polyclonal goat-anti-human igg against c1-inh  (Cedarlane)

Journal: Blood

Article Title: A mechanism for hereditary angioedema with normal C1 inhibitor: an inhibitory regulatory role for the factor XII heavy chain

doi: 10.1182/blood-2018-06-860270

Figure Lengend Snippet: Recombinant FXII. (A) Schematic diagrams of FXII, αFXIIa, and βFXIIa showing noncatalytic (white boxes) and protease (pink boxes) domains. Positions of the active site serine (Ser544) are indicated by black bars. Sites of proteolysis during activation are indicated by arrows. Cleavage after Arg353 (black arrow) converts FXII to αFXIIa. Cleavage of αFXIIa after Arg334 (white arrow) separates the noncatalytic and protease domains, forming βFXIIa. FXII noncatalytic domains are the fibronectin type 2 (F2), epidermal growth factor (EGF), fibronectin type 1 (F1), and kringle (K) domains, and a proline-rich region (PRR). (B) Schematic diagrams of FXII with lysine or arginine replacement of Thr309 (gray arrow). Cleavage after Lys/Arg309 creates 2 proteins, δHC and δFXII. (C) Nonreducing SDS-PAGE of purified FXII (∼3 μg per lane). Shown are FXII-WT, FXII-Lys309, FXII-Arg309, and FXII-3C. Reducing (D) and nonreducing (E) SDS-PAGE of the FXIIa (activated) forms of FXII shown in panel C. Proteins were activated by incubation with dextran sulfate. Positions of molecular mass standards in kilodaltons are shown to the left of the images in panels C-E. To the right of these images are markers for FXII, activated FXII (αFXIIa), HC of αFXIIa (HC), LC of αFXIIa (LC), HC of FXII-Lys309 or FXII-Arg309 cleaved after residue 309 (δHC), and FXII residues Thr310 to Ser596 (δFXII).

Article Snippet: The following antibodies were used (obtained from the companies shown in parentheses): goat horseradish peroxidase (HRP)–anti-human FXII, sheep HRP–anti-human PK, and goat HRP–anti-human C1-INH immunoglobulin Gs (IgGs) (Enzyme Research Laboratories); goat anti-human HK IgG (Nordic Immunology); and rabbit anti-plasminogen IgG 1677-1-AP (Proteintech).

Techniques: Recombinant, Activation Assay, SDS Page, Purification, Incubation, Residue

Journal: Blood

Article Title: A mechanism for hereditary angioedema with normal C1 inhibitor: an inhibitory regulatory role for the factor XII heavy chain

doi: 10.1182/blood-2018-06-860270

Figure Lengend Snippet: FXII cleavage by coagulation proteases. (A) Clotting times in an aPTT assay for normal plasma (NPP), FXII-deficient plasma (DP), or FXII-deficient plasma supplemented with recombinant FXII-WT (WT), FXII-Lys309 (309K), FXII-Arg309 (309R), or FXII-3C (3C). Each symbol indicates 1 clotting time. (B) Recombinant FXII (200 nM) incubated in standard buffer with 200 μM S-2302 at 37°C in the presence (+) or absence (−) of 70 μM polyphosphate. Left panel, FXII-Lys309; right panel, FXII-Arg309. Changes in OD 405 nm were continuously monitored. Curves are means of 3 independent runs. (C) Western blot of FXII-deficient plasma supplemented with 400 nM FXII-WT, FXII-Lys309, or FXII-Arg309 induced to clot with TF (2.5 pM) and CaCl2 (6.25 mM). Fibrin formation was prevented with H-Gly-Pro-Arg-Pro-OH. Western blots of time courses were probed with polyclonal IgG to human FXII. Positions of molecular mass standards in kilodaltons are shown to the left of the images. (D-F) FXII (200 nM) in standard buffer incubated with 25 nM plasmin (D), 10 nM plasmin, thrombin, or FXIa (E), or α-kallikrein (50 nM) (F). Western blots of time courses were probed with a mixture of monoclonal IgGs to the FXII HC (15H8) and LC (1B2). To the right of images in panels C-F are markers for FXII (FXII), the HC and LC of αFXIIa, HC of FXII-Lys309 or FXII-Arg309 cleaved after residue 309 (δHC) and FXII residues Thr310 to Ser596 (δFXII). (G) FXII (100 nM) incubated in standard buffer with (+) or without (−) 25 nM thrombin for 120 minutes at 37°C. Thrombin was inhibited with argatroban (125 μM) and FXIIa cleavage of S-2302 (500 μM) was determined. The proteins are FXII-WT (WT), FXII-Lys309 (309 Lys), and FXII-Arg309 (309 Arg). Control (XIIa) is 12 nM αFXIIa. Error bars ± 1 standard deviation (SD); n = 6.

Article Snippet: The following antibodies were used (obtained from the companies shown in parentheses): goat horseradish peroxidase (HRP)–anti-human FXII, sheep HRP–anti-human PK, and goat HRP–anti-human C1-INH immunoglobulin Gs (IgGs) (Enzyme Research Laboratories); goat anti-human HK IgG (Nordic Immunology); and rabbit anti-plasminogen IgG 1677-1-AP (Proteintech).

Techniques: Coagulation, Clinical Proteomics, Recombinant, Incubation, Western Blot, Residue, Control, Standard Deviation

Journal: Blood

Article Title: A mechanism for hereditary angioedema with normal C1 inhibitor: an inhibitory regulatory role for the factor XII heavy chain

doi: 10.1182/blood-2018-06-860270

Figure Lengend Snippet: PK activation by FXII and δFXII. (A) Western blot of FXII/PK reciprocal activation. Plasma-derived FXII (200 nM) and PK (200 nM) were incubated in standard buffer at 37°C. At indicated times, samples were removed into reducing sample buffer. Western blots were probed with goat anti-human FXII IgG or sheep anti-human PK IgG. Positions of markers for FXII, PK, and the HC and LC of αFXIIa and kallikrein are indicated to the right of each image. (B) Kallikrein generation in reciprocal reactions with FXII. FXII-WT (, ), FXII-Lys309 (, ), or FXII-Arg309 (, ), 100 nM in standard buffer was incubated with (, , ) or without (, , ) thrombin (25 nM) for 2 hours at 37°C. Reactions were stopped with argatroban (125 μM). PK (60 nM) was mixed with 12.5 nM of the preincubated FXII. At various times, aliquots were removed, FXIIa was inhibited with CTI, and kallikrein concentration was determined by chromogenic assay. (C) FXII amino acid sequence within the proline-rich region showing the amino acids inserted to create a cleavage site for 3CP in the protein FXII-3C. The image to the right of the arrow shows the N and C termini after cleavage of FXII-3C with 3CP. (D) Schematic diagrams of FXII with the sequence Leu-Glu-Val-Leu-Phe-Gln-Gly inserted between FXII residues Gln307 and Pro308 to create a cleavage site for 3C protease in FXII-3C. Cleavage of FXII-3C with 3C creates a mixture of δHC (with a slightly different C terminus than in panel B), and δFXII-3C. The latter differs from δFXII in having an additional Gly-Pro at the N terminus. Cleavage of δFXII after Arg353 creates the fully active species δFXIIa-3C. (E) Nonreducing 20% Tris–2-(N-morpholino)ethanesulfonic acid (MES) SDS-PAGE of FXII-3C cleaved by 3C protease and the purified FXII-3C δHC and δFXII-3C. Positions of molecular mass standards in kilodaltons are shown to the left of the image. (F) Kallikrein generation in reciprocal reactions with FXII-3C and δFXII-3C. PK (60 nM) was incubated in standard buffer at 37°C with 12.5 nM FXII-WT (), FXII-3C (□), FXII-3C cleaved with 3C protease () or purified δFXII-3C (), or in the absence of FXII (). Reactions were run in the absence (left panel) or presence (right panel) of 70 μM Poly-P. At the indicated times, aliquots were removed and tested for kallikrein generation by chromogenic assay. For panels B and F, error bars ± 1 SD; n = 5 or 6.

Article Snippet: The following antibodies were used (obtained from the companies shown in parentheses): goat horseradish peroxidase (HRP)–anti-human FXII, sheep HRP–anti-human PK, and goat HRP–anti-human C1-INH immunoglobulin Gs (IgGs) (Enzyme Research Laboratories); goat anti-human HK IgG (Nordic Immunology); and rabbit anti-plasminogen IgG 1677-1-AP (Proteintech).

Techniques: Activation Assay, Western Blot, Clinical Proteomics, Derivative Assay, Incubation, Concentration Assay, Chromogenic Assay, Sequencing, SDS Page, Purification

Journal: Blood

Article Title: A mechanism for hereditary angioedema with normal C1 inhibitor: an inhibitory regulatory role for the factor XII heavy chain

doi: 10.1182/blood-2018-06-860270

Figure Lengend Snippet: Properties of δFXII-3C and its active form δFXIIa-3C, and their effects on HK cleavage. (A-C) Activation of FXII. FXII-WT (), FXII-3C (□), or δFXII-3C (), 200 nM, were incubated in standard buffer at 37°C with kallikrein (10 nM) (A), plasmin (500 nM) (B), or FXIa (10 nM, subunit concentration) (C). Kallikrein was inhibited with HO3, and FXIa and plasmin were inhibited with aprotinin, and FXIIa generation was determined by chromogenic substrate cleavage. (D) PK activation by FXII-T or δFXII-T. PK (200 nM) was incubated in standard buffer at 37°C with vehicle (), 200 nM FXII-T (□), or δFXII-T (), and cleavage of chromogenic substrate was continuously monitored. (E) PK activation by FXIIa. PK (60 nM) was incubated in standard buffer at 37°C with 50 pM αFXIIa (), βFXIIa (□), or δFXIIa-3C (), or in the absence of FXIIa (). At various times, reactions were stopped with CTI, and kallikrein generation was determined by chromogenic assay. (F) FXI (30 nM) was incubated in standard buffer at 37°C with 10 nM αFXIIa (), βFXII (), or δFXIIa-3C (), or in the absence of FXIIa (). At various times, reactions were stopped with CTI, and FXIa generation was determined by chromogenic assay. For panels A and B, n = 4; C, n = 6; D, n = 3; E, n = 9; and F, n = 6. Error bars ± 1 SD for all panels. (G) HK cleavage in human plasma. Shown are western blots of human FXII-deficient plasma supplemented with FXII-WT, FXII-3C, or δFXII-3C (400 nM) in the absence (−) or presence (+) of PTT-A silica-based reagent. At indicated times, samples were removed into nonreducing sample buffer. Western blots were probed with goat anti-human HK IgG (HK) or goat anti-human C1-INH IgG (C1-INH). Positions of standards for HK, the 2 bands of cleaved HK (HKa), and kallikrein or αFXIIa in complex with C1-INH are shown on the right. Positions of molecular mass standards in kilodaltons are shown to the left of the images.

Article Snippet: The following antibodies were used (obtained from the companies shown in parentheses): goat horseradish peroxidase (HRP)–anti-human FXII, sheep HRP–anti-human PK, and goat HRP–anti-human C1-INH immunoglobulin Gs (IgGs) (Enzyme Research Laboratories); goat anti-human HK IgG (Nordic Immunology); and rabbit anti-plasminogen IgG 1677-1-AP (Proteintech).

Techniques: Activation Assay, Incubation, Concentration Assay, Chromogenic Assay, Clinical Proteomics, Western Blot

Journal: Blood

Article Title: A mechanism for hereditary angioedema with normal C1 inhibitor: an inhibitory regulatory role for the factor XII heavy chain

doi: 10.1182/blood-2018-06-860270

Figure Lengend Snippet: Effect of δFXII on HK cleavage in mice. (A) FXII-WT, FXII-3C, or δFXII-3C were administered IV to WT C57Bl/6 mice to an estimated final plasma concentration of 140 nM or 40 nM. Shown are nonreducing western blots of plasma collected 0, 15, 30 minutes, or ∼18 hours (overnight [ON]) after FXII infusion. Blots were developed with anti-murine HK IgG (anti-mHKFXI). (B) As in panel A. δFXII-3C was infused into WT, PK-deficient (Klkb1−/−) mice), or FXI-deficient (F11−/−) mice (140 nM final plasma concentration). FXII-WT mice also received an IV infusion of vehicle (Control) or monoclonal anti-human FXII IgG 1B2. Samples were analyzed as in panel A. (C-D) WT mice received IV infusions of FXII-WT (C) or FXII-Arg309 (D) (140 nM final plasma concentration). Mice were then infused with vehicle (−TF) or 70 ng/kg human TF (+TF). Samples of plasma collected 0, 15, or 30 minutes postvehicle/TF infusion were analyzed as in panel A. (E) WT mice were treated with vehicle or 70 ng/kg human TF after infusion of FXII-Arg309, as in panel D. Some mice were pretreated with the thrombin inhibitor hirudin (10 mg/kg). FXI-deficient (F11−/−) mice were treated with 70 ng/kg human TF after FXII-Arg309 infusion. The 2 left panels in E are the same as in panel D. (F) Western blot for plasminogen in plasma from WT C57Bl/6 mice before (C) or after (+ASO) subcutaneous injections of an ASO directed at the mRNA of plasminogen. (G) Western blots of plasma HK in WT mice treated with anti-plasminogen ASO. Mice received FXII-WT (left) or FXII-Arg309 (right) prior to infusion of vehicle (−TF) or 70 ng/kg human TF (+TF). For blots in panels A-E and G, arrows on the right indicate the positions of cleaved HK (HKa), and positions of molecular mass standards in kilodaltons are shown to the left of the images. Plg, the position of the band representing plasminogen.

Article Snippet: The following antibodies were used (obtained from the companies shown in parentheses): goat horseradish peroxidase (HRP)–anti-human FXII, sheep HRP–anti-human PK, and goat HRP–anti-human C1-INH immunoglobulin Gs (IgGs) (Enzyme Research Laboratories); goat anti-human HK IgG (Nordic Immunology); and rabbit anti-plasminogen IgG 1677-1-AP (Proteintech).

Techniques: Clinical Proteomics, Concentration Assay, Western Blot, Control

Journal: Blood

Article Title: A mechanism for hereditary angioedema with normal C1 inhibitor: an inhibitory regulatory role for the factor XII heavy chain

doi: 10.1182/blood-2018-06-860270

Figure Lengend Snippet: Effect of anti-FXII IgG 15H8 on protease activity and HK cleavage. (A) Effect of IgG 15H8 on activity of FXII-WT and FXII-T. PK (50 nM) was incubated in standard buffer at 37°C with 200 μM S-2302 and 50 nM FXII-WT (left) or FXII-T (right) in the absence () or presence () of 15H8 IgG (50 nM). For FXII-WT, a control reaction without FXII was run (). Continuous cleavage of S-2302 at 405 nM was followed. Data are means of 3 runs. (B) Western blot showing the effect of 15H8 antibody infusion on HK in WT mice supplemented with FXII-WT. The blot was processed as in Figure 5. Arrows on the right indicate the positions of cleaved HK (HKa), and positions of molecular mass standards in kilodaltons are shown to the left of the image. (C-E) Effect of IgG 15H8 on FXIIa generation. Plasma FXII (200 nM) was incubated in standard buffer at 37°C with vehicle (control) (C), 20 nM kallikrein (D), or 20 nM plasmin (E) in the absence () or presence () of 15H8 (400 nM). At various time points, aliquots were mixed with stop solution (containing HO3 for kallikrein, aprotinin for plasmin) and FXIIa concentration was determined by chromogenic assay. Data show means ± 1 SD.

Article Snippet: The following antibodies were used (obtained from the companies shown in parentheses): goat horseradish peroxidase (HRP)–anti-human FXII, sheep HRP–anti-human PK, and goat HRP–anti-human C1-INH immunoglobulin Gs (IgGs) (Enzyme Research Laboratories); goat anti-human HK IgG (Nordic Immunology); and rabbit anti-plasminogen IgG 1677-1-AP (Proteintech).

Techniques: Activity Assay, Incubation, Control, Western Blot, Clinical Proteomics, Concentration Assay, Chromogenic Assay

Journal: Blood

Article Title: A mechanism for hereditary angioedema with normal C1 inhibitor: an inhibitory regulatory role for the factor XII heavy chain

doi: 10.1182/blood-2018-06-860270

Figure Lengend Snippet: Properties of δFXII-3C and its active form δFXIIa-3C, and their effects on HK cleavage. (A-C) Activation of FXII. FXII-WT (), FXII-3C (□), or δFXII-3C (), 200 nM, were incubated in standard buffer at 37°C with kallikrein (10 nM) (A), plasmin (500 nM) (B), or FXIa (10 nM, subunit concentration) (C). Kallikrein was inhibited with HO3, and FXIa and plasmin were inhibited with aprotinin, and FXIIa generation was determined by chromogenic substrate cleavage. (D) PK activation by FXII-T or δFXII-T. PK (200 nM) was incubated in standard buffer at 37°C with vehicle (), 200 nM FXII-T (□), or δFXII-T (), and cleavage of chromogenic substrate was continuously monitored. (E) PK activation by FXIIa. PK (60 nM) was incubated in standard buffer at 37°C with 50 pM αFXIIa (), βFXIIa (□), or δFXIIa-3C (), or in the absence of FXIIa (). At various times, reactions were stopped with CTI, and kallikrein generation was determined by chromogenic assay. (F) FXI (30 nM) was incubated in standard buffer at 37°C with 10 nM αFXIIa (), βFXII (), or δFXIIa-3C (), or in the absence of FXIIa (). At various times, reactions were stopped with CTI, and FXIa generation was determined by chromogenic assay. For panels A and B, n = 4; C, n = 6; D, n = 3; E, n = 9; and F, n = 6. Error bars ± 1 SD for all panels. (G) HK cleavage in human plasma. Shown are western blots of human FXII-deficient plasma supplemented with FXII-WT, FXII-3C, or δFXII-3C (400 nM) in the absence (−) or presence (+) of PTT-A silica-based reagent. At indicated times, samples were removed into nonreducing sample buffer. Western blots were probed with goat anti-human HK IgG (HK) or goat anti-human C1-INH IgG (C1-INH). Positions of standards for HK, the 2 bands of cleaved HK (HKa), and kallikrein or αFXIIa in complex with C1-INH are shown on the right. Positions of molecular mass standards in kilodaltons are shown to the left of the images.

Article Snippet: Antibodies The following antibodies were used (obtained from the companies shown in parentheses): goat horseradish peroxidase (HRP)–anti-human FXII, sheep HRP–anti-human PK, and goat HRP–anti-human C1-INH immunoglobulin Gs (IgGs) (Enzyme Research Laboratories); goat anti-human HK IgG (Nordic Immunology); and rabbit anti-plasminogen IgG 1677-1-AP (Proteintech).

Techniques: Activation Assay, Incubation, Concentration Assay, Chromogenic Assay, Western Blot