Recommendations would be to consider testing for PNH by flow cytometry in those patients with unexplained thrombosis and those who:. A frequent question is whether a normal lactate dehydrogenase LDH level could exclude PNH and therefore negate the need for sending a sample for peripheral blood flow cytometric testing. There are, admittedly rare, situations in which the LDH may not be raised such as those with a predominant Type II partially deficient of GPI-linked proteins red cell population in which hemolysis may be minimal, in patients who are heavily red cell transfusion—dependent, and in some instances where the thrombosis has occurred in a patient with a small percentage of PNH cells.
Screening by flow cytometry should therefore still be considered. This is a complex area and one of continued research interest. There is an intrinsic relationship between the coagulation cascade and the complement system that is revealed by understanding some of the mechanisms thought to result in thrombosis in PNH.
Platelet activation, complement-mediated hemolysis, impaired nitric oxide NO bioavailability, impairment of the fibrinolytic system, and inflammatory mediators are all proposed mechanisms and thought to be responsible for the increased thrombotic risk in patients with PNH.
Multiple factors are likely to contribute to any one thrombotic event. Platelet activation, known to initiate blood clotting, is likely to be the main culprit of the high incidence of thrombosis associated with PNH. Although complement activation of platelets theoretically may result in lysis or removal of platelets and thereby contributes, to a minor degree, to some of the thrombocytopenia 49 , 50 the survival of platelets from PNH patients has been found to be normal.
Phosphatidylserine becomes a determinant for phagocyte recognition of senescent or apoptotic cells to be cleared and may contribute to lowering of the platelet count. One study found that the platelet count rose, 56 whereas another did not.
Therefore, rather than causing lysis of platelets, the complement attack of platelets results in morphologic changes and the release of vesiculated membrane attack complex MAC. Platelet lysis is therefore minimized by this release from the cell surface of excess MAC by exovesiculation. The externalized phosphatidylserine on the microvesicles acts as a binding site for prothrombinase 62 and tenase complexes. Activated platelets also interact with neutrophils and can promote thrombus formation by release of neutrophil serine proteases and nucelosomes, synergistically activating Factor X further and thus triggering blood coagulation primarily through the extrinsic pathway.
It also remains to be explored whether PNH neutrophils more readily release serine proteases and the interaction with tissue factor pathway inhibitor TFPI that is already thought to be altered in PNH discussed later. The activation of platelets may also in itself perpetuate or exacerbate events, in a feedback loop, in patients through continuing the activation of the alternative pathway of complement through P-selectin but also by initiating activation of the classical pathway of complement as a result of platelet-derived chondroitin sulfate.
As well as the loss of CD59, further mechanisms by which platelets are activated in PNH Table 1 are through the depletion of NO, the direct toxicity of cell-free hemoglobin, increased reactive oxygen species causing oxidative stress, the generation of thrombin, which itself further activates platelets,and as a consequence of endothelial dysfunction.
It should be mentioned that one study determined that the platelets in PNH were hyporeactive and concluded that this may be caused by chronic hyperstimulation because of continual complement system attack. Thrombotic events have been temporally associated with increased hemolysis, 16 , 26 , 39 , 72 and intravascular hemolysis is also likely to be one of the principle contributors to thromboembolism in this disorder.
Hemolysis, through factors such as toxicity of the free hemoglobin and NO depletion, has been implicated in the initiation of platelet activation and aggregation. Further disintegration of heme releases toxic species of iron, which participate in biochemical reactions, such as the Fenton reaction, that generate free radicals and thus catalyze the formation of reactive oxygen species and result in loss of membrane lipid organization.
Reactive oxygen species were higher and reduced glutathione lower when studied in patients with PNH, and the PNH cells themselves were at higher oxidative stress. NO, a free radical, binds avidly to soluble guanylate cyclase resulting in increased intracellular cyclic guanosine monophosphate cGMP 81 Figure 3. CGMP activates cGMP-dependent kinases that decrease intracellular calcium concentration in smooth muscle, producing relaxation, vasodilatation, and increased regional blood flow, primarily by suppressing platelet aggregation, expression of cell adhesion molecules on endothelial cells, and secretion of procoagulant proteins.
Close integration between the complement cascade grey and the coagulation cascade green. The relationships with red cell hemolysis, platelet activation, endothelial cells, and white blood cells are also demonstrated. Detailed information regarding each interaction is given the text.
NO also interacts with components of the coagulation cascade to downregulate clot formation. For example, NO has been shown to chemically modify and inhibit Factor XIII, which suggests that NO deficiency would enhance clot stability and reduce clot dissolution.
The reaction of NO with oxyhemoglobin is fast and irreversible. The chronic nature of the hemolysis in PNH is such that even at baseline, in between paroxysms, there is sufficient release of free hemoglobin to saturate biochemical systems in place to remove it, resulting in NO depletion.
The rate of NO depletion correlates with the severity of intravascular hemolysis of which LDH is a sensitive marker. In addition to hemoglobin decompartmentalization and NO scavenging, intravascular hemolysis also releases erythrocyte arginase, an enzyme that converts l -arginine, the substrate for NO synthesis, to ornithine, thereby further reducing the systemic availability of NO 95 Figure 3.
Circulating procoagulant microvesicles in association with red blood cells have also been described, 98 although other investigators have found this source to be very low.
It binds pro-urokinase uPA to the cell surface, which converts plasminogen to plasmin and results in clot lysis. It is possible that the absence of u-PAR from the cell surface in PNH results in an increased tendency to thrombosis as a result of impaired fibrinolysis and reduced clot dissolution. Because of a lack of anchorage of u-PAR to the cell membrane, the increased plasma levels are thought to also contribute to the increased risk of venous thrombosis by competing with membrane-bound u-PAR.
It may potentiate thrombosis but is unlikely to be a sole cause of thrombosis. Fibrinolytic defects, such as plasminogen deficiency, are not generally associated with thrombosis. Binding of antithrombin to endothelial cells is thought to be mediated by heparan sulfate, also a GPI-linked protein. Its deficiency may partly contribute to the hypercoagulable state in PNH, although there have been no studies exploring this. Heparan sulfate—deficient mice have, however, been found to have the same amount of fibrin deposition as wild-type mice, raising the possibility that there is compensation for reduction in heparan sulfate by other glycosaminoglycans.
Only complete deficiency appears to lead to thrombosis. TFPI is predominantly released by the endothelium but is also present on the surface of monocytes, within platelets, and circulating in the plasma and is anchored, most likely indirectly, through the GPI anchor. It is a potent anticoagulant protein that abrogates blood coagulation by inhibiting both factors Xa and the tissue factor—factor VIIa catalytic complex, making it the only physiologically active inhibitor of the initiation of blood coagulation.
It has been suggested that defective expression or reduced activity as TFPI is downregulated by inflammatory cytokines , potentially coexistent problems in PNH, may contribute to both arterial and venous thrombosis. Expression of TFPI on the surface of platelets after dual-agonist activation has been described.
Membrane-bound PR3 modulates thrombus formation by cleaving the thrombin receptor and thereby decreasing thrombin-mediated platelet activation. Endothelial dysfunction occurs during any thrombotic event. Tissue factor, a key initiator of coagulation, is expressed in subendothelial mural cells and adventitial fibroblasts in and around the vessel wall and closely links the coagulation and complement cascades. The endothelium has also been implicated in the pathogenesis of thrombosis in hemolytic states.
Free hemoglobin and its breakdown oxidative product heme can directly activate endothelial cells and further promote inflammation and coagulation as well as increase tissue factor production and release of high molecular weight von Willebrand factor VWF. CD, similarly to CD, is derived from a subportion of endothelial cell junctions. It has a very short half-life in the circulation; its presence in the circulation in PNH is therefore indicative of persistent endothelial damage associated with the chronic hemolysis of PNH.
A similar association has been described in sickle-cell disease. As well as direct complement activation, similarly to platelets, the endothelial expression of cell adhesion molecules is also promoted by NO depletion because NO is known to suppress their expression.
Whether endothelial cells are affected by the PIG-A mutation is of considerable research interest. If they are found to be deficient of the complement regulatory proteins, CD55 and CD59, their dysfunction in PNH would be both primary and secondary contributors to thrombosis.
Complement activation plays a major role in vascular inflammation. These will further activate the endothelium with the production of endothelial cell microparticles, potentially self-perpetuating the problem. IL-6 promotes thrombin formation. Complement activation on the surface of monocytes and neutrophils is also followed by the formation of the MAC. On these cells the MAC induces cell activation and also proliferation.
One study demonstrated that complement activation by antiphospholipid antibodies and downstream signaling via C5a receptors in neutrophils leads to the induction of tissue factor Figure 3. Much of protein S, the cofactor for activated protein C, circulates in complex with the complement protein C4b-binding protein, inhibiting its anticoagulant function. Patients with sickle-cell disease also appear to be more resistant to activated protein C, which may be a result of increased factor VIII coagulant activity as well as the reduced protein S.
Thrombosis in PNH is also seen at the time of infection and may be partly caused by the increased hemolysis that usually coincides. There are also likely contributing hemolysis-independent mechanisms. The invading pathogens or damaged host cells are recognized by antigen-presenting cells, neutrophils, monocytes, macrophages, endothelial cells, and platelets, resulting in tissue factor exposure that is sustained by cytokines and chemokines.
The pathogen can also further induce complement activation, promoting generation of more C5a and MAC. C5a feeds back to promote expression of tissue factor.
Finally, the pathway turns full circle with the knowledge that a fourth pathway separate to the classical, lectin, and alternative has been described to activate the complement system in which thrombin itself cleaves and activates C3 and C5 independent of C3. This might explain the observation that once a patient has their first thrombosis, this often heralds further thrombotic complications spiraling out of control, despite anticoagulation, until the patient eventually succumbs.
Thrombosis in a patient with PNH is a requirement for urgent intervention because of the high likelihood of mortality or significant disability and the rapid deterioration that frequently occurs.
Attention needs to be given to the balance between bleeding for example, because of the underlying bone marrow failure and the highly thrombotic tendencies.
Randomized controlled trials are lacking but experience has been gained in large PNH centers. The optimal management of acute thrombotic events requires immediate full anticoagulation in the absence of major contraindications beginning with heparin therapy aiming for anti-Xa levels between 0. Continuing anticoagulation with the vitamin K antagonists is generally recommended in the long term if there are no contraindications see discussion on secondary prophylaxis later.
Recurrent thromboses and extension of existing thromboses are frequent complications in PNH. There is no published experience of the newer oral anticoagulants in PNH. It has been reported that hemolysis may be exacerbated on the initiation of heparin because at low doses it activates the alternative complement pathway.
However, at higher concentrations, it acts as an inhibitor. Indeed, development of any thrombosis in a patient with PNH is now considered one of the primary indicators to commence eculizumab therapy, and this should be done without delay.
The management of Budd-Chiari syndrome in a patient with PNH, which may occur despite anticoagulant prophylaxis, is usually complex. As with other thrombotic events in this condition, immediate commencement of eculizumab is recommended. We have shown by our own series of patients in Leeds that urgent commencement of eculizumab can reduce mortality and long-term sequelae of Budd-Chiari syndrome. When portal hypertension is the predominant problem, a transjugular intrahepatic portosystemic shunt procedure is often helpful by decreasing portal pressure gradients, improving synthetic function, reducing transaminase levels, and controlling ascites.
Allogeneic bone marrow transplant has been previously considered but, despite improvements in other indications, the associated morbidities and mortalities in hemolytic PNH remain unsatisfactory.
Liver transplantation is contraindicated because of the risk of recurrent thrombosis found in all cases of patients with PNH who had Budd-Chiari syndrome and underwent liver transplantation, although most of the data are in the pre-eculizumab era. The previous high mortality from mesenteric vein thrombosis appeared to be associated with surgical intervention. Medical management, which would now include commencing eculizumab therapy if it is available, should be feasible when imaging demonstrates that the bowel infarction has not led to transmural necrosis and bowel perforation.
Preventing thrombosis in PNH is an important aim in the management of patients with PNH and would be expected to lead to reduced morbidity and mortality. In vitro, heparin and low-molecular-weight heparin therapy have been shown to inhibit the hemolysis in PNH.
There have been reports of an increased incidence of heparin-induced thrombocytopenia and consequent thrombosis, thought to be explained by the increased platelet activation in PNH with induced release of platelet factor 4. If there is concern, theoretically, fondaparinux may be a safer formulation. In patients who are not treated with eculizumab, consideration of primary prophylaxis should be given to reduce the risk of thrombosis if there is no contraindication, such as thrombocytopenia or other bleeding risk.
Given that eculizumab improves the management of established thrombosis in PNH, then the pros and cons of prophylactic anticoagulation needs discussion with the patient. In addition, thrombocytopenia is a relative contraindication to anticoagulation and this complication is not uncommon in patients with PNH. In addition, there are still clear cases of thromboses occurring while patients are therapeutically anticoagulated, 14 , 28 , 33 , 37 , 39 , , which is less surprising when the proposed mechanisms are considered.
After a thrombotic event, it appears that anticoagulation alone as secondary prevention is not sufficient. There are no studies of antiplatelet drugs, such as aspirin or clopidogrel, in PNH, but again, mechanistically, it is clear that they are unlikely to be of benefit and again there is a true risk of hemorrhage.
Considering that the mechanisms of thrombosis in PNH appear to lie with the role of platelet activation through direct complement activation as well as intravascular hemolysis and the release of free hemoglobin with all its consequent effects and mechanisms mediated through C5a, it might be anticipated that complement blockade should eliminate the risk of thrombosis, although there are no prospective trial data. Data for patients treated by the National PNH Centre, Leeds, UK, have recently been published supporting a continuing dramatic reduction in thrombosis rate and this is perhaps one of the important factors behind the significantly improved initial survival for patients treated with eculizumab.
An important question still to be addressed is whether anticoagulation can safely be discontinued in patients with PNH who have had a previous thrombosis and are receiving eculizumab. This has been achieved successfully and reported in 3 patients, although longer follow-up is required. Thrombosis has been well-recognized as the leading cause of death in PNH. Preventing thrombosis in this disease and effectively treating thrombosis early on in its presentation are paramount.
Appreciating the high frequency of thrombosis in PNH should lead one to thorough, and possibly multiple, investigations to exclude thrombosis. A patient presenting with thrombosis should be considered for screening for PNH if they fall into one of the 4 categories described. The tendency toward thrombosis in patients with PNH is multifactorial in etiology, involving the absence of GPI-anchored complement inhibitors on the surfaces of circulating platelets, the high levels of intravascular free plasma hemoglobin with the consequent scavenging of NO, fibrinolytic defects, and the pro-inflammatory effects of C5a.
The relative importance of each factor is not yet known but the integration between the 2 major host protection systems, coagulation and innate immunity, is obvious. The majority of the mechanisms relate to complement dysfunction and its consequences. Therefore eculizumab, which addresses these mechanisms, resulting in the reduction of thrombosis risk, has now become an important part of the management of this most feared complication.
Because thrombosis is the leading cause of death, the impact of eculizumab on thrombosis largely explains the improved survival seen with eculizumab therapy.
Contribution: A. Conflict-of-interest disclosure: A. Sign In or Create an Account. Sign In. Skip Nav Destination Content Menu. Close Abstract. Incidence and sites of thrombosis. When should we test for PNH in patients presenting with thrombosis? Proposed mechanisms of thrombosis in PNH. Management of thrombosis in PNH. Article Navigation. CME Article June 20, Thrombosis in paroxysmal nocturnal hemoglobinuria CME.
Anita Hill , Anita Hill. Department of Haematology, St. This Site. Google Scholar. Richard J. Kelly , Richard J. Peter Hillmen Peter Hillmen. Blood 25 : — Article history Submitted:.
Connected Content. Cite Icon Cite. Continuing Medical Education online. Figure 1. View large Download PPT. Figure 2. Table 1 Causes of activated platelets in PNH. Complement-mediated through loss of CD59 and assembly of C5b-9 on the surface 2. NO depletion 3. Direct effects of free hemoglobin 4. Increased levels of reactive oxygen species 5. Endothelial dysfunction 6. Thrombin activation. View Large. Table 2 Consequences of intravascular hemolysis in the mechanisms of thrombosis in PNH.
Increased levels of reactive oxygen species 2. Release of arginase resulting in NO depletion 3. Release of procoagulant red cell microparticles. Table 3 Proposed hemolysis-independent mechanisms of thrombosis in PNH.
Thrombin activation 2. Deficiency of u-PAR although may only be significant in the presence of red cell microparticles 3. Deficiency of heparan sulfate 4. Deficiency of TFPI 5. Deficiency of PR3 6. Endothelial cell activation 7. Decreased levels of protein S Thrombin activation of the complement system perpetuating mechanisms above. 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.
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