Research On The Relationship Between Platelet Glycoprotein Desialylation And The Efficacy Of The First-line Therapy For ITP

BackgroundPrimary immune thrombocytopenia (ITP) is a common clinical bleeding disorder. Currently, the pathogenesis of refractory ITP is not clear, and there is no effective treatment.Although it is not hematologic malignant diseases, but because of recurrent severe platelet reduction, poor efficacy, the ITP patients quality of life is very poor with great risk of death. The pathophysiology of ITP is multifaceted, not yet fully clear. At present the vast majority of ITP is induced by a common humoral and cellular immune-mediated platelet destruction which is widely regarded. Corticosteroids and intravenous administration of immunoglobulin G(IVIG) is the first-line treatment of ITP. The main mechanism of therapy includes inhibiting the production of autoantibodies, blocking Fc receptor signal transduction pathway, inhibiting the macrophage phagocytosis, promoting megakaryocytes to produce platelets. Ultimately the level of the platelet can be enhanced. In recent years, different research groups have found that the first-line treatment of glucocorticoid and IVIG are effective for the anti-GPⅡb/Ⅲa antibody-mediated ITP. However, the patients presented with anti-GPIb-Ⅴ-Ⅸ complex antibody often poorly respond to the first-line treatment. At present, in addition to the specific platelet clearance in an Fc-dependent pathway, are there other ways which can cause platelets elimination? The latest in vitro and in vivo study confirms the existence of platelet clearance in an Fc-independent fashion. Under the action of neuraminidase, platelet membrane glycoproteins desialylate and expose the acetylglucosamine and galactose residues. It induces the Willebrand Factor receptor clustering and changes the signal transduction. The cluster combines with the MAC-1 receptor of liver Kupffer cells or the Ashwell-Morell receptor of hepatocytes in the liver, then the platelets are cleared. This is different from the previous viewpoint of platelet clearance in the spleen. However, how the platelets change in ITP patients. It is unclear whether there is such a Fc-independent platelet clearance pathway which induces the poor respond of the first-line treatment for some ITP patients.Objective1.Detect desialylation of platelet membrane glycoprotein in ITP with FITC-labelled ECL and RCA-1, and compare the difference between the ITP and the normal.2.Compare the correlation of the desialylation level and the efficacy of first-line therapy for ITP. Provide the basis for the Fc-independent platelet clearance pathway, which is good for development of effective treatment.3.Compare the correlation of Anti GPIba antibody and the desialylation level of platelet membrane glycoprotein in ITP.4.Compare the difference desialylation of platelet membrane glycoprotein between the ITP and the other disease control groups, discuss the possibility of desialylation inducing platelet clearance in different diseases.Methods1.The ITP patients were enrolled between March 2013 and January 2015 in the department of hematology, the second affiliated hospital of anhui medical university. At the same time, the newly diagnosed rheumatic diseases with thrombocytopenia, myelodysplastic syndrome(MDS), aplastic anemia(AA) and acute myeloid leukemia(AML) patients were enrolled as the other disease control groups. Meanwhile 20 normal controls were enrolled.2. We determine the desialylation level of platelet membrane glycoprotein by detecting ECL and RCA-1 on the platelet surface with flow cytometry. The platelets of the ITP patients, the other disease control and the normal control are respectively incubated with ECL and RCA-1. The desialylation level of platelets was measured as the detected percentage of ECL and RCA-1.3. Improved monoclonal antibody immobilization of platelet antigen assay(MAIPA) was used to detect the major types of antibodies in ITP patients, including anti GPⅡb Ⅲa and GP I ba specific autoantibodies. This was used for ITP patients classified selection.4. The ITP patients receiving first-line treatment mainly:Firstly use dexamethasone(40mg/d,3-5d) combined with or without high-dose FVIG(0.4g·kg-1·d-1,3-5d), then take orally prednisone at a dose of 4g·kg-1·d-1, reduce the dose according to the efficacy, maintain treatment for a month with monitoring of blood. The efficacy criteria:① Complete response (CR):After treatment, platelet count ≥100×109/L and without bleeding. ② Response (R):After treatment, platelet count ≥30 ×109/L and at least 2-fold increase the baseline count and without bleeding. ③ No response (NR):After treatment, platelet count <30×109/L or less than 2-fold increase the baseline count or bleeding. According to the efficacy, the patients were divided into CR, R, NR three groups.Results1. In the gender and age, the normal and the ITP were not significant different (gender: χ2= 2.796, P= 0.094, age:t=-0.352, P= 0.726). But the platelet count was significantly lower in the ITP, t= 21.914, P<0.001.2. The statistical difference of the platelet membrane glycoprotein desialylation between the normal and the ITP was significant (P<0.001). The level of the platelet membrane glycoprotein desialylation of the ITP was significantly higher.3. According to the efficacy, the 61 cases of ITP patients were divided into CR, R and NR three groups. Using Kruskal-Wallis rank sum test to analyze, the ECL and RCA-1 level of the three groups were significant different,P<0.01. Correlation analysis indicated that efficacy and the desialylation level are related(P<0.01). The higher desialylation, the more poor the efficacy.4. Due to the specimen retention problem, in this experiment, only 29 patients were detected by improved MAIPA. The 29 cases of ITP patients were divided into three groups as with anti-GPIbα antibody, only with anti-GPⅡb/Ⅲa antibody and without the two antibodies. Using Kruskal-Wallis rank sum test to analyze, the ECL and RCA-1 level of the three groups were not significant different,P> 0.05.5. Analysis of the platelet membrane glycoprotein desialylation in the other disease control group:Compared with the normal, Platelet desialylation of the rheumatic diseases with thrombocytopenia, MDS and AA was higher(P<0.05). The statistical difference of desialylation between the normal and the AML was not significant(P>0.05). The desialylation level of five disease groups was different(P<0.05). Desialylation level of the ITP, rheumatic diseases with thrombocytopenia, MDS and AA group was higher.Conclusion1. The platelet membrane glycoprotein desialylation in ITP was significantly higher than normal.2. The efficacy of the first-line therapy in ITP is related with the level of desialylation. The efficacy of the patients with significant desialylation is poor. The level of desialylation can be used to prompt the treatment of ITP patients. These findings shed light on Fc-independent platelets clearance, designating desialylation as a potential therapeutic target in the treatment of refractory ITP.3. Because of small number of correlation analysis cases, there is no difference in platelet desialylation of each antibody group. But we can see a higher tendency of with anti-GPIba antibody group in the charts. This need expand the sample size for further study.4.The desialylation level of the ITP group was not different from the rheumatic diseases with thrombocytopenia, MDS and AA groups, but was significant higher than the AML group. It is need to consider that platelet desialylation also play in the role of platelets clearance in the rheumatic diseases, MDS and AA.