PB1385 - Developing a stem cell-based model for Glanzmann Thrombasthenia to explore VWF-GPIb axis during megakaryopoeisis

Background: Glanzmann Thrombasthenia (GT) is caused by germline variants in the genes coding for fibrinogen receptor integrin α2bβ3. Patients have a bleeding phenotype, and it is widely accepted that GT platelets do not aggregate in response to different agonists, except to ristocetin. We however noticed an aberrant ristocetin response in our 12 GT patients with pathogenic variants in ITGA2B or ITGB3.

Aims: This study aims to develop a disease model for GT. This is a pilot study to explore the use of the immortalized megakaryocyte cell line (imMCKL) as model for GT, and to characterise the VWF-GPIb axis during megakaryocyte (MK) and platelet formation, with as secondary aim to use this for testing variants of unknown significance.

Methods: Functional data from patients were analysed retrospectively. ITGB3 KO imMKCLs were generated using CRISPR/Cas9 and characterised using flow cytometry and immunoblot.

Results: Platelet aggregometry for 12 GT patients showed a mildly decreased response to ristocetin in six patients but a clear reversible response in all (Table 1, Figure 1). The reversible response was present on repeated testing. CD42 measurement by flow cytometry did not show a defect. Two ITGB3 KO imMKCLs were generated that showed a complete depletion of integrin α2b (MFI=0.4%) and β3 (MFI=3.9%) using flow cytometry and immunoblotting after four days of differentiation to mature MKs. A decrease in Fluo 3-AM positive MKs was detected using flow cytometry (WT=50.3%, KO=18.2%), indicating decreased calcium signalling. Surprisingly, a decreased expression in GPIb (MFI=3.8%) and GPIX (MFI=15.4%) was observed in day four ITGB3 KO MKs.

Conclusion(s): Platelets of GT patients show a decreased response to ristocetin. ITGB3 KO MK derived from imMKCL show defective GPIb/IX expression. Proplatelet assays on fibrinogen and VWF, in vivo imaging and platelet function tests of ITGB3 KO MKs are ongoing to further understand these preliminary observations.