Review article: blood platelet number and function in chronic

liver disease and cirrhosis

P. WI TTERS* , , K. FRESON, C. VERSLYPE* , , K. PEERLI NCK, , M. HOYLAERTS, F. NEVENS* , ,
C. VAN GEET, & D. CASSI MAN* ,
*Laboratory of Hepatology; Depart-
ment of Pediatrics, University Hospital
Gasthuisberg; Center for Molecular
and Vascular Biology; Department of
Hepatology, University Hospital
Gasthuisberg; Department of
Cardiology, University Hospital
Gasthuisberg, University of Leuven,
Leuven, Belgium
Correspondence to:
Dr P. Witters, Afd. Hepatologie, O.&
N. 1, Herestraat 49-bus 703, Leuven
BE-3000, Belgium.
E-mail: Peter.witters@gmail.com
Publication data
Submitted 23 January 2008
First decision 18 February 2008
Resubmitted 28 February 2008
Accepted 29 February 2008
Epub OnlineAccepted 5 March 2008
SUMMARY
Background
The liver plays a central role in coagulation and brinolysis but is also
closely intertwined with the function and number of blood platelets.
Aim
To describe and integrate all literature concerning blood platelets and
liver disease by performing a thorough literature research.
Methods
A thorough literature research on blood platelets and liver disease
was performed.
Results
Thrombocytopenia is a marked feature of chronic liver disease and cir-
rhosis. Traditionally, this thrombocytopenia was attributed to passive
platelet sequestration in the spleen. More recent insights suggest an
increased platelet breakdown and to a lesser extent decreased platelet
production plays a more important role. Besides the reduction in num-
ber, other studies suggest functional platelet defects. This platelet dys-
function is probably both intrinsic to the platelets and secondary to
soluble plasma factors. It reects not only a decrease in aggregability,
but also an activation of the intrinsic inhibitory pathways. The net
effect, nally, is a decreased platelet function in the various types of
chronic liver diseases and cirrhosis. Finally, recent data suggest that
platelets are not only affected by but can also contribute to the liver
disease process, as for instance, in viral hepatitis and cholestatic liver
disease.
Conclusion
Platelet research in liver disease is a growing area of investigation and
could provide new pathophysiological insights.
Aliment Pharmacol Ther 27, 10171029
Alimentary Pharmacology & Therapeutics
2008 The Authors 1017
Journal compilation 2008 Blackwell Publishing Ltd
doi:10.1111/j.1365-2036.2008.03674.x
I NTRODUCTI ON
Haemostasis and hepatology are closely related. It is
well-known that patients with cirrhosis show a marked
decrease in liver synthesis of coagulation factors,
which leads to a prolongation of the prothrombin
time.
1
Less well studied, but equally important in the
physiological process of clot formation, are the defects
in the primary haemostasis in patients with liver dis-
ease. Blood platelets initiate the haemostatic process
by interacting with the damaged vessel wall.
2
These
form a haemostatic plug within seconds after injury.
The secondary haemostasis (coagulation) starts simul-
taneously, but enrolls slower and strengthens this plug
by crosslinked-brin within minutes.
2, 3
Cirrhosis is the nal common end-point of many
chronic liver diseases and thrombocytopenia is a well-
known feature of cirrhosis. However, in addition to the
quantitative changes in platelets, there are qualitative
defects. These can be assessed in specialized haemo-
static laboratories. The most primitive but physiologi-
cally meaningful platelet function test is the in vivo
bleeding time assessment.
4
More sensitive and repro-
ducible tests have been developed, for instance, assays
that try to simulate the in vivo platelet function in vitro,
under ow conditions, to study shear-induced platelet
activation. These tests are technically demanding, but
reect aggregation in maximally physiological condi-
tions.
5
Less physiological, but more widely used and
currently the gold standard for platelet function
assessment is the aggregometry.
4, 6
This in vitro assay
measures platelet aggregation in response to different
sets of agonists [collagen, thrombin, adenosine diphos-
phate (ADP), etc.]. Aggregometry is performed with
platelet rich plasma (obtained by slight centrifugation
of anticoagulated blood) where optical light turbidity
is a measure of aggregation.
7
It can also be performed
with whole blood by electrical impedance measure-
ment.
4
Another sensitive assay is the quantication of
membrane molecule expression by ow cytometry
[uorescent-activated cell sorting (FACS) analysis]. By
assessing the presence of stimulation-dependent anti-
gens [e.g. CD62P (P-selectin)] or platelet-leucocyte
complexes, the degree of platelet activation can be
estimated.
4, 7
Even more specialized, on a molecular
level, the concentrations of second messengers [e.g.
calcium, cyclic adenosine monophosphate (cAMP)] and
the release of platelet granules containing pro-
aggregatory molecules [serotonin, adenosine triphos-
phate (ATP), platelet factor 4, beta-thromboglobulin,
etc.] can be assessed.
4
Finally, because of the technical
difculties with platelet function tests, several tests (as
are thromboelastography and sonoclot) have been
developed for everyday clinical use. These tests require
little or no sample preparation and give instant bed-
side results to assist in clinical decision making.
7
In
thromboelastography, for example, blood clots in a
backward and forward rotating sample cup around a
suspended pin. Because of haemostasis, the cups
motions become limited. These movements are mapped
and different variables (depending on coagulation,
brinolysis and platelet function) can be calculated.
6, 7
Although spontaneous bleeding caused by low
platelet counts in cirrhosis is an infrequent event, low
platelet counts become clinically relevant when per-
forming liver biopsies, liver transplantation or giving
myelosuppressive agents (antiviral treatment like inter-
feron or cytostatics).
8
Less well studied is the recent
observation that platelets can contribute to the pro-
gression of liver disease (e.g. in viral hepatitis and
cholestatic liver disease).
9, 10
Finally, platelet derived
serotonin has convincingly proven to be involved in
the initiation of liver regeneration.
11
The aim of this review was to give a comprehensive
overview of the literature covering both quantitative
(counts) and qualitative (function) aspects of blood
platelets in cirrhosis and chronic liver disease. Most of
this review handles cirrhotic liver disease on the
whole, as the literature contains few data on the inu-
ence of different aetiology or degree of liver brosis on
platelet (dys)function. Nevertheless, when available,
data are discussed with regard to the different types of
cirrhosis separately. We will mainly distinguish between
viral liver disease (hepatitis B or C), alcoholic liver
disease, biliary cirrhosis [primary biliary cirrhosis (PBC),
primary sclerosing cholangitis (PSC)] and vascular liver
disease (e.g. extrahepatic portal vein obstruction, non-
cirrhotic portal hypertension, Budd-Chiari syndrome).
PLATELET NUMBER I N CI RRHOSI S AND
CHRONI C LI VER DI SEASE
Theoretically, lowered platelet counts can be the result
of decreased platelet production, enhanced splenic
sequestration or platelet consumption use. Kinetic
studies with radiolabelled platelets in cirrhosis and
chronic liver disease indicate that there is a decreased
platelet survival.
1215
Depending on the ability of the
bone marrow to increase platelet production, platelet
counts can be reduced
12
or normal
14
depending on the
1018 P. WI TTERS et al.
2008 The Authors, Aliment Pharmacol Ther 27, 10171029
Journal compilation 2008 Blackwell Publishing Ltd
ability of the bone marrow to increase the platelet pro-
duction. These ndings are in accordance with the
decreased,
16, 17
normal
18
or increased
19
percentage of
reticulated platelets (young platelets) in cirrhosis. The
main site of platelet consumption is in the
spleen.
12, 13, 15
Here, we review the evidence for the three different
mechanisms leading to thrombocytopenia.
Platelet production
Thrombopoetin (TPO) is the most important growth
factor in the regulation of megakaryocyte development
and platelet production. However, its serum value is
not a reliable indicator of platelet production because
upon binding to its receptor on thrombocytes and
megakaryocytes, it is internalized and cleared from the
circulation.
8, 20
As TPO is mainly produced in the liver
and kidney at a constant rate, this clearance from the
circulation by platelets is the most important mecha-
nism regulating the concentration in the physiological
state.
21
So, serum TPO (net effect from production and
clearance) should always be interpreted with total
platelet count, including immeasurable intrasplenic
platelets,
22
to estimate platelet production.
Even 13 years after its original description, the
function of TPO in liver disease is not clear. As the
liver is the major site of TPO production
20, 21
it is
reasonable to expect a decreased plasma level and
indeed TPO mRNA levels in the liver were slightly
decreased in cirrhosis.
23
However, normal,
18, 19, 24, 25
decreased
16, 17, 26, 27
and increased
28
serum TPO levels
have also been published. These conicting results can
be explained by differences in laboratory techniques:
the use of different antibodies (more sensitive poly-
clonal vs. monoclonal antibodies) and the lack of an
international standard preparation of TPO.
20
TPO levels
have been related to Child-Pugh stage and albumin
levels in some studies,
26
but not in others,
19, 22
and to
portal haemodynamics: a decreased TPO level was
found in patients with hepatofugal portal blood ow.
25
Liver transplantation is known to resolve thrombocy-
topenia sequentially to an increase in serum TPO and
increased platelet production.
18, 22, 23
Other thrombo-
poietic cytokines (interleukin 3, interleukin 6 and
interleukin 11) do not play a major role,
27
although
therapy with recombinant human interleukin 11
increases platelet counts in cirrhotic patients.
29
Beside a decreased TPO production, there is also an
increased TPO breakdown in cirrhosis. Partial splenic
embolization or splenectomy results in increased TPO
and platelet count (presumably through diminished
platelet mediated TPO breakdown) and restores the
physiological relation between platelet count and
TPO.
22, 23, 30
The exact role of impairment of thrombopoiesis in
liver disease remains to be determined. Bone marrow
megakaryocyte density in cirrhosis was similar to the
density in idiopathic thrombocytopenic purpura (ITP)
and markedly elevated compared to aplastic anemia,
suggesting that a primary production decit is not the
only cause of thrombocytopenia.
19
Platelet sequestration (distribution)
As splenomegaly is an important feature of portal
hypertension, it is not surprising that splenic platelet
sequestration was thought to be an important mecha-
nism in the aetiology of thrombocytopenia. This the-
ory was rst suggested in 1965 by Aster.
31
Although
this theory was never proven, it was generally
accepted because of lack of other explanations.
32
Kinetic radiolabelled platelet studies do suggest splenic
pooling, but they also report shorter platelet survival
time, indicative of splenic destruction rather than mere
splenic pooling.
1214
The term congestive splenomegaly is a mechanical
oversimplication and there is no linear correlation
between spleen size and portal pressure.
33
Further-
more, decompression of the portal venous system by
transjugular portosystemic shunts could not consis-
tently demonstrate a benet in terms of platelet count
(no benet;
34, 35
some benet
3638
). In the studies that
demonstrated a benet, there was no normalization
36
38
and no correlation with achieved portal pressure or
percentage of pressure reduction.
36, 37
Most reports
also conclude that portocaval and distal splenorenal
shunts do not improve platelet counts in this set-
ting.
15, 39, 40
This argues against simple splenic pooling
caused by increased portal pressure but does not
exclude the spleen from contributing to platelet break-
down. As already stated, partial splenic embolization
can increase platelet count.
22, 23, 41
Platelet breakdown
Kinetic studies indicate that platelet breakdown, illus-
trated by decreased platelet survival time, is also an
important mechanism.
1215
There is a remarkable
homology between ITP and thrombocytopenia in
REVI EW: PLATELETS I N CHRONI C LI VER DI SEASE 1019
2008 The Authors, Aliment Pharmacol Ther 27, 10171029
Journal compilation 2008 Blackwell Publishing Ltd
cirrhosis. The reticulated platelet proportion (propor-
tion of young platelets) and glycocalicin index (a mar-
ker of platelet production) were signicantly higher in
both diseases compared with healthy controls.
19, 42
As in ITP, it has been proposed that autoantibody-
mediated platelet destruction plays a major role. In
chronic liver disease, these platelet-associated IgGs are
markedly elevated
24, 4346
as well as the number of B
cells producing antibodies against the major autoanti-
gen of platelets, GPIIb IIIa.
43
The antibody titre corre-
lates inversely with the number of platelets
24, 43, 44
and
with spleen volume.
12, 24
Furthermore, splenic emboli-
zation increased platelet survival time whereas the
platelet-associated IgG decreased.
45
However, it is
widely recognized that the specicity of platelet associ-
ated IgGs for autoantibody-mediated thrombocytope-
nia is quite low, so it is considered an inappropriate test
for the diagnosis of ITP.
43
Although not undisputedly
proven, it seems an attractive hypothesis. Moreover as
in ITP, the antibodies could not only mediate FCc-med-
iated clearance in the reticuloendothelial system, but
also to some extent suppress megakaryopoiesis.
19
Others found an increased brinogen and plasmino-
gen turnover suggesting platelet consumption.
14
Also,
increased brinogen degradation products and to a
lesser extent, increased D-dimers have been reported,
however, without a clear relation to platelet numbers,
brinogen levels or prothrombin activity.
47
Finally,
soluble P-selectin levels, a marker of in vivo platelet
activation, are increased in chronic liver disease
48
(cfr.
infra: platelet exhaustion).
Overall, it is not yet clear what the exact mecha-
nisms leading to thrombocytopenia in cirrhosis are. It
is most likely a multifactorial process combining
increased splenic platelet breakdown, splenic pooling
and the inability of the bone marrow to increase plate-
let production adequately.
Platelet count in different types of cirrhosis and
brotic liver disease
Most authors agree on a decrease in platelet count in
relation to the severity of cirrhosis.
4951
There are only
few reports that relate the platelet number to the dif-
ferent aetiologies of cirrhosis.
However, in PBC, an immune-mediated disorder,
platelet-associated IgGs seem to play an important role
in the thrombocytopenia, with recovery after cortico-
steroid administration.
52
In one patient with PBC, the
antibodies eluted from the platelets bound to GPII-
b IIIa and a 70 kDa mitochondrial antigen. Analysis of
the peptide sequences showed partial amino acid
sequence homology suggesting the possibility of a
common antibody binding site.
53
Most likely the thrombocytopenia in viral hepatitis
also represents a multifactorial process with altered
Figure 1. Platelets in normal (a) and cirrhotic (b) condition. (a) In normal platelets, stimulation by collagen, thrombin, ADP
and TXA B2 leads to activation of the PLC (in green, left side of the gure). This results in the formation of DAG and IP3.
DAG leads to activation of PLA2 and the production and release of TXA B2. This reinforces the PLC activity and stimulates
additional platelets. IP3 results in calcium release from the dense tubular system (stored through the SERCA pump). The
increase in cytosolic calcium (also aided by the ADP P2X receptor and the H Ca-Na-antiporter) moderates the platelet acti-
vation: the shape change (by actin); the release of stored pro-aggregatory molecules (ATP and 5HT from the dense bodies
and BTG, PF4 and P selectin from the alfa granules) recruiting other platelets and the conformational change of the GPII-
b IIIa receptor (together with the phosphorylation by the PKC) allowing interactions with brin reinforcing the haemostatic
plug. These mechanisms are under tight homeostatic control of the inhibitory pathways (in red, right side of the gure):
cAMP (produced by AC: stimulated by adenosine, PGI2 and PGE2 and inhibited by ADP via the P2Y2 receptor) and cGMP
(produced by the NO-stimulated GC). (b) In cirrhosis, platelet activation is decreased (in green, left side of the gure). There
is decreased PLC and PLA2 activity leading to decreased TXA B2 production and decreased IP3 mediated cytosolic calcium
increase. There is also less calcium transport by the H Ca-Na-antiporter because of diminished intraplatelet acidication.
The secondarily impaired release of the dense bodies and the alpha granules is less effective because of the storage pool
defect: less ATP and 5HT; BTG, PF4 and P-selectin-levels in the granules respectively. The inhibitory pathways are also up-
regulated (in red, right side of the gure). This results in more cAMP- and cGMP-mediated inhibition. Finally, there are also
negative plasma factors decreasing platelet activation as are the FDP and ApoE (See text for details). 5HT, serotonin; AC,
adenylate cyclase; ApoE, Apolipoprotein E; ATP, adenosine-triphosphate; BTG, b-thromboglobulin; Ca, calcium; cAMP,
cyclic adenosine monophosphate; cGMP, cyclic guanosine monophosphate; DAG, diacylglycerol; FDP, brin(ogen) degrada-
tion products; GPIIb IIIa, glycoprotein IIb IIIa; GTP, Guanosine triphosphate; H, hydrogen; IP3, inositol-triphosphate; Na,
sodium; NO, nitric oxide; P2Y1, P2X1, P2TY2, ADP receptors; PC, phosphatidylcholine; PF4, platelet factor 4; PGE2, prosta-
glandin E2; PGI2, prostacyclin; PIP2, phosphatidylinositol-bisphosphate; PKC, protein kinase C; PLA2, phospholipase A2;
PLC, phospholipase C; SERCA, sarco endoplasmic reticulum Ca-ATPase; TXA2, thromboxane A2; TXB2, thomboxane B2.
1020 P. WI TTERS et al.
2008 The Authors, Aliment Pharmacol Ther 27, 10171029
Journal compilation 2008 Blackwell Publishing Ltd
production of TPO with increasing hepatic brosis
54
and splenomegaly. As in other chronic liver disease, in
viral hepatitis, TPO increase has been documented
(compared to other causes of chronic liver disease
51
and to controls
55
). However, there is no (inverse) cor-
relation between TPO level and platelet count
55
and
RT-PCR and immunohistochemistry showed equal
hepatic expression as that of other liver diseases.
55
There is an inverse correlation between platelet count
and spleen size, suggesting platelet breakdown or
sequestration.
54, 55
However, other studies suggest that
platelet autoantibodies are common in individuals
with HCV infection and that their detection does not
assist in the diagnosis of immune thrombocytopenia.
50
Thrombocytopenia can also arise independent of
hypersplenism.
56
So, the exact mechanisms are as yet
not clear.
PLATELET FUNCTI ON
Platelet function in cirrhosis
The existence of a functional platelet defect (hypoag-
gregability) was described for the rst time by Thomas
et al.
57
Bleeding time, the paradigmal platelet function
test has been reported to be abnormal in 2.542% of
patients with cirrhosis.
5860
This correlates only weakly
with the platelet count, suggestive of an additional
functional decit. This bleeding time prolongation is
closely related to the degree of liver failure.
60
(a)
(b)
REVI EW: PLATELETS I N CHRONI C LI VER DI SEASE 1021
2008 The Authors, Aliment Pharmacol Ther 27, 10171029
Journal compilation 2008 Blackwell Publishing Ltd
Only few studies on platelets in ow conditions (the
most physiologic test in vitro) have been per-
formed.
5, 6164
These report conicting results
(decreased
5, 64
and normal
6163
platelet function) pos-
sibly because of differences in experimental setup as
varying shear rates from 600 s,
64
800 s,
5, 63
1600 s
62
to 2600 s
61
and differences in the adhesive surfaces
that were perfused. Moreover, because these studies
were performed in whole blood or reconstituted blood
(with adjusted hematocrit and platelet count), factors
other than platelet function could have played an
important role. Indeed, plasma factors and haemorhe-
ological factors dependent on the hematocrit are
important in these experiments.
62
In humans and in animal models of chronic liver dis-
ease and cirrhosis, there is a clear hypoaggregability, as
demonstrated with in vitro aggregation tests (the gold
standard)
5, 61, 6574
and with owcytometric analysis of
stimulation dependent antigens (e.g. P-selectin).
70
A
decreased response to collagen,
61, 6567, 71, 73, 75
throm-
bin,
6669
arachidonic acid,
5, 61, 67, 70, 73
ADP,
5, 61,
65, 70, 72
U46619 (an ADP-mimetic),
5, 61, 75
epineph-
rine
5, 61, 70
and ristocetin
5, 61, 76
have been reported.
These ndings are also conrmed by thromboelastog-
raphy.
77, 78
Crossover experiments (using platelet
aggregometry) with patient or control platelets in
control or patient plasma hold both an intrinsic plate-
let defect and a circulating plasma factor responsible
for this hypofunction.
72, 74
The molecular mechanisms underlying this intrin-
sic platelet defect leading to hypoaggregability have
been studied extensively. The most important defects
are depicted in Figure 1: the normal platelet (Figure 1a)
as compared to the cirrhotic platelet (Figure 1b).
There is evidence supporting a reduced transmem-
brane signalling in cirrhotic platelets after stimulation
with thrombin or collagen.
66, 67
This leads to a
decreased activation of phopholipase C, A2 and cyclo-
oxygenase thromboxane synthetase
66, 67
resulting in
decreased thromboxane production.
79
There is also
evidence for a decreased arachidonic acid availability
for prostaglandin and thromboxane production.
71, 79
Moreover, dietary supplementation with arachidonic
acid increases the membrane levels of this fatty acid
and improves (collagen-induced) platelet aggregation,
returning to pre-treatment levels 4 weeks after with-
drawal.
71
The IP3 production in response to thrombin stimula-
tion is signicantly lower
69
in platelets from patients
with cirrhosis. There is a reduced cytosolic calcium
increase, also because of a decreased release from
intracellular stores.
69
As intracellular calcium is the
nal common pathway in platelet aggregation, this
leads to platelet hypofunction. There is also a
decreased cytosolic alkalinization (Na
+
H
+
antiporter
activity), which normally facilitates calcium entry
from the extracellular environment.
69
Furthermore, there is evidence for a storage pool
defect in cirrhosis, decreasing the effect of granule
release on the aggregation. There is decreased ATP
and serotonin (5HT) in the dense bodies and decreased
PF4 (platelet factor 4), b-thromboglobulin (BTG) and
P-selectin in the alpha granules.
65, 68, 70, 80
These gran-
ules have a normal ultrastructure and normal relative
volume.
68
Plasma levels of BTG and PF4 are reported
to be elevated in relation to platelet count,
68
although
not all authors agree.
80
Finally, there is an upregulation of inhibitory path-
ways. Basal cyclic adenosine monophosphate and cyclic
guanosine monophosphate, the two main inhibitory
messengers, are upregulated in the cirrhotic platelet.
69
There was a normal upregulation upon stimulation
indicating an increased stimulation of circulating
factors in vivo like PGI2
8183
and nitric oxide (NO)
84
for
cAMP and cGMP, respectively.
69
There is one study
suggesting a locoregional difference in platelet aggre-
gation, i.e. a difference in platelet aggregation between
portal and, simultaneously drawn, systemic blood.
Splanchnic vasodilatation and enhanced generation of
PGI2 shift the collagen (but not the ADP) aggregation
curve signicantly to the right in portal blood. There
were no differences in coagulation and brinolytic
parameters.
82
As possible underlying mechanism platelet exhaus-
tion was proposed, as platelets are faced with the por-
tal hyperdynamic circulation. Platelets could be
damaged during intravascular activation (with loss of
granules), which could give rise to their subsequent
hypo-function when tested in vitro.
65
This hypothesis
is somewhat challenged by the normal microscopy of
the platelets,
68
the normal concentration of thrombin-
antithrombin complexes, D-dimers and F1+2 [brin
(ogen) degradation products] values and the absence
of stimulation-dependent antigens on the platelet
membrane.
70
The increased urinary excretion of
thromboxane A2 (TXA2) metabolites (2,3 dinor-throm-
boxane B2 (TXB2) and 11dehydro-TXB2) could be
explained by intrasplenic destruction.
70
Currently, defective transmembrane signalling (and
secondarily a decrease of the intracellular messengers)
1022 P. WI TTERS et al.
2008 The Authors, Aliment Pharmacol Ther 27, 10171029
Journal compilation 2008 Blackwell Publishing Ltd
is thought to be the most important factor in the
platelet hypo-function.
67, 70
Besides this intrinsic platelet defect numerous
plasma factors appear to play a role. Numerous nega-
tive factors are known.
High-density lipoprotein (HDL) apolipoprotein E
contents are increased in cirrhosis and this was corre-
lated with the inhibition of platelet aggregation.
85
There are also brin(ogen) degradation products,
which appear in excess in liver disease,
47
are known
to adsorb to the platelet surface and interfere with
platelet function.
86
However, their levels may not be
well correlated with reduced platelet aggregation.
87
Bile salts can also exert a negative effect. In vitro
aggregation studies show an inhibitory effect of bile
salts on platelet aggregation and serotonin release
induced by ADP or collagen.
88
Only chenodeoxycholic
acid is an aggregation-inducing bile salt.
88
In vivo
studies in rat models associated with elevated bile
acids (bile duct ligation and cholic acid feeding) show
a clear inhibitory effect on ADP-epinephrine induced
aggregation.
89
Finally, as already stated, primary and secondary
haemostasis are closely related. As defects in the coag-
ulation are well-studied (for review see
2, 90
) it is clear
that they independently contribute to the haemostatic
abnormalities in patients with liver disease. However,
their impairment may also affect platelet function. For
example, platelets play an important role in thrombin
formation and thrombin is a platelet agonist.
1
What
effect activated recombinant factor VII (rFVIIa; Novo-
seven, Novo Nordisk A S, Copenhagen, Denmark) has
on platelets or on thrombin formation remains to be
demonstrated.
1
There are also positive factors that promote platelet
activation e.g. vWF that is reported to be upregulated
in cirrhotic patients.
62
Despite the reduced number of
(more active) high molecular weight vWF multi-
mers,
42, 62
the reduced collagen binding capacity and
relative ristocetin activity (all indicative of loss of
function), it is likely that the quantitative effect
exceeds the qualitative effect.
62
Glycoprotein Ib (the
vWF receptor) has been reported to be increased
47
or
decreased
76
in cirrhosis. As already stated, these
plasma factors can contribute to the apparent discrep-
ancy between the studies under ow conditions and
the in vitro aggregation tests.
Unconjugated bilirubin is a strong inducer of plate-
let aggregation similar to ADP in isolated platelets.
This could be completely inhibited by prostaglandin I
2
or E
2
.
91
However, the physiological relevance of this
observation has to be questioned as the effect was
entirely inhibited by the addition of 0.1% bovine
serum albumin, probably because of the high afnity
of albumin to unconjugated bilirubin.
91
Tenfold concentrated ascitic uid caused irreversible
platelet aggregation and serotonin release of normal
platelet-rich plasma similar to collagen and could be
abolished by adding collagenase.
92
The coagulopathy
after peritoneovenous shunting could be the result of
direct and rapid intravenous infusion of procoagulant
collagen.
92
Overall, there is a clear platelet dysfunction. As for
the intrinsic platelet defect, several steps of platelet
activation are shown to be impaired. Underlying mech-
anisms remain speculative. Furthermore, the blood
platelets from patients with liver disease encounter
several agonists and antagonists. This could lead to
hyper- and hypoaggregability; it is, however, not clear
what the contribution of these isolated ndings is in the
complex entity of chronic liver disease and cirrhosis.
Platelet function in different types of liver
disease
As with platelet number, also platelet function seems
dependent on the degree of liver brosis.
5, 67, 68
Until now, there has been little attention on the dif-
ferent liver diseases in relation to platelet abnormalities.
Most reports are on cirrhotic platelets. Here, we review
the reports on the different liver disease entities.
In cholestatic liver disease, there is some evidence
that contrary to other types of cirrhosis, the platelets
demonstrate a hyperaggregability. Patients with
PBC PSC have a better survival after variceal bleed-
ing
93
and demonstrate less blood loss during liver
transplantation,
94, 95
although this can also be
explained by a better synthetic liver function at the
time of transplantation. Thrombosis of portal veins has
also been detected in 40% of PBC liver at the time of
transplantation.
96
There have been some platelet func-
tion studies in these patients (see Table 1) that are
indicative of hyperaggregability. This has been docu-
mented by the use of thromboelastography,
97, 98
by
sonoclot analysis
99
(two newer and less well studied
platelet tests that try to evaluate the complete haemo-
static system) and PFA-100 analysis.
98, 99
In the latter,
there is a lack of prolongation of closure time
98, 99
as
is normally seen in cirrhosis.
61, 98
On a molecular
level, there is an increased expression of CD42b (an
REVI EW: PLATELETS I N CHRONI C LI VER DI SEASE 1023
2008 The Authors, Aliment Pharmacol Ther 27, 10171029
Journal compilation 2008 Blackwell Publishing Ltd
Table 1. Platelet function in different liver diseases
References Disease (n) Authors ndings conclusions
Functional platelet studies in cholestatic liver disease
Ingelberg et al.
65
PBC (10), Alcoholic
cirrhosis (10)
In PBC and alcoholic cirrhosis, there is a decreased aggregation
to collagen and ADP
In PBC, there is a smaller release of ATP and total releasable
ATP is diminished
Platelets are probably damaged during an intravascular activation
(loss of granules)
Ben-Aril et al.
97
PBC (47), PSC (21),
non-cholestatic
cirrhosis (40),
healthy controls(40)
Hypercoagulability on thromboelastography in 28% (PBC), 43%
(PSC), 5% (non-cholestatic cirrhosis), 0% (healthy)
No correlation with platelet count or brinogen level
Not explained by protein C, S or ATIII
Pihush et al.
98
PBC PSC (37), Hepatis
C alcoholic
cirrhosis (53),
healthy controls (62)
Hypercoagulability on thromboelastography in PBC PSC
associated with higher brinogen levels
Normal PFA-100 closure time in contrast to prolonged closure
time in hepatitis C alcoholic cirrhosis
Increased expression of LIBS-1 and CD42b in PBC PSC
Biagini et al.
99
PBC (51), healthy
controls (102)
Sonoclot rate signicantly higher compared with controls
Sonoclot rate associated with increased TAT, TF and correlates
with hyperhomocysteinemia (presumably due to vitamin
deciencies and homozygous TT677 MTHFR genotype)
Functional platelet studies in alcoholic liver disease
Torres Duart et al.
102
Healthy control (9) Decreased aggregation in whole blood (to collagen) and in platelet rich
plasma (to ADP, arachidonate and collagen) by the addition of alcohol
Barrison et al.
103
Alcoholic cirrhosis (14),
healthy controls
Impaired platelet aggregation in 12 14 cirrhotics
Higher malondialdehyde production correlated to the higher
cholesterol:triglyceride ratio in cirrhotics
Hillbom et al.
104
Alcoholics (26, 13 with
fatty liver), healthy (13)
Decreased platelet aggregation in response to ADP
Prolonged bleeding time in alcoholics with fatty liver disease
Hilbom et al.
105
Active alcoholics
(14, 7 with fatty
liver)
On admission: prolonged bleeding time, decreased platelet count,
aggregability to arachidonate and decreased TXB2 formation
914 days after ethanol withdrawal: normalization of
bleeding time, increased TXB2 formation compared to
controls after stimulation with ADP
Watanabe et al.
106
Alcoholic liver disease Decreased aggregation in response to ADP, normal to collagen
possibly because of changes in fatty acid compositions of the
platelet membrane
Ouwendijk et al.
107
Alcoholic cirrhosis (16) In 12 patients frequently increased serum TXB2 levels
Functional platelet studies in viral liver disease
Panasiuk et al.
49
Chronic hepatitis (29),
viral cirrhosis (27),
healthy controls (32)
Higher serum concentrations of PF4 (sevenfold) and BTG (twofold)
Higher P-selectin expression on resting platelets and normal (viral
hepatitis) or higher (viral cirrhosis) after stimulation with
thrombin compared with healthy controls
Ferroni et al.
108
Chronic hepatitis C (39),
healthy controls
Higher soluble P-selectin in hepatitis C with higher levels in
patients with lower platelet counts
Correlation of soluble P-selectin with serum hepatitis C-RNA and
lack of correlation with platelet associated immunoglobulins
ADP, adenosine diphosphate; ATP, adenosine triphosphate; BTG, betathromboglobulin; LIBS-1, ligand-induced binding site;
PBC, primary biliary cirrhosis; PF4, platelet factor 4; PSC, primary sclerosing cholangitis; TAT, thrombin-antithrombin com-
plexes; TF, tissue factor; TXB2, thromboxane B2; MTHFR, 5,10-methylenetetrahydrofolate reductase.
See text for more details.
1024 P. WI TTERS et al.
2008 The Authors, Aliment Pharmacol Ther 27, 10171029
Journal compilation 2008 Blackwell Publishing Ltd
antigen of the gpIb V IX complex which binds to
vWF) and of ligand-induced binding sites both in
basal conditions and after stimulation with ADP or
epinephrine.
98
This suggests that platelets are preacti-
vated in PBC PSC. This is consistent with the higher
levels of thrombin-antithrombin complexes and
increased levels of homocysteinemia and tissue factor
as signs of endothelial activation.
99
In a well-studied
animal model of cholestasis (bile-duct ligated rats)
there is a rapid rise in the stimulated platelet cytosolic
calcium, the nal common pathway in platelet activa-
tion, to a greater amplitude compared with control
animals.
100
This is because of a greater release from
intracellular stores, which store more calcium as a
result of increased sacroplasmatic endoplasmatic-retic-
ulum calcium-ATPase activity.
100
Possible explana-
tions for this hyperaggregability in PBC PSC are the
marked systemic inammatory activity and the signi-
cantly higher levels of unconjugated bilirubin, which
might be pro-aggregable (cfr. supra).
98
Not all reports are unequivocal on this hyperaggrega-
bility, as this could not be demonstrated by platelet
aggregometry (the gold standard) in both patients
65
and
animal models of PBC.
75
Moreover, there is an increased
bleeding time with a trend to normalize after inhibition
of NO production (a potent platelet antagonist),
75
a
smaller amount of releasable ATP possibly caused by
loss of platelet granules during intravascular activa-
tion,
65
and a lack of increase of CD62P after stimula-
tion;
98
all indicative of decreased platelet function.
Very recently, the contribution of blood platelets to
liver damage in cholestatic liver disease was studied in
bile-duct ligated mice.
10
Platelet depletion or blockage
of the P-selectin receptor of platelets led to decreased
aggregation formation (within the liver), decreased
platelet adhesion and less leucocyte accumulation
resulting in improved liver transaminase levels.
10
Studies on platelets in alcoholic liver disease (see
Table 1) are more difcult to interpret because of
the interfering effects of the exogenic toxin (ethanol)
on the blood platelets, so active drinking should be
taken into account. Ethanol has direct effects on the
platelet lipids, the second messenger system (medi-
ated by cAMP, inositol trisphosphate and diacylglyc-
erol) and the phospholipase A2 system, all resulting
in altered platelet aggregability.
101
It is clear that
alcohol, at physiologically relevant concentration,
has an inhibitory effect on secondary platelet aggre-
gation in whole blood as well as in platelet rich
plasma.
102
In alcoholic liver disease, most studies indicate a
decreased aggregability
103105
although not all
agree.
106
Moreover, in alcoholic liver disease, the mal-
ondialdehyde production (a by-product of prostaglan-
din synthesis)
103
and TXB2 production
107
are elevated.
This could indicate an in vivo hyperaggregability,
possibly contributing to the thrombocytopenia.
In viral liver disease (see Table 1), there is evidence
for an in vivo platelet activation demonstrated by the
increased concentration of BTG and PF4 in serum.
49
P-selectin expression on resting platelets is also ele-
vated in chronic hepatitis and cirrhosis compared to
normal controls.
49
Also, plasma soluble P-selectin lev-
els where markedly elevated in chronic hepatitis C.
108
This correlated directly with serum hepatitis C virus-
RNA and was signicantly higher in patients with low
platelet counts and without a relation with platelet
associated immunoglobulin G.
108
This suggests that
hepatitis C infection might be directly responsible for
the in vivo platelet activation in patients with chronic
hepatitis C.
108
Currently platelets are increasingly
appreciated as inammatory players secreting chemo-
tactic factors and mediating cell-to-cell interactions.
In mouse models of acute viral hepatitis, it is shown
that platelet depletion reduces accumulation of virus
specic cytotoxic T lymphocytes and organ damage.
Transfusion of platelets in these mice restored accu-
mulation of cytotoxic T lymphocytes and severity of
disease.
9
In vascular liver diseases as extrahepatic portal
vein obstruction and non-cirrhotic portal brosis,
parenchymal liver function is usually very well pre-
served notwithstanding the presence of portal hyper-
tension. Platelet hypoaggregability has been
demonstrated in both diseases.
109, 110
Coagulation
disorders, suggestive of mild disseminated intravascu-
lar coagulation were common.
109
In an experimental
rat model of prehepatic portal hypertension (partial
portal vein ligation), portal hypertensive rats demon-
strated an increased hemorrhagic time and a dimin-
ished in vivo platelet activity demonstrated by an
in vivo arterial laser lesion model.
111
This could be
contradictorily corrected with ultralow dose aspirin
administration.
111
In BuddChiari syndrome some patients demon-
strate hyperaggregability and increased BTG levels.
110
Also ultrastructural abnormalities like absence of
alpha granules and clumping and fusion of the gran-
ules in the centre of the platelets have been
shown.
112
REVI EW: PLATELETS I N CHRONI C LI VER DI SEASE 1025
2008 The Authors, Aliment Pharmacol Ther 27, 10171029
Journal compilation 2008 Blackwell Publishing Ltd
CONCLUSI ONS
In chronic liver disease and cirrhosis, alterations in
primary haemostasis (platelet adhesion, activation and
aggregation) have received less attention than changes
in secondary haemostasis (coagulation).
Regarding platelet count, an increased intrasplenic
platelet breakdown with variable roles of decreased
platelet production and splenic pooling appear to be
the most important determinants. Regarding the func-
tional change, there is a decreased aggregability attrib-
utable to defective (transmembrane and intracellular)
signalling, a storage pool defect and an upregulation of
the inhibitory pathways. However, there are many con-
tradictory reports. This is mainly because of the differ-
ent inclusion criteria, different types and degree of
liver disease or the small number of patients included.
Research in established animal models of liver disease
could further help overcome these difculties.
Typical for platelet research is the technical dif-
culty, the need for fresh blood samples, and the imme-
diate execution of the different screening tests. The
more specialized the assays (for instance, platelet stud-
ies under ow conditions), the more they are prone to
artefactual ndings. This is a consequence of the very
sensitive nature of platelet tests.
The study of platelet numbers and functional
changes in relation to (cause and consequence) differ-
ent types of chronic liver disease remains an area of
interesting research. In view of the importance of
blood platelets in disease processes such as tissue
repair, inammation, vasculogenesis etc, a consider-
able impact on our understanding of the pathophysiol-
ogy of liver disease, portal hypertension, liver brosis
and therapy thereof can be expected.
ACKNOWLEDGEMENTS
Declaration of personal interests: P. W. is aspirant for
the F. W. O.-Vlaanderen. F. N. and D. C. are funda-
mental clinical researchers for the F. W. O.-Vlaander-
en. Declaration of funding interests: None.
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2008 The Authors, Aliment Pharmacol Ther 27, 10171029
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