A Clinical Study On RhTPO Management Of First-Line Treatments Resistant ITP In Pregnancy/Study On Apoptosis-Promoting Effect Of Anti-GPIb Monoclonal Antibodies

Part? A Clinical Study On rhTPO Management Of First-Line Treatments Resistant ITP In PregnancyImmune thrombocytopenia(ITP)is an acquired autoimmune disease characterized by a transient or persistent decrease in platelet count.Both increased platelet destruction caused by autoantibodies to platelet membrane glycoproteins and insufficient platelet production are involved in ITP.ITP is the most common cause of thrombocytopenia in early pregnancy,associated with 5%of all thrombocytopenia cases in pregnant women.Patients with severe thrombocytopenia(platelet count,20 × 109/L)are at risk for spontaneous bleeding,postpartum hemorrhage,and placental abruption.Platelet IgG type autoantibody can pass through the placental barrier and result in fetal or transient neonatal thrombocytopenia.From 8.9%to 14.7%newborns of pregnant women with ITP have severe thrombocytopenia,and the incidence of intracranial hemorrhage(ICH)is approximately 1.5%.Similar to nonpregnant adult patients with ITP,first-line treatments include corticosteroids and intravenous immunoglobulin(IVIG).In a recent retrospective study,Sun et al reported that the response rates of pregnant patients with ITP to either IVIG or corticosteroids were less than 40%and might be lower than that of the nonpregnant patients.The secondary options are limited if the patients fail to respond to the primary treatments.Recombinant human thrombopoietin(rhTPO)is a full-length glycosylated TPO with a molecular weight of 90 000 Daltons,expressed in Chinese hamster ovary cells and purified using bio-engineering techniques.rhTPO is a recombinant form of the c-Mp1 ligand.It maintains an amino acid sequence identical to the endogenous TPO,but further glycosylated.It has been proven to be active in both human and animal models.rhTPO was approved by the China State Food and Drug Administration for the treatment of chronic ITP refractory to first-line therapy.A multicenter randomized trial revealed that rhTPO rapidly increased platelet counts with a total res-ponse rate of 60.3%in patients with steroid-resistant ITP.However,rhTPO has not been tested for the management of patients with ITP in pregnancy.Here,we performed a prospective multicenter open-labeled study to investigate whether rhTPO is clinically applicable for ITP in pregnancy.AIMThis multicenter,open-labeled,single-arm study aimed to determine the safety and efficacy of rhTPO in patients with corticosteroid/IVIG-resistant ITP in pregnancy.METHODS1.Enrollment criteria included pregnant women between 18 and 50 years of age with bleeding manifestations and failure to respond to corticosteroids and/or IVIG(can be with stable dose of corticosteroids on enrollment)who were refractory to platelet transfusion.Patients who had 2 serial 1-hour posttransfusion corrected count increments of less than 10 × 109/L were considered as platelet refractoriness.Patients' platelet counts were below 30 x 109/L Gestational age was more than 12 weeks.2.All eligible participants received rhTPO at an initial dose of 300 U/kg once daily subcutaneously for 14 days and then received sequential maintenance therapy.To reduce the risk for thrombocytosis during maintenance,dose was tapered to 300 U/kg every other day when platelet counts exceeded 50 × 109/L,and treatment discontinued when platelet counts rose above 100 × 109/L,After delivery,the dose was further tapered to 300 U/kg every week,and adjusted if the platelet count could not maintain above 30 × 109/L.If the platelet count did not reach 30 × 109/L or above within 2 weeks or fell below 30 × 109/L and did not achieve 30 × 109/L within 2 weeks after dose adjustment,then treatment was discontinued?If patients experienced severe bleeding symptoms,platelet transfusion was permitted.Any requirements for additional ITP-specific intervention were considered treatment failure.3.The primary end point was a platelet count greater than 30 x 109/L on the 14th day of the study.The platelet counts were determined at each collaborative center.Complete response was defined as a platelet count of at least 100 × 109/L and the absence of bleeding.Response was defined as a platelet count more than 30 × 109/L and increase of at least twice the baseline.Nonresponse was defined as a platelet count below 30 × 109/L,We considered the following secondary endpoints:adverse events in mothers and neonates;neonatal platelet counts at birth,day 3 and day 7 if necessary(thrombocytopenic neonates),and day 42;and stillbirth,premature birth(before 37 weeks of gestation),and birth weight,2.5 kg.4.All patients were assessed weekly for the efficacy and safety of the treatment in pregnancy and at 4-week intervals for 24 weeks after delivery.After birth,infants' physical measures and development assessment were performed monthly by pediatricians.5.Assessment of bleeding was performed on days 1 and 14,according to the reported scoring system by the Gruppo Italiano Malattie Ematologiche dell' Adulto ITP Working Party.6.Serum anti-TPO antibodies were measured by enzyme-linked immunosorbent assay 4 weeks after the first dose,at the end of rhTPO treatment,and 6 months after delivery.7.All participants who received at least 1 dose of rhTPO were included in primary and safety analyses.Baseline characteristics of the study population were summarized using descriptive statistical methods.Correlations were analyzed using Spearman's correlation.Differences between groups were analyzed using Fisher's exact test or t test.A P value less than.05 was considered statistically significant.Data management and statistical analyses were performed using Statistical Package for the Social Sciences,version 22.0(SPSS,Chicago,IL).RESULT1.Patients32 pregnant patients with ITP were enrolled in the study.In 1 patient,the diagnosis changed from ITP to aplastic anemia before treatment initiated.All the mothers with ITP were followed up to the end of the 24th week after delivery.The median follow-up time for infants was 53(range,39-68)weeks.The median age of the pregnant ITP patients was 26 years(interquartile range[IQR],24-33 years),and 93.5%(29/31)were primigravidae.The median gestational age at the time of enrollment was 24 weeks(IQR,16-27 weeks).The median baseline platelet count was 10 ×109/L(IQR,6-12 ×109/L).Three patients had underlying pregnancy-induced hypertension,and 4 had pregnancy-related diabetes.Seventy-four percent(23/31)of these patients were diagnosed with ITP before pregnancy,and 25.8%(8/31)were diagnosed during pregnancy.None of the enrolled pregnant patients with ITP had ever responded to previous treatments.Although some patients did have temporary increase of platelet count,none of their peak platelet counts exceeded 30×109/L.All patients were heavily transfused and became refractory to platelet transfusion before enrollment-On the day of enrollment,most patients were graded with a bleeding score of 1 with petechiae or a score of 2 with hematuria.Two patients were graded with a score of 3 with gingival and vagina bleeding,respectively.2.ResponseOf these patients,74.2%(23/31)responded to the initial 14-day rhTPO therapy,including 10 with complete response and 13 with response.Eight patients were nonresponsive,although their platelet counts rose mildly.The median platelet count of responders was 100×109/L(IQR,36-160 ×109/L)on day 14.On day 14,most participants were graded 0(25/31,80.6%).It appears that the bleeding symptom of 26 patients was ameliorated.The relapse-free survival rates(platelet count at least 30x109/L)at weeks 4 and 12 after withdrawal of rhTPO were 69.6%(16/23)and 21.7%(5/23),respectively.3.Safety of mothersSafety and adverse events were evaluated in all 31 participants.rhTPO was well tolerated.Only mild previously reported adverse events were observed,including 1 case of dizziness,1 of fatigue,and 1 of pain at injection site.The safety of the 31 infants was also evaluated.No congenital disease or developmental delays of newborns were observed during a median follow-up of 53(range,39-68)weeks.ConclusionThis study demonstrates that rhTPO is a potentially safe,effective,and fast-acting treatment of pregnant patients with ITP who are refractory to fist-line therapy and platelet transfusion.Our work has paved the way for further study on the clinical application of rhTPO and other thrombopoietic agents in the management of ITP in pregnancy.Part ?:Study on Apoptosis-Promoting Effect of Anti-GPIb Monoclonal AntibodiesBackgroundPrimary immune thrombocytopenia(ITP)is the most common immunological hemorrhagic disease in clinical practice.It accounts for about 30%of hemorrhagic diseases.It is characterized by a decrease in platelet counts in peripheral blood.The symptoms can be mild without bleeding,and also can be life-threatening.ITP patients have lost immune tolerance,resulting in autoantibodies against platelet glycoprotein receptors GP?b/?a and GP?b/?.20-40%of ITP patients have autoantibodies against the complex of GPIb.Apoptosis is also an important pathway for platelet clearance.Plasma of anti-GPIb ITP patients was incubated with healthy platelets,and platelet apoptosis was observed.Thus,both GPIba initiation and apoptosis play an important role in the pathogenesis of ITP.In this study,anti-GPIba monoclonal antibody(mAb)was used to establish a passive immunized ITP animal model in Balb/c mice to study the relationship between anti-GPIba antibodies and platelet apoptosis,and to explore the possible mechanism of ITP.MethodA.Whether NIT G can cause thrombocytopenia without Fc or F(ab)2 structures 1.Does IVIG alleviate thrombocytopenia induced by anti-GPIb? monoclonal antibodyWT Balb/c mice were divided into four groups:IVIG + NIT G group,IVIG +NIT B group,PBS + NIT G group,and PBS + NIT B group.Inject 10%IVIG on the first day of experiment(DO).Anti-GPIb monoclonal antibody NIT G or NIT B was injected one day later(D1).2.Will monovalent NIT G Fab cause thrombocytopeniaWT Balb/c mice were injected intravenously with intact NIT G antibodies(1.5 ?g/mouse),NIT G Fab(1.0 ?g/mouse)and PBS(1.5 ?g/mouse),respectively.B,NIT G and monovalent NIT G Fab can induce platelet apoptosis1.Can NIT G and NIT G Fab cause PS exposure?WT Balb/c mice were injected intravenously with IgG(as a control,1.5?g/mouse)intact NIT G antibody(1.5 ?g/mouse),NIT G Fab(1.0 ?g/mouse)and NIT B(1.5 ?g/ml)respectively.24 hours later,mouse PRP were collected,use Annexin V to stain and flow cytometry to detect PS exposure.2.NIT G and NIT G Fab increase Caspase-3 activationWT Balb/c mice were treated with PBS(100 ?L/mouse),intact NIT G antibody(1.5 ?g/mouse),NIT G Fab(1.0 ?g/mouse)and NIT B(1.5 ?g/mouse)respectively.Mouse PRP was stained with FAM-FLICA caspase 3/7 kit and caspase-3 activation was measured by flow cytometry.3.Effect of NIT G on platelet protein phosphorylationAll the antibodies to be detected(1 ?L)were incubated with PRP(100 ?L)of wild-type Balb/c mice for 1 hour,then centrifuged,and using 4G10 ? detect phosphorylation.C,Can anti-GPIba antibodies cause mouse platelet activation under high shear stressThe antibody was incubated with PRP for 30 minutes and 15 ?L of PRP was loaded and sheared with a cone-plate viscometer.P-selection exposure was determined by flow cytometry.ResultA.NIT G can cause thrombocytopenia without Fc or F(ab)2 structure 1.IVIG can ameliorate NIT B-induced thrombocytopenia but can not alleviate NIT G-induced thrombocytopeniaComparing IVIG + NIT G with NIT G,IVIG + NIT B and NIT B,the results indicate that IVIG can improve thrombocytopenia caused by NIT B,but it can not alleviate the thrombocytopenia caused by NIT G.NIT G may cause thrombocytopenia by a non-Fc receptor dependent pathway.2.Monovalent NIT G Fab can cause thrombocytopeniaFour hours after injected NIT G or NIT G Fab,mice platelets began to drop.Platelets were significantly reduced after 24 hours,and gradually returned to normal on the 4th day.This shows that NIT G rely on neither the Fc segment or the F(ab)2 structure to cause thrombocytopenia.B.NIT G and monovalent NIT G Fab can cause platelet apoptosis1.NIT G and NIT G Fab can cause PS exposureNIT G and NIG Fab increased platelet surface PS exposure,whereas NIT B did not(P = 0.0162,0.0170,0.2886).PS exposure is one of the hallmarks of apoptosis.It is a "clear me" signal that causes clearance.NIT G and NIT G fab may cause apoptosis of platelets therefor thrombocytopenia.2.NIT G and NIT G Fab increase Caspase-3 activationThe results showed that compared with the control group,NIT G and NIG Fab increased the activation of caspase-3 in platelets(P = 0.0101,0.0143),while the results in NIT B group showed no significant difference(P = 0.0828).Caspase-3 It is an important molecule in the apoptotic pathway,suggesting that NIT G and NIT G Fab may induce platelet apoptosis by activating the apoptotic pathway.3.NIT G can inhibit the level of JAK2 phosphorylationIn vitro,platelets of WT Balb/c mice were incubated with different antibodies.Tyrosine phosphorylation in platelet proteins was detected by probe 4G10 and the band around 120KD was found to have a difference.The anti-phospho-JAK2 antibody pTyr1007 showed that this band was JAK2,NIT G could inhibit the phosphorylation of JAK2(P = 0.018),while NIT B,NIT E had no significant difference with the control group(P>0.05).C.anti-GPIba antibodies do not cause platelet activation in mice under high shear stressIt was found that the anti-GPIba antibodies NIT A,NIT B,NITG did not increase the platelet surface p-selectin in WT Balb/c mice under high shear stress.Conclusion1.Anti-GPIba monoclonal antibody NIT G and monovalent NIT G Fab can cause platelet apoptosis.And this ability does not depend on F(ab)2 structure or Fc segment,which means no antibody cross-linking is required.2.NIT G and NIT G Fab may promote platelet apoptosis through inhibition of JAK2 phosphorylation.