OC 73.5 - Investigations to assess Impact of Syk-inhibition on antibody-mediated desialylation: novel implications on therapy for immune thrombocytopenia

Wednesday, July 13, 2022

11:15 AM – 11:30 AM

Location: ExCel Center, ICC Capital Suite Room 14-16

Presentation Format: In-Person

Presenting Author(s)

Anurag Singh, PhD

Institute for Clinical and Experimental Transfusion Medicine, University Hospital of Tuebingen, Tuebingen, Germany

Co-Author(s)

FT

Filip Toma

PhD Student
Institute for Clinical and Experimental Transfusion Medicine, University Hospital Tübingen
Tübingen, Baden-Wurttemberg, Germany
Karina Althaus, MD

Physician, Director of Platelet Immunology Lab.
Center for Clinical Transfusion Medicine, University Hospital of Tübingen
Tübingen, Baden-Wurttemberg, Germany
Tamam Bakchoul, MD

Medical Director
Center for Clinical Transfusion Medicine, University Hospital of Tübingen
Tübingen, Baden-Wurttemberg, Germany

Background: Immune thrombocytopenia (ITP) is an autoantibody-mediated bleeding disorder with increased clearance of platelets and megakaryocyte dysfunction. Previously, we showed that antibody-mediated desialylation plays a pivotal role in pathophysiology of ITP, leading to impaired platelet adhesion and megakaryocyte differentiation via FcγRIIA receptor.

Aims: In current study, we aim to investigate whether spleen tyrosine kinase (Syk) inhibition can alter platelet dysfunction and desialylation in ITP.

Methods: Using in vitro cell culture and flow cytometry methods, we analyzed the impact of Syk inhibitors R406 and PRT318. Role of FcγRIIA was studied by crosslinking anti CD32 clone AT-10 with the secondary Fab2 antibody to stimulate the receptor. Platelet function was determined as CD62P and PAC1 expression, and apoptosis as mitochondrial-transmembrane potential (Δψ) depolarization and phosphatidylserine (PS) externalization. Human ITP AAb-induced platelet desialylation was analyzed using a lectin binding assay (LBA) after incubation of platelets with ITP or healthy donor sera and flow cytometry.

Results: Our data shows that Syk inhibition can effectively alter antibody-mediated biological effects in ITP. Activation of FcyRIIA via crosslinking resulted in significant increase in apoptosis and desialylation markers. R406 and PRT318 significantly inhibited apoptosis (mean %PS externalization ±SEM: 27.9±2.1 vs. 9.1±2.3 and 2.1±90.50 respectively, p< 0.001) and desialylation (mean FI±SEM 2.15±0.17 vs 1.25±0.10 and 0.93±0.16 respectively, p< 0.01). Sera from ITP patients induced desialylation in healthy washed platelets, which could be inhibited with both R406 and PRT318 (mean FI±SEM 1.62±0.10 vs 1.13±0.04 and 0.93±0.05 respectively, p< 0.01). In ongoing studies, we explore the opportunity to restore impaired proplatelet formation by megakaryocytes with Syk-inhibition, and investigate the possibility of reverting desialylation in an established ex vivo model of thrombopoiesis.

Conclusion(s): Our data indicates that Syk-inhibition might be a promising approach to prevent antibody-mediated platelet desialylation in ITP and might serve as a potential therapeutic approach to prevent patient complications during pathogenesis of ITP.