Antiplatelet antibody predicts platelet desialylation and apoptosis in immune thrombocytopenia

Abstract

Immune thrombocytopenia (ITP) is a bleeding disorder caused by dysregulated B- and T- cell functions, which lead to platelet destruction. A well-recognized mechanism of ITP pathogenesis involves anti-platelet and anti-megakaryocyte antibodies recognizing membrane glycoprotein (GP) complexes, mainly GPIb/IX and GPIIb/IIIa. In addition to the current view of phagocytosis of the opsonised platelets by splenic and hepatic macrophages via their Fc γ receptors, antibodyinduced platelet desialylation and apoptosis have also been reported to contribute to ITP pathogenesis. Nevertheless, the relationship between the specific thrombocytopenic mechanisms and various types of anti-platelet antibodies has not been established. In order to ascertain such association, we used sera from 61 ITP patients and assessed the capacity of anti-platelet antibodies to induce neuraminidase 1 (NEU1) surface expression, RCA-1 lectin binding and loss of mitochondrial inner membrane potential on donors' platelets. Sera from ITP patients with detectable antibodies caused significant platelet desialylation and apoptosis. Anti-GPIIb/IIIa antibodies appeared more capable of causing NEU1 surface translocation while anti-GPIb/IX complex antibodies resulted in a higher degree of platelet apoptosis. In ITP patients with anti-GPIIb/IIIa antibodies, both desialylation and apoptosis were dependent on FcγRIIa signaling rather than platelet activation. Finally, we confirmed in a murine model of ITP that destruction of human platelets induced by anti-GPIIb/IIIa antibodies can be prevented with the NEU1 inhibitor oseltamivir. A collaborative clinical trial is warranted to investigate the utility of oseltamivir in the treatment of ITP.

Figure 1.

Antibody patern 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.

Figure 2.

Effect of immune thrombo-cytopenia patient sera on platelet de-sialylation and activation. (A and B) NEU1 surface translocation and (C and D), RCA-1 lectin binding comparing patient subgroups and controls. (E) Effect of purified immune thrombocytopenia (ITP) immu-noglobulin G (IgG) (50 µg/mL) on RCA-1 lectin binding. (F and G) P-selectin expression on control and patient sera treated platelets. (H) P-selectin expression on platelets treated with GPIIb/IIIa or GPIb/IX antibodies. CTRL: control; Pt: patient; Neg: antibody-negative; Pos: antibody-positive; MFI: mean fluorescence intensity. Data shown as mean ± standard deviation. Levels of significance are expressed as P-values. ns: non-significant, *P<0.05, **, P<0.01. Mann Whitney and Kruskal-Wallis test with Dunn’s multiple comparison.

Figure 3.

Effect of immune thrombocytopenia sera on platelet apoptosis. Loss of mitochondrial inner transmembrane potential (AWm) as measured by DiOC₆ in platelets treated by (A) immune thrombocytopenia (ITP) patients’ (Pt) and controls’ sera (CTRL), as well as (B) antibody-positive (Pos) and antibody-negative (Neg) patients’ sera. (C) Effect of purified ITP immunoglobulin G (IgG) (50 µg/mL) on washed platelets. (D) Histogram representing the effect of anti-FcyRIIa antibody IV.3 on AWm despite the presence of patient sera. (E) Effect of anti-FcyRIIa antibody IV.3 on AWm of 3 patients; data point represents the mean of 3 experiments. MFI: mean fluorescence intensity. Data shown as mean ± standard deviation. Levels of significance are expressed as P-values. ns: non-significant, *P<0.05, ***P<0.001. Mann Whitney and Kruskal-Wallis test with Dunn’s multiple comparison. Repeated measures ANOVA used in 3E.

Figure 4.

Differential effect of anti-GPIIb/IIIa and anti-GPIb/IX antibodies on NEU1 surface translocation and mitochondrial inner transmembrane potential. (A) Effect on NEU1 surface translocation and (B) on mitochondrial inner transmembrane potential (Δψm). CTRL: control. Black horizontal lines represent the means for each group. Green horizontal lines indicate the cutoff for positive samples (2SD over [for NEU1] and under [for DIOC₆] the average of controls). Two patients in the anti-GPIIb/IIIa antibody subgroup and 2 patients in the anti-GPIb/IX subgroup had concomitant anti-GPV antibodies. MFI: mean fluorescence intensity.

Figure 5.

Effect of anti-GPV antibodies on platelet desialylation and apoptosis. (A) Effect on desialytion and (B) on apoptosis. CTRL: control; Pt: patient. Data shown as mean ± standard deviation. ns: non-significant, ***P<0.001. Kruskal-Wallis test with Dunn’s multiple comparison. MFI: mean fluorescence intensity.

Figure 6.

Murine model of immune thrombocytopenia with anti-GPIIb/IIIa antibodies. Effect of 3 immune thrombocytopenia (ITP) patients’ (Pt) immunoglobulin G (IgG) (black dots) and oseltamivir treatment (red dots) in the presence of patient IgG, on human platelet (plt) survival in NOD/SCID mice (n=5 for each patient group) measured as human platelet percentage at 2, 4, and 6 hours after IgG injection. Data shown as mean ± standard error of the mean. Levels of significance are expressed as P-values. ns: nonsignificant, **P<0.01, ****P<0.0001. Linear mixed model. NOD/SCID: non-obese diabetic/severe combined immunodeficient.