Hepatic Thrombopoietin Production Research Articles

Notch1 regulates hepatic thrombopoietin production

AbstractNotch signaling regulates cell-fate decisions in several developmental processes and cell functions. However, the role of Notch in hepatic thrombopoietin (TPO) production remains unclear. We noted thrombocytopenia in mice with hepatic Notch1 deficiency and so investigated TPO production and other features of platelets in these mice. We found that the liver ultrastructure and hepatocyte function were comparable between control and Notch1-deficient mice. However, the Notch1-deficient mice had significantly lower plasma TPO and hepatic TPO messenger RNA levels, concomitant with lower numbers of platelets and impaired megakaryocyte differentiation and maturation, which were rescued by addition of exogenous TPO. Additionally, JAK2/STAT3 phosphorylation was significantly inhibited in Notch1-deficient hepatocytes, consistent with the RNA-sequencing analysis. JAK2/STAT3 phosphorylation and TPO production was also impaired in cultured Notch1-deficient hepatocytes after treatment with desialylated platelets. Consistently, hepatocyte-specific Notch1 deletion inhibited JAK2/STAT3 phosphorylation and hepatic TPO production induced by administration of desialylated platelets in vivo. Interestingly, Notch1 deficiency downregulated the expression of HES5 but not HES1. Moreover, desialylated platelets promoted the binding of HES5 to JAK2/STAT3, leading to JAK2/STAT3 phosphorylation and pathway activation in hepatocytes. Hepatocyte Ashwell-Morell receptor (AMR), a heterodimer of asialoglycoprotein receptor 1 [ASGR1] and ASGR2, physically associates with Notch1, and inhibition of AMR impaired Notch1 signaling activation and hepatic TPO production. Furthermore, blockage of Delta-like 4 on desialylated platelets inhibited hepatocyte Notch1 activation and HES5 expression, JAK2/STAT3 phosphorylation, and subsequent TPO production. In conclusion, our study identifies a novel regulatory role of Notch1 in hepatic TPO production, indicating that it might be a target for modulating TPO level.

Notch1 Regulates Hepatic Thrombopoietin Production

Notch signaling is highly conserved and regulates cell-fate decisions in several developmental processes and cell functions. However, a role for Notch in hepatic thrombopoietin (TPO) production has not been described. We noted that mice with a deficiency in hepatic Notch1 had thrombocytopenia, and so investigated TPO production and other features of platelets in these mice. We found that the liver ultrastructure and hepatocyte function were comparable between control mice and Notch1-deficient mice. However, the Notch1-deficient mice had significantly lower plasma TPO and hepatic TPO mRNA levels, concomitant with lower numbers of circulating platelets and impaired megakaryocyte differentiation and maturation. Addition of exogenous TPO rescued megakaryocyte maturation and circulating platelet numbers. Additionally, JAK2/STAT3 phosphorylation was significantly inhibited in Notch1-deficient hepatocytes. RNA-seq analysis showed significantly reduced JAK-STAT signaling. JAK2/STAT3 phosphorylation and TPO production was also impaired in purified cultured Notch1-deficient hepatocytes after treatment with desialylated platelets. Interestingly, Notch1 deficiency downregulated the expression of HES5 but not HES1. Moreover, desialylated platelets promoted the binding of HES5 to JAK2/STAT3, leading to JAK2/STAT3 phosphorylation and pathway activation. Furthermore, blockage of Dll4 on desialylated platelets inhibited hepatocyte Notch1 activation and HES5 expression, JAK2/STAT3 phosphorylation and subsequent TPO production. In conclusion, our study identifies a novel regulatory role of Notch1 in hepatic TPO production, indicating that it might be a target for modulating TPO level.

Hepatic thrombopoietin gene silencing reduces platelet count and breast cancer progression in transgenic MMTV-PyMT mice

AbstractIn humans, platelet count within the normal range is required for physiological hemostasis, but, adversely, platelets also support pathological thrombosis. Moreover, by releasing growth factors, they may enhance neoplastic proliferation. We hypothesize that platelet count correlates with platelet-dependent pathologies, even within the range of hemostatic competence. Because platelet production is promoted by thrombopoietin signaling through the myeloproliferative leukemia virus oncogene (cMPL), a receptor expressed on megakaryocytes, we evaluated the feasibility of selective targeting of hepatic thrombopoietin production to test this hypothesis. We synthesized murine- and primate-specific antisense oligonucleotides (THPO-ASO) that silence hepatic thrombopoietin gene (THPO) expression without blocking extrahepatic THPO. Repeated doses of THPO-ASO were administered to mice and a baboon, causing a sustained 50% decline in plasma thrombopoietin levels and platelet count within 4 weeks in both species. To investigate whether reducing platelet count within the translationally relevant hemostatic range could alter a neoplastic process, we administered THPO-ASO to 6-week-old transgenic MMTV-PyMT mice that develop early ductal atypia that progresses into cMPL-negative fatal metastatic breast cancer within 2 to 3 months. THPO-ASO treatment increased the average time to euthanasia (primary humane endpoint) at 2 cm3 combined palpable tumor volume. Our results show that THPO-ASO reduced blood platelet count, plasma platelet factor 4, vascular endothelial growth factor, thrombopoietin levels, bone marrow megakaryocyte density, tumor growth rate, proliferation index, vascularization, platelet and macrophage content, and pulmonary metastases vs untreated controls. These findings confirm that sustained and moderate pharmacological platelet count reduction is feasible with THPO-ASO administration and can delay progression of certain platelet-dependent pathological processes within a safe hemostatic platelet count range.

Platelet storage induces accelerated desialylation of platelets and increases hepatic thrombopoietin production

BackgroundStored platelets undergo deleterious changes, referred to as platelet storage lesions (PSLs), which accelerate the desialylation of platelets and result in their phagocytosis and clearance by hepatic macrophages. Recent studies have reported that Ashwell–Morell receptor binds to desialylated platelets, thereby inducing hepatic thrombopoietin (TPO) production in a mouse model. Therefore, this study aimed to demonstrate these relationships between PSL and hepatic TPO production in human study.MethodsPlatelet concentrates (PCs) were obtained from 5 healthy volunteers and the remaining were discarded samples from the blood bank. PCs were divided into two halves, and stored either at 22 or 4 °C. Experiments were conducted using serial samples. Desialylation was assessed using flow cytometry, and structural changes were visualized using electron microscopy. Following co-culture of HepG2 cells (HB-8065, ATCC) with isolated platelets, hepatic TPO production was determined using real-time quantitative polymerase chain reaction and the supernatant TPO level was measured using a Luminex kit.ResultsFor 5 days of storage duration, platelet counts were not influenced by the storage conditions, but the degree of desialylation was proportional to the storage duration. Significant changes in the platelet surface and structure according to storage conditions were noted in electron microscopy. HepG2 cells incubated with aged platelets expressed more TPO mRNA, and supernatant TPO levels were proportional to the storage duration. Refrigeration also influenced on the results of this study, but they were not statistically significant.ConclusionsThis is the first study to demonstrate that, in vitro, aging and refrigeration affect the integrity of human platelets, resulting in induction of hepatic TPO mRNA and protein expression.

Platelet GPIba Is Important for Thrombopoietin Production and Thrombopoietin-Induced Platelet Generation

Background: Platelets are generated from megakaryocytes in the bone marrow and play a key role in hemostasis. GPIba is the major subunit in the GPIb-IX complex expressed on platelets and megakaryocytes (MKs). Deficiency of GPIba results in Bernard Soulier Syndrome (BSS), a severe bleeding phenotype characterized by thrombocytopenia and abnormal platelet morphology.The generation of megakaryocytes and platelets is regulated by the hematopoietic growth factor, thrombopoietin (TPO). In patients with thrombocytopenia, the TPO receptor agonists and recombinant TPO (rTPO) have been used to stimulate thrombopoiesis. TPO production primarily occurs in liver parenchymal cells with minor amounts synthesized in other tissues. The major clearance mechanism of TPO from circulation is via interaction between TPO and its receptor, c-Mpl, following which the c-Mpl-TPO complex undergoes internalization and degradation. However, whether it is the production or clearance of TPO the main regulator of circulating TPO levels has been debated but remains unclear.The prevailing theory was that hepatic TPO is generated constitutively, and the levels of circulating TPO are fine-tuned by its uptake and metabolism in platelets and megakaryocytes. Recently, this concept was modified by studies in Ashwell-Morell receptor (AMR) deficient mice, which highlighted the important role played by platelets in the regulation of hepatic TPO mRNA expression. As the most glycosylated platelet surface protein, GPIba contributes to chilled platelets clearance viahepatocytes, as well as antibody-mediated thrombocytopenia. However, the relationship between GPIba and plasma TPO has not been exploredMethods and Results: We observed a significant decrease (approx. 3 fold) of TPO levels in both plasma and sera of GPIba-/- mice compared to wild type (WT) controls. Given the enlarged size of GPIba-/- platelets compared to WT, we investigated whether the low circulating TPO levels could be due to increased TPO clearance. Interestingly, we found similar levels of intracellular TPO in WT and GPIba-/- platelets, and the capacity to adsorb TPO in vitro was not enhanced. Further, we observed GPIba-/- platelets, surprisingly, have less c-Mpl expression compared to WT. Therefore, the decreased circulating TPO levels in GPIba-/- mice is unlikely due to enhanced TPO clearance.We next quantified TPO mRNA from hepatic liver cells as they are known as the major source of TPO. Consistent with the low circulatory TPO levels, we found reduced hepatic TPO mRNA in GPIba-/- mice, suggesting a deficiency in hepatic TPO generation. To determine whether exogenous platelet transfusions could influence steady-state hepatic TPO production, we transfused WT or GPIba-/- mice with WT or GPIba-/- platelets. Post-transfusion of WT, but not GPIba-/- platelets elevated TPO levels to around 150% in GPIba-/- mice. Additionally, our preliminary data showed that co-culture of hepatocytes with WT, but not GPIba-/- platelets in vitro increased hepatic TPO mRNA expression, further supporting a role for GPIba in TPO generation.To test whether TPO can equally induced platelet generation in GPIba-/- and WT mice. We injected rTPO into the two groups of mice, and found that in contrast to WT controls, rTPO did not significantly increase the platelet number of GPIba-/- mice in the circulation.Conclusion: Our data demonstrated that GPIba is important for TPO generation in liver and is necessary for TPO-induced platelet generation. These discoveries may have significant implications in TPO therapy in ITP. DisclosuresNo relevant conflicts of interest to declare.

Platelet Dysfunction: Status of Thrombopoietin in Thrombocytopenia Associated with Chronic Liver Failure.

Thrombocytopenia is a common hematological abnormality in patients with chronic liver disease, and its prevalence is higher in patients with liver failure. Although the presence of thrombocytopenia has historically been associated with portal hypertension, the characterization of thrombopoietin has improved our understanding of the determinants of platelet count in patients with liver disease. In particular, the association between thrombopoietin levels and residual liver function helped disclose the multifaceted pathophysiology of thrombocytopenia in patients with chronic liver failure. In this regard, important results were provided by studies performed in patients with chronic viral hepatitis that assessed the complex interplay between thrombocytopenia induced by the myelosuppressive effect of interferon-based treatment and thrombopoietin pathophysiology. These studies showed that successful antiviral therapy is accompanied by improved hepatic thrombopoietin production. Moreover, studies that evaluated thrombopoietin and platelet count dynamics before and after liver transplantation were instrumental in describing how restoration of liver function determines a normalization of the thrombopoietin-platelet count feedback that is deranged in patients with end-stage liver disease.

Molecular mechanisms regulating platelet clearance and thrombopoietin production

The human body produces and removes 1011 platelets daily to maintain a normal steady‐state platelet count. Platelet production must be tightly regulated to avoid spontaneous bleeding if counts are low (thrombocytopenia) or arterial occlusion and organ damage if counts are high (thrombocytosis). Recent studies have highlighted the role of glycan modifications on platelet surface proteins in mediating platelet clearance. Platelets undergoing cold storage lose sialic acid and β‐galactose. Following transfusion, cold‐stored platelets are cleared via hepatic Ashwell–Morell receptor (AMR) and the macrophage (Kupffer cell) αMβ2 integrin lectin domain. New data point to the fact that platelets surface loses sialic acid during their circulatory lifetime, suggesting a novel platelet in vivo clearance mechanism via the AMR. Thrombopoietin (TPO) is the primary regulator of platelet production, supporting the survival, proliferation and differentiation of the platelet precursors, bone marrow megakaryocytes. Although it is clear that hepatocytes are the primary source of TPO, the mechanisms regulating circulating TPO levels have been subject to discussion for decades. Two major models for TPO regulation have been proposed. In one model, hepatic TPO production is constitutive and TPO serum levels are maintained solely by its uptake and metabolism by the TPO receptor c‐Mpl on platelets and megakaryocytes. In another model, hepatic TPO production is regulated. However, the physiological ligand–receptor pair capable of regulating steady‐state TPO production remains unclear. In this review, mechanisms of glycan–lectin‐mediated platelet clearance and TPO production will be discussed.

The Ashwell-Morell receptor regulates hepatic thrombopoietin production via JAK2-STAT3 signaling.

The hepatic Ashwell-Morell receptor (AMR) can bind and remove desialylated platelets. Here we demonstrate that platelets become desialylated as they circulate and age in blood. Binding of desialylated platelets to the AMR induces hepatic expression of thrombopoietin (TPO) mRNA and protein, thereby regulating platelet production. Endocytic AMR controls TPO expression through Janus kinase 2 (JAK2) and the acute phase response signal transducer and activator of transcription 3 (STAT3) in vivo and in vitro. Recognition of this newly identified physiological feedback mechanism illuminates the pathophysiology of platelet diseases, such as essential thrombocythemia and immune thrombocytopenia, and contributes to an understanding of the mechanisms of thrombocytopenia observed with JAK1/2 inhibition.

Platelets Regulate Thrombopoietin Production: The Missing Link

Humans produce and remove ~1011 platelets daily, and the rate of production can rise sharply under conditions of platelet destruction. Platelet production must be tightly regulated to avoid spontaneous bleeding if counts are low or arterial occlusion and organ damage if counts are high. Thrombopoietin (TPO), the primary regulator of megakaryopoiesis and platelet production, was first isolated and cloned in 1994. This discovery was a major milestone in medicine and led to the availability of TPO mimetics for clinical care. Hepatocytes are the primary cells responsible for the production and secretion of circulating TPO. However, mechanisms regulating circulating TPO levels have been debated for decades. In one model, circulating TPO levels are maintained solely by its uptake and metabolism by high-affinity c-Mpl receptors on platelets and megakaryocytes. In another model, circulating platelet levels are sensed, resulting in appropriately regulated levels of TPO mRNA expression in liver and bone marrow. Despite these discussions, no physiological ligand-receptor pair capable of regulating steady state TPO production has yet been identified and the mechanisms through which platelets regulate TPO production remain poorly understood. Recent studies have highlighted the role of glycan modifications on platelet surface proteins in mediating platelet clearance. The hepatic Ashwell-Morell receptor (AMR) can bind and remove platelets with reduced α2,3-linked sialic acid (desialylated platelets), such as after cold storage, during sepsis, or in St3gal4-/- mice lacking the ST3Gal-IV sialyltransferase. The AMR is a transmembrane heteroligomeric glycoprotein complex composed of ASGPR1 (also called CLEC4H1, HL-1) and ASGPR2 (CLEC4H2, HL-2) subunits, which are highly conserved among mammalian species. However, since its discovery four decades ago, the regulatory role of the hepatic AMR remains unclear. Injection of desialylated platelets into rabbits stimulates platelet production, suggesting a possible link between desialylated platelet consumption and liver TPO secretion. We report that sialic acid loss determines platelet circulatory lifespan by triggering platelet removal by the hepatic AMR. Binding of desialylated platelets to the AMR induces hepatic TPO gene transcription and translation, thereby regulating platelet production. This conclusion is based on the following results. First, desialylated platelet uptake by the AMR regulates hepatic TPO mRNA expression in situ, as evidenced by increased (~50%) hepatic TPO mRNA levels in St3gal4-/- mice and reduced (~50%) hepatic TPO mRNA levels in Asgr2-/- mice lacking the AMR ASGPR2 subunit, relative to controls. Second, injection of endogenously (isolated from St3gal4-/- and Asgr2-/- mice) and exogenously (sialidase-treated) desialylated mouse platelets into wild-type mice stimulates hepatic TPO mRNA expression by ~50% after 12h and subsequently increases plasma TPO levels and blood platelet counts. In contrast, desialylated platelets injected into Asgr2-/- mice have no effect on hepatic TPO mRNA content, plasma TPO levels and blood platelet counts. Third, incubation of desialylated human platelets with the hepatoma cell line HepG2 increases HepG2 TPO mRNA content by approximately three-fold after six hours in vitro. Recognition of this previously unrecognized physiological feedback mechanism illuminates a novel platelet-dependent regulatory mechanism for hepatic TPO production. Disclosures No relevant conflicts of interest to declare.

Quantification of hepatic thrombopoietin mRNA transcripts in patients with chronic liver diseases shows maintained gene expression in different etiologies of liver cirrhosis.

Platelet production is regulated by thrombopoietin (TPO), which is primarily synthesized in the liver. The TPO in patients with liver diseases could possibly be owing to impaired hepatic TPO production. As we reported previously, TPO serum levels are not decreased in patients with liver diseases compared with healthy controls and do not depend on the stage of cirrhosis or platelet count, but are highly elevated in patients with chronic virus hepatitis. To study possible mechanisms, we measured hepatic TPO mRNA levels in liver tissue samples from 31 liver cirrhosis patients by quantitative TaqMan real-time RT-PCR and corresponding serum TPO concentrations by ELISA. Median TPO serum levels were elevated in patients with viral hepatitis (n = 12) compared with patients with a biliary (n = 10), alcoholic (n = 6) or other (n = 3) disease etiology, while hepatic TPO mRNA levels did not differ. The TPO mRNA levels in patients with chronic liver diseases were not different from normal liver tissue sample. The TPO mRNA and TPO serum level did not correlate. We conclude that hepatic TPO gene expression appears to be maintained on a constitutive transcriptional level in patients with liver diseases and does not change dependent on disease etiology.

Plasma thrombopoietin in patients with cavernous transformation of the portal vein.

Thrombopoietin (TPO), the primary regulator of thrombopoiesis, is produced mainly in the liver. Previous studies investigating blood TPO in chronic liver diseases revealed conflicting results. It has been suggested that hepatic TPO production is regulated by the portal blood supply to the liver. However, the role of TPO in the pathobiological basis of idiopathic portal vein thrombosis (PVT) and cavernous transformation of the portal vein (CTPV) has not been elucidated. The objective of this study is to assess plasma TPO concentrations in patients with CTPV. Eleven patients (4 men and 7 women, aged 38+/-12 years) with CTPV were studied. Sixteen healthy adults served as the control group (8 men and 8 women, aged 34+/-12 years). Median plasma TPO concentration was 326 pg/mL (range, 15-1402 pg/mL) in the patients with CTPV and 62.65 pg/mL (range, 38.5-102 pg/mL) in the control group (P = .003). In this study, we found significantly higher TPO concentrations in the plasma of patients with CTPV. The higher concentrations could be a result of the altered portal hemodynamics due to thrombosis. Moreover, TPO release by activated platelets might lead to the subsequent propagation of thrombosis in PVT.

Hepatic Thrombopoietin mRNA Is Increased in Acute Inflammation

The plasma concentration of thrombopoietin (TPO) in general is inversely related to the mass of platelets and megakaryocytes. However, reactive thrombocytosis of inflammatory disease is accompanied by elevated TPO levels. To investigate whether the rate of TPO mRNA expression is altered during acute inflammation, rats were injected with bacterial lipopolysaccharide (LPS). After 6 h, total RNA from liver and kidney was reverse transcribed and analyzed by competitive PCR for TPO and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). LPS-treated rats showed a significant increase in hepatic TPO mRNA concentration. The ratio of TPO to GAPDH mRNA was 3.5 +/- 0.6% in the livers of control rats and 8.3 +/- 2.0% in the livers of LPS-treated rats (mean +/- SD). Thus, reactive thrombocytosis of inflammatory disease might result from an increase in hepatic TPO production. Since platelets are involved in the immune reaction, reactive thrombocytosis may be a mechanism of host defense.

Expression of hepatic thrombopoietin mRNA in primary cultured hepatocytes and in rats with acute liver injury or bone marrow suppression with or without cirrhosis.

The main causes of thrombocytopenia in cirrhosis are thought to be platelet destruction and the reduction of thrombopoietin (TPO) expression in the liver. The mechanisms by which levels of TPO mRNA are regulated in cirrhosis have not been elucidated. In this study, we investigated some possible mechanisms. We used three experimental models: bone marrow suppression, acute liver injury and primary cultured hepatocytes. We used northern blots to assess the kinetics of TPO mRNA expression in the livers of irradiated rats (with and without cirrhosis) in acute liver injury and in primary cultured hepatocytes treated with hepatotoxin or cytokines. Although the bone marrow was hypocellular, there was no apparent enhancement of TPO mRNA expression in the irradiated rats with cirrhotic livers compared with the unirradiated rats with cirrhotic livers. There were no conspicuous changes in hepatic TPO mRNA expression between the livers of the control rats and the three models of acute liver injury. There were no conspicuous changes in the levels of TPO mRNA between control hepatocytes and hepatocytes treated with hepatotoxin or cytokines. Our results suggest that bone marrow is not a regulator of hepatic TPO production in cirrhosis. The reduced TPO mRNA expression found in cirrhotic rats may not result merely from serious cellular damage; it may be associated with cirrhosis-specific regulatory mechanisms for the expression of the TPO gene. Further studies are needed to search for other factors that may induce reduced TPO expression.

Interleukin-6 increases thrombopoietin production in human hepatoma cells HepG2 and Hep3B.

The concentration of circulating thrombopoietin (TPO) is relatively high in patients with thrombocytosis reactive to inflammatory diseases. We investigated whether immunomodulatory cytokines stimulate TPO synthesis in cultured human hepatoma cells (lines HepG2 and Hep3B), renal proximal tubular cells, and bone marrow fibroblasts. The effects of interleukins (IL) IL-1beta, IL-6, and IL-11 and of tumor necrosis factor-a (TNF-alpha) on the rate of TPO secretion were measured by ELISA. TPO mRNA levels were quantitated by competitive reverse transcription PCR. HepG2 and Hep3B cells produced significant amounts of TPO mRNA and TPO protein. Renal tubular cells synthesized less TPO, and in bone marrow fibroblasts, neither TPO mRNA nor TPO protein was detected. Only IL-6 affected TPO protein secretion, causing a 1.5-fold stimulation in HepG2 and Hep3B cells in 24-h incubation periods. The TPO mRNA content in these cells was doubled by IL-6 after 2, 6, or 24 h of stimulation. Thus, IL-6 could cause thrombocytosis in inflammatory disease partly by increasing hepatic TPO production.

The physiological response of thrombopoietin (c-Mpl ligand) to thrombocytopenia in the rat

It has been suggested that circulating levels of thrombopoietin (TPO) are determined primarily by platelet and megakaryocyte clearance of TPO and not by changes in hepatic TPO production. The experimental evidence accumulated so far to support this hypothesis is incomplete. We have therefore developed a new model of non-immune thrombocytopenia in the rat and used it to assess the relationship of TPO (c-mpl ligand) to the platelet mass. 14 d following the administration of busulphan, the platelet count reached a nadir of <2% of its initial value and remained at this level for up to 6 d. Circulating TPO was measured by two different bioassays which were sensitive enough to measure normal levels of TPO and levels rose from 106 ± 29 pg/ml in animals with a normal platelet count to 2015 ± 544 pg/ml in those with thrombocytopenia. These elevated levels of TPO were solely a response to the low platelet count since transfusion of a normal mass of platelets into the thrombocytopenic animals returned the TPO levels exactly to normal. The increase in TPO levels in thrombocytopenic animals was not due to increased TPO production since the thrombocytopenic animals did not show any increase in TPO mRNA in total or polysome-associated hepatic RNA. Rather, rat platelets were able to bind and stoichiometrically remove TPO from thrombocytopenic plasma via high-affinity receptors (Kd = 38 ± 10 pm; 233 ± 32 receptors/platelet). These results serve as a proof that the circulating level of TPO is determined not by alterations in TPO transcription or translation but by the ability of the platelet mass to bind and remove TPO from the circulation.

The physiological response of thrombopoietin (c‐Mpl ligand) to thrombocytopenia in the rat

It has been suggested that circulating levels of thrombopoietin (TPO) are determined primarily by platelet and megakaryocyte clearance of TPO and not by changes in hepatic TPO production. The experimental evidence accumulated so far to support this hypothesis is incomplete. We have therefore developed a new model of non-immune thrombocytopenia in the rat and used it to assess the relationship of TPO (c-mpl ligand) to the platelet mass. 14 d following the administration of busulphan, the platelet count reached a nadir of <2% of its initial value and remained at this level for up to 6 d. Circulating TPO was measured by two different bioassays which were sensitive enough to measure normal levels of TPO and levels rose from 106 +/- 29 pg/ml in animals with a normal platelet count to 2015 +/- 544 pg/ml in those with thrombocytopenia. These elevated levels of TPO were solely a response to the low platelet count since transfusion of a normal mass of platelets into the thrombocytopenic animals returned the TPO levels exactly to normal. The increase in TPO levels in thrombocytopenic animals was not due to increased TPO production since the thrombocytopenic animals did not show any increase in TPO mRNA in total or polysome-associated hepatic RNA. Rather, rat platelets were able to bind and stoichiometrically remove TPO from thrombocytopenic plasma via high-affinity receptors (Kd = 38 +/- 10 pm; 233 +/- 32 receptors/platelet). These results serve as a proof that the circulating level of TPO is determined not by alterations in TPO transcription or translation but by the ability of the platelet mass to bind and remove TPO from the circulation.

Regulation of hepatic thrombopoietin production by portal hemodynamics in liver cirrhosis

Objective: This study was designed to clarify how thrombopoietin (TPO) functions in and, to some extent, causes thrombocytopenia complicating liver cirrhosis and portal hypertension. Methods: Our study population consisted of 19 cirrhotic and six noncirrhotic patients who underwent percutaneous transhepatic portography (PTP) and hepatic venography. Results: The platelet counts of the cirrhotic patients were significantly lower than those of the noncirrhotic patients (8.7 ± 4.1 vs 17.4 ± 7 × 10 4/μl; p < 0.01). The flow direction in the splenic vein was confirmed by PTP. Ten hepatofugal and nine hepatopetal flow directions in the splenic vein were noted among the cirrhotics. The hepatofugal group showed lower portal venous pressure (20 ± 10 vs 32 ± 4 cm H 2O; p < 0.01) than the hepatopetal group and had a higher incidence of hepatic encephalopathy (six of 10 vs zero of nine; p < 0.01). The hepatic vein—portal difference in TPO did not differ substantially between the cirrhotics and noncirrhotics (0.12 ± 0.04 vs 0.24 ± 0.07 fmol/ml). Comparisons of this value among the three groups showed the TPO difference to be lowest in the hepatofugal group (hepatofugal: 0.04 ± 0.03, hepatopetal: 0.21 ± 0.07, noncirrhotic: 0.24 ± 0.07; p < 0.05). Conclusions: Our findings suggest that TPO production in the cirrhotic liver is regulated by the portal blood supply to the liver. Thus, portal hemodynamics may play a critical role in the development of thrombocytopenia.