The Study Of Sensitizing TPO-RAs On Platelet Production By Rhein And Inhibiting Platelet Destruction By CD44 Mab Or Oseltamivir In ITP

BackgroundImmune thrombocytopenia(ITP)is a common clinical autoimmune hemorrhagic disease.Besides low platelet counts,ITP patients also have varying degrees of bleeding symptoms and generally lower health-related quality of life.The etiology of ITP includes genetic susceptibility,environmental factors and imbalance of immune tolerance.The excessive clearance of platelets by immune cells and insufficient production of platelets by bone marrow megakaryocytes are the main reasons for the decrease of platelet count.According to the recommendations of relevant guidelines and consensus of ITP,TPO receptor agonists are listed as the first choice for the second-line treatment of ITP.Eltrombopag and romiplostim bind with TPO receptor and then activate Janus kinase/signal transducer and activator of transcription(JAK/STAT),phosphatidylinositol 3-kinase and protein kinase B(PI3K/AKT),extracellular signal-regulated kinase(ERK)and other intracellular signal pathways to promote the production of platelet by megakaryocytes.A number of clinical studies and meta-analysis showed that about 50%of ITP patients are still nonresponsive or relapsed after using TPO receptor agonists,and increasing drug dose cannot reverse this situation,which is a huge problem in the clinical management of ITP.Diacerein is a non-steroidal anti-inflammatory drug,which has been used to treat osteoarthritis for more than 20 years.Diacerein will completely transform into the active metabolite rhein before entering the systemic circulation,and play an important role by inhibiting inflammatory cytokines such as IL-1 and NLRP3 inflammasome.In addition to anti-inflammatory effect,diacerein and rhein have a valuable addition in combination therapy as sensitizer.Compared with single treatments,celecoxib combined with diacerein can more effectively attenuate joint inflammation and improve bone cartilage metabolism by regulating JNK and MAPK signaling pathways.Our team first observed that two ITP patients,who were effective in the treatment of eltrombopag,had a significant increase in platelet counts after taking diacerein concomitantly due to the onset of osteoarthritis.In the interim analysis of a pilot clinical study done by us,fifteen ITP patients who did not respond or relapsed after using eltrombopag received a combined treatment of eltrombopag and diacerein,seven of whom(46.6%)responded again.These evidences indicated that diacerein may have sensitization effect on eltrombopag,and the combination of eltrombopag and diacerein is expected to improve platelet counts of ITP patients and reverse the clinical dilemma of no response or relapse of eltrombopag.We suspected that the sensitization effect of rhein on TPO receptor agonists may be through regulating the platelet formation of megakaryocytes.Objective1.To investigated the effect of TPO receptor agonists combined with rhein versus TPO receptor agonists on megakaryocyte maturation and platelet production.2.Transcriptomic and proteomic sequencing were used to screen the pivotal genes and proteins regulated by eltrombopag plus rhein in megakaryocytes.3.In vivo and in vitro experiments were conducted to verify whether the combination of TPO receptor agonists and rhein promote the platelet production of megakaryocytes through PI3K signal pathway,to further reveal the potential sensitization mechanism,so as to provide evidences for optimizing the second-line treatment of ITP by this combination therapy.Methods1.Patients and healthy controls:This study recruited ITP patients and healthy volunteers who met the inclusion criteria in QiLu Hospital of Shandong University.On the premise of full informed consent,clinical data and peripheral blood samples of the participants were collected.2.Preparation of serum,isolation of cord blood mononuclear cells and induction of megakaryocytes:The serum of ITP patients and healthy controls were isolated and frozen in a refrigerator at minus 80℃.Cord blood mononuclear cells were isolated and CD34+cells were sorted.Megakaryocytes were cultured in SFEM medium containing rh-TPO,rh-IL-3,rh-SCF and serum.Rhein(0-100 μM)or eltrombopag combined with rhein(0-100 μM)was added on the fourth day after culture,the cell suspension was collected on the 14th day.3.Active ITP murine model:CD61 knockout mice were immunized with platelets from wild-type mice.The splenocytes of successfully immunized CD61 knockout mice were transferred to irradiated SCID mice to construct active ITP murine model.4.RNA sequencing:Megakaryocytes were induced by serum from ITP patients and treated with eltrombopag combined with rhein or eltrombopag alone.RNAs were extracted,and then sequenced by Illumina platform.5.Proteomic sequencing:Megakaryocytes were induced by serum from ITP patients and treated with eltrombopag combined with rhein or eltrombopag alone.Proteins were extracted,and then sequenced by Q Exactive HF-x.6.Flow cytometry:To detect the proportion of CD41+megakaryocytes and CD41+ CD42+megakaryocytes in vitro,in the bone marrow and spleen of mice.To analyze the level of apoptosis,the percentage of megakaryocyte polyploidy,the phosphorylation level of PI3K and MAPK in megakaryocytes.7.Cytokine detection:The levels of nine cytokines in cell culture supernatant were detected with LEGEND plex panel 1 kit,and the level of TGF-beta was detected with ELISA kit.8.PI3K inhibition assay:To verify whether the combination of TPO receptor agonists and rhein promote platelet production of megakaryocytes through PI3K signal pathway,PI3K inhibitor LY294002 was used in cell experiment in vitro and ITP active murine model.Results1.Eltrombopag combined with rhein(10 μM)significantly increased megakaryocytes and platelets in vitro.Different concentrations of rhein(1-100 μM)had no positive effects on megakaryocyte and platelet counts.Compared with eltrombopag monotherapy,eltrombopag combined with 10 μM Rhein significantly increased megakaryocyte and platelet counts,but eltrombopag combined with other concentrations or rhein(1 μM、50 μM、100 μM)could not.2.Eltrombopag combined with rhein significantly promoted CD41+megakaryocytes polyploid maturation,apoptosis and platelet production in vitro.In order to simulate the abnormal state of megakaryocytes in ITP,serum from patients who did not respond or relapsed after using eltrombopag were added to the culture system.The proportion of CD41+,CD41+CD42+,CD41+CD61+megakaryocytes in the combination group(eltrombopag plus rhein)was significantly higher than that in the monotherapy group(eltrombopag).In addition,the combined therapy significantly increased polyploid maturation and apoptosis of CD41+megakaryocytes,and further promoted platelet production.3.Romiplostim combined with rhein significantly increased platelet counts and megakaryocyte percentage,and promoted polyploidy maturation of CD41+megakaryocytes in ITP active murine model.Since eltrombopag could not activate TPO receptor in mice,we chose romiplostim to complete vivo experiment.In ITP active model,the platelet counts in rhein monotherapy group(10 mg/kg,30 mg/kg)were not significantly improved compared with that in control group.The platelet counts in romiplostim combined with rhein(10 mg/kg)group were significantly higher than that in romiplostim monotherapy group on day 21 and day 28.The proportion of CD41+CD42+ megakaryocytes in bone marrow and spleen of mice in romiplostim plus rhein(10 mg/kg)group was significantly higher than that in romiplostim group respectively.What’ more,the polyploidy maturation of CD41+megakaryocyte in the combination group was significantly higher than that in romiplostim group.4.RNA sequencing analysis revealed the regulation of gene expression by eltrombopag combined with rhein.To assess the impact of eltrombopag plus rhein versus eltrombopag alone on gene expression in megakaryocytes,we performed mRNA sequencing analysis in 7 pairs of megakaryocytes induced by ITP patient’s serum with the treatment of eltrombopag plus rhein or eltrombopag.We found 515 significant differential genes between the two groups.GO and KEGG analysis showed that these differential genes were mainly linked with megakaryocyte differentiation,platelet formation,integrin complex(CD41,CD42 and CD61),Rho GTPase related actin cytoskeleton,TGF-β,PI3K and MAPK signal pathways.5.Proteomic sequencing analysis revealed the regulation of protein expression by eltrombopag combined with rhein.Mass spectrometric analysis and proteomic identification were performed on 4 pairs of protein samples,and 62 differential proteins were screened between the eltrombopag plus rhein group and eltrombopag group.GO and KEGG analysis showed that the differential proteins were closely linked with TGF-β,cytokine-cytokine receptor interaction,integrin complex,Rho GTPase related actin cytoskeleton and PI3K signaling pathway.6.Eltrombopag combined with rhein regulated TGF-β and inflammatory cytokines in vitro.The level of TGF-β in the eltrombopag plus rhein group was significantly higher than that in eltrombopag group.The levels of IFN-α,IFN-y,IL-12p70,IL-17A,IL-18,IL-23 and IL-33 in the combination group were significantly lower than those in the monotherapy group.There was no significant difference in TNF-α or IL-1β between two groups.7.Eltrombopag combined with rhein promoted the production of platelet by megakaryocytes through activating PI3K phosphorylation.In vitro,the phosphorylation level of PI3K in CD41+ megakaryocytes in the eltrombopag plus rhein group was significantly higher than that in eltrombopag group.However,there was no significant difference in MAPK phosphorylation between two groups.No significant difference was observed in megakaryocyte percentage,polyploid percentage as well as platelet counts between eltrombopag-LY294002 group and eltrombopag-rhein-LY294002 group.8.Romiplostim combined with rhein attenuated thrombocytopenia in active ITP mice by increasing PI3K phosphorylation.In ITP active model,the phosphorylation level of PI3K in CD41+megakaryocytes in bone marrow in the romiplostim plus rhein group was significantly increased than that in romiplostim group.However,there was no significant difference in MAPK phosphorylation between two groups.There was no significant difference in platelet counts on day 21 and day 28 between romiplostim-LY294002 group and romiplostim-rhein-LY294002 group.Conclusion1.Compared with eltrombopag monotherapy,eltrombopag combined with rhein significantly increased megakaryocyte percentage,polyploid maturation and megakaryocyte apoptosis,and then promoted platelet formation in vitro.2.Romiplostim combined with rhein significantly attenuated thrombocytopenia in ITP active mice,with higher megakaryocyte percentage and better polyploidy maturation than romiplostim alone.3.The addition of rhein to TPO receptor agonists can play a sensitizing role by activating PI3K phosphorylation signal of megakaryocytes,regulating the secretion of a variety of cytokines,promoting megakaryocyte maturation and platelet production in ITP.BackgroundImmune thrombocytopenia(ITP)is an autoimmune disorder characterized by low platelet counts with an increased risk of bleeding.At present,no clinical prognostic or diagnostic biomarkers was established.Central to the immune pathogenesis of ITP is that autoantibody-sensitized platelets are phagocytosed by monocytes/macrophages via Fcγreceptors(Fc yRs).Clinical data showed that the expression of the activating subset of FcyRs was elevated in ITP patients,thus leading to increased phagocytosis of platelets.Current research indicates that aberrant macrophage polarization towards the proinflammatory M1 phenotype is also involved in the perturbation of immune tolerance of ITP.Moreover,ligation of platelet-specific autoantibodies to the activating subset of FcyRs could promote the activation of macrophages with enhanced phagocytic functions and immune responses.It is an effective way to inhibit platelet destruction by regulating the phagocytosis function and immune response of monocyte/macrophages through correcting FcγRs expression and M1 polarization in ITP.CD44 is a transmembrane adhesion molecule preferentially expressed on cells of the innated and adaptive immune systems,such as monocytes/macrophages and lymphocytes.Substantial studies have demonstrated that CD44 is involved in phagocytosis and clearance of apoptotic cells,such as neutrophils and eosinophils.CD44 also plays a pivotal role in inflammatory responses,including cellular adhesion,migration,and activation.Additionally,numerous studies suggest that CD44 promotes proinflammatory M1 macrophage recruitment and activation,which in turn enhances adipose inflammation,systemic insulin resistance,hepatic inflammation,and downstream expression of proinflammatory cytokines.These researches emphasized that CD44 is a pivotal component involved in phagocytosis and inflammation,and led to the suggestion that CD44 could be considered as a target for novel therapy in autoimmune diseases.Under inflammatory conditions,CD44 expression is upregulated on hematopoietic cells.CD44 also exists in a soluble form;and immune activation are often associated with increased plasma level of soluble CD44.However,CD44 level and its correlation with disease severity in ITP are unknown.CD44 monoclonal antibody(mAb)treatment has shown robust anti-inflammatory effects in multiple murine models of autoimmune diseases,including rheumatoid arthritis,inflammatory bowel disease and autoimmune diabetes.Most importantly,a series of studies completed by Crow et al.demonstrated that CD44 mAbs(5035-41.1D and KM114)successfully ameliorated murine ITP and suggested that the mechanism of action for CD44 mAbs in severe combined immunodeficient mice is primarily through targeting monocytes/macrophages.These inspiring findings strengthened the potential therapeutic value of CD44 mAbs in ITP.However,further studies are needed to investigate whether the use of specific recombinant antihuman CD44 mAbs might be a candidate strategy for ITP patients.In the present study,we aimed to investigate the correlation between CD44 levels and disease severity in patients with ITP and explored the immunomodulatory mechanisms of the antihuman CD44 mAb BJ18 on platelet phagocytosis mediated by monocytes/macrophages.Objective1.In this study,we aimed to detect CD44 levels,including both the soluble form found in plasma and the surface expression on circulating monocytes/macrophages in ITP patients and healthy controls,and then investigate whether there is a correlation between CD44 levels and disease severity in ITP patients.2.Targeting CD44,we selected antihuman CD44 monoclonal antibody BJ18 and aimed to verify the effect of BJ18 on platelet phagocytosis mediated by monocytes/macrophages in ITP patients and healthy controls.3.To investigate the effect of BJ18 on FcyRs expression,proinflammatory M1 and anti-inflammatory M2 phenotypes,and to reveal the immunomodulatory mechanism of BJ18 inhibiting platelet phagocytosis,further providing a valuable basis for the treatment of ITP with CD44 mAb.Methods1.Patients and controls:From May 2017 to March 2020,ITP patients and healthy volunteers were enrolled at the Department of Hematology,Qilu Hospital,Shandong University.Based on the full informed consent,we collected participants’ clinical information and peripheral blood samples.2.Preparation of plasma,peripheral blood mononuclear cells(PBMCs),monocytes/macrophages:Whole blood was obtained from patients and volunteers and centrifuged,then plasma was collected and stored at-80℃.PBMCs were separated by density gradient centrifugation and cultured at 37℃ for 2 h.Adherent cells were considered as monocytes/macrophages.3,Macrophage polarization:Adherent cells cultured in complete medium supplemented with rh-MCSF for 7 days,followed by refreshing the culture medium with LPS plus rh-IFN-γ for M1 polarization,or IL-4 for M2 polarization.To treat monocytes/macrophages,we used mouse antihuman CD44 mAb BJ18 and mouse IgG isotype.4.Isolation,CMFDA labeling,and opsonization of platelets:Platelet-rich plasma was prepared from volunteers’ peripheral blood,and isolated platelets were incubated with CMFDA in dark for 1 hours,washed once,and resuspended in PBS.For opsonization,CMFDA-labeled platelets were incubated with 10 μg of mouse antihuman CD41 monoclonal antibody for 30 min at room temperature and then washed once.5.In vitro phagocytosis assays:Adherent monocytes/macrophages were cultured in complete medium supplemented with the antihuman CD44 mAb BJ18 or IgG control for 12 h and washed once,followed by incubation with PMA for 2 h.Subsequently,adherent cells were incubated with opsonized CMFDA-labeled platelets(macrophages:platelets,1:10)for 1 h.6.Flow cytometry analysis:Surface expression of CD44,FcγRI,FcγRⅡα,and FcγRⅢ on CD14+monocytes/macrophages was determined by flow cytometry.The phenotypes of macrophage polarization were analyzed by the M1 marker(CD86-PE)and M2 marker(CD163-APC).For intracellular staining,macrophages were permeabilized and stained with PE-Cy7 labeled anti-CD68 antibodies.7.Enzyme-linked immunosorbent assay:The level of plasma soluble CD44 from ITP patients and healthy controls were assayed by a commercial ELISA kit.8.Quantitative real-time PCR(RT-qPCR):RT-qPCR was performed for FcyRIIa,FcγRⅡb,TNFα,IL-6 and endogenous control GAPDH on a LightCycler? 480 System.Results1.Elevated plasma CD44 concentration in ITP patientsPlasma soluble CD44 concentration was significantly increased in ITP patients with platelet count below 30×109/L compared to healthy donors,and ITP patients with platelet count above or equal to 30×109/L.The level of soluble CD44 in enrolled ITP patients was negatively correlated with platelet count and positively correlated with bleeding risk.2.Increased expression of CD44 on circulating monocytes/macrophages in ITP patientsThe expression of CD44 on circulating CD 14+monocytes/macrophages from ITP patients with platelet count below 30×109/L was significantly higher than that from healthy controls and from ITP patients with platelet above or equal to 30×109/L.Furthermore,the expression of CD44 on monocytes/macrophages was negatively correlated with platelet count and positively correlated with bleeding risk.3.Antihuman CD44 mAb BJ18 inhibited FcyR-mediated platelet phagocytosisWe found that the pretreatment of monocytes/macrophages with BJ18 inhibited platelet phagocytosis in a dose-dependent manner,reaching maximum inhibition at lug/mL mAb compared with control group and 0.1ug/mL BJ18 group.There was no significant difference in the surface binding of platelets to monocytes/macrophages from ITP patients by the treatment with BJ18 or isotype control.4.Effect of BJ18 on gene expression of FcγRⅡa and FcγRⅡb on monocytes/macrophagesThe expression of FcγⅡa mRNA on monocytes/macrophages from ITP patients was remarkably dampened by the pretreatment of BJ18.The expression of FcγRⅡb mRNA on monocytes/macrophages was increased by BJ18,though no significant changes were observed.5.Protein expression of FcγRⅠ,FcγRⅡa and FcγRⅢ on monocytes/macrophagesIn flow cytometry analysis,expression of activating FcγRs(FcγRⅠ,FcγRⅡa,and FcγRⅢ)on monocytes/macrophages from ITP patients were significantly decreased after the treatment of BJ18,respectively.Similarly,BJ18 significantly dampened the expression of FcγRⅡa and FcγRⅢ on monocytes/macrophages from healthy controls.The expression of FcγRⅠ was decreased by BJ18,but didn’t reach significant difference.6.Regulation of Ml but not M2 polarization by BJ18The percentage of M1 macrophages induced from ITP patients or healthy controls was significantly reduced by BJ18.TNF-α and IL-6 mRNA expression in M1 macrophages induced from patients or controls were significantly decreased after the treatment of BJ18,respectively.However,no significant difference was observed in the percentage of M2 macrophages induced from ITP patients or healthy controls after the treatment of BJ18.Conclusion1.Our results demonstrated that CD44 levels,including soluble form in plasma and surface expression on circulating monocytes/macrophages,were significantly elevated in ITP patients.Liner correlations were verified between CD44 levels and major clinical characteristics(platelet count and bleeding risk).2.Antihuman CD44 antibody BJ18 successfully inhibited platelet phagocytosis by monocytes/macrophages from ITP patients in vitro.3,Further studies indicated that BJ18 corrected abnormal FcγRs expression on monocytes/macrophages by reducing activating FcγRⅠ,FcγRⅡa,FcγRⅢ expression.Moreover,the polarization of proinflammatory Ml macrophages could be inhibited by BJ18.4.Our data indicated that CD44 levels have potential predictive value for the disease severity of ITP,and antihuman CD44 mAb BJ18 may be a promising therapy for ITP patients.BackgroundPrimary immune thrombocytopenia is an autoimmune disorder characterized by decreased platelet counts and increased bleeding risks.Classical mechanisms of ITP mainly involve autoantibody and cytotoxic T lymphocyte mediated platelet destruction,as well as impaired megakaryopoiesis and thrombopoiesis.Corticosteroids have been the standard initial treatment of ITP for more than 30 years.Dexamethasone(DXM)and prednisone are the most commonly used corticosteroid options.Reported initial response rates of corticosteroids varied from 50%to 85%in ITP;however,sustained response rates were relatively low when administered for a limited duration.Additionally,adverse effects from long-term exposure outweigh their benefits.Instead,combination treatments targeting multiple pathological mechanisms might offer prolonged efficacy and limited toxicity.Recently,desialylation has been identified as a novel mechanism of Fc-independent platelet clearance.In ITP,anti-GPIbα antibodies or cytotoxic T cells induced the translocation of neuraminidase-1 and the desialylation of platelets,which resulted in Fc-independent platelet clearance via hepatic Ashwell-Morell receptors.At the same time,autoantibody-mediated desialylation mediated impaired platelet adhesion,megakaryocyte differentiation and proplatelet production in ITP.Most importantly,sialidase inhibitor successfully ameliorated thrombocytopenia in multiple ITP murine models.These inspiring findings indicate that the prevention of desialylation by a sialidase inhibitor might be a promising therapeutic alternative for ITP patients.Oseltamivir is a sialidase inhibitor widely used for prophylaxis and treatment of influenza virus A or B infection.In two separate retrospective studies,oseltamivir increased platelet counts,seemingly independent of influenza condition.In patients with thrombocytopenia during sepsis,a randomized controlled trial suggested that the addition of oseltamivir could significantly increase platelet response,shorten platelet recovery time,and reduce platelet transfusion.Along those lines,sporadic case reports described that oseltamivir significantly increased platelet counts in two patients with primary ITP,and in two additional patients with HIV-related ITP.Furthermore,a pilot study confirmed that oseltamivir plus standard therapy induced remarkably higher initial responses than oseltamivir monotherapy in several multi-refractory ITP patients.Although these findings further strengthened the potential therapeutic value of oseltamivir in ITP,larger prospective studies are needed to investigate whether the combination of oseltamivir and standard corticosteroids might be a candidate strategy for initial management of ITP patients.Considering oseltamivir’s abilities to reduce platelet clearance and increase proplatelet production by inhibiting desialylation,we hypothesize that DXM plus oseltamivir will offer complementary mechanisms to optimize outcomes.Therefore,we aimed to evaluate the activity and safety of DXM plus oseltamivir vs dexamethasone in newly diagnosed treatment-naive adult ITP patients.ObjectiveBased on the RCT research method,we aimed to compare the initial response rate and time to response between DXM plus oseltamivir and DXM monotherapy as the first-line initial treatment of adult newly diagnosed ITP;to compare the sustained response rate and overall duration of response between the two arms;to evaluate the incidence of adverse events and safety between the two arms;to evaluate the improvement of bleeding risk and Health-related quality of life between the two arms;to explore the prognostic value of platelet specific autoantibodies.Methods1.Study design and participants:This multicenter,open-label,parallel group,individually randomized controlled study was conducted in five tertiary medical centers in China.We enrolled newly diagnosed ITP patients,who were aged 18 years or older and had either a baseline platelet count below 30×109/L or were experiencing bleeding manifestations.Eligible patients were randomized 1:1 to receive either DXM-oseltamivir(combination group)or DXM(monotherapy group).2.Treatment:DXM was administered orally at 40 mg daily to both groups for 4 days(days 1-4).If platelet counts remained less than 30×109/L or there were bleeding symptoms by day 10,another course of DXM was given(days 11-14).Oseltamivir was given orally at 75 mg bid concomitantly for 10 days(days 1-10)in the combination group.For the combination group,patients who relapsed after an initial response were allowed an immediate supplemental course of oseltamivir single agent(75 mg bid for 10 days)according to investigators’ advice and the patients’ decision.3.Outcomes:The primary endpoints of this trial were initial response(IR)and sustained response(SR).A response(CR+R)lasting for at least 6 months without any additional ITP-specific intervention was defined as a SR.The secondary endpoints included time to response(TTR),bleeding scores,duration of response(DOR),and adverse events.4.Statistical analysis:Analyses were conducted using IBM SPSS Statistics,version 23.0.Differences in IR,SR,bleeding event,and adverse event were compared between two groups by Fisher’s exact test.Bleeding scores and time to response were compared between two groups with Mann-Whitney test.The Kaplan-Meier method and log-rank test were used to compare differences in duration of response between groups.Platelet counts at different follow-up visits and change scores of each ITP-PAQ subscale were compared between two arms with Mann-Whitney test.Two-sided p values less than 0.05 were considered statistically significant.Results1.Baseline characteristicsBetween February 1,2016 and May 1,2019,120 patients underwent screening for eligibility,96 underwent randomization(47 combination,49 monotherapy),and 90 were included in modified intention-to-treat analysis(43 combination,47 monotherapy).Baseline characteristics were balanced between the two groups.2.Initial responseIn the modified intention-to-treat population,the DXM-oseltamivir regimen achieved a significantly higher IR rate compared with DXM at day 14(37[86%]of 43 vs 31[66%]of 47;OR 3.18,95 CI%1.13-9.23,P=0.030).The combination therapy also induced a significantly higher initial CR rate than DXM monotherapy(27[63%]of 43 vs 18[38%]of 47;OR 2.72,95 CI%1.19-6.12,P=0.034).Both therapies took effect quickly,and there was no significant difference in median TTR between the two groups.3.Long-term outcomesThe modified intention-to-treat analysis indicated that the 6-month SR rate in the DXM-oseltamivir group was significantly higher than that in the DXM group(23[54%]of 43 vs 14[30%]of 47;OR 2.17,95 CI%1.16-6.13,P=0.032).There was no significant difference in the 12-month SR between the two groups.During the 18-month observational period,overall duration of response was similar between two groups by the Kaplan-Meier analysis(HR 1.42,95 CI%0.86-2.35,log rank P=0.176).Eight(19%)of 43 patients in the combination group received retreatment with oseltamivir single agent,five(63%)of whom responded again with a prolonged median DOR of 4 months.4.Bleeding riskAt day 14,DXM-oseltamivir therapy induced comparable bleeding scores and bleeding events compared with DXM monotherapy.At 6 months,the combination therapy resulted in significantly lower bleeding scores and significantly fewer bleeding events than monotherapy.At 12 months,there was no statistical difference in bleeding events or bleeding scores between the two groups.5.Antiplatelet autoantibodiesIn the DXM group,regardless of anti-GPⅡb/Ⅲa antibody status,patients with detectable anti-GPⅠb/Ⅸ antibodies had significantly poorer IR rates than those without anti-GPIb/Ⅸantibodies.For patients with anti-GPⅠb/Ⅸ antibodies(including single and double positive),we did not find a significant difference in the incidence of IR between DXM-oseltamivir and DXM monotherapy.6.Health-related quality of lifeDXM-oseltamivir induced significantly enhanced improvement in ITP-related symptoms and overall quality of life compared to DXM monotherapy.7.Adverse eventsAll adverse events reported in this study fall within the product summaries of DXM and oseltamivir.Most of the adverse events were mild to moderate(graded 1 to 2)and resolved spontaneously after treatment was completed.Conclusion1.Our results demonstrated that the inclusion of oseltamivir to DXM-based therapy yielded significantly improved initial and sustained response than DXM monotherapy in the management of newly-diagnosed ITP.Multiple cycles of oseltamivir as modification of current first-line treatment,may be more effective in maintaining platelet response.2.Generally,both regimens were well tolerated.3.This oral and less expensive combination therapy provides a new,promising strategy for better and steadier response in ITP patients.