PB1317 - Cytockeleton features of native human megakaryocytes from healthy donors and patients with myeloproliferative neoplasms

Tuesday, June 25, 2024

13:45 – 14:45 ICT

Room: Exhibition Hall

Presenting Author(s)

Anna Boldova, PhD

PhD student
Center for Theoretical Problems of Physicochemical Pharmacology
Moscow, Moskva, Russia

Co-Author(s)

SK

Sofya Kuznetsova

Research fellow
Center for Theoretical Problems of Physicochemical Pharmacology
Moscow, Moskva, Russia
OF

Olga Fedyanina

Research fellow
Center for Theoretical Problems of Physicochemical Pharmacology
Moscow, Moskva, Russia
AP

Alexey Pshonkin

Hematologist, Head of day hospital department, P.h.D.
Dmitry Rogachev National Medical Research Center Of Pediatric Hematology, Oncology and Immunology
Moscow, Moskva, Russia
MP

Mikhail Panteleev

Director
Center for Theoretical Problems of Physico-Сhemical Pharmacology
Moscow, Moskva, Russia
NS

Natalia S. Smetanina

Professor
Dmitry Rogachev National Research Center of Pediatric Hematology, Oncology and Immunology
Moscow, Moskva, Russia
AS

Anastasia N. Sveshnikova

Head of Laboratory
Dmitry Rogachev National Medical Research Center Of Pediatric Hematology, Oncology and Immunology, Moscow, Russia
Moscow, Moskva, Russia

Background: Megakaryocyte (MK) maturation depends on their interactions with extracellular matrix, particularly with collagen. Mature MKs give rise to platelets via tubulin-dependent protrusions. Other actin-based protrusions, named podosomes, are important for extracellular matrix degradation and MK motility. In patients with myeloproliferative neoplasms (MPN) high platelet counts are caused by increased MK numbers, but the details of MK morphology and cytoskeleton structure in MPN remain unexplored.

Aims: Evaluation of MK cytoskeleton features in patients with MPN.

Methods: MKs were isolated from bone marrow by incubation of mononuclear fraction of human bone-marrow aspirate with a protein microarray with immobilized monafram (F(ab')2 fragment of anti-GP IIb-IIIa monoclonal antibody), type I or type III collagen, fibrinogen alone or in combination with monafram. After incubation the unbound cell were washed away and the captured MK were fixed with PFA, permeabilized, stained by fluorescently-labeled antibodies against β1-tubulin and phalloidin-Alexa647 and analyzed by Nikon-AX scanning confocal microscope.

Results: Monafram-captured MKs of healthy donors show actin stress fibers, single or clustered podosomes and convoluted microtubules in line with published data. For MKs from patients with MPN podosomes were observed on all coatings and frequently formed complicated structures like rosettes and belts. The number of rosettes and single podosomes were higher on both collagens compared to fibrinogen. The rosette size was significantly lower on fibrinogen. No differences in cytoskeleton features were observed between collagen type I and type III coatings.

Conclusion(s): MKs isolated from the bone marrow of MPN patients have similar cytoskeleton structure to MKs of healthy donors, namely, they form complex structures of podosomes, the number of which increased on collagen coatings compared to fibrinogen.

Funding: The study was supported by the endowment foundation "Science for Children".
Ethics. The study was performed in accordance with the Declaration of Helsinki and approved by the NMRC PHOI ethics committee.