Page 199 - Haematologica Vol. 107 - September 2022

ARTICLE - ITP antibody predicts desialylation and apoptosis S.S. Zheng et al.
AB
Figure 1. Antibody pattern by indirect flow cytometry and monoclonal antibody immobilization of platelet-specific antigen assay.
(A) Focusing on GPIIb/IIIa and GPIb/IX in 61 immune thrombocytopenia (ITP) patients, 43% had no detectable antibodies by flow cytometry (Ab negative); of the 35 patients with positive antibody by flow cytometry, 15% had antibodies against GPIIb/IIIa (IIb/IIIa); 8% against GPIb/IX (Ib/IX); 11% had antibodies against both complexes (IIb/IIIa & Ib/IX). (B) Examination of anti-GPV antibody by monoclonal antibody immobilization of platelet-specific antigen assay (MAIPA) in 31 patients with available sera, in relation to anti GPIIb/IIIa and GPIb/IX antibodies. GP: glycoprotein.
metry. NEU1 expression was significantly increased in ITP sera-treated platelets compared to controls (Figure 2A). Further scrutiny of the patient subgroups, determined by the antibody status, demonstrated that NEU1 transloca- tion was significantly different from controls only in pla- telets treated with sera from patients with detectable APA (Figure 2B). Indeed, when we examined the level of pla- telet desialylation, as assessed by RCA-1 binding, we found that only antibody-positive sera could induce de- tectable desialylation (Figure 2C). Of note, a significant difference in RCA-1 binding was not detected when com- paring the whole ITP group (antibody-positive and - negative sera) to normal controls (Figure 2D). This is likely due to the weak binding of lectins (such as RCA-1) to their target sugars.46
We further examined whether the IgG fraction was re- sponsible for platelet desialylation. We used purified IgG
Table 1. Summary of anti-platelet antibodies found in sera as determined by monoclonal antibody immobilization of platelet-specific antigens assay.
Patient N
Patient %
GPIIb/IIIa
6
28.6
GPIb/IX
3
14.3
GPV
2
9.5
GPIIb/IIIa+GPIb/IX
5
23.8
GPIIb/IIIa+GPV
2
9.5
GPIb/IX+GPV
2
9.5
GPIIb/IIIa+GPIb/IX+GPV
1
4.8
Total
21
100.00
GP: glycoprotein.
from two ITP patients containing anti-GPIIb/IIIa antibodies to treat donor’s platelets. Figure 2E shows that the IgG fraction causes significant platelet desialylation. This re- sult confirmed the findings by Marini and colleagues, that ITP patients’ IgG leads to cleavage of platelets’ sialic acid and hence, platelet desialylation.36 Together, these data show that ITP autoantibodies induce NEU1 translocation and platelet desialylation.
Given the previous observation of platelet activation by monoclonal antibodies against GPIba causing platelet de- sialylation,28 we examined the ability for ITP patients’ sera to induce P-selectin externalization. Compared to the control group, there was no enhanced anti-CD62P binding to treated platelets (Figure 2F). Unlike NEU1 expression and desialylation, no difference in platelet activation was noted between sera from patients with or without detect- able APA (Figure 2G). Interestingly, the three sera with the highest P-selectin expression were all from secondary ITP patients (systemic lupus erythematosus [SLE], anti-phos- pholipid syndrome [APS] and B-cell lymphoma), raising the likelihood of distinct disease biology in secondary ITP. Finally, no difference was seen when patients with GPIIb/IIIa APA were compared with those with anti-GPIb/IX antibodies (Figure 2H).
Anti-platelet antibodies predict platelet apoptosis
In order to determine the role of ITP auto-antibodies in platelet apoptosis, washed platelets were treated with either ITP or control sera. Like NEU1 expression, upon treatment with patients’ sera, platelets showed signifi- cantly reduced DiOC6 fluorescence compared to the con- trol (Figure 3A). This denotes the loss of mitochondrial inner membrane potential, ΔΨm, indicating the presence of platelet apoptosis. Consistent with the observations for
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