Thrombopoietic cytokines and reversal of thrombocytopenia after liver transplantation

OBJECTIVES: Thrombopoietin (TPO), the key regulator of platelet production, is mainly produced by the liver and reduced expression of TPO could cause thrombocytopenia in liver cirrhosis. Reversal of thrombocytopenia by orthotopic liver transplantation seems to be mediated through an increase in TPO plasma levels after transplantation, but other cytokines with thrombopoietic activity could augment the actions of TPO on post transplant platelet recovery. DESIGN: Measurement of thrombopoietic cytokines before and for 14 days post liver transplantation in a cohort of thrombocytopenic liver transplant patients. METHODS: TPO, interleukin-3 (IL-3), IL-6, and IL-11 plasma levels as well as peripheral platelet count were analysed in thrombocytopenic patients with liver disease undergoing orthotopic liver transplantation (17 patients) and followed for 14 days after the intervention. RESULTS: Before liver transplantation, TPO plasma levels were undetectable and IL-3, IL-6, and IL-11 levels were normal. Sixteen out of 17 patients showed a significant rise of TPO levels within 2 days after transplantation, with a peak between days 4 and 6, while IL-3 and IL-6 levels did not show a significant rise. IL-11 levels remained normal. Platelet counts were significantly higher than pretransplantation levels by day 14 post transplantation. CONCLUSION: Restitution of normal TPO production by liver replacement seems to be of key importance for reversal of thrombocytopenia in liver disease. The early acting thrombopoietic factor IL-3 and the late acting factors IL-6 and IL-11 do not play a major role for recovery of peripheral platelet count after orthotopic liver transplantation.     

The role of thrombopoietin in the thrombocytopenia of patients with liver cirrhosis

OBJECTIVES: Thrombocytopenia is a common disorder among cirrhotics that has been traditionally explained by splenic platelet pooling and destruction. Thrombopoietin (TPO), the main stimuli for thrombopoiesis is produced primarily in the liver and degraded by circulating platelets, but its role in the thrombocytopenia of liver cirrhosis is not well understood. The main goal of this study is to clarify the role of TPO in the pathogenesis of thrombocytopenia in cirrhosis. METHODS: The relation among TPO, platelet count, spleen size, portal hypertension, and liver function was studied in 33 cirrhotic patients before and after either partial splenic embolization or liver transplantation. RESULTS: Cirrhotics with thrombocytopenia had lower serum TPO levels than healthy controls (median values (interquartile range: ICR) were 120.7 (42.0-191.6) vs 756.4 (527.0-965.1) pg/mL, respectively; p<0.001). Among cirrhotics with thrombocytopenia, serum TPO was related to spleen size (rho=-0.387, p=0.046), but not to platelet count as occurs physiologically. After partial splenic embolization, TPO and platelet count increased significantly and the physiological relation between TPO and platelet count was restored (rho=-0.665, p=0.026). Similar results were observed after liver transplantation. CONCLUSIONS: Our results suggest that besides impaired production in the failing liver, an increased TPO degradation by platelets sequestered in the congested spleen may contribute to thrombocytopenia in cirrhotic patients.     

The biology of thrombopoietin and thrombopoietin receptor agonists

Thrombopoietin (TPO) is the major physiological regulator of platelet production. TPO binds the TPO receptor, activates JAK and STAT pathways, thus stimulating megakaryocyte growth and platelet production. There is no “sensor” of the platelet count; rather TPO is produced in the liver at a constant rate and cleared by TPO receptors on platelets. TPO levels are inversely proportional to the rate of platelet production. Early recombinant TPO molecules were potent stimulators of platelet production and increased platelets in patients with immune thrombocytopenia, chemotherapy-induced thrombocytopenia, myelodysplastic syndromes and platelet apheresis donors. Neutralizing antibodies formed against one recombinant protein and ended their development. A second generation of TPO receptor agonists, romiplostim and eltrombopag, has been developed. Romiplostim is an IgG heavy chain into which four TPO agonist peptides have been inserted. Eltrombopag is an oral small molecule. These activate the TPO receptor by different mechanisms to increase megakaryocyte growth and platelet production. After administration of either to healthy volunteers, there is a delay of 5 days before the platelet count rises and subsequently reaches a peak after 12–14 days. Both have been highly effective in treating ITP and hepatitis C thrombocytopenia. Studies in a wide variety of other thrombocytopenic conditions are underway.     

Thrombopoietin concentrations in peripheral blood correlated with platelet numbers in two patients with thrombocytopenia by chronic graft-versus-host disease

Thrombocytopenia is well known to be one of the clinical manifestations of chronic graft-versus-host disease (cGVHD). However, there exist cases in which the cause of thrombocytopenia has been unexplained. Recently, thrombopoietin (TPO) from bone marrow (BM) stromal cells and transforming growth factor (TGF)-beta from platelets and megakaryocytes have been identified as strong positive and negative regulators of megakaryopoiesis in vivo. We hypothesized that the decreased TPO production from BM could be one of the causes of thrombocytopenia in the patients with cGVHD. In the present study, therefore, TPO and TGF-beta concentrations in peripheral blood (PB) and BM were measured serially in two patients with acute leukemia who had received fully matched stem cell transplantation from relatives and subsequently developed extensive cGVHD with thrombocytopenia. The results showed that platelet numbers correlated well with the TPO concentrations, which were consistently higher in BM than in PB. The difference in TPO concentrations between BM and PB was decreased when the platelet levels were low, indicating that the amount of TPO production from BM decreased throughout the duration of thrombocytopenia. TGF-beta concentrations were normal during all periods in which measurements were carried out. Thus, our results suggest that one mechanism of thrombocytopenia in patients with cGVHD is low TPO production by BM cells.     

Impaired liver function and retroviral activity are risk factors contributing to HIV-associated thrombocytopenia. Swiss HIV Cohort Study.

OBJECTIVE: To investigate the relationship between thrombopoetin (TPO) serum levels and HIV-associated thrombocytopenia. DESIGN AND METHODS: The relationship between TPO levels and severity of HIV-associated thrombocytopenia was investigated. Thirty-eight patients (19 patients with 30-96x10(9) platelets/l and 19 patients with <10x10(9) platelets/l) were matched with 38 HIV-positive non-thrombocytopenic patients (>150x10(9) platelets/l). RESULTS: HIV-positive patients with normal platelet counts had a median TPO serum level of 137 pg/ml. Patients with 30-96x10(9) platelets/l had decreased TPO levels with a median of 90 pg/ml (P = 0.016), and were more likely to have elevated serum aspartate-transferase levels (P<0.001) and hepatomegaly by palpation or ultrasound imaging (P = 0.005). The median TPO serum level of HIV-infected patients with severe thrombocytopenia was 110 pg/ml (non-significant). All patients with severe thrombocytopenia were positive for antibodies against hepatitis B virus core antigen, compared with 80% of HIV-infected persons without thrombocytopenia. Patients with severe thrombocytopenia were more likely to have high HIV replication compared to patients with normal platelet counts (P = 0.02), and reduction of plasma HIV-1 RNA levels was associated with increasing platelet counts. Severe thrombocytopenia was not associated with liver disease. CONCLUSIONS: Liver disease predisposes for low TPO serum levels and mild thrombocytopenia. High retroviral activity predisposes for severe, immune thrombocytopenic purpura-like thrombocytopenia. At least two distinct categories of severe HIV-associated thrombocytopenia exist, one responsive to antiretroviral treatment and one non-responsive to antiretroviral treatment.     

Transient thrombopoietin peak after liver transplantation for end-stage liver disease

Knowledge about synthesis of thrombopoietin (TPO) by human liver cells is now well established and makes the study of TPO serum levels and interdependency with platelet concentrations important before and after liver transplantation. We followed these variables in 14 patients suffering from primary biliary cirrhosis (group A), 10 with hepatitis B (group B) and nine with hepatitis C (group C) infections, as well as 11 patients suffering from alcoholic cirrhosis (group D). In the pretransplant serum samples, TPO levels and platelet counts revealed median values (range) of 112.75 pg/ml (5.0-349.3) in group A, 67.8 pg/ml (14.8-249.9) in B, 68.6 pg/ml (31.4-147.3) in C and 57.9 pg/ml (25.2-264.2) in D for TPO, and 138 x 10(9)/l (36-243) in A, 48 x 10(9)/l (22-129) in B, 105 x 10(9)/l (32-176) in C and 109 x 10(9)/l (33-285) in D for platelets, the latter levels being abnormally reduced. Within 2-3 d of transplantation, the TPO levels started to increase to transient peaks of mostly 5-10 times baseline, which were followed by continuous correction of thrombocytopenia. Splenomegaly was identified to be an important co-factor of thrombocytopenia in groups A and D. We conclude that decreased TPO production in patients with end-stage liver diseases is reverted by orthotopic liver transplantation.     

New insights into the thrombopoietic status of patients on dialysis through the evaluation of megakaryocytopoiesis in bone marrow and of endogenous thrombopoietin levels

The thrombopoietic status of patients with uremia remains unclear. This issue was addressed with particular reference to marrow megakaryocytopoiesis and endogenous thrombopoietin (TPO) levels. A study was conducted in 114 patients on hemodialysis, 43 patients on continuous ambulatory peritoneal dialysis, and 48 age-matched controls. Reticulated platelets, a marker of marrow megakaryocytopoiesis, were measured by flow cytometry. Serum TPO levels, platelet-associated IgG (PAIgG) levels, and hepatitis C virus (HCV) antibody titers were also measured by enzyme-linked immunosorbent assay. Circulating and reticulated platelet counts were significantly lower in the patients on dialysis than in the controls. Thrombocytopenia (less than 150 x 10(9)/L) was most frequent in the HCV-positive hemodialysis patients, who had a higher incidences and higher PAIgG titers. The following results were obtained in the HCV-negative dialysis patients: (1) platelet counts chronologically decreased with years on hemodialysis; (2) platelet counts were associated with the reticulated platelet counts; (3) serum TPO levels were significantly elevated in the dialysis patients, responding to the decrease of reticulated platelets; (4) hematocrits had a positive correlation with serum TPO levels, and serum TPO levels were significantly higher in the patients on hemodialysis who did not require recombinant human erythropoietin therapy than in the other patients. In conclusion, thrombocytopenia is a frequent finding in patients on dialysis. The failure of megakaryocyte production could be the principal cause of the platelet reduction, and the peripheral destruction and sequestration of platelets may be concomitantly involved. Elevation of serum TPO may in part serve as an aid to erythropoiesis in dialysis patients.     

Thrombopoietin levels and peripheral platelet counts following living related donor liver transplantation

BACKGROUND/AIMS: Thrombopoietin is the primary hematopoietic growth factor. Thrombopoietin deficiency may cause thrombocytopenia in advanced liver disease. The aim of our study was to investigate the relevance of thrombopoietin levels to peripheral platelet counts in patients with liver disease who underwent LRDLT (living related donor liver transplantation). METHODOLOGY: We divided the six patients who underwent LRDLT into two groups. Group 1 had thrombocytopenia and group 2 had normal platelet counts. We measured serum thrombopoietin and peripheral platelet counts before and after LRDLT. RESULTS: Pre-LRDLT thrombopoietin and peripheral platelet counts were lower in group 1 than in group 2. Thrombopoietin in group 1 significantly increased on the first day after LRDLT and peripheral platelet counts in group 1 increased following the rise in thrombopoietin (p < 0.05). Moreover, a marked increase in thrombopoietin and peripheral platelet counts was found in splenectomized patients during LRDLT. CONCLUSIONS: These findings suggested inadequate thrombopoietin production in advanced stage liver disease which caused thrombocytopenia. Improvement of thrombopoietin production in graft liver function may contribute to increase of peripheral platelet counts.     

Thrombocytopenia in HIV infection: impairment of platelet formation and loss correlates with increased c-Mpl and ligand thrombopoietin expression

Thrombocytopenia is a common hematologic disorder in patients infected with the human immunodeficiency virus (HIV) and represents a risk for bleeding which is further deleterious during surgery. The major causes of the thrombocytopenia include accelerated peripheral platelet destruction by antiplatelet antibodies and insufficient production of platelets from the infected megakaryocytes. Additionally, at an earlier stage of platelet development, HIV may inhibit megakaryopoiesis at multiple stages of pluripotent CD34+ progenitor stem cell differentiation possibly contributing to decreased levels of platelets in circulation. In HIV infected patients, both the serum thrombopoietin (TPO) levels and theTPO-c-Mpl complexes on the platelet surface were significantly elevated. Therapeutic infusion of HIV infected patients with pegylated recombinant human megakaryocyte growth development factor (PEG-rHu-MGDF) restores platelet counts to normal levels and reduces the c-Mpl expression per platelet. In vitro aggregation of platelets treated with TPO and agonist, adenosine diphosphate (ADP), decrease the dose of ADP that is required for half-maximum aggregation. In vivo dosing does not effect platelet aggregation showing that the metabolism of TPO following its internalization through TPO-c-Mpl complex is rapid and that dosing within the therapeutic range does not constitute increased risk of thrombotic disease.     

淋巴瘤患者自体外周血干细胞移植中血浆TPO水平动态检测及其价值

采用多抗夹心酶联免疫吸附 (EL ISA)全程动态检测 11例恶性淋巴瘤患者 PBSCT中血浆促血小板生成素 (TPO)水平 ,探讨 TPO动态检测对淋巴瘤患者外周血干细胞移植 (PBSCT)后血小板恢复的预测价值。结果 :相关分析表明预处理化疗后淋巴瘤患者血浆 TPO水平随血小板计数减少呈进行性升高 ,其回落变化明显早于血小板数量回升 ,血浆 TPO水平与血小板计数呈显著负相关。认为 TPO在 PBSCT后骨髓巨核细胞系重建及血小板生成中具有重要作用 ;血浆 TPO水平动态检测对临床预测 PBSCT后骨髓巨核系统重建具有实际意义     

Recombinant human thrombopoietin: basic biology and evaluation of clinical studies

Thrombocytopenia is a common medical problem for which the main treatment is platelet transfusion. Given the increasing use of platelets and the declining donor population, identification of a safe and effective platelet growth factor could improve the management of thrombocytopenia. Thrombopoietin (TPO), the c-Mpl ligand, is the primary physiologic regulator of megakaryocyte and platelet development. Since the purification of TPO in 1994, 2 recombinant forms of the c-Mpl ligand--recombinant human thrombopoietin (rhTPO) and pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF)--have undergone extensive clinical investigation. Both have been shown to be potent stimulators of megakaryocyte growth and platelet production and are biologically active in reducing the thrombocytopenia of nonmyeloablative chemotherapy. However, neither TPO has demonstrated benefit in stem cell transplantation or leukemia chemotherapy. Other clinical studies have investigated the use of TPO in treating chronic nonchemotherapy-induced thrombocytopenia associated with myelodysplastic syndromes, idiopathic thrombocytopenic purpura, thrombocytopenia due to human immunodeficiency virus, and liver disease. Based solely on animal studies, TPO may be effective in reducing surgical thrombocytopenia and bleeding, ex vivo expansion of pluripotent stem cells, and as a radioprotectant. Ongoing and future studies will help define the clinical role of recombinant TPO and TPO mimetics in the treatment of chemotherapy- and nonchemotherapy-induced thrombocytopenia.     

Reticulated platelets as a marker of megakaryopoiesis in liver cirrhosis; relation to thrombopoietin and hepatocyte growth factor serum concentration

BACKGROUND/AIMS: Thrombocytopenia often accompanies chronic liver diseases. It can occur due to the decrease in blood platelet production by megakaryocytes, the increase in peripheral destruction, or splenic sequestration. METHODOLOGY: We estimate the reticulated platelets by use of flow cytometry in patients with liver cirrhosis with thrombocytopenia (n-24, platelets median (M)-77g/L), with normal platelet count (n-16, platelets M-193g/L) and in healthy (n-27, platelets M-242g/L). The level of reticulated platelets was determined in whole peripheral blood stained with thiazole orange and incubated with monoclonal antibodies anti-CD41. RESULTS: Patients with liver cirrhosis and thrombocytopenia revealed significantly lower reticulated platelet levels than patients without thrombocytopenia and healthy subjects (M-1.0% vs. 1.5% vs. 2.0% respectively). The correlation between reticulated platelet level and platelet count, serum level of thrombopoietin and hepatocyte growth factor in liver cirrhosis was not established. An inverse correlation was noted between reticulated platelets and thrombopoietin (r - 0.6, p<0.01) and hepatocyte growth factor (r - 0.5, p<0.01) in the control group. A positive correlation between platelet count in liver cirrhosis and serum level of thrombopoietin (r - 0.35, p<0.05) and hepatocyte growth factor (r - 0.48, p<0.01) was observed. CONCLUSIONS: Our studies showed that decreased production of platelets by megakaryocytes due to low thrombopoietin concentration could be a possible cause of thrombocytopenia in liver cirrhosis.     

In vivo adenovirus vector-mediated transfer of the human thrombopoietin cDNA maintains platelet levels during radiation-and chemotherapy- induced bone marrow suppression

Thrombopoietin (TPO, c-mpl ligand) has emerged as a major hematopoietic cytokine stimulating megakaryocyte proliferation, endomitosis, and platelet production. This study shows that a single administration of an adenovirus (Ad) vector encoding TPO (AdCMV.TPO) abrogates thrombocytopenia induced in mice by carboplatin and irradiation. Normal Balb/c mice receiving the vector had increased platelet counts peaking at 7 days and returning to baseline by day 15. Mice rendered pancytopenic with 500 rads and 1.2 mg of carboplatin had a nadir platelet count of five percent of the baseline. Mice receiving AdCMV.TPO 3 days before receiving irradiation and chemotherapy achieved a platelet nadir fourfold higher, and had significant reduction in duration of thrombocytopenia, than mice receiving the control Ad vector. Introduction of AdCMV.TPO the same day of chemotherapy and irradiation was equally effective in acceleration of platelet recovery, but administration of AdCMV.TPO 3 days after chemotherapy-radiation had little effect on platelet recovery. At 30 days after therapy bone marrow and spleen of mice treated with AdCMV.TPO were populated with a large number of polyploid megakaryocytes, but there was no evidence of circulating megakaryocytes in the liver or lungs and no pathologic bone abnormalities such as osteosclerosis or myelofibrosis. These observations suggest that an Ad vector may be an excellent delivery system to provide adequate TPO production to maintain platelet levels in circumstances associated with life-threatening thrombocytopenia.     

Thrombopoietin in Plasmodium falciparum malaria

Thrombopoietin (TPO) is the key growth factor for platelet production and is elevated in states of platelet depletion. As thrombocytopenia is a common finding in malaria, we analysed TPO regulation before, during and after antimalarial treatment. Before treatment, TPO serum levels were significantly higher in patients with severe malaria (n = 35) than in patients with uncomplicated malaria (n = 44; P = 0.024), normalizing within 14-21 d of therapy. The rapid normalization of TPO levels and increase in low peripheral platelet counts after treatment indicate that the biosynthesis of TPO and its regulation in malaria patients are normal.     

Thrombopoietic cytokines in relation to platelet recovery after bone marrow transplantation

In order to evaluate the importance of different thrombopoietic stimulatory cytokines in accelerating platelet recovery after bone marrow transplantation (BMT), we assayed serial plasma concentrations of three cytokines, thrombopoietin (TPO), interleukin-6 (IL-6), and IL-11 through the course of platelet nadir and recovery after BMT. Both mean TPO and IL-6 levels showed a marked rise and later fall preceding or coincident with the platelet nadir and recovery, suggesting their potential role as circulating regulators or stimulators of thrombopoiesis. In contrast, IL-11 levels remained remarkably constant through the whole course suggesting that this cytokine, though capable of stimulating thrombopoiesis, does not serve as a circulating regulator of platelet production. Additionally, we assayed the levels of these three cytokines following initial platelet transfusion to assess the capacity of transfused platelets to adsorb these thrombopoietic cytokines from the plasma and reduce their circulating levels, thus potentially modifying their availability for stimulating megakaryocyte proliferation. No consistent falls in TPO, IL-6 or IL-11 levels were observed following the initial two platelet transfusions. These data support the importance of circulating TPO and IL-6 as hormones capable of stimulating platelet production. Their physiologic relevance as in vivo regulators of thrombopoiesis and clinical utility for therapy of thrombocytopenia need further investigation.     

The role of the liver in the production of thrombopoietin compared with erythropoietin

The liver plays an important role in the production of haemopoietic hormones. It acts as the primary site of synthesis of erythropoietin (EPO) in the fetal stage, and it is the predominant thrombopoietin (TPO)-producing organ for life. In contrast to that of EPO and other liver proteins, the hepatic synthesis of TPO is influenced little by external signals. Hepatocytes express the TPO gene in a constitutive way, i.e. irrespective of the level of platelets in blood. Megakaryocytes and platelets remove the hormone from blood by means of their high-affinity TPO receptors. Normally, the plasma level of TPO is relatively low ( approximately 10(-12) mol/l). However, in thrombocytopenic states due to marrow failure or bleeding, the concentration of circulating TPO may increase greatly. The simple feedback regulation by TPO and its target cells is efficient in maintaining constant platelet numbers in healthy people. Persisting thrombocytopenia develops only in severe liver or marrow failure. On the other hand, an increase in circulating TPO and interleukin 6 (IL-6) may cause reactive thrombocytosis in inflammatory diseases, including cancer. The indications for recombinant human thrombopoietin (rHuTPO) therapy and its impact on transfusion medicine are still under investigation.