PB1281 - Static, turbulent and 3D models to study platelet formation and function using genetically modified imMKCL to mimic Glanzmann Thrombasthenia

Tuesday, June 25, 2024

13:45 – 14:45 ICT

Room: Exhibition Hall

Presenting Author(s)

Kato Ramaekers, MA

PhD Candidate
KU Leuven
Leuven, Vlaams-Brabant, Belgium

Co-Author(s)

ML

Marco Lunghi

PhD Student
University of Pavia
Pavia, Lombardia, Italy
CT

Chantal Thys

Technician
KU Leuven
Leuven, Vlaams-Brabant, Belgium
Cv

Chris van Geet

professor
KU Leuven
Leuven, Vlaams-Brabant, Belgium
KP

Kathelijne Peerlinck

professor
KU Leuven
Leuven, Vlaams-Brabant, Belgium
KE

Koji Eto

professor
Kyoto University
Kyoto, Kyoto, Japan
Alessandra Balduini, MD

MD, PhD
University of Pavia, Pavia, Italy
Pavía, Lombardia, Italy
CD

Christian Di Buduo

professor
University of Pavia
Pavia, Lombardia, Italy
Veerle Labarque

Professor
Department of Pediatric Hematology and Oncology, University Hospital Leuven Gasthuisberg, Leuven
ฺฺBrussels, Brussels Hoofdstedelijk Gewest, Belgium
Kathleen Freson

Professor
Department of Cardiovascular Sciences, Centre for Molecular and Vascular Biology, University of Leuven, Leuven, Belgium
Leuven, Vlaams-Brabant, Belgium

Background: The main limitation of next generation sequencing studies for inherited platelet disorders (IPD) remains the detection of numerous variants of unknown significance (VUS) and variants in novel genes with unknown roles in platelets and megakaryocytes (MK). A cell-based model that allows gene/variant characterisation is required that mimics MK/platelet formation and function defects found in IPD patients.

Aims: We aim to develop such a model using genetically modified immortalized megakaryocyte cell lines (imMCKLs) (1). This is a proof-of-concept study for Glanzmann Thrombasthenia (GT), a well-known IPD caused by integrin α2bβ3 defects. We have studied MK/platelet formation and function from ITGB3-/- imMKCLs.

Methods: MKs and platelets from 3 ITGB3-/- imMKCLs were produced in static, turbulent (shaking flasks) and 3D (silk bone marrow model (2)) conditions and characterised using FACS, immunoblotting, and immunostaining. Data from 12 GT patients were analysed retrospectively.

Results: GT MK showed no expression of α2bβ3 and absent binding to fluorescent-labelled fibrinogen. These MK showed normal differentiation, polyploidisation, and expression of GPIb/IX/V in static cultures. Platelet formation from imMKCLs could only be studied in turbulent and 3D conditions. GT imMKCLs seem to produce less platelets in turbulent condition (61±19%) and 3D environment (60±25%), compared to WT (Figure 1A). Likewise, we also observed a platelet count in the lower normal range ( < 180 G/L) in 8 out of 12 GT patients. As expected, there is a trend towards lower PAC1 binding in GT platelets formed in 3D environment (Figure 1B). Interestingly, GT MK secrete significantly less VWF in the medium (Figure 1C). GT proplatelets lack staining of α-granules along their shafts (Figure 1D). Further studies must be undertaken if this also relates to their reduced platelet aggregometry response to ristocetin.

Conclusion(s): This study shows that genetically modified imMKCL can serve as a disease model to study IPD. More studies are needed to define platelet functionality.