Abstract

IntroductionPrimary immune thrombocytopenia (ITP) is an autoimmune disorder with reduced platelet production, and increased platelet destruction is considered the main pathogenic mechanism. However, some ITP patients present resistance to treatment with corticosteroids or relapse after a short period of remission. The existence of these patients led us to consider the possibility of other non-immune mechanisms of ITP pathogenesis. It has been reported that in some patients with persistent or chronic ITP, the pro-platelet formation of megakaryocytes (MKs) is intrinsically impaired (Rivière et al, British Journal of Haematology 2015; 170: 408). Many MT-associated proteins (MAPs) are required for megakaryopoiesis and thrombopoiesis, but the roles of MAPs in ITP patients have rarely been reported. The hypothesis of this study is that the expression and function of MAPs in corticosteroid-resistant/relapse ITP patients are abnormal and that this abnormality may lead to the impaired pro-platelet formation (PPF) and contribute to the pathogenesis in these ITP patients.MethodsThirty corticosteroid-resistant/relapse ITP patients and 10 healthy donors were enrolled in this study. MKs were isolated by cell sorting from bone marrow samples obtained by aspiration from the posterior iliac crest, and single-cell microarray and bioinformatics analysis were performed. CD34+ cells were isolated from bone marrow samples by cell sorting for in vitro culture with TPO, and on day 8, differentiated MKs were enriched. Polymerase chain reaction (PCR) and Western blot (WB) were performed to verify the expression level of MAPs in differentiated MKs. Immunofluorescence staining was performed for tubulin and MAPs and observed by inverted confocal fluorescence microscopy. In the ITP group, all trans retinoic acid (ATRA) was added to the differentiating culture process, and in the donor group, lentivirus-expressing MAP-specific siRNAs were transferred into the CD34+ cells to knock down MAP expression before culture. Proliferation, GPIIb/IIIa-GPIbIX expression, ploidy distribution and PPF were observed in the different groups. Finally, the isolated megakaryocyte progenitors (MKPs) from ITP patients and donors were transferred into irradiated NOD/SCID mice.ResultsThe bioinformatics analysis of the results showed that the expression of MAPs in corticosteroid-resistant/relapse ITP patients was relatively decreased and that the Pak2 signaling pathway may be the upstream regulator of the abnormal MAPs. The mRNA and protein levels of MAPs in cultured differentiated MKs from ITP patients were decreased compared with controls, which is consistent with the bioinformatics analysis results. The phosphorylation level of the Pak2 signaling pathway was also decreased in the ITP group. The in vitro cultured differentiated MKs from ITP patients displayed normal proliferation, GPIIb/IIIa-GPIbIX expression, and ploidy distribution, but impaired and decreased PPF compared with control groups. Immunofluorescence results of the differentiated MKs showed that the expression of MAPs and the distribution of microtubules were abnormal both in MKs and in pro-platelets from ITP patients. In the MAP-silenced donor group, the percentage of proplatelet-displaying megakaryocytes was significantly decreased. In the ITP group, when ATRA was added to the culture, MAP expression and Pak2 phosphorylation were recovered, and as a result, the PPF was increased. When transferred into the irradiated NOD/SCID mice, the platelet reconstitution of MKPs from corticosteroid-resistant/relapse ITP patients was low. This finding supported the conclusion that the abnormal MAPs in MKs could lead to decreased platelet production.ConclusionMAP expression was decreased in corticosteroid-resistant/relapse ITP patients, and this abnormality was responsible for the decreased PPF of MKs from these patients. After treatment with ATRA, the Pak2 pathway activity, the MAP expression level, and the MK function was recovered. The abnormality of the expression and function of MAPs in corticosteroid-resistant/relapse ITP patients may be a new pathogenic mechanism in these patients. DisclosuresNo relevant conflicts of interest to declare.

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