Signal pathways involved in glycoprotein-specific antibody-induced platelet apoptosis

Introduction: In immune thrombocytopenia (ITP), antibodies targeting platelet surface glycoproteins, particularly anti-GPIIb/IIIa and anti-GPIb/IX, are major contributors to platelet destruction. While it is well-established that these antibodies induce platelet clearance, the precise signaling pathways leading to platelet apoptosis remain poorly understood. This study investigates the signaling pathways involved in antibody-induced platelet apoptosis.

Method: Washed human platelets were pretreated for 30 minutes with inhibitors, including IV.3 (an FcgammaRIIA-blocking antibody) and Syk inhibitors (Lanraplenib and R406). Platelets were then stimulated in vitro with monoclonal antibodies targeting GPIIb/IIIa (anti-CD41) or GPIb/IX (anti-CD42b) for 1 hour and 6 hours. Isotype antibodies and dimethyl sulfoxide (DMSO) were used as controls. Apoptosis was assessed using flow cytometry with CD62P, Annexin V, and mitochondrial membrane potential.

Results: Anti-CD41 and anti-CD42b monoclonal antibodies targeting the epitopes on GPIIb and GPIbα, respectively, induced platelet apoptosis (isotype control 1.61%±2.18% vs anti-CD41 17.74%±8.85%, P<0.01; isotype control 10.93%±0.89% vs Anti-CD42b 39.73%±10.07%, P<0.01). Anti-CD41 triggered significant apoptosis after 1 hour of incubation, while anti-CD42b-induced apoptosis was more pronounced after 6 hours. Pretreatment with IV.3, a monoclonal antibody that inhibits the FcgammaRIIA signaling in platelets, reduced anti-CD41-induced apoptosis (isotype control 6.45%±3.79% vs IV.3 1.79%±0.20%, P<0.05) but did not affect apoptosis induced by anti-CD42b monoclonal antibody (isotype control 37.65%±4.52% vs IV.3 43.35%±4.57%, P>0.05). Similarly, Syk inhibitors significantly reduced anti-CD41-mediated apoptosis (vehicle 17.74%±8.85% vs Lanraplenib 1.81%±0.93%, P<0.05; vehicle 23.44%±7.95% vs R406 7.64%±3.90%, P<0.05 ) but had no effect on anti-CD42b-induced apoptosis (vehicle 29.08%±21.02% vs Lanraplenib 39.96%±18.88%, P>0.05; vehicle 29.08%±21.02% vs R406 29.92%±10.48%, P>0.05 ) [1] [2] [3].

Conclusion: Platelet apoptosis triggered by anti-CD41 and anti-CD42b monoclonal antibodies involves distinct signaling pathways. Understanding these distinct mechanisms could inform therapeutic strategies in ITP.

• References

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• 2 Patel P., Shaik N.F., Zhou Y., Golla K., McKenzie S.E., Naik U.P.. 2020; Apoptosis signal-regulating kinase 1 regulates immune-mediated thrombocytopenia, thrombosis, and systemic shock. J Thromb Haemost 18: 11
• 3 Marini I., Zlamal J., Faul C., Holzer U., Hammer S., Pelzl L., Bethge W., Althaus K., Bakchoul T.. 2021; Autoantibody-mediated desialylation impairs human thrombopoiesis and platelet lifespan. Haematologica 106 (01) 196-207