THE SPECTRUM OF JAK2-POSITIVE MYELOPROLIFERATIVE NEOPLASMS: COMPLICATIONS AND THERAPEUTIC ADVANCES

Thrombotic disease in the myeloproliferative neoplasms

Anna Falanga1 and Marina Marchetti1
1Division

of Immunohematology and Transfusion Medicine, Ospedali Riuniti di Bergamo, Bergamo, Italy

Thrombosis is a leading cause of morbidity and mortality in patients with Philadelphia chromosomenegative
myeloproliferative neoplasms (MPNs), particularly polycythemia vera and essential thrombocythemia. Mechanisms
involved in the pathogenesis of the acquired thrombophilic state associated with these diseases include abnormalities
of MPN clonederived blood cells, which display prothrombotic features, and abnormalities of normal vascular cells,
which become procoagulant in response to inflammatory stimuli. Ultimately, the release into the blood of elevated
levels of procoagulant microparticles by platelets and vascular cells and the increase in the global thrombin generation
due to an acquired activated protein C resistance result in a highly prothrombotic scenario in patients with
polycythemia vera and essential thrombocythemia. The acquired point mutation in the pseudokinase domain of JAK2
(JAK2V617F) in these disorders is variably associated with thrombosis and, more consistently, with elevations in WBC
counts and alterations in biomarkers of blood-clotting abnormalities. The predictive value of these biomarkers for
thrombosis remains to be established to identify subsets of patients at elevated risk who may benefit from prophylaxis
with antithrombotic drugs.

Introduction
Myeloproliferative neoplasms (MPNs) are clonal disorders of
hematopoietic stem cells characterized by proliferation of one or
more myeloid cell lines (granulocytic, erythroid, megakaryocytic,
and mast cells). According to the World Health Organization
(WHO) classification, these include classic MPN Philadelphia
chromosome (Ph)negative diseases which include polycythemia
vera (PV), essential thrombocythemia (ET), and myelofibrosis
(MF). The discovery of recurrent molecular abnormalities such as
JAK2V617F and MPL W515L/K has reinforced the original idea of
Dameshek in 1951 that these diseases share a common pathogenetic
mechanism and presumably belong to a single disorder. Clonality
studies have established that PV, ET, and MF are diseases that
originate from a common hematopoietic stem cell that is hypersensitive to the action of growth factors. The result is an autonomous
proliferation of hematopoietic colonies, which leads to overproduction of red blood cells (RBCs), platelets, and leukocytes. Among
these disorders, ET has the most favorable outcome. Patients with
ET have a lifespan that nearly rivals that of a healthy population
matched by age and sex. However, the life expectancy of patients
with PV and ET is strongly affected by disease-related hemostatic
complications, including thrombosis and, to a lesser extent, hemorrhages. Reported incidence of thrombosis ranges from 12%-39% in
PV and from 11%-25% in ET.1 Clinical manifestations of thrombosis vary from mild microcirculatory disturbances to more serious
complications such as arterial and venous thrombosis (Figure 1).
Arterial thrombosis, which accounts for 60%-70% of events related
to MPNs, includes ischemic stroke, acute myocardial infarction, and
peripheral arterial occlusion. The high incidence of heart valvular
lesions in MPNs raises the possibility that a significant proportion of
these may be related to emboli of cardiac origin. Events involving
the venous system are represented by deep venous thrombosis of the
lower extremities, pulmonary embolism, intraabdominal (hepatic,
portal, and mesenteric) and cerebral vein thrombosis. In PV, venous
thromboses are relatively common and constitute approximately
one-third of total events. The prevalence of splanchnic and cerebral

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vein thrombosis is unusually high among patients with MPN, and

these events are often the presenting feature of the disease before
diagnosis.2 MPNs constitute the most common cause of splanchnic
venous thromboses, accounting for approximately 50% of Budd
Chiari syndrome (BCS) cases and 25% of portal vein thromboses
(PVT). MPNs associated with BCS and PVT have unique features
compared with classic MPNs, including the onset at a younger
age, female predominance in PV, and the presence of normal blood
counts, which sometimes renders the diagnosis of MPN quite
challenging.3
Typical of, but not exclusive to, ET is the involvement of the
microcirculatory system, which manifests as erythromelalgia, transient ischemic attacks, visual or hearing transitory defects, recurrent
headache, and peripheral paresthesia. Focal symptoms such as
dysarthria, transient monocular blindness, or transient mono- or
hemiparesis are less common. Visual dysfunction may also manifest
as transient diplopia and sudden reversible attacks of blurred vision.
ET and PV management remains highly dependent on the patients
thrombotic risk.4 Because the use of myelosuppressive drugs (eg,
hydroxyurea) can reduce the rate of thromboses and hemorrhages in
these patients, but can also accelerate the rate of leukemic transformation, a risk-oriented management strategy is highly recommended. Older age ( 60 years) and previous thrombosis are well
established cardiovascular risk factors for thrombosis in these
patients. The absence of both of these 2 risk factors identifies
low-risk patients. There is great attention being given to moving
beyond these recognized risk factors, particularly in the young or
asymptomatic low- and intermediate-risk patients not without risk
of thrombosis. Recently, the impact of new risk factors, such as
leukocytosis and JAK2V617F mutational status and/or mutational
burden, has begun to be under active investigation.
Even in the absence of thrombotic manifestation, ET and PV
patients present with a hypercoagulable state, which is a laboratory
finding of increased levels of plasma biomarkers of hemostatic
system activation.5-6 Therefore, an acquired thrombophilic state

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Figure 1. The spectrum of thrombotic manifestation in ET and PV. Thrombophilia, which severely affects the morbidity and mortality of PV and ET,
is variably characterized by microcirculatory disturbances and arterial and venous thromboses that often precede disease recognition. Thrombotic
occlusions of large arteries most commonly involve cerebral or coronary vessels. Ischemic stroke constitutes 30%-40% of all thrombotic events in PV
patients. Acute coronary syndromes are more rare, particularly during followup of treated patients. Acute vascular occlusions in other areas are not
uncommon both in PV and ET patients. Venous thrombosis usually manifests with an increased incidence of deep venous thromboses of the lower
limbs, which may cause pulmonary embolism. Superficial phlebitis of the legs is also common and venous thromboses at unusual sites are not as rare as
in the general population. Thromboses of cerebral sinuses and of splanchnic (portal and hepatic) veins have been repeatedly reported in relatively young
female patients. Microcirculatory disturbances are the most peculiar thrombotic manifestations in PV and ET patients and are responsible for a wide
range of clinical symptoms arising from the formation of platelet thrombi in the end-arterial circulation of the peripheral, cerebral, coronary, skin, and
abdominal vessels.

develops in these patients, who are prone to vascular complications,

but the mechanisms ultimately responsible for blood activation
coagulation and the increased thrombotic tendency in ET and PV
have not been fully elucidated.

Pathogenesis of thrombosis
The pathogenesis of thrombosis in ET and PV is multifaceted.
However, 2 main mechanisms recapitulate the origin of the hypercoagulable state in these disorders. One relies on the abnormalities of
the blood cells (ie, platelets, erythrocytes, and leukocytes) arising
from the clonal hematopoietic progenitor cells, which express a
prothrombotic phenotype, and the other involves the inflammatory
response of host vascular cells to the insult of cytokines and other
mediators released by malignant cells (Figure 2).
The alterations of circulating MPN clone derived blood cells entail
not only quantitative changes in the number of circulating blood
cells, with consequent hyperviscosity, but also qualitative changes
that lead to the expression of procoagulant characteristics. The
prothrombotic factors expressed by transformed vascular cells (ie,

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platelets, RBCs, and leukocytes) include the production of procoagulant and proteolytic molecules, secretion of inflammatory cytokines,
and expression of cell adhesion molecules. In addition, blood
hyperviscosity and increased levels of leukocyte-derived proteases
(ie, elastase, cathepsin-G, and myeloperoxidase) may contribute to
the thrombophilic state by affecting the integrity of endothelial cell
monolayer. Abnormalities of the endothelium do occur in MPN Ph
diseases. Activated/injured endothelial cells express higher levels of
adhesion molecules on their surface, which favors platelet and
leukocyte arrest, allowing the localized secretion of thrombogenic
and angiogenic peptides by inflammatory cells. In this scenario, the
increased production of procoagulant microparticles from activated
platelets and vascular cells and the increased thrombin generation
due to an acquired resistance to activated protein C (APC) represent
very important mechanisms that trigger systemic hypercoagulation
and ultimately thrombosis in ET and PV patients.

Abnormalities of MPN vascular cells

Platelets. The role of thrombocytosis in the pathogenesis of
thrombotic events is controversial. Although clinical improvement

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Figure 2. Pathogenesis of thrombophilia in MPN. The pathogenesis of the acquired thrombophilic state in ET and PV is multifaceted. However,
2 main mechanisms recapitulate the origin of hypercoagulation in these disorders. One relies on the abnormalities of blood cells (ie, platelets,
erythrocytes, and leukocytes) arising from the clonal proliferation of hematopoietic progenitor cells, which acquire a prothrombotic phenotype. The
other generates from the host inflammatory response to the insult of cytokines and other mediators by the malignant cells. The latter mechanism also
contributes to thrombosis in nonmalignant conditions.

of microcirculatory disturbances and/or improved platelet function

after control of thrombocytosis have been reported, no clear
correlation of thrombocytosis with risk of major cardiovascular
events has been demonstrated.7-8 For example, in both the
Polycythemia Vera Study Group (PVSG)9 and the European
Collaboration on Low-Dose Aspirin in Polycythemia Vera
(ECLAP)10 prospective trials, platelet count did not predict for
thrombosis. The results of the only randomized clinical trial to date,
the Primary Thrombocythaemia 1 (PT1) study, which randomized
high-risk ET patients to hydroxyurea (a global myelosuppressive
agent) or anagrelide (a platelet-only reducing agent), showed that,
despite a similar control of platelet count by either drug, the
composite primary end point (ie, arterial or venous thrombosis,
serious hemorrhage, or death from vascular causes) occurred more
frequently in recipients of anagrelide plus aspirin than in those
receiving hydroxyurea plus aspirin.11 The global effects of hydroxyurea on blood cell populations other than platelets (eg,
leukocytes) might underlie the correlation between control of
platelet count and reduction of thrombosis rate observed in the first
prospective study in high-risk ET patients by Cortelazzo et al.12 In
contrast, in MF, a condition characterized by less frequent cardiovascular events than in PV or ET, a correlation between thrombocytosis
and thrombosis was found.13
Conversely, extreme thrombocytosis (ie, platelets 1500 109/L)
can favor hemorrhagic rather than thrombotic manifestations in

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ET patients.12 This paradox has been attributed to the possible

occurrence of an acquired VWD due to an increased clearance by
platelets of the large circulating VWF multimers.14 Platelet count
reduction proved successful in normalizing the plasma VWF
multimer pattern and in reducing bleeding tendency.7,8,15
Despite the inconclusive data on the role of thrombocytosis, several
other lines of evidence support a contribution of platelets to the
pathogenesis of thrombosis in ET and PV. For example, the prompt
relief of symptoms with aspirin, together with the normalization of
platelet activation tests, suggest a role of platelets in mediating
microvessel occlusions (ie, erythromelalgia) in ET patients.16 In
contrast to the inefficacy of the oral anticoagulant warfarin, control
of platelet function with low-dose aspirin and normalization of
platelet counts prevents the recurrence of microvascular circulation
disturbances in the end-arterial microvasculature of the cerebral,
coronary, and peripheral circulation. Furthermore, low-dose aspirin
significantly reduces the risk of cardiovascular events in PV, as
demonstrated by the ECLAP randomized clinical trial. Patients
randomized to receive aspirin had a 60% reduction of combined end
point of nonfatal acute myocardial infarction, nonfatal stroke, or
death from cardiovascular causes.10
The characteristics of thrombohemorrhagic diathesis in ET and PV
prompted the design of many in vitro studies to demonstrate and
characterize possible platelet abnormalities. In the past, numerous

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Figure 3. Role of platelet abnormalities in MPN-associated thrombophilia. Many studies have investigated the contribution of platelets in the onset
of the thrombophilic state in MPNs, and it is now clear that the increased platelet count is not a major element in the risk of thrombosis. Rather, platelet
qualitative abnormalities have been implicated in the pathogenesis of hypercoagulability in ET and PV patients. Increased expression of P-selectin,
thrombospondin, and the activated fibrinogen receptor GPIIb/IIIa by platelets has been found to be correlated with thrombosis. The formation of
platelet-leukocyte aggregates, platelet activation, and microparticle shedding are also implicated in the pathogenesis of thrombosis in these patients.
Microparticles expose the anionic phosphatidylserine, providing a catalytic surface for the generation of thrombin, which further amplifies platelet
activation.

platelet defects have been identified in ET and PV patients. The

majority of these observations were related to a decreased functionality and included abnormal platelet aggregation, reduced levels of
membrane adhesion molecules (ie, glycoprotein Ib [GPIb], GPIIbIIIa, GPIV, and GPVI), acquired storage pool disease, and defective
platelet metabolism (ie, abnormal arachidonic acid metabolism).17,18
In contrast, more recent studies show that platelets from these
patients circulate in an activated status, as assessed by the detection
of increased expression on their surface of P-selectin and tissue
factor (TF).19,20 It may be that the permanent activation status of the
MPN platelets may exhaust their functions, leading to a reduced
response to the stimuli in vitro. We found that although the
circulating ET platelets express high surface P-selectin, their
response to ADP and epinephrine stimulation is inferior to that of
healthy control platelets. Similar dysfunctions in ET and PV
platelets were also reported by others.21 Furthermore, some platelet
aggregation abnormalities in ET may be caused by laboratory
artifacts due to the extensive dilution of platelet-rich plasma needed
in the aggregation assay.22 The evidence of enhanced in vivo
platelet activation in ET and PV patients is further demonstrated by
the finding of increased release of platelet activation products both
in plasma (ie, beta-thromboglobulin and platelet factor 4) and urine
(ie, the thromboxane A2 [TxA2] metabolites 11-dehydro-TxB2 and
2,3-dinor-TxB2).21,23 Once activated, platelets expose on their
surface the anionic phosphatidylserine, usually kept on the inner
leaflet (ie, the cytosolic side) of cell membrane, providing a catalytic

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surface for the generation of thrombin, which further amplifies

platelet activation (Figure 3).
Recently, our group demonstrated in patients with ET and PV, and
particularly in carriers of the JAK2V617F mutation, an increased
thrombin generation capacity of platelets, which was associated
with the occurrence of platelet activation. Cytoreductive therapy
with hydroxyurea significantly affected this prothrombotic phenotype.24 We also showed that immature platelets, which are more
reactive than their mature counterparts, are also increased in patients
with ET or PV (Figure 4) and are significantly down-regulated by
hydroxyurea.25 No studies have been performed to compare, in the
same patient, platelets with and without JAK2V617F mutation.
Conversely, there are data comparing platelets from JAK2V617F
with those from JAK2V617F patients and platelets from subjects
with different allele mutation burden. In a study of MPN patients by
our group, a direct association between JAK2V617F allele burden
and increased platelet-associated thrombin generation potential was
observed.24
It is relevant to discuss whether thrombopoietin (TPO) and its
receptor (c-MPL) may play a role on platelet function relative to the
presence of the JAK2V617F mutation. A decreased expression of
c-MPL on platelets and megakaryocytes surface is an established
characteristic of PV and MF, but not of ET. Very recently, it was
shown that c-MPL expression is down-regulated in JAK2V617F

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Figure 4. Immature platelets are increased in JAK2V617F ET and PV patients. Figure shows data on immature platelet fraction (IPF) values
according to disease type, JAK2V617F mutation, and hydroxyurea (HU) treatment. Both ET and PV patients positive for the JAK2V617F mutation and
not receiving HU showed significantly higher IPF count compared with control subjects (P .05). In addition, JAK2V617F ET and PV patients not
treated with HU showed significantly higher IPF count compared with JAK2V617F and HU-treated JAK2V617F and JAK2V617F patients not
treated with HU. *P .05 compared with controls. Modified with permission from Panova-Noeva et al.24

platelets in MF due to an increased c-MPL degradation, leading to a

megakaryocytic cell enhanced cell survival and proliferation. However, very little information is available on the possible relationship
between TPO/cMPL and the hemostatic platelet functions. There is
evidence that TPO induces a potentiation of platelet aggregation and
dense-granule secretion after standard stimuli (eg, collagen, ADP,
and epinephrine) in patients with MPN.26 However, relationships
between c-MPL levels, TPO levels, JAK2V617F mutation, and
platelet hemostatic function have not been explored as yet. It may be
possible that the constitutive activation of c-MPL in platelets
positive for JAK2V617F may render these cells more reactive to
stimuli and determine an increased expression of P-selectin.

RBCs. The prothrombotic effect of an elevated hematocrit has been

clearly demonstrated in PV by the observation that a progressively
higher hematocrit corresponds to an increase in the thrombotic
risk.27 Hematocrit levels around the upper limit of normal values
may be an important factor in the causation of occlusive vascular
diseases, particularly in the cerebral circulation, as a consequence of
an increased blood viscosity. Indeed, at high hematocrit values
(47%-53%), the cerebral blood flow is significantly slower than at
hematocrit values in the lower range (36%-46%). In untreated
PV subjects, most thrombotic accidents occur in the cerebral
circulation, which is particularly sensitive to blood hyperviscosity.
At high shear rates, the raise of RBC mass displaces platelets toward
the vessel wall, thus facilitating shear-induced platelet activation
and enhancing platelet-to-platelet interactions. In addition, at low
shear rates, as in the venous bed, hyperviscosity can increase the
thrombotic risk by causing a major disturbance to the blood flow. A
proper management of blood hyperviscosity is essential but does not
abolish the in vivo platelet activation and the increased thrombotic
risk in PV subjects.23 In addition to an increased RBC count,
biochemical changes in cell membrane and intracellular content of
RBCs are also found in ET and PV.28 These changes may
independently impair blood flow through the formation of RBC
aggregates that have the potential to block blood flow in small

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vessels directly and facilitate platelet-leukocyte interaction. This

likely contributes to ischemia and infarct, especially in the cerebral
blood flow.

Leukocytes. Many retrospective studies have identified leukocytosis as a potential risk factor for arterial and venous thrombosis
in patients with ET and PV.29-32 Leukocytosis was also a risk factor
for recurrent arterial thrombosis in young (ie, 60 years) ET and
PV patients (hazard ratio for arterial recurrence 3.35, 95% confidence interval [CI], 1.22-9.19).33 In contrast, leukocytosis at
diagnosis (defined by a cutoff level of either 15 or 9.4 109/L) did
not appear to influence the risk of thrombosis in a retrospective
cohort of low-risk ET or PV patients.34 Prospective clinical studies
with stratification of patients according to their baseline leukocyte
counts are needed to definitely classify leukocytosis as a prognostic risk factor. Leukocytes can contribute to the pathogenesis of
thrombosis in ET and PV through recently discovered mechanisms
of activation and interaction with platelets, endothelial cells, and the
coagulation system. In addition, leukocytes contribute to inflammatory processes in atherosclerotic plaques and in this way increase
the probability of vascular events. Because neutrophils represent
the most abundant proportion of the circulating leukocytes, a role
for neutrophils in thrombosis of MPN has been hypothesized.
Neutrophils have a central role in the inflammatory response and
also in linking the inflammatory response to the activation of blood
coagulation.35 Once activated, neutrophils produce reactive oxygen
species, release proteolytic enzymes from their cytoplasmic azurophilic granules (ie, elastase and cathepsin G), and express higher
and functional levels of the 2 integrin Mac 1 (or CD11b) on their
cell surface. All of these molecules can affect the hemostatic system
and induce a prothrombotic condition.36 The fact that activated
neutrophils can induce a hypercoagulable state in vivo has been well
demonstrated by a study of a group of healthy donors administered
G-CSF for the mobilization and collection of peripheral blood
progenitor cells.37 G-CSF caused activation of neutrophils in these
subjects, which was associated with a parallel increment in plasma

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levels of biomarkers of activation of blood coagulation and endothelium. These effects were transient, persisting only as long as the
growth factor was administered (ie, 5-6 days), and normalized after
G-CSF withdrawal.
In ET and PV patients, neutrophil activation has been demonstrated
by detection of specific phenotypical changes (by increments in
membrane-associated CD11b) and of increased plasma concentration of neutrophil granule derived proteases (ie, elastase and
myeloperoxidase).5,19 These abnormalities are directly correlated
with increased plasma levels of biomarkers of both blood clotting
and endothelium activation, supporting a possible involvement of
neutrophils in the pathogenesis of the hypercoagulable state in these
disorders. Several studies described increased levels of circulating
platelet-neutrophil aggregates in ET and PV patients and attributed
this phenomenon to both platelet and neutrophil activation.38
Interestingly, in patients receiving aspirin, the increments in CD11b
expression and neutrophil/platelet aggregates induced by in vitro
stimulation were significantly lower compared with nonaspirintreated subjects, suggesting that aspirin treatment may inhibit the
interaction between neutrophils and platelets.19 Because the G-CSF
receptor is linked to the JAK2 pathway, it is possible that the
constitutive activation of signaling through this receptor in the
presence of JAK2V617F mutation can be in part responsible for the
activated phenotype of neutrophils in patients with ET and PV.
However, our data on neutrophil activation analyzed according to
the JAK2V617F mutation39 show that the levels of only some of the
activation markers are more altered in the JAK2V617F than in the
JAK2V617F ones (ie, CD14 and leukocyte alkaline phosphatase),
whereas others, (ie, CD11b and plasma elastase) are equally
elevated in both groups. It is likely that mechanisms independent of
the JAK2V617F mutation are present in non-JAK2V617F mutation
carriers.

Abnormalities of host vascular cells

Endothelium. Physiologically, the endothelium facilitates blood
flow by providing an antithrombotic surface that inhibits platelet
adhesion and coagulation activation. Several factors may perturb the
resting state of endothelium in patients with MPN and turn it into a
proadhesive and procoagulant surface. Reactive oxygen species and
intracellular proteases released by activated neutrophils can induce
detachment or lysis of endothelial cells, affecting functions involved
in thromboregulation. It has also been demonstrated that damage of
endothelium determines the release in circulation of specific markers including thrombomodulin, selectins, and VWF. This is of
particular relevance in the pathogenesis of thrombosis in MPN
because once platelets bind to VWF, they become activated and able
to aggregate and strengthen the clot.40 Selectins are a family of
adhesion molecules expressed by endothelial cells (P-selectin and
E-selectin), platelets (P-selectin), and leukocytes (L-selectin).41
Because they are released into the circulation, their presence has
been used as an index of endothelial, platelet, and leukocyte
activation. In patients with ET and PV, we measured increased
plasma levels of soluble P-, E-, and L-selectins42 and soluble
thrombomodulin.39 High levels of membrane-bound and plasmasoluble P-selectin and E-selectin have been found in ET patients
with thrombosis, suggesting that sustained endothelium and platelet
activation might contribute to the pathogenesis of thrombosis in
these diseases.43 In addition to releasing substances that stimulate
thrombus formation after injury, endothelial cells release the platelet
inhibitor nitric oxide (NO), providing a negative feedback mechanism for the propagation of thrombus formation.44 NO is a free
radical product generated through the oxidation of L-arginine to

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L-citrulline by NO synthases. The endothelial-derived NO is one of

the main mediators influencing vascular hemodynamic and the
interaction of leukocytes and platelets with endothelial cells. In fact,
NO mediates vascular relaxation in response to vasoactive substances and shear stress; inhibits platelet adhesion, activation,
secretion, and aggregation; and promotes platelet disaggregation.
Moreover, NO inhibits the expression of P-selectin on platelets and
impairs leukocyte adhesion to the endothelium. Clinical conditions
have been reported in which a deficiency of endogenous NO
production may contribute to a thrombotic event. Our group
recently studied circulating NO in MPN patients42 and found
reduced plasma levels of NO in patients with ET compared with
controls. This confirms the previous observation that in MPN
patients with thrombocytosis, the production of NO by platelets is
impaired.45 However, in the same study and for the first time, we
observed that ET patients treated with hydroxyurea presented with
the highest levels of plasma NO. A similar effect of hydroxyurea on
NO plasma levels was reported in patients with sickle cell anemia46
and may contribute to the known ability of hydroxyurea to prevent
thromboembolic complications in ET patients.12 In the same study,
PV patients showed high plasma NO levels compared with controls
and these levels were not affected by hydroxyurea treatment. This
result was as expected, because a high hematocrit level is associated
with increased NO in the blood and may represent a compensatory
mechanism in a high-thrombotic-risk situation.
Recent studies have shown that angiogenesis plays an important
role in the biology of hematological malignancies including MPN.47
Levels of circulating endothelial cells, together with serum levels of
VEGF and other proangiogenic cytokines, have been studied in
MPN as markers of angiogenetic activity. The number of circulating
endothelial cells (resting, activated, apoptotic, and circulating
precursor endothelial cells) was repeatedly found to be increased in
patients with MPN regardless of JAK2V617F status and was not
affected by cytoreductive treatment.48-50
All of these lines of evidence suggest that endothelium in MPN is
activated, suggesting an important endothelial contribution to the
hypercoagulable state. In addition, angiogenesis may have a role in
the pathophysiology of MPN.

Role of the JAK2V617F mutation

The demonstration of the acquired gain-of-function V617F mutation in the tyrosine kinase JAK-2 gene has greatly influenced the
diagnostic and therapeutic approach in MPN patients. Several
studies have implicated the JAK2V617F mutation in the increased
thrombotic tendency observed in ET and PV patients and
3 independent meta-analyses were published recently.51-53 In the
analysis by Ziakas et al involving 2905 ET patients,51 the
JAK2V617F mutation was associated with an increased risk of both
venous (odds ratio [OR] 2.09; 95% CI, 1.44-3.05) and arterial
thrombosis (OR 1.96; 95% CI, 1.43-2.67), as well as thrombosis
at presentation (OR 1.88; 95% CI, 1.38-2.56). The meta-analysis
by Dahabreh et al showed in 2436 ET patients that the risk of arterial
(OR 1.68; 95% CI, 1.31-2.15) and venous (OR 2.5; 95% CI,
1.71-3.66) thromboses were significantly increased in JAK2V617F
compared with wild-type patients.52 Similarly, the most recent
meta-analysis of 21 studies involving ET patients and 6 studies with
MF, showed that in ET patients, the JAK2V617F mutation is
associated with a significant 2-fold increased risk of thrombosis
(OR 1.92; 95% CI, 1.45-2.53) of both venous (OR 2.49; 95%
CI, 1.71-3.61) and arterial (OR 1.77; 95% CI, 1.29-2.43) vessels;
its role in PMF patients is uncertain.53 The association between the

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Figure 5. Hemostatic alterations in JAK2V617F and JAK2V617F MPN patients. Abnormalities of platelets, erythrocytes, leukocytes, and
endothelial cells lead to systemic hypercoagulability as represented by an increased production of procoagulant microparticles and the occurrence of
an acquired APC resistance. Studies addressing whether the JAK2V617F mutation may specifically affect the hemostatic system indicate that both
cellular (ie, platelets and leukocytes) and plasma compartments of hemostasis are more activated in MPN patients positive for the JAK2V617F mutation.
Therefore, the expression of the JAK2V617F mutation may represent a molecular lesion relevant to promote the cellular procoagulant phenotype.
N indicates not different from healthy control subjects; m, elevated compared with healthy control subjects; mm, elevated compared with
JAK2V617F patients.

JAK2V617F mutation and thrombosis was recently confirmed in a

retrospective multicenter study from Korea involving 239 patients
with ET.54 In that study, previous thrombotic history and the
JAK2V617F mutation were associated with a higher 10-year
cumulative incidence rate of thrombohemorrhagic events.
Although many studies and 3 meta-analysis have been published on
the role of JAK2V617F mutation in the increased thrombotic
tendency in ET, few data are available in PV. In one prospective
study by Vannucchi et al including 173 PV patients, those subjects
with a mutant allele burden 75% had a 3.56-fold higher relative
risk (95% CI, 1.47-7.1) of total thrombosis, particularly during the
follow-up (relative risk 7.1; 95% CI, 1.6-10.1).55 A retrospective
analysis of patients with either PV or ET found that the frequency of
thrombosis progressively increased according to the allele burden in
both types of diseases, with the highest rate of vascular complications in patients with an allele burden 50%.56 However, in a
recent prospective study of patients with PV, no significant relationship between the mutant allele burden and the risk of thrombosis
was observed.57

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Few studies have been published addressing whether the JAK2V617F

mutation may specifically affect the hemostatic system.20,39,58,59
These studies indicate that both cellular (ie, platelets and leukocytes) and plasma compartments of hemostasis were more activated
in those patients positive for the JAK2V617F mutation (Figure 5).
Regarding cellular components, P-selectin was found to be increased on platelets from JAK2V617F ET patients20; CD14 and
LAP were demonstrated to be more highly expressed on neutrophils
from ET JAK2V617F mutation carriers39; and a significantly higher
expression of CD11b was observed on neutrophils and monocytes
from JAK2V617F PMF patients.59 In addition, our group were
able to demonstrate an elevated expression of TF in platelets and
increased levels of circulating platelet/neutrophil aggregates from
JAK2V617F ET patients compared with JAK2V617F patients.39
The evidence that both platelets and neutrophils from JAK2V617F
patients expressed increased activation features is in good agreement with the findings of increased mixed-cell aggregate formation
in ET patients carrying the JAK2V617F mutation. Among hypercoagulation parameters, plasma levels of soluble thrombomodulin
were found to be elevated in JAK2V617F ET patients,39 and

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soluble P-selectin levels were significantly elevated in JAK2V617F

ET, PV, and PMF patients compared with JAK2V617F patients.58
In a study by Marchetti et al, an involvement of the JAK2V617F
mutation was observed in the presence of an acquired APC
resistance phenotype.60
Circulating platelets are heterogeneous in size and structure. In
healthy subjects, a small percentage of circulating platelets (2%) are
the so-called reticulated or immature platelets recently released
from the BM. Their number, reflecting the rate of thrombopoiesis, is
directly correlated with megakaryocyte proliferating activity. In
vitro studies show that newly formed platelets have a higher
hemostatic activity compared with mature platelets, as demonstrated by the increased response to thrombin and higher expression
of surface P-selectin.61
Recently, in 46 ET and 38 PV consecutive patients, we found a
positive correlation between the JAK2V617F mutation and the
quantity of immature platelets.25 Furthermore, we observed that the
levels of circulating immature platelets are susceptible to myelosuppressive treatment, which may explain the favorable effect of
hydroxyurea therapy on MPN outcome, as well as the associated
thrombotic risk.
Few studies have evaluated the molecular mechanisms by which
JAK2V617F mutation can affect the prothrombotic phenotype of a
cell. The JAK2V617F-activating mutation can cause an increase in
RBC adhesiveness through modification of surface adhesion molecules, thus facilitating thrombosis, and may render platelet hyperresponsive through altered expression of c-MPL signal transduction
for TPO-induced platelet priming.62 Activation of JAK2 is also
involved in TF expression by neutrophil and monocyte through the
MAPK and PI3K pathway.63
Finally, the presence of JAK2V617F in both endothelial cells and
hematopoietic cells belonging to BCS patients with PV has been
demonstrated. This indicates that endothelial cells from these
patients are involved in the malignant process and suggests that in
this subpopulation of patients, the disease may originate from a cell
common to the hematopoietic and endothelial cell systems.64
Other somatic mutations have been characterized in MPN, including
several mutations in JAK2 exon 12 ( 5% of JAK2V617F PV
patients), and a mutation in the MPL gene (MPL W515) described
in approximately 10% of PMF patients and 1% of ET patients, but
not in PV patients. No information is available on the thrombophilic
state of MPN patients carrying these rare mutations. The JAK2 exon
12 and MPL W515 mutations have not been detected in patients
with BCS and PVT,3 whereas MPL-mutated ET has been associated
with high platelet count, microvascular symptoms, and a high risk of
postdiagnosis arterial thrombosis.65,66

Main prothrombotic features in the blood

In patients with ET and PV, the main prothrombotic features
that manifest in the blood as a consequence of the activation of
MPN clone derived blood cells and host normal vascular cells
are an increased thrombin generation sustained by an acquired
APC resistance, and the appearance in the circulation of high
levels of procoagulant microparticles derived from platelets and
vascular cells.

578

Acquired APC resistance

PC, a serine protease activated by thrombin bound to the endothelial
receptor thrombomodulin, is one of the major physiological anticoagulant in humans. APC, in complex with its cofactor, protein S
(PS), reduces blood clotting activation through the proteolytic
inactivation of coagulation factor V and factor VIII. A resistance to
inactivation by APC, inherited or acquired, is associated with an
increased risk of thrombosis in many situations, including pregnancy, oral contraceptive use, hormone replacement therapy, and
cancer. Decreased levels of PC and PS can be responsible for the
occurrence of an APC-resistant phenotype, and studies have reported a reduction in the concentration of natural anticoagulants in
patients with MPN.67 By using the thrombin-generation assay, we
demonstrated the occurrence of an acquired APC resistance phenotype in ET and PV patients.60 The results analyzed according to the
presence (ie, positivity or negativity) and status (ie, heterozygosity
or homozygosity) of the JAK2V617F mutation indicated that
JAK2V617F mutation carriers are more APC resistant than noncarriers, especially if they are homozygous (ie, JAK2V617F allele
burden 50%). This suggests a progression to the APC-resistant
phenotype determined by the JAK2V617F status and, together with
previous observations, supports the hypothesis of a more hypercoagulable condition in JAK2V617F carriers. Prothrombin, factor V,
free PS, and tissue factor pathway inhibitor levels were significantly
reduced in patients and mainly in JAK2V617F carriers. Multiple
regression analysis indicated that the low free PS level is a major
determinant of the increased APC resistance. A high prevalence of
acquired APC resistance, determined as the classical prolongation
of activated partial thromboplastin time after the addition of APC,
was reported by Arellano-Rodrigo et al in patients with ET6 and was
found more frequently in those patients with previous history of
thrombosis; again, decreased levels of free PS were detected and
were inversely correlated with JAK2V617F allele burden.
Several lines of research indicate that a protease from platelets is
able to cleave PS in plasma.68 On the basis of the finding of
decreased PS levels in MPN, we decided to investigate the
relationship between platelet-associated PS cleaving activity and in
vivo PS cleavage in a group of ET patients.69 PS cleavage was found
to be significantly increased in patients with ET, along with elevated
levels of platelets, and returned to normal values in ET patients on
hydroxyurea treatment with normal platelet counts. Therefore,
proteases from platelets seem to contribute to the presence of
cleaved PS in the circulation of ET patients and may enhance the
coagulation response in vivo by down-regulating the anticoagulant
activity of PS.

Plasma microparticles
Microparticles are membrane fragments ranging in size from
0.1-1 m that are released by most cell types, including blood
and vascular cells, upon activation. Microparticles are known to
be elevated in thromboembolic diseases and malignancy,70
including patients with ET.71 The levels and cellular origin of
microparticles were determined by flow cytometric analysis, and
the microparticle-associated procoagulant activity was measured
using a thrombin-generation assay. A significantly higher number of circulating microparticles positive for platelet and endothelial markers, as well as for TF, were found in ET subjects
compared with controls. In addition, microparticle-rich plasma
from patients with ET showed higher thrombin-generation
potential, which was significantly correlated with the total
number of microparticles. A subsequent study by Duchemin et al

American Society of Hematology

has shown the presence of an acquired thrombomodulinresistant phenotype in PV and ET patients that is partially
determined by circulating microparticles.72

5.

Conclusions and future perspectives

Thrombotic complications significantly affect the morbidity and
mortality of patients with MPN. In addition, MPN patients commonly present with abnormalities in laboratory coagulation tests
that are consistent with a hypercoagulable state. The pathogenesis of
blood activation in these diseases is complex and multifactorial, and
involves the abnormalities of platelets, erythrocytes, and leukocytes
arising from the clonal proliferation of hematopoietic progenitor
cells and abnormalities of endothelial cells. These alterations
include not only quantitative changes in the number of circulating
blood cells, but also qualitative changes in cellular molecular
characteristics. These properties lead to an increased production of
procoagulant microparticles and the occurrence of an APC-resistant
phenotype and very likely contribute to clotting system activation.
Clinical data indicate an association of the JAK2V617F mutation
with disease severity. Biological data also show an association of
this mutation with the expression of cellular and soluble biomarkers
of coagulation activation. However, future clinical research should
focus on the evaluation of the role of these biomarkers in identifying
MPN patients at higher risk of thrombosis, who may benefit from
primary thromboprophylaxis. Finally, a better understanding of the
molecular events leading to the development of the hypercoagulable
state in Ph MPN patients may provide appropriate tools for
targeted therapies to reverse coagulopathy.

6.

7.

8.

9.

10.

11.

12.

Acknowledgments
The work conducted at the Laboratory of the Division of Immunohematology and Transfusion Medicine, Ospedali Riuniti di Bergamo
(Bergamo, Italy) was partially supported by grants from the
Associazione Italiana per la Ricerca sul Cancro and from the
National Institutes of Health Myeloproliferative Disorders Research
Consortium (both to A.F.).

13.

14.

Disclosures
Conflict-of-interest disclosure: The authors declare no competing
financial interests. Off-label drug use: None disclosed.

Correspondence

15.
16.

Anna Falanga, MD, Division of Immunohematology and Transfusion Medicine, Ospedali Riuniti di Bergamo, Italy, Largo Barozzi,
1, 24128 Bergamo, Italy; Phone: 39-035-266578; Fax: 39-035266659; e-mail: annafalanga@yahoo.com.
17.

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