| Part I:TWS119 promotes megakaryocytes apoptosis andplatelets formation in immune thrombocytopeniaBACKGROUND:Primary immune thrombocytopenia(ITP)is one of the most common bleeding diseases,characterized by reduced platelet count in peripheral blood and autoimmune disorder is the initiating factor of ITP.No obvious bleeding symptoms are observed in mild patients,while some severe patients encounter bleeding symptoms of skin or mucous membrane,menorrhagia,even life-threatening viscera and intracranial hemorrhage.Increased platelet destruction and/or reduced platelet production are prominent features of ITP.It has been known that platelets arise from proplatelets,which are developed from long and thin cytoplasmic extensions of mature megakaryocytes.Mature megakaryocyte apoptosis is temporally associated with proplatelet formation.Previous studies demonstrated that Caspase activation within megakaryocytes was required for platelet production.They presented evidence that in vitro-grown megakaryocytes,during their terminal stages of maturation,exhibited the activation of Caspases-3 and Caspase-9.The pan-Caspase inhibitor as well as more specific inhibitors of Caspase-3 and Caspase-9 blocked proplatelet formation,whereas an inhibitor of calpeptin had no effect.Overexpression of Bcl-2 also inhibited proplatelet formation in maturing MKs.They concluded that proplatelet formation was regulated by a Caspase activation limited to only some cellular compartments.Ucar et al.examined megakaryocyte apoptosis in bone marrow aspiration of children with acute and chronic ITP and investigated the role of megakaryocytes apoptosis in ITP pathophysiology.The ultrastructural abnormalities could be reversed by in vivo treatment with prednisone and intravenous immunoglobulin.When suspension culture of CD34+ cells were performed with ITP plasma,morphology compatible with para-apoptosis could be induced in megakaryocytes.Therefore,we suppose that decreased megakaryocyte apoptosis or other forms of programmed cell death are responsible for persistent thrombocytopenia.Glycogen synthase kinase-3(GSK-3)is a multifunctional serine/threonine protein kinase.It was originally discovered that GSK-3 is one of the major rate-limiting enzymes involved in glucose metabolism.However,GSK-3 has been involved in other important physiological processes in the cell,such as cell differentiation,proliferation and apoptosis.The GSK-3? inhibitor TWS119 is a class of 4.6-disubstituted pyridopyrimidines,which has been shown to enhance megakaryocyte production and platelet release in human bone marrow cells in vitro.Recent studies have shown that GSK-3? inhibitors may be effective in promoting platelet release by promoting megakaryocyte production and maturation.In the present study,we observed that megakaryocyte production,platelet release,and megakaryocyte apoptosis were increased in the in vitro culture system of ITP patients with plasma at the median concentration of TWS119.We further investigated the effects of TWS119 on megakaryocyte production and platelet production in a mouse model.The TRAIL expression of the TWS119 treated group was enhanced compared to the control group.The promotion of TWS119 on megakaryocy'te apoptosis and platelet release suggests that it may have potential therapeutic effects in ITP.OBJECTIVE:By investigating the effect of low-dose TWS119 on the maturation,apoptosis and thrombocytopenia of megakaryocytes in the presence of ITP patients,the possibility of promoting functional platelet production in ITP patients was explored.METHODS:(1)ITP patients and healthy controls:Plasma samples were obtained from 62 ITP patients and 23 healthy controls.(2)CD34+ cells were separated from cord blood and cultured in 1mL serum-free expansion medium(SFEM)supplemented with recombinant human thrombopoietin(rhTPO),stem cell factor(SCF)and recombinant human interleukin-3(rhIL-3).In order to detennine the optimal concentration of TWS119 in promoting megakaryocyte maturation and platelet release,different concentration of TWS119 were added,following with 14 days culturing.(3)After the optimal concentration of TWS 119 was determined,CD34+ cells were cultured in the presence of 100 ?L plasma from ITP patients or healthy controls and 900 ?L SFEM supplemented with rhTPO,SCF,rhIL-3 and TWS119 for 14 days.The quantity,apoptosis ratio and quality of megakaryocytes and number of platelets release were examined by flow cytometry.(4)Expression levels of TRAIL,TRAIL-R2,Bcl-2,Bcl-xL,TNF-?,Fas and Fas-L mRNA in megakaryocytes were analyzed by RT-qPCR.(5)Expression levels of TRAIL,TRAIL-R2,Bcl-2,Bcl-xL,TNF-?,Fas,Fas-L,active Caspase-3 and active Caspase-8 were analyzed by flow cytometry.Expression levels of TRAIL,Caspase-3 and Caspase-8 protein were detected by western blot.(6)CD61 knockout(KO)mice were transfused with platelets from wild-type(WT)C57BL/6 mice.Spleens of CD61-KO mice were removed and prepared into a splenocyte suspension,injecting intraperitoneally to severe combined immune deficient(SCID)mice.(7)The SCID mice were divided into 4 groups.Group a subjected to irradiation without any cell transfer.Group b received an intraperitoneally injection of TWS119 and irradiation.Group c received an intraperitoneally of splenocyte and irradiation.Group d received the same volume of splenocyte transfer and TWS119 injection in addition to irradiation.Platelet counts were recorded weekly to evaluate the response of TWS119 on SCID mice.RESULTS:(1)TWS119 could induce the megakaryocyte maturation and platelet production in a dose-dependent manner.Our data showed a concentration-dependent of TWS119 on megakaryocyte production and platelet release,and the optimal concentration was 1 ?M.TWS 119 induced the highest megakaryocyte numbers,platelet production and polyploidy(?4N)in culture systems at l ?M.Conversely,10 ?M or higher concentrations of TWS119 suppressed megakaryocyte and platelet production.TWS119 showed a toxic effect over 100 ?M,in which cases TWS119 induced a high megakaryocyte apoptosis but low megakaryocyte numbers and platelet release,indicating that an invalid megakaryocyte apoptosis occurred.(2)Different effect of ITP plasma on megakaryocyte maturation and platelet release.Sixty-two ITP patients and twenty-three healthy donors were enrolled in this study.These ITP patients were divided into three groups according to the effect their plasma had on megakaryocyte production.Group A plasma treatment showed an increased megakaryocy,te compared with that of healthy controls,however,numbers of megakaryocyte in Group B treatment was markedly decreased and Group C showed no significant change.The platelet release values in group A and Group B were significantly decreased compared with that of healthy controls,but no remarkable differences between Group C and healthy controls.Apoptosis of group A was lower than healthy controls,and no remarkable difference between Group B,Group C and healthy controls.(3)Effects of TWS119 on megakaryocyte production,maturation,apoptosis and platelet release cultured with ITP plasma.The number of megakaryocytes,platelet release,megakary'ocyte apoptosis and percentage of polyploidy were significantly increased in Group A after TWS119 treatment.Similar results were observed in healthy control group.However,such effects of TWS119 were not performed in the presence of plasma from Group B or C.(4)Abnormal expression of Bcl-xL,Bcl-2,TRAIL,Caspase-3 and Caspase-8 in megakaryocytes cultured with Group A patient plasma.Our results indicated the expression of Bcl-xL and Bcl-2 in megakaryocytes displayed an elevated level in Group A cultures.At day 14,both Bcl-xL and Bcl-2 expressions in Group A were significantly higher than in controls.When TWS119 was added to the culture system,both Bcl-xL and Bcl-2 expressions decreased significantly in Group A.The expression of TRAIL,active Caspase-8 and active Caspase-3 in GroupA was remarkably lower than that of controls at day 14,which was consistent with inhibited megakaryocyte apoptosis and decreased platelet production.We next added TWS119 to the culture system,the results showed that TRAIL,active Caspase-8 and active Caspase-3 expressions in Group A were significantly boosted.No detectable difference on the expression of active Caspase-8 in megakaryocytes of healthy controls was observed after TWS119 treatment.We next examined the mRNA expression of Bcl-xL and Bcl-2 in megakaryocytes.Bcl-2 showed a higher expression in Group A than in healthy controls.However,there was no obvious difference between Group A and control group of Bcl-xL expression.When TWS119 was added to the culture system,both Bcl-xL and Bcl-2 decreased.The mRNA expression of TRAIL was lower in Group A than in control aroup.TWS119 could increase the TRAIL mRNA expression in both groups.Further experiments were undertaken to investigate the protein levels of TRAIL,Caspase-8 and Caspase-3 by western blot.The results indicated that TWS119 could enhance TRAIL,cleaved Caspase-8 and cleaved Caspase-3 in megakaryocytes cultured with ITP plasma.(5)TWS119 treatment increased peripheral platelet counts and alleviated thrombocytopenia in an ITP murine model.The SCID mice were separated into 4 groups.Group a received only irradiation without any cell transfer at all.Group b received irradiation and an intraperitoneal injection of 1mg/kg TWS119 by the day of irradiation,and continuous treated with TWS119 injection once every other day.Group c received irradiation and splenocyte transfer.Group d were treated with irradiation,TWS119 and splenocyte transfer.The results showed that the number of platelets in Group a and Group b increased at the second week,indicating the recovery of transient thrombocytopenia caused by irradiation.The success of the active ITP mouse model was demonstrated by the fact that platelet counts of mice in Group c and Group d injected with splenocyte remained low levels during the monitoring period.The number of platelets in Group d increased slowly by day 7,and was higher than that in Group c after day 14,following with a significantly increasing than that in Group c by day 21,suggesting that intraperitoneal injection of TWS119 can effectively improve the survival rate of mice and reduce the number of platelets at the same time.CONCLUSION:TWS119 could promote megakaryocyte maturation and platelet production in vitro in a dose-dependent manner.Low-dose TWS119 could effectively promote megakaryocyte maturation,apoptosis and platelet production.TWS119 could partly improve the impaired megakaryocyte maturation,apoptosis and insufficient platelet production which induced by plasma of ITP patients and increase the expression of TRAIL in megakaryocytes,while increasing the sensitivity of TRAIL.The role of Low-dose TWS119 in promoting megakaryocyte maturation and platelet production might be beneficial for the treatment of thrombocytopenic diseases.Part II:Anti-c-Mpl antibodies in immune thrombocytopeniasuppress thrombopoiesis and decrease response to rhTPOBACKGROUND:Primary immune thrombocytopenia(ITP)is a common immune-mediated hematologic disorder characterized by a low platelet count(peripheral blood platelet count<100×109/L),about 30%of all bleeding disorders.Detectable autoantibodies binding to platelet glycoproteins(GP)and normal or increased number of megakaryocytes in bone marrow are the major clinical characteristics.At present,the research focus on the pathogenesis of ITP is mainly divided into two parts,increased platelets destruction and decreased platelets production.Excessive platelet destruction is mainly due to direct lysis of platelets by Cytotoxic T lymphocytes(CTLs)and/or autoantibody-mediated excessive platelet clearance;whereas insufficient platelet production is mediated by the immune system.Mature disorders and abnormalities caused by apoptosis.The pathogenetic mechanism of ITP is not completely clear yet.It has been long believed that thrombocytopenia is mediated by autoantibodies.Previous study indicated that autoantibodies produced by autoreactive B cells against self-antigens,specifically immunoglobulin G(IgG)antibodies that recognize platelet surface glycoproteins.The antibody-coated platelets are destructed either by phagocytic cells or by autoantibody-induced activation of complement.However,autologous platelet survival studies showed that in approximately two thirds of ITP patients,platelet turnover is either reduced or normal,not increased as expected if platelet destruction were the only mechanism causing thrombocytopenia.Since megakaryocytes also express GP Ib/IX and GPIIb/IIIa on their surfaces during maturation and most ITP autoantibodies react with one or both of the these glycoprotein complexes,it follows that autoantibodies binding to megakaryocytes could interfere with platelet production and release from the bone marrow either by causing intramedullary megakaryocyte or platelet destruction or by interfering with megakaryocyte maturation.It has been known that platelets arise from proplatelets,which are developed from long and thin cytoplasmic extensions of mature megakaryocytes.Several reports indicated a close relationship among apoptosis,megakaryocyte maturation,and platelet release.Therefore,impaired megakaryocyte apoptosis may lead to persistent platelet reduction in ITP patients.Recently,although ITP patients have received new treatments such as rituximab and thrombopoietin(TPO)receptor agonists,about 30%failed to respond or relapsed shortly after the treatment.The mechanism of action of TPO is to form a TPO/c-Mpl complex with megakaryocyte surface receptors to promote megakaryocyte differentiation,maturation and platelet production.The anti-c-Mpl antibody binds to c-Mpl on the surface of megakaryocytes,and the spatial inhibition results in the inability of TPO to bind to c-Mpl,making megakaryocytes unable to mature,leading to reduced platelet production.Studies have reported that patients with positive anti-c-Mpl antibodies are accompanied by elevated serum TPO concentrations,so it is speculated that anti-c-Mpl antibodies may compete with TPO to bind to c-Mpl,block TPO/c-Mpl pathway,and cause megakaryocyte differentiation,development,maturity disorders,subsequently resulting in thrombocytopenia.At the same time,anti-c-Mpl antibodies may also bind to c-Mpl on the surface of megakaryocytes in the bone marrow or consume platelets by antigen-antibody reaction,but the specific mechanism remains to be further studied.There are cases of preventive or over-treatment in patients with ITP whose TPO treatment effect is unclear.To further confirm the diagnosis,invasive examination into bone marrow puncture is often to be used.This study will explore the significance and value of plasma anti-c-Mpl antibody in the evaluation of TPO in the treatment of ITP through a prospective multicenter,double-blind trial,and provide a clinically scalable non-invasive laboratory test for the clinical application of TPO.OBJECTIVE:Evaluate the feasibility of anti-c-Mpl antibody in the diagnosis of ITP by comparing the difference of anti-c-Mpl antibody between patients with ITP and other patients with thrombocytopenia.Moreover,detecting the level of anti-c-Mpl antibody in the serum of patients with ITP was used to investigate the value of rhTPO,dexamethasone,rituximab and eltrombopag in the treatment of prognosis.METHODS:(1)Specimen collection:Peripheral blood of 187 patients with ITP(31 newly diagnosed and 156 chronic ITP patients),31 patients with aplastic anemia(AA),17 patients with myelodysplastic syndrome(MDS),22 patients with rheumatic diseases(RD),25 acute myeloid leukemia(AML)and 59 healthy donor(HD)were collected with detection of platelet counts,and plasma was separated for use.(2)Demographic and clinical symptoms of ITP patients were statistically recorded atthe time of blood collection.Age,sex,platelet count and laboratory examination results were recorded.The treatment methods,response,hematological examination and prognosis of the patients were investigated and statistically analyzed.(3)Sample detection:Enzyme-linked immunosorbent assay(ELISA)was used todetect the expression level of anti-c-Mpl antibody in serum.(4)Bone marrow aspiration was performed from the posterior superior iliac spine andthe bone marrow smears were stained in accordance with the Wright-Giemsa procedure.(5)Serum TPO levels were measured using ELISA kits.(6)Modified monoclonal antibody-specific immobilization of platelet antigens(MAIPA)was performed to detect anti-platelet glycoprotein autoantibodies of ITP patients.(7)Obtaining CD34+ cells:CD34+ cells were purified from healthy umbilical cordblood mononuclear cells and cultured in a 24-well plate,adding stem cell factor(SCF)and recombinant human interleukin-3(rhIL-3).Each well was supplemented with 100 ng rhTPO or 10 ?M eltrombopag in the presence of purified IgG of ITP patients or healthy controls.(8)Flow cytometry was used to detect the number of megakaryocytes and apoptosis,platelet production and megakaryocyte ploidy in the culture system.RESULTS:(1)Anti-c-Mpl antibodies were detected in various thrombocytopenic disorders.Serum samples from 59 HD,187 ITP,31 AA.17 MDS,22 RD and 25 AkML patients were tested by ELISA assay.The anti-c-Mpl-antibody-positive(anti-c-Mpl+)group was designated as those with levels over 2187.93 ng/ml.Anti-c-Mpl antibodies were detected in 54 ITP(28.9%),1 MDS(5.9%),7 RD(31.8%)and 2 AML(8%)patients.The fraction of anti-c-Mpl+ patients was significantly greater in ITP than in the MDS,AA,or AML patient groups.(2)Anti-c-Mpl+ITP patients have decreased megakaryocyte and platelet counts compared to anti-c-Mpl-antibody-negative(anti-c-Mpl-)patients.Among these patients,the anti-c-Mpl+ group showed a significant lower peripheral platelet count than the anti-c-Mpl-.The newly diagnosed group had a higher ratio of anti-c-Mpl+ patients than the chronic group.Moreover,a larger proportion of anti-c-Mpl+ patients were also detected of anti-GPIb/IX and anti-GP?b/?a double positive antibodies compared to anti-c-Mpl-patients.Furthermore,the anti-c-Mpl+group had fewer bone marrow megakaryocytes compared with the anti-c-Mpl-group.(3)Anti-c-Mpl antibodies are correlated with higher TPO levels in ITP patients.The AA group presented with higher serum TPO levels than ITP,MDS.AML and RD groups,while the HD possessed the lowest level.Furthermore,TPO levels were higher in the anti-c-Mpl+ than the anti-c-Mpl-ITP group.(4)Anti-c-Mpl antibody positivity is associated with decreased therapeutic rhTPO efficacy.At the end of the third month of the initial treatment,27(50.0%)received rhTPO among the 54 anti-c-Mpl+ patients,of whom 14(51.9%)achieved clinical CR and R.Of the 133 anti-c-Mpl-patients,83(62.4%)received rhTPO,62(74.7%)of whom achieved clinical CR and R.We also evaluated the correlation between anti-c-Mpl antibody and treatment effect under the logistic regression model,which showed that presence of anti-c-Mpl antibody was inversely correlated with the achievement of a clinical response after rhTPO treatment.In patients who had achieved CR and R with rhTPO treatment,the median TTR was 16 days in the anti-c-Mpl+ group compared with 9 days in the anti-c-Mpl-group.Our data demonstrated that anti-c-Mpl+ patients had a poorer TTR to rhTPO treatment than those who were anti-c-kMpl-.In addition,two of three patients in the anti-c-Mpl+ group with high anti-c-Mpl antibody titres were NR following eltrombopag treatment,while two anti-c-Mpl-patients both acquired CR after eltrombopag treatment.(5)Anti-c-Mpl antibodies titres decreased following rhTPO treatment in ITP patients.Patients treated with rhTPO experienced significantly decreased anti-c-Mpl titres post-treatment.There were no significant differences between anti-c-Mpl antibody titres pre-and post-treatment in the RTX,DXM and eltrombopag groups.(6)Purified anti-c-Mpl IgG from ITP patients suppresses megakaryocyte platelet production in the presence of TPO-RAs in vitro.In the presence of rhTPO and eltrombopag,megakaryopoiesis from CD34+ cells were significantly lower in the anti-c-Mpl+ group compared with anti-c-Mpl-,which led to lower platelet generation.A lower percent of polyploidy was observed in anti-c-Mpl+group compared with the anti-c-Mpl-group with rhTPO treatment.However,there were no significant differences in percent polyploidy in the presence of eltrombopag.and megakaryocyte apoptosis between anti-c-Mpl+ and anti-c-Mpl-in the presence of rhTPO or eltrombopag.In addition,the anti-c-Mpl+ group had significantly lower megakaryopoiesis,apoptosis,and platelet generation compared to the HD group.CONCLUSION:In summary,the present work has identified the presence of anti-c-Mpl antibodies in ITP patients both newly diagnosed and chronic.The presence of anti-c-Mpl antibodies was correlated with worse prognosis and poorer response to rhTPO treatment in chronic ITP.Thus,anti-c-Mpl antibodies might be a potential functional biomarker and therapeutic target in ITP patients. |
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