Journal: The Journal of Immunology Author Choice

Article Title: A Conformational Change of Complement C5 Is Required for Thrombin-Mediated Cleavage, Revealed by a Novel Ex Vivo Human Whole Blood Model Preserving Full Thrombin Activity

doi: 10.4049/jimmunol.2001471

Figure Lengend Snippet: Cleavage of purified C5 by thrombin, as detected by protein staining and Western blot. (A) SDS-PAGE on purified C5 (500 nM) from Comptech, Quidel, and our MgCl2 affinity-purified C5 eluted under mild conditions using a 2 M MgCl2 solution, incubated with thrombin (100 nM) for 0, 15, and 60 min at 37°C before the addition of lepirudin (1 μM). At 0 min, lepirudin was added before thrombin. Intact and cleaved α-chain is indicated by α and α′ respectively, β-chain is indicated with β. The gel was stained with Coomassie brilliant blue. (B) Purified C5 (500 nM) from Comptech was incubated with thrombin (100 nM) for different time intervals (7.5 min to 20 h), as indicated on top of a representative SDS-PAGE stained with Coomassie brilliant blue. Reference proteins are shown in separate lanes; thrombin (Thr), lepirudin (Lep), and C5 (C5), and an m.w. reference (M). C5 α-chain (α) and β-chain (β) are indicated in the label on the right-hand side of the figure, so are the primary (α′) and secondary (α′′ cleavage fragments, together with thrombin and C5a. (C) Purified Comptech C5 (500 nM) was incubated for 1 h at 37°C with PBS, thrombin (100 nM), thrombin (100 nM) in the presence of lepirudin (1 μM), or cobra venom factor (CVF). C5a-containing fragments were detected by Western blot using a polyclonal antibody against C5a. (D) GPRP-plasma was incubated with thrombin (400 nM) alone or in the presence of lepirudin (7 μM), purified Comptech C5 (60 μg/ml) alone or in the presence of thrombin, or the combination of purified C5 (60 μg/ml), thrombin (400 nM), and lepirudin (7 μM), for 16 h at 37°C. C5a-containing fragments were immunoprecipitated using a monoclonal antibody against C5a (clone 137-26) and detected by Western blot as described for (B). (E) Identical conditions as described for (C), but incubations were performed in serum from a C5-deficient individual instead of in GPRP-plasma. All samples were run under reducing conditions (A–E). M, m.w. markers with size indicated in kDa.

Article Snippet: Proteins were transferred onto an immunoblot polyvinylidene fluoride membrane (Bio-Rad) and blotted using rabbit polyclonal IgG anti-prothrombin (Molecular innovations; Novi, MI) followed by an HRP-linked goat anti-rabbit-IgG (Southern Biotech, Birmingham, AL).

Techniques: Purification, Staining, Western Blot, SDS Page, Affinity Purification, Incubation, Combined Bisulfite Restriction Analysis Assay, Immunoprecipitation

Journal: The Journal of Immunology Author Choice

Article Title: A Conformational Change of Complement C5 Is Required for Thrombin-Mediated Cleavage, Revealed by a Novel Ex Vivo Human Whole Blood Model Preserving Full Thrombin Activity

doi: 10.4049/jimmunol.2001471

Figure Lengend Snippet: C5a Ag exposure and C5 cleavage in hydrochloric acid–acidified GPRP-plasma and serum. (A) Conformationally selective ELISA for detecting a C5a neoepitope of C5 in GPRP-plasma and C5-deficient serum including reconstitution with purified C5 (60 μg/ml) from Comptech. The plasma pH was adjusted with hydrochloric acid to 6.4, 6.8, or kept at 7.4, and incubated for 15 min at 37°C. Exposure of the C5a neoepitope in C5 was detected by ELISA, combining capturing and detecting Abs specific for the C5a neoepitope and C5b, respectively. Data are shown as the optical density mean values ± SD (n = 4), *p < 0.001. (B) GPRP-plasma was pH-adjusted to 6.4 and 6.8 with hydrochloric acid or kept at 7.4, and incubated with 0.9% NaCl, lepirudin (Lep) and/or thrombin (Thr, 400 nM) for 1 h at 37°C. C5a-containing fragments were specifically enriched by immunoprecipitation using a mAb against C5a (clone 137-26) and detected by Western blot, under reduced conditions, using a polyclonal Ab against C5a. Intact and cleaved C5 α-chain are indicated by α and α′, respectively, on the representative Western blot from two replicates. M, m.w. markers with size indicated in kDa.

Article Snippet: Proteins were transferred onto an immunoblot polyvinylidene fluoride membrane (Bio-Rad) and blotted using rabbit polyclonal IgG anti-prothrombin (Molecular innovations; Novi, MI) followed by an HRP-linked goat anti-rabbit-IgG (Southern Biotech, Birmingham, AL).

Techniques: Enzyme-linked Immunosorbent Assay, Purification, Incubation, Immunoprecipitation, Western Blot

Journal: The Journal of Immunology Author Choice

Article Title: A Conformational Change of Complement C5 Is Required for Thrombin-Mediated Cleavage, Revealed by a Novel Ex Vivo Human Whole Blood Model Preserving Full Thrombin Activity

doi: 10.4049/jimmunol.2001471

Figure Lengend Snippet: C5a Ag exposure and C5 cleavage in hydrochloric acid– and lactic acid–acidified GPRP-plasma. (A and B) Conformationally selective ELISA for the detection of C5a neoepitope of C5 in normal GPRP-plasma (pH 7.4) and plasma acidified to pH 6.8 with hydrochloric acid (HCl) (A) or lactic acid (B), with or without neutralization to pH 7.4 with NaOH 1 min after acidification. Exposure of the C5a neoepitope in C5 was detected by ELISA, combining capturing and detecting Abs specific for the C5a neoepitope and C5b, respectively. Purified C5 (Comptech) at 60 μg/ml was included as a control. Data are shown as the optical density mean values ± SD (n = 3), *p < 0.001. (C) GPRP-plasma with and without lepirudin was pH-adjusted to 6.8 with hydrochloric acid (HCl) or lactic acid, with or without neutralization to pH 7.4 with NaOH 1 min after acidification. All samples, including GPRP-plasma, were kept at 7.4, and purified C5 with thrombin (400 nM) +/− lepirudin were incubated for 1 h at 37°C. C5a-containing fragments were specifically enriched by immunoprecipitation using a mAb against C5a (clone 137-26) and detected by Western blot, under reduced conditions, using a polyclonal Ab against C5a. Samples, as indicated on top of the membrane, are GPRP-plasma pH 7.4 (lanes 1 and 2), GPRP-plasma acidified to pH 6.8 with either hydrochloric acid (HCl) (lanes 3–6) or with lactic acid (lanes 7–10). Samples in lanes 4, 6, 8, and 10 are neutralized to pH 7.4 with NaOH after acidification. It is indicated which samples contain lepirudin. Intact and cleaved C5 α-chain are indicated by α and α′, respectively, on the representative Western blot from three replicates. M, m.w. markers with size indicated in kDa.

Article Snippet: Proteins were transferred onto an immunoblot polyvinylidene fluoride membrane (Bio-Rad) and blotted using rabbit polyclonal IgG anti-prothrombin (Molecular innovations; Novi, MI) followed by an HRP-linked goat anti-rabbit-IgG (Southern Biotech, Birmingham, AL).

Techniques: Enzyme-linked Immunosorbent Assay, Neutralization, Purification, Incubation, Immunoprecipitation, Western Blot

Journal: The Journal of Immunology Author Choice

Article Title: A Conformational Change of Complement C5 Is Required for Thrombin-Mediated Cleavage, Revealed by a Novel Ex Vivo Human Whole Blood Model Preserving Full Thrombin Activity

doi: 10.4049/jimmunol.2001471

Figure Lengend Snippet: Cleavage of C5 in clotting blood and thrombin-mediated cleavage of C5b in the C5b6 complex. (A) Human whole blood was collected in additive-free glass serum tubes. The blood was immediately acidified with 5% (v/v) lactic acid (0.165, 0.330, 0.500 M) or hydrochloric acid (HCl) (0.165, 0.330, 0.500 M) or added physiologic NaCl for volume control. The blood was let to clot for 60 min at 37°C and then centrifuged to serum. C5a-containing fragments were specifically enriched by immunoprecipitation using a mAb against C5a (clone 137-26) and detected by Western blot, under reduced conditions, using a polyclonal Ab against C5a. Intact and cleaved C5 α-chain are indicated by α and α′, respectively, on the representative Western blot from two replicates. (B) SDS-PAGE on purified C5b6 (50 μg/ml) from Comptech incubated with and without thrombin (400 nM) in PBS at pH 7.4, 6.8, and 6.4 for 60 min at 37°C. The samples were run on an SDS-PAGE under reduced conditions and stained with SYPRO Ruby Protein Gel Stain. Intact and cleaved α-chain is indicated by α and α′, respectively, β-chain is indicated with β, and C6 with C6. M, m.w. markers with size indicated in kDa.

Article Snippet: Proteins were transferred onto an immunoblot polyvinylidene fluoride membrane (Bio-Rad) and blotted using rabbit polyclonal IgG anti-prothrombin (Molecular innovations; Novi, MI) followed by an HRP-linked goat anti-rabbit-IgG (Southern Biotech, Birmingham, AL).

Techniques: Coagulation, Immunoprecipitation, Western Blot, SDS Page, Purification, Incubation, Staining