Thrombopoietin ameliorates doxorubicin-induced toxicities in H9c2 myocardiocytes by inhibiting oxidative stress through the SIRT1/p38 MAPK signaling pathway

Objective:To explore whether thrombopoietin can exert a protective effect against doxorubicin-induced cardiotoxicity by modulating the sirtuin 1(SIRT1)signaling pathway.Methods:H9c2 cell viability was determined by CCK-8 and cardiomyocyte apoptosis was detected by TUNEL assay.The protein expressions of SIRT1 and p38 MAPK were measured by Western blot.RT-qPCR was also used to determine SIRT1 mRNA expression.In addition,intracellular reactive oxygen species levels and antioxidant enzyme activities were evaluated.Results:Thrombopoietin treatment reversed doxorubicin-induced decline in H9c2 cell viability.It also increased SIRT1 and decreased p-p38 MAPK protein expressions.In addition,thrombopoietin significantly attenuated doxorubicin-induced apoptosis and oxidative stress,and enhanced antioxidant enzyme activities.However,silencing SIRT1 abrogated the protective effects of thrombopoietin,as evidenced by reduced cell viability and increased oxidative stress and reactive oxygen species levels.Conclusions:Thrombopoietin alleviates doxorubicin-induced cardiomyocyte injury by reducing oxidative stress and apoptosis via the SIRT1/p38 MAPK pathway.However,its protective effects need to be further verified in animal tests.

血小板生成素thrombopoietin对神经保护作用的体外研究

目的体外研究血小板生成素(TPO,一种调控巨核细胞和血小板生成的因子),对神经祖细胞C17.2的保护作用。方法建立无血清条件下C17.2细胞凋亡的模型,用TPO不同浓度处理C17.2细胞,利用MTT、Western blotting、流式细胞技术等方法检测TPO对C17.2细胞的保护作用。结果不同浓度的TPO(0、1、10、50、100、200ng/ml)都能促进C17.2细胞增殖,并且具有剂量依赖性。TPO使磷酸化AKT水平增加,促进神经祖细胞增殖,LY294002可以阻止细胞的增殖。用流式细胞仪方法检测表达Annexin V的细胞减少。结论体外研究显示TPO通过激活PI3K/AKT信号通路,对神经祖细胞C17.2起保护作用,这一发现为临床上神经损伤的治疗提供了新的思路。

Trombinol,a bioactive fraction of Psidium guajava,stimulates thrombopoietin expression in HepG2 cells

Objective:To study the regulation of trombinol on thrombopoietin,an essential regulator of thrombocyte production.Methods:Effect of trombinol on thrombopoietin regulation was evaluated at the m RNA and protein levels in human hepatoma Hep G2 cells.The m RNA expressions were revealed by PCR and real-time PCR,while the protein expressions were analyzed using western blotting and human ELISA kit.Statistical differences between the test were determined by student's t-test with P < 0.05 was considered statistically significant.Results:Trombinol significantly increased the expression of thrombopoietin at the level of m RNA and protein secretion in Hep G2 cell lines.Trombinol with the concentration of15 mg/m L,positively induces 2.5-fold of thrombopoietin expression.Up-regulation of GABP,a transcription factor of thrombopoietin,is suggested to be involved in cellular regulatory mechanisms of trombinol.Here,our result shows convincing evidence that trombinol affects the thrombopoietin productions in vitro.This molecular explanation of thrombopoietin's stimulating function is in line with the traditional use of Psidium guajava for treatment of diseases involving thrombocytopenia.Conclusions:Thrombopoietin stimulating function of trombinol could be potentially considered as one of alternative treatment for thrombocytopenia-related cases,including post chemotherapy shock,dengue fever and liver failure.

Recombinant human thrombopoietin treatment in patients with chronic liver disease-related thrombocytopenia undergoing invasive procedures:A retrospective study

BACKGROUND Chronic liver disease(CLD)related thrombocytopenia increases the risk of bleeding and poor prognosis.Many liver disease patients require invasive procedures or surgeries,such as liver biopsy or endoscopic variceal ligation,and most of them have lower platelet counts,which could aggravate the risk of bleeding due to liver dysfunction and coagulation disorders.Unfortunately,there is no defined treatment modality for CLD-induced thrombocytopenia.Recombinant human thrombopoietin(rhTPO)is commonly used to treat primary immune thrombocytopenic purpura and thrombocytopenia caused by solid tumor chemotherapy;however,there are few reports on the use of rhTPO in the treatment of CLD-related thrombocytopenia.AIM To evaluate the efficacy of rhTPO in the treatment of patients with CLDassociated thrombocytopenia undergoing invasive procedures.METHODS All analyses were based on the retrospective collection of clinical data of patients with CLD who were treated in the Department of Infectious Diseases at The First Affiliated Hospital of Soochow University between June 2020 and December 2021.Fifty-nine male and 41 female patients with liver disease were enrolled in this study to assess the changes in platelet counts and parameters before and after the use of rhTPO for thrombocytopenia.Adverse events related to treatment,such as bleeding,thrombosis,and disseminated intravascular coagulation,were also investigated.RESULTS Among the enrolled patients,78(78%)showed a platelet count increase after rhTPO use,while 22(22%)showed no significant change in platelet count.The mean platelet count after rhTPO treatment in all patients was 101.53±81.81×10^(9)/L,which was significantly improved compared to that at baseline(42.88±16.72×10^(9)/L),and this difference was statistically significant(P<0.001).In addition,patients were further divided into three subgroups according to their baseline platelet counts(<30×10^(9)/L,30-50×10^(9)/L,>50×10^(9)/L).Subgroup analyses showed that the median platelet counts after treatment were significantly higher(P<0.001,all).Ninety(90%)patients did not require platelet transfusion partially due to an increase in platelet count after treatment with rhTPO.No serious adverse events related to rhTPO treatment were observed.Overall,rhTPO demonstrated good clinical efficacy for treating CLD-associated thrombocytopenia.CONCLUSION rhTPO can improve platelet count,reduce the risk of bleeding,and decrease the platelet transfusion rate,which may promote the safety of invasive procedures and improve overall survival of patients with CLD.

Thrombopoietin induced proliferation and differentiation of fetal liver CD34^+ cells with phenotype change from hemopoiesis to neurogenesis

BACKGROUND： Previous studies have reported a neurotrophin-like motif in the N-terminal receptor binding region of the thrombopoietin （TPO） molecule, and have described localization of TPO and TPO receptor in the brain. Therefore, it is believed that TPO may be involved in regulation of neurogenesis. OBJECTIVE： To validate the effect of TPO on trans-differentiation, or differentiation from hematopoietic stem cells （HSCs） to neural stem cells （NSCs）. DESIGN, TIME AND SETTING： Comparative studies were performed from March 2004 to April 2007 at the Department of Experimental Medicine, Northern Hospital, and the Department of Immunology, Fourth Military Medical University of Chinese PLA. MATERIALS： Human fetal liver （FL） was obtained from fetuses after water-balloon abortion. Gestational age ranged from 16 to 20 weeks. The study was approved by the Institutional Review Board and Ethics Committee of the Northern Hospital. TPO was kindly provided by Genentech Inc （USA）. Iscove＇s Modified Dulbecco＇s Medium （IMDM） and neurobasalTM medium were purchased from Invitrogen （USA）. MACS CD34 multisort kit was purchased from Miltenyi Biotec （Germany）. METHODS： CD34^＋ cells were isolated from human FL mononuclear cells using MACS CD34 multisort kit and cultured at 1 × 10^5/mL in IMDM, containing TPO for 60 days with weekly changes of half of the medium. After culturing for 30 and 60 days, the TPO-induced cells were resuspended in neurobasalTM medium containing 10% fetal brain extracts and plated in an 8-well BIOCOAT poly-D-Lysine Culture Slide and cultured for another 7 days. MAIN OUTCOME MEASURES： Cell number, viability, phenotype and expression of hemopoiesis-related and neurogenesis-related proteins were examined by trypan blue exclusion with hemocytometer, immunoblot, immunocytochemistry and flow cytometry. RESULTS： After 60 days of induction with TPO, the cell number increased by 4.6-fold compared to the initial culture. Although the proportion of the cells expressing the hemopoietic stem cell associated antigen （CD34） decreased steadily, both proportions of the cultured FL-derived CD34^＋cells expressing CD41a and CD61 remained unchanged, which still accounted for 10%. Noticeably, the proportions of the cells expressing nestin and epidermal growth factor receptor increased significantly （both 〉 50%）, whereas the expression of more mature neural or glial proteins [microtubule-associated protein-2 （MAP2）, glial fibrillary acidic protein （GFAP）, oligodendrocyte marker 04 （04）] markers on the cultured fetal liver derived-CD34^＋ cells were at lower levels. After another 7 days incubation in neurobasalTM medium, these TPO-induced cells formed neurospheres, which were labeled with nestin, and differentiated into cells with morphological characteristics of neurons, astrocytes and oligodendrocytes, which were labeled with MAP-2, GFAE and 04, respectively. CONCLUSION： TPO can induce FL-derived HSCs to differentiate or trans-differentiate into NSCs and its progenitors.

Effects of thrombopoietin pre-treatment on peri-liver transplantation thrombocytopenia in a mouse model of cirrhosis with hypersplenism

BACKGROUND During cirrhosis,the liver is impaired and unable to synthesize and clear thrombopoietin properly.At the same time,the spleen assumes the function of hemofiltration and storage due to liver dysfunction,resulting in hypersplenism and excessive removal of platelets in the spleen,further reducing platelet count.When liver function is decompensated in cirrhotic patients,the decrease of thrombopoietin(TPO)synthesis is the main reason for the decrease of new platelet production.This change of TPO leads to thrombocytopenia and bleeding tendency in cirrhotic patients with hypersplenism.AIM To investigate the clinical efficacy of recombinant human TPO(rhTPO)in the treatment of perioperative thrombocytopenia during liver transplantation in cirrhotic mice with hypersplenism.METHODS C57BL/6J mice and TPO receptor-deficient mice were used to establish models of cirrhosis with hypersplenism.Subsequently,these mice underwent orthotopic liver transplantation(OLT).The mice in the experimental group were given rhTPO treatment for 3 consecutive days before surgery and 5 consecutive days after surgery,while the mice in the control group received the same dose of saline at the same frequency.Differences in liver function and platelet counts were determined between the experimental and control groups.Enzyme-linked immunosorbent assay was used to assess the expression of TPO and TPO receptor(c-Mpl)in the blood.RESULTS Preoperative administration of rhTPO significantly improved peri-OLT thrombocytopenia in mice with cirrhosis and hypersplenism.Blocking the expression of TPO receptors exacerbated peri-OLT thrombocytopenia.The concentration of TPO decreased while the concentration of c-Mpl increased in compensation in the mouse model of cirrhosis with hypersplenism.TPO pre-treatment significantly increased the postoperative TPO concentration in mice,which in turn led to a decrease in the c-Mpl concentration.TPO pre-treatment also significantly enhanced the Janus kinase(Jak)/signal transducers and activators of transcription pathway protein expressions in bone marrow stem cells of the C57BL/6J mice.Moreover,the administration of TPO,both before and after surgery,regulated the levels of biochemical indicators,such as alanine aminotransferase,alkaline phosphatase,and aspartate aminotransferase in the C57BL/6J mice.CONCLUSION Pre-treatment with TPO not only exhibited therapeutic effects on perioperative thrombocytopenia in the mice with cirrhosis and hypersplenism,who underwent liver transplantation but also significantly enhanced the perioperative liver function.

THE STIMULATING EFFECT OF HEPARIN IN SYNERGY WITHTHROMBOPOIETIN ON MEGAKARYOCYTOPOIESISAND THROMBOPOIESIS

Objective To demonstrate whether heparin could act synergically with thrombopoietin (TPO) instimulating megakaryocytopoiesis and thrombopoiesis. Methods Megakaryocytic leukemia cell line M- 07e,human cord blood CD34+ cells and Balb/c mice were used for studies. The effectS of hoparin and/or TPO onmegakaryocytopoiesis and thrombopoiesis were studied by [ 3H ] - TdR incoroperation assay, CFU- MK plasmicsemi- solid culture and experiment in Balb/c mice in vivo. In addition, M- 07e cells were used as targets tofurther investigate the possible molecular mechanism by which heparin might act synergically with TPO throughNorthern and Western blot analyses. Results Heparin can act synergically with TPO in stimulating theproliferation of leukemia cell line M- 07e, growth of CFU- MK from human cord blood CD34+ cells andmegakaryocytopoiesis and thrombopoiesis in vivo in mice, possibly by antagonizing downregulation of c- mplexpression by TPO at the level of mRNA transcription, and increasing the phosphotyrosine content Of Jak2,triggered by TPO. Conclusion Heparin participated in positive regulation of megakaryocytopoiesis andthrombopoiesis by enhancing the megakaryocytopoietic activities of TPO, and the mechanism of which might beinvolved in the modification of the molecules c- mpl and Jak2 on the way of signal transduction triggered by TPO.

Correlation between thrombopoietin and inflammatory factors,platelet indices,and thrombosis in patients with sepsis:A retrospective study

BACKGROUND Thrombopoietin(TPO)is a primary regulator of thrombopoiesis in physiological conditions.TPO,in combination with its specific cytokine receptor c-Mpl,drives platelet production by inducing the proliferation and differentiation of megakaryocytes.However,the role of TPO in sepsis is not well determined.The elevated levels of TPO are often accompanied by a decrease of platelet count(PLT)in systemic infected conditions,which is contrary to the view that TPO promotes platelet production under physiological conditions.In addition,whether TPO mediates organ damage in sepsis remains controversial.AIM To explore the relationships between TPO and inflammatory factors,platelet indices,and thrombotic indicators in sepsis.METHODS A total of 90 patients with sepsis diagnosed and treated at the emergency medicine department of The First People’s Hospital of Foshan between January 2020 and March 2021 were enrolled in this study.In addition,110 patients without sepsis who came to the emergency medicine department were included as controls.Clinical and laboratory parameters including age,gender,TPO,blood cell count in peripheral blood,platelet indices,inflammatory factors such as high-sensitivity Creactive protein(hs-CRP),interleukin(IL)-21,and IL-6,organ damage indicators,and thrombotic indicators were collected and analyzed by using various statistical approaches.RESULTS The results showed that the TPO levels were higher in the sepsis group than in controls[86.45(30.55,193.1)vs 12.45(0.64,46.09)pg/mL,P<0.001],but PLT was lower(P<0.001).Multivariable analysis showed that white blood cell count(WBC)[odds ratio(OR)=1.32;95%confidence interval(CI):1.01-1.722;P=0.044],TPO(OR=1.02;95%CI:1.01-1.04;P=0.009),IL-21(OR=1.02;95%CI:1.00-1.03;P=0.019),troponin I(OR=55.20;95%CI:5.69-535.90;P=0.001),and prothrombin time(PT)(OR=2.24;95%CI:1.10-4.55;P=0.027)were independent risk factors associated with sepsis.TPO levels were positively correlated with IL-21,IL-6,hs-CRP,creatinine,D-dimer,PT,activated prothrombin time,international normalized ratio,fibrinogen,WBC count,and neutrophil count,and negatively correlated with PLT,thrombin time,red blood cell count,and hemoglobin concentration(P<0.05).Receiver operating characteristic analysis showed that TPO had fair predictive value in distinguishing septic patients and non-septic patients(the area under the curve:0.788;95%CI:0.723-0.852;P<0.001).With an optimized cutoff value(28.51 pg/mL),TPO had the highest sensitivity(79%)and specificity(65%).CONCLUSION TPO levels are independently associated with sepsis.High TPO levels and low PLT suggest that TPO might be an acute-phase response protein in patients with infection.

Thrombopoietin-receptor agonists in perioperative treatment of patients with chronic liver disease

Thrombocytopenia is a multifactorial disorder that is common in patients with chronic liver disease(CLD),leading to challenging perioperative planning.As thrombocytopenia in CLD is associated with thrombopoietin(TPO)deficiency,the use of TPO-receptor agonists(TPO-RAs)to increase platelet counts is a promising approach.This has led to the development of various TPO-RAs,including romiplostim,eltrombopag,avatrombopag,and lusutrombopag.Of these,only avatrombopag and lusutrombopag are approved by the United States Food and Drug Administration for the perioperative treatment of thrombocytopenia in patients with CLD.Platelet transfusion is commonly used for the clinical management of thrombocytopenia in patients with CLD undergoing invasive procedures.However,the limitations and possible risks of transfusion,including short duration of efficacy,development of antiplatelet antibodies,risk of infections and such complications as transfusion-related acute lung injury or circulatory overload,and possibility of refractoriness,limit its use.Moreover,there is no consensus among guidelines as to the platelet count at which transfusions are indicated.Results from studies using TPO-RAs perioperatively in patients with thrombocytopenia and CLD are promising and provide an alternative to platelet transfusions in the pre-and post-operative setting.These TPO-RAs are the subject of this review,with focus on their use in the perioperative setting in patients with thrombocytopenia,associated supporting clinical trials,efficacy and safety data,and their use with respect to platelet transfusions.

Recombinant human thrombopoietin in alleviating endothelial cell injury in sepsis

Background To evaluate the effect of recombinant human thrombopoietin(rhTPO)on clinical prognosis by exploring changes in endothelial cell injury markers and inflammatory factors in patients with sepsis after treatment with rhTPO.Methods This retrospective observational study involved patients with sepsis(diagnosed according to Sepsis 3.0)admitted to Shanghai General Hospital intensive care unit from January 1,2019 to December 31,2022.Patients were divided into two groups(control and rhTPO)according to whether they received rhTPO.Baseline information,clinical data,prognosis,and survival status of the patients,as well as inflammatory factors and immune function indicators were collected.The main monitoring indicators were endothelial cell-specific molecule(ESM-1),human heparin-binding protein(HBP),and CD31;secondary monitoring indicators were interleukin(IL)-6,tumor necrosis factor(TNF)-α,extravascular lung water index,platelet,antithrombin III,fibrinogen,and international normalized ratio.We used intraperitoneal injection of lipopolysaccharide(LPS)to establish a mouse model of sepsis.Mice were randomly divided into four groups:normal saline,LPS,LPS+rhTPO,and LPS+rhTPO+LY294002.Plasma indicators in mice were measured by enzyme-linked immunosorbent assay.Results A total of 84 patients were included in the study.After 7 days of treatment,ESM-1 decreased more significantly in the rhTPO group than in the control group compared with day 1(median=38.6[interquartile range,IQR:7.2 to 67.8]pg/mL vs.median=23.0[IQR:−15.7 to 51.5]pg/mL,P=0.008).HBP and CD31 also decreased significantly in the rhTPO group compared with the control group(median=59.6[IQR:−1.9 to 91.9]pg/mL vs.median=2.4[IQR:−23.2 to 43.2]pg/mL;median=2.4[IQR:0.4 to 3.5]pg/mL vs.median=−0.6[IQR:−2.2 to 0.8]pg/mL,P<0.001).Inflammatory markers IL-6 and TNF-αdecreased more significantly in the rhTPO group than in the control group compared with day 1(median=46.0[IQR:15.8 to 99.1]pg/mL vs.median=31.2[IQR:19.7 to 171.0]pg/mL,P<0.001;median=17.2[IQR:6.4 to 23.2]pg/mL vs.median=0.0[IQR:0.0 to 13.8]pg/mL,P=0.010).LPS+rhTPO-treated mice showed significantly lower vascular von Willebrand factor(P=0.003),vascular endothelial growth factor(P=0.002),IL-6(P<0.001),and TNF-α(P<0.001)than mice in the LPS group.Endothelial cell damage factors vascular von Willebrand factor(P=0.012),vascular endothelial growth factor(P=0.001),IL-6(P<0.001),and TNF-α(P=0.001)were significantly elevated by inhibiting the PI3K/Akt pathway.Conclusion rhTPO alleviates endothelial injury and inflammatory indices in sepsis,and may regulate septic endothelial cell injury through the PI3K/Akt pathway.

Plasma thrombopoietin levels in patients with aplastic anemia and idiopathic thrombocytopenic purpura

OBJECTIVE: To evaluate the role of thrombopoietin (TPO) in the pathology of chronic thrombocytopenic disease. METHODS: We measured the endogenous plasma concentration of TPO in 40 patients with acquired aplastic anaemia (AA) and in 32 patients with idiopathic thrombocytopenic purpura (ITP) by a sensitive Sandwich enzyme-linked immunosorbent assay (ELISA) and compared the results. RESULTS: Plasma TPO concentrations were significantly higher in AA patients (774 +/- 393 pg/ ml) in comparison with healthy control subjects (55 +/- 34 pg/ml, P 0.05). There was also no relationship between their plasma TPO levels and platelet counts. CONCLUSIONS: TPO levels may be regulated not only by platelets but also by megakaryocytes in AA and ITP, and measurement of TPO levels is useful for diagnosing thrombocytopenia and understanding the pathophysiology of thrombocytopenia.

Recombinant human thrombopoietin in combination with cyclosporin A as a novel therapy in corticosteroid-resistant primary immune thrombocytopenia

Background The management of patients with refractory immune thrombocytopenia （ITP） is challenging, as there is no standard treatment option. The aim of this study was to investigate the efficacy of recombinant human thrombopoietin （rhTPO） in combination with cyclosporin A （CsA） for the management of patients with corticosteroid-resistant primary ITP.

Construction of a fusion protein between N-terminal 153 peptide of thrombopoietin and erythropoietin

Thrombopoietin (TPO) functions as a regulator of megakaryocytes in their differentiation and maturation, and is a candidate pharmaceutical for the curing of thrombocytopenia. Erythropoietin (EPO) is a hematopoietic cytokine that regulates the level of red blood cell. It is widely used in renal anemia and tumor associated anemia, and is proved to be safe and effective. In order to study the possibility of using TPO EPO fusion protein for the curing of anemia and thrombocytopenia induced by high dose chemotherapy, a fusion gene is constructed by linking TPO N terminal 153 peptide and EPO mature peptide coding region. The fusion gene is expressed in mammalian cells, revealing that the expression product can support the growth of TPO responsive Ba/F3 mpl cells and EPO dependent Bet 2 cells in the absence of any other stimulating cytokine. It also stimulates the formation of erythroid colonies and megakaryocytic colonies in semi solid bone marrow cultures. These results indicate that the fusion protein has both the in vitro activities of TPO and EPO. The preliminary in vivo experiment reveals that the TPO EPO fusion protein containing cell supernatant raises the platelet level by 37% in mice, while its function on erythroid hematopoiesis remains to be determined. These results indicate that the construction of TPO EPO fusion protein and the further study of its use in high dose chemotherapy are possible

The equivalents of human blood and spleen dendritic cell subtypes can be generated in vitro from human CD34 ＋ stem cells in the presence of fins-like tyrosine kinase 3 ligand and thrombopoietin

Dendritic cells （DCs） are immune cells specialized to capture, process and present antigen to T cells in order to initiate an appropriate adaptive immune response. The study of mouse DC has revealed a heterogeneous population of cells that differ in their development, surface phenotype and function. The study of human blood and spleen has shown the presence of two subsets of conventional DC including the CDlb/c＋ and CD141＋CLEC9A＋ conventional DC （cDC） and a plasmacytoid DC （pDC） that is CD304＋CD123＋. Studies on these subpopulations have revealed phenotypic and functional differences that are similar to those described in the mouse. In this study, the three DC subsets have been generated in vitrofrom human CD34＋ precursors in the presence of fins-like tyrosine kinase 3 ligand （FIt3L） and thrombopoietin （TPO）. The DC subsets so generated, including the CDlb/c＋ and CLEC9A＋ cDCs and CD123 ＋ pDCs, were largely similar to their blood and spleen counterparts with respect to surface phenotype, toll-like receptor and transcription factor expression, capacity to stimulate T cells, cytokine secretion and cross-presentation of antigens. This system may be utilized to study aspects of DC development and function not possible in vivo.

Switching between eltrombopag and recombinant human thrombopoietin in patients with immune thrombocytopenia: an observational study

Background:Recombinant human thrombopoietin(rh-TPO)and eltrombopag are two distinct TPO receptor agonists(TPO-RAs)with different mechanisms.During the pandemic,when immunosuppressive medications are controversial,switching to another TPO-RA may be worth exploring in patients who do not benefit from their first TPO-RA.We investigated the outcomes of switching from rh-TPO to eltrombopag or vice versa in immune thrombocytopenia(ITP)patients.Methods:This prospective,open-label,observational investigation included 96 adult ITP patients who needed to switch between rh-TPO and eltrombopag between January 2020 and January 2021 at Peking University People’s Hospital in China.The study evaluated response rates and platelet counts at different time points after the switch,bleeding events,time to response,duration of response,and adverse events.Results:At 6 weeks after switching,response was observed in 21/49 patients(43%)who switched for inefficacy and 34/47 patients(72%)who switched for non-efficacy-related issues.In the inefficacy group,9/27 patients(33%)responded to eltrombopag,and 12/22 patients(55%)responded to rh-TPO.In the non-efficacy-related group,21/26(81%)and 13/21(62%)patients in the eltrombopag and rh-TPO groups maintained their response rates at 6 weeks after switching,respectively.Response at 6 months was achieved in 24/49 patients(49%)switching for inefficacy and 37/47 patients(79%)switching for non-efficacy issues.In the inefficacy group,13/27 patients(48%)responded to eltrombopag,and 11/22 patients(50%)responded to rh-TPO.In the non-efficacy-related group,22/26 patients(85%)and 15/21 patients(71%)in the eltrombopag and rh-TPO groups maintained their response rates at 6 months after switching,respectively.Both eltrombopag and rh-TPO were well tolerated.Conclusions:Our study confirmed the safety and effectiveness of switching between rh-TPO and eltrombopag for ITP patients who had no response to or experienced adverse events with their first TPO-RA.When the switch was motivated by other reasons,including patient preference and platelet count fluctuations,the probability of response was high.Registration:ClinicalTrials.gov,NCT04214951.

口服海曲泊帕与皮下注射重组人血小板生成素用于单倍体造血干细胞移植

背景:异基因造血干细胞移植是治疗恶性血液病的重要手段,术后血小板植入延迟是常见并发症,严重影响患者生存质量,然而,目前并无标准方案来提高血小板植入率和预防血小板植入延迟。目的:对比分析口服海曲泊帕与皮下注射重组人血小板生成素促进恶性血液病患者单倍体造血干细胞移植后血小板植入的安全性及有效性。方法:回顾性分析2016年1月至2022年10月进行单倍体造血干细胞移植的163例恶性血液病患者的临床资料。+2 d开始皮下注射重组人血小板生成素的患者共72例,归为重组人血小板生成素组;+2 d开始口服海曲泊帕的患者共27例,归为海曲泊帕组;未应用海曲泊帕及重组人血小板生成素的64例患者归为空白对照组。对3组植入情况、100 d内Ⅱ-Ⅳ度急性移植物抗宿主病发生率、1年生存率、1年复发率及安全性进行分析。结果与结论:(1)中位随访时间52(12-87)个月,空白对照组、重组人血小板生成素组、海曲泊帕组患者中性粒细胞植入时间分别为(12.95±3.88)d,(14.04±3.71)d,(13.89±2.74)d,差异无显著性意义(P=0.352);血小板植入时间分别为(15.16±6.27)d,(17.67±6.52)d,(17.00±4.75)d,差异无显著性意义(P=0.287);(2)空白对照组、重组人血小板生成素组、海曲泊帕组第60天血小板完全植入率分别为64.06%,90.28%,92.59%,差异有显著性意义(P<0.001);亚组分析显示,空白对照组与重组人血小板生成素组比差异有显著性意义(P<0.001),空白对照组与海曲泊帕组比差异有显著性意义(P=0.004),重组人血小板生成素组与海曲泊帕组比差异无显著性意义(P=0.535);(3)空白对照组、重组人血小板生成素组、海曲泊帕组100 dⅡ-Ⅳ度急性移植物抗宿主病发生率分别为25.00%,30.56%,25.93%,差异无显著性意义(P=0.752);(4)巨细胞病毒血症、巨细胞病毒肺炎、肝功能损伤发生率在3组间无显著性差异(P>0.05);(5)随访期内,3组患者均未发生血栓事件;(6)结果表明,重组人血小板生成素、海曲泊帕均可提高恶性血液病患者单倍体造血干细胞移植后血小板的植入率,疗效相当且安全性良好。

Study of thrombopoietin for gene therapy of thrombocytopenia

Thrombopoietin (TPO) is likely to be a potent, specific and reliable medication in the treatment of thrombocytopenia. A TPO-highly-expressed plasmid pcDNA3-TPO was constructed and a primary study was made on the expression of TPO cDNA in vitro and gene transfer study for thrombocytopenia in vivo. rhTPO showed complete and stable bioactivity by a series of indicators. High expression of TPO was detected in plasma from healthy mice or thrombocytopenia mice model receiving direct intramuscular injection of pcDNA3-TPO. And the platelet level of healthy mice peaked to 1.9-fold of baseline. Mice with CTX-induced thrombocytopenia achieved profound nadirs, acceleration of recovery, even 1.8—2.0-fold supranormal levels of peripheral platelet counts. The results offered experimental support for clinical application of gene therapy for thrombocytopenia via direct intramuscular injection of TPO cDNA.

Screening of proteins that interact with human thrombopoietin receptor c-Mpl using yeast two-hybrid system

Thrombopoietin (TPO) is the major cytokine involved in platelet production and exerts its effects via the receptor c-Mpl. The yeast two-hybrid system has been used to screen the proteins interacting with c-Mpl. First, the cDNA fragment of c-Mpl intracellular domain was cloned into two-hybrid vector pAS2, and the resulting plasmid is designated as pASMM. Then a human placenta cDNA library was screened using the pASMM as a target plasmid. Seven positive clones were isolated from 150 000 independent transformants. Sequence analysis of one of the positive clones demonstrates that a part of coding sequence of vimentin from 611 bp to 3’ end and flanking non-translation region was obtained. Therefore, there is an interaction between vimentin and TPO receptor. The results suggest that cytoskeletal protein may play an important role in TPO signal transduction pathway.

Recombinant human thrombopoietin prior to mobilization chemotherapy facilitates platelet recovery in autologous transplantation in patients with lymphoma:Results of a prospective randomized study

Background:Chemotherapy plus granulocyte colony-stimulating factor (GCSF) regimen is one of the available approaches to mobilize peripheral blood progenitor cells (PBPCs).It causes thrombocytopenia and delays leukapheresis.This study aimed to evaluate the role of recombinant human thrombopoietin (rhTPO) before mobilization chemotherapy in facilitating leukapheresis in patients with lymphoma.Methods:In this randomized open-label phase 2 trial, patients were randomly assigned in a 1:2 ratio to receive mobilization with rhTPO plus GCSF in combination with chemotherapy (the rhTPO plus GCSF arm) or GCSF alone in combination with chemotherapy (the GCSF alone arm).The recovery of neutrophils and platelets and the amount of platelet transfusion were monitored.Results:Thirty patients were enrolled in this study between March 2016 and August 2018. Patients in the rhTPO plus GCSF arm (n = 10) had similar platelet nadir after mobilization chemotherapy (P=0.878) and similar amount of platelet transfusion (median 0 vs.1 unit,P=0.735) when compared with the GCSF alone arm (n = 20). On the day of leukapheresis, the median platelet count was 86 ×10^(9)/L (range 18-219) among patients who received rhTPO and 73 ×10^(9)/L (range 42-197) among those who received GCSF alone (P=0.982). After the use of rhTPO, the incidence of platelet count <75 ×10^(9)/L on the day of leukapheresis did not decrease significantly (30.0% vs. 50.0%,P=0.297).Platelet recovery after PBPC transfusion was more rapid in the rhTPO plus GCSF arm (median 8.0 days [95% confidence interval 2.9-13.1] to platelets ≥50 ×10^(9)/L vs. 11.0 days [95% confidence interval 8.6-13.4],P=0.011).The estimated total cost of the mobilization and reconstitution phases per patient was similar between the two treatmtent groups (P=0.362 andP=0.067,respectively).Conclusions:Our findings indicate that there was no significant clinical benefit of rhTPO use in facilitating mobilization of progenitor cells,but it may promote platelet recovery in the reconstitution phase after high-dose therapy.Trial registration:This trial has been registered in Clinicaltrials.gov as NCT03014102.

Protective Effect of Thrombopoietin on Myocardial Cell in Vitro

Objectives To investigate the protective effect of thrombopoietin (TPO) on myocardial cells in vitro. Methods H9C2 cell line was maintained in Iscove’s modified Dulbecco’s medium (IMDM) supplemented with 10% calf serum. Beating cells from heart ventricles of neonatal heart were cultured at an in vitro system. Apoptosis of the cell line above was induced by treatment of doxorubicin (DOX) and was blocked by TPO. Cell survival rate of H9C2 cell was measured by the MTT assay. Changes of beating rate of neonatal myocardial cells were captured by digital camera and beating rate was calculated. Flow cytometry was employed to study anti-apoptotic effect of TPO by staining JC-1 protein to H9C2 cell. Results MTT assay demonstrated that doxorubicin reduced cell survival rate by 73.8%±1.1%, 50 ng·mL-1 and 100 ng·mL-1 TPO increased cell survival rate by 84.6%±3.6% (P<0.05), 86%±4% (P<0.01) at a dose-dependent manner. Beating rate of primary neonatal myocardial cells also decreased to 15%±8% at 48 h, 100 ng·mL-1 TPO improved beating rate to 48%±11% (P<0.01). TPO decreased apoptotic rate from 19%±9% to 11%±6% (P<0.05). Conclusions TPO has protective effect on myocardial cells in vitro. Anti-apoptosis is one of the mechanisms by which TPO protects injured heart.