Although many case reports and observational studies have reported a correlation between iron deficiency anaemia (IDA) and thrombotic events, the mechanism for this is poorly understood. To evaluate this, we examined the change in coagulability in patients receiving treatment for IDA. Adult patients with IDA were recruited for this study and treated with intravenous iron. The change in coagulability was assessed by thrombin generation using the calibrated automated thrombogram method. The change in factor VIII (FVIII) activity was later examined as a possible link. Forty‐eight participants received intravenous iron and were included in this study. After treatment with intravenous iron, endogenous thrombin potential and peak height decreased in IDA patients by a mean of 122·4 nmol/l/min (95% confidence interval [CI]: 17·9–227, P = 0·023) and 51·9 (95% CI: 26·6–77·2, P < 0·001) respectively. Time to peak (peak time) increased by a mean of 23·6 s (95% CI: 5·4–41·9, P = 0·012). FVIII activity was reduced by a mean of 9·6% (95% CI: 2·54–16·7, P = 0·009). In conclusion, treating IDA reduces the blood's coagulability, as evidenced by the change in thrombin generation and FVIII activity levels. No correlation was found between the degree of iron deficiency correction and thrombogram parameters.

The global prevalence of anaemia in 2010 was estimated to be about a third of the world's population, accounting for 68·4 million years lived with disability (Kassebaum et al, 2014). Of the many aetiologies, iron deficiency anaemia (IDA) is the most prevalent by far, affecting approximately one in five females (Kassebaum et al, 2014). Although anaemia is the most common disorder of the blood and is recognized to cause cardiovascular and cerebrovascular morbidity, it is not generally regarded as a factor affecting the blood's coagulability (Lawler et al, 2013). The assumption that there seems to be a connection between IDA and coagulability was postulated due to several case reports (Belman et al, 1990; Benedict et al, 2004; Keung & Owen, 2004; Ogata et al, 2008; Batur Caglayan et al, 2013) that described both venous and arterial thrombosis in patients with IDA. This assumption was strengthened by the first case‐control study to investigate the association between IDA and ischaemic strokes (Maguire et al, 2007). The study showed that previously healthy children diagnosed with a stroke (both arterial ischaemic stroke and sinovenous thrombosis) were 10 times more likely to have a diagnosis of IDA than healthy children without a diagnosis of stroke. Furthermore, children with IDA accounted for more than 50% of all stroke cases. These findings suggest that IDA is a significant risk factor for ischaemic stroke in otherwise healthy young children. Another case‐control study, which included more than 50 000 cases from the Taiwanese Longitudinal Health Insurance Database, showed that a previous diagnosis of IDA had an odds ratio of 1·49 (95% confidence interval [CI]: 1·39–1·6; P < 0·01) that of controls in patients with a diagnosis of ischaemic stroke (Chang et al, 2013) and, using the same database, it was shown that the odds ratio of previously diagnosed IDA for subjects with a venous thromboembolism (VTE) was 1·43 (95% CI: 1·10–1·87; P < 0·001) compared with controls (Hung et al, 2015). Many postulations come to mind about the pathogenesis in which IDA could lead to ischaemic strokes or other thrombotic events, three of which are more prominent than the others. The first suggests that IDA leads to an increase in coagulability by increasing the activity of coagulation factors, such as factor (F) VIII, as demonstrated in hereditary haemorrhagic telangiectasia patients with low serum iron levels (Livesey et al, 2012). The second, through reactive thrombocytosis secondary to IDA (Hartfield et al, 1997). And the third is due to erythrocyte deformability caused by the low mean corpuscular volume (Brandão et al, 2009). In order to understand whether coagulability has a role to play in the pathogenesis of thrombotic events in patients with IDA, the present study aimed to examine the change in coagulability in patients receiving treatment for IDA. To do so we used the calibrated automated thrombogram (CAT); a test that could quantify thrombin generation in real time during the clotting cascade. Thrombin generation is the result of an intricate set of enzymatic reactions initiated by the exposure of tissue factor and its interaction with activated FVII (Rapaport & Rao, 1995). At first, through the activation of the prothrombinase complex, a small amount of thrombin is generated but this is insufficient to cause a definite conversion of fibrinogen to fibrin, yet enough to cause a positive feedback through the activation of FV, FVIII and FXI (FXI), leading to what is known as the thrombin burst (Camire & Bos, 2009). These two stages are known as the initiation phase and the amplification phase of thrombin generation (Mann et al, 2006). Moreover, not only does thrombin accelerate its own generation via positive feedback but it also inhibits its production through interaction with thrombomodulin and the activation of protein C, which, in turn, inhibits activated FV and activated FVIII (Walker & Fay, 1992). Indeed, the capacity to generate thrombin reflects the end result of the complex interaction at play between coagulation enzymes and their inhibitors. As opposed to traditional coagulation tests, which use clot formation as their endpoint and is present when 5% of all physiologically thrombin is formed (Hemker & Béguin, 1995), thrombin generation assays are more useful at reflecting thrombotic or haemorrhagic phenotypes (van Veen et al, 2008). Plotting the concentration of thrombin against time produces the thrombin generation curve (Fig 1), which comprises four parameters. The first is lag time, which is defined as the moment that the concentration deviates by more than two standard deviations from the horizontal baseline. Lag time is proportional to clotting time and reflects the initiation phase of thrombin generation. Peak height and peak time (or time to peak) are the second and third parameters. The fourth is the endogenous thrombin potential (ETP) which is the area under the curve and is proportional to the concentration of thrombin formed and to the time it has been active. In recent years, studies have showed that higher thrombin generation indicates higher incidence of thrombosis, less bleeding and vice versa (Al Dieri et al, 2012). Both retrospective (Tripodi et al, 2007; ten Cate‐Hoek et al, 2008) and prospective (Besser et al, 2008) studies have demonstrated that higher thrombin generation is correlated with VTE events. However, a less profound correlation was found with arterial thrombotic events (Ulrich‐Möckel et al, 2009; Carcaillon et al, 2011). These studies suggest a place for the use of thrombin generation in clinical practice rather than in basic research only. On this basis, we chose to use the thrombin generation assay in this study as it could quantify the change in coagulability, together with the fact that it holds a phenotypic significance. Figure 1 Open in figure viewer PowerPoint A screenshot of the Thrombinoscope software showing the analysis of the thrombogram parameters of one participant after treatment with intravenous iron. Lag time is the time thrombin concentration deviates more than two standard deviation from baseline. Peak time is the time in minutes it takes to reach peak concentration of thrombin and endogenous thrombin potential is the area under the curve reflecting the activity of the overall thrombin generated. [Colour figure can be viewed at wileyonlinelibrary.com

Material and methods The study was conducted in two medical centres, HaEmek Medical Centre and Nazareth Towers’ outpatient clinics, in the north‐eastern area of Israel. The study was approved by the local institutional review board and ethics committee. All participants gave written informed consent prior to the study. Study population and data collection Individuals who were eligible for enrolment in the study were adults diagnosed with IDA who were scheduled for intravenous iron replacement therapy. IDA was defined as a haemoglobin lower than 130 g/l in males or 120 g/l in females, together with a ferritin level lower than 15 μg/l. Inclusion criteria were age older than 18 years, IDA unresponsive to oral iron replacement therapy or otherwise such therapy was contraindicated and a written informed consent form. Exclusion criteria included chronic inflammatory disease, chronic kidney disease, diagnosed malignancy (current or past), concurrent infection, pregnancy, drugs interfering with coagulability (such as anticoagulants, contraceptives, hormone replacement therapy, etc.) and personal history of past VTE. Patients who were not compliant with suggested therapy were excluded later. Data obtained from the participants’ interview included demographic characteristics (age at the time of the interview, gender and ethnicity). Smoking habits were classified into three categories (current, past and never), weight and height were measured at the time of the interviews and body mass index (BMI) was calculated (weight [kg]/height2 [cm]). Blood samples were collected at the time of enrolment (from July 2014 to October 2015 to assess baseline blood count, perform iron studies, basic metabolic panel and thrombin generation assay. An additional citrated plasma samples was stored as backup. All participants were treated with intravenous iron sucrose. Each participant received a total dose of elemental iron in milligrams according to the haemoglobin iron deficit calculated as body weight × (140 − haemoglobin [g/l]) × 0·2145 + 500. This was given in 200 mg doses each 2–3 weeks. Once completed, blood was drawn in the same manner in a follow‐up session scheduled 2–3 weeks later, to assess change in blood count, iron studies and thrombin generation. Complete blood counts and basic metabolic panels were analysed by the ADVIA 2120i haematology system (Siemens Healthcare, Erlangen, Germany) and the AU5800 series chemistry analyser (Beckman Coulter, Brea, CA, USA), respectively. For thrombin generation, blood drawn into citrated collection tubes was centrifuged and stored at −70°C until analysed. Thrombin generation was tested on platelet‐poor plasma with the CAT technique using 5 pmol/l tissue factor and 4 pmol/l phospholipids. No corn trypsin inhibitor was used. Results were read on a microtitre plate fluorometer with the Fluoroskan Ascent analyser (Vantaa, Finland). The thrombogram comprises four parameters (Fig 1): (i) lag time, (ii) peak height, (iii) time to peak and (iv) ETP. Longer lag time and time to peak alongside lower ETP and peak height suggests lower coagulability and shorter lag time and time to peak alongside higher ETP and peak height suggests higher coagulability. We used the HemosIL assay by Instrumentation Laboratory (Bedford, MA, USA) for the quantitative determination of FVIII activity in citrated plasma using the backup citrated plasma collection tubes. Statistical analyses Normal distribution was assessed by visual inspection of histograms with normal curves and using the Shapiro–Wilk test. Categorical variables were presented as frequency and percentage and continuous variables were presented as mean ± SD. For each participant, the change (i.e. difference) in laboratory test results (such as haemoglobin, platelet count etc.) and thrombogram parameters (such as ETP, lag time etc.), before and after intravenous iron treatment, was calculated. The paired t‐test (or Wilcoxon sign‐rank test) was used to explore significant change before and after intravenous iron treatment. The association between the change in IDA parameters (haemoglobin and ferritin) and the change in thrombogram parameters was examined using Pearson correlation (or Spearman correlation). The statistical analyses were performed using SAS 9.4 software (SAS Institute, Cary, NC, USA) and R version 3.4.0 (The R Core Foundation, Vienna, Austria). Statistical significance was considered when P < 0·05.

Results A total of 82 participants diagnosed with IDA and about to start an intravenous iron replacement therapy were enrolled in the study. Of these, 23 participants were not compliant with the suggested therapy and were excluded during the enrolment period, 7 were excluded for screening failure, 3 were excluded because of faulty blood test tubes and one patient was excluded because of pregnancy discovered during treatment. The recruitment for this study stopped after 50 patients had finished their treatment; of these, 2 were screening failure discovered during data processing, resulting in 48 participants who were the candidates for statistical analysis (Fig 2). Figure 2 Open in figure viewer PowerPoint Study flow diagram. This was a two‐centre study that recruited 82 patients. Seven of these patients were screening failures, discovered in the follow‐up period. During treatment, 23 patients were lost to follow‐up and were then excluded. One patient became pregnant during treatment and was excluded. Upon analysis, three blood samples were faulty, could not be analysed and were subsequently excluded. Thus, 48 patients remained for statistical analysis. The mean age at enrolment was 47·0 ± 14·2 years, 45 (93·7%) were females and 44 (91·7%) had never smoked before. The baseline characteristics of the participants, including their medical history at the time of enrolment, are summarized in Table 1. The aetiologies of IDA in the study population are summarized in Table 2 and are similar to the reported epidemiology in resource‐rich countries (Short & Domagalski, 2013; DeLoughery, 2017). The most common aetiology for IDA was menorrhagia, accounting for almost half of the patients enrolled. 12 patients had no definite diagnosis at enrolment and were still under evaluation, none of them had overt gastrointestinal bleeding and 6 of them had a normal upper and lower endoscopy prior to enrolment. Table 1. Demographic, anthropometric and medical history data at moment of enrolment Parameter* Participants (n = 48) Age, years 47·0 ± 14·2 Gender Females 45 (93·7%) Males 3 (6·3%) Smoking status Current smokers 3 (6·2%) Past smokers 1 (2·1%) Never smokers 44 (91·7%) Body mass index, kg/m2 27·4 ± 6·0 Medical illness Ischaemic heart disease 2 (4·2%) Cerebrovascular disease 1 (2·1%) Chronic lung disease 0 (0·0%) Diabetes 10 (20·8%) Hypertension 11 (22·9%) Heart failure 0 (0·0%) Cirrhosis 0 (0·0%) Table 2. Aetiologies of iron deficiency anaemia at enrolment Aetiology Cases, n (%) Chronic blood loss 31 (64·6%) Menorrhagia 23 (47·9%) Upper gastrointestinal bleeding* 4 (10·4%) Lower gastrointestinal bleeding† 3 (6·3%) Recurrent epistaxis 1 (2·1%) Reduced iron absorption 5 (10·4%) Bariatric surgery 4 (10·4%) Helicobacter pylori infection 1 (2·1%) No definite diagnosis at enrollment 12 (25%) On average, a total dose of 1248 ± 277 mg of iron sucrose was administered, after which haemoglobin levels increased by an average of 25 ± 14 g/l (P < 0·001), ferritin levels increased by an average of 112·5 ± 78 μg/l (P < 0·001) and platelet counts decreased by an average of 51·5 ± 49·4 109/l (P < 0·001) after correcting for secondary reactive thrombocytosis (Deray et al, 1984), and red blood cell distribution width (RDW) increased by an average of 2·2 ± 2·9% (P < 0·001) due to the relative reticulocytosis seen in the early period after iron reservoirs are replete (Bessman, 1977). It is safe to say that the aforementioned results support an adequate treatment of IDA. The complete blood count and iron studies before and after intervention are summarised in Table 3 and Fig 3. Table 3. Comparison of complete blood count and iron studies at baseline and after treatment with intravenous iron Parameters N * Baseline (mean ± SD) N * After treatment (mean ± SD) Difference (after − before) P value Haemoglobin, g/l 48 93 ± 14 46 117 ± 12 25 ± 14 <0·001 Platelet count, 109/l 48 297·9 ± 69·8 46 246·9 ± 58·0 −51·5 ± 49·4 <0·001 Leucocytes, 109/l 48 5·9 ± 1·6 46 5·8 ± 1·7 0·0 ± 1·3 0·853 RDW, % 48 16·8 ± 1·6 46 19·0 ± 3·7 2·2 ± 2·9 <0·001 Ferritin, μg/l 48 5·8 ± 5·5 41 118·5 ± 79·9 112·5 ± 78·0 <0·001 Transferrin, g/l 44 3·46 ± 0·49 32 2·63 ± 0·47 −0·77 ± 0·3 <0·001 Iron, μmol/l 46 4·42 ± 2·45 37 10·33 ± 4·03 5·76 ± 4·31 <0·001 Figure 3 Open in figure viewer PowerPoint t‐test. [Colour figure can be viewed at Adequate treatment of iron deficiency anaemia. Shown are box plots for (A) complete blood count parameters [haemoglobin, platelet and leucocyte counts, red cell distribution width (RDW)] and (B) iron studies (ferritin, free iron and transferrin) at baseline and after treatment with intravenous iron. The horizontal line inside each box indicates the median, the filled circle indicates the mean, and the bottom and top of each box indicate the 25th percentile and 75th percentile, respectively. The bars indicate the upper adjacent value [75th percentile plus 1·5 × interquartile range IQR)] and the lower adjacent value (25th percentile minus 1·5 × IQR), empty circles indicate outliers and asterisks indicated statistical significance using the paired‐test. [Colour figure can be viewed at wileyonlinelibrary.com As for thrombin generation parameters (Table 4): ETP decreased by an average of 122·4 nmol/l/min (95% CI: 17·9–227, P = 0·023), peak height decreased by an average of 51·9 (95% CI: 26·6–77·2, P < 0·001) and peak time increased by an average of 23·6 s (95% CI: 5·4–41·9, P = 0·012). Lag time did not significantly change after intervention (P = 0·777). Three out of the four parameters showed a statistically significant change, all in the direction of a lower coagulability. However, none of the thrombogram parameters showed a statistically significant correlation with the change in haemoglobin or ferritin levels (Table 5). Furthermore, the participants were divided into two subgroups according to the level of haemoglobin increase after completing treatment: 40 had an increase of at least 10 g/l and 28 had an increase of at least 20 g/l. However, no statistically significant correlation in participants who “responded better” to treatment was found (Table 6). Table 4. Comparison of thrombogram parameters at baseline and after treatment with intravenous iron Thrombogram parameters Baseline (n = 48) After treatment (n = 48) Difference (95% CI) P value Lag time, min 3·35 ± 0·57 3·33 ± 0·66 −0·02 (−0·15 to 0·19) 0·777 ETP, nmol/l/min 1776·45 ± 366·91 1654·03 ± 279·45 −122·41 (−226·96 to −17·86) 0·023 Peak height, nmol/l 347·32 ± 50·07 295·38 ± 72·55 −51·94 (−77·24 to −26·65) 0·001 Peak time, min 5·75 ± 0·7 6·14 ± 1·15 0·39 (0·09–0·7) 0·012 Table 5. The correlation between both the change in haemoglobin and ferritin and the change in thrombogram parameters Parameters Δ Haemoglobin (n = 46)* Δ Ferritin (n = 41)* Correlation P value Correlation P value ΔLag time, min −0·24 0·113 0·23 0·146 ΔETP, nmol/l/min −0·23 0·126 0·16 0·312 ΔPeak height, nmol/l −0·25 0·098 0·27 0·089 ΔPeak time, min 0·06 0·714 −0·1 0·529 Table 6. The correlation between the degree of change in haemoglobin and thrombogram parameters Parameters Δ Hb ≥ 10 g (n = 40) Δ Hb ≥ 20 g (n = 28) Correlation P value Correlation P value ΔLag time, min −0·29 0·069 −0·26 0·185 ΔETP, nmol/l/min −0·12 0·452 −0·04 0·427 ΔPeak height, nmol/l −0·26 0·099 −0·1 0·703 ΔPeak time, min 0·09 0·595 −0·08 0·982 We evaluated the change of FVIII activity after our initial evaluation demonstrated a reduction in coagulability. We used the stored backup citrated plasma samples in all cases except one, which was not available as it was used for a backup thrombin generation assay. For the remaining 47 patients, the mean baseline FVIII activity was 124·9 ± 42%. Following intravenous iron therapy, the mean FVIII activity was reduced to 115·3 ± 38% (Fig 4). The difference of means between baseline and post‐treatment FVIII activity was 9·6% and was statistically significant (95% CI: 2·54–16·7, P = 0·009). Figure 4 Open in figure viewer PowerPoint t‐test was achieved (P = 0·008). [Colour figure can be viewed at A reduction in FVIII levels after treatment with intravenous iron. Shown are box plots for FVIII activity at baseline and after treatment with intravenous iron. The horizontal line inside each box indicates the median, the filled circle indicates the mean, and the bottom and top of each box indicate the 25th percentile and 75th percentile, respectively. The bars indicate the upper adjacent value [75th percentile plus 1·5 × interquartile range IQR)] and the lower adjacent value (25th percentile minus 1·5 × IQR), empty circles indicate outliers. Statistical significance using the paired‐test was achieved (= 0·008). [Colour figure can be viewed at wileyonlinelibrary.com

Discussion Iron deficiency anaemia seems to be a novel risk factor for thrombosis, and while the mechanism for this is not fully understood, there is evidence that coagulation has an important role to play. For example, a recent study showed that the tendency of the blood to coagulate was increased in patients with IDA, as evidenced by rotational thromboelastometry, when compared to a control group (Özdemir et al, 2018). This shows that thrombosis in patients with IDA is not limited to reactive thrombocytosis or hyperviscosity due to microcytosis, as mentioned in the introduction, but rather to an increase in coagulation. Examining the change in coagulability in patients being treated for IDA could reinforce the postulation that IDA affects coagulability, and a better understanding of this association could encourage a more aggressive treatment in patients with risk factors for thrombotic events. Moreover, the suggested association between coagulation and iron deficiency is rational in evolutionary terms, as evolutionary fitness would be improved by an individual capability to limit blood loss by increasing coagulability and haemostasis at sites of vascular injury. In this study we have shown, for the first time, that treating patients with IDA using intravenous iron replacement therapy reduces the coagulability of their blood as indicated by three out of the four thrombogram parameters (Fig 5): the ETP, which has the strongest correlation to clinical phenotype (Lipets & Ataullakhanov, 2015), and the peak height both decreased, and the time to peak increased; all pointing towards a reduction in coagulability. This further strengthens the postulation that coagulation plays a significant role in the pathogenesis of thrombosis in patients with IDA. Figure 5 Open in figure viewer PowerPoint t‐test. [Colour figure can be viewed at A reduction in thrombin generation after treatment with intravenous iron. Shown are box plots for thrombin generation parameters [endogenous thrombin potential (ETP), lag time, peak height and peak time] at baseline and after treatment with intravenous iron. The horizontal line inside each box indicates the median, the filled circle indicates the mean, and the bottom and top of each box indicate the 25th percentile and 75th percentile, respectively. The bars indicate the upper adjacent value (75th percentile plus 1·5 × interquartile range [IQR]) and the lower adjacent value (25th percentile minus 1·5 × IQR), empty circles indicate outliers and asterisk statistical significance using the paired‐test. [Colour figure can be viewed at wileyonlinelibrary.com A possible explanation for this is the association between iron deficiency and elevated plasma levels of FVIII (Livesey et al, 2012), which is an essential protein in the clotting cascade and a strong risk factor for VTE (Kyrle et al, 2000). Until now, this association did not necessarily mandate causality, but in this study, we have demonstrated a reduction in FVIII levels after treating IDA, suggesting a causality between FVIII activity and iron stores. These findings are in line with a previous study that evaluated the change in FVIII in 11 children with IDA that were treated with iron (Ozsoylu & Gürsel, 1980). The change in FVIII can explain why lag time was spared, because FVIII has no role in the initiation phase but is active in the amplification where a positive feedback loop is responsible for the thrombin burst. A reduction in FVIII levels would explain a reduction in peak height and ETP and would increase the peak time. A suggested mechanism for the change in FVIII is, interestingly enough, the predicted presence of iron response elements that regulate the transcription of the FVIII gene (F8) (Campillos et al, 2010). This needs further evaluation and future research in molecular biology. There are several limitations to this study. First, there are no standardized reference values for the thrombogram parameters due to high interlaboratory variability, estimated at 58–118% (Dargaud et al, 2007). This makes it difficult to interpret whether the baseline thrombogram parameters in this study population are different compared to a healthy population and enables interpretation only with studies made using the same laboratory and measurement methods. Having said that, Saliba et al (2016) conducted their study in the same laboratory used here, in which they aimed to measure the thrombogram parameters using the CAT method on 73 healthy individuals. After comparing our data with the healthy individuals using the unpaired Student's t‐test we showed a significantly higher coagulability profile in our patients as the ETP was higher by 231·8 nmol/l/min (95% CI: 110·5–353·0, P < 0·001), the peak height was higher by 74·3 nmol/l (95% CI: 55·1–93·6, P < 0·001), the peak time was lower by 51 s (95% CI: 31·2–60·2, P < 0·001) and the lag time was, again, not changed. This suggests that IDA patients have increased coagulability compared to the healthy population. Nevertheless, this comparison should be interpreted with extreme caution as the groups identifiers are not similar; the healthy individuals were younger compared to the IDA patients (36 years vs. 47 years), more than half of them were males while the IDA patients were mostly females, and more than 50 per cent of them are current smokers while the majority of IDA patients have never smoked. Second, although we have demonstrated a statistically significant change in thrombin generation, it is not known whether this change is clinically significant and is yet to be tested. Third, the vast majority of the study population were females (93·7%); rendering generalisability to males problematic. Finally, the findings of this study prompt future follow‐up studies. One of those should compare the incidence of thromboembolic events in patients, particularly those with risk factors for VTE, receiving aggressive iron replacement therapy versus a more lenient therapy (e.g., intravenous iron versus oral iron). Another would study the mechanism in which IDA affects coagulability and how iron affects the transcription of other coagulation factors. Lastly, although statistical significance was achieved, a larger sample is needed in the future to prove a higher statistical power and to investigate the clinical outcome for venous and arterial thrombosis in patients with IDA.

Author contributions All five authors have made a substantial contribution (as stated below) and have given their final approval of the version submitted. The study was conceived and designed by J. Nashashibi and M. Elias. The acquisition of the data was undertaken by J. Nashashibi, G. R. Avraham and Y. Awni. J. Nashashibi, M. Elias and N. Shwartz analysed and interpreted the data. The manuscript was drafted by J. Nashashibi and revised by M. Elias.