Iron salts are used in the treatment of iron deficiency anemia. Diabetic patients are frequently anemic and treatment includes administration of iron. Anemic patients on hemodialysis are at an increased risk of thromboembolic coronary events associated with the formation of dense fibrin clots resistant to fibrinolysis. Moreover, in chronic kidney disease patients, high labile plasma iron levels associated with iron supplementation are involved in complications found in dialyzed patients such as myocardial infarction. The aim of the present study was to investigate whether iron treatment is involved in the formation of the fibrin clots. Clotting of citrated plasma supplemented with Fe3+ was investigated by thromboelastometry and electron microscopy. The results revealed that iron modifies coagulation in a complex manner. FeCl3 stock solution underwent gradual chemical modification during storage and altered the coagulation profile over 29 days, suggesting that Fe3+ interacts with both proteins of the coagulation cascade as well as the hydrolytic Fe3+ species. Iron extends clotting of plasma by interacting with proteins of the coagulation cascade. Fe3+ and/or its hydrolytic species interact with fibrinogen and/or fibrin changing their morphology and properties. In general FeCl3 weakens the fibrin clot while at the same time precipitating plasma proteins immediately after application. Fe3+ or its derivatives induced the formation of insoluble coagulums in non-enzymatic reactions including albumin and transferrin. Iron plays a role in coagulation and can precipitate plasma proteins. The formation of coagulums resistant to lysis in non-enzymatic reactions can increase the risk of thrombosis, and extending clotting of plasma can prolong bleeding.
Iron salts are used in the treatment of iron deficiency anemia, as a supplemental intake of iron during pregnancy and in multivitamin preparations. In the majority of cases it is safe to use but toxic effects begin to appear at doses >10–20 mg/kg of elemental iron, and ingestions of >50 mg/kg are associated with severe toxicity or lethality (
The role of iron treatment in the formation of fibrin clots should therefore be investigated. Of note is that in humans divalent iron, Fe+2, is rapidly oxidized to trivalent iron, Fe3+, by ferroxidase (
In the present study, we investigated clotting of citrated plasma supplemented with Fe3+ (and calcium Ca2+ to initiate clotting) by thromboelastometry and electron microscopy. The results showed that iron changes plasma clotting characteristics, kinetics and the dynamics of clot formation in plasma. More changes were observed as the time of storing stock solution of FeCl3 increased, possibly due to different derivatives of Fe3+ being formatted over 29 days. Additionally, the morphology of clotted fibrin in the Ca2+- and Fe3+-treated plasma was different than the untreated, normal, control-clotted fibrin.
Kaolin, CaCl2 solution, pins and cups were purchased from Haemoscope Co. (Neils, IL, USA). Fully active human tissue plasminogen activator (tPA), product number HTPA-TC was purchased from Molecular Innovations, Inc. (Novi, MI, USA). Ferric chloride, fibrin and thrombin were purchased from Sigma-Aldrich Co. LLC (St. Louis, MO, USA).
Lyophilized specialty assayed reference plasma, cat. no. 5185 (S.A.R.P., 10×1 ml) purchased from Helena Laboratories (Beaumont, TX, USA) was prepared from a frozen pool of citrated plasma obtained from healthy donors. S.A.R.P. has normal PT and aPTT clotting times and may be used as reference data based on the following parameters: fibrinogen**, factor II*, factorV**, factor VII*, factor VIII*, factor IX*, factor X*, factor XI**, ristocetin cofactor*, vWF:Ag*, factor XII, protein C*, protein S - total, free) where (*) denotes samples standardized according to World Health Organization (WHO) regulations, and (**) denotes samples calibrated against ISTH reference material. Plasma was stored at 4°C and reconstituted by adding 1 ml of deionized water, followed by a 3-min rest. Plasma for electron microscopy experiments was obtained from healthy subjects aged between 20 and 25 years, both males and females. Ethical approval was obtained from the University of Pretoria Human Ethics Committee, and this study conforms to the principles of the Declaration of Helsinki.
Thromboelastography allows measurement of a total coagulation profile and yields data on the kinetics and dynamics of clot formation in plasma (
Purified fibrinogen (cat. no. F3879-250MG; Sigma-Aldrich), human albumin (cat. no. A9511, Sigma-Aldrich) samples were treated with 5 μl 0.2 M CaCl2, followed by the addition of 5 μl of freshly prepared 0.2 M FeCl3. After mixing, thrombin was added, to create an extensive fibrin network. Human platelet rich plasma (PRP) samples were treated (addition of CaCl2 and FeCl3) in the same manner, but without thrombin. The samples were fixed immediately in 2.5% glutaraldehyde/formaldehyde in PBS solution, pH 7.4, for 30 min. The samples were then left for 16 min and 3 h, followed by fixing in order to obtain a time-dependent analysis of the effect of FeCl3 and CaCl2 on PRP. Smears were then fixed followed by rinsing three times with PBS for 5 min prior to being fixed for 30 min with 1% osmium tetraoxide (OsO4). The samples were again rinsed three times with PBS for 5 min and were dehydrated serially with 30, 50, 70 and 90% ethanol, and three times with 100% ethanol. The material was mounted and coated with carbon. A Zeiss ULTRA plus FEG-SEM with InLens capabilities (Microscopy and Microanalysis Unit of the University of Pretoria, Pretoria, South Africa) was used to study the surface morphology of fibrin and micrographs were taken at 1 kV.
FeCl3 water solution was diluted at 1:1,000 from 0.2 M stock solution with or without DMSO and analyzed on a UV/VIS spectrometer at a range of 230–800 nm. Samples were analyzed at day 0 and periodically up to day 29 after FeCl3 preparation.
UV/VIS spectra of FeCl3 were altered over the 29 days (
There are no normal ranges of TEG parameters for control plasma. However, the results yielded in this study were very consistent: R (sec): 408, K (sec): 84, An (°): 70.6, MA (mm): 27.2, LY30 (%): 0 (all parameters ±10%) (
While FeCl3 stock solution with DMSO was used all described effects of Fe3+ were less evident confirming our previous suggestion that the free radicals were playing role in coagulation (
Electron microscopy images revealed that iron-treated plasma forms structures different from those of the control fibrin where fibrin strands formed a solid and thick mesh (
The aim of this study was to investigate the effects of Fe3+ on coagulation. However, during initial experiments we observed that the stock solution of FeCl3 changed color and some changes were evident in the thromboelastogram. Therefore, we prepared FeCl3 stock solution and analyzed the samples obtained by spectroscopy from day 0 to 29. Spectrometric data strongly indicated that FeCl3 undergoes gradual chemical modification. Our results suggest that, for example, the concentration of Fe2(OH)24+ (λmax at 335 nm) increases while that of Fe(OH)2+ (λmax at 297 nm) decreases, which is in agreement with observations from previous studies (
Simultaneously, we detected gradual changes in the plasma clotting parameters. Divalent and trivalent metals can change the clotting characteristics of plasma and blood (
In the present study, we have established that coagulation parameters change as FeCl3 storing time increases. Thus, Fe3+ as well as the hydrolytic Fe3+ species interact with proteins of the coagulation cascade. We also observed clot lysis following its initial formation. This happened a few days after the preparation of FeCl3 stock solution and was more evident (
Free radicals are known to affect coagulation and fibrinolysis, and free radical scavengers normalize these processes (
In the Fe3+-treated samples instantaneous formation of insoluble coagulums was observed. This effect was more prominent over time and was the effect of Fe3+ and its hydrolytic species (
We also attempted to identify the proteins that were precipated following iron addition. Albumin is the most abundant protein in the circulation and represents 52–60% of the total plasma protein. It plays an important role in the transportation and storage of hormones, fatty acids and drugs, and in the transportation of essential metal ions. Both Fe2+ and Fe3+ ions bind to heme serum albumin through the heme iron complex but only Fe3+ binds to heme-free albumin. Fe3+ ions are transported in plasma mainly by a non-heme iron-binding glycoprotein transferrin, which composes ~7–10% of plasma protein (
FeCl3 is used in animal models to study early arterial thrombus formation as a result of rapid endothelial injury, and the associated thrombotic formation. FeCl3 application is a valuable model for investigation into thrombosis and atherosclerosis. However, caution should be applied since iron interacts with various proteins from the coagulation cascade and its effects depend on storage of the stock solution (
In conclusion, trivalent iron is involved in coagulation in a complex manner. It extends the clotting of plasma by interacting with proteins of the coagulation cascade. Fe3+ and/or its hydrolytic species interact with fibrinogen and/or fibrin, changing their morphology and properties. Moreover, when stored, FeCl3 produces derivatives that potentiate changes in plasma clotting, some of which can be attributed to free radicals formed during FeCl3 storage. In general FeCl3 is able to weaken the fibrin clot while precipitating plasma proteins immediately after application. This property can be exploited therapeutically in stanching the flow of blood from wounds when optimum concentrations of FeCl3 are found.
This study was supported in part by grants from the Frank Stranahan Endowed Chair and Children Miracle Network.
UV/VIS spectra of FeCl3 stock solution diluted at 1:1,000 at day 0 until day 29.
Typical thromboelastogram of clotted plasma at day 0: (a) plasma treated with Ca2+, control, solid blue line; plasma treated with Ca2+ and Fe3+, dashed orange line; plasma treated with Ca2+, Fe3+ and tPA at 0.5 μg/ml, dashed dotted green line. Thromboelastogeram of plasma at day 29: (b) plasma treated with Ca2+, control, solid blue line; plasma treated with Ca2+ and Fe3+, dashed orange line.
Morphology of plasma treated with Ca2+ and Fe3+ at (a) ~0, (b) ~960 and (c) ~4000 sec. (d) Control plasma treated with Ca2+. (e) Pure fibrinogen treated with thrombin and Ca2+ and Fe3+. (f) Human serum albumin treated with Ca2+ and Fe3+.
TEG pin and cup of plasma treated with Ca2+ and Fe3+. Arrows point to coagulums formed subsequent to Fe3+ addition.