Although viral hepatitis treatments have evolved over the years, the resultant liver cirrhosis still does not completely heal. Platelets contain proteins required for hemostasis, as well as many growth factors required for organ development, tissue regeneration and repair. Thrombocytopenia, which is frequently observed in patients with chronic liver disease (CLD) and cirrhosis, can manifest from decreased thrombopoietin production and accelerated platelet destruction caused by hypersplenism; however, the relationship between thrombocytopenia and hepatic pathogenesis, as well as the role of platelets in CLD, is poorly understood. In this paper, experimental evidence of platelets improving liver fibrosis and accelerating liver regeneration is summarized and addressed based on studies conducted in our laboratory and current progress reports from other investigators. In addition, we describe our current perspective based on the results of these studies. Platelets improve liver fibrosis by inactivating hepatic stellate cells, which decreases collagen production. The regenerative effect of platelets in the liver involves a direct effect on hepatocytes, a cooperative effect with liver sinusoidal endothelial cells, and a collaborative effect with Kupffer cells. Based on these observations, we ascertained the direct effect of platelet transfusion on improving several indicators of liver function in patients with CLD and liver cirrhosis. However, unlike the results of our previous clinical study, the smaller incremental changes in liver function in patients with CLD who received eltrombopag for 6 mo were due to patient selection from a heterogeneous population. We highlight the current knowledge concerning the role of platelets in CLD and cancer and anticipate a novel application of platelet-based clinical therapies to treat liver disease.

Chronic liver disease (CLD) refers to a long-term pathological process of continuous destruction of liver parenchyma and its gradual substitution with fibrous tissue, which ultimately results in liver cirrhosis associated with a fatal outcome. CLD has diverse etiologies, which include hepatotrophic viruses, chemicals, alcohol and drug abuse, autoimmune disorders, cholestasis and metabolic diseases[1,2], and it is a major cause of morbidity and mortality in many countries[3,4]. Hepatocellular carcinoma (HCC) is a dominant complication of CLD and cirrhosis, with the third highest death rate among malignancies in the world[5].

HCC was shown to significantly correlate with advanced fibrosis, as a sustained wound-healing response to hepatitis C virus infection; however, despite the progress in viral hepatitis therapies, they are unable to completely heal hepatocellular injuries and prevent liver cirrhosis[6]. At present, liver transplantation is the only treatment option for end-stage liver failure, but its clinical availability is hindered by serious problems such as donor shortage, surgical complications, graft rejection and high cost[1,7-11]. Therefore, other therapeutic approaches are being looked into; among them, measures to resolve liver fibrosis have been investigated.

Liver fibrosis is caused by a continuous excessive deposition of the extracellular matrix (ECM) in response to chronic liver injury[1,2]. Although advanced liver fibrosis has been considered irreversible, resulting in permanent substitution of hepatocytes with the ECM components, recent reports indicate that certain immunotherapies may promote partial resolution of liver cirrhosis[12-15]. These studies encourage the development of novel approaches to treat patients with advanced CLD.

Thrombocytopenia, i.e., the reduction of platelet count in blood, is a common hematological complication of CLD caused by decreased production of hormone thrombopoietin (TPO) in the damaged liver and/or increased destruction of platelets through phagocytosis in the enlarged spleen, as well as the loss of hematopoietic function in bone marrow due to alcohol abuse or viral infection[16-19]. Platelets are anuclear blood cells derived from TPO-stimulated megakaryocytes; they not only play a critical role in hemostasis, but also secrete several growth factors, including platelet-derived growth factor (PDGF) and hepatocyte growth factor (HGF), which promote liver regeneration[20-25]. As a feedback response, thrombocytopenia further aggravates hepatic destruction and contributes to the pathogenesis of CLD and cirrhosis, suggesting that measures to prevent platelet loss may be useful as a therapeutic approach to treat CLD.

Indeed, our previous studies in experimental animal models revealed that platelets play a crucial role in promoting liver regeneration after hepatectomy by inducing hepatocyte proliferation[26-29] and stopping the progression of liver fibrosis[30-32]. Furthermore, in patients with CLD and cirrhosis, platelet transfusion and splenectomy used as a platelet increment therapy, positively affected hepatic function[33-35]. Along these lines, we have investigated the safety of long-term administration of the TPO receptor (TPO-R) agonist eltrombopag for treating patients with CLD.

However, the effect of thrombocytopenia on liver damage and exact mechanisms underlying thrombocytopenia in patients with CLD and cirrhosis are still unclear and require further study to confirm the clinical utility of increasing platelets in CLD.

The aim of this editorial is to summarize the current perspectives of novel approaches to treat liver cirrhosis based on augmentation of platelet counts using TPO-R agonists.

Liver fibrogenesis is triggered by destruction of hepatic cells and represents a wound-healing process leading to excessive deposition of the matrix proteins collagens and elastin, glycoproteins, proteoglycans and carbohydrates; in the context of chronic liver injury, fibrosis ultimately results in the substitution of liver tissue with ECM, formation of scar tissue, and gradual ceasing of hepatic functions[1,36]. Histologically, liver consists of parenchymal hepatocytes (70%-80%) and non-parenchymal cells such as Kupffer cells, sinusoidal endothelial cells and stellate cells. Hepatic stellate cells (HSCs) reside in the perisinusoidal space of the liver, also known as the space of Disse, between hepatocytes and sinusoidal endothelial cells and are the major fibrogenic cell type in the liver as they produce a large number of ECM components and secrete transforming growth factor-β (TGF-β), a key mediator of liver fibrogenesis[1,36].

In the normal liver, HSCs have a star-like morphology corresponding to a quiescent state, and their primary function is the storage of vitamin A as retinol ester in lipid droplets[37,38]. In response to liver injury, HSCs undergo activation and change into contractile myofibroblastic cells, which proliferate, secrete TGF-β, and increase matrix production. As a result, collagens IV and VI in the space of Disse are progressively replaced by fibrous collagens I and III and fibronectin, characteristic for ECM remodeling and fibrosis[39,40].

In our previous study, we revealed a link between the activation of human HSCs and platelets by showing that platelets and platelet-derived extracts suppressed transdifferentiation of quiescent HSCs into the myofibroblast-like phenotype as well as the production of collagen type I via cAMP signaling[31]. The underlying mechanism is based on the increase of adenosine concentration in the HSC milieu due to breakdown of ADP and ATP, which are abundant in platelet-dense granules[31]. As a result, adenosine entering HSCs through its cognate receptors prevents their activation and down-regulates their ability to secrete TGF-β and deposit the ECM. In addition, interaction between HSCs and platelets promotes the release of platelet-derived HGF, which was shown to inhibit the expression of type I collagen in cultured HSCs[41] and to attenuate liver fibrosis in mice by decreasing hepatic TGF-β secretion and blocking myofibroblast activation[42]. However, although these findings indicate that platelets can reduce hepatic fibrogenesis through inhibition of HSC activation, it is unclear whether they can be translated to the clinical situation, as the production of HGF by human platelets is lower than that by rodent platelets[43].

TPO is the most important factor in the regulation of megakaryocyte proliferation and differentiation into platelets through activation of its cognate receptor c-Mpl, also known as TPO-R[44]. Several agonists of the c-Mpl receptor, such as eltrombopag and romiplostim, are approved for clinical application as agents by which to increase platelet counts in chronic immune thrombocytopenia[44,45]. Moreover, they are currently undergoing clinical trials as treatment options to reduce thrombocytopenia in patients with CLD and liver cirrhosis[46-48], as the increase in platelet counts could make these patients eligible for interferon-based antiviral therapy[49,50].

The strategy to treat liver fibrosis in CLD through inhibition of thrombocytopenia was proved feasible in studies showing that TPO improved both platelet counts and liver fibrosis, even in conditions of hepatic cirrhosis[30,32]. Thus, in cirrhotic rats with dimethylnitrosamine-induced liver fibrosis and 70% hepatectomy, platelet increase by a single intravenous injection of TPO correlated with the inhibition of HSC activation and decrease of the fibrotic area in the liver, while antiplatelet serum attenuated hepatic regeneration[30]. In another study, mice with liver fibrosis induced by carbon tetrachloride (CCl₄) showed improvement after weekly intraperitoneal administration of TPO for 5-8 wk[32].

Although mechanistic insights into the correlation of increased platelet counts with the reversal of liver fibrosis are yet to be provided, it can be suggested that platelets may promote hepatocyte proliferation by secreting HGF, which is a potent mitogen for hepatocytes through activation of the MET receptor that is essential for organogenesis and wound healing. Moreover, HGF may contribute to the resolution of fibrosis by modulating levels of TGF-β and matrix metalloproteinases (MMPs), which are the main ECM enzymes degrading collagen.

The suggested association between platelets, HGF, and hepatic fibrosis is supported by the findings of Takahashi et al[51], who showed that transfused human platelets improved CCl₄-induced liver fibrosis in severe combined immune deficiency mice by increasing HGF levels in the mouse liver, which suppressed HSC activation, induced MMP-9 expression and inhibited hepatocyte apoptosis.

Thrombocytopenia is typically treated by platelet transfusion, which is suggested to improve liver function. However, it has not been established whether platelet transfusion can benefit CLD patients with thrombocytopenia since the pathogenesis of thrombocytopenia in CLD is multifactorial; therefore, the published guidelines on platelet transfusion do not cover CLD-related platelet loss[49]. As animal experiments indicate feasibility of using blood transfusion for thrombocytopenia associated with chronic liver injuries, clinical trials have been conducted.

A recent study included patients with CLD and cirrhosis (Child-Pugh class A or B) who demonstrated thrombocytopenia with platelet counts between 50000 and 100000/μL; the patients were treated with 10 U of platelet concentrate weekly for 12 wk and followed up for 9 mo after the last transfusion[33]. Although the platelet count did not show a significant increase, a marked improvement of liver function was observed, as evidenced by higher serum albumin levels 1 and 3 mo post-transfusion and higher serum cholinesterase concentration 9 mo post-transfusion; at the same time, serum hyaluronic acid levels indicative of liver fibrosis tended to decrease. However, this clinical trial was a non-controlled, non-randomized study based on a small sample size (6 patients); therefore, randomized controlled trials using larger patient cohorts need to be conducted in order to conclusively determine clinical value of platelet transfusion in CLD.

However, on the other hand, platelet preparations, despite recent prolongation of the preservation period, can only be used for 4 d due to the need to prevent proliferation of bacteria by preservation at room temperature. Furthermore, in patients with hematologic diseases who require repeated blood transfusions, alloantibodies are produced for human leukocyte antigens (HLAs) and human platelet antigens (HPAs) that differ from the patient’s own antigens, thereby triggering platelet transfusion refractoriness (PTR). Patients with PTR caused by anti-HLA antibodies or anti-HPA antibodies require HLA/HPA-matched platelet transfusions; however, securing donors is extremely difficult[81].

These factors result in an imbalance between supply and demand, thus generating interest in research that seeks to develop an alternative transfusion source to blood donation. Despite research in which hematopoietic stem cells (which are somatic stem cells) are used as a source to manufacture platelets ex vivo, this research has not been put to practical use due to the absence of an ex vivo human hematopoietic stem cell amplification method in which the cells’ functions can be retained.

Using our independently developed human mesenchymal stromal cells, we have succeeded in cultivating larger numbers of megakaryocytes, the sources of platelet production, than reported in previous studies; however, this method has not produced enough platelets to serve as a substitute for donated blood[82]. Similarly, studies in which umbilical cord blood-derived CD34+ cells are used as a source for red blood cell production have been unable to amplify these cells ex vivo; thus, no technique has yet been developed that can produce 10¹² red blood cells, the number of red blood cells needed for a single transfusion[83].

Alternatively, pluripotent embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells)[84] can be grown semipermanently in vitro and can resolve the crucial issue of “yielding the number of original cells”. In iPS cell-based regenerative medicine, post-transplantation cancer is a major concern. In response to this concern, safety for platelets and red blood cells can easily be ensured with a combination of anucleated cells, removal of nucleated cells with a filter prior to transfusion, and radiation to eliminate mixed-in lymphocytes. In addition, banking of iPS cells with various HLAs enables the construction of a system for stable provision of HLA-matched platelets[85].

Furthermore, Ono-Uruga et al[86] have discovered that pre-adipocytes endogenously possess the megakaryocyte inducer p45NF-E2 and can induce differentiation of megakaryocytes into platelets while increasing their own expression of p45NF-E2; they are striving to stably and safely produce platelets for transfusion from pre-adipocytes for medical applications. However, the number of platelets produced by this method is currently too low for practical use.

Although platelet increment therapies such as splenectomy and platelet transfusion can ameliorate CLD and liver cirrhosis, they may also cause serious side effects[48,87-90]. Thus, portal vein thrombosis[48,87], hemorrhage, infection and injury to the pancreatic tail[88] are among surgical complications encountered during splenectomy, while the platelet activation frequently observed in platelet transfusion may result in proinflammatory responses, febrile non-hemolytic reactions, and acute lung injury[89,90]. Besides, as we indicated earlier, there are reports about detrimental effects of platelets on hepatocytes[73-75]. Therefore, other approaches to treat thrombocytopenia in CLD should be considered. Among them, TPO-R agonists eltrombopag and romiplostim, already approved as therapeutics for chronic immune thrombocytopenic purpura, may also hold promise as treatment for CLD and liver cirrhosis.

Eltrombopag is a low molecular weight, synthetic nonpeptidyl drug, whereas romiplostim is a peptide containing an IgG Fc fragment in its structure[91]. Eltrombopag has already been tested in a phase II clinical trial involving patients with hepatitis C-associated cirrhosis and concurrent thrombocytopenia to increase platelet counts before starting anti-viral therapy with peg-interferon and ribavirin, which showed promising results[47]. Although eltrombopag is a TPO-R ligand, it caused little effect on platelet function and did not activate the PI3K/Akt pathway, in contrast to TPO, which caused significant platelet activation[92].

The safety of using TPO-R (c-Mpl) agonists was examined in c-Mpl-expressing leukemia cells. Similar to TPO, eltrombopag did not increase but rather inhibited proliferation of leukemia cells in vitro, suggesting the lack of tumorigenic side effects[93]. Moreover, TPO had no proliferative effect on HCC either in vitro or in vivo[94], while eltrombopag induced cell cycle arrest in HCC, demonstrating that it may act as a chemotherapeutic agent to inhibit the progression of HCC developed as a complication of CLD and liver cirrhosis[95]. Thus, eltrombopag represents a promising novel candidate drug to treat both CLD-related thrombocytopenia and associated malignant neoplasm.

To increase platelets in a continuous manner and avoid platelet transfusion refractoriness, we conducted an exploratory clinical trial and administered eltrombopag for 6 mo. The study included 5 patients with both CLD and a hepatitis C virus infection (Child-Pugh class A) who presented with thrombocytopenia (average platelet count 54 × 10⁹/L). All of the patients administered eltrombopag maintained platelet counts between 10.0 and 15.0 × 10¹⁰/L during the 6-mo study, and their serum albumin, cholinesterase, alanine aminotransferase, T-bilirubin, hyaluronic acid and type IV collagen levels, as well as the platelets (%) and liver volumes, were stable throughout the clinical trial. The liver volumes calculated by computed tomography during the clinical trial were also stable, and no new cancerous lesions were observed.

However, in contrast to our previous clinical study[33], we predicted that the increment values would be small because the liver function was representative of a heterogeneous population (Figure 1). Additionally, recent reports stated that the aging of platelets controls TPO production[96]; thus, we concluded that the aging and activation of platelets are involved in liver regeneration. Therefore, we are planning a novel therapy with TPO receptor agonists by using a desialylated formulation. In other words, increasing the platelet count by using TPO and aging the platelets via administration of a desialylated formulation to promote liver regeneration could be an effective treatment of CLD and liver cirrhosis (Figure 1).

Open in New Tab   Full Size Figure   Download Figure

Figure 1 Flow charts of our previous study and our prospective study.

Together with the antitumor effects of eltrombopag on hepatocellular carcinoma (HCC) that we previously reported[95], we are preparing for eltrombopag clinical trials for HCC patients with unmet medical needs and who cannot use sorafenib (Figure 2).

Open in New Tab   Full Size Figure   Download Figure

Figure 2 Representation of the mechanism of eltrombopag regarding its effects on liver regeneration, liver fibrosis and hepatocellular carcinoma.

Since Trousseau[97] first reported the excessive blood coagulation in cancer patients with elevated platelet counts in 1865, a large number of studies have been conducted on cancer and platelets[98].

Cancer development involves the following progression: (1) separation from the primary tumor and infiltration of blood vessels; (2) transportation through blood flow; (3) adhesion to the capillary walls of distant organs; (4) extravascular migration; and (5) proliferation at the new site to form a metastatic lesion. Of course, platelets are involved in almost every step of this process.

This editorial suggests the current perspective of novel treatments for liver cirrhosis by using TPO-R agonists to increase platelet counts in a clinical setting.

There is significant evidence that platelets play a role in improving fibrosis. Upon their release, the ATP and ADP within platelet dense granules are degraded by HSCs into adenosine, which is incorporated into the HSCs.

There are three distinct mechanisms of liver regeneration induced by platelets: a cooperative effect with LSECs; a cooperative effect with Kupffer cells; and a direct effect on hepatocytes. Additionally, platelet transfusion improves liver function in patients with CLD and cirrhosis. Despite its safety and maintenance in increasing platelet counts, administration of the TPO-R agonist eltrombopag for 6 mo did not result in improvements of liver function in patients with CLD. Therefore, we are planning a new approach to develop novel strategies with TPO-R agonists and a desialylated formulation for treating liver diseases for which there are currently no effective treatments except transplantation. Of course, it is necessary to pay sufficient attention to the onset of thrombosis by excessively increasing platelets.

Editorial support, in the form of medical writing was provided by Cactus Communications.