Iron and thrombosis.

Although essential for cell physiology, an increase or depletion of body iron has harmful effects on health. Apart from iron deficiency anemia and iron overload-related organ tissue damage, there are increasing evidences that body iron status is implicated in atherosclerotic cardiovascular diseases. The hypothesis formulated in 1981 that iron depletion may protect against cardiovascular events is intriguing and has generated a significant debate in the last two decades. Indeed, to study this phenomenon, several investigators have tried to design appropriate experimental and clinical studies and to identify useful biochemical and genetic markers of iron status. The results of the literature on the effect of iron deficiency and overload on vascular health are critically reviewed in this study from a pathogenic and clinical point of view.

Introduction

Iron is an essential nutrient for living cells because of its role as a cofactor for enzymes in the mitochondrial respiration chain, in the DNA synthesis, and being the central molecule for binding and transport of oxygen by hemoglobin and myoglobin.

While the lack of iron leads to growth arrest and anemia, an increased accumulation of this metal is associated with toxic radical formation and progressive tissue damage. It is interesting to note that both iron deficiency and excess have been associated with an increased risk of developing thromboembolic events [1-7].

This review will analyze, from a clinical and pathogenic point of view, the existing literature data on the relationship between iron and arterial and venous thrombosis.

Iron deficiency and thrombosis

There are several reports in the literature on thrombotic complications in iron-deficient children and adults [8-26]. Secondary thrombocytosis has been implicated in many cases. Indeed, iron deficiency is a cause of reactive thrombocytosis, usually mild [27]. For instance, within a study group of children with iron deficiency, reactive thrombocytosis was found in up to one-third of them [28].

Nagai et al. [29] reported a case of severe iron deficiency with marked thrombocytosis (1,020 × 109/l) that was complicated by central retinal vein occlusion. By contrast, Kinoshita et al. [26] described two cases of cerebral venous sinus thrombosis associated with iron deficiency and normal platelet count. Hartfield et al. [13] reported six children with iron deficiency who developed an ischemic stroke or venous thrombosis. Four of them had a concomitant thrombocytosis. In a prospective case–control study, Stolz et al. [30] found that severe anemia, along with thrombophilia and hypercholesterolemia, were independent risk factors for cerebral venous thrombosis.

The mechanisms causing reactive thrombocytosis in iron deficiency anemia are not completely understood. Iron is an important regulator of thrombopoiesis [31, 32]. Whereas normal iron levels are required to prevent thrombocytosis by inhibiting thrombopoiesis, a minimum amount of iron is required to maintain platelet production. Thus, while thrombocytosis is usually associated with a mild iron deficiency and is the result of a lack of inhibition of thrombopoiesis, a severe defect of this metal may be accompanied by thrombocytopenia. However, studies on thrombopoietic cytokines failed to show any effect on reactive thrombocytosis in iron deficiency [27]. For instance, Akan et al. [33] assayed the serum levels of thrombopoietin, erythropoietin, leukemia inhibitor factor, IL-6, and IL-11 in patients with iron-deficient anemia with or without elevated platelet count. Only erythropoietin level was elevated, correlated with thrombocytosis, and decreased with iron replacement. The other cytokines remained unchanged after therapy, suggesting that they probably do not play any significant role in iron deficiency-associated reactive thrombocytosis. Recently, Bilic and Bilic reported that the amino acid sequence homology of thrombopoietin and erythropoietin may explain the thrombocytosis in children with iron deficiency anemia [34]. By contrast, two other reports suggested that the relationship between iron deficiency and reactive thrombocytosis is more complex than a mere consequence of a crossreactivity between erythropoietin and thrombopoietin [35, 36]. In addition to the increased thrombotic risk associated with high platelet count, other authors have suggested that the decrease in antioxidant defense in iron deficiency anemia may cause increased oxidant stress, which in turn may result in a tendency toward platelet aggregation [37]. Thus, the abnormal platelet count and function observed in iron deficiency anemia could act synergistically to promote thrombus formation, especially in the setting of an underlying atherosclerotic disease [23].

However, as not all cases of iron-related thrombotic events occur in patients with concomitant high platelet count [13, 26], other pathogenic mechanisms have been proposed in these last years. Thus, iron deficiency may contribute to a hypercoagulable state by affecting blood flow patterns within the vessels because of reduced deformability and increased viscosity of microcytic red blood cells [13]. Furthermore, anemic hypoxia secondary to iron deficiency could precipitate situations of increased metabolic stress (i.e., infections) in particularly vulnerable areas of the brain supplied by end arteries, such as the basal ganglia, thalamus, and hypothalamus [38]. This phenomenon could explain the association between iron-deficient anemia and reversible focal deficits and stroke found by some authors [1, 13, 39, 40].

Iron overload and thrombosis

Accumulation of iron in excess of physiologic requirements has been implicated in the development of several chronic illnesses, including cardiovascular diseases [7]. As previously reported, iron is a prooxidant cofactor associated with an increased production of hydroxyl radical in cardiovascular tissues and increased progression of atherosclerosis in experimental models [41-44]. Indeed, in an experimental mouse thrombosis model, a moderate iron overload markedly accelerated thrombus formation, impaired vasoreactivity, and enhanced the production of reactive oxygen species and systemic markers of oxidative stress [41]. It is interesting to note that the administration of dl-cysteine, a reactive oxygen species scavenger, completely abrogated the iron load-induced thrombus formation thus corroborating the hypothesis that iron accelerates thrombosis through a prooxidant mechanism. Similarly, laboratory investigations have demonstrated iron-dependent generation of reactive oxygen species in endothelial cell cultures and increased aortic atherosclerosis in the apolipoprotein E-deficient mice and cholesterol-fed rabbits with increased iron intake [42-44].

The research in this field has focused on the evaluation of the impact of iron depletion and iron overload on cardiovascular outcomes. Sullivan first postulated in 1981 the “iron hypothesis” by which the chronic iron depletion has a protective effect against ischemic heart disease and may account for the reduced risk of cardiovascular events in menstruating women [45-47]. Basing on these observations, some investigators have studied the effect of serial blood donations on the coronary heart disease risk, but their results were inconsistent [48-53]. However, an important support to the hypothesis of a potential link between blood donation and reduced cardiovascular risk came from a recent study conducted by Zheng et al. [54] who found that high-frequency blood donors had decreased serum ferritin levels, a marker of body iron stores; decreased serum 3-nitrotyrosine levels, a marker of oxidative stress; and greater flow-mediated dilation in the brachial artery, a marker of vascular function. It is interesting to note that in a clinical study, the iron chelation with deferoxamine improved endothelial function in patients with coronary artery disease [55]. By contrast, in a multicenter, randomized controlled trial (the iron [Fe] and Atherosclerosis Study [FeAST]) on 1,277 patients with symptomatic peripheral arterial disease, the reduction of body iron stores by phlebotomy did not significantly decrease all-cause mortality or death plus nonfatal myocardial infarction and stroke [56]. Similarly, in a prospective analysis of the second National Health and Nutrition Examination Study (NHANES II), Sempos et al. [57] observed either no association (in Caucasian men) or a possible nonsignificant increased risk (in Caucasian women) of cardiovascular or coronary heart disease death among individuals with low ferritin concentrations.

On the other hand, the association between biochemical markers of body iron load and the risk of developing cardiovascular disease have been investigated by several studies [7, 58–72]. In the Kuopio Ischemic Heart Disease Risk Factor Study (KIHD), serum ferritin levels were found to be one of the strongest risk factors for acute myocardial infarction among Finnish men [61]. Similar findings were reported from a Canadian study [65] that observed an increased risk of myocardial infarction among subjects in the highest serum iron category and from the Bruneck study [64] in which a positive association between serum ferritin levels and ultrasound measures of progression of carotid atherosclerosis over a 5-year follow-up period was found. A relationship between serum ferritin levels and carotid atherosclerosis was also identified by a recent study conducted by Wolff et al. [71]. Haidari et al. [60] observed a significant correlation between serum ferritin levels and risk of coronary heart disease in male Iranian patients. However, a number of epidemiological studies did not find an association between iron status and coronary artery disease [61, 69, 70, 72]. For instance, Bozzini et al. [61] found that the mean serum ferritin concentrations were slightly higher in coronary heart disease patients than in controls, but this difference disappeared after adjusting for sex and C-reactive protein.

The 1996 discovery of HFE gene mutations responsible for most cases of hereditary hemochromatosis has led to the use of genetic markers of iron overload, which are not influenced by external factors such as inflammation, in epidemiologic studies. Thus, several authors have investigated in recent years the relationship between C282Y and H63D mutations in the HFE gene and the risk of cardiovascular diseases [73-88]. Three prospective population-based studies have reported an association between heterozygotes and vascular events [75-77].

The first study was from a subgroup of the original Finnish KIHD cohort [75]. Of 68 individuals, 8 (11.8%) were diagnosed with acute myocardial infarction, and of 1,150 noncoronary heart disease participants, 77 (6.7%) were carriers of C282Y. The crude relative risk of myocardial infarction was 2.0 (95%CI = 0.9–4.1) and the adjusted relative risk was 2.3 (95%CI = 1.1–4.8). In a cohort of 12,239 Dutch postmenopausal women, the C282Y carrier status was assessed among 531 women who died of cardiovascular disease and 555 randomly selected women who did not die of cardiovascular disease [76]. This study reported a relative risk of 1.6 (95%CI = 1.1–2.4) for total cardiovascular death. Finally, in the United States Atherosclerosis Risk in Communities (ARIC) study [77], a C282Y carrier frequency of 9.9% among 243 coronary heart disease cases and 6.1% among 535 controls was reported. The crude relative risk of coronary heart disease associated with C282Y carrier status was 1.6 (95%CI = 0.9–3.0) and was 2.7 (95%CI = 1.2–6.0) after being controlled for other risk factors. To support of these findings, Gaenzer et al. [78] found an association between increased iron stores and impaired endothelial function (measured as endothelium-dependent dilation and intima-media thickness) in patients homozygous for C282Y mutation. Iron-depletion therapy normalized the endothelial function in such patients thus reducing the increased risk of cardiovascular events.

However, the majority of the studies disagreed with these results [61, 79–89]. Indeed, Bozzini et al. [61] found a similar rate of carriers of C282Y mutation among patients with coronary atherosclerotic disease and controls. In a case–control study on 1,098 subjects, Rossi et al. [80] found that C282Y mutation was not a predictor of asymptomatic carotid atherosclerosis. Franco et al. [81] reported that the HFE genes were not associated with coronary or peripheral atherosclerosis in patients aged less than 50 years. Similarly, in the West of Scotland Coronary Prevention Study (WOSCOPS), Gunn et al. [86] found that the presence of a C282Y mutation in the HFE gene did not predict the occurrence of coronary events over a mean follow-up of 4.9 years. It is interesting to note that Yunker et al. [89] analyzed the relationship between biochemical and genetic markers of iron overload and carotid intima-media thickness and brachial flow-mediated vasodilation by high-resolution ultrasound in 907 males, but neither ferritin nor hemochromatosis genotype were related to brachial endothelial function and carotid atherosclerosis. In addition, a recent large study from Denmark found no increased risk of coronary heart disease among carriers of the C282Y mutation or individuals who had compound heterozygosity for the C282Y and H63D mutations [79].

Other investigation have focused on the association between genetic markers of iron overload and idiopathic dilated cardiomyopathy and stroke, but their results were conflicting [90-94].

Finally, some authors have suggested that HFE C282Y could interact with other predisposing factors for venous thromboembolism, such as factor V Leiden, thus exacerbating their prothrombotic effect [95].

Table 1 summarizes the most important studies on the association between genetic markers of iron overload and cardiovascular diseases.

Table 1

Summary of the most important studies on the association between HFE gene mutations (C282Y and H63D) and the risk of cardiovascular diseases

Authors [reference]	Study design	Population	Results
Tuomainen et al. [75]	Prospective	1,150 individuals	C282Y heterozygosity is associated with a 2.3 RR for AMI compared with noncarriers
Roest et al. [76]	Prospective	12,239 postmenopausal women	C282Y heterozygosity is associated with a 1.6 RR for TCD compared with noncarriers
Rasmussen et al. [77]	Prospective	243 CHD cases and 535 controls	C282Y heterozygosity is associated with a 2.7 RR for CHT compared with noncarriers
Gaenzer et al. [78]	Case–control	41 C282Y/C282Y cases and 51 controls	C282Y homozygosity is associated with impaired endothelial function
Bozzini et al. [61]	Case–control	546 CHD cases and 303 controls	C282Y mutation is not associated with CHD
Rossi et al. [80]	Case–control	1,098 subjects	C282Y mutation is not a risk factor for asymptomatic carotid atherosclerosis
Franco et al. [81]	Case–control	256 CHD cases and 272 controls	C282Y and H63D mutations are not associated with CHD
Ellervik et al. [79]	Prospective	9,178 individuals	C282Y and H63D mutations are not associated with CHD
	Case–control	2,441 CHD and 1,113 AMI cases vs 8,080 controls	C282Y and H63D mutations are not associated with CHD
Gunn et al. [86]	Case–control	482 CHD cases and 1,104 controls	C282Y mutation is not associated with CHD
Campbell et al. [88]	Case–control	924 AMI cases and 1,029 controls	C282Y mutation is not associated with CHD
Yunker et al. [89]	Case–control	907 individuals	HFE genotype is not related to brachial endothelial function and carotid atherosclerosis

AMI: acute myocardial infarction, CHD: coronary heart disease, RR: relative risk, TCD: total cardiovascular death

Conclusions

It is interesting to note that although with different pathogenic mechanisms, both iron deficiency and overload have been associated with an increased thrombotic risk in experimental and clinical studies.

However, several aspects need to be still elucidated in this field. In particular, large prospective controlled trials are needed to elucidate the role of genetic markers of iron stores and the impact of long-term iron depletion on morbidity and mortality from cardiovascular events.