Hypercoagulability in congenital haemolytic anaemias

Introduction

Thalassaemia, a congenital haemolytic anaemia caused by partial or complete lack of synthesis of one of the α- or β-globin chains of haemoglobin A, is the most common genetic disorder worldwide¹. Regular red blood cell transfusion is the mainstay of treatment in β-thalassaemia major (TM), the most severe form. In the past 30 years, the adoption of transfusion regimens tailored to keep haemoglobin closer to normal levels has dramatically improved the life expectancy of patients with this disease. Advances in the understanding of the molecular and pathophysiological mechanisms underlying the disease process have led to the identification of three main determinants of the natural history of this disorder: ineffective erythropoiesis, chronic anaemia/haemolysis and iron overload¹. This last, caused not only by multiple transfusions but also by increased intestinal absorption, causes continuous oxidative stress and massive cardiac and hepatic deposition of this metal, which ultimately results in multiple organ failure and premature death¹. The introduction of efficacious chelation therapy by means of parenterally administered desferrioxamine and, more recently, of oral agents such as deferiprone and deferasirox has been a major step forward in the reduction of the complications of iron overload^2,3. Among the clinical complications of this haematological disorder, thromboembolic episodes are particularly important and frequent, especially in patients with thalassemia intermedia. Like patients with thalassaemia, patients affected by sickle cell disease, which is the most prevalent genetic disorder in the African American population and results from a single amino acid mutation of the β-globin chain of haemoglobin A, also have an increased incidence of thrombotic events in addition to the well-known haemolytic and vaso-occlusive complications^4,5. This review summarises the main clinical, pathogenic and therapeutic aspects of thrombotic complications in these congenital haemolytic anaemias.

Hypercoagulability in thalassemia

Clinical findings

In addition to clinical symptoms typical of their congenital disease, patients with thalassaemia may develop thrombotic complications and both arterial and venous thromboses have been reported in several case series. For instance, a study on survival and causes of death in TM, carried out in Italy at the end of the 1980s, indicated thromboembolic events as the primary cause of death in four of 159 (2.5%) transfusion-dependent patients⁶. Thromboembolic events were reported in another Italian survey involving patients with TM and thalassemia intermedia (TI), with incidences of 4% (27/683) and 9.6% (5/52), respectively⁷. The thromboembolic events had a significant association with cardiac, metabolic or endocrine dysfunction. The most frequent localisation (in 2.2% of TM patients) was the central nervous system, giving a clinical picture of headache, seizures and hemiparesis. Similarly, Logothetis and colleagues described a “stroke syndrome” and neurological deficits compatible with transient ischaemic attacks in about 20% of 138 cases of TM in Greece⁸. These clinical findings were supported by a further study, carried out to assess the rate of silent brain damage in patients with benign haemoglobinopathies, in which it was found that 37.5% of patients with TI showed asymptomatic brain damage on magnetic resonance imaging⁹. A large study conducted by Taher and colleagues¹⁰, analysing data from 8,860 thalassaemic patients (6,670 with TM and 2,190 with TI), demonstrated that thromboembolic events occurred 4.38 times more frequently in patients with TI than in those with TM, with more venous thrombotic events occurring in TI and more arterial thrombotic events in TM. Patients with TI who developed a thromboembolic event were more frequently splenectomised females and had haemoglobin levels below 9 g/dL. Finally, in another series of TI patients, 29% of them developed either deep vein thrombosis, pulmonary embolism or portal vein thrombosis during a 10-year follow-up¹¹. All but one patient had undergone splenectomy. A recent meta-analysis conducted by Dentali and colleagues¹² on the association between β-thalassaemia trait and arterial cardiovascular disease identified eight case-control studies and found that this inherited condition, usually asymptomatic, exerts a protective effect in male subjects against the development of atherothrombotic cardiovascular events (OR 0.45; 95% CI 0.45–0.60). This unexpected finding is thought to be due to low serum cholesterol levels, consequent to liver iron overload and lower pressure, often measured in such people.

Pathophysiology

Several factors contributing to the hypercoagulable state in patients with β-thalassaemia have been identified, the most important including cellular factors (i.e., platelets, red blood cells, endothelial cells and monocytes) and acquired coagulation defects¹³.

The role played by cells in the hypercoagulability of patients with β-thalassaemia was recently investigated by Tripodi and colleagues¹⁴ by means of thromboelastometry and thrombin generation tests. All the thromboelastometry parameters determined in whole blood, including shortened clotting time and clot formation time and increased maximum clot firmness, were consistent with hypercoagulability, especially in splenectomised patients. However, thrombin generation determined in platelet-poor plasma was not significantly different from that of healthy individuals, suggesting that red blood cells, platelets and possibly other cellular elements play a more significant role than plasma alterations in the hypercoagulability observed in thalassaemic patients.

There is increasing evidence that thalassemic patients have activated platelets¹³. Indeed, platelet activation studies have revealed impaired platelet aggregation, increased platelet adhesion and circulating platelet aggregates, shortened survival and enhanced excretion of urinary metabolites of thromboxane A₂ and prostacyclin^15–17. The status of chronic platelet activation has also been confirmed by flow cytometric studies, which showed enhanced P-selectin (a platelet activation marker) expression on intact platelets in patients with thalassaemia, and is correlated with pre-β lipoproteins¹⁸. These platelet abnormalities are more pronounced in splenectomised patients, especially those affected by TI^19,20. The increased platelet aggregation and high platelet count following splenectomy contributes significantly to the increased susceptibility to thrombosis observed in this subset of patients²¹. Another fundamental mechanism of platelet activation is oxidative stress, caused by iron overload that catalyses hydroxyl radical generation from activated oxygen species²². Beside platelet abnormalities, oxidative injury is also responsible for the majority of the alterations observed in thalassaemic red blood cells²³. Indeed, as a consequence of the continuous oxidative stress, the membrane of thalassaemic red blood cells expresses abnormally high levels of negatively charged phospholipids, particularly phosphatidylserine. The negatively charged membrane surface assembles the prothrombinase complex, resulting in increased thrombin generation which in turn promotes fibrin clot formation²⁴. These pathogenic mechanisms leading to thromboembolic complications are, for several reasons, more frequently observed in splenectomised patients with the milder TI than in those with the more severe TM. Firstly, patients with TM are usually regularly transfused and thus the great majority of their circulating red blood cells are donor-derived normal allogeneic cells. Secondly, the spleen, when present, helps to remove the structurally abnormal and rigid red cells that cause hypercoagulability by playing as platelets, whereas after splenectomy there is a larger burden of circulating abnormal red cells, particularly in non-transfused or rarely transfused patients.

On the whole, these data provide good evidence that splenectomy in patients with TI must be considered a risk factor for venous thromboembolism²². Thrombophilic mutations do not appear to play an important role in the pathogenesis of thrombosis observed in β-thalassemia. In two studies from the Eastern Mediterranean region the presence of factor V Leiden, the G20210A prothrombin mutation and methylene tetrahydrofolate reductase mutations was not significantly correlated with thrombotic risk^25,26. Strong evidence for the presence of a chronic hypercoagulable state in thalassaemia also stems from measurements of coagulation factors and inhibitors in patients. Prothrombin fragment 1+2, a marker of thrombin generation, is elevated in TI¹¹. The levels of protein C and protein S in b-thalassaemia have been investigated in many studies and were generally decreased, especially in the splenectomised patients^11,27. The presence of anti-phospholipid antibodies has been reported in the serum of β-thalassemia patients, but none of the patients developed any complications²⁸.

Management

As venous thrombosis is more prevalent in splenectomised patients with β-TI, a number of strategies can be applied to prevent this clinical complication. First, the indications for splenectomy should be reviewed. In the past, the attempt to limit or not give transfusions to these patients by performing splenectomy was driven by the risk of transmission of blood-borne viral infections and by the consequences of iron overload. Both these concerns are now much smaller, because the blood supply is safer following appropriate donor testing, and the new oral chelating agents are more effective and easier to use (albeit more expensive) than the time-honoured parenteral desferal. Hence, the main thrust of prevention is to have fewer splenectomised patients with TI, and the practice of recommending splenectomy should be much more limited than it was until recently. The use of medications such aspirin and anticoagulants could be considered in the presence of independent thrombotic risk factors such as splenectomy, high platelet count and surgery.

Hypercoagulability in sickle cell disease

Clinical findings

Patients with sickle cell disease are at risk of a variety of thrombotic complications²⁹. Data from a case-control study of 1,070 patients and a retrospective cohort study of 65,000 consecutive hospital admissions of black men suggested that individuals with sickle cell trait had higher rates of venous thromboembolic events (deep vein thrombosis or pulmonary embolism) than a control group of blacks with normal haemoglobin^30,31. In the case-control study, black men with sickle cell trait had an approximately 4-fold increased risk of pulmonary embolism (OR 3.9; 95% CI, 2.2–6.9) and an approximately 2-fold risk of combined deep vein thrombosis or pulmonary embolism (OR 1.8; 95% CI, 1.2–2.9)³⁰. In the retrospective study, pulmonary embolism occurred in 2.2% of patients with sickle cell trait and in 1.5% of patients with normal haemoglobin³¹. A recent retrospective study based on reported discharge diagnoses showed that patients with sickle cell anaemia younger than 40 years were more likely to be diagnosed with pulmonary embolism than were African Americans without this congenital chronic anaemia (0.44% versus 0.12%), although, interestingly, the prevalence of deep vein thrombosis was similar in the two groups³². Finally, sickle cell anaemia also appears to be a significant risk factor for pregnancy-related venous thromboembolism, with an odds ratio of 6.7 (95% CI: 4.4–10.1)³³.

Pathophysiology

Sickle cell disease is characterised by chronic haemolysis and recurrent ischaemia due to micro-vascular occlusion following the adhesion of erythrocytes and leucocytes to the vascular endothelium³⁴. In addition, sickle cell anaemia is complicated by chronic activation of coagulation and of vascular endothelial cells, resulting in a hypercoagulable state³⁵. Although this hypercoagulability is considered multi-factorial chronic haemolysis plays a pivotal role, as it does in other chronic haemolytic anaemias such as paroxysmal nocturnal haemoglobinuria and β-thalassaemia^12,36. In particular, the external exposure of phosphatidylserine, which alters the adhesive properties of red blood cells, appears to play a key role in the haemostatic changes. Indeed, the number of phosphatidylserine-positive red blood cells was significantly correlated with plasma markers of thrombin generation, such as prothrombin fragment 1+2, D-dimer and plasmin-antiplasmin complexes, suggesting a role for red blood cells in coagulation activation^37,38. Similarly to what occurs in thalassaemia, platelets in sickle cell anaemia are activated and their activation is directly correlated with measures of haemolysis^5,39. Other studies have suggested a possible mechanistic role of circulating microparticles (small membrane-derived vesicles released by cells following activation or apoptosis), derived from red blood cells, platelets, endothelial cells and monocytes, in the hypercoagulable state seen in chronic haemolytic anaemias^40–42. The total number of microparticles, total tissue factor-positive microparticles, monocyte-derived tissue factor-positive microparticles and erythrocyte-derived microparticles are correlated with the levels of D-dimer, thrombin-antithrombin complexes and prothrombin fragment 1+2 in patients with sickle cell disease⁴⁰. The strong association of erythrocyte-derived microparticles with markers of fibrinolysis and coagulation activation as well as with haemolytic markers further confirms a role for haemolysis in the coagulation activation that is observed in patients with haemolytic anaemias. This finding is similar to those observed in a study by Ataga and colleagues⁴³, in which associations were observed between markers of coagulation activation (prothrombin fragment 1+2, thrombin-antithrombin complexes and D-dimer) and measures of haemolysis in a cohort of patients with sickle cell disease.

Management

A number of studies have been conducted in the past to determine the effects of antiplatelet and anticoagulant drugs on vaso-occlusive and thrombotic complications in sickle cell disease, but most of the studies were small and/or uncontrolled³⁶. Based on limited data and poorly designed trials, no convincing evidence exists at present to recommend drugs such as aspirin, dipyridamole, ticlopidine, heparin, or vitamin K antagonists for the prevention of vaso-occlusive and thrombotic complications in sickle cell disease⁴⁴. For this reason, sickle cell disease should not be an independent indication for treatment with antiplatelet or anticoagulant agents. Effective thromboprophylaxis (with low-molecular-weight heparin, low-dose unfractionated heparin, fondaparinux, or intermittent pneumatic compression) should be reserved to selected cases, such as hospitalised sickle cell disease patients undergoing surgical procedures. Indeed, prophylactic red blood cell transfusion significantly reduces the risk of strokes in black children with sickle cell anaemia⁴⁵, probably due to a beneficial effect in part related to the normalisation of prothrombin fragment 1+2 levels⁴⁶. Treatment with both hydroxyurea and decitabine also appears to decrease plasma markers of thrombin generation⁴⁷.

Conclusions

Based on the data available in the literature there is strong evidence that a hypergulable state is associated with inherited haemolytic anaemias, especially thalassaemia intermedia and sickle cell disease. Although the exact pathogenic mechanism needs to be clarified, there is increasing information on the crucial role of the presence of abnormal red blood cells in the development of hypercoagulability. This finding could have important therapeutic consequences and pave the way to the development of novel approaches to decrease the occurrence of thrombotic complications in haemolytic anaemias. Finally, well-controlled clinical studies of anticoagulants and/or antiplatelet agents employing appropriate clinical end-points in inherited haemolytic anaemias are warranted.