INTERNAL MEDICINE INTERNAL MEDICINE
Volume 46 No. 1, 2008
R. TĂNĂSESCU, ADRIANA NICOLAU, MARINA ŢICMEANU, DIMELA LUCA, SIMONA CARAIOLA, INIMIOARA
MIHAELA COJOCARU, A. FRĂSINEANU, R. IONESCU, ADRIANA HRISTEA, AMALIA ENE, RUXANDRA
TĂNĂSESCU, C. BĂICUŞ, An Immunological Approach to Cerebral Ischemia. Immune Cells and Adhesion Molecules
ANA MARIA VLĂDĂREANU, CRISTINA CIUFU, H. BUMBEA, MINODORA ONISÂI, S. ARAMĂ, An Update on the
Platelet Dysfunction in Chronic Myeloproliferative Syndromes ......................................................................................
D. ZDRENGHEA, LAURA POANTĂ, DANA POP, V. ZDRENGHEA, M. ZDRENGHEA, Physical Training – Beyond
Increasing Exercise Capacity ...........................................................................................................................................
MARIA DOROBANŢU, ELISABETA BĂDILĂ, S. GHIORGHE, ROXANA DARABONT, M. OLTEANU, P. FLONDOR,
Total Cardiovascular Risk Estimation in Romania. Data from the Sephar Study .............................................................
D.M. DUDA-SEIMAN, SILVIA MANCAŞ, D. GAIŢĂ, SIMONA DRĂGAN, DANA VELIMIROVICI, STELA IURCIUC,
C.A. SARĂU, M. IURCIUC, MARIA RADA, BILEANA PETCOV, Lifestyle, Cardio-metabolic Risk and Arterial
O.S. PLEASCAR, D. COZMA, L. PETRESCU, DANIELA POPA, M. SLOVENSKI, S.I. DRĂGULESCU, Left Atrial
Surface Cut Point to Detect Trapezoidal Shape for Characterisation of Atrial Anatomical Remodeling .........................
LUCICA AGOŞTON-COLDEA, L.D. RUSU, RALUCA PAIS, ADRIANA ALBU, M.L. RUSU, TEODORA MOCAN,
S. PERNA, The Impact of the Metabolic Syndrome on the Patients with Acute Coronary Syndrome ............................
MARIA ILEA, D. ZDRENGHEA, G. BODISZ, ADINA MĂLAI, LILIANA GHEORGHIU, D. PREDESCU, MARIA
BEUDEAN, VICTORIA OSSIAN, ELENA GLIGOR, CRISTINA VLAD, DANA POP, E. BOGDAN, R. ROŞU,
Cardiac Peptides During Exercise Test in Ischemic and Non-ischemic Heart Failure Patients ........................................
SILVIA MANCAŞ, GEORGETA MIHALAŞ, D. GAIŢĂ, SIMONA DRĂGAN, D.M. DUDA-SEIMAN, C.A. SARĂU,
LAVINIA NOVEANU, BILEANA PETCOV, GEORGIANA MANCAŞ, V. IONESCU, MONICA PĂCURAR,
Environmental Factors and Cardiovascular Risk in Young Individuals ...........................................................................
INIMIOARA MIHAELA COJOCARU, M. COJOCARU, R. TĂNĂSESCU, CECILIA BURCIN, ADINA NICOLETA
ATANASIU, ANA MARIA PETRESCU, ANDREEA CRISTINA MITU, IULIA ILIESCU, LAURA DUMITRESCU,
Changes in Plasma Levels of Complement in Patients with Acute Ischemic Stroke ........................................................
T. MOGOŞ, CARMEN DONDOI, Which Dose of Omega-3 Fatty Acids Must be Taken in Different Cases of
I. COPACI, MIHAELA ENACHE, C. JURCUŢ, M. ŞOTCAN, C. CONSTANTINESCU, Polycystic Hepatic Disease ..........
A. MIRONIUC, LAVINIA COMES, IOANA CONSTANTINESCU, CLARA MIRONIUC, DANA BONTEA, Cryoglobulinemic
Vasculitis with Multiple Digital Necrosis in Viral Hepatitis ............................................................................................
ROM. J. INTERN. MED., 2008, 46, 1, 1–96
An Immunological Approach to Cerebral Ischemia (I).
Immune Cells and Adhesion Molecules
R. TĂNĂSESCU1*, ADRIANA NICOLAU2, MARINA ŢICMEANU1, DIMELA LUCA1, SIMONA CARAIOLA2*,
INIMIOARA MIHAELA COJOCARU1, A. FRĂSINEANU1, R. IONESCU2*, ADRIANA HRISTEA3, AMALIA ENE4,
RUXANDRA TĂNĂSESCU3, C. BAICUŞ2*
1Department of Neurology, Colentina Hospital, Bucharest, Romania
2Department of Internal Medicine, Colentina Hospital, Bucharest, Romania
3“Prof. Matei Balş” Institute of Infectious Diseases, Bucharest, Romania
4Department of Neurology, University Hospital, Bucharest
*Réseau d’Epidémiologie Clinique International Francophone (RECIF)
Ischemic stoke is a major cause of death and an important source of disability in industrialized
countries. Since there is no ideal treatment for cerebral ischemia, any approach aiming to limit the
devastating consequences of the ischemic process is justified. Concerning immune responses, it has
become clear in the latest years that actors of the immune system are involved in multiple and various
neurobiological processes such as cerebral ischemia, neurodegeneration, neuroprotection and
neuroregeneration. An immunological approach to cerebral ischemia can distinguish, besides the
implication of inflammation in the developing of atherothrombosis thus leading to stroke, the clear
involvement of immune cells and mediators in processes continuing the initial stage of ischemia,
having consequences on recovery or lesion extent. Cerebral infarctions develop within minutes to
hours of cessation of the cerebral blood flow, but may expand over subsequent days. There is
increasing evidence that leukocytes, cytokines, cell adhesion molecules, and other immune mediators
contribute to secondary infarction growth, but inflammatory cytokines are also involved in signaling
pathways leading to neuroprotection related to ischemic pre-conditioning. The aim of this review is to
show some aspects concerning the complex and diverse functions of immune modifications occurring
in cerebral ischemia. This first part will focus on the involvement of immune cells, adhesion
molecules and immunological transcription factors in the development of ischemic lesion.
Since ischemic stoke is, in industrialized
countries, the third leading cause of death and an
important source of disability, any approach aiming
to limit the devastating consequences of cerebral
ischemia is justified. Even if immune responses in
the nervous system have been mainly studied in the
context of autoimmunity and infections, it has
become clear in recent years that actors of the
immune system are involved in multiple and
various neurobiological processes such as cerebral
ischemia, neurodegeneration, neuroprotection. An
immunological approach to cerebral ischemia can
distinguish, on the one hand, the implication of
inflammation in the developing of atherothrombosis.
Inflammation of the vessel wall is an essential part
of atherosclerosis, in general, but immune interactions
play a decisive role in the destabilization of
atherosclerotic plaque. This leads to local formation
of thromboemboli that may transiently or
permanently occlude intracranial arteries and
induce focal ischemia. On the other hand, cerebral
infarctions develop within minutes to hours after
cessation of cerebral blood flow and may expand
over days. There is increasing evidence that cells
and mediators are involved in immunological
cascades continuing the initial stage of ischemia
with processes apparently silent, but with
consequences on recovery or lesion extent.
Leukocytes, cytokines, cell adhesion molecules, and
other immune mediators contribute to secondary
infarction growth. At the same time, inflammatory
cytokines are involved in neuroprotection related to
ischemic pre-conditioning. This review is aiming to
present some aspects concerning the immune
modifications occurring in central nervous system
(CNS) ischemia. The present section will focus
on the involvement of immune cells (neutrophils,
macrophages, microglia, T cells), adhesion molecules
and immunological transcription factors in the
development of ischemic lesion. Cytokine participation
ROM. J. INTERN. MED., 2008, 46, 1, 3–8
4 R. Tănăsescu et al. 2
in brain ischemia will be treated separately in a future
MECHANISMS OF NEURAL DEATH. THE EXTENT
OF INFARCTION IS DEPENDENT ON THE
DURATION OF BLOOD FLOW CESSATION
Ischemic impairment or cessation of cerebral
focal blood flow restricts the delivery of oxygen
and glucose, leading to an impaired energy-
dependent maintenance of ionic gradients. This
conducts to neuron and glia depolarization leading
to extracellular accumulation of Ca2+ and release of
glutamate, an excitatory aminoacid . Ca2+
accumulation activates proteolytic enzymes and
generates the production of free-radical species,
and release of glutamate. These processes induce
rapid necrotic cell death of the cells most affected
by ischemia, located in the core of the ischemic
territory, where the flow reduction is the most severe.
On the other hand, a significant proportion of neurons
die by an internal program of self-destruction
(apoptosis or programmed cell death), ongoing for
several days after the initial insult. It must be
emphasized that this must be differentiated by the
physiological apoptosis processes, occurring in the
natural evolution of the cell. Apoptosis involves
activation of caspases and requires partially preserved
protein synthesis. Other key injury mechanisms
include the production of oxygen- and nitrogen-based
free radicals, inflammatory mediators such as tumor
necrosis factor (TNF), interleukin (IL)-1, inducible
nitric oxide synthase (iNOS), cyclo-oxygenase-2,
protease activation (calpains, caspases, extracellular
proteases), mitochondrial and endoplasmic reticulum
Initially during cerebral ischemia, developing
infarctions are surrounded by an ischemic penumbra
that can be salvaged by reperfusion. This ischemic
penumbra means cortical areas where the blood
flow is reduced, but still exceeds a flow and
duration threshold that produces an irreversible
state of ischemic neuronal injury. In order to study
progression and mechanisms of focal ischemic
brain injury, several animal models have been used,
in which surgical occlusion of major arteries was a
widely performed method . In the model of
permanent occlusion of the middle cerebral artery
(MCA) at proximal sites, leading to complete
infarctions of the basal ganglia and the neocortex,
the infarction volume has been observed to change
significantly during the first 3 days. Early reperfusion
reconstitutes perfusion and significantly modifies
stroke development: ischemia lasting less than
30 minutes leads to a restricted infarction with
pannecrosis of neurons and glial cells in the
caudatoputamen, but only partly affects the neocortex.
Further delay in reperfusion produces an increasing
neocortical infarction. Depending on the timing of
reperfusion, neocortical infarctions can be surrounded
by areas of selective neuronal death characterized
by loss of large pyramidal neurons but preservation of
endothelial and glial structures .
IMMUNE CELLS IN FOCAL ISCHEMIA
Focal cerebral ischemia leads to local activation
of microglia and astroglia and to an important influx
of blood leukocytes . Granulocytes are the first
blood cells that appear in the brain in response to
focal ischemia . They are attracted by chemokines
released from ischemic tissue and accumulate in the
cerebral vessels within hours before they invade the
infarction and its boundary zone, with a peak at
24 hours after infarction onset, thereafter rapidly
decreasing in number. Within the second week after
infarction, granulocytes have mostly disappeared. It
was suggested that in transient ischemia models
accumulation of intravascular granulocyte probably
reduces the blood flow in the reperfusion phase thus
contributing to infarction extent .
The main population of leukocytes that enter
the brain after focal cerebral ischemia are monocytes/
macrophages, attracted into areas of pannecrosis
and the border zones by chemokines . The
infiltrates demarcate the necrotic brain tissue, and
rapidly remove debris leaving a glial scar, helped
additionally by resident microglia which contribute
to the phagocytic response. These microglia
become indistinguishable from macrophages upon
Trying to distinguish between hematogenous
macrophage infiltration and local microglial responses,
super-paramagnetic iron particles were injected
into the circulation. They are rapidly taken up by
circulating macrophages. When these iron-laden
macrophages infiltrate lesions they become detectable
as areas of signal loss on T2-weighted magnetic
resonance (MR) images. With this imaging technique
3 An immunological approach to cerebral ischemia
it was demonstrated that hematogenous macrophages
were recruited in the rat model with a delay of several
days to ischemic brain lesions, and that macrophage
invasion was temporally unrelated to the breakdown
of the blood-brain barrier (BBB) . In a pilot study
of human stroke, this late recruitment was recently
confirmed . These data suggest that macrophages
could play a role in tissue remodeling and
repair, rather than in aggravating the neuronal injury
because they appear at a stage in which delayed
neuronal cell death has already ceased. On the
other hand, microglial activation appears to play a
detrimental role as it occurs early. It is maybe in this
respect that minocyclin (a tetracyclin derivative) was
shown to attenuate microglial responses and reduce
infarction volume .
Important to note is that macrophage/microglial
responses to cerebral ischemia are diverse. In the
murine model two different populations are
described . On the one hand, CD4+ macrophages/
microglia that are also present in other models of
CNS lesion are gradually increasing from day 2 with
a peak at day 14 when they covered the entire area of
infarction. Secondly, macrophages/microglia expressing
the T- and NK-cell surface molecule, CD8,
constitute a separate and unusual population .
This population of CD8+ macrophages/ microglia
was exclusively located in the border zone and the
core of necrotic brain tissue transiently between days
3 and 6 and had almost disappeared at day 14. Their
functional role in brain ischemia is not completely
clarified at present. These CD8+ cells could
contribute to exacerbation of ischemic brain damage
as well as to tissue remodeling and healing
processes. It was shown on alveolar macrophages
studied in vitro that signaling via the CD8 molecule
led to expression of TNF, IL-1β, and iNOS. It is
important to emphasize that CD8+ macrophages/
microglia appear to be relatively specific to areas
of ischemic pannecrosis (necrotic lesion of all brain
structures) and are not seen in areas of selective
neuronal death or degeneration .
T cells preferentially infiltrate in a large
amount the border zones of infarctions within the
first 7 days after stroke . This was demonstrated
in both experimental and human stroke studies.
One important point is that T-cell CNS recruitment
is usually observed in autoimmune and infectious
conditions, but not in degenerative disorders. In
experimental autoimmune encephalomyelitis (EAE),
systemic immunization with myelin basic protein
generates CD4+ helper/inducer T cells that are
antigen specific and accumulate within the CNS
after 10 to 12 days to induce myelin destruction by
macrophages . In cerebral ischemia, the T-cell
response is likely to be antigen non-specific
because the period between lesion induction and
T-cell infiltration is too short for generation of
systemic, antigen-specific immune responses .
The signals that attract T cells and keep them in the
CNS parenchyma for many days are unknown, but
studies suggest that infiltration of tolerized T cells
into ischemic brain lesions can be beneficial. Naive
rats fed with myelin basic protein, a procedure
known to induce immunological tolerance to EAE
induction, had reduced infarction volumes at 24
and 96 hours after MCA occlusion (10). This effect
was attributed to the infiltration with T cells that
produced transforming growth factor (TGF)-β1 of
evolving ischemic brain lesions. TGF-β1 is known
as having neuroprotective effects and being a
strong immunosuppressant. It was shown that the
neuroprotective effect of myelin basic protein
tolerance in cerebral ischemia can be transferred to
naive animals by infusion of tolerized lymphocytes
(adoptive transfer) .
CELL ADHESION MOLECULES AND THEIR
FUNCTIONAL ROLE IN CEREBRAL ISCHEMIA
Cell adhesion comprises multiple steps
during the inflammatory response in focal
ischemia, providing the traffic signals for entry of
leukocytes into the brain . Selectins (E-selectin,
P-selectin, L-selectin) are supporting leukocyte
rolling on luminal endothelium, thus reducing the
velocity of leukocytes in the blood flow. Integrins
are mediating firm adhesion of leukocytes to the
endothelium. There are different molecules of the
integrin family directed to the adhesion of leukocyte
subsets. In this respect, lymphocytes have on their
surface the CD11a/CD18 (leukocyte function
associated antigen-1; LFA-1) and the very late
antigen-4 (VLA-4) complex, monocytes LFA-I,
VLA-4, and the CD11b/CD18 complex, and
granulocytes LFA-1 and the CD11b/CD18 complex
. The corresponding endothelial counter-receptors
are intercellular adhesion molecule-1 (ICAM-1) for
LFA-1 and C11lb/CD18, and the vascular cellular
6 R. Tănăsescu et al. 4
adhesion molecule-1 (VCAM-1) for VLA-4. ICAM-1
is induced on brain endothelial cells 3 hours after
focal ischemia, have a peak of expression at 6 till
12 hours, and persists for several days. Similar to
ICAM-1 are E-selectin (endothelial leukocyte adhesion
molecule-1; ELAM-1) and VCAM-1 .
The importance from a functional point of view
of cell adhesion processes in stroke development was
proved in transient focal ischemia [12–14].
Treatments with antibodies or with a
recombinant neutrophil inhibiting factor directed
against the CD11b/CD18 complex present on
granulocytes and monocytes/macrophages were
used. Antibodies against the CD11b/CD18 complex
in a murine model subjected to 2 hours of transient
occlusion of MCA led to a significant reduction in
infarction volume and to a decrease in the number
of apoptotic cells. At the same time, infiltration by
granulocytes was reduced. Similar results were
obtained with a recombinant neutrophil inhibiting
factor directed against the CD11b/CD18 complex.
Blocking of the corresponding ligand on endothelial
cells, ICAM-1, reduced stroke volumes on day 2 by
80% . Accordingly, in the ICAM-1 knockout
mice it was shown a five-fold decrease in infarction
size . Blocking of lymphocyte and monocyte
entry by antibodies to the α4 integrin in the rat
model decreased infarction volume and improved
neurological outcome even when instituted after
onset of focal ischemia .
The effects seen in transient focal ischemia by
inhibiting leukocyte adhesion are not reproducible in
permanent ischemia models: the use of antileukocyte
antibodies in models of permanent MCA occlusion
was ineffective . This discrepancy can be explained
by the fact that granulocytes adhere to the microvascular
endothelium via the ICAM-1/ CD11b/CD18 adhesion
pathway thereby realizing a mechanic disconnection
of the dependent parenchyma from reperfusion (“no
reflow phenomenon”). This prolonged hypoxia period
leads thereafter to the extension into the penumbra
zone of the infarction area. A relatively recent study
in human stroke using a rat monoclonal antibody
against ICAM-1 conjugated with polyethylene
glycol in order to reduce immunogenicity,
Enlimolab, failed and led to unacceptable side-
effects by activating human neutrophils .
Adverse effects of Enlimomab may have been
caused by immunological factors, maybe by
activation of the complement, neutrophils, and
endothelial cells, as seen in murine model in which
monoclonal anti-ICAM-1 was administered .
TRANSCRIPTION FACTORS AND NITRIC OXIDE:
IMPLICATIONS IN PRO-INFLAMMATORY AND
NEUROTOXIC PATHWAYS IN CEREBRAL
It has been proved that immune cascades
contribute to significant infarction growth beyond
24 hours after focal cerebral ischemia .
The cellular responses in focal cerebral ischemia
are accompanied by the expression of immunological
transcription factors, iNOS, and cytokines.
Interferon regulatory factor 1 (IRF) is a
transcription factor that can be activated by TNF
and IL-1β. IRF gene expression is markedly up-
regulated at 12 hours after focal ischemia, with a
peak on day 4. It was shown that transgenic mice
lacking the IRF gene were protected from ischemic
brain damage and developed smaller infarctions
. The molecular mechanisms underlying
ischemic neuroprotection in IRF knockout mice
have not been completely understood, but they can
imply lack of nitric oxide induction . It is
known that IRF can induce gene transcription of
interleukin-1 converting enzyme (ICE or caspase 1)
and inducible NO synthase (iNOS). Lack of nitric
oxide (NO) induction is a likely mechanism since
macrophages from IRF knockout mice produced
virtually no NO and synthesized only low levels of
iNOS mRNA in vitro. NO is a small molecule but
that exerts pleiotropic actions. NO is synthesized
by oxidation of L-arginine by the enzyme NO
synthase (NOS) which exists in three isoforms,
neuronal NOS (nNOS), endothelial NOS (eNOS),
and inducible NOS (iNOS) . The three ways of
NO production are different in some aspects: the
NO secretion via eNOS and nNOS is calcium-
dependent and small amounts of NO are produced
(nano), while iNOS that is expressed by astrocytes
and microglia is implied in a NO production that
typically occurs only in an inflammatory setting.
NO production via iNOS is calcium-independent
and in higher concentrations (micro). NO
production is enhanced at all stages of cerebral
ischemia, but the effects on brain parenchyma can
vary in function of the concentration . Early
during ischemia NO produced through eNOS
activation in endothelial cells is beneficial and
induces an increase of the cerebral blood flow by
vasodilatation as well as anti-adhering effects on
leukocytes . In contrast, iNOS appears to be
detrimental, inducing synthesis of large amounts of
NO continuously for long periods, then reacts with
superoxide to form peroxynitrite, which has cytotoxic
5 An immunological approach to cerebral ischemia
actions. In the murine model with MCA occlusion,
iNOS mRNA expression in the postischemic brain
started between 6 and 12 hours, with a peak at
96 hours, and subsided after 7 days . Conversely,
disruption of the iNOS gene in mice led to smaller
infarctions and reduced motor deficits after focal
ischemia. It is very important to note that this
reduction in ischemic damage was not observed in
the first 24 hours from stroke onset, but it was
found at day four. This supports the idea that iNOS
expression is one of the critical factors participating
in the delayed expansion of brain damage .
Accidentul vascular ischemic reprezintă o cauză importantă de dizabilitate şi
deces în ţările industrializate. Cunoaşterea aspectelor neuroimunologice care
privesc ischemia cerebrală este importantă, câtă vreme implicarea sistemului imun
în geneza şi evoluţia leziunilor ischemice este astăzi o certitudine. Infarctul
cerebral se instalează în intervale de la minute la ore după oprirea fluxului
sanguin cerebral, dar la nivelul zonelor afectate există procese care durează zile
sau săptămâni. Actorii sistemului imun (leucocite, citokine, molecule de adeziune,
alţi neuromediatori) vor contribui la majorarea sau limitarea zonelor infarctizate.
Citokinele inflamatorii sunt implicate, pe lângă posibile acţiuni deletare asupra
parenchimului cerebral, în procese de neuroprotecţie asociate fenomenului de
precondiţionare ischemică. Articolul de faţă face o trecere în revistă a principalelor
aspecte legate de implicarea sistemului imun asociată ischemiei cerebrale.
Abordarea subiectului a fost împărţită în două secţiuni. Cea de faţă se referă la
implicarea versantului celular (macrofage, microglii, limfocite T) în modelarea
leziunii ischemice, urmând ca participarea citokinelor la procesele legate de
leziunea ischemică cerebrală să fie abordată într-o secţiune separată.
Correspondence author: R. Tănăsescu, MD, PhD, AFS
“Carol Davila” University of Medicine and Pharmacy, Bucharest
Department of Neurology, Colentina Hospital
19–21 Şos. Ştefan cel Mare, 020125, Bucharest, Romania
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Received January 16, 2008
An Update on the Platelet Dysfunction in Chronic Myeloproliferative Syndromes
ANA MARIA VLĂDĂREANU1, CRISTINA CIUFU1, H. BUMBEA1, MINODORA ONISÂI1, S. ARAMĂ2
1Department of Hematology, Emergency Universitary Hospital, “Carol Davila” University of Medicine and Pharmacy
2Department of Physiology, “Carol Davila” University of Medicine and Pharmacy, Bucharest, Romania
The thrombotic and hemorrhagic diathesis represents a frequent complication in myelo-
proliferative disorders (CMPD). They are correlated with the number of platelets, but also with their
qualitative disorders, such as membrane glycoprotein changes. The latter are revealed by many
platelet essays including flow-cytometry and include modified activation, secretion and aggregation
patterns. The thrombopoietin platelet receptor (cMPL), affected by the JAK2 V617 mutation
encountered in CMPD, may be associated with a prothrombotic status. Its implication reveals the
importance of the molecular genetics profile in defining molecular diagnostic hallmarks and makes it
a candidate in the early diagnosis of myeloproliferative disorder and a predictor of thrombotic
complications in this group of diseases.
Key words: myeloproliferative syndromes, thrombosis, hemorrhage, JAK2 V617 mutation.
The clonal stem cell disorders, including
chronic myeloproliferative disorders (CMPD), are
associated with qualitative and quantitative disorders
of the erythroid, granulocyte, and thrombocyte
These dysfunctions are thought to cause
bleeding and thrombotic complications. This is
the reason why thrombotic and hemorrhagic events
occur quite frequently in patients suffering from
myeloproliferative syndromes and are also considered
important causes of morbidity and mortality .
The qualitative defects of the platelets together
with their quantitative alterations such as thrombocytosis
(which occurs mainly in myeloproliferative syndromes)
produce important disorders of hemostasis.
Chronic myeloproliferative disorders are stem-
cell disorders, in this group being included essential
thrombocythemia, polycythemia vera, myeloid
metaplasia with myelofibrosis and chronic myeloid
leukemia. In all of these diseases there are
described hemorrhagic diathesis, thrombo-embolic
complications and qualitative defects of the
platelets. Although these diseases have many common
features, the spectrum of platelets’ disorders and
their clinical picture is different in CMPD. The
thrombotic complications are more frequent in
essential thrombocythemia and polycythemia vera,
and the hemorrhagic complications are more
frequent in MMM, especially when there is an
32% of the patients with CMPD may develop
arterial or microvascular thrombotic complications;
venous thrombosis is reported as well .
The thrombotic risk is increasing with age, a
previous thrombosis history and the occurrence of
general associated risk factors such as hyper-
cholesterolemia, smoking, diabetes mellitus, arterial
THE PLATELET ROLE IN THE
ETHIOPATHOGENESIS OF THROMBOSIS IN
CHRONIC MYELOPROLIFERATIVE DISORDERS
The importance of the platelet’s role in the
etiopathogenesis of thrombotic events in CMPD is
multifactor through: their number, their qualitative
defects, the pattern of platelet activation, the
formation of platelet leukocyte aggregates, the
appearance of platelet micro-particles, and the
impact of JAK mutation .
1. In CMPD, especially in polycythemia vera,
erythrocytosis leads to increased blood viscosity
which may rheologically contribute to the
thrombosis tendency. But, this rheologic effect of
erythrocytosis is not the only mechanism explaining
the thrombotic tendency in MPD .
2. Thrombocytosis is a contributing factor in
the ethiopathogenesis of thrombosis in PV and ET
. There is a relative correlation between the
ROM. J. INTERN. MED., 2008, 46, 1, 9–15
Ana Maria Vlădăreanu et al. 2
degree of thrombocytosis and the thrombotic
complications in chronic myeloproliferative disorders
 and there is evidence that platelet count control
reduces the incidence of thrombosis.
Even if the control of thrombocytosis is
decreasing the frequency of thrombotic complications,
especially in patients with previous thrombosis, the
degree of the thrombocytosis is not correlated with
the thrombotic risk all the time .
In the ECLAP analysis, antiplatelet therapy,
but not cytoreductive treatment, was significantly
associated with a lower risk of cardiovascular
3. Qualitative disorders of the platelets in
chronic myeloproliferative disorders
Optic microscopy reveals heterogeneity in
size and morphology of platelets, which is
expressed by the large mean platelet volume 
and by anisocytosis and anisochromia on blood
smears as shown in Fig. 1.
Electronic microscopy revealed the decrease
of alpha granules and mitochondria and also
changes of the dense tubular and canalicular system
. A decrease of the ADP, ATP and serotonin
content of dense granules was also proved .
Some patients suffering from chronic myelo-
proliferative disorder have a prolonged bleeding
time, this change being more frequent in MMM
than in the other chronic myeloproliferative diseases.
The prolonged bleeding time is not correlated with
high bleeding risk .
Waddell et al.  were the first to describe
the membrane glycoprotein platelet changes in
chronic myeloproliferative disorders, such as: the
decrease of the alfaIIb betaIII and Ib receptors and
a low fibrinogen surface binding, indicating a
decrease of alfaIIb betaIII receptor. Besides the
quantitative decrease, the surface receptors alfaIIb
betaIII were also proved to have an altered
activation pattern .
Other qualitative platelet defects in CMPD
include: impaired dense granule release, defective
Ca mobilization, lipoxygenase accumulation .
4. The pattern of platelet activation
The pattern of platelet activation and the
changes in the surface receptors are presented in Fig. 2.
The platelet secretion and aggregation as a response
to epinephrine, ADP, collagen is decreased in chronic
myeloproliferative disorders and in myelodysplastic
syndromes. This aggregation defect is expressed by
the absence of the first aggregation wave .
The pathogenesis of platelet activation pattern
in chronic myeloproliferative syndromes is not quite
defined. It is multifactorial and includes: the lipo-
oxygenase deficiency, the disturbances in bone
marrow microenvironment, the hematocrit range, the
implication of activated leucocytes, the impaired
platelet NO synthetase, elevated thrombopoietin
level, the effect of JAK2 mutation .
In CMPD, especially in polycythemia vera,
the rise of haematocrit increases blood viscosity.
The axial migration of the erythrocytes in the
bloodstream “pushes” the platelets to the wall of
the vessel, increasing the platelet-vessel interaction,
especially in high shear conditions – in small
arteries and capillaries – contributing to platelet’s
As reported by Schafer, a large number of
patients have a lipooxygenase deficiency, which
could increase the capacity of endoperoxidasis to
produce thromboxan A2 .
Cooper et al. were the first to prove the
decrease of adenylate-cyclase activation GPD2
induced, correlated with a decrease of 50% of PGD2
receptors on the platelets, and normal responses to
PGE2 and PGI2 . This is suggestive for an
inhibitor mechanism defect in MPD.
It was recently established that platelets of the
PV and MMM patients, but not of ET and CML
patients, have a low expression of the thrombopoietin
receptor and the tyrosin-phosphorylation thrombopoietin
induced is also decreased; tyrosin-phosphorylation
thrombin induced is preserved .
In normal platelets, PGD2 increases AMP
because of adenylate-cyclase stimulation, leading
to platelet response inhibition .
Fujimoto et al.  proved the decrease of
Ca mobilization agonist-induced, the signaling by
thromboxan-receptor and the proteic phosphorylation
because of GMPc protein-kinase deficiency .
According to their observations platelet calcium
influx was lower than in control patients. Their
conclusion was that GP IIb/IIIa anomalies are
involved in the decrease of platelet calcium influx
in chronic myeloproliferative diseases patients .
The increase of P selectin expression,
thrombospondin and GP IIb/IIIa was correlated
with thorombosis. In chronic myeloproliferative
syndrome patients it has been shown an expression
and functional decrease of GP IIb/IIIa (CD41/
CD61) receptor, also of fibrinogen receptor, fibronectin
receptor, vitronectine receptor, thrombospondin
receptor, von Willebrand factor receptor. Also it
3 An update on the platelet dysfunction
has been found a low von Willebrand factor
binding on the platelet .
Some studies  showed an increase of
VEGF level that demonstrates increased endothelial
activation. The leukocyte activation (especially
monocytes and neutrophils) allows the release of
the granules content which activate the coagulation
pathways, induce platelet aggregation, release
inflammatory cytokines and oxygen superoxide, all
of the above contributing to the endothelial
The interaction between neutrophils and
platelets becomes apparent after the adhesion
cascade, when CD62P binds to P-selectin on the
neutrophil. The adhesion is stabilised by two other
complexes: CD11b of the beta-integrin complexed
with CD18 with GP Ib of the platelet and the
binding of fibrinogen with the platelet GP IIb/IIIa.
Some studies noticed an increase of the CD62P,
CD11b, CD42b expression in patients with
polycythemia vera and essential thrombocythemia
; the level of CD11b/CD42b and CD62P/
CD11b complexes was also increased.
Falanga et al.  underline that the
expression of JAK2 mutation in ET patients
confers to neutrophils a different haemostatic
property in terms of increased interaction with
platelets and increased expression of surface TF
and fibrinogen, suggesting a new link of the
mutation with the prothrombotic state.
It was also described a deficiency of GP Ia-IIa
and an abnormal response to collagen stimulation;
GP IV (CD 36) – a membrane glycoprotein
implicated in platelet-collagen interaction and
platelet-thrombospondin interaction – is known to
be decreased in essential thrombocythemia .
In chronic myeloproliferative disorders, there
are also other alterations of the platelet membrane
receptors – GP IV (CD36), thrombospondin receptor
which is released by platelet secretion. Its effect is
on the strength of the platelet aggregates, making
this process irreversible. The expression of this
receptor is increased in chronic myeloproliferative
disorders – all the patients with essential thrombo-
cythemia presented a proteolysed form of thrombo-
spondin (TSP) and this form was not detected in
reactive thrombocytosis . A study showed that
he level of normal GP IV and the associated
proteins may become normal during the treatment
with Interferon, but the altered form of TSP
receptor still persists .
It was proved over twenty years ago that the
Fc-IgG platelet receptors are increased in
chronic myeloproliferative diseases .
The variability of the platelet receptors is
thought to be genetically determined and there are
few studies establishing that platelet’s glycoproteins
polymorphism could contribute to thrombosis.
PLA2 allele of GPIIIa was correlated to the
increased incidence of arterial thrombosis, but this
conclusion must be confirmed by more studies .
5. The acquired von Willebrand disease
The acquired von Willebrand disease seems
to be an important factor contributing to the
bleeding diathesis in patients with thrombocytosis
in MPD. The von Willebrand factor multimers
analysis on agarosis gel electrophoresis has shown
the decrease of ultra-large multimers, similar to
von Willebrand disease type 2. It is correlated to
the degree of thrombocytosis and could be
corrected by cytoreduction therapy .
Possible mechanisms of production: high
clearance of ultra-large multimers by selective
platelet binding, or increased proteolysis of von
Willebrand factor, perhaps by the exposure of von
Willebrand factor cleavage loci to ADAMT13.
Because this acquired defect was also reported in
secondary thrombocytosis, it represents more a
favoring factor and not the cause of bleeding
complications in chronic myeloproliferative diseases,
especially in severe thrombocytosis .
6. The formation of platelet leucocyte
aggregates and the appearance of platelet
The formation of platelet leucocyte aggregates
(Fig. 3) is increased in MPD patients . They
form after platelet and neutrophil activation and
contribute to thrombus formation by the release of
granule contents, by inducing tissue factor expression
on monocytes, by the release of superoxide and
cytokines, by enhancing platelet activation and
endothelial activation and damage .
Harrison, C.N. (ASH-2005)  reported that
the increased level of highly thrombogenic platelet
microparticles in ET and PV was correlated with
the thrombotic tendency.
7. The impact of JAK mutation
An alternative explanation of modified platelet
activation includes the effect of JAK2 mutation
(Fig. 4). Recent data suggest that JAK2 mutation
affects cMPL platelet receptor of thrombopoietin .
Ana Maria Vlădăreanu et al. 4
The JAK2V617F represents a G to T somatic
mutation of Jak2 at nucleotide 1849, in exon 14,
resulting in the substitution of valine to phenyl-
alanine at codon 617.
In chronic myeloproliferative syndromes, the
mutation occurs in multipotent stem-cell . The
homozygous progenitors are more frequent in PV
patients and very rare in ET patients .
The JAK2V617F mutations occur in 90–95%
of PV patients, 50–70% of ET patients and 40–
50% of MMM patients . This mutation has
also been demonstrated in other myeloproliferative
disorders, including atypical MPD and also in
myelodysplastic syndromes .
The presence of JAK2 mutation has also been
revealed in elderly AML patients and it underlines
the possibility that these patients have had
previously undiagnosed chronic myeloproliferative
syndromes. Recent data revealed that JAK2
mutation occurs in a significant number of AML
secondary to chronic myeloproliferative syndromes
patients. In de novo AML patients the incidence of
this mutation is low .
The intracytoplasmic domain of this receptor
is correlated to JAK2, member of kinase family
Janus. The correlation between the JAK mutation
and the thrombopoietin receptor is shown in Figure 5.
The thrombopoietin stimulates the phosphorylation
of some proteins, including Janus kinase JAK2 and
TYK2. The thrombopoietin and its receptor
represent the control-key to regulate the platelet
number; the most of PV and MMM patients have a
decrease of thrombopoietin receptors and an
incomplete glycosylation of these receptors. This
defect cannot be found in other platelet membrane
glycoproteins (GPIIb), but it is related to the disease
duration and extramedullar hematopoiesis .
According to Campbell  the presence of
the JAK2V617 mutation divides essential thrombo-
cythemia into two distinct subtypes. Patients with
the mutation present higher hemoglobin levels,
higher white cell counts and bone marrow
hypercellularity . The risk of thrombosis in
mutated ET patient has not been reported by all
A group of investigators from Italy –
Bergamo  have reported that JAK2 mutation in
ET brings up a distinct clinical entity with a
biological phenotype intermediate between JAK2
wild-type ET and PV. For the first time they
present a comparison between the thrombotic risk
of ET and PV patients defined on the basis of their
JAK2 mutational status .
They have reported for JAK2 mutation ET
and PV that hemoglobin and hematocrit levels as
well as white cells number and activation parameters
(PRV1 and LAP)  increase whereas platelet
number decreases as compared with those with
JAK2 wild-type ET.
The association between increased granulocyte
PRV-1 and LAP expression and thrombosis is
noteworthy. Their recent data showing that
leukocytes of JAK2 mutated ET patients present a
prothrombotic state indicated by a significantly
increased expression of surface tissue factor and
fibrinogen and a tendency to form higher numbers
of leukocyte-platelet aggregates . These results
explain why hydroxyurea is more effective in
reducing the thrombotic events particularly in
JAK2 mutated ET patients and anagrelide (a
megakaryocyte restricted inhibitory agent) is not
, and also hydroxyurea possibly reduces
the leukocytes-platelet aggregates by endothelin
1gene and ICAM-1 overexpression and by
increasing the NO level .
J. Smalberg et al. reported the association
between thrombosis of the hepatic veins – the
Budd-Chiari syndrome – BCS and the JAK2V617
mutation status. The mutation occurred in 59% of
BCS patients  and may be used to characterize
occult MPD – as a molecular diagnostic hallmark –
and could be included in the early diagnosis for
MPD in BCS.
Tefferi  underlines that it is reasonable to
consider the screening of JAK2V617 mutation in
the initial evaluation of unexplained thrombocytosis,
unusual thrombotic complication including abdominal
or cerebral vein thrombosis, arterial events at
young age and other MPD characteristic clinical
manifestations including erythromelalgia .
Despite the rare occurrence of thrombocytopenia,
some patients with MPD may sometimes develop life
threatening thrombotic complications like PTE
(pulmonary thromboembolism), DVT (Deep vein
thrombosis)  highlighting the importance of the
functional study of the platelet mainly through the
flow-cytometric study of their activation.
Since the platelet number is not the only
factor that controls the thrombotic or hemorrhagic
5 An update on the platelet dysfunction
diathesis in MPD, the importance of studying
platelet function is increasing. Outlining the genetic
profile (hence the molecular changes) may provide
early predictors for potentially vital complications
in patients diagnosed or susceptible of MPD.
Acknowledgements. The current article was written
based on the data collected during a National Research Grant
in the Program “Excellence in Research” – MULTRO (A
complex, multidiscipline study of platelet in myelodysplastic
and myeloproliferative syndromes with 5 partners) sponsored
by the Romanian Ministry of Research and Development.
Diateza trombotică şi cea hemoragică reprezintă complicaţii frecvente în
sindroamele mieloproliferative cronice (SPMC) şi în sindroamele mielodisplazice
(SMD). Aceste modificări sunt corelate atât cu numărul absolut de trombocite, cât
şi cu prezenţa anumitor defecte calitative, precum alterări ale glicoproteinelor
membranare, care pot fi evidenţiate prin diferite teste plachetare, inclusiv examenul
flowcytometric. Modificările glicoproteinelor membranare induc alterarea activării,
secreţiei şi agregării plachetare. Receptorul pentru trombopoietina (cMPL) afectat
de mutaţia JAK2 V617, întâlnită în sindroamele mieloproliferative, poate fi asociat
cu apariţia unui status protrombotic. Implicaţia sa relevă importanţa profilului
genetic molecular în definirea unor markeri moleculari de diagnostic, făcându-l
astfel candidat pentru diagnosticul precoce al sindroamelor mieloproliferative şi
predictor pentru complicaţiile trombotice apărute în acest grup de boli.
Author correspondence: Ana-Maria Vlădăreanu, Assistant Professor
Department of Hematology, Emergency Universitary Hospital
169, Splaiul Independenţei, 050098, Bucharest, Romania
Phone no: +4021 318 05 22, Fax: +4021 318 05 70
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Received February 7, 2008