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Iron deficiency and thrombosis
bo (0.9% NaCl) at weeks 3, 4 and 5. Control animals (Con) were given a normal diet and placebo injections (Figure 1A). At 3 weeks, Def animals were anemic (hemo- globin: 9.4±0.8 g/dL) in comparison to Con animals (hemoglobin: 13.5±0.2 g/dL). Both mean corpuscular vol- ume (Def: 53.1 [IQR: 52.7-54] fL vs. Con: 62 [IQR: 61.8- 62.2] fL) and mean corpuscular hemoglobin (Def: 16.5±0.6 g/dL vs. Con: 20.4±0.5 g/dL) were likewise lower, consis- tent with the microcytic and hypochromic anemia of ID. As in previous studies,16 platelet counts were higher in Def rats (1288.8±403.2x103/mL) than in Con animals (776.3±171.6 x103/mL) (Figure 1B and C, Online Supplementary Table S1).
Over the following weeks, there were dose- and time- dependent improvements in the affected erythroid and platelet hematologic parameters, with no changes in white blood cell counts (Figure 1B and C, Online Supplementary Figure S1A, Online Supplementary Table S1). Weight and food consumption were comparable between groups (Online Supplementary Figure S1B). We found a strong correlation between hemoglobin concentration and platelet count at the end of the experiment (r=0.812, P<0.001) (Figure 1D). A dose of 20 mg/kg BW ferric car- boxymaltose was the most effective at normalizing hema- tologic parameters, as those of Def+Fe20 animals were closest to those of Con rats at the end of the experiment. Def animals remained anemic, with elevated platelet counts (Figure 1B and C, Online Supplementary Table S1).
After Prussian blue staining, iron and could be found in cells lining liver sinusoids, with staining extending further towards central veins (Figure 1E) with increasing dose. In spleens, stainable iron was found in the red pulp of Con animals but was completely absent in Def rats. Accumulation in the marginal zone increased with higher iron dosage. Among Def animals, positive staining for iron in the red pulp, the site of erythropoiesis, was most prominent in Def+Fe20 rats.
The levels of hepcidin, the central regulator of systemic iron homeostasis, are low during ID. The expression of liver hepcidin, as determined by real-time quantitative polymerase chain reaction, was low in all Def animals, except for Def+Fe20 ones, in which it approached the val- ues in Con animals (Figure 1F). Based on these results, the dose of ferric carboxymaltose selected for subsequent experiments was 20 mg/kg BW, as this was the dose most effective at reversing ID.
Our animal model clearly showed that ID causes throm- bocytosis; however it was not clear whether this has any mechanistic consequences. We first evaluated hemostasis using the tail bleeding assay. Def rats had a shorter bleed- ing time and less blood loss as compared to Con rats (Figure 1G). Iron replacement therapy increased blood loss in a dose-dependent manner, with the loss approaching levels of Con animals at higher concentrations of iron. This supports the notion that ID-induced thrombocytosis is an adaptive mechanism to counter potential blood loss. On the other hand, ID-induced thrombocytosis may con- tribute to an increased tendency to thrombosis.
Iron deficiency enhances thrombotic tendency in a venous model of thrombosis
To evaluate a thrombotic tendency in ID, we utilized a model of venous thrombosis induced by stenosis of the IVC. We initially evaluated the effect of ID alone (Experiment 1, Figure 2), and then reversed ID with the
established dose of ferric carboxymaltose (Experiment 2, Figure 3). As expected, in experiment 1 Def animals devel- oped microcytic hypochromic anemia alongside thrombo- cytosis (Figure 2A-D, Online Supplementary Table S2).
After induction of thrombosis, high frequency ultra- sonography was performed along the full length of the thrombus in vivo (Online Supplementary Figure S2A, Online Supplementary Video 1). The thrombus circumference was outlined on multiple cross-sectional planes (Online Supplementary Figure S2B), enabling three-dimensional reconstruction and calculation of thrombus dimensions (Online Supplementary Figure S2D and E)
In preliminary experiments with untreated animals, we observed that thrombus volume did not increase in size beyond 24 h after ligation and shrank, likely reflecting thrombus resolution (Online Supplementary Figure S3A). As we were interested in thrombus formation, we evaluated thrombus size in the hours shortly after IVC stenosis and found that thrombus size values formed a plateau from 5 h (Online Supplementary Figure S3B). Hypothesizing that thrombus formation would be accelerated in ID, we selected 4 h after stenosis as our endpoint for comparing iron-treated animals to Con animals.
Upon induction of venous thrombosis, resultant throm- biwerelargerinDefratsthaninConratsat3hand4h, as determined by both ultrasound, and histology (Figure 2E-K, Online Supplementary Figure S4A). The increase in thrombus volume from 3 to 4 h was significantly larger in Def rats (73.4 ± 38.7 mm3 vs. Con: 21 ± 9.2 mm3, P=0.019) (Figure 2G) and the final volume at 4 h was likewise larger in these animals (Def: 208.2 ± 74.9 mm3 vs. Con: 81.6 ± 20.5 mm3, P=0.008) (Figure 2H). Thrombus length fol- lowed a similar pattern although the increase in length from 3 to 4 h was not statistically significant (Def: 9.87 [IQR: 9.09-12.3] mm vs. Con: -0.35 [IQR: -0.85-3.3] mm, P=0.117) (Figure 2I and J), because of one Def animal whose thrombus did not increase in length within the period of measurement. The final thrombus was, howev- er, significantly longer in Def rats (30.4 ± 82 mm, vs. Con: 15 ± 3.4 mm, P=0.004). Thrombus area was also measured by B-mode transverse ultrasound and found to be larger in Def animals (62.9 [IQR:56.5-74.7] mm2 vs. Con: 39.4 [IQR: 33.6-54.6] mm2; P=0.007) (Online Supplementary Figure S4B, C). On histology, the transverse area of the thrombi was larger in Def rats (41.5 [IQR: 39.9-55.8] mm2 vs. Con: 25.3 [IQR: 9.1-21.5] mm2, P=0.001) (Figure 2L). There was a strong correlation between histologically determined area and ultrasound-measured volume (r=0.912, P≤0.001) (Figure 2M). The length of the thrombi determined histo- logically was also larger in Def animals (19.2 ± 4.3 mm vs. Con: 13 ± 3.5 mm, P=0.005), and likewise correlated strongly with the length measured by ultrasound (r=0.764, P=0.001) (Figure 2O). Histologically determined area also correlated strongly with ultrasound-measured area (r=0.761, P=0.003) (Online Supplementary Figure S4D). Thus, histological evaluation of thrombus dimensions post-mortem was a reliable readout for the actual situa- tion in vivo.
We then evaluated whether iron supplementation could reverse the increased thrombus size in ID (Experiment 2). Def animals that received placebo (Def) were anemic, with higher platelet counts as compared to control rats fed a normal diet and given the placebo (Con). Administration of 20 mg/kg BW ferric carboxymaltose to iron-deficient animals (Def+Fe) reversed anemia and normalized platelet
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