ALBERT

Description: Background: The low platelet count in autoimmune thrombocytopenia (ITP) is caused by enhanced destruction of opsonised platelets in the spleen upon binding of the anti-platelet autoantibodies (AAbs) to the glycoproteins (GPs) express on PLT's surface. Data from animal model suggested that desialylation may contribute to PLT destruction in ITP. However, accumulating evidence suggests that reduction of PLT generation from megakaryocytes (MKs) in bone morrow is also responsible thrombocytopenia in ITP. Based on these considerations, we hypothesized that AAb-mediated desialylation of the GPs expressed on PLT and MKs may interfere with PLT formation and life span. Methods: Sera from 100 ITP patients were investigated in this study. AAb-induced desialylation was detected using a lectin binding assay (LBA) by flow cytometry (FC). To investigate the impact of desialylation on the life-span of human PLTs, the NSG mouse model was used. PLTs and MKs functions were assessed after AAb treatment using proplatelet formation test and adhesion assays on different surfaces. Results: Sera from 35/100 (35%) ITP patients induced cleavage of sialic acid from PLT surface. Injection of desialylating AAbs in vivo resulted in accelerated clearance of human PLTs which was significantly reduced by a specific sialidase inhibitor that prevents desialylation on the PLT surface (survival after 5h: 29%, range 22-40% vs. 48%, range 41-53%, p=0.014, respectively). Desialylating AAbs caused a significant reduction in PLT adhesion to fibrinogen and von Willebrand factor (mean of % adherent PLTs compared to control IgG: 34±6%, p=0.004 and 26±2%, p=0.001, respectively). Interestingly, PLT adhesion was recovered in the presence of a sialidase inhibitor (mean of % adherent PLTs: 86±6%, p=0.001 and 67±10, p=0.020, respectively). IgG fractions from 7/10 (70%) ITP-sera were able to cleave sialic acid and induce exposure of ß-galactose residues on CD34+-derived MKs. Desialylating AAbs induced lower ability to form proplatelet extensions compared to control IgG, which was significantly increased in the presence of the sialidase inhibitor (mean of % proplatelet forming MKs: 42±11% vs. 90±9%, p=0.032, respectively). Conclusion: Our findings show that AAbs from a subgroup of ITP patients are not only able to cleave sialic acid on surface of human PLTs, but also on MKs leading to accelerate PLT destruction and impaired thrombopoiesis, respectively. In addition, we observed that AAb-mediated receptor desialyation interferes with cell interaction with extracellular matrix proteins leading to impaired PLT adhesion, MK differentiation and thrombopoiesis. These novel findings highlight the multiple effects of AAbs in ITP and add to the existing evidence that ITP is rather a group of disorders sharing common characteristics, namely loss of immune tolerance toward PLT and MK antigens and increased bleeding tendency. Disclosures No relevant conflicts of interest to declare.

Description: The pathophysiology of COVID-19 associated thrombosis seems to be multifactorial. We hypothesized that COVID-19 is accompanied by procoagulant platelets and platelet apoptosis with subsequent alteration of the coagulation system. We investigated depolarization of mitochondrial inner transmembrane potential (ΔΨm), cytosolic calcium (Ca2+) concentration, and phosphatidylserine (PS) externalization by flow cytometry. Platelets from intensive care unit (ICU) COVID-19 patients (n=21) showed higher ΔΨm depolarization, cytosolic Ca2+ concentration and PS externalization, compared to healthy controls (n=18) and COVID-19 non-ICU patients (n=4). Moreover significant higher cytosolic Ca2+ concentration and PS was observed compared to septic ICU control group (ICU control). In ICU control group (n=5; ICU non-COVID-19) cytosolic Ca2+ concentration and PS externalization was comparable to healthy control, with an increase in ΔΨm depolarization. Sera from ICU COVID-19 patients induced significant increase in apoptosis markers (ΔΨm depolarization, cytosolic Ca2+ concentration and PS externalization) compared to healthy volunteer and septic ICU control. Interestingly, immunoglobulin G (IgG) fractions from COVID-19 patients induced an Fc gamma receptor IIA dependent platelet apoptosis (ΔΨm depolarization, cytosolic Ca2+ concentration and PS externalization). Enhanced PS externalization in platelets from ICU COVID-19 patients was associated with increased sequential organ failure assessment (SOFA) score (r=0.5635) and D-Dimer (r=0.4473). Most importantly, patients with thrombosis had significantly higher PS externalization compared to those without. The strong correlations between procoagulant platelet and apoptosis markers and increased D-Dimer levels as well as the incidence of thrombosis may indicate that antibody-mediated platelet apoptosis potentially contributes to sustained increased thromboembolic risk in ICU COVID-19 patients.