Page 198 - Haematologica Vol. 107 - September 2022

ARTICLE - ITP antibody predicts desialylation and apoptosis S.S. Zheng et al.
and incubation with antibodies against GPIIb/IIIa complex (AP2; Beckman Coulter, USA), GPIX (FMC25, Millipore, USA) or GPV (G-11, Santa Cruz Biotechnology, Inc. USA). The pla- telets were washed, solubilized and incubated in goat anti-mouse IgG Fc fragment-specific antibody (Jackson ImmunoResearch, USA) precoated microtiter plates (ThermoFisher, USA). The reactions were washed and further incubated with goat anti-human IgG Fc fragment- specific-horseradish peroxidase-conjugated antibody (Sigma-Aldrich, USA). SureBlueTM TMB–microwell peroxi- dase substrate (KPL Inc. USA) was then added and stopped at 10 min with 0.18 M sulphuric acid. The absorb- ance was read at dual wavelength (450 nm and 492 nm).
Platelet neuraminidase expression, desialylation and activation
In order to examine sera-induced platelet neuraminidase expression, 1x106 washed donor platelets were incubated with patient or control sera (1:25) for 30 min at 37°C fol- lowed by washing and incubation with anti-NEU1 mouse monoclonal antibody (1:25, Santa Cruz Biotechnology, Inc. USA) for 20 min at room temperature and washing. Simi- larly, for platelet desialylation, the washed sera-donor platelet reactions were incubated with FITC-labeled Rici- nus communis lectin (RCA-1, Vector Laboratories, USA, 0.5 μg/mL). In order to determine the effect of patients’ IgG, control experiments were performed using purified IgG from healthy donors and ITP patients (50 μg/mL). Platelet activation was assessed by flow cytometry with anti-P- selectin monoclonal antibody APC (1:5, eBioscienceTM, Ger- many).
Platelet apoptosis
In order to evaluate ITP patient sera’s effect on platelet mitochondrial inner membrane depolarization potential, 1x106 washed donor platelets in phosphate-buffered saline containing 1 mM MgCl2, 5.6 mM glucose, 10 mM HEPES and 0.1% bovine serum albumin were incubated with patients’ or controls’ sera (1:10) for 30 min at 37°C, followed by washing and incubation with 100 nM DiOC6 (Molecular Probes) for 15 min in the dark. In order to examine the role of FcγR in platelet apoptosis, platelets were pre-incubated with anti-FcγRIIa antibody (clone IV.3, 2.5 μg/mL) for 10 min at 37°C prior to treatment with patients’ sera. In order to determine the effect of patients’ IgG on platelet apop- tosis, experiments were performed using 50 μg/mL of IgG, followed by washing and incubation with 50 nM DiOC6.
NOD/SCID mouse model of immune thrombocytopenia
Human platelets (450x106) were transfused via the tail vein into the non-obese diabetic/severe combined immuno- deficient (NOD/SCID) mice and allowed to stabilize for 2.5 hours (h). Forty μg/g of patients’ or controls’ IgG was then administered intravenously.42 Oseltamivir-treated mice re-
ceived intraperitoneal (IP) injection of 10 μg/g oseltamivir immediately prior to human platelet infusion and again, immediately before IgG injection. Mouse blood was col- lected at time 0 (prior to IgG injection), 2, 4 and 6 h fol- lowing IgG injection. Human platelets were identified using anti-human CD41a V450 (BD Biosciences, USA) by flow cytometry. The percentage of human platelets at time 0 was set at 100%.
Statistical analysis
Flow cytometry data were processed with FlowJo soft- ware (LCC, USA). Data were analyzed with GraphPad Prism version 8 (GraphPad Software, USA). The significance level was set at less than 0.05. Kruskal-Wallis test with Dunn’s multiple comparison was carried out for in vitro desialy- lation and apoptosis experiments. Linear mixed model was used to examine the effect of neuraminidase inhibitor on platelet survival in vivo. Mann Whitney test was applied in other analyses.
Results
Patient characteristics
Of the 61 patients (aged between 18-90 years old) in- cluded in this study, 61% were female. The majority (84%) had primary ITP. Thirty-five patients (57%) had detectable APA in their sera as determined by flow cytometry. MAIPA demonstrated that nine patients had sole anti-GPIIb/IIIa antibodies, five patients had sole anti-GPIb/IX antibodies while seven other patients had antibodies against both complexes. The rest (14 patients, 23%) had no detectable anti-GPIIb/IIIa or anti-GPIb/IX antibodies by MAIPA (Figure 1A).38 Compared to the direct testing,43 the lower detection rate of dual antibody positive patients may reflect the lower sensitivity in antibody determination using indirect method, which is consistent with a prior MAIPA report.41 Recent work demonstrated that GPV is an important tar- get of APA in ITP.44,45 We additionally interrogated patient samples with positive indirect APA (sera from 31 patients were available) for the presence of anti-GPV antibodies. Using a cutoff of 3 standard deviations (SD) over the mean of 20 normal controls, 21 patients were positive for anti- bodies against GPIIb/IIIa, GPIb/IX and/or GPV (Table 1). Seven were found to have anti-GPV antibodies in their sera. However, two patients have co-existing anti- GPIIb/IIIa antibodies, two others have co-existing anti- GPIb/IX antibodies, and one patient had all three antibodies (Table 1; Figure 1B).
Anti-platelet antibodies predict platelet desialylation
In order to determine whether ITP patients’ sera can in- duce desialylation, we treated donor platelets with ITP or control sera and measured NEU1 expression by flow cyto-
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