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 chromosome–negativemyeloproliferative neoplasms (MPNs), particularly polycythemia vera and essential thrombocythemia. Mechanismsinvolved in the pathogenesis of the acquired thrombophilic state associated with these diseases include abnormalitiesof MPN clone–derived 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 elevatedlevels of procoagulant microparticles by platelets and vascular cells and the increase in the global thrombin generationdue to an acquired activated protein C resistance result in a highly prothrombotic scenario in patients withpolycythemia 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 WBCcounts and alterations in biomarkers of blood-clotting abnormalities. The predictive value of these biomarkers forthrombosis remains to be established to identify subsets of patients at elevated risk who may benefit from prophylaxiswith antithrombotic drugs.

IntroductionMyeloproliferative neoplasms (MPNs) are clonal disorders ofhematopoietic stem cells characterized by proliferation of one ormore myeloid cell lines (granulocytic, erythroid, megakaryocytic,and mast cells). According to the World Health Organization(WHO) classification, these include classic MPN Philadelphiachromosome (Ph)–negative diseases which include polycythemiavera (PV), essential thrombocythemia (ET), and myelofibrosis(MF). The discovery of recurrent molecular abnormalities such asJAK2V617F and MPL W515L/K has reinforced the original idea ofDameshek in 1951 that these diseases share a common pathogeneticmechanism and presumably belong to a single disorder. Clonalitystudies have established that PV, ET, and MF are diseases thatoriginate from a common hematopoietic stem cell that is hypersensi-tive to the action of growth factors. The result is an autonomousproliferation of hematopoietic colonies, which leads to overproduc-tion of red blood cells (RBCs), platelets, and leukocytes. Amongthese disorders, ET has the most favorable outcome. Patients withET have a lifespan that nearly rivals that of a healthy populationmatched by age and sex. However, the life expectancy of patientswith PV and ET is strongly affected by disease-related hemostaticcomplications, including thrombosis and, to a lesser extent, hemor-rhages. Reported incidence of thrombosis ranges from 12%-39% inPV and from 11%-25% in ET.1 Clinical manifestations of thrombo-sis vary from mild microcirculatory disturbances to more seriouscomplications such as arterial and venous thrombosis (Figure 1).

Arterial thrombosis, which accounts for 60%-70% of events relatedto MPNs, includes ischemic stroke, acute myocardial infarction, andperipheral arterial occlusion. The high incidence of heart valvularlesions in MPNs raises the possibility that a significant proportion ofthese may be related to emboli of cardiac origin. Events involvingthe venous system are represented by deep venous thrombosis of thelower extremities, pulmonary embolism, intraabdominal (hepatic,portal, and mesenteric) and cerebral vein thrombosis. In PV, venousthromboses are relatively common and constitute approximatelyone-third of total events. The prevalence of splanchnic and cerebral

vein thrombosis is unusually high among patients with MPN, andthese events are often the presenting feature of the disease beforediagnosis.2 MPNs constitute the most common cause of splanchnicvenous thromboses, accounting for approximately 50% of BuddChiari syndrome (BCS) cases and 25% of portal vein thromboses(PVT). MPNs associated with BCS and PVT have unique featurescompared with “classic” MPNs, including the onset at a youngerage, female predominance in PV, and the presence of normal bloodcounts, which sometimes renders the diagnosis of MPN quitechallenging.3

Typical of, but not exclusive to, ET is the involvement of themicrocirculatory system, which manifests as erythromelalgia, tran-sient ischemic attacks, visual or hearing transitory defects, recurrentheadache, and peripheral paresthesia. Focal symptoms such asdysarthria, transient monocular blindness, or transient mono- orhemiparesis are less common. Visual dysfunction may also manifestas transient diplopia and sudden reversible attacks of blurred vision.

ET and PV management remains highly dependent on the patient’sthrombotic risk.4 Because the use of myelosuppressive drugs (eg,hydroxyurea) can reduce the rate of thromboses and hemorrhages inthese patients, but can also accelerate the rate of leukemic transfor-mation, a risk-oriented management strategy is highly recom-mended. Older age (� 60 years) and previous thrombosis are wellestablished cardiovascular risk factors for thrombosis in thesepatients. The absence of both of these 2 risk factors identifieslow-risk patients. There is great attention being given to movingbeyond these recognized risk factors, particularly in the young orasymptomatic low- and intermediate-risk patients not without riskof thrombosis. Recently, the impact of new risk factors, such asleukocytosis and JAK2V617F mutational status and/or mutationalburden, has begun to be under active investigation.

Even in the absence of thrombotic manifestation, ET and PVpatients present with a hypercoagulable state, which is a laboratoryfinding of increased levels of plasma biomarkers of hemostaticsystem activation.5-6 Therefore, an acquired thrombophilic state

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

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develops in these patients, who are prone to vascular complications,but the mechanisms ultimately responsible for blood activationcoagulation and the increased thrombotic tendency in ET and PVhave not been fully elucidated.

Pathogenesis of thrombosisThe pathogenesis of thrombosis in ET and PV is multifaceted.However, 2 main mechanisms recapitulate the origin of the hyperco-agulable state in these disorders. One relies on the abnormalities ofthe blood cells (ie, platelets, erythrocytes, and leukocytes) arisingfrom the clonal hematopoietic progenitor cells, which express aprothrombotic phenotype, and the other involves the inflammatoryresponse of host vascular cells to the insult of cytokines and othermediators released by malignant cells (Figure 2).

The alterations of circulating MPN clone–derived blood cells entailnot only quantitative changes in the number of circulating bloodcells, with consequent hyperviscosity, but also qualitative changesthat lead to the expression of procoagulant characteristics. Theprothrombotic factors expressed by transformed vascular cells (ie,

platelets, RBCs, and leukocytes) include the production of procoagu-lant and proteolytic molecules, secretion of inflammatory cytokines,and expression of cell adhesion molecules. In addition, bloodhyperviscosity and increased levels of leukocyte-derived proteases(ie, elastase, cathepsin-G, and myeloperoxidase) may contribute tothe thrombophilic state by affecting the integrity of endothelial cellmonolayer. Abnormalities of the endothelium do occur in MPN Ph�

diseases. Activated/injured endothelial cells express higher levels ofadhesion molecules on their surface, which favors platelet andleukocyte arrest, allowing the localized secretion of thrombogenicand angiogenic peptides by inflammatory cells. In this scenario, theincreased production of procoagulant microparticles from activatedplatelets and vascular cells and the increased thrombin generationdue to an acquired resistance to activated protein C (APC) representvery important mechanisms that trigger systemic hypercoagulationand ultimately thrombosis in ET and PV patients.

Abnormalities of MPN vascular cellsPlatelets. The role of thrombocytosis in the pathogenesis ofthrombotic events is controversial. Although clinical improvement

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. Thromboticocclusions of large arteries most commonly involve cerebral or coronary vessels. Ischemic stroke constitutes 30%-40% of all thrombotic events in PVpatients. Acute coronary syndromes are more rare, particularly during followup of treated patients. Acute vascular occlusions in other areas are notuncommon both in PV and ET patients. Venous thrombosis usually manifests with an increased incidence of deep venous thromboses of the lowerlimbs, which may cause pulmonary embolism. Superficial phlebitis of the legs is also common and venous thromboses at unusual sites are not as rare asin the general population. Thromboses of cerebral sinuses and of splanchnic (portal and hepatic) veins have been repeatedly reported in relatively youngfemale patients. Microcirculatory disturbances are the most peculiar thrombotic manifestations in PV and ET patients and are responsible for a widerange of clinical symptoms arising from the formation of platelet thrombi in the end-arterial circulation of the peripheral, cerebral, coronary, skin, andabdominal vessels.

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of microcirculatory disturbances and/or improved platelet functionafter control of thrombocytosis have been reported, no clearcorrelation of thrombocytosis with risk of major cardiovascularevents has been demonstrated.7-8 For example, in both thePolycythemia Vera Study Group (PVSG)9 and the EuropeanCollaboration on Low-Dose Aspirin in Polycythemia Vera(ECLAP)10 prospective trials, platelet count did not predict forthrombosis. The results of the only randomized clinical trial to date,the Primary Thrombocythaemia 1 (PT1) study, which randomizedhigh-risk ET patients to hydroxyurea (a global myelosuppressiveagent) or anagrelide (a platelet-only reducing agent), showed that,despite a similar control of platelet count by either drug, thecomposite primary end point (ie, arterial or venous thrombosis,serious hemorrhage, or death from vascular causes) occurred morefrequently in recipients of anagrelide plus aspirin than in thosereceiving hydroxyurea plus aspirin.11 The global effects of hy-droxyurea on blood cell populations other than platelets (eg,leukocytes) might underlie the correlation between control ofplatelet count and reduction of thrombosis rate observed in the firstprospective study in high-risk ET patients by Cortelazzo et al.12 Incontrast, in MF, a condition characterized by less frequent cardiovas-cular events than in PV or ET, a correlation between thrombocytosisand thrombosis was found.13

Conversely, extreme thrombocytosis (ie, platelets � 1500 � 109/L)can favor hemorrhagic rather than thrombotic manifestations in

ET patients.12 This paradox has been attributed to the possibleoccurrence of an acquired VWD due to an increased clearance byplatelets of the large circulating VWF multimers.14 Platelet countreduction proved successful in normalizing the plasma VWFmultimer pattern and in reducing bleeding tendency.7,8,15

Despite the inconclusive data on the role of thrombocytosis, severalother lines of evidence support a contribution of platelets to thepathogenesis of thrombosis in ET and PV. For example, the promptrelief of symptoms with aspirin, together with the normalization ofplatelet activation tests, suggest a role of platelets in mediatingmicrovessel occlusions (ie, erythromelalgia) in ET patients.16 Incontrast to the inefficacy of the oral anticoagulant warfarin, controlof platelet function with low-dose aspirin and normalization ofplatelet counts prevents the recurrence of microvascular circulationdisturbances in the end-arterial microvasculature of the cerebral,coronary, and peripheral circulation. Furthermore, low-dose aspirinsignificantly reduces the risk of cardiovascular events in PV, asdemonstrated by the ECLAP randomized clinical trial. Patientsrandomized to receive aspirin had a 60% reduction of combined endpoint of nonfatal acute myocardial infarction, nonfatal stroke, ordeath from cardiovascular causes.10

The characteristics of thrombohemorrhagic diathesis in ET and PVprompted the design of many in vitro studies to demonstrate andcharacterize possible platelet abnormalities. In the past, numerous

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. Theother generates from the host inflammatory response to the insult of cytokines and other mediators by the malignant cells. The latter mechanism alsocontributes to thrombosis in nonmalignant conditions.

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platelet defects have been identified in ET and PV patients. Themajority of these observations were related to a decreased function-ality and included abnormal platelet aggregation, reduced levels ofmembrane adhesion molecules (ie, glycoprotein Ib [GPIb], GPIIb-IIIa, GPIV, and GPVI), acquired storage pool disease, and defectiveplatelet metabolism (ie, abnormal arachidonic acid metabolism).17,18

In contrast, more recent studies show that platelets from thesepatients circulate in an activated status, as assessed by the detectionof increased expression on their surface of P-selectin and tissuefactor (TF).19,20 It may be that the permanent activation status of theMPN platelets may exhaust their functions, leading to a reducedresponse to the stimuli in vitro. We found that although thecirculating ET platelets express high surface P-selectin, theirresponse to ADP and epinephrine stimulation is inferior to that ofhealthy control platelets. Similar dysfunctions in ET and PVplatelets were also reported by others.21 Furthermore, some plateletaggregation abnormalities in ET may be caused by laboratoryartifacts due to the extensive dilution of platelet-rich plasma neededin the aggregation assay.22 The evidence of enhanced in vivoplatelet activation in ET and PV patients is further demonstrated bythe finding of increased release of platelet activation products bothin plasma (ie, beta-thromboglobulin and platelet factor 4) and urine(ie, the thromboxane A2 [TxA2] metabolites 11-dehydro-TxB2 and2,3-dinor-TxB2).21,23 Once activated, platelets expose on theirsurface the anionic phosphatidylserine, usually kept on the innerleaflet (ie, the cytosolic side) of cell membrane, providing a catalytic

surface for the generation of thrombin, which further amplifiesplatelet activation (Figure 3).

Recently, our group demonstrated in patients with ET and PV, andparticularly in carriers of the JAK2V617F mutation, an increasedthrombin generation capacity of platelets, which was associatedwith the occurrence of platelet activation. Cytoreductive therapywith hydroxyurea significantly affected this prothrombotic pheno-type.24 We also showed that immature platelets, which are morereactive than their mature counterparts, are also increased in patientswith ET or PV (Figure 4) and are significantly down-regulated byhydroxyurea.25 No studies have been performed to compare, in thesame patient, platelets with and without JAK2V617F mutation.Conversely, there are data comparing platelets from JAK2V617F�

with those from JAK2V617F� patients and platelets from subjectswith different allele mutation burden. In a study of MPN patients byour group, a direct association between JAK2V617F allele burdenand increased platelet-associated thrombin generation potential wasobserved.24

It is relevant to discuss whether thrombopoietin (TPO) and itsreceptor (c-MPL) may play a role on platelet function relative to thepresence of the JAK2V617F mutation. A decreased expression ofc-MPL on platelets and megakaryocytes surface is an establishedcharacteristic of PV and MF, but not of ET. Very recently, it wasshown that c-MPL expression is down-regulated in JAK2V617F�

Figure 3. Role of platelet abnormalities in MPN-associated thrombophilia. Many studies have investigated the contribution of platelets in the onsetof 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, plateletqualitative 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 ofplatelet-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 plateletactivation.

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platelets in MF due to an increased c-MPL degradation, leading to amegakaryocytic cell–enhanced cell survival and proliferation. How-ever, very little information is available on the possible relationshipbetween TPO/cMPL and the hemostatic platelet functions. There isevidence that TPO induces a potentiation of platelet aggregation anddense-granule secretion after standard stimuli (eg, collagen, ADP,and epinephrine) in patients with MPN.26 However, relationshipsbetween c-MPL levels, TPO levels, JAK2V617F mutation, andplatelet hemostatic function have not been explored as yet. It may bepossible that the constitutive activation of c-MPL in plateletspositive for JAK2V617F may render these cells more reactive tostimuli and determine an increased expression of P-selectin.

RBCs. The prothrombotic effect of an elevated hematocrit has beenclearly demonstrated in PV by the observation that a progressivelyhigher hematocrit corresponds to an increase in the thromboticrisk.27 Hematocrit levels around the upper limit of normal valuesmay be an important factor in the causation of occlusive vasculardiseases, particularly in the cerebral circulation, as a consequence ofan increased blood viscosity. Indeed, at high hematocrit values(47%-53%), the cerebral blood flow is significantly slower than athematocrit values in the lower range (36%-46%). In untreatedPV subjects, most thrombotic accidents occur in the cerebralcirculation, which is particularly sensitive to blood hyperviscosity.At high shear rates, the raise of RBC mass displaces platelets towardthe vessel wall, thus facilitating shear-induced platelet activationand enhancing platelet-to-platelet interactions. In addition, at lowshear rates, as in the venous bed, hyperviscosity can increase thethrombotic risk by causing a major disturbance to the blood flow. Aproper management of blood hyperviscosity is essential but does notabolish the in vivo platelet activation and the increased thromboticrisk in PV subjects.23 In addition to an increased RBC count,biochemical changes in cell membrane and intracellular content ofRBCs are also found in ET and PV.28 These changes mayindependently impair blood flow through the formation of RBCaggregates that have the potential to block blood flow in small

vessels directly and facilitate platelet-leukocyte interaction. Thislikely contributes to ischemia and infarct, especially in the cerebralblood flow.

Leukocytes. Many retrospective studies have identified leukocyto-sis as a potential risk factor for arterial and venous thrombosisin patients with ET and PV.29-32 Leukocytosis was also a risk factorfor recurrent arterial thrombosis in young (ie, � 60 years) ET andPV patients (hazard ratio for arterial recurrence � 3.35, 95% con-fidence interval [CI], 1.22-9.19).33 In contrast, leukocytosis atdiagnosis (defined by a cutoff level of either 15 or 9.4 � 109/L) didnot appear to influence the risk of thrombosis in a retrospectivecohort of low-risk ET or PV patients.34 Prospective clinical studieswith stratification of patients according to their baseline leukocytecounts are needed to definitely classify leukocytosis as a prog-nostic risk factor. Leukocytes can contribute to the pathogenesis ofthrombosis in ET and PV through recently discovered mechanismsof activation and interaction with platelets, endothelial cells, and thecoagulation system. In addition, leukocytes contribute to inflam-matory processes in atherosclerotic plaques and in this way increasethe probability of vascular events. Because neutrophils representthe most abundant proportion of the circulating leukocytes, a rolefor neutrophils in thrombosis of MPN has been hypothesized.Neutrophils have a central role in the inflammatory response andalso in linking the inflammatory response to the activation of bloodcoagulation.35 Once activated, neutrophils produce reactive oxygenspecies, release proteolytic enzymes from their cytoplasmic azuro-philic granules (ie, elastase and cathepsin G), and express higherand functional levels of the �2 integrin Mac 1 (or CD11b) on theircell surface. All of these molecules can affect the hemostatic systemand induce a prothrombotic condition.36 The fact that activatedneutrophils can induce a hypercoagulable state in vivo has been welldemonstrated by a study of a group of healthy donors administeredG-CSF for the mobilization and collection of peripheral bloodprogenitor cells.37 G-CSF caused activation of neutrophils in thesesubjects, which was associated with a parallel increment in plasma

Figure 4. Immature platelets are increased in JAK2V617F� ET and PV patients. Figure shows data on immature platelet fraction (IPF) valuesaccording to disease type, JAK2V617F mutation, and hydroxyurea (HU) treatment. Both ET and PV patients positive for the JAK2V617F mutation andnot receiving HU showed significantly higher IPF count compared with control subjects (P � .05). In addition, JAK2V617F� ET and PV patients nottreated with HU showed significantly higher IPF count compared with JAK2V617F� and HU-treated JAK2V617F� and JAK2V617F� patients nottreated with HU. *P � .05 compared with controls. Modified with permission from Panova-Noeva et al.24

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levels of biomarkers of activation of blood coagulation and endothe-lium. These effects were transient, persisting only as long as thegrowth factor was administered (ie, 5-6 days), and normalized afterG-CSF withdrawal.

In ET and PV patients, neutrophil activation has been demonstratedby detection of specific phenotypical changes (by increments inmembrane-associated CD11b) and of increased plasma concentra-tion of neutrophil granule–derived proteases (ie, elastase andmyeloperoxidase).5,19 These abnormalities are directly correlatedwith increased plasma levels of biomarkers of both blood clottingand endothelium activation, supporting a possible involvement ofneutrophils in the pathogenesis of the hypercoagulable state in thesedisorders. Several studies described increased levels of circulatingplatelet-neutrophil aggregates in ET and PV patients and attributedthis phenomenon to both platelet and neutrophil activation.38

Interestingly, in patients receiving aspirin, the increments in CD11bexpression and neutrophil/platelet aggregates induced by in vitrostimulation were significantly lower compared with nonaspirin-treated subjects, suggesting that aspirin treatment may inhibit theinteraction between neutrophils and platelets.19 Because the G-CSFreceptor is linked to the JAK2 pathway, it is possible that theconstitutive activation of signaling through this receptor in thepresence of JAK2V617F mutation can be in part responsible for theactivated phenotype of neutrophils in patients with ET and PV.However, our data on neutrophil activation analyzed according tothe JAK2V617F mutation39 show that the levels of only some of theactivation markers are more altered in the JAK2V617F� than in theJAK2V617F� ones (ie, CD14 and leukocyte alkaline phosphatase),whereas others, (ie, CD11b and plasma elastase) are equallyelevated in both groups. It is likely that mechanisms independent ofthe JAK2V617F mutation are present in non-JAK2V617F mutationcarriers.

Abnormalities of host vascular cellsEndothelium. Physiologically, the endothelium facilitates bloodflow by providing an antithrombotic surface that inhibits plateletadhesion and coagulation activation. Several factors may perturb theresting state of endothelium in patients with MPN and turn it into aproadhesive and procoagulant surface. Reactive oxygen species andintracellular proteases released by activated neutrophils can inducedetachment or lysis of endothelial cells, affecting functions involvedin thromboregulation. It has also been demonstrated that damage ofendothelium determines the release in circulation of specific mark-ers including thrombomodulin, selectins, and VWF. This is ofparticular relevance in the pathogenesis of thrombosis in MPNbecause once platelets bind to VWF, they become activated and ableto aggregate and strengthen the clot.40 Selectins are a family ofadhesion molecules expressed by endothelial cells (P-selectin andE-selectin), platelets (P-selectin), and leukocytes (L-selectin).41

Because they are released into the circulation, their presence hasbeen used as an index of endothelial, platelet, and leukocyteactivation. In patients with ET and PV, we measured increasedplasma levels of soluble P-, E-, and L-selectins42 and solublethrombomodulin.39 High levels of membrane-bound and plasma-soluble P-selectin and E-selectin have been found in ET patientswith thrombosis, suggesting that sustained endothelium and plateletactivation might contribute to the pathogenesis of thrombosis inthese diseases.43 In addition to releasing substances that stimulatethrombus formation after injury, endothelial cells release the plateletinhibitor nitric oxide (NO), providing a negative feedback mecha-nism for the propagation of thrombus formation.44 NO is a freeradical product generated through the oxidation of L-arginine to

L-citrulline by NO synthases. The endothelial-derived NO is one ofthe main mediators influencing vascular hemodynamic and theinteraction of leukocytes and platelets with endothelial cells. In fact,NO mediates vascular relaxation in response to vasoactive sub-stances and shear stress; inhibits platelet adhesion, activation,secretion, and aggregation; and promotes platelet disaggregation.Moreover, NO inhibits the expression of P-selectin on platelets andimpairs leukocyte adhesion to the endothelium. Clinical conditionshave been reported in which a deficiency of endogenous NOproduction may contribute to a thrombotic event. Our grouprecently studied circulating NO in MPN patients42 and foundreduced plasma levels of NO in patients with ET compared withcontrols. This confirms the previous observation that in MPNpatients with thrombocytosis, the production of NO by platelets isimpaired.45 However, in the same study and for the first time, weobserved that ET patients treated with hydroxyurea presented withthe highest levels of plasma NO. A similar effect of hydroxyurea onNO plasma levels was reported in patients with sickle cell anemia46

and may contribute to the known ability of hydroxyurea to preventthromboembolic complications in ET patients.12 In the same study,PV patients showed high plasma NO levels compared with controlsand these levels were not affected by hydroxyurea treatment. Thisresult was as expected, because a high hematocrit level is associatedwith increased NO in the blood and may represent a compensatorymechanism in a high-thrombotic-risk situation.

Recent studies have shown that angiogenesis plays an importantrole in the biology of hematological malignancies including MPN.47

Levels of circulating endothelial cells, together with serum levels ofVEGF and other proangiogenic cytokines, have been studied inMPN as markers of angiogenetic activity. The number of circulatingendothelial cells (resting, activated, apoptotic, and circulatingprecursor endothelial cells) was repeatedly found to be increased inpatients with MPN regardless of JAK2V617F status and was notaffected by cytoreductive treatment.48-50

All of these lines of evidence suggest that endothelium in MPN isactivated, suggesting an important endothelial contribution to thehypercoagulable state. In addition, angiogenesis may have a role inthe pathophysiology of MPN.

Role of the JAK2V617F mutationThe demonstration of the acquired gain-of-function V617F muta-tion in the tyrosine kinase JAK-2 gene has greatly influenced thediagnostic and therapeutic approach in MPN patients. Severalstudies have implicated the JAK2V617F mutation in the increasedthrombotic tendency observed in ET and PV patients and3 independent meta-analyses were published recently.51-53 In theanalysis by Ziakas et al involving 2905 ET patients,51 theJAK2V617F mutation was associated with an increased risk of bothvenous (odds ratio [OR] � 2.09; 95% CI, 1.44-3.05) and arterialthrombosis (OR � 1.96; 95% CI, 1.43-2.67), as well as thrombosisat presentation (OR � 1.88; 95% CI, 1.38-2.56). The meta-analysisby 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 recentmeta-analysis of 21 studies involving ET patients and 6 studies withMF, showed that in ET patients, the JAK2V617F mutation isassociated 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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JAK2V617F mutation and thrombosis was recently confirmed in aretrospective multicenter study from Korea involving 239 patientswith ET.54 In that study, previous thrombotic history and theJAK2V617F mutation were associated with a higher 10-yearcumulative incidence rate of thrombohemorrhagic events.

Although many studies and 3 meta-analysis have been published onthe role of JAK2V617F mutation in the increased thrombotictendency in ET, few data are available in PV. In one prospectivestudy by Vannucchi et al including 173 PV patients, those subjectswith a mutant allele burden � 75% had a 3.56-fold higher relativerisk (95% CI, 1.47-7.1) of total thrombosis, particularly during thefollow-up (relative risk � 7.1; 95% CI, 1.6-10.1).55 A retrospectiveanalysis of patients with either PV or ET found that the frequency ofthrombosis progressively increased according to the allele burden inboth types of diseases, with the highest rate of vascular complica-tions in patients with an allele burden � 50%.56 However, in arecent prospective study of patients with PV, no significant relation-ship between the mutant allele burden and the risk of thrombosiswas observed.57

Few studies have been published addressing whether the JAK2V617Fmutation may specifically affect the hemostatic system.20,39,58,59

These studies indicate that both cellular (ie, platelets and leuko-cytes) and plasma compartments of hemostasis were more activatedin those patients positive for the JAK2V617F mutation (Figure 5).Regarding cellular components, P-selectin was found to be in-creased on platelets from JAK2V617F� ET patients20; CD14 andLAP were demonstrated to be more highly expressed on neutrophilsfrom ET JAK2V617F mutation carriers39; and a significantly higherexpression of CD11b was observed on neutrophils and monocytesfrom JAK2V617F� PMF patients.59 In addition, our group wereable to demonstrate an elevated expression of TF in platelets andincreased levels of circulating platelet/neutrophil aggregates fromJAK2V617F� ET patients compared with JAK2V617F� patients.39

The evidence that both platelets and neutrophils from JAK2V617F�

patients expressed increased activation features is in good agree-ment with the findings of increased mixed-cell aggregate formationin ET patients carrying the JAK2V617F mutation. Among hyperco-agulation parameters, plasma levels of soluble thrombomodulinwere found to be elevated in JAK2V617F� ET patients,39 and

Figure 5. Hemostatic alterations in JAK2V617F� and JAK2V617F� MPN patients. Abnormalities of platelets, erythrocytes, leukocytes, andendothelial cells lead to systemic hypercoagulability as represented by an increased production of procoagulant microparticles and the occurrence ofan acquired APC resistance. Studies addressing whether the JAK2V617F mutation may specifically affect the hemostatic system indicate that bothcellular (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 withJAK2V617F� patients.

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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 JAK2V617Fmutation was observed in the presence of an acquired APCresistance phenotype.60

Circulating platelets are heterogeneous in size and structure. Inhealthy subjects, a small percentage of circulating platelets (2%) arethe so-called reticulated or immature platelets recently releasedfrom the BM. Their number, reflecting the rate of thrombopoiesis, isdirectly correlated with megakaryocyte proliferating activity. Invitro studies show that newly formed platelets have a higherhemostatic activity compared with mature platelets, as demon-strated by the increased response to thrombin and higher expressionof surface P-selectin.61

Recently, in 46 ET and 38 PV consecutive patients, we found apositive correlation between the JAK2V617F mutation and thequantity of immature platelets.25 Furthermore, we observed that thelevels of circulating immature platelets are susceptible to myelosup-pressive treatment, which may explain the favorable effect ofhydroxyurea therapy on MPN outcome, as well as the associatedthrombotic risk.

Few studies have evaluated the molecular mechanisms by whichJAK2V617F mutation can affect the prothrombotic phenotype of acell. The JAK2V617F-activating mutation can cause an increase inRBC adhesiveness through modification of surface adhesion mol-ecules, thus facilitating thrombosis, and may render platelet hyperre-sponsive through altered expression of c-MPL signal transductionfor TPO-induced platelet priming.62 Activation of JAK2 is alsoinvolved in TF expression by neutrophil and monocyte through theMAPK and PI3K pathway.63

Finally, the presence of JAK2V617F in both endothelial cells andhematopoietic cells belonging to BCS patients with PV has beendemonstrated. This indicates that endothelial cells from thesepatients are involved in the malignant process and suggests that inthis subpopulation of patients, the disease may originate from a cellcommon to the hematopoietic and endothelial cell systems.64

Other somatic mutations have been characterized in MPN, includingseveral mutations in JAK2 exon 12 (� 5% of JAK2V617F� PVpatients), and a mutation in the MPL gene (MPL W515) describedin approximately 10% of PMF patients and 1% of ET patients, butnot in PV patients. No information is available on the thrombophilicstate of MPN patients carrying these rare mutations. The JAK2 exon12 and MPL W515 mutations have not been detected in patientswith BCS and PVT,3 whereas MPL-mutated ET has been associatedwith high platelet count, microvascular symptoms, and a high risk ofpostdiagnosis arterial thrombosis.65,66

Main prothrombotic features in the bloodIn patients with ET and PV, the main prothrombotic featuresthat manifest in the blood as a consequence of the activation ofMPN clone– derived blood cells and host normal vascular cellsare an increased thrombin generation sustained by an acquiredAPC resistance, and the appearance in the circulation of highlevels of procoagulant microparticles derived from platelets andvascular cells.

Acquired APC resistancePC, a serine protease activated by thrombin bound to the endothelialreceptor thrombomodulin, is one of the major physiological antico-agulant in humans. APC, in complex with its cofactor, protein S(PS), reduces blood clotting activation through the proteolyticinactivation of coagulation factor V and factor VIII. A resistance toinactivation by APC, inherited or acquired, is associated with anincreased risk of thrombosis in many situations, including preg-nancy, oral contraceptive use, hormone replacement therapy, andcancer. Decreased levels of PC and PS can be responsible for theoccurrence of an APC-resistant phenotype, and studies have re-ported a reduction in the concentration of natural anticoagulants inpatients with MPN.67 By using the thrombin-generation assay, wedemonstrated the occurrence of an acquired APC resistance pheno-type in ET and PV patients.60 The results analyzed according to thepresence (ie, positivity or negativity) and status (ie, heterozygosityor homozygosity) of the JAK2V617F mutation indicated thatJAK2V617F mutation carriers are more APC resistant than noncar-riers, especially if they are homozygous (ie, JAK2V617F alleleburden � 50%). This suggests a progression to the APC-resistantphenotype determined by the JAK2V617F status and, together withprevious observations, supports the hypothesis of a more hyperco-agulable condition in JAK2V617F carriers. Prothrombin, factor V,free PS, and tissue factor pathway inhibitor levels were significantlyreduced in patients and mainly in JAK2V617F carriers. Multipleregression analysis indicated that the low free PS level is a majordeterminant of the increased APC resistance. A high prevalence ofacquired APC resistance, determined as the classical prolongationof activated partial thromboplastin time after the addition of APC,was reported by Arellano-Rodrigo et al in patients with ET6 and wasfound more frequently in those patients with previous history ofthrombosis; again, decreased levels of free PS were detected andwere inversely correlated with JAK2V617F allele burden.

Several lines of research indicate that a protease from platelets isable to cleave PS in plasma.68 On the basis of the finding ofdecreased PS levels in MPN, we decided to investigate therelationship between platelet-associated PS cleaving activity and invivo PS cleavage in a group of ET patients.69 PS cleavage was foundto be significantly increased in patients with ET, along with elevatedlevels of platelets, and returned to normal values in ET patients onhydroxyurea treatment with normal platelet counts. Therefore,proteases from platelets seem to contribute to the presence ofcleaved PS in the circulation of ET patients and may enhance thecoagulation response in vivo by down-regulating the anticoagulantactivity of PS.

Plasma microparticlesMicroparticles are membrane fragments ranging in size from0.1-1 �m that are released by most cell types, including bloodand vascular cells, upon activation. Microparticles are known tobe elevated in thromboembolic diseases and malignancy,70

including patients with ET.71 The levels and cellular origin ofmicroparticles were determined by flow cytometric analysis, andthe microparticle-associated procoagulant activity was measuredusing a thrombin-generation assay. A significantly higher num-ber of circulating microparticles positive for platelet and endothe-lial markers, as well as for TF, were found in ET subjectscompared with controls. In addition, microparticle-rich plasmafrom patients with ET showed higher thrombin-generationpotential, which was significantly correlated with the totalnumber of microparticles. A subsequent study by Duchemin et al

578 American Society of Hematology

has shown the presence of an acquired “thrombomodulin-resistant” phenotype in PV and ET patients that is partiallydetermined by circulating microparticles.72

Conclusions and future perspectivesThrombotic complications significantly affect the morbidity andmortality of patients with MPN. In addition, MPN patients com-monly present with abnormalities in laboratory coagulation teststhat are consistent with a hypercoagulable state. The pathogenesis ofblood activation in these diseases is complex and multifactorial, andinvolves the abnormalities of platelets, erythrocytes, and leukocytesarising from the clonal proliferation of hematopoietic progenitorcells and abnormalities of endothelial cells. These alterationsinclude not only quantitative changes in the number of circulatingblood cells, but also qualitative changes in cellular molecularcharacteristics. These properties lead to an increased production ofprocoagulant microparticles and the occurrence of an APC-resistantphenotype and very likely contribute to clotting system activation.Clinical data indicate an association of the JAK2V617F mutationwith disease severity. Biological data also show an association ofthis mutation with the expression of cellular and soluble biomarkersof coagulation activation. However, future clinical research shouldfocus on the evaluation of the role of these biomarkers in identifyingMPN patients at higher risk of thrombosis, who may benefit fromprimary thromboprophylaxis. Finally, a better understanding of themolecular events leading to the development of the hypercoagulablestate in Ph� MPN patients may provide appropriate tools fortargeted therapies to reverse coagulopathy.

AcknowledgmentsThe work conducted at the Laboratory of the Division of Immunohe-matology and Transfusion Medicine, Ospedali Riuniti di Bergamo(Bergamo, Italy) was partially supported by grants from theAssociazione Italiana per la Ricerca sul Cancro and from theNational Institutes of Health Myeloproliferative Disorders ResearchConsortium (both to A.F.).

DisclosuresConflict-of-interest disclosure: The authors declare no competingfinancial interests. Off-label drug use: None disclosed.

CorrespondenceAnna Falanga, MD, Division of Immunohematology and Transfu-sion Medicine, Ospedali Riuniti di Bergamo, Italy, Largo Barozzi,1, 24128 Bergamo, Italy; Phone: 39-035-266578; Fax: 39-035-266659; e-mail: annafalanga@yahoo.com.

References1. Tefferi A. Polycythemia vera and essential thrombocythemia:

2012 update on diagnosis, risk stratification, and management.Am J Hematol. 2012;87(3):285-293.

2. Reikvam H, Tiu RV. Venous thromboembolism in patientswith essential thrombocythemia and polycythemia vera. Leuke-mia. 2012;26(4):563-571.

3. Kiladjian JJ, Cervantes F, Leebeek FW, et al. The impact ofJAK2 and MPL mutations on diagnosis and prognosis ofsplanchnic vein thrombosis: a report on 241 cases. Blood.2008;111(10):4922-4929.

4. Tefferi A. Annual Clinical Updates in Hematological Malignan-cies: a continuing medical education series: polycythemia veraand essential thrombocythemia: 2011 update on diagnosis,

risk-stratification, and management. Am J Hematol. 2011;86(3):292-301.

5. Falanga A, Marchetti M, Evangelista V, et al. Polymorphonu-clear leukocyte activation and hemostasis in patients withessential thrombocythemia and polycythemia vera. Blood.2000;96(13):4261-4266.

6. Arellano-Rodrigo E, Alvarez-Larran A, Reverter JC, et al.Platelet turnover, coagulation factors, and soluble markers ofplatelet and endothelial activation in essential thrombocythe-mia: relationship with thrombosis occurrence and JAK2 V617Fallele burden. Am J Hematol. 2009;84(2):102-108.

7. Elliott MA, Tefferi A. Thrombosis and haemorrhage in polycyth-aemia vera and essential thrombocythaemia. Br J Haematol.2005;128(3):275-290.

8. Harrison CN. Platelets and thrombosis in myeloproliferativediseases. Hematology Am Soc Hematol Educ Program. 2005:409-415.

9. Berk PD, Goldberg JD, Donovan PB, Fruchtman SM, BerlinNI, Wasserman LR. Therapeutic recommendations in polycythe-mia vera based on Polycythemia Vera Study Group protocols.Semin Hematol. 1986;23(2):132-143.

10. Landolfi R, Marchioli R, Kutti J, et al. Efficacy and safety oflow-dose aspirin in polycythemia vera. N Engl J Med. 2004;350(2):114-124.

11. Harrison CN, Campbell PJ, Buck G, et al; United KingdomMedical Research Council Primary Thrombocythemia 1 Study.Hydroxyurea compared with anagrelide in high-risk essentialthrombocythemia. N Engl J Med. 2005;353(1):33-45.

12. Cortelazzo S, Finazzi G, Ruggeri M, et al. Hydroxyurea forpatients with essential thrombocythemia and a high risk ofthrombosis. N Engl J Med. 1995;332(17):1132-1136.

13. Cervantes F, Alvarez-Larran A, Arellano-Rodrigo E, GranellM, Domingo A, Montserrat E. Frequency and risk factors forthrombosis in idiopathic myelofibrosis: analysis in a series of155 patients from a single institution. Leukemia. 2006;20(1):55-60.

14. Landolfi R, Cipriani MC, Novarese L. Thrombosis and bleed-ing in polycythemia vera and essential thrombocythemia:pathogenetic mechanisms and prevention. Best Pract Res ClinHaematol. 2006;19(3):617-633.

15. Schafer AI. Thrombocytosis. N Engl J Med. 2004;350(12):1211-1219.

16. Michiels JJ, Berneman Z, Van Bockstaele D, van der PlankenM, De Raeve H, Schroyens W. Clinical and laboratory features,pathobiology of platelet-mediated thrombosis and bleedingcomplications, and the molecular etiology of essential thrombo-cythemia and polycythemia vera: therapeutic implications.Semin Thromb Hemost. 2006;32(3):174-207.

17. Landolfi R, Rocca B, Patrono C. Bleeding and thrombosis inmyeloproliferative disorders: mechanisms and treatment. CritRev Oncol Hematol. 1995;20(3):203-222.

18. Schafer AI. Bleeding and thrombosis in the myeloproliferativedisorders. Blood. 1984;64(1):1-12.

19. Falanga A, Marchetti M, Vignoli A, Balducci D, Barbui T.Leukocyte-platelet interaction in patients with essential throm-bocythemia and polycythemia vera. Exp Hematol. 2005;33(5):523-530.

20. Arellano-Rodrigo E, Alvarez-Larran A, Reverter JC, VillamorN, Colomer D, Cervantes F. Increased platelet and leukocyteactivation as contributing mechanisms for thrombosis in essen-tial thrombocythemia and correlation with the JAK2 mutationalstatus. Haematologica. 2006;91(2):169-175.

21. Jensen MK, de Nully Brown P, Lund BV, Nielsen OJ,

Hematology 2012 579

Hasselbalch HC. Increased platelet activation and abnormalmembrane glycoprotein content and redistribution in myelopro-liferative disorders. Br J Haematol. 2000;110(1):116-124.

22. Grignani C, Noris P, Tinelli C, Barosi G, Balduini CL. In vitroplatelet aggregation defects in patients with myeloproliferativedisorders and high platelet counts: are they laboratory artefacts?Platelets. 2009;20(2):131-134.

23. Landolfi R, Ciabattoni G, Patrignani P, et al. Increased throm-boxane biosynthesis in patients with polycythemia vera: evi-dence for aspirin-suppressible platelet activation in vivo. Blood.1992;80(8):1965-1971.

24. Panova-Noeva M, Marchetti M, Spronk HM, et al. Platelet-induced thrombin generation by the calibrated automatedthrombogram assay is increased in patients with essentialthrombocythemia and polycythemia vera. Am J Hematol.2011;86(4):337-342.

25. Panova-Noeva M, Marchetti M, Buoro S, et al. JAK2V617Fmutation and hydroxyurea treatment as determinants of imma-ture platelet parameters in essential thrombocythemia andpolycythemia vera patients. Blood. 2011;118(9):2599-2601.

26. Pecquet C, Diaconu CC, Staerk J, et al. Thrombopoietinreceptor down-modulation by JAK2 V617F: restoration ofreceptor levels by inhibitors of pathologic JAK2 signaling andof proteasomes. Blood. 2012;119(20):4625-4635.

27. Adams BD, Baker R, Lopez JA, Spencer S. Myeloproliferativedisorders and the hyperviscosity syndrome. Hematol OncolClin North Am. 2010;24(3):585-602.

28. Turitto VT, Weiss HJ. Red blood cells: their dual role inthrombus formation. Science. 1980;207(4430):541-543.

29. Landolfi R, Di Gennaro L, Barbui T, et al; European Collabora-tion on Low-Dose Aspirin in Polycythemia Vera (ECLAP).Leukocytosis as a major thrombotic risk factor in patients withpolycythemia vera. Blood. 2007;109(6):2446-2452.

30. Carobbio A, Antonioli E, Guglielmelli P, et al. Leukocytosisand risk stratification assessment in essential thrombocythemia.J Clin Oncol. 2008;26(16):2732-2736.

31. Carobbio A, Finazzi G, Guerini V, et al. Leukocytosis is a riskfactor for thrombosis in essential thrombocythemia: interactionwith treatment, standard risk factors, and Jak2 mutation status.Blood. 2007;109(6):2310-2313.

32. Palandri F, Polverelli N, Catani L, Ottaviani E, Baccarani M,Vianelli N. Impact of leukocytosis on thrombotic risk andsurvival in 532 patients with essential thrombocythemia: aretrospective study. Ann Hematol. 2011;90(8):933-938.

33. De Stefano V, Za T, Rossi E, et al; GIMEMA ChronicMyeloproliferative Neoplasms Working Party. Leukocytosis isa risk factor for recurrent arterial thrombosis in young patientswith polycythemia vera and essential thrombocythemia. Am JHematol. 2010;85(2):97-100.

34. Gangat N, Wolanskyj AP, Schwager SM, Hanson CA, TefferiA. Leukocytosis at diagnosis and the risk of subsequentthrombosis in patients with low-risk essential thrombocythemiaand polycythemia vera. Cancer. 2009;115(24):5740-5745.

35. Falanga A, Marchetti M, Barbui T, Smith CW. Pathogenesis ofthrombosis in essential thrombocythemia and polycythemiavera: the role of neutrophils. Semin Hematol. 2005;42(4):239-247.

36. Afshar-Kharghan V, Thiagarajan P. Leukocyte adhesion andthrombosis. Curr Opin Hematol. 2006;13(1):34-39.

37. Falanga A, Marchetti M, Evangelista V, et al. Neutrophilactivation and hemostatic changes in healthy donors receivinggranulocyte colony-stimulating factor. Blood. 1999;93(8):2506-2514.

38. Marchetti M, Falanga A. Leukocytosis, JAK2V617F mutation,and hemostasis in myeloproliferative disorders. PathophysiolHaemost Thromb. 2008;36(3-4):148-159.

39. Falanga A, Marchetti M, Vignoli A, et al. V617F JAK-2mutation in patients with essential thrombocythemia: relation toplatelet, granulocyte, and plasma hemostatic and inflammatorymolecules. Exp Hematol. 2007;35(5):702-711.

40. Friedenberg WR, Roberts RC, David DE. Relationship ofthrombohemorrhagic complications to endothelial cell functionin patients with chronic myeloproliferative disorders. Am JHematol. 1992;40(4):283-289.

41. Carlos TM, Harlan JM. Leukocyte-endothelial adhesion mol-ecules. Blood. 1994;84(7):2068-2101.

42. Cella G, Marchetti M, Vianello F, et al. Nitric oxide derivativesand soluble plasma selectins in patients with myeloproliferativeneoplasms. Thromb Haemost. 2010;104(1):151-156.

43. Karakantza M, Giannakoulas NC, Zikos P, et al. Markers ofendothelial and in vivo platelet activation in patients withessential thrombocythemia and polycythemia vera. Int J Hema-tol. 2004;79(3):253-259.

44. Freedman JE, Loscalzo J. Nitric oxide and its relationship tothrombotic disorders. J Thromb Haemost. 2003;1(6):1183-1188.

45. Rejto L, Huszka M, Kaplar M, Udvardy M. Effects of in vitroplatelet activation on platelet derived nitric oxide production inhealthy humans and in chronic myeloproliferative diseases withelevated platelet counts. Platelets. 2003;14(5):283-286.

46. Glover RE, Ivy ED, Orringer EP, Maeda H, Mason RP.Detection of nitrosyl hemoglobin in venous blood in thetreatment of sickle cell anemia with hydroxyurea. Mol Pharma-col. 1999;55(6):1006-1010.

47. Di Raimondo F, Palumbo GA, Molica S, Giustolisi R. Angio-genesis in chronic myeloproliferative diseases. Acta Haematol.2001;106(4):177-183.

48. Trelinski J, Wierzbowska A, Krawczynska A, et al. Plasmalevels of angiogenic factors and circulating endothelial cells inessential thrombocythemia: correlation with cytoreductivetherapy and JAK2-V617F mutational status. Leuk Lymphoma.2010;51(9):1727-1733.

49. Belotti A, Elli E, Speranza T, Lanzi E, Pioltelli P, Pogliani E.Circulating endothelial cells and endothelial activation inessential thrombocythemia: results from CD146� immunomag-netic enrichment-flow cytometry and soluble E-selectin detec-tion. Am J Hematol. 2012;87(3):319-320.

50. Alonci A, Allegra A, Bellomo G, et al. Evaluation of circulatingendothelial cells, VEGF and VEGFR2 serum levels in patientswith chronic myeloproliferative diseases. Hematol Oncol. 2008;26(4):235-239.

51. Ziakas PD. Effect of JAK2 V617F on thrombotic risk inpatients with essential thrombocythemia: measuring the uncer-tain. Haematologica. 2008;93(9):1412-1414.

52. Dahabreh IJ, Zoi K, Giannouli S, Zoi C, Loukopoulos D,Voulgarelis M. Is JAK2 V617F mutation more than a diagnos-tic index? A meta-analysis of clinical outcomes in essentialthrombocythemia. Leuk Res. 2009;33(1):67-73.

53. Lussana F, Caberlon S, Pagani C, Kamphuisen PW, Buller HR,Cattaneo M. Association of V617F Jak2 mutation with the riskof thrombosis among patients with essential thrombocythaemiaor idiopathic myelofibrosis: a systematic review. Thromb Res.2009;124(4):409-417.

54. Lee HS, Park LC, Lee EM, et al. Incidence rates and risk factorsfor vascular events in patients with essential thrombocythemia:

580 American Society of Hematology

a multicenter study from Korea. Clin Lymphoma MyelomaLeuk. 2012;12(1):70-75.

55. Vannucchi AM, Antonioli E, Guglielmelli P, et al. Prospectiveidentification of high-risk polycythemia vera patients based onJAK2(V617F) allele burden. Leukemia. 2007;21(9):1952-1959.

56. Carobbio A, Finazzi G, Antonioli E, et al. JAK2V617F alleleburden and thrombosis: a direct comparison in essential throm-bocythemia and polycythemia vera. Exp Hematol. 2009;37(9):1016-1021.

57. Passamonti F, Rumi E, Pietra D, et al. A prospective study of338 patients with polycythemia vera: the impact of JAK2(V617F) allele burden and leukocytosis on fibrotic or leukemicdisease transformation and vascular complications. Leukemia.2010;24(9):1574-1579.

58. Robertson B, Urquhart C, Ford I, et al. Platelet and coagulationactivation markers in myeloproliferative diseases: relationshipswith JAK2 V6I7 F status, clonality, and antiphospholipidantibodies. J Thromb Haemost. 2007;5(8):1679-1685.

59. Alvarez-Larran A, Arellano-Rodrigo E, Reverter JC, et al.Increased platelet, leukocyte, and coagulation activation inprimary myelofibrosis. Ann Hematol. 2008;87(4):269-276.

60. Marchetti M, Castoldi E, Spronk HM, et al. Thrombin genera-tion and activated protein C resistance in patients with essentialthrombocythemia and polycythemia vera. Blood. 2008;112(10):4061-4068.

61. Harrison P, Robinson MS, Mackie IJ, Machin SJ. Reticulatedplatelets. Platelets. 1997;8(6):379-383.

62. Kubota Y, Tanaka T, Ohnishi H, et al. Constitutively activatedphosphatidylinositol 3-kinase primes platelets from patientswith chronic myelogenous leukemia for thrombopoietin-induced aggregation. Leukemia. 2004;18(6):1127-1137.

63. Rafail S, Ritis K, Schaefer K, et al. Leptin induces theexpression of functional tissue factor in human neutrophils andperipheral blood mononuclear cells through JAK2-dependent

mechanisms and TNFalpha involvement. Thromb Res. 2008;122(3):366-375.

64. Sozer S, Hoffman R. Hoffman, et al. Laser-capture microdissec-tion and analysis of liver endothelial cells from patients withBudd-Chiari syndrome. Methods Mol Biol. 2011;755:405-415.

65. Beer PA, Campbell PJ, Scott LM, et al. MPL mutations inmyeloproliferative disorders: analysis of the PT-1 cohort.Blood. 2008;112(1):141-149.

66. Vannucchi AM, Antonioli E, Guglielmelli P, et al. Characteris-tics and clinical correlates of MPL 515W�L/K mutation inessential thrombocythemia. Blood. 2008;112(3):844-847.

67. Bucalossi A, Marotta G, Bigazzi C, Galieni P, Dispensa E.Reduction of antithrombin III, protein C, and protein S levelsand activated protein C resistance in polycythemia vera andessential thrombocythemia patients with thrombosis. Am JHematol. 1996;52(1):14-20.

68. Brinkman HJ, Mertens K, van Mourik JA. Proteolytic cleavageof protein S during the hemostatic response. J Thromb Hae-most. 2005;3(12):2712-2720.

69. Dienava-Verdoold I, Marchetti MR, te Boome LC, et al.Platelet-mediated proteolytic down regulation of the anticoagu-lant activity of protein S in individuals with haematologicalmalignancies. Thromb Haemost. 2012;107(3):468-476.

70. Zwicker JI, Liebman HA, Neuberg D, et al. Tumor-derivedtissue factor-bearing microparticles are associated with venousthromboembolic events in malignancy. Clin Cancer Res. 2009;15(22):6830-6840.

71. Trappenburg MC, van Schilfgaarde M, Marchetti M, et al.Elevated procoagulant microparticles expressing endothelialand platelet markers in essential thrombocythemia. Haemato-logica. 2009;94(7):911-918.

72. Duchemin J, Ugo V, Ianotto JC, Lecucq L, Mercier B, AbgrallJF. Increased circulating procoagulant activity and thrombingeneration in patients with myeloproliferative neoplasms.Thromb Res. 2010;126(3):238-242.

Hematology 2012 581