Fibrinogen

Manufactured by Agilent Technologies

Sourced in United States, Denmark

Fibrinogen is a key component of the blood clotting process. It is a plasma glycoprotein that is converted into fibrin during blood clot formation.

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Lab products found in correlation

β actin, by Cell Signaling Technology (2 mentions) P0260, by Agilent Technologies (1 mentions) Maxisorp plate, by Thermo Fisher Scientific (1 mentions) Mars software, by BMG Labtech (1 mentions) Spectrostar nano microplate reader, by BMG Labtech (1 mentions) A0080, by Agilent Technologies (1 mentions) Serotonin, by Merck Group (1 mentions) Adenosine diphosphate adp, by Merck Group (1 mentions) Sodium citrate tube, by BD (1 mentions) Kaluza 2, by Beckman Coulter (1 mentions) Glut1, by Merck Group (1 mentions) Glut1, by Thermo Fisher Scientific (1 mentions) Pdgfrβ, by Thermo Fisher Scientific (1 mentions) Af2335, by R&D Systems (1 mentions) β actin, by AbFrontier (1 mentions) Enhanced chemiluminescence, by Merck Group (1 mentions) Cd42b, by Abcam (1 mentions) Cy3 conjugated donkey anti mouse igg, by Jackson ImmunoResearch (1 mentions) Horseradish peroxidase hrp linked secondary antibody, by Cell Signaling Technology (1 mentions) Aβ 1 16, by BioLegend (1 mentions)

19 protocols using fibrinogen

Citrullination of Fibrinogen Assay

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As described¹¹ (link), Maxisorp plates (Nunc, Roskilde, Denmark) were coated overnight at 4 °C with 1 µg/ml fibrinogen (Calbiochem, Darmstadt, Germany). Wells were washed thrice and blocked in Tris-buffered saline (TBS) buffer containing 0.05% Tween-20, pH 7.4. Hereafter, the wells were incubated for 3 h at room temperature (RT) with PAD-containing samples with or without GSH, H₂O₂, and EDTA, as stated in figure legends. Purified leukocytes were applied in RPMI 1640 containing 5% AB serum and, when relevant, 15 nM PMA, 25 µM DPI, which inhibits NOX2 and other flavoenzymes by interacting with the flavin part³⁰ (link), or H₂O₂ at various concentrations. After three washes in washing buffer (PBS, 0.05% Tween-20, pH 7.4), mouse anti-citrullinated fibrinogen antibody (0.5 µg/ml) and hereafter horse radish peroxidase-conjugated polyclonal rabbit-anti mouse immunoglobulin antibodies (P0260, Dako, Glostrup, Denmark) were added and developed as described¹¹ (link). Optical density (OD) was measured at 490–650 nm using the SPECTROstar nano Microplate Reader (BMG Labtech, Ortenberg, Germany). Data were processed using MARS software (BMG Labtech).

Damgaard D., Bjørn M.E., Jensen P.Ø, & Nielsen C.H. (2017). Reactive oxygen species inhibit catalytic activity of peptidylarginine deiminase. Journal of Enzyme Inhibition and Medicinal Chemistry, 32(1), 1203-1208.

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Baboon Cerebrovascular Occlusion Protocol

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Postmortem brain tissues were obtained from adult baboons (Papio Anubis) weighing 16–20 Kg (7–12 years old), housed at the Institute of Primate Research (IPR), National Museums of Kenya. The IPR internal review board of the National Museums granted ethical approval and permission for this entire study. As described previously (13), the animals were subjected to permanent occlusion of both the internal carotid arteries and a left vertebral artery for survival periods of 1, 3, 7, 14, 21 and 28 days, as well as sham operated animals (n = 4–8 each group). We evaluated densities of capillary endothelium using H&E and COL4 immunohistochemistry as described above, and the BBB leakage with fibrinogen (1:2000 dilution, A0080, Dako, Cambridge, UK) (13). Pericytes in the deep WM were determined in coronal sections at the level of the frontal lobe in the same manner as described above in human brains. Percentage of fibrinogen stained area (%) in the WM was calculated to evaluate BBB leakage.

Ding R., Hase Y., Ameen‐Ali K.E., Ndung'u M., Stevenson W., Barsby J., Gourlay R., Akinyemi T., Akinyemi R., Uemura M.T., Polvikoski T., Mukaetova‐Ladinska E., Ihara M, & Kalaria R.N. (2020). Loss of capillary pericytes and the blood–brain barrier in white matter in poststroke and vascular dementias and Alzheimer’s disease. Brain Pathology, 30(6), 1087-1101.

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Platelet Reactivity Assessment by Flow Cytometry

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Whole blood samples were collected inside a 3.2% sodium citrate tube (Becton Dickinson, Franklin Lakes, NJ, USA) and processed between 1 and 3 hours after blood collection using flow cytometry-based assay as described before.^{49 (link)} Platelet reactivity was assessed under basal (unstimulated) conditions and after stimulation with adenosine diphosphate (ADP; 1.2 and 125uM; Sigma-Aldrich, Zwijndrecht, The Netherlands), cross-linked collagen-related-peptide (CRP-XL; a kind gift from Prof. Farndale, Cambridge, UK), and co-stimulation between ADP with 50 uM serotonin (Sigma-Aldrich, Zwijndrecht, The Netherlands). Platelet reactivity was defined by median fluorescence intensity (MFI) of α-granule protein P-selectin (Biolegend, San Diego, CA, USA) expression and fibrinogen (DAKO, Santa Clara, CA) binding to the activated integrin αIIbβ3. All data were extracted using Kaluza 2.1 software (Beckman Coulter, France) and normalized against quality controls to ensure measurement stability.

Vadaq N., Zhang Y., Meeder E., Van de Wijer L., Gasem M.H., Joosten L.A., Netea M.G., de Mast Q., Matzaraki V., Schellekens A., Fu J, & van der Ven A.J. (2022). Microbiome-Related Indole and Serotonin Metabolites are Linked to Inflammation and Psychiatric Symptoms in People Living with HIV. International Journal of Tryptophan Research : IJTR, 15, 11786469221126888.

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Immunohistochemical Analysis of Brain Sections

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Brains were sectioned sagittally using a vibratome at either 50 μm (one-hour post-fixed tissue) or 30 μm (tissue post-fixed overnight). At least three non-adjacent sections (> 100 μm apart) were analysed per animal per condition. Sections were processed as floating sections. Sections which had been post-fixed overnight underwent antigen retrieval (incubated in 0.25% trypsin for 5 min at 37 °C, followed by 3 × 5 min washes in PBS). Sections were blocked for 2 h at room temperature on an orbital shaker in blocking solution (5% donkey serum, 0.1% triton in PBS). Sections were incubated with primary antibodies [Fibrinogen (1:5000; Dako; A0080), Glut-1 (1:500; ThermoFisher; MA5–11315), Glut-1 (1:10,000; Millipore; 07–1401), PDGFR-β (1:500; eBioscience; 14–1402-82), Aminopeptidase-N/CD13 (1:250; R&D Systems; AF2335)] in blocking solution overnight at 4 °C on a shaker. Sections were then washed in PBS (3 × 5 min) before being incubated with Alexa Fluor secondary antibodies for 4 h at room temperature on a shaker. Sections were washed in PBS (3 × 5 min), incubated with DAPI (1 min at room temperature), further washed in PBS (3 × 5 min), then mounted on slides with Dako Mounting Medium.

Rajani R.M., Ratelade J., Domenga-Denier V., Hase Y., Kalimo H., Kalaria R.N, & Joutel A. (2019). Blood brain barrier leakage is not a consistent feature of white matter lesions in CADASIL. Acta Neuropathologica Communications, 7, 187.

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RT-PCR, Western Blot, and Antibody Analysis

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RT–PCR and western blot analysis were performed as described previously [28 (link)]. The following primary antibodies were used for the immunoblotting assay: β-actin (AbFrontier, Seoul, Korea), ERRγ (Perseus Proteomics, Tokyo, Japan), and fibrinogen (Dako, Carpinteria,CA, USA). All primer sequences are described in S1 Table.

Zhang Y., Kim D.K., Lu Y., Jung Y.S., Lee J.M., Kim Y.H., Lee Y.S., Kim J., Dewidar B., Jeong W.I., Lee I.K., Cho S.J., Dooley S., Lee C.H., Li X, & Choi H.S. (2017). Orphan nuclear receptor ERRγ is a key regulator of human fibrinogen gene expression. PLoS ONE, 12(7), e0182141.

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Real-time Cell Adhesion Assay on Fibrinogen

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Cell adhesion was performed using flow conditions and real-time video microscopy as previously described [26 (link)]. Briefly, glass coverslips were precoated with 10 μg/ml fibrinogen (from Dako, Glostrup, Denmark), purified and depleted of contaminant fibronectin and vWF, and mounted onto a flow chamber of 200 μm height filled with IMDM. CHO cells resuspended in IMDM at a concentration of 1.25x10⁶/ml were perfused over a fibrinogen-coated coverslip at flow rates from 60–200 μl/min to produce wall shear rates from 30–100 s^-1. In some experiments, cells were preincubated 20 min with 250–500 μM ^pYMESRADR or ^pYMESRAAR peptides before perfusion at 30 s^-1. Cells were visualized using a 10x Hoffman Modulation Contrast objective together with an inverted microscope. Real-time images were recorded using a charged-coupled-device camera. The number of adherent cells was counted over a 12 min time course using Histolab and Archimed software (Microvision Instruments, Evry, France).

Lee W.H., Schaffner-Reckinger E., Tsoukatos D.C., Aylward K., Moussis V., Tsikaris V., Trypou P., Egot M., Baruch D., Kieffer N, & Bachelot-Loza C. (2015). Inhibition of αIIbβ3 Ligand Binding by an αIIb Peptide that Clasps the Hybrid Domain to the βI Domain of β3. PLoS ONE, 10(9), e0134952.

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Dinitrophenylhydrazine-Based Protein Oxidation

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Following IEF, IPG strips were incubated in 2N HCl with 10 mM DNPH at 25 °C for 20 min. Next, samples were washed with 2M Tris-Base/30% glycerol for 15 min and the protein was separated according to molecular weight as described above. The 2-DE gels were then transferred to a PVDF membrane which was incubated overnight at 4 °C with the primary antibody for DNPH (Molecular Probes, Eugene, Oregon, USA) or fibrinogen (DAKO, Santa Clara, CA, USA). The blots were washed and incubated with goat anti-rabbit IgG conjugated HRP for 2 h. Enhanced chemiluminescence (Millipore, Burlington, MA, USA) was applied for detection [40 (link)].

Wang P.W., Lin T.Y., Hung Y.C., Chang W.N., Yang P.M., Chen M.H., Yeh C.T, & Pan T.L. (2019). Characterization of Fibrinogen as a Key Modulator in Patients with Wilson’s Diseases with Functional Proteomic Tools. International Journal of Molecular Sciences, 20(18), 4528.

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Fluorescent Immunostaining of CD42b and Fibrinogen

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The slides were dried by heating to 56˚C for 2 hours in an oven, deparaffinized in Xylene (2× 10mins) followed by rehydration through alcohols and in distilled water. Sections were pretreated with 0.1 mol/L citric acid buffer in a microwave for 15 minutes. Slides were left to cool at room temperature for 30 minutes before being rinsed in Tris-buffered saline (TBS). The slides were incubated with 4% normal donkey serum in TBS buffer for 30 minutes at 37°C, followed by incubation with primary antibodies CD42b (mouse monoclonal antibody, pre-diluted; Abcam) and Fibrinogen (rabbit polyclonal antibody, 1:200; Dako) in TBS buffer for 1 hour at 37°C, then 4°C overnight. After incubation, the sections were rinsed in TBS buffer, followed by incubation with secondary antibodies (Cy3 conjugated donkey anti-mouse IgG (1:200); Alexa-Flour 488–conjugated donkey anti-rabbit IgG, (1:100); Jackson ImmunoResearch Inc.) for 2 hours at room temperature. The sections were rinsed in TBS (4× 5mins) and counterstained with DAPI (1:250). Finally, the sections were rinsed in TBS and then dehydrated through alcohols, cleared in xylene, and mounted with EZ-Mount. Negative controls were performed with non-immune normal serum used instead of the primary antibody. The sections were viewed and imaged with a fluorescence confocal microscope.

Fitzgerald S., Wang S., Dai D., Douglas A., Kadirvel R., Gounis M.J., Chueh J.Y., Puri A.S., Layton K.F., Thacker I.C., Hanel R.A., Sauvageau E., Aghaebrahim A., Almekhlafi M., Demchuk A.M., Nogueira R.G., Pereira V.M., Kvamme P., Kayan Y., Almandoz J.E., Yoo A.J., Kallmes D.F., Doyle K.M, & Brinjikji W. (2019). Platelet-rich clots as identified by Martius Scarlett Blue staining are isodense on NCCT. Journal of neurointerventional surgery, 11(11), 1145-1149.

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Platelet Activation by Aβ1-40 and CRP

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Platelets were activated with CRP (Richard Farndale, University of Cambridge, United Kingdom) or soluble Aβ40 (1-40; Bachem Peptide, cat no 4014442.1000) sequence single-letter code (DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV). Aβ1-40 stock solutions with a concentration of 1 mg/ml, were solved in sterile H2O and stored at -20 • C. Apyrase (grade II, from potato) and prostacyclin from Calbiochem were used for isolation.
Antibodies against phosphotyrosine (Millipore clone 4G10; cat no 05-321), phospho-LAT (Tyr 200 ; Abcam cat no ab68139); Aβ1-16 (Biolegend, 6E10, cat no SIG-39320) and fibrinogen (Dako cat no A0080) were used for immunoblotting. The antibodies to LAT (cat no 9166), βactin (cat no 4967), α-tubulin (cat no 2144), and horseradish peroxidase (HRP)-linked secondary antibodies (cat no 7074 and cat no 7076) were from Cell Signaling Technology.

Donner L., Toska L.M., Krüger I., Gröniger S., Barroso R., Burleigh A., Mezzano D., Pfeiler S., Kelm M., Gerdes N., Watson S.P., Sun Y, & Elvers M. (2020). The collagen receptor glycoprotein VI promotes platelet-mediated aggregation of β-amyloid. Science signaling, 13(643).

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Insulin Signaling in Mice Tissues

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For insulin signaling experiments, mice were injected through the tail vein with 0.75 U/kg of human insulin or saline and sacrificed 10 min later. Liver, epididymal adipose tissue (EAT), and muscle were collected, for western blot analysis with antibodies to Akt, phospho-Akt, and β-actin (Cell Signaling Technology). For UCP-1, UCP-2 and fibrin analysis relevant tissues were collected for western blot analysis with antibodies to UCP-1 (Abcam, Waltham, MA), UCP2 (Proteintech, Rosemont, IL) and fibrinogen (Dako, Santa Clara, CA). Tissues were lysed in RIPA buffer (Sigma) with protease and phosphatase inhibitors (Thermo Fisher Scientific), electrophoresed and transferred to nitrocellulose membranes. Membranes were incubated with primary antibodies, washed with PBS-containing-0.1% Tween-20 and incubated with species specific IRDye®680RD/800CW-conjugated secondary antibodies. Immunoreactive bands were visualized using the Odyssey Imaging System (LI-COR) and densitometric quantification performed using Image Studio™ Lite Software 5.2 (LI-COR).

Samad F., Bai H., Baik N., Haider P., Zhang Y., Rega-Kaun G., Kaun C., Prager M., Wojta J., Bui Q., Chakrabarty S., Wang J., Parmer R.J, & Miles L.A. (2021). The plasminogen receptor Plg-RKT regulates adipose function and metabolic homeostasis. Journal of thrombosis and haemostasis : JTH, 20(3), 742-754.

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