Cleaved kininogen as a biomarker for bradykinin release in hereditary angioedema
2017; Elsevier BV; Volume: 140; Issue: 6 Linguagem: Inglês
10.1016/j.jaci.2017.07.012
ISSN1097-6825
AutoresZonne L. M. Hofman, Steven de Maat, Chiara Suffritti, Andrea Zanichelli, Cassandra L. R. van Doorn, Silvie Sebastian, Nóra Veszeli, Dorottya Csuka, Thomas Renné, Gerard Pasterkamp, Marco Cicardi, Henriette Farkas, C. Erik Hack, Coen Maas,
Tópico(s)Autoimmune Bullous Skin Diseases
ResumoBradykinin is a vasoactive inflammatory mediator that plays a key role in hereditary angioedema (HAE). It is released from its precursor high-molecular weight kininogen (HK) by plasma kallikrein, with cleaved HK (cHK) as a byproduct. Direct determination of bradykinin has proven valuable, but is technically complicated because of instability and small peptide size.1Cugno M. Tedeschi A. Nussberger J. Bradykinin in idiopathic nonhistaminergic angioedema.Clin Exp Allergy. 2017; 47: 139-140Crossref PubMed Scopus (12) Google Scholar, 2Nussberger J. Cugno M. Amstutz C. Cicardi M. Pellacani A. Agostoni A. Plasma bradykinin in angio-oedema.Lancet. 1998; 351: 1693-1697Abstract Full Text Full Text PDF PubMed Scopus (637) Google Scholar Western blotting studies have shown elevated cHK production in HAE,3Suffritti C. Zanichelli A. Maggioni L. Bonanni E. Cugno M. Cicardi M. High-molecular-weight kininogen cleavage correlates with disease states in the bradykinin-mediated angioedema due to hereditary C1-inhibitor deficiency.Clin Exp Allergy. 2014; 44: 1503-1514Crossref PubMed Scopus (27) Google Scholar, 4Banerji A. Busse P. Shennak M. Lumry W. Davis-Lorton M. Wedner H.J. et al.Inhibiting plasma kallikrein for hereditary angioedema prophylaxis.N Engl J Med. 2017; 376: 717-728Crossref PubMed Scopus (116) Google Scholar underpinning its potential value as a clinical biomarker for bradykinin-mediated pathology. However, western blotting is a laborious and semi-quantitative method, which motivates the development of new bioassays.5Faucette R. Conley G. Cosic I. Kopacz K. Chikwamba K. Sexton D.J. An ELISA for the detection of cleaved high molecular weight kininogen in human plasma.Allergy. 2016; 71: 528-591Crossref Google Scholar Here, we describe the development of an immunoassay for clinical evaluation of cHK levels. Experimental procedures are described in this article's Online Repository at www.jacionline.org. Fig 1, A, shows a Western blot of the purified HK and cHK preparations that were used for assay development (reduced; antibody reacts with HK light chain). As reported earlier, cHK contains 2 light-chain fragments.6Reddigari S. Kaplan A.P. Cleavage of human high-molecular weight kininogen by purified kallikreins and upon contact activation of plasma.Blood. 1988; 71: 1334-1340PubMed Google Scholar We selected a monoclonal variable domain of a heavy-chain-only antibody (VHH-D1) against cHK, as well as a monoclonal VHH (H4) against total (c)HK and used these as capture antibody and detection antibody, respectively. In a microtiter plate assay setup, purified cHK is detected approximately 5 times better than purified HK (Fig 1, B). When normal pooled plasma (NPP) is incubated with βFXIIa (1 μg/mL, synonymous to FXIIf), all HK is converted into cHK (see Fig E1, A, in this article's Online Repository at www.jacionline.org). Initially, the assay setup detected cHK with low, but significant signals (Fig E1, B). We next established that the anionic polymer dextran sulfate (average Mr 500 kDa; DXS) increases assay sensitivity in the presence of inhibitors that prevent contact activation (Fig 1, C; enzyme inhibition controls in Fig E1, D and E). Furthermore, DXS enhances assay sensitivity in the absence of plasma enzymes (Fig E1, C). Next, we prepared an assay standard by mixing NPP with activated NPP (in the presence of inhibitors) at varying ratios. At a plasma sample dilution of 1:32, changes of 2.5% in cHK levels within a range of approximately 0% to 20% from baseline levels in NPP can be detected (Fig 1, D; Western blot Fig 1, E). At sample dilutions 1:64 and 1:128, detection ranges are approximately 10% to 70% (data not shown) and approximately 20% to 100%, respectively (see Fig E2, A, in this article's Online Repository at www.jacionline.org; Western blot, Fig E2, B). Further investigation showed that this immunoassay setup captures complexes composed of (c)HK and DXS as a function of plasma cHK levels (see Fig E3 in this article's Online Repository at www.jacionline.org). Next, we investigated cHK levels in patients with C1 inhibitor hereditary angioedema (C1-INH-HAE). The index cohort consisted of 71 patients (from Hungary), diagnosed with C1-INH-HAE due to C1-INH deficiency (n = 60, type I) or dysfunction (n = 11, type II), and 44 healthy controls. We analyzed 78 remission samples (71 patients) and 28 samples (19 patients) collected during angioedema attacks. Median cHK levels in healthy controls were 6.4% (range, 2.7%-15.0%; Fig 2, A), 15.2% (range, 1.8%-74.7%) during remission (P < .0001, vs controls) and 29.6% (range, 6.3%-58.6%) during angioedema attacks (P < .0001 and P = .0027, vs controls and remission, respectively). We then measured cHK levels in a validation cohort of 62 patients with C1INH-HAE (from Italy) diagnosed with type I (n = 58) or type II (n = 4) C1INH-HAE and 26 healthy controls. We analyzed 64 remission samples (62 patients) and 14 samples collected during angioedema attacks (12 patients). Median cHK levels in healthy controls were 6.4% (range, 3.4%-17.4%; Fig 2, B), 17.7% (range, 3.8%-64.3%) during remission (P < .0001, vs controls) and 37.2% (range, 10.9%-74.7%) during angioedema attacks (P < .0001 and P = .0039, vs controls and remission, respectively). Based on a selected cutoff value of the 97.5 percentile of healthy controls (dotted lines in Fig 2, A, B, and D), sensitivity, specificity, and positive predictive value were determined for each cohort (see Table E1 in this article's Online Repository at www.jacionline.org). Repeated determinations of cHK levels (9 samples with cHK levels ranging from 2.4% to 30%; 3 experiments on separate days) showed an SD of 1.8% (range, 0.8%-3.7%). For 33 patients, plasma was collected during both remission and angioedema attacks. Paired analysis showed that cHK levels increase by 10% (average) during angioedema attacks (P < .0001; Fig 2, C). We next investigated the role of preanalytical contact activation.7Kaplan A.P. Joseph K. Complement, kinins, and hereditary angioedema: mechanisms of plasma instability when C1 inhibitor is absent.Clin Rev Allergy Immunol. 2016; 51: 207-215Crossref PubMed Scopus (42) Google Scholar Blood of 48 patients was simultaneously collected in sodium citrate tubes, and in collection tubes with enzyme inhibitors (p100). cHK levels in inhibitor tubes were slightly, but not significantly, lower than those in citrated plasma (P = .0785; median difference, 3.0%; Fig 2, E). A subset (5 of 48) of samples had high cHK levels in citrated plasma (>25%), but more than 10% lower in plasma with enzyme inhibitors. This suggests a contribution of ex vivo cHK formation. Median cHK levels in samples collected in tubes with enzyme inhibitors were 7.3% for healthy controls (n = 6) (Fig 2, D; range, 4.1%-9.7%). In samples of patients with HAE, these were 13.5% during remission (n = 43; range, 3.3%-47.4%; P = .0610 vs controls) and 19.8% during angioedema attacks (n = 3; range, 12.9%-40.4%; P = .0214 and .4063, vs controls and remission, respectively). Similar results were obtained with samples that were collected in a previously published combination of inhibitors (see Fig E4 in this article's Online Repository at www.jacionline.org).3Suffritti C. Zanichelli A. Maggioni L. Bonanni E. Cugno M. Cicardi M. High-molecular-weight kininogen cleavage correlates with disease states in the bradykinin-mediated angioedema due to hereditary C1-inhibitor deficiency.Clin Exp Allergy. 2014; 44: 1503-1514Crossref PubMed Scopus (27) Google Scholar, 4Banerji A. Busse P. Shennak M. Lumry W. Davis-Lorton M. Wedner H.J. et al.Inhibiting plasma kallikrein for hereditary angioedema prophylaxis.N Engl J Med. 2017; 376: 717-728Crossref PubMed Scopus (116) Google Scholar Data on yearly angioedema attack frequency were available for patients in the Hungarian cohort. We found that remission cHK levels did not correlate with angioedema attack frequency (P = .3618; see Fig E5, E, in this article's Online Repository at www.jacionline.org). In 96 samples from the Italian cohort, C1-INH antigen levels and function and C4 and C1q levels were determined. All parameters had a weak to intermediate negative correlation with cHK levels (Fig E5, A-D). HK cleavage was previously analyzed by Western blot for 19 samples from the Italian cohort.3Suffritti C. Zanichelli A. Maggioni L. Bonanni E. Cugno M. Cicardi M. High-molecular-weight kininogen cleavage correlates with disease states in the bradykinin-mediated angioedema due to hereditary C1-inhibitor deficiency.Clin Exp Allergy. 2014; 44: 1503-1514Crossref PubMed Scopus (27) Google Scholar We compared these data to our immunoassay. There was a strong positive correlation between cHK levels detected by the 2 methods (P = .0013; r = .6836; Fig E5, F). However, baseline cHK levels are much lower when estimated by immunoassay (5.2% ± 1.1%) in comparison to estimation by Western blot under these specific conditions (∼35%).3Suffritti C. Zanichelli A. Maggioni L. Bonanni E. Cugno M. Cicardi M. High-molecular-weight kininogen cleavage correlates with disease states in the bradykinin-mediated angioedema due to hereditary C1-inhibitor deficiency.Clin Exp Allergy. 2014; 44: 1503-1514Crossref PubMed Scopus (27) Google Scholar In line with previous studies in HAE (and idiopathic nonhistaminergic acquired angioedema),3Suffritti C. Zanichelli A. Maggioni L. Bonanni E. Cugno M. Cicardi M. High-molecular-weight kininogen cleavage correlates with disease states in the bradykinin-mediated angioedema due to hereditary C1-inhibitor deficiency.Clin Exp Allergy. 2014; 44: 1503-1514Crossref PubMed Scopus (27) Google Scholar, 4Banerji A. Busse P. Shennak M. Lumry W. Davis-Lorton M. Wedner H.J. et al.Inhibiting plasma kallikrein for hereditary angioedema prophylaxis.N Engl J Med. 2017; 376: 717-728Crossref PubMed Scopus (116) Google Scholar, 8Giard C. Nicolie B. Drouet M. Lefebvre-Lacoeuille C. Le Sellin J. Bonneau J. et al.Angio-oedema induced by oestrogen contraceptives is mediated by bradykinin and is frequently associated with urticaria.Dermatology. 2012; 225: 62-69Crossref PubMed Scopus (19) Google Scholar, 9Baroso R. Sellier P. Defendi F. Charignon D. Ghannam A. Habib M. et al.Kininogen cleavage assay: diagnostic assistance for kinin-mediated angioedema conditions.PLoS One. 2016; 11: e0163958Crossref PubMed Scopus (19) Google Scholar we found elevated cHK levels in plasma of patients with HAE during remission. This is not directly attributable to ex vivo contact activation and indicates that the baseline rate of HK cleavage in patients with HAE is increased, but also suggests the presence of additional disease modifiers. In conclusion, we developed a sensitive immunoassay for cHK detection. This method might be implemented in C1INH-HAE patient care. Determination of cHK may be useful for investigation of bradykinin in HAE and beyond.8Giard C. Nicolie B. Drouet M. Lefebvre-Lacoeuille C. Le Sellin J. Bonneau J. et al.Angio-oedema induced by oestrogen contraceptives is mediated by bradykinin and is frequently associated with urticaria.Dermatology. 2012; 225: 62-69Crossref PubMed Scopus (19) Google Scholar, 9Baroso R. Sellier P. Defendi F. Charignon D. Ghannam A. Habib M. et al.Kininogen cleavage assay: diagnostic assistance for kinin-mediated angioedema conditions.PLoS One. 2016; 11: e0163958Crossref PubMed Scopus (19) Google Scholar Blood was collected from healthy volunteers after written informed consent. According to standard procedures, 9 volumes of blood were collected in 1 volume of sodium citrate (3.2% w/v). Samples were centrifuged twice at 2000g for 10 minutes shortly after collection. Plasma samples of ±50 healthy volunteers was pooled (referred to as NPP) and stored in aliquots at −80°C until use. This protocol was approved by University Medical Center Utrecht ethical committee. Blood from patients with C1-INH-HAE was collected as part of routine procedure during the annual control visits at the 3rd Department of Internal Medicine, Semmelweis University, Budapest, Hungary. The diagnosis of HAE was confirmed by clinical and laboratory criteria (positive family history, clinical symptoms of angioedema, low functional C1-INH level, low C4, normal C1q concentrations as advised by international guidelinesE1Cicardi M. Aberer W. Banerji A. Bas M. Bernstein J.A. Bork K. et al.Classification, diagnosis, and approach to treatment for angioedema: consensus report from the Hereditary Angioedema International Working Group.Allergy. 2014; 69: 602-616Crossref PubMed Scopus (482) Google Scholar). Samples were collected from patients during remission; in each case, at least 2 weeks elapsed since the date of the last attack. Samples obtained during angioedema attacks were collected before acute treatment and within 6 hours after the onset of the symptoms. According to standard procedures, 9 volumes of blood to 1 volume of sodium citrate (3.2% w/v) were centrifuged at 3500g for 10 minutes. Thereafter, plasma was stored at −80°C until use. In addition, citrated plasma from healthy family members (8 samples) and from healthy donors (who had been referred for routine medical evaluation, and volunteered for the study) was collected with the same procedure and after written consent. The study protocol was approved by the institutional review board of Semmelweis University, Budapest. To corroborate the data obtained in this cohort, we analyzed plasma samples of a second cohort of patients with C1-INH-HAE. Blood was collected in the course of routine diagnostic procedures and all patients gave oral informed consent that the remaining plasma could be used for research purposes. The Ethical Committee of the University of Milan approved that written informed consent is not necessary if the plasma had been obtained during routine diagnostics and that this plasma could be used for research investigating the pathophysiology of hereditary angioedema. The diagnosis of HAE was based on the presence of at least 1 recommended clinical and 1 recommended laboratory criteria. Blood samples were taken from all the patients at least 8 days from an angioedema attack (remission samples), and from selected patients within the first 6 hours of the beginning of an angioedema attack (attack samples). According to standard procedures, blood was collected using sodium citrate as anticoagulant (3.2% w/v). After centrifugation at 2000g for 20 minutes at room temperature, the plasma was divided into aliquots and stored at −80°C until tested. Citrated plasma from healthy donors was collected with the same procedure. To evaluate the effect of blood collection on cHK levels, plasma samples were collected in sodium citrate containers as well as BD Bioscience EDTA-P100 tubes (Vianen, The Netherlands) containing inhibitors to prevent preanalytic contact activationE2Yi J. Kim C. Gelfand C.A. Inhibition of intrinsic proteolytic activities moderates preanalytical variability and instability of human plasma.J Proteome Res. 2007; 6: 1768-1781Crossref PubMed Scopus (87) Google Scholar for direct comparison. A subset of samples was collected in tubes containing 100 mM benzamidine, 400 μg/mL hexadimethrine bromide, 2 mg/mL soybean trypsin inhibitor, 263 μM leupeptin, and 20 mM aminoethylbenzene-sulphonyl fluoride dissolved in acid/citrate/dextrose (100 mM sodium citrate, 67 mM citric acid, and 2% dextrose, pH 4.5).E3Suffritti C. Zanichelli A. Maggioni L. Bonanni E. Cugno M. Cicardi M. High-molecular-weight kininogen cleavage correlates with disease states in the bradykinin-mediated angioedema due to hereditary C1-inhibitor deficiency.Clin Exp Allergy. 2014; 44: 1503-1514Crossref PubMed Scopus (68) Google Scholar Aprotinin, bromophenol blue, dextran sulfate molecular (average) weight 500.000, DL-dithiothreitol (DTT), glycine, glycerol, isopropyl[1]-d-1-thiogalactopyranoside, KCl, mouse monoclonal anti–c-Myc antibody, skimmed milk powder, NaCl, Na2HPO4, NaH2PO4, triethylamine, and Tween-20 were from Sigma-Aldrich (St Louis, Mo). Talon Superflowbeads for His-tag purification were from GE Healthcare (Little Chalfont, UK). Imidazole was from Merck (Darmstadt, Germany). PolySorp and MaxiSorp microtiter plates, NeutrAvidin Protein, and PageBlue were from Thermo Scientific (Waltham, Mass). Chromogenic assays were performed in Costar "V" Vinyl microtiter plates (Corning, NY). Spectra/Por dialysis membranes (MWCO 3.5 kD) were purchased from Spectrumlabs (Rancho Dominguez, Calif). Bolt 4% to 12% Bis-Tris Plus Gels, 3-(N-morpholino)propanesulfonic acid (MOPS) buffer, Alexa Fluor 680 Donkey anti-sheep IgG, EZ-Link Sulfo-NHS-LC-Biotin, and One-shot TOP10 Chemically Competent E coli were from Life Technologies (Carlsbad, Calif). Immobilon-FL and ethanol were from Merck-Millipore (Amsterdam, The Netherlands). Polyclonal affinity-purified HK antibody was from Affinity Biologicals (Ancaster, Ontario, Canada). Peroxidase-conjugated polyclonal rabbit anti-sheep and anti-mouse antibodies were from Dako (Heverlee, Belgium). HK, cHK, and β-FXIIa were from Enzyme Research Laboratories (South Bend, Ind). HK-immunodepleted plasma was from Stago BNL (Leiden, The Netherlands). Odyssey Blocking Reagent was from LI-COR (Hamburg, Germany). 3,3′,5,5′-Tetramethyl-benzidine was from Tebu Bio (Heerhugowaard, The Netherlands). Streptavidin-poly-horseradish peroxidase (HRP) was from Sanquin Blood Supply (Division Reagents, Amsterdam, The Netherlands). Phe-Pro-Arg-chloromethylketone (PPACK) was purchased from Haematologic Technologies (Essex Junction, Vt). Ampicillin was obtained from Carl Roth GmbH (Karlsruhe, Germany). 2x Yeast Tryptone capsules were from MP Biomedicals (Santa Ana, Calif). H-D-Pro-Phe-Arg-pNA (L-2120) was from Bachem (Bubendorf, Switzerland). Tris-HCl was from Roche (Woerden, The Netherlands). A phage-display library of Llama glama variable fragments of heavy-chain–only antibody fragments against cHK was obtained by immunization of 2 Llamas, with human cHK and generation of a phage-display library as previously described by de Maat et al.E4De Maat S. van Dooremalen S. de Groot P.G. Maas C. A nanobody-based method for tracking factor XII activation in plasma.Thromb Haemost. 2013; 110: 458-468Crossref PubMed Scopus (37) Google Scholar Animal experiments were approved by the ethical committee of the University of Utrecht, in compliance with European guidelines for animal research. Phagemid vector pUR8100 containing an myc-tag, a his-tag, ampicillin resistance, and an lacI repressor gene was used. TG1 E coli were used for the production of phages and Top10 E coli for the production of VHHs. cHK and HK were biotinylated using EZ-Link Sulfo-NHS-LC-Biotin according to the manufacturer's instructions. A polysorp microtiter plate was coated with 5 μg/mL NeutrAvidin protein in PBS, pH 7.4, overnight at 4°C and blocked with filtered 1% skimmed milk powder and 0.1% Tween-20 (w/v) in PBS (mPBSt), pH 7.4. Next, 500 ng/mL biotinylated cHK in mPBSt, pH 7.4, was incubated on the NeutrAvidin-coated plate for 1 hour at room temperature (RT). Isolated phages were blocked with mPBSt, pH 7.4, for 1 hour and incubated on the plate. After washing thoroughly with PBSt, pH 7.4, cHK-bound phages were eluted with 0.1 mol trimethylamine and added to 1 mol Tris-HCl. TG1 E coli were infected using the cHK-selected phage-pool, and single colonies were obtained to produce monoclonal VHHs. Culture supernatants containing monoclonal cHK VHHs were incubated on a NeutrAvidin-coated plate containing 500 ng/mL biotin-immobilized cHK or HK for 1 hour. After washing, bound VHHs were detected via myc-tag using anti-myc antibodies. VHHs with the highest specificity toward cHK compared with HK were selected for production and purification. VHHs were produced and purified as described previously.E4De Maat S. van Dooremalen S. de Groot P.G. Maas C. A nanobody-based method for tracking factor XII activation in plasma.Thromb Haemost. 2013; 110: 458-468Crossref PubMed Scopus (37) Google Scholar In brief, selected VHHs were produced in 2xYT medium, and production was induced with 0.1 mol isopropyl β-D-1-thiogalactopyranoside. Cell pellets of the production cultures were lysed by freeze-thawing. Talon beads, binding the VHH his-tag, were added to the PBS suspension of the cell lysate. Talon bead–bound VHH was eluted by 150 mM imidazole in PBS, pH 7.4, and dialyzed overnight in PBS. In selection studies, 9 selected VHHs were coated at a concentration of 5 μg/mL in PBS (pH 7.4) on maxisorp plates overnight (o/n) at 4°C. Plates were blocked with 200 μL PBS supplemented with 1% (w/v) skimmed milk powder and 0.1% (w/v) Tween-20, pH 7.4, for 1 hour at RT, and incubated with 250 ng/mL cHK or HK for 1 hour at RT. After washing, bound cHK was detected with polyclonal sheep anti-human HK IgG, and polyclonal rabbit antisheep HRP-conjugated IgG. PBS consisted of 137 mM NaCl, 2.7 mM KCl, 9.2 mM Na2HPO4, and 1.76 mM KH2PO4, pH 7.4. A second type of PBS with lower pH consisted of 127.9 mM NaCl, 6.2 mM Na2HPO4, and 3.7 mM NaH2PO4, pH 7.0. PBSt was supplemented with 0.1% (w/v) Tween-20. mPBST was PBSt with 1%, w/v, skimmed milk powder filtered with a 0.45-μm filter before use. Inhibitor mix was mPBSt containing 200 μM PPACK and 100 KIU/mL aprotinin. Diluted inhibitor mix was inhibitor mix containing 20 μM PPACK and 10 KIU/mL aprotinin. VHH-D1 was coated on 96-well maxisorp plates at 5 μg/mL in 50 μL PBS (pH 7.4) per well o/n at 4°C. During all the following steps, the plates were incubated on a plate shaker (600 rpm) at RT. First, plates were blocked with 200 μL mPBSt, pH 7.0, for 1 hour. Samples were prepared by dilution in inhibitor mix, pH 7.0, whereafter DXS (0.5 μg/mL final concentration; 5 μL DXS solution was added to 200 μL sample) was added to the inhibitor mix to enhance (c)HK binding to the plates. After 10 minutes incubation and mixing, 50 μL sample per well was added. Plates were incubated for 1 hour at RT, and washed 3 times with 200 μL PBSt, pH 7.0. Next, 50 μL 2 μg/mL biotinylated VHH-H4 was added in diluted inhibitor mix, pH 7.0, for 1 hour, and plates were washed 3 times with 200 μL PBSt, pH 7.0. Next, 50 μL 0.5 μg/mL streptavidin poly-HRP was added in diluted inhibitor mix, pH 7.0, for 1 hour, and plates were washed 3 times with 200 μL PBSt, pH 7.0. 3,3′,5,5′-Tetramethyl-benzidine substrate conversion (100 μL) was allowed up to 10 minutes and stopped by adding 0.3 mol H2SO4 and analyzed with a microplate reader at 450 nm (Versamax, Molecular Devices, Sunnyvale, Calif). Samples tested in the assay included 250 ng/mL purified cHK or HK in diluted inhibitor mix, pH 7.0, as well as plasma samples in inhibitor mix. For some experiments, NPP was activated with 1 μg/mL βFXIIa. Before activation both NPP and activators were prewarmed to 37°C. To stop activation, NPP was diluted 1:32, 1:64, or 1:128 in inhibitor mix, pH 7.0. Calibration standards were made by activating NPP with 1 μg/mL βFXIIa for 6 minutes. Western blotting of HK confirmed that HK was fully cleaved under these conditions. Activated NPP was mixed with NPP at various ratios to obtain a calibration standard. NPP was activated with βFXIIa as described above and sampled 1:40 in reducing sample buffer (15.5% glycerol, 96.8 mM Tris-HCl, 3.1% SDS, 0.003% bromophenol blue, and 25 mM DTT). Samples were incubated for 10 minutes at 100°C, and 4 μL sample per lane was run on a 4% to 12% Bis-Tris gradient gel in 1X MOPS buffer. For purified HK and cHK, 7 ng per lane was loaded (reduced). Proteins were transferred onto Immobilon-FL membranes. After blocking with Odyssey Blocking Reagent, membranes were incubated with polyclonal sheep anti-human HK IgG (1:4000, reacts with light-chain HK) in Odyssey Blocking Reagent and, after washing with tris buffered saline (50 mM Tris, 150 mM NaCl, pH = 7.4, TBS) 1% Tween-20 (w/v), with Alexa 680 donkey anti-sheep IgG (1:5000). Membranes were scanned using infrared fluorescence detection system (Odyssey). LI-COR Odyssey V3.0 software was used to quantify signal density. To quantify HK cleavage, the density of the 120-kD band of noncleaved HK was compared with that of the 50- and 40-kD fragment bands of cHK. Density of the fragments in fully contact-activated plasma was generally lower than the density of the 120-kD zymogen band. To analyze the relative amount of fragment present compared with zymogen, fragment density was multiplied by (density zymogen in plasma/density fragments in fully contact-activated plasma). The kininogen content captured by VHH-D1 on maxisorp plates was analyzed by first incubating the sample on VHH-D1–coated plates as described above; only in this case 500 μL instead of 200 μL per condition was prepared. Eight wells per condition were incubated for 1 hour; after washing thoroughly, 40 μL sample buffer containing 25 mM DTT was added and transferred from well to well; to concentrate the kininogen content in the sample buffer added, 15 μL per condition, per lane, was loaded on gel. To analyze kininogen cleavage in NPP in PBSt, pH 7.0, buffer in the presence or absence of 200 μM PPACK and 100 KIU/mL aprotinin, NPP was diluted 1:32 and incubated at 37°C for 60 minutes. Next, the samples were diluted one-third in sample buffer containing DTT, and 15 μL per lane was loaded on gel. Western blotting of healthy control and patients with C1INH-HAE was performed under nonreducing conditions as described by Suffritti et al.E3Suffritti C. Zanichelli A. Maggioni L. Bonanni E. Cugno M. Cicardi M. High-molecular-weight kininogen cleavage correlates with disease states in the bradykinin-mediated angioedema due to hereditary C1-inhibitor deficiency.Clin Exp Allergy. 2014; 44: 1503-1514Crossref PubMed Scopus (68) Google Scholar NPP was diluted 1:32 in mPBSt, pH 7.0, or inhibitor mix, pH 7.0, and DXS with a final concentration of 0.5 μg/mL was added. After 30 minutes, chromogenic substrate H-D-Pro-Phe-Arg-pNA (0.5 mM final concentration) was added and substrate conversion was measured with a microplate reader at 405 nm (Versamax, Molecular Devices). C1-INH antigen levels and functionality and C4 and C1q levels were determined in plasma from C1INH-HAE patient remission samples and healthy controls of the validation cohort as described before.E3Suffritti C. Zanichelli A. Maggioni L. Bonanni E. Cugno M. Cicardi M. High-molecular-weight kininogen cleavage correlates with disease states in the bradykinin-mediated angioedema due to hereditary C1-inhibitor deficiency.Clin Exp Allergy. 2014; 44: 1503-1514Crossref PubMed Scopus (68) Google Scholar GraphPad Prism 7 software (GraphPad Software, La Jolla, Calif) was used for data analysis. D'Agostino's K2 test was used to determine sample distribution. Mann-Whitney t test was used to compare groups of technical replicates. Kruskal-Wallis test was used for difference in cHK levels per disease state and intercohort differences corrected for multiple testing with Dunn's multiple comparison's test. Wilcoxon-paired rank test was used for comparison of disease state within 1 patient and sample collection in citrated tubes and inhibitor tubes obtained at the same time. Correlations were analyzed using Spearman r test. Standards were included on each plate and plotted in GraphPad Prism 7.0 software (GraphPad Software) using a sigmoidal 4PL fit model to which sample values were related. Baseline levels of cHK in NPP were extrapolated from the OD of NPP and OD of NPP mixed with set amounts of activated NPP (0%-15%) of 3 individual experiments. Before extrapolation, graphs of the experiments were controlled for linearity using GraphPad software (GraphPad Software; R2: 0.95, 0.99, and 0.97, respectively). Next, the following calculation was used:BaselinecHK=ODNPP/(ODNPP−ODx%activatedNPP/x%). The determined baseline cHK level of 5.2% ± 1.1% represents the mean and SD of 21 samples.Fig E2Cleaved high molecular weight kininogen (cHK) detection in plasma between the range of 20% and 100%. Plasma samples with increasing levels of cHK were prepared by mixing NPP with fully activated NPP in the presence of enzyme inhibitors. A, Immunoassay (1:128 dilution). B, Corresponding Western blot in the same sample range (reduced). Data represent mean ± SD of 3 separate experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig E3Assembly of (cleaved) high molecular weight kininogen on dextran sulfate improves assay sensitivity. A, Western blot (reduced) of "input" NPP and products that are captured by VhH-D1. B, Densitometric quantification of HK and light-chain fragments (normalized for "input" signals). C, Detection of cHK by immunoassay (1:64 dilution) in NPP, or kininogen-depleted plasma (ΔHK). When cHK levels are low, HK acts as a signal amplifier. Data represent mean ± SD of 3 separate experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig E4cHK detection in C1-INH-HAE patient plasma samples with hexadimethrine bromide. Data points represent patient or control plasma sample, lines indicate medians. Samples were analyzed at 1:32 dilution, in the presence of 8 μg/mL DXS, to overcome hexadimetrine bromide. Kruskal-Wallis test was used for multiple comparison.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig E5Correlation of cHK levels with C1-INH activity, complement parameters, and yearly angioedema attack frequency. Comparison of cHK detection by immunoassay or Western blot (nonreduced). Plasma cHK levels of patients with C1-INH-HAE or healthy controls are plotted against C1-INH function (A), C1-INH antigen level (B), C4 (C) or C1q (levels expressed as a percentage of normal values) (D), yearly angioedema attack frequency (E), and previously determined cHK levels previously analyzed by Western blot in the Italian cohort (F).E3Suffritti C. Zanichelli A. Maggioni L. Bonanni E. Cugno M. Cicardi M. High-molecular-weight kininogen cleavage correlates with disease states in the bradykinin-mediated angioedema due to hereditary C1-inhibitor deficiency.Clin Exp Allergy. 2014; 44: 1503-1514Crossref PubMed Scopus (68) Google Scholar Correlation was calculated with Spearman r.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table E1Sensitivity, specificity, and positive predictive value of the cHK immunoassay per cohortHungarian cohortItalian cohortRemissionAttackRemissionAttackSensitivity54%83%53%93%Specificity98%98%100%100%Positive predictive value98%96%100%100%Cutoff values used: 97th percentile values of healthy control samples were used, which were 14.7% cHK for the Hungarian cohort and 17.7% for the Italian cohort. Open table in a new tab Cutoff values used: 97th percentile values of healthy control samples were used, which were 14.7% cHK for the Hungarian cohort and 17.7% for the Italian cohort.
Referência(s)