Autoantibody-induced desialylation of platelets in immune thrombocytopenia: Potential impact on platelet function and survival

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Dokumentart: PhDThesis
Date: 2023-12-06
Language: English
Faculty: 4 Medizinische Fakultät
Department: Medizin
Advisor: Bakchoul, Tamam (Prof. Dr.)
Day of Oral Examination: 2023-05-24
DDC Classifikation: 610 - Medicine and health
Other Keywords: Thrombozyten
Anitköper-vermittelte Desialinisierung
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Introduction: Immune thrombocytopenia (ITP) is an autoimmune disease that is characterized by a low platelet (PLT)-count and increased bleeding tendency. Antibody-induced PLT desialylation, the loss of sialic acid on the surface of PLT glycoproteins (GPs) has been recently suggested to contribute to an increased PLT depletion in ITP. Based on this previous finding, the presence of desialylating autoantibodies (AAbs) in a larger human ITP cohort as well as the potential impact of ITP AAb-induced PLT desialylation on PLT functionality and PLT survival were investigated in this study. Methods: In a first step, sera from well characterized ITP patients were tested using a lectin binding assay (LBA). After the incubation of PLTs from healthy individuals with ITP or control sera, glycan changes were analyzed via flow cytometry (FC). To investigate the impact of desialylation on PLT functionality, an adhesion assay on the extracellular matrix proteins fibrinogen as well as von Willebrand factor (vWF) was established. To prove whether ITP AAb-induced desialylation affects PLT survival, an NSG mouse model was utilized. Results: 100 ITP sera were investigated in this study. 28/100 sera led to a significant elevation in the exposure of the sugar residues ß-Galactose as well as 21/100 sera to an increased exposure of N-Acetylglucosamine (mean fold FI: 3.50, range: 1.79-13.61, p value 0.0001; FI: 2.41, range: 1.54-5.47, p value 0.0001, respectively). Human ITP AAb-induced PLT desialylation was further identified being not only restricted to AAb targeting GP Ib-IX as also anti-GP IIb/IIIa ITP AAbs were identified to induce desialylation that was significantly reduced via PLT FcγRIIA blockade (% desialylation mean±SEM: 100±0% vs. 52±11%, p value 0.011; 100±0% vs. 60±9%, p value 0.0190, respectively). Moreover, ITP AAb-induced desialylation led to an impaired PLT adhesion function on fibrinogen and vWF, whereas these changes were only moderate in PLTs that were incubated with non-desialylating AAbs (% adherent cells/field mean±SEM: 74±8% vs. 34±7%, p value 0.0021 and 67±5% vs. 26±2%, p value 0.0027, respectively). Notably, the impaired PLT function was significantly reduced in the presence of a sialidase inhibitor (% of adherent cells/field mean±SEM: 36±5% vs. 86±6%, p value 0.0001 and 26±2% vs. 67±11%, p value 0.0203, for fibrinogen and vWF, respectively). Interestingly, the presence of desialylating ITP AAbs was identified to be also associated with low PLT-counts and an increased bleeding tendency in this ITP patient cohort. The biological relevance of PLT desialylation was demonstrated as the injection of desialylating human ITP AAbs resulted in an accelerated clearance of human PLTs in a murine model of ITP. Importantly, the increased PLT depletion was significantly reduced by the pretreatment of PLTs with a sialidase inhibitor that prevents desialylation on the PLT surface (survival of human PLTs after 300 min: 28±5% vs. 45±3%, p value 0.0190, respectively). Conclusion: Our findings indicate that desialylating AAbs, that have the potential to induce significant changes in the glycan pattern of PLTs, are present in a subgroup of ITP patients. Furthermore, this study demonstrates that ITP AAb-induced desialylation seems not be a solely Fc-independent mechanism as also human anti-GP IIb/IIIa ITP AAbs showed the potential to induce significant PLT desialylation in a Fc-dependent pathway. Moreover, the presence of desialylating ITP AAbs was also associated with clinical overt thrombocytopenia, increased bleeding tendency and impaired PLT function. These data suggest that desialylation might result in an increased bleeding phenotype in ITP patients. Most importantly, human ITP AAb-induced changes in the glycan pattern of PLTs were identified to contribute to an increased PLT destruction in an apoptosis independent pathway in vivo, which was significantly reduced via sialidase inhibition. Taken together, findings of this study suggest that a routinely performed screening for the presence of desialylating AAbs and the use of sialidase inhibitors might further dissect the complex pathomechanisms present in ITP patients and be promising for novel ITP treatment approaches.

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