Glycoprotein-specific antibodies differentially induce cell death, alter autophagy and proplatelet formation in human megakaryocytes in vitro

Introduction Immune thrombocytopenia (ITP) is an autoimmune disorder characterized by platelet destruction and impaired platelet production due to autoantibodies (AAbs) targeting megakaryocyte (MK) and platelet specific glycoproteins. While AAbs against CD41/CD61 (GPIIb/IIIa) and CD42a/CD42b (GPIX/GPIbα) are frequently detected in ITP, their direct effects on MK fate remain incompletely understood. We previously demonstrated that antibody binding to these receptors triggers apoptosis in human MKs. Here, we extend these findings by investigating how these antibodies modulate autophagy and proplatelet formation, key processes in MK maturation and platelet biogenesis. Method Human CD34+ hematopoietic stem cells were isolated and differentiated into MKs in serum-free expansion medium supplemented with thrombopoietin (TPO), stem cell factor (SCF), and interleukins (IL-3, IL-6, IL-11). Mature MKs were incubated with different antibodies targeting CD41, CD61, CD42a, and CD42b. Apoptosis was tested by Annexin V/PI flow cytometry, autophagy was analyzed using commercially available autophagy detection kit, and proplatelet formation was evaluated microscopically on fibrinogen-coated surfaces. Results Whereas Antibodies targeting CD42b reduced autophagy levels to 55.3±11.1% of the negative control (set as 100%; P=0.02), corresponding to an approximate 45% reduction in autophagic activity. In contrast, antibodies targeting CD61 showed no consistent effect on autophagy (101.5±17.9% of negative control, P=0.98). We have further tested the effect of antibodies on proplatelet formation in vitro. Preliminary data indicated reduced proplatelet formation after treatment with antibodies that induced apoptosis in human MKs, compared to negative control (9.38±2.1% vs. 16.78±7.4%, respectively). Further experiments are planned to confirm these findings. Conclusion Our findings reveal that specific glycoprotein-targeting antibodies not only induce apoptosis but also alter autophagy and compromise proplatelet formation in human MKs in vitro. These results highlight distinct antibody-mediated pathways affecting MK viability and maturation, offering new insights into impaired thrombopoiesis in ITP. Publication History Article published online: 17 February 2026 © 2026. Thieme. All rights reserved. Georg Thieme Verlag KG Oswald-Hesse-Straße 50, 70469 Stuttgart, Germany