Whether the liver is also involved in the metabolism of TPO is currently unclear. Previous studies on the role of the liver in the catabolism of EPO have provided evidence that the rate of hepatic catabolism of this glycoprotein hormone is minor at best. Dinkelaar et al. have shown that the plasma disappearance half-time of endogenous EPO is little elevated in rats in which liver failure is induced by the administration of the hepatotoxic agent D-galactosamine-HCl .
The present immunoassays for circulating TPO have not been standardized. Thus, while intrastudy differences in TPO concentrations may provide valuable scientific and diagnostic information, the comparison of data between different studies and laboratories is usually not valid. Median or mean values reported for the concentration of plasma or serum TPO have ranged from 20 to 240 pg/ml in normal people, independently of gender. Values in the lower range were generally measured by commercial enzyme-linked immunosorbent assays with monoclonal antibodies [76–78], whereas values in the upper range were reported by most [79–83], but not all [84,85], investigators who developed assay systems based on polyclonal antibodies from rabbits immunized with rHuTPO or rHuMGDF. Possibly, assays utilizing polyclonal antibodies are also sensitive to truncated forms of TPO in blood. Still other authors have expressed their results in molar units (normal value ∼1 fmol/ml) [86,87]. Folman et al. have reported that TPO levels in serum are on average 3.4 times higher than in plasma , but other investigators measured much smaller differences when serum and plasma values were compared [81,82,85,89,90]. It is recommended to use plasma rather than serum for assay of TPO, because platelets release TPO during blood clotting. Finally, it is worth noting that children have higher plasma TPO levels than adults .
As mentioned above, the concentration of circulating TPO is normally elevated in thrombocytopenic states not associated with hepatic disease, e.g. after chemotherapy. However, TPO levels are increased less significantly in thrombocytopenic patients with severe acute or chronic liver failure [42–44,91–95]. Evidence for impaired TPO synthesis has been provided by the demonstration of lowered TPO mRNA levels in cirrhotic liver tissue from children  and adults [43,44]. In the study by Wolber et al. it was shown that hepatic TPO mRNA levels, blood platelet counts and the concentration of plasma proteins of liver origin were lowest in acute liver failure, intermediate in decompensated cirrhosis, and close to normal in compensated cirrhosis . Ishikawa et al. have reported that the decrease in the TPO mRNA content of the liver of rats treated with the hepatotoxic agent dimethylnitrosamine is due in part to reduced TPO mRNA expression and in part to the loss of hepatocytes .
In humans suffering from end-stage liver failure, the concentration of TPO in blood increases after successful orthotopic liver transplantation reaching a maximum 5–6 days after surgery [42,43,93,96]. The peripheral platelet count reaches normal values 2 weeks after orthotopic liver transplantation [43,91,93]. Although some investigators have claimed that plasma TPO is not lowered in cirrhosis [92,95], one has to conclude that the respective TPO production rates are lowered, taking into account the slowed metabolism of the hormone at reduced platelet and megakaryocyte mass. The restitution of normal peripheral platelet counts following liver transplantation is not associated with reduced platelet destruction and spleen size, as shown by computed tomography volumetry . If patients do not survive their liver transplantation, TPO levels reach extremely high values in association with the postoperative thrombocytopenia . Non-transplanted thrombocytopenic patients with cirrhosis who undergo only portal decompression by insertion of a transjugular intrahepatic portosystemic stent shunt show no rise in the concentration of circulating TPO or platelets . Thus, the thrombocytopenia associated with liver disease is due at least partly to impaired TPO production. Accordingly, plasma TPO increases in response to the thrombocytopenia caused by interferon (alpha or beta) therapy for treatment of chronic hepatitis C infection in non-cirrhotic patients [99,100], but not in patients with cirrhosis . Lack of TPO due to impaired liver function is also considered a risk factor contributing to HIV-associated thrombocytopenia . Lowered plasma TPO levels in patients with veno-occlusive liver disease following high-dose chemotherapy have been noted in one report , but not in another .
Compared with TPO, less interest has been paid to changes in EPO production related to acute or chronic non-malignant liver diseases. In patients with end-stage renal failure, serum EPO may increase after hepatitis B or C infection, resulting in an improvement of red cell status . The mechanism of this increase still needs to be identified. Clinically relevant lack of EPO has not been reported in cirrhotic patients , which is probably associated with the secondary role of the liver in the synthesis of this haemopoietic hormone.
Erythrocytosis has been observed in 3–12% of patients with hepatocellular carcinoma [108–110]. In several other cases, the development of erythrocytosis may have been prevented by the factors causing anaemia of inflammation in tumour patients [111,112]. Kew and Fisher measured a mean serum EPO concentration of 77 U/l in 65 patients with hepatocellular carcinoma, compared with a normal value of 22 U/l . By immunohistochemistry, EPO could be demonstrated in the carcinoma cells but not in the surrounding normal hepatic tissue .
Patients with non-hepatic solid tumours, infections or autoimmune diseases are often anaemic. This complication has been called ‘anaemia of chronic disease’ or ‘anaemia of inflammation', and been explained by increased haemolysis, bleeding, lowered EPO production, impaired iron mobilization, and reduced proliferation of erythrocytic progenitors due to the action of distinct pro-inflammatory cytokines [111,112]. In turn, patients with malignancies and inflammations often present with reactive thrombocytosis. The combination of anaemia, thrombocytosis and leucocytosis has been termed ‘haematological stress syndrome’ . Bioassay  and immunoassay [76,78–82,87,116] measurements have shown that the concentration of circulating TPO is abnormally high in reactive thrombocytosis associated with cancer or autoimmune diseases, such as inflammatory bowel disease. TPO levels also increase greatly in patients with fulminant septicaemia . Basically, the elevated TPO levels could be due to an increased lifespan of the hormone or due to stimulation of its production. Support for the latter concept is provided by our finding that the immunomodulatory peptide IL-6 enhances TPO gene expression in human hepatoma cell cultures . Furthermore, an analysis of the published sequence of the 5′-flanking region of the human TPO gene  shows that there are several potential IL-6 responsive elements. Thus, TPO resembles acute phase proteins [66,90,117]. In rat hepatocytes in primary culture, hepatocyte growth factor/scatter factor, but not IL-6 or other cytokines, was found to increase TPO mRNA expression . A recent study shows that the majority of carcinoma cell lines of various origins express TPO mRNA variants , thus indicating that the thrombocytosis in tumour patients could be a paraneoplastic syndrome.
While serious episodes of bleeding are usually not to be expected until the blood platelet count falls below 5% of the normal, anaemic people suffer from symptoms of tissue hypoxia, such as shortness of breath, tachycardia and angina pectoris, if the blood haemoglobin concentration falls below just 50% of normal. In healthy people, the decrease in blood haemoglobin concentration will be prevented by an increase in EPO gene expression primarily in the kidneys. This regulation is missing in patients with chronic renal failure whose plasma EPO concentration is by one to two orders of magnitude lower than in people with normal renal function at similar blood haemoglobin concentrations. Here, the treatment with rHuEPO is a true replacement therapy. rHuEPO corrects the anaemia in virtually all predialysis and dialysis patients with chronic renal failure  provided the iron supply is sufficient . Apart from impaired availability of iron, infectious and inflammatory diseases may reduce responsiveness to rHuEPO, because various immunomodulatory cytokines inhibit the proliferation of erythrocytic progenitors [111,125]. Measurements of C-reactive protein and baseline fibrinogen concentrations in serum may provide early recognition of the probability of response to rHuEPO .
During rHuEPO therapy, storage iron is mobilized for use in red cell production, which leads to a decline in serum ferritin levels. Combined with phlebotomy, this effect has been considered a tool for treatment of iron overload associated with frequent blood transfusion . A histological study has proven the regression of the deposited iron in liver biopsies . With respect to liver disease, it is also of interest that the treatment with rHuEPO can increase antibody titres after hepatitis B vaccination in dialysis patients .
The results of preclinical studies in normal animals and in myelosuppressed thrombocytopenic mice, dogs and monkeys have been reviewed previously [6,7,130]. To sum up, the administration of recombinant TPO or MGDF produces an increase in the concentration of circulating platelets beginning after 3–5 days. Thus, the action of TPO appears to be due to a stimulation of the proliferation and differentiation of megakaryocytic progenitors rather than an immediate sequestration of platelets from preformed megakaryocytes. TPO increases the number, size and fluidity of the megakaryocytes in the bone marrow. In addition, it increases the growth of more primitive pluripotent haemopoietic progenitor cells.
rHuTPO and PEG-rHuMGDF have been administered to tumour patients before and after chemotherapy in phase-I/II trials, which are described in more detail elsewhere . Vadhan-Raj et al. treated 12 sarcoma patients 3 weeks before chemotherapy with a single intravenous dose of rHuTPO (0.3–2.4 μg/kg body weight) . This treatment resulted in a dose-dependent increase in the concentration of circulating platelets. The patients’ platelets were morphologically and functionally unaltered. Haematocrit and white blood cell numbers were unaffected. Note, however, that the drug did not only promote the proliferation and differentiation of cells in the megakaryocytic lineage; it also expanded the pool of multilineage progenitors in bone marrow and their mobilization into peripheral circulation . Very similar observations were made when PEG-rHuMGDF was given to patients with solid tumours before [134,135] or after  chemotherapy. In an additional study, granulocyte colony-stimulating factor (G-CSF, 5 μg/kg body weight) was combined with PEG-rHuMGDF at doses of 0.03–5 μg/kg body weight for daily subcutaneous injection in a randomized, blinded phase-I trial in 41 patients with advanced cancer after dose-intensive chemotherapy with carboplatin and cyclophosphamide . The platelet nadir and the recovery to baseline platelet count occurred earlier in the patients given PEG-rHuMGDF than in the placebo control group. Furthermore, the degree of the mobilization of peripheral blood progenitor cells (PBPC) on day 15 after chemotherapy was significantly greater in patients treated with PEG-rHuMGDF in combination with G-CSF, suggesting that this treatment might allow for more efficient collection of stem cells for autologous or allogeneic transplantation . In the studies by Fanucchi et al.  and Basser et al. , thrombotic complications were seen in two patients, but the relation of these events to the treatment with PEG-rHuMGDF was not clarified.
The safety and activity of rHuTPO as a PBPC mobilizer in combination with G-CSF was evaluated recently in 29 breast cancer patients treated with high-dose chemotherapy followed by PBPC reinfusion. This regimen resulted in an accelerated granulocyte and platelet recovery and decreased blood cell transfusion requirements. In the majority of patients, only a single apheresis procedure was needed .
The author's studies are supported by the Deutsche Forschungsgemeinschaft (DFG, SFB 367-C8). The expert secretarial work of Ms Lisa Zieske in the preparation of this manuscript is gratefully acknowledged.
• Of special interest
•• Of outstanding interest
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