Summary of the multiple factors thought to contribute to the prothrombotic state in paroxymal nocturnal hemoglobinuria PNH and interaction. Further details are provided in the text. As previously described, thrombosis is the most serious complication associated with PNH. Hall et al. The mechanisms behind thrombus formation in PNH are complex and subject to continued research. Interactions between the complement system, platelets and coagulation likely explain some of the increased risk of thrombosis.
Due to the multifactorial and variable nature of the disease, it is likely that a combination of several factors may contribute to the increased incidence of thrombus formation and associated mortality Figure 2.
Figure 2. Hemostasis mechanisms in paroxymal nocturnal hemoglobinuria PNH patients are imbalanced towards thrombosis. Platelets have been reported to play a significant role in the formation of thrombus in PNH patients, by both contributing to a prothrombotic state and initiating clot formation. However, this is not the case, as the lifespan of platelets in PNH patients is normal.
The exact role of PMPs is not fully understood, however, they are considered to play a role in the generation of a prothrombotic state in PNH. However, further studies are necessary to analyze and quantify their specific role in PNH-induced risk of thrombosis. Phosphatidylserine is expressed on the surface of platelet-derived microparticles as a result of MAC binding-induced morphological changes.
The prothrombotic properties of residual activated platelets platelets post microparticle production is still a matter of discussion. Activated platelets in PNH patients have been shown to possess greater than ten times the factor V binding sites compared with those from normal controls. As well as contributing to a wide array of symptoms in PNH, hemolysis is thought to contribute to a prothrombotic state, but its role is becoming increasingly scrutinized. Excess free hemoglobin is a further possible mechanism that may underpin the prothrombotic state in PNH.
There is increasing evidence supporting possible prothrombotic effects of free hemoglobin on platelets and the vascular endothelium. Anti-thrombin is enhanced by binding to the heparan sulphate receptor, a GPI-linked protein expressed on endothelial cells and hypothesized to be lost in patients with PNH.
No studies have fully investigated the significance of the heparan sulphate receptor, however, a compensatory mechanism has been suggested after there was no change in fibrin deposition in animal studies of heparan sulphate deficiency.
Free hemoglobin has also been demonstrated to produce reactive oxygen species ROS via two mechanisms: 23 the amphipathic heme interacts with the phospholipid membrane, and via the Fenton reaction catalyzes the production of ROS, 78 and extracellular hemoglobin autoxidizes to methemoglobin catalyzed by peroxidase enzymes which further generates ROS. The formation of ROS is well-documented to produce phospholipid disorganisation, induce cytotoxicity and promote inflammation, 81 23 and studies have also shown that ROS can directly enhance platelet activation.
Neutrophils have also been reported to contribute to the prothrombotic mechanisms in PNH. This mechanism may explain the high prevalence of thrombi in veins at atypical sites and warrants further study in PNH.
Thrombin, the generation of which is increased by many of the mechanisms described above, has been observed to independently activate complement proteins C3 and C5. This may begin to highlight a mechanism in which the MAC contributes to plasmin-induced synthesis of PAF in endothelial cells, however, it is unclear whether this would contribute to thrombosis or platelet hyporeactivity.
Hemoglobin and nitric oxide NO bind in an irreversible reaction, the rate of which is suggested to increase by up to — times as a result of intravascular hemolysis and the loss of heme compartmentalization. There is a growing body of evidence in the literature that individuals with an increased risk of thrombosis form fibrin clots with an altered 3-dimensional structure.
The dense clot structures are more resistant to fibrinolysis due to the increased number of fibers that need to be lysed and a reduced permeation of the lytic enzymes into the denser clot structure.
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. Additionally, a deficiency of GPI-anchored proteins involved in hemostasis may be implicated. Abstract 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.
Publication types Review. Substances Antibodies, Monoclonal, Humanized eculizumab.
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