Detection of Neprilysin-Derived BNP Fragments in the Circulation: Possible Insights for Targeted Neprilysin Inhibition Therapy for Heart Failure.

BACKGROUND: Entresto™ is a new heart failure (HF) therapy that includes the neprilysin (NEP) inhibitor sacubitril. One of the NEP substrates is B-type natriuretic peptide (BNP); its augmentation by NEP inhibition is considered as a possible mechanism for the positive effects of Entresto. We hypothesized that the circulating products of BNP proteolysis by NEP might reflect NEP impact on the metabolism of active BNP. We suggest that NEP-based BNP cleavage at position 17-18 results in BNP ring opening and formation of a novel epitope with C-terminal Arg-17 (BNP-neo17 form). In this study, we use a specific immunoassay to explore BNP-neo17 in a rat model and HF patient plasma. METHODS: We injected BNP into rats, with or without NEP inhibition with sacubitril. BNP-neo17 in plasma samples at different time points was measured with a specific immunoassay with neglectable cross-reactivity to intact forms. BNP-neo17 and total BNP were measured in EDTA plasma samples of HF patients. RESULTS: BNP-neo17 generation in rat circulation was prevented by NEP inhibition. The maximum 13.2-fold difference in BNP-neo17 concentrations with and without sacubitril was observed at 2 min after injection. BNP-neo17 concentrations in 32 HF patient EDTA plasma samples ranged from 0 to 37 pg/mL (median, 5.4; interquartile range, 0-9.1). BNP-neo17/total BNP had no correlation with total BNP concentration (with r = -0.175, P = 0.680) and showed variability among individuals. CONCLUSIONS: BNP-neo17 formation is NEP dependent. Considering that BNP-neo17 is generated from the active form of BNP by NEP, we speculate that BNP-neo17 may reflect both the NEP activity and natriuretic potential and serve for HF therapy guidance.

Full-Size and Partially Truncated Cardiac Troponin Complexes in the Blood of Patients with Acute Myocardial Infarction.

BACKGROUND: The measurement of cardiac isoforms of troponin I (cTnI) and troponin T (cTnT) is widely used for the diagnosis of acute myocardial infarction (AMI). However, there are conflicting data regarding what forms of cTnI and cTnT are present in the blood of AMI patients. We investigated cTnI and cTnT as components of troponin complexes in the blood of AMI patients. METHODS: Gel filtration techniques, sandwich fluoroimmunoassays, and Western blotting were used. RESULTS: Plasma samples from patients with AMI contained the following troponin complexes: (a) a cTnI-cTnT-TnC complex (ITC) composed of full-size cTnT of 37 kDa or its 29-kDa fragment and full-size cTnI of 29 kDa or its 27-kDa fragments; (b) ITC with lower molecular weight (LMW-ITC) in which cTnT was truncated to the 14-kDa C-terminal fragments; and (c) a binary cTnI-cTnC complex composed of truncated cTnI of approximately 14 kDa. During the progression of the disease, the amount of ITC in AMI samples decreased, whereas the amounts of LMW-ITC and short 16- to 20-kDa cTnT central fragments increased. Almost all full-size cTnT and a 29-kDa cTnT fragment in AMI plasma samples were the components of ITC. No free full-size cTnT was found in AMI plasma samples. Only 16- to 27-kDa central fragments of cTnT were present in a free form in patient blood. CONCLUSIONS: A ternary troponin complex exists in 2 forms in the blood of patients with AMI: full-size ITC and LMW-ITC. The binary cTnI-cTnC complex and free cTnT fragments are also present in patient blood.

Monoclonal antibodies with equal specificity to D-dimer and high-molecular-weight fibrin degradation products.

Fibrin degradation results in the formation of fibrin degradation products (FDPs) of different molecular weights, which include D-dimer. Commercial D-dimer assays recognize multiple forms of FDP with different specificity. As a result, the absence of an international D-dimer standard and the marked discrepancy in the D-dimer values in the same samples measured by assays from different manufacturers have become the primary problems that clinicians face in the D-dimer determination. We consider that an assay with equal specificity to all FDP forms regardless of their molecular weights could help to solve these problems. We aimed to produce mAbs that could equally recognize high-molecular-weight FDP (HMW FDP) and D-dimer. mAbs against D-dimer were produced. The HMW FDP/D-dimer ratios in plasma samples were analyzed following protein separation by gel filtration using the developed fluoroimmunoassay. A sandwich immunoassay with equal specificity to HMW FDP and D-dimer was developed and applied to determine HMW FDP/D-dimer ratios in patients with different diseases. Although the HMW FDP levels prevailed in thrombotic patients, the FDP and D-dimer levels were comparable in septic patients. Meanwhile, the D-dimer levels often exceeded the HMW FDP levels in patients who had undergone surgery. The 'D-dimer' levels that were detected by different assays also varied greatly depending on the assay specificities to FDP and D-dimer. Our findings show that the introduction of assays with equal specificities to FDP and D-dimer in clinical practice is a possible way of standardizing D-dimer measurements.

Processing of pro-brain natriuretic peptide is suppressed by O-glycosylation in the region close to the cleavage site.

BACKGROUND: Processing of the brain natriuretic peptide (BNP) precursor, proBNP, is a convertase-dependent reaction that produces 2 molecules--the active BNP hormone and the N-terminal part of proBNP (NT-proBNP). Although proBNP was first described more than 15 years ago, very little is known about the cellular mechanism of its processing. The study of proBNP processing mechanisms is important, because processing impairments could be associated with the development of heart failure (HF). METHODS: The biochemical properties of recombinant proBNP and NT-proBNP and the same molecules derived from the blood of HF patients were analyzed by gel-filtration chromatography, site-directed mutagenesis, and different immunochemical methods with a panel of monoclonal antibodies (MAbs). RESULTS: Part of the proBNP molecule (amino acid residues 61-76) located near the cleavage site was inaccessible to specific MAbs because of the presence of O-glycans, whereas the same region in NT-proBNP was completely accessible. We demonstrated that a convertase (furin) could effectively cleave deglycosylated (but not intact) proBNP. Of several mutant proBNP forms produced in a HEK 293 cell line, only the T71A variant was effectively processed in the cell. CONCLUSIONS: Only proBNP that was not glycosylated in the region of the cleavage site could effectively be processed into BNP and NT-proBNP. Site-directed mutagenesis enabled us to ascertain the unique suppressing role of T71-bound O-glycan in proBNP processing.