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Compstatin

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Compstatin is a synthetic cyclic peptide that functions as a complement inhibitor. It inhibits the activation of the complement system by binding to the complement component C3. Compstatin helps regulate the immune response and can be used in research applications.

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Lab products found in correlation

Cd11b, by BD (1 mentions) Rabbit complement sera, by Merck Group (1 mentions) 0.22 μm syringe filter, by Pall Corporation (1 mentions) Monensin, by Merck Group (1 mentions) Cytomics fc500 flow cytometer, by Beckman Coulter (1 mentions) Magnet activated cell sorting, by Miltenyi Biotec (1 mentions) Fitc conjugated goat anti rabbit igg, by Vector Laboratories (1 mentions) Fitc conjugated goat anti human igg, by Jackson ImmunoResearch (1 mentions) Flowlogic, by Inivai Technologies (1 mentions) Prostaglandin e1, by Merck Group (1 mentions) Human thrombin, by Enzyme Research (1 mentions) Complement c3, by Merck Group (1 mentions) Loxoribine, by InvivoGen (1 mentions) Pam3csk4, by InvivoGen (1 mentions) Gm csf, by STEMCELL (1 mentions) C5ari, by Merck Group (1 mentions) W 54011, by Merck Group (1 mentions) Dimethyl sulfoxide (dmso), by Merck Group (1 mentions) Clone mopc 21, by BioLegend (1 mentions)

8 protocols using compstatin

1

Compstatin Inhibition of Complement Activation

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compstatin is a 13-amino acid cyclic peptide (ICVVQDWGHHRCT) isolated from a phage-displayed random peptide library that binds to C3, inhibiting its proteolytic cleavage by C3 convertase. The 50% inhibitory concentrations (IC50s) required for inhibition of classical and alternative pathway-mediated haemolytic activities for compstatin are 63 and 12 μM, respectively. Reduction and alkylation of compstatin results in a control peptide (IAVVQDWGHHRAT), which completely lacks complement-inhibitory activity (17 (link)).
Herein, 720 μL of the blood samples were pre-incubated with 140 μL of diluent buffer (PBS), compstatin (Tocris Bioscience, Bristol, UK; 200 μM final concentration) or control peptide (Tocris Bioscience; 200 μM final concentration) for 30 min at 37°C. Then, 140 μL of PBS, LPS (5 μM final concentration) or C-SVMP (1 μM final concentration) were added and incubated for 30 min in a water bath at 37°C under agitation. Plasma and leukocytes were processed for analysis, as described below.
Luchini L.S., Pidde G., Squaiella-Baptistão C.C, & Tambourgi D.V. (2019). Complement System Inhibition Modulates the Pro-Inflammatory Effects of a Snake Venom Metalloproteinase. Frontiers in Immunology, 10, 1137.
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2

Compstatin Inhibition of Leukocyte-mediated Mf Survival

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For the compstatin inhibition studies, autologous serum was pretreated with either 100μM compstatin (Tocris Bioscience, Avonmouth, Bristol, U.K.) or 100μM compstatin control peptide (Tocris Bioscience, Avonmouth, Bristol, U.K.) for 30 min at 37°C. The compstatin treated serum and antibody treated leukocytes were added directly to the PMN attachment and Mf survival assays described above. Inhibition of Cd11b activity was implemented by incubating 2g of the monoclonal antibody clone M1/70 purified specifically for use with live cells (BioLegend, San Digeo, CA, cat #101248) with PMNs for 2 hours at room temperature prior to adding B.malayi Mf and incubating for 5 days at 37°C prior to assessing survival as described above. Block of ICAM-1 function was carried out as for Cd11b using 2g of anti-ICAM1 antibody [MEM-11] (Abcam, Cambridge, MA).
McCoy C.J., Reaves B.J., Giguère S., Coates R., Rada B, & Wolstenholme A.J. (2017). Human Leukocytes Kill Brugia malayi Microfilariae Independently of DNA-Based Extracellular Trap Release. PLoS Neglected Tropical Diseases, 11(1), e0005279.
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3

Neutrophil Activation by Complement Factors

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Neutrophil activation was determined by mixing freshly purified neutrophils (5 × 105 cells) with either NICC (1000 IEQ) alone, NICC and PPP (200 μL), or NICC, PPP and 200 μM Compstatin (Tocris Bioscience, UK). Complement-specific neutrophil activation was determined by mixing neutrophils with NICC alone or in combination with 0.8, 1.2, 2.0, 3.0, or 4.0 mg of rabbit complement sera (Sigma, USA). Neutrophil populations were gated by size and positive selection on CD45 and CD16 (BD Biosciences). Neutrophil activation was measured by median fluorescence intensity (MFI) of CD66b and CD11b (BD Biosciences). Change in the MFI of neutrophil-bound TF (Affinity Biologicals, Canada) indicated neutrophil expression of TF. Cumulative analysis on all neutrophils (CD45+ and CD16+) was performed, as well as independent analyses on single cells and doublets (Figure 5). Neutrophil activation assay using freshly isolated PRP (~300-500 × 109 cells/L) did not induce higher levels of CD66b, CD11b, or TF, therefore all subsequent assays used only PPP to simplify the interpretation of the findings.
Liuwantara D., Chew Y.V., Favaloro E.J., Hawkes J.M., Burns H.L., O'Connell P.J, & Hawthorne W.J. (2016). Characterizing the Mechanistic Pathways of the Instant Blood-Mediated Inflammatory Reaction in Xenogeneic Neonatal Islet Cell Transplantation. Transplantation Direct, 2(6), e77.
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4

Conditioned Media Characterization and Modification

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MSC- or MDF-derived CM was harvested 3 days post incubation. For some experiments, CM was subjected to some modifications; CM was filtered using a 0.22-μm syringe filter (Pall Corporation, Port Washington, NY, USA); filtered CM was heat-inactivated at 100 °C for 5 min; CM was treated with different doses (15–60 μM) of compstatin (TOCRIS, Bristol, UK), which is a C3 inhibitor; CM was separated using filter units with a molecular weight cutoff size of 50 or 100 kDa (Millipore, Billerica, MA, USA). For FPLC separation and western blot analysis, 200 ml of filtered CM, which was harvested 3 days post incubation in the absence of serum, were concentrated (200 × ) using filter units with a molecular weight cutoff size of 50 kDa. For ER/Golgi block, cells were incubated for 24 h with 2 μM of monensin (Sigma, St. Louis, MO, USA), which blocks the protein transport from the endoplasmic reticulum to the Golgi apparatus. Then, CM was harvested and filtered by using a 0.22-μm syringe filter.
Lee D.S., Yi T.G., Lee H.J., Kim S.N., Park S., Jeon M.S, & Song S.U. (2014). Mesenchymal stem cells infected with Mycoplasma arginini secrete complement C3 to regulate immunoglobulin production in b lymphocytes. Cell Death & Disease, 5(4), e1192-.
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5

Complement Deposition on Purified Malaria-Infected Erythrocytes

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Late trophozoite-stage IEs were purified by magnet-activated cell sorting (Miltenyi Biotec) in PBS supplemented with BSA (1%) (28 (link)). The purified IEs (2 × 106 cells/mL) were incubated (30 min, 4°C) with mAbs (10 μg/mL) or purified pooled human IgG (1 mg/mL), followed by incubation (1 h, 37°C) with NHS (1%) and compstatin (6 nM; Tocris) (to inhibit cleavage of C3). As positive and negative controls, type A and 0 erythrocytes were incubated with type 0 NHS and compstatin.
Complement components were detected by incubation (4°C, 30 min) with the same antibodies as above, but at different concentrations (anti-C1q: 30 μg/mL; anti- C4c: 0.5 μg/mL), followed by incubation with FITC-conjugated goat anti-rabbit IgG (1:150; Vector) and ethidium bromide (2 μg/mL; to visualize parasite DNA). Binding of human IgG to the IEs was detected in a similar way, using FITC-conjugated goat anti-human IgG (1:150; Jackson Immuno Research) (28 (link)). All incubations, dilutions, and washes were done in PBS supplemented with BSA (1%), except for the incubation with NHS and compstatin, which was done in BSA-supplemented VBS++ buffer containing Ca2+ and Mg2+ (Complement Technology). Samples were run on a Cytomics FC500 flow cytometer (Beckman Coulter). Single ethidium bromide-positive cells were analyzed for complement components and antibody labeling by FlowLogic (Inivai Technologies).
Larsen M.D., Quintana M.D., Ditlev S.B., Bayarri-Olmos R., Ofori M.F., Hviid L, & Garred P. (2019). Evasion of Classical Complement Pathway Activation on Plasmodium falciparum-Infected Erythrocytes Opsonized by PfEMP1-Specific IgG. Frontiers in Immunology, 9, 3088.
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6

Investigating Immune Cell Activation Compounds

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This study used the following compounds: loxoribine (InvivoGen, CA, USA, cat# tlrl-lox), Pam3CSK4 (InvivoGen, CA, USA, cat# tlrl-pms), human thrombin (Enzyme Research Laboratories, IN, USA, cat# HIIa), prostaglandin E1 (PGE1, Millipore, MA, USA, cat# 538903-1MG), complement C3 (Millipore, cat #204885); GM-CSF (Stemcell Technologies, MA, USA, cat #78015.1), and compstatin (Tocris, MN, USA, cat# 2585). Thrombin and Pam3CSK4 were dissolved in water and blood or isolated cells were treated with 10 μg/mL of Pam3CSK4 or 0.05 U/mL of thrombin. Pam3CSK4 concentration is based on previously known mediation of platelet–neutrophil aggregates8 (link); low concentration of thrombin was used in order to activate platelets without making them form a thrombus. Platelets do not tolerate DMSO at concentrations higher than 0.05%, thus, loxoribine was dissolved in 700 μL DMSO4 and 770 μL water. Cells were treated with 1 mM loxoribine; complement C3 and compstatin were dissolved in HEPES-modified Tyrode’s buffer.
Koupenova M., Corkrey H.A., Vitseva O., Manni G., Pang C.J., Clancy L., Yao C., Rade J., Levy D., Wang J.P., Finberg R.W., Kurt-Jones E.A, & Freedman J.E. (2019). The role of platelets in mediating a response to human influenza infection. Nature Communications, 10, 1780.
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7

Complement Inhibition and Thromboelastography

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Changes in TEG were evaluated following complement inhibition. Two receptor inhibitors were used, one for the C3a receptor (SB 290157, C3aRI, Cayman Chemical Company, Ann Arbor, MI) and another for the C5a receptor (W54011, C5aRI, EMD Millipore, Billerica, MA).(32 (link), 33 (link)) The C3aRI and C5aRI were solubilized in dimethyl sulfoxide (DMSO) and further diluted in phosphate-buffered saline (PBS) to obtain the experimental solution per supplier instructions. An equivalent carrier (0.05% v/v DMSO in PBS) was used as a control. The C3aRI and C5aRI inhibitors were added to 500 μL of whole blood (WB) for a final concentration of 300 nM and 6 nM, respectively. These doses are comparable to the inhibitors’ respective IC50 doses of the C3aRI (IC50 of 200 nM) and C5aRI (IC50 of 3.1 nM), respectively. (32 (link), 33 (link)) To evaluate inhibition of upstream alternative complement pathway, the C3 and C3b inhibitor Compstatin (Tocris Bioscience, Bristol, UK) was added to whole blood to a final concentration of 25 μM and 50 μM. After the addition of inhibitors, samples were incubated for five minutes in 37°C water bath prior to running TEG using the same methodology as described previously.
Samuels J.M., Coleman J.R., Moore E.E., Bartley M., Vigneshwar N., Cohen M., Silliman C.C., Sauaia A, & Banerjee A. (2020). Alternative Complement Pathway Activation Provokes a Hypercoagulable State with Diminished Fibrinolysis.. Shock (Augusta, Ga.), 53(5), 560-565.
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8

Complement Modulation in Dendritic Cells

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Blocking of complement receptors was performed by incubating DC for 1 hour with the indicated concentrations of a specific C3aR antagonist (C3aRA) or C5aR antagonist (C5aRA; both from Calbiochem, now Sigma-Aldrich; dissolved in dimethyl sulfoxide [DMSO]) prior to treatment with pdFVIII plus LPS. As a control, DC were incubated with DMSO only (Sigma-Aldrich). Complement proteins were blocked by treating DC with pdFVIII plus LPS in the presence of the following neutralizing monoclonal antibodies: anti-C3/C3a/C3adesArg (clone K13/16), anti-C5/C5a/C5adesArg (clone G25/2, both isotype IgG1, BioLegend, Fell, Germany), eculizumab (anti-C5, isotype IgG2/4, Alexion, Paris, France), or the C3-targeting peptide compstatin (Tocris Bioscience, Bristol, UK). An IgG1 isotype control antibody (clone MOPC-21, BioLegend) and ovalbumin (OVA)323-339 (ISQAVHAAHAEINEAGR, AnaSpec, Fremont, USA) served as controls.
Ringler E., Iannazzo S.O., Herzig J., Weiss L.M., Anzaghe M., Miller L, & Waibler Z. (2023). Complement protein C3a enhances adaptive immune responses towards FVIII products. Haematologica, 108(6), 1579-1589.
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