Implication of microvesicles in thrombosis associated with

Paroxysmal nocturnal hemoglobinuria

Literature reviewOctober 2012

B DEVALET

history

definition

diagnosis

clinical aspects

pathophysiology of disease

thrombotic events

pathophysiology of thrombosis

risk factors for thrombosis

treatment

treatment of subclinical PNH and AAPNH

treatment of classic PNH

prophylaxis of thrombosis

treatment of thrombosis

treatment during pregnancy

Literature

PNH History

First description by a german physicianPaul Struumlbing in 1889

More detailed description by Dr Ettore Marchiafava and Dr Alessio Nazari in 1911 then Dr Ferdinando Micheli in 1931 (Marchiafava-Micheli syndrome)

Identification of the molecular basis of PNH helped to define the physiology of the complement system in humans (47)

PNH Definition

Rare acquired disorder of hematopoietic stem cells

Incidence not really known but estimated 1-21000000 inhabyear

Somatic mutation in X-linked gene the phosphatidylinositol glycan class A (PIG-A)

PIG-A si required for synthesis of glycosyl phosphatidylinositol (GPI) anchor

Deficiency in GPI anchored proteins (many)

Lack of CD59 laquo MIRL raquo and CD55 laquo DAF raquo (complement regulatory proteins) leads to complement mediated intravascular hemolysis (47)

PNH Definition

Non malignant clonal expansion 2 steps hypothesis (53)

step 1 PIG-A mutation

no clonal expansion asymptomatic

step 2 injury to the normal bone marrow

T cells and NK cells attack

normal hematopoietic cells destroyedbut PNH cells escape (absence of GPI anchored targeted peptides or accessory molecules required for T cells attack)

bone marrow failure clonal expansion

PNH Diagnosis

(Ham and sucrose tests)

Flow cytometry with monoclonal antibodies that bind to specific GPI-anchored proteins (precentage of PNH cells measured)(38)

clone size

use at least 2 different monoclonal antibodies directed against 2 different GPI-anchored proteins(rare congenital deficiencies of CD59 or CD55)

on at least 2 different cells lineages(risk of falsely negative tests if only red cells are screened ndash recent hemolytic crisis or recent transfusion)

phenotypic mosaicism

PNH III cells completely deficient in GPI anchored-proteins

PNH II cells partly deficient

laquo PNH raquo I cells express GPI-anchored proteins at normal density

PNH Diagnosis

FLAER (fluorescein-labeled proaerolysin)(34)

Aerolysin = the principal virulence factor of the bacterium Aeromonas hydrophila

binds selectively and with high affinity to the GPI anchor

more accurate assessment of the GPI anchor deficit in PNH (PNH clones lt1 can be detected)

complete blood count reticulocytes LDH bilirubin haptoglobin iron stores BM aspirate and biopsy cytogenetics

PNH Clinical aspects

3 different forms (clinical polymorphism)(3 4 34)

Classic PNH

young adults

intravascular hemolytic anemia (with crisis)

Hb lt12 gdL

hemoglobinuria (in the morning)

jaundice

general symptoms (asthenia malaise)

worsening of hemolysis during infection (complement activation above basal level)

no evidence of bone marrow failure (neutro gt1500microL platelets gt120 000microL)

dysphagia abdominal pain erectile dysfunction (35)

thrombosis

reccurent infectious diseases (ORL lungs) 40 2d cause of death

risk of MDSAML 5 25 at 10y (x100)

PNH associated with aplastic anemia

small PNH clone (majority of patients lt10)

clinicalbiochemical evidence of hemolysis

2 or 3 cytopenias (Hb lt10 gdL neutro lt1000microL platelets lt80 000microL)

BM failure dominates the clinical picture

Subclinical PNH

clone size lt1

no hemolysis

We find PNH clone in 60 of AA and in 20 of low risk MDS(47)

associated to AA high probability of response to immunosuppressive therapy

associated to low risk MDS (refractory anemia)

less pronounced morphological abnormalities of the blood cells

more severe thrombocytopenia

lower rate of karyotypic abnormalities

higher incidence of HLA-DR15

lower rate of progression to AML

higher probability of reponse to cyclosporine

Hypothesis that aplastic anemia and a subgroup of low risk MDS are immune-mediated diseases

immunity provides the selection pressure that favors the outgrowth of PNH clone

PNH Clinical aspects (3-4)

Classic PNH Aplastic anemia-PNH

Age 40y 30y

Abdominal pain 35 20

Median clone size 916 168

Median survival 18y gt22y

Thrombotic events (at 10y) 379 278

Medullar aplasia 20 at 10y 100

PNH Pathophysiology of disease (1)

Intravascular hemolysis

free Hb in the plasma

NO fixation to Hb

NO depletion

smooth muscle dystonias

dysphagia

abdominal pain

erectile dysfunction

arterial constriction

reduced blood flow to the kidneys

arterial hypertension

pulmonary hypertension (+undiagnosed thrombosis)

PNH Thrombotic events (1-2)

Occurs in 40 of PNH

Cause of death in 40-67 of patients (major cause of death)

Arterial (15) and venous (85) thrombosis

Involve more than one site (205)

Usual sites

deep veins thrombosis

pulmonary embolism

myocardial infarction

CNS arteries

and unusual sites

hepatic veins (Budd-Chiari syndrome the most frequent thrombotic complication of PNH 407)

cavernous sinus

CNS veins

mesenteric veins

dermal veins

PNH Thrombotic events

Age of onset lower than in general population (46y vs 73y)

Recurrences despite anticoagulant therapy described(3)

Thrombosis during pregnancy and post-partum

increased risk of thrombosis

maternal mortality 20

perinatal mortality 10

PNH Pathophysiology of thrombosis (1)

Unknown - multifactorialhellip

Role of hemolysis controversialhellip

no link between

severity of hemolysis and thrombosis

severity of anemia and thrombosis

NO depletion(40)

causes arterial constriction

increases platelets activation and aggregation

enhances TF expression

ADP release induce platelets activation(5)

human RBC stroma activate the alternate complement pathway(7)

interraction of activated complement components with platelets

release of PF-3

activation of coagulation

Mechanisms and clinical implications of thrombosis in paroxysmal nocturnal hemoglobinuria

Journal of Thrombosis and Haemostasis Volume 10 Issue 1 pages 1-10 4 JAN 2012 DOI 101111j1538-7836201104562x httponlinelibrarywileycomdoi101111j1538-7836201104562xfullf1

PNH Pathophysiology of thrombosis

CD59 and CD55-deficient platelets

complement can induce platelet activation

one study showed increased adherence of patelets to the abdominal vessels(1)

PNH platelets after MAC (membrane attack complex) stimulation expose more FVa-binding sites and increase thrombin generation more than normal platelets(40)

BUT other studies showed impaired function of PNH platelets (reactive downregulation in response to chronic hyperstimulation)

Endothelial cells(40)

direct toxicity of free hemoglobin released

endothelial precursor cells seem to be bone marrow-derived cells

probable same GPI-anchored proteins deficiency

more susceptible to complement injury

thrombosis in unusual sites

damage resulting from the uptake of monocyte-derived MVs (released from GPI-deficient monocytes upon complement damage)

These MVs contain tissue factor thus increasing TF expression on endothelial cells

increased number of EMVs with a prothrombotic and proinflammatory phenotype in PNH

increased levels of endothelial cells activation markers (VWF sVCAM-1)

Possible role of C5a protein

binds to a receptor on granulocytes

recruitement and activation of granulocytes and monocytes (enhanced in GPI-deficient white blood cells)

release of inflammatory molecules

damage the endothelium

TF expression

release of monocyte-derived MVs containing TF

= link between inflammation and thrombosis

Tissue factor pathway inhibitor (TFPI)

TFPI limits coagulation initiation by inhibiting TF formation

mainly produced by the endothelium of the microvasculature

bound to GPI-anchored protein (definciency in PNH)

loss of TFPI expression has been associated with increased risk of thrombosis

Neutrophil proteinase-3 (PR-3)(19)

membrane bound protein co-localized with GPI- anchored neutrophil antigen 2a

absence of neutrophil antigen 2a in PNH leads to loss of expression of neutrophil proteinase-3

PAR-1 (the predominant human platelet thrombin receptor) is a substrate for PR-3

impared down regulation of PAR-1

increased

propensity for platelet activation

PR-3 also modulates coagulation in various other ways

Urokinase-type plasminogen activator receptor (u-PAR)(10131517)

central role in the regulation of haemostatic processes on cell surface

binding of u-PA (urokinase plasminogen activator) to u-PAR converts plasminogen into plasmin

involved in fibrinolysis make it localized pericellularly

GPI-anchored protein

underexpressed on cellular surface in PNH (granulocytes and monocytes)

increased concentrations of soluble uPAR (released from PNH hematopoietic cells)

impaired and displaced fibrinolytic system

Other fibrinolysis parameters

conflicting results

Increased circulating MVs in PNH patients(18)

procoagulant properties of MVs (exposing PS containing TF) have been demonstrated in vitro

complement activation may stimulate the release of procoagulant MVs

no link between clone size and number of MVs

majority of them come from platelets(8)

EMVs levels are also increased in PNH patients with prothrombotic and proinflammatory phenotypes(9)

similar level of erythrocyte MVs in vivo but PNH erythrocytes release higher amounts of procoagulant MVs upon complement stimulation in vitro (may suggest rapid clearance from the circulation)(40)

Increase of procoagulant activity (fact V fibrinogen fact VIII fact IX fact X and vWF)(10)

positive correlation with clone size

Frequency of congenital thrombophila factors is not increased in PNH(40)

testing for such factors may identify PNH patients at additional risk

its value for treatment decision in PNH is unknown

authors do not recommend routine testing

PNH Risk factors for thrombosis (1-2)

Large WBC clone(4)

clone gt50 10y cumulative incidence of thrombosis = 345

clone lt50 10y cumulative incidence of thrombosis = 53

Previous history of thrombosis

Old age (gt55y)

Use of transfusions(3)

Other Elevation of D-dimers

Indications of prophylactic anticoagulation

PNH Treatment of subclinical PNH and AAPNH (47)

Subclinical PNH

no symtoms no treatment

close monitoring (every 6-12 months) because expansion of the clone may occur

treatment of the underlying bone marrow failure syndrome (allogenic transplantation immunosuppressive therapy)

PNH Treatment of classic PNH

Eculizumab (Solirisreg Alexion Pharmaceuticals)(6 18 31)

humanized monoclonal antibody

PNH Treatment of classic PNH eculizumab

blocks the activation of terminal complement C5 and prevents the formation of C5a and C5b-9

administration schedule

25-45 min IV infusion

induction dose of 600 mg every 7 days for 4 doses

then 900 mg 7 days later

followed by a maintenance dose of 900 mg every 14 days

vaccination against Neisseria meningitidis at least 14 days prior to receiving eculizumab

05y risk of neisserial sepis (even after vaccination)(34)

revaccinate every 3 to 5 years

medical attention if patient develops signs of neisserial infection

TRIUMPH (randomized placebo-controlled phase 3 study)

in transfusion-dependant PNH patients with good bone marrow reserve

reduces hemolysis transfusion requirements

improves anemia fatigue and measures of quality of life

SHEPHERD (open-label phase 3 study)(6)

to investigate the long-term safety and efficacy of eculizumab

97 patients enrolled median age 41 y follow up 52 weeks

8997 pat got a fast response and maintained complete inhibition of hemolysis

efficient

most common adverse events headache nasopharyngitis upper respiratory tract infection

83 of infections possibly related to treatment

44 SAEs (none was considered probably or definely related to treatment)

well tolerated

Impact on overall survival

Survival for patients on eculizumab appears to be similar to that of the normal population(50)

Reasons for suboptimal response(47-48)

breakthrough intravascular hemolysis near the end of a treatment cycle

cycles of 13 or 12 days

increase dose of eculizumab

extravascular hemolysis

Frequently persistant elevation of reticulocytes bilirubin and LDH levels presistant reduction of haptoglobin

A small group of eculizumab-treated patients little or no improvement in either anemia or constitutional symptoms

inhibition of intravascular lysis by eculizumab

increase of PNH red blood cells loaded with early components of the complement system (C3 opsonins)

eculizumab does not block the C3 convertase (not down regulated by CD55)

recognized by reticuloendothelal cells

hemolysis in the spleen (and the liver)

What to do

No treatment if no constitutional or anemia symptoms or transfusion dependance

Corticosteroids or splenectomy

Reduction of thombosis with eculizumab

Number of TE events and incidence rates determined in patients from 3 clinical studies with eculizumab(31)

87-94 reduction of the thrombotic events rate during eculizumab treatment

Decrease in plasma D-dimers levels with eculizumab(44)

suppression of thrombin generation

Study of markers of thrombin generation and fibrinolysis in 23 PNH patients(18)

before and after 5 and 11 w of eculizumab treatment

eculizumab reduces the activation of the plasma hemostatic system and the vascular endothelium

role of the endothelial cell activation in the pathogenesis of thrombosis in PNH

no effect of eculizumab on the number of EMVs on MVs procoagulant activity or on the number of ICAM-1 expressing EMVs

Rapid and sustained decrease in the markers of thrombin generation and inflammation during eculizumab therapy(44)

significant reduction in D-dimers thrombin- antithrombin complex IL-6 soluble P-selectin (expressed by activated platelets and endothelial cells) TFMP (tissue factor bearing microparticles)

no link between hemolysis (LDH) and markers of thrombin generation and inflammation

the decrease of TFMP didnrsquot correlate with changes in markers of thrombin generation and inflammation

What happens if we stop eculizumab

risk of hemolytic crisis

increased number of PNH red blood cells during eculizumab treatment

risk of thrombosis

one case described of a first thrombotic event 3 weeks after the last eculizumab dose (women died)

during anticoagulant prophylaxy (therapeutically dosed phenprocoumonMarcoumarreg)

no hemolysis

mechanism

PNH Treatment of classic PNH (2)

Bone marrow transplantation

the only curative option

important graft-versus-PNH effect

lack of suitable donnors

high rates of treatment-related morbiditymortality

2-year survival probability 56(34)

acute and chronic graft versus host disease 15 and 20

only for patients with life threatning cytopenias or disabling hemolysis or thrombosis that is not controlled with eculizumab

Supportive care

transfusions if needed (efficient may lessen hemolysis by suppressing erythropoiesis)

folate supplements

iron supplements if needed (hemoglobinuria hemosiderinuria) rare during eculizumab therapy (sometimes iron depletion therapy needed)

EPO increases hemolysis symptoms may be used when patients remain anemic with eculizumab and have low EPO level

corticosteroids long term toxicity use for acute hemolysis crises

androgens (Danazol) no data

PNH Prophylaxis of thrombosis (1-2)

Primary prophylactic anticoagulation

no randomized trials

some data suggest that warfarin effectively reduces thrombotic risk

anticoagulation in PNH patients is difficult

thrombocytopenia (aplasia liver failure)

risk of bleeding

difficult to maintain a therapeutic INR with warfarin

oesophagal spasm

mesenteric insufficiency

irregular feeding

recommendation of the international PNH interest group

consider vitamin K antagonist prophylaxis in patients with PNH granulocyte clone gt50 and no contraindications for prophylaxis

other authors say

No proven benefit for primary prophylactic anticoagulation (except pregnancy)

aspirin glycoprotein IIb-IIIa receptor antagonists

platelets may have a role in the pathophysiology of thrombosis in PNH

most thrombotic events occur in the venous system

New oral anticoagulants (rivaroxaban dabigatran hellip)

Immunosuppressive therapy

need to define subgroups of PNH patients who could benefit from anticoagulation (risk factors)

Eculizumab is the best preventive treatment for thrombosis

PNH Treatment of thrombosis (1-2)

If life-threatening thrombosis

thrombolytic therapy

Anticoagulation therapy

foundation of treatement

warfarin LMWH NOAC

for how long lifelong

PNH Treatment during pregnancy (34)

greater need for folate and iron supplementation

prophylactic full anticoagulation with LMWH if no contraindication

example enoxaparin 1mgkg12h SC

starting when pregnancy is confirmed

monitor anti-factor Xa levels

sometimes need to switch to unfractionated heparin if a cesarean section is planned

anticoagulation has to be continued during the post-partum period

eculizumab

listed as category C pharmaceuticals (drug with little or no information available)

important potential benefit of eculizumab in reducing thrombosis

IgG2 isotypes do not cross the placenta probably little impact on the fetus

single case report eculizumab was administered to a pregnant PNH patient in week 30 of her pregnancy (twins) No complications were reported

Literature

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2) Ziakas PD Poulou LS Rokas GI Bartzoudis D Voulgarelis M Thrombosis in paroxysmal nocturnal hemoglobinuria sites risks outcome An overview J Thromb Haemost 20075(3)642-5

3) Peffault de Latour R Mary JY Salanoubat C Terriou L Etienne G Mohty M et al Paroxysmal nocturnal hemoglobinuria natural history of disease subcategories Blood 2008112(8)3099-106

4) Moyo VM Mukhina GL Garrett ES Brodsky RA Natural history of paroxysmal nocturnal haemoglobinuria using modern diagnostic assays Br J Haematol 2004126(1)133-8

5) Hall C Richards S Hillmen P Primary prophylaxis with warfarin prevents thrombosis in paroxysmal nocturnal hemoglobinuria (PNH) Blood 2003102(10)3587-91

6) Brodsky RA Young NS Antonioli E Risitano AM Schrezenmeier H Schubert J et al Multicenter phase 3 study of the complement inhibitor eculizumab for the treatment of patients with paroxysmal nocturnal hemoglobinuria Blood 2008111(4) 1840-7

7) Poskitt TR Fortwengler HP Jr Lunskis BJ Activation of the alternate complement pathway by autologous red cell stroma J Exp Med 1973138(3)715-22

8) Hugel B Socie G Vu T Toti F Gluckman E Freyssinet JM et al Elevated levels of circulating procoagulant microparticles in patients with paroxysmal nocturnal hemoglobinuria and aplastic anemia Blood 199993(10)3451-6

9) Simak J Holada K Risitano AM Zivny JH Young NS Vostal JG Elevated circulating endothelial membrane microparticles in paroxysmal nocturnal haemoglobinuria Br J Haematol 2004125(6)804-13

10) Grunewald M Siegemund A Grunewald A Schmid A Koksch M Schopflin C et al Plasmatic coagulation and fibrinolytic system alterations in PNH relation to clone size Blood Coagul Fibrinolysis 200314(7)685-95

11) Wiedmer T Hall SE Ortel TL Kane WH Rosse WF Sims PJ Complement-induced vesiculation and exposure of membrane prothrombinase sites in platelets of paroxysmal nocturnal hemoglobinuria Blood 199382(4)1192-6

12) Gralnick HR Vail M McKeown LP Merryman P Wilson O Chu I et al Activated platelets in paroxysmal nocturnal haemoglobinuria Br J Haematol 199591(3)697-702

13) Ploug M Eriksen J Plesner T Hansen NE Dano K A soluble form of the glycolipid-anchored receptor for urokinase-type plasminogen activator is secreted from peripheral blood leukocytes from patients with paroxysmal nocturnal hemoglobinuria Eur J Biochem 1992208(2)397-404

14) Ronne E Pappot H Grondahl-Hansen J Hoyer-Hansen G Plesner T Hansen NE et al The receptor for urokinase plasminogen activator is present in plasma from healthy donors and elevated in patients with paroxysmal nocturnal haemoglobinuria Br J Haematol 199589(3)576-81

15) Plesner T Behrendt N Ploug M Structure function and expression on blood and bone marrow cells of the urokinase-type plasminogen activator receptor uPAR Stem Cells 199715(6)398-408

16) Ninomiya H Hasegawa Y Nagasawa T Abe T Excess soluble urokinase-type plasminogen activator receptor in the plasma of patients with paroxysmal nocturnal hemoglobinuria inhibits cellassociated fibrinolytic activity Int J Hematol 199765(3)285-91

Literature

17) Sloand EM Pfannes L Scheinberg P More K Wu CO Horne M et al Increased soluble urokinase plasminogen activator receptor (suPAR) is associated with thrombosis and inhibition of plasmin generation in paroxysmal nocturnal hemoglobinuria (PNH) patients Exp Hematol 200836(12)1616-24

18) Helley D Peffault de Latour R Porcher R Rodrigues CA Galy-Fauroux I Matheron J Duval A Schved J-F Fischer A-M and Socieacute G on behalf of the French Society of Hematology Evaluation of hemostasis and endothelial function in patients with paroxysmal nocturnal hemoglobinuria receiving eculizumab Haematologica 201095574-581

19) Jankowska AM Szpurka H Calabro M Mohan S Schade AE Clemente M Silverstein RL Maciejewski JP Loss of expression of neutrophil proteinase-3 a contributory factor in thrombotic risk in paroxysmal nocturnal hemoglobinuria Haematologica 2011 Jul96(7)954-62

20) Burnier L Fontana P Kwak BR Angelillo-Scherrer A Cell-derived microparticles in haemostasis and vascular medicine Thromb Haemost 2009 Mar101(3)439-51

21) Williams JC Mackman N MPs or ICs Blood 2011 Jan 27117(4)1101-2

22) Beyer C Pisetsky DS The role of microparticles in the pathogenesis of rheumatic diseases Nat Rev Rheumatol 2010 Jan6(1)21-9 Epub 2009 Dec 1

23) Owens AP 3rd Mackman N Microparticles in hemostasis and thrombosis Circ Res 2011 May 13108(10)1284-97

24) van der Pol E Hoekstra AG Sturk A Otto C van Leeuwen TG Nieuwland R Optical and non-optical methods for detection and characterization of microparticles and exosomes J Thromb Haemost 2010 Dec8(12)2596-607

25) Lacroix R Robert S Poncelet P Dignat-George F Overcoming limitations of microparticle measurement by flow cytometry Semin Thromb Hemost 2010 Nov36(8)807-18 Epub 2010 Nov 3 Review

26) Yuana Y Oosterkamp TH Bahatyrova S Ashcroft B Garcia Rodriguez P Bertina RM Osanto S Atomic force microscopy a novel approach to the detection of nanosized blood microparticles J Thromb Haemost 2010 Feb8(2)315-23 Epub 2009 Oct 19

27) Freyssinet JM Toti F Membrane microparticle determination at least seeing whats being sized J Thromb Haemost 2010 Feb8(2)311-4 Epub 2009 Oct 30

28) Lacroix R Robert S Poncelet P Kasthuri RS Key NS Dignat-George F ISTH SSC Workshop Standardization of platelet- derived microparticle enumeration by flow cytometry with calibrated beads results of the International Society on Thrombosis and Haemostasis SSC Collaborative workshop J Thromb Haemost 2010 Nov8(11)2571-4

29) Mullier F Bailly N Chatelain C Dogneacute JM Chatelain B Needs interests and limitations of calibrated polystyrene beads for flow cytometry-based quantification of biological microparticles J Thromb Haemost 2011 Jun 4

30) Jy W Ricci M Shariatmadar S Gomez-Marin O Horstman LH Ahn YS Microparticles in stored red blood cells as potential mediators of transfusion complications Transfusion 2011 Apr51(4)886-93

31) Hillmen P Muus P Duhrsen U Risitano AM Schubert J Luzzatto L et al Effect of the complement inhibitor eculizumab on thromboembolism in patients with paroxysmal nocturnal hemoglobinuria Blood 2007110(12)4123-8

32) Ziakas PD Poulou LS Rokas GI Bartzoudis D Voulgarelis M Thrombosis in paroxysmal nocturnal hemoglobinuria sites risks outcome An overview J Thromb Haemost 20075 642ndash5

33) Borowitz MJ Craig FE Digiuseppe JA Illingworth AJ Rosse W Sutherland DR Wittwer CT Richards SJ Clinical Cytometry Society Guidelines for the diagnosis and monitoring of paroxysmal nocturnal hemoglobinuria and related disorders by flow cytometry Cytometry B Clin Cytom 2010 Jul78(4)211-30

Literature

34) Brodsky RA How I treat paroxysmal nocturnal hemoglobinuria Blood 20091136522-6527

35) Hardij J Mejia J Gheldof D Varin E Michiels C Chatelain B Lucas S Mullier F Dogneacute JM The CPS disc centrifuge a new original method to detect microparticles (MPs) International Society of Thrombosis and Hemostasis July 2011 Abstract 01827

36) Kozuma Y Sawahata Y Takei Y Chiba S Ninomiva H Procoagulant properties of micrparticles released from red blood cells in paroxysmal nocturnal haemoglobinuria Br J Haematol 2011 Mar152(5)631-9 Epub 2011 Jan 17

37) Piccin A Murphy WG Smith OP Circulating microparticles pathophysiology and clinical implications Blood Rev 2007 May21(3)157-71

38) Peffault de Latour R Amoura Z Socieacute G Lrsquoheacutemoglobinurie paroxystique nocturne La revue de meacutedecine interne 31(2010) 200-7

39) Weitz IC Thrombosis in paroxysmal nocturnal hemoglobinuria- insights into the role of complement in thrombosis Thromb Res 2010 Apr125 Suppl2S106-7

40) Van Bijnen ST Van Heerde WL Muus P Mechanisms and clinical implications of thrombosis in paroxysmal nocturnal hemoglobinuria J Thromb Haemost 2012101-10

41) Raghupathy R Derman O Response of paroxysmal nocturnal hemoglobinuria clone with aplastic anemia to rituximab Case Report Hematol 20122012106182 Epub 2012 May 7

42) Bellido M van der Velden VH Leebeek FW Te Boekhorst PA Thrombotic complications without evidence of hemolysis in paroxysmal nocturnal hemoglobinuria is eculizumab indicatedAnn Hematol 2012 Jul 4

43) Peffault de Latour R Schrezenmeier H Bacigalupo A Blaise D de Souza CA Vigouroux S Willemze R Terriou L Tichelli A Mohty M de Guibert S Marsh J Passweg J Mary JY Socie GAllogeneic stem cell transplantation in paroxysmal nocturnalhemoglobinuria Haematologica 2012 Jun 11

44) Weitz IC Razavi P Rochanda L Zwicker J Furie B Manly D Mackman N Green R Liebman HAEculizumab therapy results in rapid and sustained decreases in markers of thrombin generation and inflammation in patients with PNH independent of its effects on hemolysis and microparticle formation Thromb Res 2012 Sep130(3)361-8 Epub 2012 Apr 28

45) Liebman HA Feinstein DI Thrombosis in patients with paroxysmal noctural hemoglobinuria is associated with markedly elevated plasma levels of leukocyte-derived tissue factor Thromb Res 2003111(4-5)235-8

46) van Bijnen ST van Rijn RS Koljenovic S te Boekhorst P de Witte T Muus PPossible high risk of thrombotic events in patients with paroxysmal nocturnal haemoglobinuria after discontinuation of eculizumab Br J Haematol 2012 Jun157(6)762- 3 Epub 2012 Feb 29

47) Parker CJ Management of paroxysmal nocturnal hemoglobinuria in the era of complement inhibitory therapy Hematology Am Soc Hematol Educ Program 2011201121-9 Review

48) Roumlth A Hock C Konik A Christoph S Duumlhrsen U Chronic treatment of paroxysmal nocturnal hemoglobinuria patients with eculizumab safety efficacy and unexpected laboratory phenomena Int J Hematol 2011 Jun93(6)704-14 Epub 2011 May 25

Literature

50) Kelly RJ Hill A Arnold LM Brooksbank GL Richards SJ Cullen M Mitchell LD Cohen DR Gregory WM Hillmen PLong- term treatment with eculizumab in paroxysmal nocturnal hemoglobinuria sustained efficacy and improved survival Blood 2011 Jun 23117(25)6786-92 Epub 2011 Apr 1

51) Pu JJ Brodsky RA Paroxysmal nocturnal hemoglobinuria from bench to bedside Clin Transl Sci 2011 Jun4(3)219-24

PNH History

PNH Definition

Diapositive numeacutero 5

PNH Definition

PNH Diagnosis

PNH Clinical aspects(3-4)

PNH Pathophysiology of disease(1)

PNH Thrombotic events(1-2)

PNH Pathophysiology of thrombosis(1)

PNH Risk factors for thrombosis(1-2)

PNH Treatment of subclinical PNH and AAPNH(47)

PNH Treatment of classic PNH(2)

PNH Prophylaxis of thrombosis(1-2)

PNH Treatment of thrombosis(1-2)

PNH Treatment during pregnancy(34)

Literature

history

definition

diagnosis

clinical aspects

pathophysiology of disease

thrombotic events

pathophysiology of thrombosis

risk factors for thrombosis

treatment

treatment of subclinical PNH and AAPNH

treatment of classic PNH

prophylaxis of thrombosis

treatment of thrombosis

treatment during pregnancy

Literature

PNH History

PNH Definition

PNH Diagnosis

clone size

PNH Diagnosis

Classic PNH

young adults

Hb lt12 gdL

jaundice

thrombosis

Subclinical PNH

clone size lt1

no hemolysis

Age 40y 30y

NO fixation to Hb

NO depletion

dysphagia

abdominal pain

Occurs in 40 of PNH

Usual sites

pulmonary embolism

CNS arteries

and unusual sites

cavernous sinus

CNS veins

mesenteric veins

dermal veins

no link between

NO depletion(40)

release of PF-3

TF expression

increased

conflicting results

Large WBC clone(4)

Old age (gt55y)

Subclinical PNH

efficient

well tolerated

What to do

risk of thrombosis

no hemolysis

mechanism

Supportive care

folate supplements

risk of bleeding

oesophagal spasm

irregular feeding

other authors say

warfarin LMWH NOAC

eculizumab

Literature

PNH History

PNH Definition

PNH Diagnosis

Literature

PNH History

PNH Definition

PNH Diagnosis

clone size

PNH Diagnosis

Classic PNH

young adults

Hb lt12 gdL

jaundice

thrombosis

Subclinical PNH

clone size lt1

no hemolysis

Age 40y 30y

NO fixation to Hb

NO depletion

dysphagia

abdominal pain

Occurs in 40 of PNH

Usual sites

pulmonary embolism

CNS arteries

and unusual sites

cavernous sinus

CNS veins

mesenteric veins

dermal veins

no link between

NO depletion(40)

release of PF-3

TF expression

increased

conflicting results

Large WBC clone(4)

Old age (gt55y)

Subclinical PNH

efficient

well tolerated

What to do

risk of thrombosis

no hemolysis

mechanism

Supportive care

folate supplements

risk of bleeding

oesophagal spasm

irregular feeding

other authors say

warfarin LMWH NOAC

eculizumab

Literature

PNH History

PNH Definition

PNH Diagnosis

Literature

PNH Definition

PNH Diagnosis

clone size

PNH Diagnosis

Classic PNH

young adults

Hb lt12 gdL

jaundice

thrombosis

Subclinical PNH

clone size lt1

no hemolysis

Age 40y 30y

NO fixation to Hb

NO depletion

dysphagia

abdominal pain

Occurs in 40 of PNH

Usual sites

pulmonary embolism

CNS arteries

and unusual sites

cavernous sinus

CNS veins

mesenteric veins

dermal veins

no link between

NO depletion(40)

release of PF-3

TF expression

increased

conflicting results

Large WBC clone(4)

Old age (gt55y)

Subclinical PNH

efficient

well tolerated

What to do

risk of thrombosis

no hemolysis

mechanism

Supportive care

folate supplements

risk of bleeding

oesophagal spasm

irregular feeding

other authors say

warfarin LMWH NOAC

eculizumab

Literature

PNH History

PNH Definition

PNH Diagnosis

Literature

PNH Definition

PNH Diagnosis

clone size

PNH Diagnosis

Classic PNH

young adults

Hb lt12 gdL

jaundice

thrombosis

Subclinical PNH

clone size lt1

no hemolysis

Age 40y 30y

NO fixation to Hb

NO depletion

dysphagia

abdominal pain

Occurs in 40 of PNH

Usual sites

pulmonary embolism

CNS arteries

and unusual sites

cavernous sinus

CNS veins

mesenteric veins

dermal veins

no link between

NO depletion(40)

release of PF-3

TF expression

increased

conflicting results

Large WBC clone(4)

Old age (gt55y)

Subclinical PNH

efficient

well tolerated

What to do

risk of thrombosis

no hemolysis

mechanism

Supportive care

folate supplements

risk of bleeding

oesophagal spasm

irregular feeding

other authors say

warfarin LMWH NOAC

eculizumab

Literature

PNH History

PNH Definition

PNH Diagnosis

Literature

PNH Diagnosis

clone size

PNH Diagnosis

Classic PNH

young adults

Hb lt12 gdL

jaundice

thrombosis

Subclinical PNH

clone size lt1

no hemolysis

Age 40y 30y

NO fixation to Hb

NO depletion

dysphagia

abdominal pain

Occurs in 40 of PNH

Usual sites

pulmonary embolism

CNS arteries

and unusual sites

cavernous sinus

CNS veins

mesenteric veins

dermal veins

no link between

NO depletion(40)

release of PF-3

TF expression

increased

conflicting results

Large WBC clone(4)

Old age (gt55y)

Subclinical PNH

efficient

well tolerated

What to do

risk of thrombosis

no hemolysis

mechanism

Supportive care

folate supplements

risk of bleeding

oesophagal spasm

irregular feeding

other authors say

warfarin LMWH NOAC

eculizumab

Literature

PNH History

PNH Definition

PNH Diagnosis

Literature

PNH Diagnosis

Classic PNH

young adults

Hb lt12 gdL

jaundice

thrombosis

Subclinical PNH

clone size lt1

no hemolysis

Age 40y 30y

NO fixation to Hb

NO depletion

dysphagia

abdominal pain

Occurs in 40 of PNH

Usual sites

pulmonary embolism

CNS arteries

and unusual sites

cavernous sinus

CNS veins

mesenteric veins

dermal veins

no link between

NO depletion(40)

release of PF-3

TF expression

increased

conflicting results

Large WBC clone(4)

Old age (gt55y)

Subclinical PNH

efficient

well tolerated

What to do

risk of thrombosis

no hemolysis

mechanism

Supportive care

folate supplements

risk of bleeding

oesophagal spasm

irregular feeding

other authors say

warfarin LMWH NOAC

eculizumab

Literature

PNH History

PNH Definition

PNH Diagnosis

Literature

Classic PNH

young adults

Hb lt12 gdL

jaundice

thrombosis

Subclinical PNH

clone size lt1

no hemolysis

Age 40y 30y

NO fixation to Hb

NO depletion

dysphagia

abdominal pain

Occurs in 40 of PNH

Usual sites

pulmonary embolism

CNS arteries

and unusual sites

cavernous sinus

CNS veins

mesenteric veins

dermal veins

no link between

NO depletion(40)

release of PF-3

TF expression

increased

conflicting results

Large WBC clone(4)

Old age (gt55y)

Subclinical PNH

efficient

well tolerated

What to do

risk of thrombosis

no hemolysis

mechanism

Supportive care

folate supplements

risk of bleeding

oesophagal spasm

irregular feeding

other authors say

warfarin LMWH NOAC

eculizumab

Literature

PNH History

PNH Definition

PNH Diagnosis

Literature

Subclinical PNH

clone size lt1

no hemolysis

Age 40y 30y

NO fixation to Hb

NO depletion

dysphagia

abdominal pain

Occurs in 40 of PNH

Usual sites

pulmonary embolism

CNS arteries

and unusual sites

cavernous sinus

CNS veins

mesenteric veins

dermal veins

no link between

NO depletion(40)

release of PF-3

TF expression

increased

conflicting results

Large WBC clone(4)

Old age (gt55y)

Subclinical PNH

efficient

well tolerated

What to do

risk of thrombosis

no hemolysis

mechanism

Supportive care

folate supplements

risk of bleeding

oesophagal spasm

irregular feeding

other authors say

warfarin LMWH NOAC

eculizumab

Literature

PNH History

PNH Definition

PNH Diagnosis

Literature

Subclinical PNH

clone size lt1

no hemolysis

Age 40y 30y

NO fixation to Hb

NO depletion

dysphagia

abdominal pain

Occurs in 40 of PNH

Usual sites

pulmonary embolism

CNS arteries

and unusual sites

cavernous sinus

CNS veins

mesenteric veins

dermal veins

no link between

NO depletion(40)

release of PF-3

TF expression

increased

conflicting results

Large WBC clone(4)

Old age (gt55y)

Subclinical PNH

efficient

well tolerated

What to do

risk of thrombosis

no hemolysis

mechanism

Supportive care

folate supplements

risk of bleeding

oesophagal spasm

irregular feeding

other authors say

warfarin LMWH NOAC

eculizumab

Literature

PNH History

PNH Definition

PNH Diagnosis

Literature

Age 40y 30y

NO fixation to Hb

NO depletion

dysphagia

abdominal pain

Occurs in 40 of PNH

Usual sites

pulmonary embolism

CNS arteries

and unusual sites

cavernous sinus

CNS veins

mesenteric veins

dermal veins

no link between

NO depletion(40)

release of PF-3

TF expression

increased

conflicting results

Large WBC clone(4)

Old age (gt55y)

Subclinical PNH

efficient

well tolerated

What to do

risk of thrombosis

no hemolysis

mechanism

Supportive care

folate supplements

risk of bleeding

oesophagal spasm

irregular feeding

other authors say

warfarin LMWH NOAC

eculizumab

Literature

PNH History

PNH Definition

PNH Diagnosis

Literature

NO fixation to Hb

NO depletion

dysphagia

abdominal pain

Occurs in 40 of PNH

Usual sites

pulmonary embolism

CNS arteries

and unusual sites

cavernous sinus

CNS veins

mesenteric veins

dermal veins

no link between

NO depletion(40)

release of PF-3

TF expression

increased

conflicting results

Large WBC clone(4)

Old age (gt55y)

Subclinical PNH

efficient

well tolerated

What to do

risk of thrombosis

no hemolysis

mechanism

Supportive care

folate supplements

risk of bleeding

oesophagal spasm

irregular feeding

other authors say

warfarin LMWH NOAC

eculizumab

Literature

PNH History

PNH Definition

PNH Diagnosis

Literature

Occurs in 40 of PNH

Usual sites

pulmonary embolism

CNS arteries

and unusual sites

cavernous sinus

CNS veins

mesenteric veins

dermal veins

no link between

NO depletion(40)

release of PF-3

TF expression

increased

conflicting results

Large WBC clone(4)

Old age (gt55y)

Subclinical PNH

efficient

well tolerated

What to do

risk of thrombosis

no hemolysis

mechanism

Supportive care

folate supplements

risk of bleeding

oesophagal spasm

irregular feeding

other authors say

warfarin LMWH NOAC

eculizumab

Literature

PNH History

PNH Definition

PNH Diagnosis

Literature

no link between

NO depletion(40)

release of PF-3

TF expression

increased

conflicting results

Large WBC clone(4)

Old age (gt55y)

Subclinical PNH

efficient

well tolerated

What to do

risk of thrombosis

no hemolysis

mechanism

Supportive care

folate supplements

risk of bleeding

oesophagal spasm

irregular feeding

other authors say

warfarin LMWH NOAC

eculizumab

Literature

PNH History

PNH Definition

PNH Diagnosis

Literature

no link between

NO depletion(40)

release of PF-3

TF expression

increased

conflicting results

Large WBC clone(4)

Old age (gt55y)

Subclinical PNH

efficient

well tolerated

What to do

risk of thrombosis

no hemolysis

mechanism

Supportive care

folate supplements

risk of bleeding

oesophagal spasm

irregular feeding

other authors say

warfarin LMWH NOAC

eculizumab

Literature

PNH History

PNH Definition

PNH Diagnosis

Literature

TF expression

increased

conflicting results

Large WBC clone(4)

Old age (gt55y)

Subclinical PNH

efficient

well tolerated

What to do

risk of thrombosis

no hemolysis

mechanism

Supportive care

folate supplements

risk of bleeding

oesophagal spasm

irregular feeding

other authors say

warfarin LMWH NOAC

eculizumab

Literature

PNH History

PNH Definition

PNH Diagnosis

Literature

TF expression

increased

conflicting results

Large WBC clone(4)

Old age (gt55y)

Subclinical PNH

efficient

well tolerated

What to do

risk of thrombosis

no hemolysis

mechanism

Supportive care

folate supplements

risk of bleeding

oesophagal spasm

irregular feeding

other authors say

warfarin LMWH NOAC

eculizumab

Literature

PNH History

PNH Definition

PNH Diagnosis

Literature

TF expression

increased

conflicting results

Large WBC clone(4)

Old age (gt55y)

Subclinical PNH

efficient

well tolerated

What to do

risk of thrombosis

no hemolysis

mechanism

Supportive care

folate supplements

risk of bleeding

oesophagal spasm

irregular feeding

other authors say

warfarin LMWH NOAC

eculizumab

Literature

PNH History

PNH Definition

PNH Diagnosis

Literature

TF expression

increased

conflicting results

Large WBC clone(4)

Old age (gt55y)

Subclinical PNH

efficient

well tolerated

What to do

risk of thrombosis

no hemolysis

mechanism

Supportive care

folate supplements

risk of bleeding

oesophagal spasm

irregular feeding

other authors say

warfarin LMWH NOAC

eculizumab

Literature

PNH History

PNH Definition

PNH Diagnosis

Literature

increased

conflicting results

Large WBC clone(4)

Old age (gt55y)

Subclinical PNH

efficient

well tolerated

What to do

risk of thrombosis

no hemolysis

mechanism

Supportive care

folate supplements

risk of bleeding

oesophagal spasm

irregular feeding

other authors say

warfarin LMWH NOAC

eculizumab

Literature

PNH History

PNH Definition

PNH Diagnosis

Literature

increased

conflicting results

Large WBC clone(4)

Old age (gt55y)

Subclinical PNH

efficient

well tolerated

What to do

risk of thrombosis

no hemolysis

mechanism

Supportive care

folate supplements

risk of bleeding

oesophagal spasm

irregular feeding

other authors say

warfarin LMWH NOAC

eculizumab

Literature

PNH History

PNH Definition

PNH Diagnosis

Literature

conflicting results

Large WBC clone(4)

Old age (gt55y)

Subclinical PNH

efficient

well tolerated

What to do

risk of thrombosis

no hemolysis

mechanism

Supportive care

folate supplements

risk of bleeding

oesophagal spasm

irregular feeding

other authors say

warfarin LMWH NOAC

eculizumab

Literature

PNH History

PNH Definition

PNH Diagnosis

Literature

Large WBC clone(4)

Old age (gt55y)

Subclinical PNH

efficient

well tolerated

What to do

risk of thrombosis

no hemolysis

mechanism

Supportive care

folate supplements

risk of bleeding

oesophagal spasm

irregular feeding

other authors say

warfarin LMWH NOAC

eculizumab

Literature

PNH History

PNH Definition

PNH Diagnosis

Literature

Large WBC clone(4)

Old age (gt55y)

Subclinical PNH

efficient

well tolerated

What to do

risk of thrombosis

no hemolysis

mechanism

Supportive care

folate supplements

risk of bleeding

oesophagal spasm

irregular feeding

other authors say

warfarin LMWH NOAC

eculizumab

Literature

PNH History

PNH Definition

PNH Diagnosis

Literature

Large WBC clone(4)

Old age (gt55y)

Subclinical PNH

efficient

well tolerated

What to do

risk of thrombosis

no hemolysis

mechanism

Supportive care

folate supplements

risk of bleeding

oesophagal spasm

irregular feeding

other authors say

warfarin LMWH NOAC

eculizumab

Literature

PNH History

PNH Definition

PNH Diagnosis

Literature

Subclinical PNH

efficient

well tolerated

What to do

risk of thrombosis

no hemolysis

mechanism

Supportive care

folate supplements

risk of bleeding

oesophagal spasm

irregular feeding

other authors say

warfarin LMWH NOAC

eculizumab

Literature

PNH History

PNH Definition

PNH Diagnosis

Literature

efficient

well tolerated

What to do

risk of thrombosis

no hemolysis

mechanism

Supportive care

folate supplements

risk of bleeding

oesophagal spasm

irregular feeding

other authors say

warfarin LMWH NOAC

eculizumab

Literature

PNH History

PNH Definition

PNH Diagnosis

Literature

efficient

well tolerated

What to do

risk of thrombosis

no hemolysis

mechanism

Supportive care

folate supplements

risk of bleeding

oesophagal spasm

irregular feeding

other authors say

warfarin LMWH NOAC

eculizumab

Literature

PNH History

PNH Definition

PNH Diagnosis

Literature

efficient

well tolerated

What to do

risk of thrombosis

no hemolysis

mechanism

Supportive care

folate supplements

risk of bleeding

oesophagal spasm

irregular feeding

other authors say

warfarin LMWH NOAC

eculizumab

Literature

PNH History

PNH Definition

PNH Diagnosis

Literature

efficient

well tolerated

What to do

risk of thrombosis

no hemolysis

mechanism

Supportive care

folate supplements

risk of bleeding

oesophagal spasm

irregular feeding

other authors say

warfarin LMWH NOAC

eculizumab

Literature

PNH History

PNH Definition

PNH Diagnosis

Literature

What to do

risk of thrombosis

no hemolysis

mechanism

Supportive care

folate supplements

risk of bleeding

oesophagal spasm

irregular feeding

other authors say

warfarin LMWH NOAC

eculizumab

Literature

PNH History

PNH Definition

PNH Diagnosis

Literature

What to do

risk of thrombosis

no hemolysis

mechanism

Supportive care

folate supplements

risk of bleeding

oesophagal spasm

irregular feeding

other authors say

warfarin LMWH NOAC

eculizumab

Literature

PNH History

PNH Definition

PNH Diagnosis

Literature

What to do

risk of thrombosis

no hemolysis

mechanism

Supportive care

folate supplements

risk of bleeding

oesophagal spasm

irregular feeding

other authors say

warfarin LMWH NOAC

eculizumab

Literature

PNH History

PNH Definition

PNH Diagnosis

Literature

What to do

risk of thrombosis

no hemolysis

mechanism

Supportive care

folate supplements

risk of bleeding

oesophagal spasm

irregular feeding

other authors say

warfarin LMWH NOAC

eculizumab

Literature

PNH History

PNH Definition

PNH Diagnosis

Literature

risk of thrombosis

no hemolysis

mechanism

Supportive care

folate supplements

risk of bleeding

oesophagal spasm

irregular feeding

other authors say

warfarin LMWH NOAC

eculizumab

Literature

PNH History

PNH Definition

PNH Diagnosis

Literature

risk of thrombosis

no hemolysis

mechanism

Supportive care

folate supplements

risk of bleeding

oesophagal spasm

irregular feeding

other authors say

warfarin LMWH NOAC

eculizumab

Literature

PNH History

PNH Definition

PNH Diagnosis

Literature

risk of thrombosis

no hemolysis

mechanism

Supportive care

folate supplements

risk of bleeding

oesophagal spasm

irregular feeding

other authors say

warfarin LMWH NOAC

eculizumab

Literature

PNH History

PNH Definition

PNH Diagnosis

Literature

Supportive care

folate supplements

risk of bleeding

oesophagal spasm

irregular feeding

other authors say

warfarin LMWH NOAC

eculizumab

Literature

PNH History

PNH Definition

PNH Diagnosis

Literature

risk of bleeding

oesophagal spasm

irregular feeding

other authors say

warfarin LMWH NOAC

eculizumab

Literature

PNH History

PNH Definition

PNH Diagnosis

Literature

warfarin LMWH NOAC

eculizumab

Literature

PNH History

PNH Definition

PNH Diagnosis

Literature

warfarin LMWH NOAC

eculizumab

Literature

PNH History

PNH Definition

PNH Diagnosis

Literature

eculizumab

Literature

PNH History

PNH Definition

PNH Diagnosis

Literature

PNH History

PNH Definition

PNH Diagnosis

Literature

PNH History

PNH Definition

PNH Diagnosis

Literature

PNH History

PNH Definition

PNH Diagnosis

Literature

PNH History

PNH Definition

PNH Diagnosis

Literature

Documents

Implication of microvesicles in thrombosis associated with