The Level And Clinical Significance Of Platelet Glycoprotein Desialylation In Primary Immune Thrombocytopenia

BackgroundPrimary immune thrombocytopenia (ITP) is an autoimmune hemorrhagic disease characterized by increased platelet destruction and impaired platelet production. Autoantibodies produced by autoreactive B cells, mainly against platelet surface glycoprotein, GP Ⅱ b/Ⅲa-integrin (70-80%) and/or GPⅠb/Ⅸ-complex(20-40%), play critical roles in the pathophysiology of ITP. Platelets are opsonized by autoantibodies and destructed in the reticuloendothelial and complement system via Fcγ-receptors results in thrombocytopenia and bleeding disorders. Additionally, CD8+cytotoxic T lymphocytes-mediated platelet lysis and decreased platelet production also contribute to thrombocytopenia in ITP. However, a recent study in murine ITP-models suggested that there is an other mechanism can induce thrombocytopenia via an Fc-independent pathway, which is association with the platelet glycoprotein desialylation. Platelet suface-glycoproteins, the sugar ehain terminal of which are covered-by sialic acid, have important effects in activation, aggregation and other functions of platelets. Desialylation of platelet GPs, the process of cleaving terminal sialic acids at the glycosidic bond from platelet GPs, was shown to be involved in platelet clearance. It resulted in the exposure of glycan ligands including beta-galactose(β-Gal) and beta(1-4)-N-acetylglucosamine (β-GlcNAc), which could initiate platelet recognition by asialoglycoprotein receptors (ASGPRs)-expressing hepatocytes, leading to platelet destruction in liver, thereby affecting platelet lifetime. Desialylation was proved to be responsible for rapid clearance of chilled platelets after transfusion and thrombocytopenia caused by free radicals or infection. Hypothesize that in addition to immune-mediated platelet cleared, the platelet membrane glycoprotein desialylation may also be involved in the pathogenesis of ITP. In this study, we analyzed the desialylation level of platelets in ITP by flow cytometry. The anti-platelet autoantibody specificity by modified monoclonal antibody-specific immobilization of platelet antigens (MAIPA) was used to detect the specific anti-platelet GPⅡb/Ⅲa and/or GP Ⅰb/Ⅺ autoantibodies as described previously. Patients’responses to treatment were also assessed. Here, we will confirm the level and clinical significance of platelet glycoprotein desialylation in primary immune thrombocytopenia. However, desialylation status of platelets and its roles in ITP have not been elucidated so far.ObjectiveTo investigate the expression level and clinical significance of platelet glycoprotein desialylation in patients with primary immune thrombocytopenia(ITP).①Desialylation level of platelets between patients with ITP and controls.②Whether there is a correlation between the desialylation level of platelets and anti-platelet autoantibody specificity.③Desialylation level of platelets in ITP between post treatment with those before treatment. MethodsFifty-six newly diagnosed or relapsed ITP patients (36females and20males) were enrolled between August2013and January2014in the Department of Hematology, Qilu Hospital, Shandong University, Jinan, China. They all met the diagnostic criteria of ITP. Patients with chronic diseases, infections, pregnancy or other autoimmune diseases were excluded. Peripheral blood samples were obtained from all patients prior to treatment. For some of them, blood samples were also gained after treatment. At the same time,24controls (14females and10males) were enrolled. Informed consent was gained from each participant. Ethical approval was obtained from the Medical Ethical Committee of Qilu Hospital. The desialylation level of platelets was measured as follows:Ethylenediaminetetraacetic acid (EDTA) anti-coagulated whole blood was obtained from ITP patients and controls. Platelet-rich plasma (PRP) was isolated by centrifugation at200g for10minutes. Fresh platelets were separated from PRP by centrifugation at850g for5minutes, washed and resuspended as described previously. All procedures were performed at room temperature. Fluorescein isothiocyanate (FITC) conjugated Ricinus communis agglutinin I (RCA-I) and Succinyl Triticum vulgare lectin (sWGA) were used to analyze beta-galactose and beta(1-4)-N-acetylglucosamine exposure on platelet surface, respectively. In addition, phycoerythrin-cyano dyes5(PE-cy5) mouse anti-human CD41a was used to define platelets. Platelets (1*107/mL) were resuspended, then incubated with20μL PE-Cy5mouse anti-human CD41a, plus5μg/mL RCA-I or0.1μg/mL sWGA for20minutes. Then, platelets were washed twice and resuspended. Cell acquisition was performed on a Beckman GalliosTM Flow Cytometer and events were analyzed using GalliosTM Cytometry List Mode Data Acquisition&Analysis Software. A total of30,000events were gated for platelets on the basis of their CD41constitutive expression and side scatter, and data were analyzed using the FCS Express4Plus software. Mean fluorescence intensity (MFI) of RCA-1and sWGA was used to represent for their binding levels.Plasma samples were separated from EDTA anti-coagulated whole blood and stored at-20℃until use. MAIPA was used to detect the specific anti-platelet GPIIb/IIIa and/or GPIb/IX autoantibodies as described previously.Results㏑esults showed that the exposure levels of β-Gal and P-GlcNAc on platelets in ITP patients were significantly higher than those in healthy controls (RCA-I:median,4.87; range,1.11-14.90vs. median,2.08; range,0.70-4.48, P<0.001; and sWGA: median,0.36; range,0.26-0.69vs. median,0.29; range,0.25-0.37, P<0.001).②Anti-platelet autoantibodies were determined by MAIPA. Among56ITP patients,7were positive for anti-GP Ⅱ b/Ⅲa,12were positive for anti-GP Ⅰ b/Ⅸ,13were positive for both, and24were negative for autoantibodies. There was no difference in the binding level of RCA-I or sWGA to platelets between patients with autoantibodies and those without autoantibodies. Furthermore, the desialylation levels were similar between patients with anti-GP Ⅰ b/Ⅸ and those without anti-GP I b/Ⅸ antibodies, as well as between patients with anti-GP Ⅱ b/Ⅲa and those without anti-GP II b/IIIa antibodies.③Blood samples after treatment were collected from25ITP patients among whom7achieved CR,7achieved R and11achieved NR. In CR group, posttreatment exposure levels of β-galactose and β-GlcNAc on platelets decreased significantly compared with those before treatment (RCA-I:median,1.96; range,0.60-9.24vs. median,4.46; range,1.47-14.9, P=0.016; and sWGA:median,0.30; range,0.28-0.32vs. median,0.34; range,0.31-0.53, P=0.022). In R group, RCA-I binding to platelets was significantly reduced after treatment (median,3.17; range,1.93-5.31vs. median,6.64; range,4.02-14.08, P=0.016), while binding of sWGA showed a trend to decrease (median,0.33; range,0.31-0.40vs. median,0.40; range,0.33-0.66, P=0.075). However, in NR group, the binding levels didn’t vary. ConclusionThe platelet membrane glycoprotein desialylation involved in the pathogenesis of primary immune thrombocytopenia, which went to enriches the thoughts for the treatment of the primary immune thrombocytopenia in the future.In conclusion, the results suggest that platelet desialylation is involved in the pathogenesis of ITP and not associated with anti-platelet autoantibody specificity. As desialylation level of platelets in ITP is markedly elevated, we suppose that it is a novel mechanism leading to platelet clearance through ASGPRs-mediated clearance in ITP.In addition, the level of platelet desialylation was downregulated in patients who showed response (both CR and R) to undergoing treatment, while no change of platelet desialylation level was observed in nonresponders. These findings suggest that downregulated platelet desialylation potentially alleviate ASGPRs-mediated platelet destruction to some extent in therapeutic responders (both CR and R). That may provide a novel therapeutic target for ITP.