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Iron deficiency and thrombosis
study in patients with unprovoked VTE, recurrence of VTE was more likely in the presence of ID.12 A recent large, retrospective study found that patients with IDA- associated thrombocytosis had a 2-fold higher risk of thrombosis in comparison to those with a normal platelet count.13 In cancer, a high platelet count is an independent risk factor for VTE.14 Erythropoiesis-stimulating agents increase thrombotic risk in cancer, but the simultaneous administration of intravenous iron lowers the incidence of VTE and the platelet count, ostensibly through prevention of iron-restricted erythropoiesis.15
Collectively, these studies suggest that ID-associated thrombocytosis may not be entirely benign. Given the widespread prevalence of ID, the impact of this complica- tion could be significant. Furthermore, ID may be co-mor- bid to conditions which already predispose towards thrombosis, such as inflammatory bowel disease, chronic kidney disease, chronic heart failure, or cancer. Chronic ill- ness leads to the induction of hepcidin, which causes the sequestration of iron from the circulation.1,2 The combina- tion of the disease and ID may thus further increase thrombotic risk.
We previously showed, in an animal model, that ID leads to thrombocytosis by augmenting megakaryo- poiesis.16,17 Our group has also shown that iron replace- ment therapy reverses ID-associated thrombocytosis in inflammatory bowel disease, without altering inflamma- tory parameters.18,19 In the current study, we investigated whether ID alone could increase thrombotic tendency. We found that ID consistently induced thrombocytosis, aug- mented some aspects of platelet function, and ultimately increased thrombus size in both venous and arterial mod- els of thrombosis. Iron administration reversed these changes, underscoring the direct relationship with ID and emphasizing the importance of maintaining iron homeo- stasis.
Methods
Ethical approval was obtained from the Austrian Federal Ministry for Science, Research, and Economy and experi- ments followed guidelines for Good Scientific Practice of the Medical University of Vienna.
Male 4- to 5-week old Sprague-Dawley rats (Himberg, Austria and Charles River Laboratories, Germany) were fed either a control diet (Con: E15510-04, 196 mg/kg Fe, Ssniff) or iron-deficient diet (Def: <10 mg/kg Fe, E15510- 2404, Ssniff). Ferric carboxymaltose at a dose of 5, 10, or 20 mg/kg body weight (BW) (+Fe5, +Fe10, +Fe20, respec- tively) or placebo (0.9% NaCl) was administered intraperitoneally to Def animals in the third, fourth and fifth weeks to determine the optimal dosage. Con animals received placebo. Subsequent experiments used 20 mg/kg BW ferric carboxymaltose (Def+Fe).
Prior to and during surgery, animals were anesthetized (see Online Supplementary Material). In order to induce venous thrombosis, inferior vena cava (IVC) branches between the left renal and caudal and iliac veins were lig- ated or cauterized to avoid collateral flow disruption of thrombus formation.20 The IVC and an interposing 2-pro- lene suture were ligated below the left renal vein using a 7-0 silk suture. The 2-prolene suture was removed, allow- ing residual flow. High frequency ultrasound was per- formed using a Vevo 2100 imaging station (Fujifilm
Visualsonics, Amsterdam, the Netherlands), with an MS250 transducer (13-24 MHz) and three-dimensional motor stage. Thrombi were circumscribed at multiple two-dimensional cross-sectional views for three-dimen- sional reconstruction. Four hours after ligation, IVC con- taining thrombi were collected for histology.
To induce arterial thrombosis, a filter paper soaked in 10% FeCl3 was placed on the right common carotid artery for 5 min, and then removed. Flow was monitored using a distally placed flow probe (Transonic, Ithaca, NY, USA) until occlusion.21 Thereafter, arteries with thrombi were excised for histology.
For histology, Carstairs staining was performed as previ- ously described.22 Immunohistochemistry was performed for ITGA2B (1:200, Abcam, Cambridge, UK), and von Willebrand factor (1:1000, Sigma-Aldrich). Thrombi were imaged with an Olympus BX61US Slide Scanner and ana- lyzed using Fiji/ImageJ.23,24 Liver and spleen were stained with the standard Perls Prussian blue stain and imaged using an Olympus BX41 microscope.
Platelet function tests
Blood was collected via cardiac puncture from surgically naïve animals. Platelet function was assessed by aggrega- tion to ADP or collagen (Multiplate aggregometer, Cobas, Roche, Switzerland), adhesion under flow using the Collagen/ADP kit (Platelet Function Analyzer 100 [PFA- 100], Siemens, Germany), and thromboelastography (TEG 5000, Haemonetics, MA, USA). Washed platelets were used to test static adhesion to wells coated with 1 mg/mL fibrinogen (Sigma-Aldrich) or 1 mg/mL collagen (Thermofisher, MA, USA).1
Flow cytometry was conducted on sublingual anticoag- ulated blood.25 Platelets were labeled with CD61-FITC (ebioscience, Thermofisher) and their activity measured using CD62P-PE (P-selectin, ebioscience, Thermofisher). The expression of CD62P was evaluated at baseline and after stimulation with thrombin (0.016 U/mL, 0.25 U/mL, Sigma) on a Cell Lab Quanta SC (Beckman Coulter, CA, USA).
Statistical analysis
A t-test for independent samples or analysis of variance with Tukey post-hoc testing was used to analyze variables with a normal distribution. Otherwise, Kruskall-Wallis and Mann-Whitney U tests with Bonferonni-Holm correc- tion for multiple comparisons were used. Correlations were tested using the Pearson correlation or Spearman rho as appropriate. Statistical testing was performed using SPSS 23.
Results
Iron deficiency causes thrombocytosis, which is reversed by iron replacement therapy in a dose-dependent fashion
Our group previously established a model of diet- induced ID that leads to anemia and thrombocytosis.16 Here, we evaluated the reversibility of these changes upon administration of a commonly used parenteral iron, ferric carboxymaltose.
Animals were placed on an iron-deficient diet (Def) for 3 weeks, and then given weekly injections of ferric car- boxymaltose at a dose of 5, 10, or 20 mg/kg BW or place-
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