Platelets and Liver Regeneration

Kazuhiro Takahashi, Soichiro Murata and Nobuhiro Ohkohchi*

Department of Surgery, University of Tsukuba, Japan

*Corresponding author: Nobuhiro Ohkohchi, Department of Surgery, Division of Gastroenterological and Hepatobiliary Surgery and Organ transplantation, University of Tsukuba, 1-1-1 Tennoudai, Tsukuba, Ibaraki, 305-8575, Japan

Received: July 14, 2014; Accepted: Aug 01, 2014; Published: Aug 05, 2014

Abstract

Platelets contain growth factors and cytokines such as hepatocyte growth factor, tumor necrosis factor-α, interleukin-6, serotonin, insulin-growth factor-1, transforming growth factor-α, endothelial growth factor, sphingosine 1-phosphate, etc. Platelets are activated by various types of stimulation and release these physiologically active substances depending on each context. In addition to primary roles as hemostasis and thrombosis, recent studies have reported the roles of platelets on promoting liver regeneration, improving liver fibrosis, and attenuating liver damage. In this article, we reviewed the recent advances in knowledge of the role of platelets in accelerating liver regeneration.

Keywords: Platelet; Hepatectomy; Liver regeneration; Hepatocytes; Growth factor; Cytokine

Introduction

Platelets are anucleated cytoplasmic fragments originating from mega karyocytes in the bone marrow [1]. After leaving the bone marrow, platelets circulate in the blood for about ten days in the body. Platelets contain three specific granule populations, i.e., dense granules, lysosomal granules, and α-granules which store various type of growth factors and cytokines such as Hepatocyte Growth Factor (HFG), insulin-like growth factor-1 (IGF-1), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), transforming growth factor-α (TGF-α), endothelial growth factor (EGF), vascular endothelial growth factor (VEGF), sphingosine 1-phosphate (S1P) [2,3], platelet derived growth factor (PDGF), etc [4-7]. Platelets are activated by physical and physiological stimulation and release these biologically active substances depending on each context [8]. Platelets are involved in various processes including stopping hemorrhage following vascular injury [9-11], fighting against microbial infections [12-14], tissue repairing [15-17], promoting metastasis [18-20] etc. In recent years, platelets are highlighted to have role in promoting liver regeneration [21-25], improving liver fibrosis [26-30], and attenuating liver damage [31-33]. In this review, we described the most-updated experimental and clinical evidences of platelets in accelerating liver regeneration.

Platelet and Liver Regeneration

Partial hepatectomy is a feasible and relatively safe procedure for benign and malignant liver tumors and living liver donor liver transplantation. Despite substantial improvements in surgical techniques and perioperative care, liver failure after hepatectomy is a devastating complication with considerable high morbidity and mortality, and it still remains an important concern after surgery [34- 37]. The incidence of liver failure ranges from 0-13% [35] or 0.7-9.1% [36]. The presence of comorbid conditions, pre-existing underlying parenchymal disease, and extent resection are the major risk factors. Preventive methods should be applied, and adequate preoperative risk evaluation and an optimal postoperative treatment are essential. Postoperative management principles resemble those applied to acute liver failure, focusing on support of liver and end-organ function, such as plasma exchange [36,38,39], molecular absorbent re circulating system such as (MARS®, Gambro, Lund Sweden) [40] and Prometheus (Fresenius Medical Care, st, wendel, Germany) [41], bio artificial liver, [42,43] and liver transplantation [44]. On the other hand, one of the recent strategies for liver failure after hepatectomy is to augment the remnant liver regeneration [45-47]. In the past, several attempts have been made in this field, i.e., stem cell injection therapy [48], erythropoietin and granulocyte macrophage colony stimulating factor administration therapy [49], gene therapy [50-54], hepatocyte transplantation [45] etc. However, there are few reports on their clinical application.

Liver regeneration after partial hepatectomy is very complex phenomenon, which is carried out by the all mature liver cell types. Intercellular interaction by growth factors and cytokines, such as HGF, EGF, TNF-α, IL-6, and serotonin, which play important roles during this process [55-57]. Hepatocytes are the first cells to enter into the cell cycle and undergo proliferation. Hepatocytes produce mitogenic signals for other liver cell types, and each mediator leads activations of downstream cascades, which transfer hepatocytes from the quiescent status into cell cycle [55,56,58]. TNF-α/nuclear factor-kappa B (NF-κB) [59,60], IL-6/signal transducer and activator of transcription 3 (STAT3) [61], phosphatidylinositol-3-kinase (PI3K)/Akt pathways [62], HGF/HFG receptor (cMet) pathway [63], and extracellular signal-regulated kinase 1/2 (ERK1/2) [64], are the major cascades during liver regeneration. Serotonin signaling is mediated through a number of specific receptors, most of them coupled with G-proteins [65]. G-proteins link the receptors to a variety of downstream pathways that elicits cellular responses. These pathways include PI3K/Akt pathways, MAPK pathways, and STAT-JAK pathway [65]. Through these proliferative signals, hepatocyte undergoes DNA synthesis, peaking at 24 hours for the rat and at approximately 36 hours for the mouse [66]. More than 95% of hepatocyte go through cell proliferation during the first 48 hours and normal liver weight is reestablished within 5-7 days for the rodents and 8-15 days for the human [56,66].

Relationship between platelets and liver regeneration was first described by Murata et al. [67], in 2004. After this report, there has been piling evidence of platelets contributing in liver regeneration from clinical and experimental data.

Experimental Evidences

In vivo

Murata et al. [22], induced thrombocytosis by thrombopoietin administration and conducted 70% partial hepatectomy to the mice and liver regeneration was evaluated. Liver/body weight increased significantly in the thrombocytotic condition compared with the normal platelet conditions. Akt was strongly phosphorylated under thrombocytosis. The authors described that platelets affect liver regeneration after hepatectomy and PI3K/Akt was the main signaling pathway involved in platelet-mediated liver regeneration.

Lesurtel et al. [21], focused that platelets are the major carriers of serotonin in the blood. They found that expression of serotonin receptors in the liver increased after hepatectomy and antagonist of serotonin receptors inhibited liver regeneration. Liver regeneration was blunted by inhibiting synthesis of peripheral serotonin, which was rescued by serotonin administration. They conclude that platelet derived serotonin is involved in the initiation of liver regeneration.

Shimabukuro et al. [68], performed 70% partial hepatectomy on rats given thrombopoietin. They compared liver regeneration, DNA synthesis, and HGF mRNA expressions in the liver. They reported that liver regained the pre-hepatectomy weight levels much faster when treated with thrombopoietin. Thrombopoietin significantly enhanced the DNA synthesis and HGF mRNA expressions.

Matsuo et al. [46], transfused platelet-rich plasma to rats after 70% partial hepatectomy. Platelet-rich plasma infusion increased liver/body weight ratio and Ki-67 labeling index after hepatectomy. Platelet-rich plasma infusion accelerated Akt activation and prolonged ERK 1/2 phosphorylation. These results indicated that platelet-rich plasma infusion had a positive impact on accelerating liver regeneration.

Myronovych et al. [69], examined using 90% partial hepatectomy model, which was considered as fatal. They induced thrombocytosis by thrombopoietin administration, and evaluated survival rate and liver regeneration. They found that all mice with the normal platelet level died early after hepatectomy, whereas half with thrombocytosis survived after hepatectomy. Liver/body weight ratio was significantly increased in the mice with thrombocytosis compared to the mice with normal platelet counts. Phosphorylation of Akt and STAT3 were earlier and stronger under thrombocytotic conditions. These results implied that thrombocytosis promoted liver regeneration through early activation of PI3K/Akt and IL-6/STAT3 pathways.

Lopez et al. [70], implanted platelets in the peritoneum of the rats after 90% partial hepatectomy. They found that although there were no difference in hepatocyte mitosis and DNA synthesis, platelets implanted group showed higher survival rate. They concluded that platelets enhanced survival by an early protective effect on hepatocyte.

Murata et al. [71], reported that liver regeneration in the cirrhotic liver after hepatectomy was significantly decreased when compared to the normal liver, and liver regeneration in the cirrhotic liver with thrombocytosis was the same level as those of the normal liver. The authors further reported that fibrosis decreased significantly in the cirrhotic liver under thrombocytotic conditions. The authors proved that these effects were due to increased number of platelets, not by thrombopoietin administration itself, by using antiplatelet serum in the thrombocytotic condition.

In vitro

Matsuo et al. [72], reported that DNA synthesis of the hepatocyte was increased by co-culturing with platelets. DNA synthesis was depended on the platelet concentration. In order to determine which fragment of platelets exert hepatocyte proliferative effect, they separated platelet extract and assessed mitogenic activity. DNA synthesis was strong in the fragment of HGF, IGF-1, and VEGF, implying that these growth factors are the key mediators for liver regeneration.

Hoshi et al. [73] reported that freeze-dried platelets preserved stored adenine nucleotides, PDGF, and IGF-1, which were the same levels as those of fresh platelets. The authors described that freeze-dried platelets induced stimulatory effect on the DNA synthesis of hepatocyte. They proved usefulness of freeze-dried platelets on promoting liver regeneration.

Mechanisms of liver regeneration induced by platelets

Murata et al. [22], reported that platelets accumulated in the liver immediately after hepatectomy. Transmission electron microscopy revealed platelet migration from the sinusoidal space into the space of Disse and platelets were in direct contact with hepatocyte in the thrombocytotic condition. Matsuo et al. [72], clarified the necessity of direct contact between hepatocyte and platelets using co-culturing chamber system, which separates upper and lower chambers by a permeable membrane. They indicated that upon direct contact with hepatocytes, platelets release soluble factors that induce hepatocyte proliferation. Through these two studies, it was considered that platelets accumulate in the liver immediately after hepatectomy, translocation from the liver sinusoids to the space of Disse and release growth factors through direct contact with hepatocyte. Growth factors stimulate initiation of hepatocyte mitosis, which eventually promotes liver regeneration.

Kawasaki et al. [74], evaluated the role of platelets in relation to liver sinusoidal endothelial cells. They proved that IL-6 concentration was increased in the supernatant of the liver sinusoidal endothelial cells co-cultured with platelets, which was cancelled by application of S1P receptor antagonist. DNA synthesis and STAT3 phosphorylation of the hepatocyte were enhanced when applying supernatant of liver sinusoidal endothelial cells co-cultured with platelets. The authors concluded that platelets induced IL-6 release from liver sinusoidal endothelial cells, which accelerated hepatocyte DNA synthesis through IL-6/STAT3 pathway. In addition, they proved that S1P induced IL-6 secretion from liver sinusoidal endothelial cells.

Takahashi et al. [75,76], clarified that hepatic expressions of TNF-α and IL-6 were enhanced by platelet transfusion after hepatectomy. Since TNF-α and IL-6 are predominantly produced by Kupffer cells [56,77], these results indicated that platelet transfusion enhanced TNF-α and IL-6 secretion from Kupffer cells. Furthermore, the authors indicated that although only a few transfused platelets were adhering to the Kupffer cells without hepatectomy, the majority of platelets transfused were adhering to the surface of Kupffer cells without being phagocytosed after hepatectomy. Based on the findings, it was assumed that platelets promoted liver regeneration by interactions with Kupffer cells.

From these studies, three different mechanisms are clarified, i.e., i) the direct effect on hepatocyte, ii) the cooperative effect with liver sinusoidal endothelial cells, and iii) the collaborative effect with Kupffer cells.

Clinical Evidences

Partial hepatectomy

Kaneko et al. [78], described that preoperative platelet counts less than 100 x 10³/μl was the strongest independent factor for postoperative mortality among patients with hepatocellular carcinoma who underwent hepatectomy. Alkozai et al. [79], reported that immediate post-operative platelet count below 100 x 103/μl was an independent risk factor for delayed postoperative liver function recovery and was associated with increased risk of postoperative mortality among patients who underwent partial hepatectomy for colorectal metastasis. They described that there was no association between preoperative platelet counts and delayed postoperative liver function recovery. Sratlinger et al. [80,81], described that patients suffering from postoperative liver dysfunction and morbidity were found to have reduced intra-platelet serotonin levels during the entire perioperative period and reduced preoperative intra-platelet serotonin was associated with and increased incidence of postoperative liver dysfunction and morbidity. They demonstrated that intra-platelet serotonin levels were an independent predictor of poor clinical outcome. On the other hand, Kim et al. [82], described that substantial portion of patients exhibited decrease in platelet counts after hepatectomy. However, such change did not affect postoperative liver function, liver regeneration, or overall complications.

Liver transplantation

Marubashi et al. [83], reported that there was a positive correlation between graft size and post-transplant thrombocytosis after living donor liver transplantation. They mentioned that splenectomy is an option in cases with a small graft. Kim et al. [84], described that total units of platelet concentrate transfused was found to be significantly associated with the graft regeneration after living donor liver transplantation. Lesurtel et al. [85], described that platelet counts < 6.0 × 10⁹ on postoperative day five was associated with a higher risk of severe complication graft loss, and decreased chance of patient survival after liver transplantation.

Perspectives

In this review, we demonstrated the most-updated evidences of platelets promoting liver regeneration and their mechanisms. Thrombopoietin receptor agonist [86-88], artificial platelets [89,90], and freeze-dried platelets [73,91,92], are developing, and some of them are beginning to be utilized in the clinical practices. However, there are still side effects and unsolved problems such as production of anti-platelet antibodies following platelet transfusion [93], and of anti-thrombopoietin antibodies after thrombopoietin administration [94], as well as the several-day delay between thrombopoietin-receptor agonist administration and increases in platelet counts. We hope platelets can add strategies for the surgical challenges, such as post-hepatectomy liver failure after massive hepatectomy and small-graft syndrome after liver transplantation [95,96].