. (eds.). Platelets in Thrombotic and Non-Thrombotic Disorders. Pathophysiology, Pharmacology and Therapeutics: an Update. Springer International Publishing, Cham; 2017. Рp. 353–366.19. Heemskerk J.W.M., Cosemans J.M.E.M., van der Meijden P.E.J. Platelets and Coagulation. In: Gresele P., Kleiman N.S., Lopez J.A., Page C.P. (eds.). Platelets in Thrombotic and Non-Thrombotic Disorders. Pathophysiology, Pharmacology and Therapeutics: an Update. Springer International Publishing, Cham; 2017. Рp. 447–462.Heemskerk J.W.M., Cosemans J.M.E.M., van der Meijden P.E.J. Platelets and Coagulation. In: Gresele P., Kleiman N.S., Lopez J.A., Page C.P. (eds.). Platelets in Thrombotic and Non-Thrombotic Disorders. Pathophysiology, Pharmacology and Therapeutics: an Update. Springer International Publishing, Cham; 2017. Рp. 447–462.20. Aslan J.E. Platelet Shape Change. In: Gresele P., Kleiman N.S., Lopez J.A., Page C.P. (eds.). Platelets in Thrombotic and Non-Thrombotic Disorders. Pathophysiology, Pharmacology and Therapeutics: an Update. Springer International Publishing, Cham; 2017. Рp. 321–336.Aslan J.E. Platelet Shape Change. In: Gresele P., Kleiman N.S., Lopez J.A., Page C.P. (eds.). Platelets in Thrombotic and Non-Thrombotic Disorders. Pathophysiology, Pharmacology and Therapeutics: an Update. Springer International Publishing, Cham; 2017. Рp. 321–336.21. Polokhov D.M., Ershov N.M., Ignatova A.A., Ponomarenko E.A., Gaskova M.A., Zharkov P.A., et al. Platelet function and blood coagulation system status in childhood essential thrombocythemia. Platelets 2019; 31 (8): 1001–11.Polokhov D.M., Ershov N.M., Ignatova A.A., Ponomarenko E.A., Gaskova M.A., Zharkov P.A., et al. Platelet function and blood coagulation system status in childhood essential thrombocythemia. Platelets 2019; 31 (8): 1001–11.22. Chen M.H., Yanek L.R., Backman J.D., Eicher J.D., Huffman J.E., Ben-Shlomo Y., et al. Exome-chip meta-analysis identifies association between variation in ANKRD26and platelet aggregation. Platelets 2019; 30: 164–73.Chen M.H., Yanek L.R., Backman J.D., Eicher J.D., Huffman J.E., Ben-Shlomo Y., et al. Exome-chip meta-analysis identifies association between variation in ANKRD26and platelet aggregation. Platelets 2019; 30: 164–73.23. Mazepa M., Hoffman M., Monroe D. Superactivated platelets: Thrombus regulators, thrombin generators, and potential clinical targets. Arterioscler Thromb Vasc Biol 2013; 33: 1747–52.Mazepa M., Hoffman M., Monroe D. Superactivated platelets: Thrombus regulators, thrombin generators, and potential clinical targets. Arterioscler Thromb Vasc Biol 2013; 33: 1747–52.24. Daskalakis M., Colucci G., Keller P., Rochat S., Silzle T., Biasiutti F.D., et al. Decreased generation of procoagulant platelets detected by flow cytometric analysis in patients with bleeding diathesis. Cytom Part B Clin Cytom 2014; 86: 397–409.Daskalakis M., Colucci G., Keller P., Rochat S., Silzle T., Biasiutti F.D., et al. Decreased generation of procoagulant platelets detected by flow cytometric analysis in patients with bleeding diathesis. Cytom Part B Clin Cytom 2014; 86: 397–409.25. Prodan C.I., Vincent A.S., Padmanabhan R., Dale G.L. Coated-platelet levels are low in patients with spontaneous intracerebral hemorrhage. Stroke 2009; 40: 2578–80.Prodan C.I., Vincent A.S., Padmanabhan R., Dale G.L. Coated-platelet levels are low in patients with spontaneous intracerebral hemorrhage. Stroke 2009; 40: 2578–80.26. Prodan C.I., Vincent A.S., Dale G.L. Coated platelet levels correlate with bleed volume in patients with spontaneous intracerebral hemorrhage. Stroke 2010; 41: 1301–3.Prodan C.I., Vincent A.S., Dale G.L. Coated platelet levels correlate with bleed volume in patients with spontaneous intracerebral hemorrhage. Stroke 2010; 41: 1301–3.27. Prodan C.I., Vincent A.S., Dale G.L. Coated-platelet levels are elevated in patients with transient ischemic attack. Transl Res 2011; 158: 71–5.Prodan C.I., Vincent A.S., Dale G.L. Coated-platelet levels are elevated in patients with transient ischemic attack. Transl Res 2011; 158: 71–5.28. Prodan C.I., Joseph P.M., Vincent A.S., Dale G.L. Coated-platelets in ischemic stroke: Differences between lacunar and cortical stroke. J Thromb Haemost 2008; 6: 609–14.Prodan C.I., Joseph P.M., Vincent A.S., Dale G.L. Coated-platelets in ischemic stroke: Differences between lacunar and cortical stroke. J Thromb Haemost 2008; 6: 609–14.29. Prodan C.I., Stoner J.A., Cowan L.D., Dale G.L. Lower coated-platelet levels are associated with early hemorrhagic transformation in patients with non-lacunar brain infarction. J Thromb Haemost 2010; 8: 1185–90.Prodan C.I., Stoner J.A., Cowan L.D., Dale G.L. Lower coated-platelet levels are associated with early hemorrhagic transformation in patients with non-lacunar brain infarction. J Thromb Haemost 2010; 8: 1185–90.