The presence of heat-labile factors interfering with binding analysis of fibrinogen with ferritin in horse plasma
© Takahashi et al.; licensee BioMed Central Ltd. 2013
Received: 14 February 2013
Accepted: 12 September 2013
Published: 22 September 2013
Horse fibrinogen has been identified as a plasma specific ferritin-binding protein. There are two ways in the binding of ferritin-binding protein with ferritin: one is direct binding and the other is indirect binding which is heme-mediated. The aim of this study was to analyze the binding between horse fibrinogen and ferritin.
Although fibrinogen in horse plasma did not show the binding to ferritin coated on the plate wells, after following heat-treatment (60°C, 30 min) of horse plasma, plasma fibrinogen as well as purified horse fibrinogen bound to plates coated with horse spleen ferritin, but not with its apoferritin which lost heme as well as iron after the treatment of reducing reagent. Binding of purified or plasma fibrinogen to ferritin was inhibited by hemin and Sn-protoporphyrin IX (Sn-PPIX), but not by PPIX or Zn-PPIX.
Heat-treatment of horse plasma enabled plasma fibrinogen to bind to plate well coated with holo-ferritin. From the binding analysis of fibrinogen and ferritin, it is suggested that horse fibrinogen recognized iron or tin in complexed with the heme- or the hemin-ring, and also suggest that some fibrinogens circulate in the form of a complex with ferritin and/or heat-labile factors which inhibit the binding of fibrinogen with ferritin.
Ferritin is a ubiquitous iron-binding protein found in animals, plants, and bacteria [1–3]. Mammalian ferritin stores iron atoms in the apoferritin shell that is composed of Heavy and Light subunits [1–3]. Intracellular ferritin also functions in storing a nontoxic form of iron to prevent its use in the production of hydroxyl radicals through the iron-mediated Fenton reaction [2–4]. Serum ferritin is present in relatively low concentrations (<1 μg/mL), and serum ferritin is the indicator of body iron stores . Serum ferritin is also a biomarker of several inflammatory diseases as in human arthritis and bovine intramammary mastitis [3, 5].
The following ferritin-binding proteins have been identified: H-kininogen [3, 6], α2-macroglobulin [7, 8], anti-ferritin autoantibodies , fibrinogen , apolipoprotein B , and α-casein . With the exception of α-casein, these proteins most likely function in the removal of circulating ferritin [3, 8]. Ferritin-binding proteins and ferritin may bind directly (e.g., binding between ferritin and H-kininogen or anti-ferritin autoantibody) [3, 6] or indirectly (e.g., heme-mediated binding between ferritin and apolipoprotein B or α-casein) [3, 11].
The purpose of this study was to determine the mechanism of horse plasma fibrinogen-ferritin binding. Horse plasma fibrinogen results in lower concentrations of ferritin in the plasma than in the serum, but heating at 75°C for 15 min results in equalization of both concentrations of ferritin [9, 11], suggesting that fibrinogen binds circulating ferritin. However, in this study, the results of this binding analysis suggested the existence of heat-labile factors that inhibits binding between fibrinogen and ferritin.
Blood samples were collected from six thoroughbred horses (age 9–28 years) kept for the research at Kitasato University and the Equine Research Center, Japan Racing Association (Tochigi, Japan). Plasma was prepared from heparinized blood and was kept at 4°C in the presence of 0.1% sodium azide. All experiments were conducted by the following established guidelines for animal welfare and were approved by the Committee on the Ethics of Animal Experiments of the Kitasato University (Permit Number: 11–091).
Commercial horse spleen ferritin (Sigma Chemicals, St. Louis, MO, USA) was further purified  and its apoferritin was prepared by dialysis of holoferritin against 100 mmol/L thioglycolic acid in 100 mmol/L acetate buffer (pH 5.5). Horse plasma samples were diluted 100-fold with phosphate buffered saline (PBS; 150 mmol/L NaCl, 15 mmol/L dibasic and 5 mmol/L monosodium phosphate, pH 7.2) containing 0.1% Tween 20 and 0.1% gelatin (buffer A) and heated at 60°C for 30 min and then centrifuged at 14,000 × g for 15 min. The resulting supernatant was used as heat-treated plasma. Aliquots (100 μL) of holo- or apo-ferritin solutions in PBS (10 nmol/L each) were added to wells of a Maxisorp F96 immunoplate (Nunc, Roskilde, Denmark) and kept overnight at 4°C. The plate wells were washed three times with PBS containing 0.05% Tween 20 (PBST) after every step. After washing with PBST, 300 μL of buffer A was added to each well and masked with gelatin for 1 h to prevent nonspecific binding. To each well was also added 100 μL of horse fibrinogen (10 μg/mL) in ELISA buffer containing 10 mmol/L EDTA (buffer B) or the heat-treated plasma samples and the plate was incubated at 37°C for 2 h. After washing, 100 μL of goat anti-human fibrinogen antibody (GenWay Biotech, Inc., San Diego, CA, USA) diluted 1000-fold with buffer B was added to each well and the plate was incubated at 37°C for 1.5 h. The plate was then washed and 100 μL of alkaline phosphatase (ALP)-labeled rabbit anti-goat IgG antibody (EMD Merck Millipore, Billerica, MA, USA), which diluted 1000-fold with buffer A, was added to well and the plate was incubated at 37°C for 1.5 h. After washing, the enzyme reaction was performed using 3 mmol/L disodium p- nitrophenyl phosphate, and absorbance of each well at 405 nm was measured with Molecular Devices VersaMax™ Absorbance Tunable Microplate Reader.
Hemin, Sn-protoporphyrin IX (Sn-PPIX), Zn-PPIX, and metal-free PPIX were prepared as preciously described . These inhibitors were simultaneously added to fibrinogen (10 μg/mL) in buffer B or heat-treated plasma (1:100 v/v) to a final concentration of 10 μmol/L. Aliquots (100 μL) of each mixture were added to holoferritin-coated wells (1 pmol/well) as described above. The detection of fibrinogen bound to the wells was performed as described above except for the use of buffer A in place of buffer B in every step. All data are expressed as the mean ± SD, and significant differences between data from the two groups was assessed using the Student’s t-test. The significant differences for multiple comparisons was assessed using one-way ANOVA followed by Tukey’s test. A P-value below 0.01 was considered statistically significant.
Why fibrinogen in untreated plasma did not bind ferritin-coated wells also remains to be determined. Watanabe et al.  reported that the structure of adenylate kinase changes due to denaturation as coated on plates, resulting in the antigenicity change. This finding led to the hypothesis that coating plasma proteins on the wells may release heat-labile factors which inhibit the binding of fibrinogen to coated ferritin. The fibrinogen concentration in this study (10 μg/mL) was estimated to be physiological concentration from plasma dilution (100-fold) and the normal range (1–2 mg/mL) . This interference of heat-unstable proteins was observed even after at least one year storage of plasma sample at 4°C (data not shown), suggesting that plasma storage did not affect interaction between fibrinogen and them. It is known that fibrinogen binds IgG [20, 21]. We detected binding between immunoglobulins in untreated plasma and fibrinogen trapped on the plate by coated anti-fibrinogen antibody (Additional file 2: Data S2). Horse IgGT and IgGb showed higher binding activity with fibrinogen. However, although an attempt to detect complexes formed between ferritin, fibrinogen, and immunoglobulin as fibrinogen-binding protein was not successful, this findings suggest that fibrinogen partly circulate as a complex with ferritin and/or a heat-labile fibrinogen-binding protein, probably IgG.
The high redox potential of iron and heme cause oxidative damage [3, 22]. Ferritin and/or fibrinogen in horse circulation may protect iron- or heme-mediated oxidative stress. Additionally, the binding between them is likely to be involved in local blood coagulation as in H-kininogen . This study may also provide preliminary data on physiological relation between iron metabolism and blood coagulation system.
In conclusion, plasma fibrinogen bound to coated ferritin only after plasma was heated (60°C, 30 min). Horse fibrinogen bound holo-ferritin, but not apo-ferritin also having no heme. Binding of fibrinogen to ferritin was inhibited by hemin and Sn-PPIX, but not by metal-free PPIX or Zn-PPIX. This study demonstrates that horse fibrinogen binds ferritin through heme-mediation, and that plasma heat-labile factors inhibit the binding between them.
This study was supported in part by a Grant-in-Aid from the Morinaga Foundation for Health and Nutrition.
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