Page 197 - Haematologica Vol. 107 - September 2022

ARTICLE - ITP antibody predicts desialylation and apoptosis S.S. Zheng et al.
Antibody-mediated platelet apoptosis has also been sug- gested in ITP. In 2006, Leytin and colleagues reported in the mouse model that monoclonal anti-GPIIb antibody in- jection induced thrombocytopenia, caspase-3 activation, enhanced phosphatidylserine (PS) exposure and mito- chondrial inner transmembrane potential (ΔΨm) depolar- isation.24 In 2012, Winkler et al. demonstrated similar findings in pediatric patients with ITP.25 More recently, pla- telet apoptosis was also confirmed in adult ITP patients by Goette and colleagues.26 However, the relationship be- tween platelet apoptosis and ITP antibody specificity was unclear, as only one patient carried sole anti-GPIb/IX anti- bodies in this study.
Another ITP pathway that has also been previously27 and recently28 described is Fc-independent platelet clearance. Li and co-workers reported platelet desialylation through antibody-induced platelet activation by treating donor platelets with monoclonal anti-GPIb/IX antibodies and ITP sera.28 Using a murine model of ITP secondary to mono- clonal anti-GPIba antibodies, the group demonstrated evi- dence of platelet removal via Ashwell-Morell receptors on hepatocytes, and the use of sialidase inhibitors to attenu- ate thrombocytopenia.28 Although the model is not di- rectly relevant to human disease due to the absence of platelet surface FcγRIIA on mouse platelets and the poly- clonal nature of primary ITP,9,29 these findings still have potential therapeutic implications: patients with anti- GPIb/IX antibodies may respond to sialidase inhibitors while patients with anti-GPIIb/IIIa antibodies were con- sidered unlikely to respond to this novel treatment. Conversely, patients who harbor anti-GPIIb/IIIa antibodies possibly respond to IVIg therapy better than those with sole anti-GPIb/IX antibodies, because anti-GPIIb/IIIa anti- bodies could drive ITP in an Fc-dependent fashion.23,30-32 Similarly, splenectomy may be ineffective in patients with only anti-GPIb/IX autoantibodies as desialylated platelets are removed by the liver.28 This differential effect and, therefore, determining antibody specificity, may influence treatment decision and ultimately patient outcomes. Nevertheless, these views have been recently challenged by Cantoni et al., who studied 93 ITP patients and found no predictability of anti-platelet antibody (APA)-specificity on the site of platelet clearance.33 Thus, ongoing investi- gations to determine whether antibody specificity pre- dicts therapeutic response is vital.
The true extent to which platelet desialylation and/or pla- telet apoptosis are involved in ITP pathology remains unclear. Notably, both desialylated and apoptotic platelets were reported to be removed by the liver.28,34,35 Yet, platelet apoptosis was observed in a murine ITP model induced by monoclonal anti-GPIIb antibodies,24 but desialylation has been demonstrated in ITP patients with anti-GPIba28 and anti-GPIIb/IIIa antibodies.36,37 Whether there is a link between platelet desialylation and platelet intrinsic apop-
tosis in ITP caused by these two antibodies is still not de- fined. In addition, the possibility of using neuraminidase inhibitors in the treatment of ITP is also to be investigated. We recently examined the sera from 61 ITP patients for the presence of APA and their specificities.38 Here, we further scrutinize the sera’s capability to induce platelet desialylation and apoptosis, and studied these two po- tential thrombocytopenic mechanisms’ relationship with the antibody subtypes. Additionally, we established a mouse model to elucidate the therapeutic effect of neur- aminidase inhibitor in preserving human platelet number in the presence of patients’ antibody and provide in vivo data on the feasibility of this agent in the treatment of ITP.
Methods
Patient sample collection
The study was approved by the Human Research and Ani- mal Care Ethics Committees of the University of New South Wales (Sydney, Australia). Sera were obtained from 61 adult ITP patients (aged between 18-90 years) and 21 healthy controls with written informed consent. All 61 pa- tients were diagnosed with ITP according to the inter- national working group’s criteria,39 which was also consistent with the 2019 updated consensus report.40 Whole blood was centrifuged at 860xg for 10 minutes (min). The sera and plasma were stored in aliquots at - 80°C until required for analysis. In some experiments, IgG fraction, purified using Protein G affinity chromatography (Sigma-Aldrich, USA), was used. The purity was over 95% as determined by gel densitometry (ImageJ, Version 2.1.0/1.53c).
Anti-platelet antibody detection and specificity determination
The indirect detection of APA and the determination of antibody specificities in this cohort of patients have re- cently been published.38 Briefly, venous blood was col- lected into 3.2% trisodium citrate. Platelet pellets were washed with wash buffer (pH 6.0) containing 140 mM NaCl, 5 mM KCl, 12 mM trisodium citrate, 10 mM glucose, 12.5 mM sucrose, followed by resuspension in buffer (pH 7.4) containing 140 mM NaCl, 3 mM KCl, 0.5 mM MgCl2, 5 mM NaHCO3, 10 mM glucose and 10 mM HEPES. Washed platelets (1x106) were incubated with patients’ samples at various dilutions for 30 min at 37°C, washed twice, incu- bated with Alexa Fluor 488 or 647-labeled anti-human IgG (Invitrogen, 1:100), washed and analyzed by flow cytometry (LSRFortessaTM X-20 [BD, USA]).
For antibody specificity determination, monoclonal anti- body immobilization of platelet specific antigens assay (MAIPA) was performed as previously described.38,41 Donor platelets were incubated with sera, followed by washing
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