Contributions
Abstract: S889
Type: Oral Presentation
Presentation during EHA23: On Saturday, June 16, 2018 from 16:45 - 17:00
Location: Room A9
Background
Primary immune thrombocytopenia (ITP) is an autoimmune disorder with reduced platelet production, and increased platelet destruction is considered to be the main pathogenic mechanism. However, the mechanism of corticosteroid-resistant ITP is still unclear. It is reported that single nucleotide polymorphisms (SNPs) in tubulin β1 in patients with ITP is related to the response of corticosteroid therapy (Paul A. Basciano et. al. 2013) and proplatelet formation (PPF) (Shinji Kunishima et. al. 2013). Our study revealed that all-trans retinoic acid (ATRA), which has demonstrated to be a promising candidate for corticosteroid-resistant/relapsed ITP patients in our previous study (Feng FE et. al. 2017), could correct the abnormal function of tubulin β1 in ITP patients with SNP and promote megakaryocyte maturation and PPF.
Aims
To explore the effect of ATRA on megakaryocyte maturation and PPF in ITP patients with tubulin β1 SNP R307H.
Methods
Thirty 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 bone marrow samples were collected again after ITP patients received a 16-week oral ATRA (10 mg twice daily) therapy. The identification of R307H substitution in TUBB1 (the gene encoding tubulin β1) was performed by polymerase chain reaction (PCR) and Sanger sequencing. 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. Western blot (WB) was performed to verify the expression level of tubulin β1 in differentiated MKs. Immunofluorescence staining was performed for tubulin β1 and observed by inverted confocal fluorescence microscopy. In the ITP group, ATRA was added to the differentiating culture process. Proliferation, GPIIb/IIIa–GPIbIX expression, ploidy distribution and PPF were observed in the different groups.
Results
The allelic frequencies between a group of 30 ITP patients and the healthy control group showed no difference. The protein levels of tubulin β1 in cultured differentiated MKs from ITP patients with homozygote SNPs were significantly decreased compared with patients with wild type tubulin β1, and heterozygote patients also showed a similar trend, but no significant difference. The in vitro culture-differentiated MKs from ITP patients with homozygote SNPs displayed normal proliferation but decreased GPIIb/IIIa–GPIbIX expression and ploidy distribution, as well as impaired and decreased PPF compared with the control groups and patients with WT tubulin β1. Immunofluorescence results of the differentiated MKs showed that the expression of tubulin β1 and the distribution of microtubules were abnormal both in MKs and in pro-platelets from ITP patients with homozygote SNPs. In the homozygote SNP group, when ATRA was added to the culture, tubulin β1 expression was recovered, and as a result, the GPIIb/IIIa–GPIbIX expression, ploidy distribution and PPF was increased. Additionally, after receiving ATRA therapy, the expression and distribution of tubulin β1 in megakaryocytes and proplatelets returned to nearly normal compared with those before therapy.
Conclusion
The megakaryocytes of ITP patients with homozygote SNPs showed a decreased expression and abnormal distribution, which might lead to its dysmaturity and impaired PPF. ATRA might correct the function of impaired tubulin β1 and promote megakaryocyte maturity and PPF.
Session topic: 33. Platelets disorders
Keyword(s): Corticosteroids, Immune thrombocytopenia (ITP)
Abstract: S889
Type: Oral Presentation
Presentation during EHA23: On Saturday, June 16, 2018 from 16:45 - 17:00
Location: Room A9
Background
Primary immune thrombocytopenia (ITP) is an autoimmune disorder with reduced platelet production, and increased platelet destruction is considered to be the main pathogenic mechanism. However, the mechanism of corticosteroid-resistant ITP is still unclear. It is reported that single nucleotide polymorphisms (SNPs) in tubulin β1 in patients with ITP is related to the response of corticosteroid therapy (Paul A. Basciano et. al. 2013) and proplatelet formation (PPF) (Shinji Kunishima et. al. 2013). Our study revealed that all-trans retinoic acid (ATRA), which has demonstrated to be a promising candidate for corticosteroid-resistant/relapsed ITP patients in our previous study (Feng FE et. al. 2017), could correct the abnormal function of tubulin β1 in ITP patients with SNP and promote megakaryocyte maturation and PPF.
Aims
To explore the effect of ATRA on megakaryocyte maturation and PPF in ITP patients with tubulin β1 SNP R307H.
Methods
Thirty 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 bone marrow samples were collected again after ITP patients received a 16-week oral ATRA (10 mg twice daily) therapy. The identification of R307H substitution in TUBB1 (the gene encoding tubulin β1) was performed by polymerase chain reaction (PCR) and Sanger sequencing. 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. Western blot (WB) was performed to verify the expression level of tubulin β1 in differentiated MKs. Immunofluorescence staining was performed for tubulin β1 and observed by inverted confocal fluorescence microscopy. In the ITP group, ATRA was added to the differentiating culture process. Proliferation, GPIIb/IIIa–GPIbIX expression, ploidy distribution and PPF were observed in the different groups.
Results
The allelic frequencies between a group of 30 ITP patients and the healthy control group showed no difference. The protein levels of tubulin β1 in cultured differentiated MKs from ITP patients with homozygote SNPs were significantly decreased compared with patients with wild type tubulin β1, and heterozygote patients also showed a similar trend, but no significant difference. The in vitro culture-differentiated MKs from ITP patients with homozygote SNPs displayed normal proliferation but decreased GPIIb/IIIa–GPIbIX expression and ploidy distribution, as well as impaired and decreased PPF compared with the control groups and patients with WT tubulin β1. Immunofluorescence results of the differentiated MKs showed that the expression of tubulin β1 and the distribution of microtubules were abnormal both in MKs and in pro-platelets from ITP patients with homozygote SNPs. In the homozygote SNP group, when ATRA was added to the culture, tubulin β1 expression was recovered, and as a result, the GPIIb/IIIa–GPIbIX expression, ploidy distribution and PPF was increased. Additionally, after receiving ATRA therapy, the expression and distribution of tubulin β1 in megakaryocytes and proplatelets returned to nearly normal compared with those before therapy.
Conclusion
The megakaryocytes of ITP patients with homozygote SNPs showed a decreased expression and abnormal distribution, which might lead to its dysmaturity and impaired PPF. ATRA might correct the function of impaired tubulin β1 and promote megakaryocyte maturity and PPF.
Session topic: 33. Platelets disorders
Keyword(s): Corticosteroids, Immune thrombocytopenia (ITP)