Platelet production and destruction in liver cirrhosis .pdf



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Titre: Platelet production and destruction in liver cirrhosis
Auteur: Paola Pradella

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Research Article

Platelet production and destruction in liver cirrhosis
Paola Pradella1, Stefania Bonetto2, Stefano Turchetto1, Laura Uxa1, Consuelo Comar2,
Francesca Zorat2, Vincenzo De Angelis1, Gabriele Pozzato2,⇑
1

Blood Bank Service, Azienda Ospedaliero-Universitaria Ospedali Riuniti di Trieste, Trieste, Italy; 2Struttura Complessa Seconda
Medicina/Ematologia, Azienda Ospedaliero-Universitaria Ospedali Riuniti di Trieste, Trieste, Italy

Background & Aims: Thrombocytopenia is common in liver
cirrhosis (LC) but the mechanisms are not fully understood. The
purpose of our work was to evaluate platelet kinetics in LC with
different etiologies by examining platelet production and
destruction.
Methods: Ninety-one consecutive LC patients (36 HCV, 49 alcoholics, 15 HBV) were enrolled. As controls, 25 subjects with idiopathic thrombocytopenic purpura, 10 subjects with aplastic
anemia, and 40 healthy blood donors were studied. Plasma
thrombopoietin (TPO) was measured by ELISA. Reticulated platelets (RP) were determined using the Thiazole Orange method.
Plasma glycocalicin (GC) was measured using monoclonal antibodies. Platelet associated and serum antiplatelet antibodies
were detected by flow cytometry. B-cell monoclonality in PBMC
was assessed by immunoglobulin fingerprinting.
Results: Serum TPO was significantly lower in LC (29.9 ± 18.1
pg/ml) compared to controls (82.3 ± 47.6 pg/ml). The GC levels
were higher in LC (any etiology) than in healthy cases. Conversely, the absolute levels of RP were lower in LC (any etiology)
than in healthy controls. The platelet-associated and serum
anti-platelet antibodies were higher in HCV+ LC compared to
healthy subjects (p <0.0064), alcoholic LC (p <0.018), and HBV+
LC (p <0.0001). B-cell monoclonality was found in 27% of the
HCV + LC, while it was not found in HBV+ or alcoholic LC.
Conclusions: Patients with LC present decreased plasma TPO,
accelerated platelet turnover, and reduced platelet production.
This indicates that LC thrombocytopenia is a multifactorial condition involving both increased platelet clearance and impaired
thrombopoiesis.
Ó 2010 European Association for the Study of the Liver. Published
by Elsevier B.V. All rights reserved.

Keywords: Liver cirrhosis; Glycocalicin; Thrombopoietin; HCV; HBV; Aplastic
anemia; Idiopathic thrombocytopenic purpura.
Received 12 December 2009; received in revised form 2 August 2010; accepted 9
August 2010; available online 22 December 2010
⇑ Corresponding author. Address: Struttura Complessa Seconda Medicina, Ospedale ‘‘Maggiore’’, Piazza Ospedale 1, 34100 Trieste, Italy. Tel.: +39 040 3992002;
fax: +39 040 3993560.
E-mail address: g.pozzato@fmc.units.it (G. Pozzato).
Abbreviations: LC, liver cirrhosis; HCV, hepatitis C virus; HBV, hepatitis B virus;
TPO, thrombopoietin; RP, reticulated platelets; GC, glycocalicin; ITP, idiopathic
thrombocytopenic purpura; AA, aplastic anemia; PBMC, peripheral blood mononuclear cells; TBS, Tris-buffered saline; PRP, platelet-rich plasma; BSA, bovin
serum albumin; ACD, acid Na-citrate dextrose.

Introduction
Thrombocytopenia is a frequent feature in patients with liver
cirrhosis (LC). It has traditionally been attributed to the sequestration of platelets by the spleen [1–3], a situation known as
‘‘hypersplenism’’ secondary to portal hypertension. Nevertheless,
no clear correlation between the portal pressure and the platelet
count has ever been observed. Furthermore, in some cirrhotic
patients thrombocytopenia may persist even after splenectomy
or after portal decompression [4–9]. Since the return to a normal
platelet count has been observed following liver transplantation
[10], it is likely that other mechanisms apart from hypersplenism,
such as reduced thrombopoietin (the cytokine which regulates
megakaryocyte maturation and platelet production) release by
the liver [11–14] or bone marrow suppression [15,16], are
involved in the thrombocytopenia of cirrhotic patients.
To increase the complexity of this problem, the etiology of the
liver disease should be taken into account since HBV, HCV, and
ethanol (the most common causes of liver cirrhosis) all induce
liver damage via different mechanisms; therefore, even if the final
outcome is the same (cirrhosis), the biochemical and immunological abnormalities are quite different, especially in HCV-infected
patients. In addition, the possibility for studying platelet turnover
‘‘in vivo’’ is limited. In this study, we assessed reticulated platelet
counts and glycocalicin levels as measures of platelet production
and destruction, respectively. We also quantified levels of serum
thrombopoietin and anti-platelet antibodies in groups of patients
affected by cirrhosis of different etiologies (HBV-related, HCVrelated or alcohol induced-LC) and in two control groups of
patients affected by hematological diseases known to affect the
platelet count, such as idiopathic thrombocytopenic purpura
(ITP) and aplastic anemia (AA), as well as in healthy control subjects. In a fraction of the patients, we assessed for the presence
of an expanded monoclonal B-cell population in the peripheral
blood mononuclear cells (PBMC) and for immunoglobulin gene
usage via isotype-specific immunoglobulin fingerprinting.

Patients and methods
Hepatological patients
Ninety-one consecutive patients with liver cirrhosis and platelet counts less than
100 109/L were enrolled in the study. Of these patients, 36 had a HCV-related
liver disease, 40 were alcoholics, and 15 had a HBV-related liver disease. None
of the patients had previously undergone interferon therapy or were taking drugs

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JOURNAL OF HEPATOLOGY
known to interfere with bone marrow function. The diagnosis of cirrhosis was
based on the grounds of either liver biopsy (58 cases, 64%) or on findings resulting
from a physical examination of the patient, results of laboratory tests, imaging
studies, and the presence of portal hypertension (splenomegaly, ascites, and
esophageal varices). The liver biopsies were placed in buffered formalin and
stained with haematoxylin and eosin or with Gomori methenamine silver for
reticulum staining. In HCV and HBV patients, the disease activity and fibrosis
were assessed according to the METAVIR scoring system [17]. Patients carrying
anti-nuclear, anti-mitochondrial, or anti-smooth muscle antibodies were discarded from the study.
The study protocol was approved by the ethical committee and conforms to
the ethical guidelines of the 1975 Declaration of Helsinki. All patients gave written informed consent for participation in this medical research.
Hematological patients
Twenty-five cases of idiopathic thrombocytopenic purpura (ITP) were enrolled in
the study. Each subject presented the active phase of the disease but had not yet
initiated immunosuppressive treatment. This disease is characterized by a high
platelet turnover and the presence of anti-platelet antibodies. The diagnosis of
ITP was based on commonly adopted criteria [18] involving: the patient’s medical
history, physical examination report, complete blood cell count, and cytomorphologic examination of the peripheral-blood smear in which no alterations of erythrocytic and leukocytic series should be present. To confirm the diagnosis, bone
marrow aspiration was performed on all patients and only those with the presence of a normal or increased number of megakaryocytes without pathologic
alterations of the erythroblastic, granuloblastic, or lymphocytic series were
included in the study. Moreover, we had the opportunity to study 10 patients
affected by severe aplastic anemia, a disease exhibiting a very low platelet count
due to a reduced platelet production. The definition of ‘‘severe’’ aplastic anemia
was determined according to currently used criteria [19]: the disease was considered severe if at least two of the following were noted: a neutrophil count less
than 0.5 109/L, a platelet count less than 20 109/L, and a reticulocyte count
less than 60 109/L with hypocellular (<20%) bone marrow.
Controls
Forty volunteer blood donors, serologically negative for HIV, HBV, and HCV, with
normal platelet counts were used as normal healthy control subjects. Informed
consent was obtained prior to all blood donations.

Methods
In this observational study, we had the opportunity to apply the same up-to-date
methods for studying platelet turnover to different groups of patients. The antiHCV antibodies were detected by a third-generation enzyme immunoassay (Ortho
HCV SAVe 3.0, Raritan, NJ). Positivity for anti-HCV antibodies was confirmed by
strip immunoblot assay (RIBA HCV 3.0, Chiron Corp., Emeryville, CA). In antiHCV-positive patients, the serum HCV-RNA was assessed by means of nested
reverse transcription polymerase chain reaction. Commercially available
enzyme-linked immunoassays were used to measure HBeAg, anti-HBe, HbsAg,
and anti-HBs (Abbott Laboratories, USA). The Serum HBV-DNA was measured using
the TaqMan polymerase chain reaction assay (COBAS TaqMan, Roche Molecular
System, lowest limit of detection: 70 copies/ml).
The maximum longitudinal diameter of the spleen was estimated by means of
an ultrasound scan by an experienced operator. Ultrasonography was carried out
using an AU 570 Asynchronous (Esaote Biomedica, Genoa, Italy).

healthy subject control samples. The same threshold was used for the analysis of
all samples. The percentage of reticulated platelets was obtained by subtracting
the percentage of reticulated platelets of unstained preparations from that of
stained samples. A normal reference range (5.9 ± 2.25) was obtained using blood
samples from 60 healthy normal subjects.
Glycocalicin assay
Blood was collected in EDTA at a 50 mM final concentration. The samples were processed within 2 h following blood collection since preliminary tests showed that no
change in glycocalicin values could be observed within this time period. The plasma
was centrifuged at 12,000g for 5 min and the supernatant was stored at –70 °C. The
monoclonal antibodies LJ-P3 and LJ-P19 were kindly given by Z.M. Ruggeri (Scripps
Clinics, La Jolla, CA, USA). LJ-P3 and LJ-P19 monoclonal antibodies interact with epitopes only present in the native glycoproteins located within residues 1-290. The
epitopes for the two monoclonal antibodies are distinct since no cross-reactivity
was observed. To determine the specificity of each monoclonal antibody, we measured glycocalicin (GC) concentration in pooled control plasma from normal subjects by co-incubating the plasma with various concentrations of mouse IgG and
LJ-P19. There was no decrease in the concentration of GC following co-incubation
of the plasma at different dilutions (1:500–1:25,000) of mouse IgG at room temperature for 1 h. By contrast, the GC concentration was decreased in plasma co-incubated in concentrations of nonbiotinylated LJ-P19 ranging between 3.3 and
0.066 lg/ml. GC was purified as previously described by Vicente et al. [20]. GC concentration was determined by the Micro BCA-method (Pierce, Rockford, USA) using
albumin as a standard. The GC value could be underestimated by approximately
30–40% in the protein assay because of the high glycosylation of GC. Five to eight
fresh random platelet concentrates, to which a 15% acid Na-citrate dextrose
(ACD) solution was added, were centrifuged at 800g for 20 min at room temperature. The platelet pellet was then washed twice with TBS, pH 7.4, containing
2 mM EDTA and 15% ACD, resuspended in TBS containing 2 mM CaCl2 and
0.1 mM phenyl-methyl-sulphonyl-fluoride and sonicated on ice. The suspension
was then stirred (2 h at RT and 18 h at 4 °C) and centrifuged at 23,000g for
30 min. The supernatant was applied to an affinity column of wheat germ agglutinin-Sepharose 6 MB (Pharmacia, Uppala, Sweden), and the bound proteins were
eluted with TBS containing 1 mM EDTA, 0.1 mM PMSF, 3 mM NaN3, and 100 mM
N-acetyl-glucosamine. The eluate was then immunopurified with an LJ-P3 Sepharose column (Pharmacia, Uppala, Sweden). GC was eluted with 50 mM diethylamine, 1 mM EDTA, and 0.1 mM PMSF. Glycine 0.55 M was added and the eluted
protein was dialysed for 18 h against TBS and concentrated with Centiprep 100.
Purified GC was stored at –80 °C. GC was assayed in plasma by ELISA following
the methods of Kunishima et al. [21] and Beer et al. [22], with some modifications.
Briefly, a flat-bottomed microtitre plate (Costar, Cambridge, USA) was coated with
the monoclonal antibody LJ-P3 (1.0 lg/ml) overnight at 4 °C, washed 3 times with
PBS containing 0.05% Tween (PBS-T), blocked with PBS containing 2% BSA and
washed again with PBS-T. The standard curve was prepared using immunopurified
GC ranging from 0.05 to 1 lg/ml in PBS-T. The plasma samples were then diluted 4fold and 8-fold in PBS-T and 100 ll of each added to individual wells. After incubation for 60 min at RT, 100 ll of the biotin-labelled LJ-P19 (1.5 lg/ml in PBS-T) were
added and incubated for a further 60 min. After three washes, each well was filled
with 50 ll of the streptavidin-HRPO (Caltag, South San Francisco, CA, USA), diluted
1000-fold in PBS-T, and incubated for 60 min at RT. After three washes, 100 ll of the
o-phenylenediamine dihydrochloride reaction was stopped by adding 50 ll of
2NH2SO4 and the absorbance was measured at 492 nm with a microtitre plate
reader. The results were plotted and expressed as GC concentration in lg/ml
and as GC indices. The GC index represents the standardization of the GC value
for a platelet count of 250 109/L and is calculated as follows: GC index = GC
lg/ml (250 109/L/patient’s platelets).
Thrombopoietin

Reticulated platelets
Peripheral blood was collected using ACD-A (at the ratio 8.5/1.5 v/v) and centrifuged at 100g for 10 min at 22 °C to obtain platelet-rich plasma (PRP). 10 ll PRP
from normal subjects and 20–50 ll from all other patients were added to 1 mL of
Thiazole Orange diluted 1:4 in Tris-buffered saline, pH 7.4, 10 mM EDTA, 0.1%
bovine serum albumin (TBS/EDTA/BSA), and incubated for 60 min at 22 °C in
the dark. PRP incubated with TBS/EDTA/BSA without Thiazole Orange was used
as a blank for each sample. Fluorescence was analysed using an Epics-Elite flow
cytometer (Coulter, USA). Logarithmic amplification was used for both light scatter and fluorescence signals. For each sample, fluorescence signals were collected
from 10,000 cells and a frequency histogram created. Three normal controls were
run in each assay; a threshold was positioned giving 1% of fluorescent platelets in

Serum thrombopoietin (TPO) levels (pg/ml) were measured by means of ELISA,
(Quantikine. R&D Systems Europe Ltd., Oxon, UK) according to the manufacturer’s
instructions. The lower limit of detection of the kit is 15 pg/ml. Sera were collected
in a serum-separator tube and allowed to clot for approximately 45–60 min; they
were then centrifuged, chilled rapidly, and stored at 20 °C until TPO determination. In our laboratory the intra-assay coefficient of variation was 8.7%.
Detection of antiplatelet antibodies by flow cytometry
The direct labeling procedures for platelet-associated Ig, and the indirect labeling
procedures for serum circulating Ig, were performed according standard
procedures.

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Using an EPICS-Elite flow cytometer (Coulter) equipped with a 488 nm argonlaser, data were acquired as 5000 ungated events and a flow rate of 150–300
events was maintained. Platelets were gated using forward- and side-angle light
scatter and log amplification with the gains adjusted to include the whole platelet
population. Fluorescent signals were then obtained with platelets sensitized to
surface IgG (direct method), patients, and control samples (indirect method).
B-cell monoclonality

Glycocalicin

Total cellular RNA was isolated from PBMC using the procedure described by
Chomczynski and Sacchi [23]. PBMC was obtained by fractionation of whole
blood (or bone marrow) on the Ficoll/Hypaque gradient. One lg of mRNA was
reverse transcribed (RT) using random examers. The entire RT product was next
subjected to PCR with 50 pmol of a degenerate VH primer placed in FW1
(50 -AGGTGCAGCTGGA(T)GG(C)AGT C(G)T(G)GG-30 ) and a specific primer for the
constant region of the gene located eight nucleotides downstream from the
beginning of the CH1 exon (50 -GAAAGGGTTGGGGC GGA T-30 ). The amplified
PCR fragments were purified, ligated into a Sma site of a pUC18 plasmid, and used
to transform the Escherichia coli strain DH5a. After expansion, the clones were
randomly picked and double-stranded DNA templates were sequenced using
the T7 sequencing kit. The procedure was performed in 30 HCV and 10 HBV cirrhosis patients, and 10 patients with alcoholic cirrhosis.
Statistics
Statistical analysis was carried out using the Chi-square-test to compare proportions and the Mann–Whitney U-test to compare means. Spearman’s rank correlation tests were used to look for correlations between the various parameters. Data
are shown as the mean ± SD. A p value of 0.05 or less was considered statistically
significant.

Results
The main characteristics of the 80 patients with liver cirrhosis are
given in Table 1. As shown, the three groups of patients had similar clinical, hematological, and biochemical characteristics. The
clinical data setting of the hematological patients is indicated in
Table 2.
Reticulated platelets
In the patients affected by alcoholic cirrhosis, the mean percentage
of reticulated platelets was very similar to that found in normal
healthy subjects (5.8 ± 3.2% vs. 5.9 ± 2.2% p: NS). Given the reduced
platelet count (76.826 ± 26.020 109/L) the absolute levels of reticulated platelets were significantly higher in normal subjects compared to patients with alcoholic cirrhosis (4.233 ± 2.367 109/L
vs. 14.666 ± 5.999 109/L p <0.0000000012). Similar results have
been found in HBV+ liver cirrhosis (percentage of reticulated platelets 5.5 ± 4.1% with absolute levels of 4.996 ± 3.143 109/L). In
HCV+ liver cirrhosis, despite a similar mean platelet count
(67.811 ± 19.181 109/L p: NS), the percentage of reticulated
platelets was significantly higher (11.3 ± 4.7%) than in patients with
alcoholic cirrhosis (5.8 ± 3.2% p <0.043) and HBV+ cirrhosis
(5.5 ± 4.1% p <0.05). Consequently, the absolute counts of reticulated platelets were higher in HCV patients (6.629 ± 7.409 109/
L) than in either patients of alcoholic cirrhosis (4.233 ± 2.367
109/L) or HBV (4.966 ± 3.143 109/L); given the high standard deviation of the HCV patients, the difference (with HBV of alcoholic cirrhosis) does not reach statistical significance (p = 0.068). In the HCV
patients, two groups can be identified, the first one (27 cases) with
low reticulated platelet count (4.3 ± 1.7% i.e. 3.171 ± 1.572 109/L)
and second one (nine cases) with normal-high reticulated platelet
count (31.6 ± 17.6% i.e. 16.640 ± 9.377 109/L p <0.0000000027).
The patients affected by ITP showed very high levels of reticulated platelets (25.7 ± 11.2%), while very low levels were found
896

in the patients with aplastic anemia (1.0 ± 0.9%). The results
obtained from these hematological cases are in line with the
pathophysiology of the underlying diseases: increased platelet
production for accelerated destruction in the former and very
low production in the latter.

In the normal healthy subjects, given the slow rate of platelet
turnover, the glycocalicin index was within normal limits
(0.9 ± 0.2), while in the patients with liver cirrhosis, independent
of etiology, the glycocalicin index was high: 1.96 ± 1.40 in HCV+
cirrhosis (p <0.0005 vs. healthy controls), 1.79 ± 1.51 in the alcoholics (p <0.006 vs. healthy controls) and 1.71 ± 1.69 in HBV+ cirrhosis (p <0.006 vs. healthy controls). In the HCV patients, even
for the glycocalicin index, two groups can be identified: the first
one (27 cases) with a value comparable to alcoholics and HBV+
cirrhosis (1.76 ± 1.48) and a second one (nine cases) with high
glycocalicin index (2.46 ± 0.84; p <0.02). The mean glycocalicin
level and index were significantly greater in patients affected
by ITP (12.9 ± 4.4, p <0.000002 vs. healthy controls). In the
patients with aplastic anemia, the glycocalicin index was normal
(0.8 ± 0.1), in accordance with the normal platelet turnover.
Thrombopoietin
The serum thrombopoietin levels were significantly (p <0.0003)
lower in patients with cirrhosis (29.9 ± 18.1 pg/ml) compared to
healthy controls (94.7 ± 35.9 pg/ml). No differences were found
on the basis of the etiology of the liver cirrhosis (29.2 ± 16.2 in
alcoholics, 30.6 ± 22.0 in HCV+ and 31.1 ± 19.9 in HBV+). On the
contrary, levels above the normal limits were found in the
patients with aplastic anemia (507.7 ± 86.1 pg/ml) and in
patients affected by ITP (155 ± 76 pg/ml). The thrombopoietin
serum levels were inversely correlated to platelet counts in ITP
patients (r: 0.87), while this correlation was not found in
patients with liver cirrhosis. Moreover, no difference was
observed in TPO serum levels between patients with or without
splenomegaly, and no correlation was observed between TPO
serum levels and either spleen longitudinal diameter or surface
area, or liver histological inflammatory activity (in both HCV
and HBV subjects) (data not shown).
Spleen size
Seventy percent of the cirrhosis patients presented splenomegaly
(i.e. a spleen longitudinal diameter over 12 cm). The level of
thrombocytopenia was not correlated to either spleen size (considering both spleen longitudinal diameter and spleen surface
area) or liver histological necro-inflammatory activity (in both
HCV and HBV patients). The spleen size was normal in all patients
with ITP and aplastic anemia.
Anti-platelet antibodies
Normal healthy subjects were not found to express any detectable levels of anti-platelet antibodies As expected, most patients
(85%) affected by ITP presented platelet-associated antibodies,
and a large fraction of them (65%) also expressed serum circulating anti-platelet antibodies. None of the patients with aplastic
anemia expressed anti-platelet antibodies. The frequencies of

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Table 1. Demographic, biochemical, and virological profiles of the patients with HCV-, HVB-, and alcoholic cirrhosis.

ALT, alanine-aminotransferase; AFP, a-fetoprotein; HBV, hepatitis B virus; HCV, hepatitis C virus.

platelet-associated antibodies in patients with liver cirrhosis (all
etiologies combines) and patients with ITP were significantly
higher compared to healthy controls (p <0.005 and 0.0001,
respectively). Among the patients with HCV-related cirrhosis,
platelet-associated antibodies were found in seven cases
(19.4%) and serum circulating anti-platelet antibodies in nine
cases (25.0%). However, five patients had both platelet-associated
and circulating anti-platelet antibodies, while four patients only
expressed serum circulating antibodies and two cases expressed
platelet-associated antibodies only. In alcoholic cirrhosis, only
one case was found to have platelet-associated antibodies
(2.5%) and two cases had circulating anti-platelet antibodies
(5.0%); even in HBV-related cirrhosis, only one patient showed
serum anti-platelet antibodies (6.6%), while no cases presenting
platelet-associated antibodies were found. Statistical analysis
showed that the prevalence of platelet-associated antibodies
was significantly higher in HCV+ cirrhosis compared to healthy
subjects (p <0.0064), alcoholic cirrhosis (p <0.018), and HBV+ cirrhosis (p <0.0001). Even the prevalence of circulating anti-platelet antibodies was significantly higher in HCV+ cirrhosis

compared to normal controls (p <0.0021), alcoholic cirrhosis
(p <0.0152), and HBV+ cirrhosis (p <0.006).
B-cell monoclonality
B-cell monoclonality was found in eight (27%) of the HCV-positive patients, whereas no monoclonality was found in HBV
(p <0.004) or alcoholic patients (p <0.003). The five HCV+ patients
with both serum and platelet-associated antibodies and one out
of the four patients with serum circulating antibodies showed
B-cell monoclonality, while the remaining two HCV+ cases with
B-cell monoclonality also suffered from other autoimmune diseases i.e. Coombs-positive hemolytic anemia and autoimmune
thyroiditis. In all these cases, a skewed immunoglobulin usage
was found; in fact, these cases presented a preferential usage of
the monoclonal 51p1 gene. The sequence of the variable regions
(Complementary Determinant Regions) of the immunoglobulins
showed a region of a different length with a different nucleotide
and amino acid sequence in each case.
Discussion

Table 2. Clinical and hematological profiles of the patients with ITP or aplastic
anemia.

ALT, alanine-aminotransferase. n.d., not determined. All patients were HCV, HBV,
and HIV negative.

The possibility to compare hematological and hepatological
patients allowed us to better understand the complex mechanisms of platelet turnover. Thrombocytopenia is the main hematological disorder observed in liver cirrhosis, and this condition
has been traditionally attributed to hypersplenism [24], a condition accompanied by an increase in the sequestration and an
accelerated destruction of platelets. However, improvements in
thrombocytopenia have not been achieved through the use of
portal decompression, including portosystemic shunting, or even
after splenectomy. Alternatively, the insufficient production of
thrombopoietin has also been proposed to be involved in the
pathogenesis of thrombocytopenia in patients with LC. Indeed,
thrombopoietin, the principal stimulating factor of megakaryothrombopoiesis, is produced by the liver and its production is
impaired in advanced liver failure [25–28]. The circulating level
of thrombopoietin is known to be regulated by a ‘‘sponge effect’’;
this refers to the fact that free levels are controlled through its

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Research Article
binding to the thrombopoietin receptor that is mainly expressed
on bone marrow megakaryocytes and circulating platelets, while
thrombopoietin continues to be produced at constant rate by the
liver [29]. Thus, the circulating levels of thrombopoietin depend
upon total receptor numbers (at least in subjects with normal
liver function). Indeed, it has been observed that following liver
transplantation, serum thrombopoietin concentrations return to
normal levels in conjunction with an increase in platelet count
[30–32]. However, several studies have showed that some
patients with liver cirrhosis have normal serum thrombopoietin
levels despite low platelet counts. Our data support the hypothesis of a role of low thrombopoietin levels in cirrhosis thrombocytopenia, since its serum levels were found to be below normal
levels in most cases. On the contrary, in a recent paper, Kajihara
et al. [33] did not find any significant differences in the thrombopoietin levels between patients with liver cirrhosis and controls.
These different findings could be explained by the different cutoff limit used for the platelets (below 100 109/L in our cases
and 150 109/L in the others) and by the different prevalence
of hepatocellular carcinoma (1% in our cases vs. 63% in those of
Kajihara et al.). There are also some reports indicating that
thrombopoietin receptors are expressed in fetal liver and in some
hepatocellular carcinoma (HCC) cell lines [34]; however, other
authors have observed increased platelet levels in HCC [35], thus
such a high prevalence of HCC should be avoided in physiological
studies.
Some rather old observations [36–39] exist indicating
increased levels of immunoglobulin G (IgG) bound to platelets
in patients with chronic liver disease that suggest the presence
of autoantibodies reactive to platelets. Immunoglobulin bound
to platelets is also found in idiopathic thrombocytopenic purpura
(ITP) [40,1], a typical autoimmune disease characterized by
increased platelet destruction mediated by autoantibodies
against several platelet surface antigens, including GPIIb-IIIa
(being the most common) and GPIb-IX [42,43]. ITP is characterized by accelerated platelet turnover (high glycocalicin levels)
and thus, in turn, by an increased platelet production (high reticulated platelet count). Glycocalicin is a fragment of the platelet
membrane glycoprotein Ib, and the glycocalicin index (GC index:
the plasma GC level corrected for the platelet count) is considered
to be a parameter of peripheral platelet turnover [44]. Since it
was recently found that most patients with either liver cirrhosis
(99% of cases) or ITP (94% of cases) present circulating anti-GPIIbIIIa antibody-producing B cells [45], the immune-mediated
destruction of platelets could be considered as the main factor
determining thrombocytopenia in cirrhosis. Accordingly, we also
found that mean GC levels and GC indices were abnormally high
in patients with liver cirrhosis, suggesting an accelerated platelet
turnover, though not at the same levels as in ITP. In patients subgrouped according to the etiology of the cirrhosis, the glycocalicin
index was significantly higher in the patients affected by HCV
compared to patients with other aetiologies. In line with these
data, the prevalence of anti-platelet antibodies was significantly
higher in HCV+ cirrhosis than in HBV+ or alcoholic cirrhosis. This
difference is not surprising as it has been known for many years
that chronic HCV infection determines several immunological
(thyroid dysfunctions, Sjogren disease, and lichen ruber planus)
[46,47] and hematological disorders (monoclonal gammopathies
of undetermined significance, non-Hodgkin’s lymphoma, Waldenström macroglobulinemia) [48–50]; although the strongest
association has been found with mixed cryoglobulinemia

898

[51,52]. The interaction of HCV envelope proteins and CD81, a
tetraspanin mainly expressed on B-lymphocytes, seems to be
the major mechanism of naïve B-cell activation [53]. Note, the
absence of any difference between HCV-positive and negative
cases in auto-antibody production in Japanese patients can be
explained by the ethnical difference in the immunological
response. In fact, while HCV infection determines B-cell monoclonality in about 25% of chronically infected Caucasian patients,
this does not occur in Japanese subjects [54]. Though the presence of detectable anti-platelet antibodies does not necessarily
imply an accelerated platelet destruction, in this series of cases
the subjects expressing anti-platelet antibodies (16 cases: seven
with detectable platelet-associated antibodies and nine cases
with serum circulating anti-platelet antibodies) showed a very
high GC index (2.51 ± 1.26, p <0.06 vs. HCV+ cirrhosis without
anti-platelet antibodies) and a very high level of reticulated
platelets (11,962 ± 10,258/mmc, p <0.05 vs. HCV+ cirrhosis without anti-platelet antibodies). On the basis of these results, as previously indicated in the ‘‘Results’’ section, in the HCV+ cirrhosis,
two groups can be identified: in the first one (75% of the cases)
the platelet production and the platelet turnover overlaps HBV+
and alcoholic cirrhosis, while the second one (25% of the cases)
is characterized by high reticulated platelet, high GC index, high
incidence of anti-platelet antibodies, and high incidence of B-cell
monoclonality. Given the comparable severity of liver disease, the
serum thrombopoietin levels were low in both groups
(31.1 ± 16.7 vs. 30.5 ± 23.8 pg/ml; p: NS), this determines a platelet count significantly (p <0.02) lower in the cases with high
platelet turnover (53,889 ± 11.241 109/L) than in the cases with
standard platelet turnover (72,631 ± 19,131 109/L).
A quantitative analysis of megakaryocyte concentrations in
bone marrow was not performed in this study. The identification
of megakaryocytic cells based on morphological studies at the
light microscopy is difficult and results are therefore often unreliable. In fact, while the identification of the mature megakaryocytes at different stages of development (given their large size,
complex nuclear appearance, and typical cytoplasmic staining)
is easy, even using light microscopy, the identification of immature megakaryocytic cells, which are small and do not have the
classical and obvious features of megakaryocytes, generally
requires ultrastructural studies and the use of electron microscopy for quantification. Alternatively, dual-color immunofluorescence staining and flow cytometry seems to be a reliable method
to quantify bone marrow megakaryocytes at any stage of differentiation [55]. On the basis of these considerations, megakaryocyte quantification is even more difficult when the platelet
turnover is elevated and, as a consequence, the immature fraction
of small megakaryocytes is likely to be larger than in normal subjects. Therefore, the analysis of marrow megakaryocytes by the
conventional light microscopy method is not reliable and the
comparison of marrow megakaryocyte density in liver cirrhosis
to that in ITP is unreliable.
In conclusion, patients with liver cirrhosis presented a normal
or decreased plasma TPO, an accelerated platelet turnover (on
the basis of a high glycocalicin index), and low or normal platelet
production (on the basis of the absolute reticulated platelet count).
Taken together, these findings indicate that cirrhotic thrombocytopenia is a multifactorial condition, involving both increased
platelet clearance in the periphery and impaired thrombopoiesis.
This is similar to what may happen in ITP since recent studies have
shown that anti-GPIIb/IIIa autoantibodies were able to suppress

Journal of Hepatology 2011 vol. 54 j 894–900

JOURNAL OF HEPATOLOGY
the production and maturation of megakaryocytes ‘‘in vitro’’, suggesting that the thrombocytopenia in ITP may not only result from
Fcc receptor-mediated platelet clearance but also from the suppression of megakaryogenesis [56,57]. This could explain the
unexceptional finding of low levels of reticulated platelets and normal glycocalicin indices in ‘‘classical’’ ITP subjects. In these cases,
the growth factor-mediated stimulation of megakaryopoiesis
might be expected to increase the platelet count. Indeed, initial
clinical trials of the investigational thrombopoietic agent
Eltrombopag (SB-497115, GlaxoSmithKline) a small-molecule,
non-peptide that acts as a thrombopoietin-receptor agonist inducing proliferation and differentiation of megakaryocytes, were
found to show promising responses in adults with chronic ITP
refractory to other treatments [58,59]. In liver cirrhosis, this agent
was found to increase the platelet count in HCV-related cirrhosis
and to facilitate antiviral treatment with fairly good results [60].
This treatment might also be effective in advanced liver failure
exhibiting thrombocytopenia and bleeding.
Finally, at least in Caucasians, a fraction of HCV-related liver
cirrhosis is characterized by an increased prevalence of autoimmune phenomena, including detectable levels of anti-platelet
antibodies [61] and this should be taken into account during
the follow-up of these patients.

Conflict of interest
The authors who have taken part in this study declared that they
do not have anything to disclose regarding funding or conflict of
interest with respect to this manuscript.
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