• eculizumab;
  • paroxysmal nocturnal hemoglobinuria;
  • thrombosis


  1. Top of page
  2. Abstract
  3. Introduction
  4. Epidemiology of thrombosis in PNH
  5. Why is the risk of thrombosis increased in PNH?
  6. Prevention and treatment of thrombosis in PNH
  7. Eculizumab
  8. Summary and conclusion
  9. Disclosure of Conflict of Interests
  10. References

Summary.  Paroxysmal nocturnal hemoglobinuria (PNH) is a rare acquired disease characterized by a clone of blood cells lacking glycosyl phosphatidylinositol (GPI)-anchored proteins at the cell membrane. Deficiency of the GPI-anchored complement inhibitors CD55 and CD59 on erythrocytes leads to intravascular hemolysis upon complement activation. Apart from hemolysis, another prominent feature is a highly increased risk of thrombosis. Thrombosis in PNH results in high morbidity and mortality. Often, thrombosis occurs at unusual locations, with the Budd–Chiari syndrome being the most frequent manifestation. Primary prophylaxis with vitamin K antagonists reduces the risk but does not completely prevent thrombosis. Eculizumab, a mAb against complement factor C5, effectively reduces intravascular hemolysis and also thrombotic risk. Therefore, eculizumab treatment has dramatically improved the prognosis of PNH. The mechanism of thrombosis in PNH is still unknown, but the highly beneficial effect of eculizumab on thrombotic risk suggests a major role for complement activation. Additionally, a deficiency of GPI-anchored proteins involved in hemostasis may be implicated.


  1. Top of page
  2. Abstract
  3. Introduction
  4. Epidemiology of thrombosis in PNH
  5. Why is the risk of thrombosis increased in PNH?
  6. Prevention and treatment of thrombosis in PNH
  7. Eculizumab
  8. Summary and conclusion
  9. Disclosure of Conflict of Interests
  10. References

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare disease characterized by chronic intravascular hemolysis and hemoglobinuria, an increased risk of thrombosis, and a variable degree of bone marrow failure. The disease is caused by an acquired mutation of the X-linked PIGA gene in the hematopoietic stem cell (HSC). The PIGA gene product is essential for correct assembly of glycosyl phosphatidylinositol (GPI) anchors, linking several proteins to the cell membrane. Therefore, this mutation results in a clone of blood cells deficient in GPI-anchored proteins (GPI-APs). Lack of the GPI-anchored complement inhibitors CD55 and, particularly, CD59 on erythrocytes results in increased sensitivity to complement-mediated lysis. CD55 inhibits C3 convertases, and CD59 prevents the assembly of the membrane attack complex (MAC) at the cell surface.

The clinical spectrum of PNH is highly variable. It ranges from classic PNH with large PNH clone sizes, massive hemolysis, and a high risk of thrombosis, to patients with relatively small clone sizes and only mild or subclinical hemolysis. Patients in the latter group often have an underlying bone marrow failure disease such as aplastic anemia (AA), resulting in more prominent pancytopenia [1]. Particularly classic PNH patients, suffer from intravascular hemolysis, resulting in anemia, fatigue, and hemoglobinuria. Paroxysms result from complement activation above basal levels, as may occur during infection. Such a hemolytic crisis may enhance hemoglobinuria, and elicit acute renal failure and thrombotic events. Thrombosis is one of the most severe complications, seriously affecting quality of life, and is a major cause of death in PNH [2]. Many patients suffer from multiple thromboses in vital organs, such as the liver, brain, or gut, sometimes even during anticoagulant prophylaxis [2,3].

The mechanism of thrombosis in PNH is still not elucidated. In this review, we report on the current state of knowledge of the pathogenesis of thrombosis and its treatment in PNH. Various mechanisms that are proposed to play a role in thrombosis in PNH are discussed (summarized in Fig. 1). Special attention is given to eculizumab, a mAb directed to complement factor C5 that efficiently blocks intravascular hemolysis and its sequelae. Importantly, strong retrospective evidence suggests that eculizumab reduces thrombotic risk in PNH [3], and it is therefore currently regarded as the best known prophylaxis.


Figure 1.  Hypothetical mechanisms for thrombosis in paroxysmal nocturnal hemoglobinuria (PNH). The hemostatic balance is maintained by coagulation and fibrinolysis, and is influenced by factors derived from the vessel wall and blood cells. Several mechanisms have been suggested to determine the direction of the balance towards a prothrombotic state in PNH. These include the release of free hemoglobin, which activates the endothelium and scavenges nitric oxide (NO). In addition, complement-mediated damage of glycosyl phosphatidylinositol (GPI)-deficient blood cells may result in the release of procoagulant microparticles into the circulation and platelet activation. Finally, deficiency of GPI-anchored fibrinolytic factors such as urokinase plasminogen activator receptor (u-PAR), and anticoagulant factors such as tissue factor pathway inhibitor (TFPI) and, potentially, proteinase 3 (PR3), may further disturb the hemostatic balance.

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Epidemiology of thrombosis in PNH

  1. Top of page
  2. Abstract
  3. Introduction
  4. Epidemiology of thrombosis in PNH
  5. Why is the risk of thrombosis increased in PNH?
  6. Prevention and treatment of thrombosis in PNH
  7. Eculizumab
  8. Summary and conclusion
  9. Disclosure of Conflict of Interests
  10. References

The cumulative 10-year incidence of thrombosis in a retrospective study of 460 PNH patients was 31% [2]. In another retrospective series of 80 patients, 39% had one or more episodes of either venous or arterial thrombosis [4]. However, these series included PNH patients diagnosed by less sensitive methods than flow cytometry, leading to overrepresentation of PNH patients with larger clones, and thus a potential overestimation of thrombotic risk [4,5]. On the other hand, as shown by Hill et al., subclinical thrombosis is frequent, which implies an underestimation of thrombosis risk. They found evidence of subclinical pulmonary embolism or myocardial ischemia in six of 10 patients [6].

The risk of venous thrombosis correlates with PNH granulocyte clone size. The study of Hall et al. reported a 44% 10-year risk of venous thrombosis in patients with a PNH granulocyte clone of > 50%. In patients with smaller clone sizes, this was only 5.8%, which is, however, still higher than in healthy controls [7]. Moyo et al. [8] confirmed the association between PNH granulocyte clone size and thrombosis, and estimated an odds ratio of 1.64 for every 10% increase in clone size. Whether PNH clone size in other lineages also correlates with thrombotic risk is unknown. However, for PNH platelet clone size this is expected, as it correlates strongly with PNH granulocyte clone size [9]. Another open question is whether PNH clone size independently increases thrombotic risk or does so by affecting the level of hemolysis. The proportion of thrombotic events that occur during hemolytic crises has never been systematically studied. However, case reports provide evidence that thrombosis may occur in patients with large clones even when little or no hemolysis is present [10,11].

A higher risk of venous thrombosis was reported in patients of African-American or Latin-American descent, and a lower risk in Chinese and Japanese patients [12–14]. In Japanese patients, this can probably be explained by a significantly lower PNH granulocyte clone size than in Western patients [14].

The risk of arterial thrombosis is probably also increased as compared with age-matched healthy controls. Ziakas et al. described 38 reports of arterial thrombosis, mainly in the central nervous system or coronary arteries, occurring in relatively young patients, with a median age of 35 years (range: 22–47 years) for acute coronary syndromes, and 37–41 years (range: 11–76 years) for stroke [15,16]. Three arterial thrombotic events were reported in a cohort of 209 Japanese patients (1.4%) [14], none in the series of 220 patients of Sociéet al. [5], eight in the cohort of 80 patients of Hillmen et al. (10%) [4], and seven in our own cohort of 97 patients (7.2%) diagnosed between 1990 and 2011 (unpublished data). In the classic PNH patient population that participated in the various eculizumab trials, 15% of pretreatment thrombotic events were arterial, located in either the cerebral vasculature (13.6%) or coronary arteries (1.4%) [3]. The international PNH registry (, a prospective follow-up study now including over 1000 patients worldwide, may provide more definite information on the incidence and prevalence of arterial and venous thrombosis in PNH [17].

Thrombosis is the most important prognostic factor affecting survival. This is even more the case for the subcategory of PNH patients in whom bone marrow failure is prominent (hazard ratio [HR] for classic PNH, 7.8; HR for AA-PNH 33) [2]. Data from several older retrospective studies showed that in 22.2% of PNH patients the cause of death was related to thrombosis, and in Western European patients this proportion was even higher (37.2%) [18]. The extremely high incidence of thrombosis in PNH and its major effects on morbidity and mortality underline its clinical importance.

Why is the risk of thrombosis increased in PNH?

  1. Top of page
  2. Abstract
  3. Introduction
  4. Epidemiology of thrombosis in PNH
  5. Why is the risk of thrombosis increased in PNH?
  6. Prevention and treatment of thrombosis in PNH
  7. Eculizumab
  8. Summary and conclusion
  9. Disclosure of Conflict of Interests
  10. References

Multiple mechanisms have been proposed to explain thrombophilia in PNH (Fig. 1); however, none of these mechanisms on its own sufficiently explains the extremely high thrombotic risk in PNH. Below, each mechanism and its proposed relative contribution will be discussed in detail.

Role of the endothelium

Endothelial cell (EC) damage is an important factor that can contribute to thrombosis. Free hemoglobin released from lysed PNH erythrocytes may be directly toxic to ECs [19]. Alternatively, EC damage could result from the uptake of monocyte-derived microparticles, which may be released from GPI-deficient monocytes upon complement damage. Such microparticles contain tissue factor (TF), thus increasing TF expression on ECs, as demonstrated by Aharon et al. [20]. In the studies of Simak et al. (n = 9) and Helley et al. (n = 23), the numbers of EC microparticles with a prothrombotic and proinflammatory phenotype, indicating EC damage, were significantly increased in PNH patients as compared with healthy controls [21]. Levels of the EC activation markers von Willebrand factor (VWF) and soluble VCAM-1 (sVCAM-1) were increased [22,23], whereas others found normal VWF levels and activity in a smaller study [24]. These results suggest endothelial damage or stimulation in PNH, either indirectly by free hemoglobin, or directly by complement-mediated damage.

It is not yet known whether PNH patient ECs harbor the PIGA mutation, and thus are more susceptible to complement damage than normal ECs. Nevertheless, even in normal ECs, the MAC upregulates TF and adhesion molecules [25]. Endothelial progenitor cells can arise from the bone marrow (reviewed in [26]). In myelodysplastic syndrome, it has been shown that circulating ECs (CECs) and hematopoietic progenitor cells harbor identical chromosomal abnormalities, indicating a common origin [27]. Preliminary evidence suggests that this may also be the case in PNH. Helley et al. [23] cultured endothelial colony-forming cells (ECFCs) from PNH patient mononuclear cells, and demonstrated CD55-deficient and CD59-deficient populations within these ECFCs. Further research is required to determine whether PNH CECs or ECFCs do indeed harbor PIGA mutations, and whether and where such cells are incorporated into the endothelium. A recent study on the frequency of donor-derived ECs in various tissues in allogeneic transplant recipients demonstrated that only a minority of ECs originate from donor HSCs [28]. A high frequency of GPI-deficient ECs in PNH thus seems unlikely, suggesting that direct complement damage to GPI-deficient ECs is relatively less important in endothelial activation. However, studies in liver transplant patients showed that recipient-derived ECs do repopulate the liver allograft [29]. Also, this study showed that, in bone marrow-transplanted mice, the liver endothelium, in contrast to other organs, is partially or even completely composed of donor-derived ECs. These results indicate that, particularly in the liver, there might be a role for GPI-deficient bone marrow-derived ECs in PNH.

Deficiency of TF pathway inhibitor (TFPI) and other GPI-APs involved in coagulation

TFPI limits coagulation initiation by inhibiting TF formation. TFPI forms a quaternary complex with TF, activated factor VII (FVIIa) and activated FX (FXa). It is mainly produced by the endothelium of the microvasculature (85%), but other sources include activated platelets, monocytes, and plasma. The full-length isoform TFPIα is most abundant. It is bound either to glycosaminoglycans or to the cell membrane via an as-yet unidentified GPI-anchored cofactor [30,31]. The alternatively spliced TFPIβ also binds to the membrane via a GPI anchor; however, this variant is absent in platelets [32].

Both TFPI isoforms are upregulated in monocytes upon lipopolysaccharide stimulation. Blocking TFPI enhances monocyte procoagulant properties [33], as may also occur in TFPI-lacking GPI-deficient monocytes. If GPI-deficient ECs are indeed present in PNH, lack of TFPI may render such ECs procoagulant. However, as HSC-derived ECs are supposedly infrequent, the contribution of TFPI-deficient endothelium in PNH-related thrombosis is probably limited. On quiescent platelets, TFPIα is not expressed. Only upon simultaneous stimulation with collagen and thrombin do these highly activated platelets express TFPIα and several other procoagulant proteins, and release TFPIα into microvesicles [32]. GPI-deficient platelets probably lack surface expression of TFPIα upon stimulation, which may further enhance their procoagulant properties.

Another protein expressed on neutrophils that is involved in hemostasis is proteinase 3 (PR3). This enzyme binds to the cell membrane by using the GPI-anchored cofactor NB1 (CD177) [34]. Jankowska et al. demonstrated that PR3 is absent on GPI-deficient neutrophils, and that circulating PR3 levels were inversely correlated with PNH granulocyte clone size. Furthermore, PR3 reduced thrombin-induced platelet activation, suggesting that a lacking PR3 on PNH platelets may promote platelet activation [35]. PR3 also modulates coagulation in various other ways, for example via cleavage of the endothelial protein C receptor, degradation of TFPI, upregulation of EC TF expression, and cleavage of VWF [36–39]. The net effect of deficient PR3 expression in PNH therefore requires further study.

The exact contribution of missing GPI-APs to PNH-related thrombosis is unclear. Despite a significant reduction in thrombosis risk during eculizumab treatment, the proportion of leukocytes and platelets lacking GPI-APs remains unchanged, or even increases in erythrocytes, arguing against a major role for GPI-AP deficiency itself. Alternatively, patients with congenital deficiency of PIGM, another gene that is essential in GPI anchor synthesis, frequently suffer from thrombosis but do not have hemolysis [40]. This would suggest an important contribution of GPI-deficient cells to thrombosis that is independent of hemolysis; however, in these patients, all ECs are supposedly GPI-deficient, and thus these may be the culprits.

Effects of free hemoglobin and nitric oxide (NO) depletion

Intravascular hemolysis increases free hemoglobin levels. Normally, free hemoglobin is rapidly cleared from the circulation by several scavenging mechanisms, such as binding to haptoglobin. Excessive intravascular hemolysis saturates these scavengers, resulting in free hemoglobin in plasma, which mediates direct proinflammatory, proliferative and pro-oxidant effects on ECs [41]. Free hemoglobin irreversibly reacts with NO to form nitrate and methemoglobin. Lysed erythrocytes release arginase, which catalyzes the conversion of arginine, the substrate for NO synthesis, to ornithine. Both processes decrease NO availability. NO normally maintains smooth muscle cell (SMC) relaxation, inhibits platelet activation and aggregation, and has anti-inflammatory effects on the endothelium. Through these mechanisms, decreased NO levels may increase the thrombotic tendency in PNH (reviewed in [19]). Levels of free hemoglobin in PNH patients do indeed strongly correlate with NO consumption and arginase levels. Correlation of thrombotic events with low NO levels was, however, not tested in this study, as this would require large patient numbers [42]. In venous thrombosis though, a role for NO depletion is doubtful, as veins lack SMCs and platelets contribute relatively little to venous thrombosis. Its definitive role, particularly in arterial thrombosis, still requires further research.

Platelet function

Platelet activation has been proposed to play a role in PNH-related thrombosis. Whereas normal platelets express both CD55 and CD59, these complement inhibitors are absent on PNH platelets [43,44]. However, while complement destroys PNH erythrocytes, it probably does not directly destroy PNH platelets, owing to their ability to shed the MAC [45]. Although thrombocytopenia is frequent in PNH, it is generally attributed to concomitant bone marrow failure and not to complement-mediated damage. In two small studies of 16 patients, the lifespan of the total platelet population was normal in the majority of patients [46,47]. Furthermore, the observation that, during eculizumab treatment, both PNH platelet clone size and platelet count remain stable fits the notion that platelets survive complement-mediated platelet destruction [9,48,49].

Although complement does not directly destroy platelets, it can induce platelet activation. Even on normal platelets, assembly of the MAC results in FV secretion from α-granules, increased prothrombinase activity, and the release of platelet microvesicles in vitro [50]. As blocking CD59 on normal platelets enhances these procoagulant responses [51], a similar response to MAC assembly would be expected in GPI-deficient PNH platelets. In vitro studies by Wiedmer et al. [52] showed that PNH platelets did indeed expose more FVa-binding sites and increased thrombin generation more than normal platelets upon MAC stimulation. Ex vivo studies provided some evidence for in vivo platelet activation [24], but these findings were not confirmed by others [43,53]. Adhesion and aggregation studies performed by Grünewald et al. [53] showed impaired function of PNH platelets, which can be potentially explained by reactive downregulation in response to chronic hyperstimulation. Together with shedding of the MAC from the platelet membrane by vesiculation, other receptors may also be lost, resulting in reduced function.

Microparticle formation

Microparticles are small membrane-derived vesicles that are shed upon activation, inflammation, or cell damage. Under normal conditions, the plasma membrane is composed of anionic phospholipids such as phosphatidylserine (PS) on the inner leaflet, and choline-based phospholipids (sphingomyelin and phosphatidylcholine) on the outer leaflet. Membrane asymmetry is lost upon cell stimulation, leading to PS exposure on the outer leaflet of the cell membrane, followed by cytoskeletal degradation and microparticle release. PS exposure on the surface of either microparticles or the cell membrane provides a surface for the assembly of the procoagulant enzyme complexes prothrombinase (FVa/FXa) and tenase (FVIIIa/FIXa), which catalyze coagulation [54,55].

Complement activation at the cell surface of GPI-deficient cells may stimulate the release of procoagulant microparticles, increasing the risk of thrombosis. Several studies have investigated this hypothesis. In vivo, the total level of microparticles exposing PS, as measured by a prothrombinase-based assay, was higher in PNH patients than in healthy controls. These microparticles were predominantly of platelet origin. No correlation was found with PNH clone size in any lineage [56]. In contrast, Simak et al. [21] used flow cytometry to enumerate PS-exposing microparticles, but did not confirm increased levels in PNH. A drawback of these studies is that they did not quantify microparticle TF content, which strongly enhances their procoagulant properties [57]. C5a was shown to induce monocyte TF expression and release of TF-containing microparticles, a phenomenon that might be enhanced on GPI-deficient leukocytes. Simak et al. [21], however, found normal leukocyte-derived microparticle levels, although monocyte origin was not specified. A case report of two PNH patients with severe recurrent thrombosis did show increased levels of circulating leukocyte-derived TF as compared with healthy controls [58].

In vitro, PNH platelets release significantly more microparticles than normal platelets upon MAC stimulation, as was shown by Wiedmer et al. [52]. In vivo though, platelet microparticle numbers were not significantly different from those in healthy controls, although the variability between patients was consistently higher than between healthy donors. This implies that a subgroup of patients may have higher microparticle concentrations [21,56].

Remarkably, the levels of erythrocyte microparticles were similar in PNH patients and healthy controls [21,56]. However, in vitro experiments did show that PNH erythrocytes release higher amounts of procoagulant microparticles upon complement stimulation [59,60]. The fact that these microparticles are not readily detected in vivo may suggest rapid clearance from the circulation, implying that their clinical relevance is doubtful.

The predictive value of microparticle levels for thrombosis in PNH is unknown. In patients with deep vein thrombosis and pulmonary embolism, the levels of TF-containing microparticles were not elevated [61,62]. In cancer patients, however, TF-containing, but not PS-expressing, microparticles did predict thrombosis [63,64]. If relevant in PNH, this probably applies most to TF-containing microparticle levels; however, these levels are unknown so far.

Fibrinolysis and anticoagulation

Impairment of fibrinolysis or anticoagulation can increase the tendency for thrombosis. Such impairment may result from deficiency of the GPI-anchored urokinase plasminogen activator (u-PA) receptor (u-PAR, CD87). u-PA converts plasminogen into plasmin and, in doing so, is involved in fibrinolysis and the degradation of extracellular matrix during tissue remodeling and cell migration. Binding of u-PA to u-PAR enhances plasmin formation and, via this mechanism, fibrinolysis remains localized pericellularly. u-PAR is indeed deficient on PNH leukocytes and platelets [65,66]. Plasma levels of soluble u-PAR are higher than in healthy controls, and correlate with PNH granulocyte clone size [65–67], suggesting that u-PAR without the GPI anchor cannot bind to the cell membrane. Thus, it may be released from GPI-deficient cells and compete with membrane-bound u-PAR for binding of u-PA [68]. The resulting decrease in local u-PA availability could increase the thrombotic risk in PNH. In a study of 78 patients, high soluble u-PAR levels were independently associated with thrombotic risk [66]. Arguing against a role for u-PAR deficiency, however, is the finding that u-PAR-deficient mice do not display spontaneous thrombosis [69].

Studies that have measured other fibrinolysis parameters in PNH report conflicting results and do not unanimously fit with global fibrinolysis impairment, as may result from u-PAR deficiency [22–24]. Gralnick et al. and Helley et al. found normal levels of the fibrinolysis inhibitors α2-antiplasmin, plasmin–antiplasmin (PAP) complexes, and plasminogen activator inhibitor-1 (PAI-1), and of the fibrinolysis activator tissue-type plasminogen activator (t-PA) [23,24]. In contrast, Grünewald et al. demonstrated a slightly lower level of plasminogen, and higher levels of D-dimer and the fibrinolysis activation markers PAP and tPA–PAI-1 complex, suggesting active fibrinolysis. These changes were inversely correlated with clone size, pointing to progressive impairment in patients with higher clone sizes [22]. Levels of the thrombin-activatable fibrinolysis inhibitor thrombomodulin and the anticoagulant proteins antithrombin, protein C and protein S were normal in PNH patients [22–24].

Role of the various mechanisms in the localization of thrombosis in PNH

An intriguing but still unresolved question in the pathophysiology of PNH-related thrombosis is its predilection for the venous system, in particular in the abdomen and brain. The most frequent manifestation is Budd–Chiari syndrome (41–44% of PNH patients with thrombosis). Other frequently affected sites include other intra-abdominal veins, intradermal veins, the central nervous system, and the limbs [2,5,15,18].

The answer to this question probably involves EC-specific procoagulant and anticoagulant properties, which are highly variable in different vascular beds (reviewed in [70,71]). The specific localization of thrombosis in PNH supports the idea that the endothelium is probably a major contributor. For example, TFPI is preferentially expressed in the microvasculature. High TFPI mRNA levels are found in human lung and liver tissue, and murine brain ECs [70,72]. If GPI-deficient ECs are present in these tissues, a lack of TFPI may have a great effect.

The various mechanisms involved in PNH-related thrombosis may differentially affect vascular beds. NO depletion probably promotes arterial thrombosis via its effects on SMC and platelet activation and not, or to a lesser extent, venous thrombosis. Bacterial and food antigens present in the mesenteric and portal veins may locally activate complement, resulting in a higher hemolytic rate and consequent endothelial damage, platelet activation, and microparticle release, and possibly also in direct complement-mediated damage to GPI-deficient ECs.

Frequency of congenital and acquired thrombophilia factors is not increased in PNH

There is no evidence that the prevalence of known genetic factors predisposing to thrombosis is increased in PNH patients. The frequency of FV Leiden in 66 PNH patients was similar to that in healthy controls [73]. Other studies reported normal levels of antithrombin, protein C, protein S and homocysteine in small series of PNH patients, and also similar frequencies of FV Leiden and methylenetetrahydrofolate reductase (MTHFR) and prothrombin mutations [74,75]. In another study of 16 patients, one had FV Leiden, one had a heterozygous prothrombin mutation, and two had a homozygous MTHFR mutation [22]. Of these patients with congenital thrombophilia factors, two experienced thrombotic events, as compared with two of 12 patients without thrombophilia. Although the frequency of genetic thrombophilia factors in PNH was apparently not increased in these relatively small studies, testing for such factors may identify PNH patients at additional risk. However, its value in unselected patients with venous thrombosis for prediction of thrombosis recurrence is limited [76]. Its value for treatment decisions in PNH is unknown, and we therefore do not recommend routine testing.

Lupus anticoagulant was not found in two studies of, in total, 26 patients, including patients with a history of venous thrombosis [22,77]. Dragoni et al. found antiphospholipid (APL) antibodies in five patients with and in three patients without thrombosis in a series of 13 PNH patients, whereas in the study of Darnige et al., APL antibodies were present in only three of 20 patients [75,77]. Again, patient numbers were small, and do not allow definitive conclusions to be drawn.

The JAK2-V617F mutation is frequently found in myeloproliferative diseases (MPDs). Like PNH, MPD is associated with a high risk of thrombosis at similar locations, raising the question whether the JAK2-V617F mutation is involved in PNH as well. No JAK-V617F mutations were found by Fouassier et al. [78] in 11 PNH patients with varying clone sizes (range: 0.5–92%), including three patients with thrombosis, whereas Sugimori et al. [79] reported three classic PNH patients with JAK-V617F mutations among 21 PNH patients with Budd–Chiari syndrome. Interestingly, the JAK-V617F mutation was found in PIGA-mutated but not in normal cells. More research on this aspect is warranted.

Prevention and treatment of thrombosis in PNH

  1. Top of page
  2. Abstract
  3. Introduction
  4. Epidemiology of thrombosis in PNH
  5. Why is the risk of thrombosis increased in PNH?
  6. Prevention and treatment of thrombosis in PNH
  7. Eculizumab
  8. Summary and conclusion
  9. Disclosure of Conflict of Interests
  10. References

Anticoagulant prophylaxis

Owing to the rarity of PNH, no randomized trials evaluating the effect of anticoagulant treatment on the risk of thrombosis have been performed. Hall et al. retrospectively compared the frequency of thrombosis in PNH patients with large granulocyte clone sizes (> 50%) between those with and without primary prophylaxis with warfarin. They found no thromboembolic events in 30 patients taking warfarin, whereas in 39 patients without prophylaxis the 10-year thrombosis rate was 36.5% [7]. Although these data suggest that warfarin effectively reduces thrombotic risk in PNH, it also represents a risk of bleeding in PNH patients, who frequently have concomitant thrombocytopenia. In the study of Hall et al. [7], two serious hemorrhages were reported in 39 patients on warfarin. Nevertheless, this study was the basis for the recommendation of the international PNH interest group to consider vitamin K antagonist (VKA) prophylaxis in patients with a PNH granulocyte clone > 50% and no contraindications for prophylaxis [1]. Literature on the efficacy of antiplatelet agents such as acetylsalicylic acid and glycoprotein IIb–IIIa receptor antagonists in preventing arterial thrombosis in PNH is non-existent, although PNH platelets may have a role in the pathophysiology of thrombosis in PNH.

Treatment of thrombosis in PNH patients

PNH patients with a proven venous thrombosis should be treated initially with low molecular weight heparin and VKA, according to the regular practice for other patients with venous thrombosis. Up to now, lifelong anticoagulation has been recommended for such patients. In rare cases, radiologic intervention has been considered in patients with acute onset of Budd–Chiari syndrome [1]. In a series of 15 Budd–Chiari patients, a transjugular intrahepatic portosystemic shunt (TIPS) was placed successfully in six of seven eligible patients [80]; however, TIPS placement may be associated with additional complement activation, and does not resolve thrombosis. Thrombolytic therapy with t-PA can be an alternative in potentially life-threatening thrombotic events, especially when a response to conventional anticoagulation is lacking. Araten et al. described nine patients in whom t-PA treatment resulted in resolution of the thrombus. However, in one patient, bleeding may have contributed to a fatal outcome [81,82]. As eculizumab has been observed to abrogate a cascade of thrombotic events, prompt initiation of eculizumab may be attempted, as this is a much safer strategy in view of the substantial bleeding risk associated with thrombolysis [83]. The role of other anticoagulants, such as the newly developed thrombin inhibitors, in the treatment or secondary prophylaxis of thrombosis has not been investigated.


  1. Top of page
  2. Abstract
  3. Introduction
  4. Epidemiology of thrombosis in PNH
  5. Why is the risk of thrombosis increased in PNH?
  6. Prevention and treatment of thrombosis in PNH
  7. Eculizumab
  8. Summary and conclusion
  9. Disclosure of Conflict of Interests
  10. References

The availability of the humanized anti-C5 mAb eculizumab has dramatically changed the treatment and prognosis of PNH. It effectively reduces hemolysis, hemoglobinuria, transfusion requirements, and anemia. In addition, symptoms attributed to hemolysis, such as smooth muscle dystonias, pulmonary hypertension, and kidney function, have been shown to improve as well, leading to an increased quality of life [42,48,84–88]. Preliminary data also suggest increased life-expectancy [49]. Although it was only retrospectively studied with limited follow-up, the rate of thromboembolic events was also highly significantly reduced during eculizumab treatment in comparison with pretreatment rates in the same cohort of PNH patients [3]. The event rate in eculizumab-treated patients was 1.07 per 100 patient-years, vs. 7.37 per 100 patient-years in the same patients pretreatment (P < 0.001).

Proposed effect of eculizumab on hemostasis in PNH

Eculizumab treatment lowered the plasma levels of coagulation activation markers (prothrombin fragments 1 and 2), markers of fibrinolysis (D-dimer and PAP complex), and markers of EC activation (t-PA, sVCAM-1, VWF, and total and free TFPI) [23,89]. The reduction in thrombotic risk in eculizumab-treated PNH patients suggests a major role for complement activation in the pathogenesis of thrombosis in PNH. By blocking C5, PNH platelet activation, damage and microparticle release may be reduced. Moreover, eculizumab effectively reduces intravascular hemolysis, and thus free hemoglobin levels and NO consumption. It may therefore prevent the detrimental effects of free hemoglobin on the endothelium, and restore the inhibition of platelet activation and aggregation by NO. Unexpectedly, however, eculizumab treatment did not change the total numbers of either PS-exposing microparticles or EC-derived microparticles, further supporting the idea that microparticles probably do not play a major role [23]. However, a decrease in the number of TF-containing microparticles may have been missed with the use of a prothrombinase-based assay to enumerate microparticles.

Eculizumab in the prevention and treatment of thrombosis in PNH

The major decrease in the thromboembolic event rate and the improvement in several hemostatic parameters in eculizumab-treated patients raises the question of whether patients receiving eculizumab without a history of thrombosis still require additional anticoagulant prophylaxis, especially thrombocytopenic patients at risk for bleeding. A randomized controlled clinical trial comparing the incidence of thrombosis in eculizumab-treated patients without a history of thrombosis with and without VKA treatment was never performed. Kelly et al. reported having stopped warfarin prophylaxis in 21 patients receiving eculizumab who had never had thrombosis. None of these patients developed thrombosis while receiving eculizumab (mean follow-up: 10.8 months) [49]. Although few clinical data are currently available, discontinuation of VKA in patients without a history of thrombosis may be justified. The international PNH registry may provide more data on this topic.

A thrombotic event in PNH is now generally considered to be a strong indication for prompt initiation of eculizumab treatment [81,90]. Whether, after a thrombotic event, additional anticoagulant prophylaxis should be continued lifelong in eculizumab-treated patients to prevent recurrence is still a matter of debate. Until now, only anecdotal evidence that discontinuation of anticoagulant treatment is safe has been reported for three patients receiving eculizumab with a history of venous and arterial thrombosis (follow-up: 10–42 months) [91]. On the other hand, recurrence of thrombosis during treatment with both eculizumab and warfarin in a patient with a history of thrombosis prior to eculizumab was also described [49]. Such cases again illustrate that neither VKA nor eculizumab offer full protection from recurrent thrombosis, and VKA discontinuation should be carefully considered.

Summary and conclusion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Epidemiology of thrombosis in PNH
  5. Why is the risk of thrombosis increased in PNH?
  6. Prevention and treatment of thrombosis in PNH
  7. Eculizumab
  8. Summary and conclusion
  9. Disclosure of Conflict of Interests
  10. References

Thrombosis risk is substantially increased in PNH and correlates with PNH clone size. The development of thrombosis is one of the most important factors negatively influencing survival. Thrombotic events in PNH, for reasons that are not yet understood, have a predilection for unusual locations in the venous system, such as the abdomen and the central nervous system. Its pathogenesis is still not understood, but is likely multifactorial. A major contributor is probably endothelial damage caused by free hemoglobin and possibly by complement itself. Though difficult to investigate, the presence and localization of GPI-deficient ECs is a key question to be answered in future research. Deficiencies of other GPI-APs involved in coagulation, such as u-PAR, and less well-characterized proteins, such as TFPI and PR3, possibly add to the thrombotic risk. Although extensively investigated, the role of complement-mediated procoagulant microparticle release is less well established. Finally, NO depletion is probably particularly relevant in arterial thrombosis.

Treating thrombosis in PNH is difficult, as prospective studies are lacking and, even during anticoagulant treatment, some patients develop multiple events. Eculizumab has dramatically improved the quality of life for PNH patients, and probably reduces the thrombotic risk, highlighting the major role for complement or complement-mediated hemolysis in thrombosis in PNH. Although great progress has been made, important questions remain unanswered: how can we predict which patients will suffer from thrombosis, and why does thrombosis occur at unusual locations? Future research is urgently needed to provide answers to these questions.

Disclosure of Conflict of Interests

  1. Top of page
  2. Abstract
  3. Introduction
  4. Epidemiology of thrombosis in PNH
  5. Why is the risk of thrombosis increased in PNH?
  6. Prevention and treatment of thrombosis in PNH
  7. Eculizumab
  8. Summary and conclusion
  9. Disclosure of Conflict of Interests
  10. References

P. Muus has served on advisory boards of Alexion Pharmaceuticals. The other authors state that they have no conflict of interest.


  1. Top of page
  2. Abstract
  3. Introduction
  4. Epidemiology of thrombosis in PNH
  5. Why is the risk of thrombosis increased in PNH?
  6. Prevention and treatment of thrombosis in PNH
  7. Eculizumab
  8. Summary and conclusion
  9. Disclosure of Conflict of Interests
  10. References
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