Characterization of Cardiac Troponin Fragment Composition Reveals Potential for Differentiating Etiologies of Myocardial Injury.

Background: Increased cardiac troponin (cTn) concentrations occur in acute myocardial injury and chronic diseases. Characterization of cTn composition in the circulation may assist in differentiating etiologies of myocardial injury. Our goal was to study cTn composition and kinetics in patients following type 1 myocardial infraction (T1MI), cardiac procedures, and chronic heart diseases to establish the relationship between cTn composition and clinical diagnosis.

Design and Analytical Evaluation of Novel Cardiac Troponin Assays Targeting Multiple Forms of the Cardiac Troponin I-Cardiac Troponin T-Troponin C Complex and Fragmentation Forms.

Background: Current studies suggest that cardiac troponin (cTn) forms in the circulation may vary in different clinical scenarios. Our aim was to design a combination of cTn assays specific to the main cTn forms and to evaluate their analytical performance.

Methods: We developed immunoassays specific for measuring (1) long-cTnT cTnI-cTnT-TnC (ITC) ternary complex, with cTnT in long form without cleavage at the C-terminal amino acids residue 189-223, designated "long-cTnT ITC complex assay;" (2) both the long-cTnT ITC complex plus short-cTnT ITC complex, designated "hs-total ITC complex assay;" (3) the central part of cTnT of both the long-cTnT ITC complex and free cTnT, designated "hs-cTnT assay.

Specific cleavage of IGFBP-4 by papp-a in nervous tissue.

Astrocytes are subtypes of glial cells involved in metabolic, structural, homeostatic, and neuroprotective processes that help neurons maintain viability. Insulin-like growth factors IGF-1 and IGF-2 are known to have neuroprotective effects on neurons and glial cells through interaction with specific receptors. IGF forms a complex with IGF-binding proteins (IGFBP) in nervous tissue and is released from the complex via IGFBP proteolysis by specific proteases.

Influence of Anticoagulants on the Dissociation of Cardiac Troponin Complex in Blood Samples.

Immunodetection of cardiac isoforms of troponin I (cTnI) and troponin T (cTnT) in blood samples is widely used for the diagnosis of acute myocardial infarction. The cardiac troponin complex (ITC-complex), comprising cTnI, cTnT, and troponin C (TnC), makes up a large portion of troponins released into the bloodstream after the necrosis of cardiomyocytes. However, the stability of the ITC-complex has not been fully investigated.

Interaction of heparin with human cardiac troponin complex and its influence on the immunodetection of troponins in human blood samples.

Objectives: Heparin is a highly charged polysaccharide used as an anticoagulant to prevent blood coagulation in patients with presumed myocardial infarction and to prepare heparin plasma samples for laboratory tests. There are conflicting data regarding the effects of heparin on the measurement of cardiac isoforms of troponin I (cTnI) and troponin T (cTnT), which are used for the immunodiagnosis of acute myocardial infarction. In this study, we investigated the influence of heparin on the immunodetection of human cardiac troponins.

Redox differences between rat neonatal and adult cardiomyocytes under hypoxia.

It is generally accepted that oxidative stress plays a key role in the development of ischemia-reperfusion injury in ischemic heart disease. However, the mechanisms how reactive oxygen species trigger cellular damage are not fully understood. Our study investigates redox state and highly reactive substances within neonatal and adult cardiomyocytes under hypoxia conditions.

PAPP-A-Specific IGFBP-4 Proteolysis in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

The insulin-like growth factors IGF-I and IGF-II-as well as their binding proteins (IGFBPs), which regulate their bioavailability-are involved in many pathological and physiological processes in cardiac tissue. Pregnancy-associated plasma protein A (PAPP-A) is a metalloprotease that preferentially cleaves IGFBP-4, releasing IGF and activating its biological activity. Previous studies have shown that PAPP-A-specific IGFBP-4 proteolysis is involved in the pathogenesis of cardiovascular diseases, such as ischemia, heart failure, and acute coronary syndrome.

The Influence of the Nucleotide Composition of Genes and Gene Regulatory Elements on the Efficiency of Protein Expression in Escherichia coli.

Recombinant proteins expressed in Escherichia coli are widely used in biochemical research and industrial processes. At the same time, achieving higher protein expression levels and correct protein folding still remains the key problem, since optimization of nutrient media, growth conditions, and methods for induction of protein synthesis do not always lead to the desired result. Often, low protein expression is determined by the sequences of the expressed genes and their regulatory regions.

IGF-Binding Proteins and Their Proteolysis as a Mechanism of Regulated IGF Release in the Nervous Tissue.

Insulin-like growth factors 1 and 2 (IGF-1 and IGF-2) play a key role in the maintenance of the nervous tissue viability. IGF-1 and IGF-2 exhibit the neuroprotective effects by stimulating migration and proliferation of nervous cells, activating cellular metabolism, inducing regeneration of damaged cells, and regulating various stages of prenatal and postnatal development of the nervous system. The availability of IGFs for the cells is controlled via their interaction with the IGF-binding proteins (IGFBPs) that inhibit their activity.

Fragmentation of human cardiac troponin T after acute myocardial infarction.

Background: Blood measurement of cardiac troponin T (cTnT) is one of the most widespread methods of acute myocardial infarction (MI) diagnosis. cTnT degradation may have a significant influence on the precision of cTnT immunodetection; however, there are no consistent data describing the level and sites of cTnT proteolysis in the blood of MI patients. In this study, we bordered major cTnT fragments and quantified their relative abundance in the blood at different times after MI.

IGFBP-4 Proteolysis by PAPP-A in a Primary Culture of Rat Neonatal Cardiomyocytes under Normal and Hypertrophic Conditions.

Cardiovascular diseases (CVD) are among the leading causes of death and disability worldwide. Pregnancy-associated plasma protein-A (PAPP-A) is a matrix metalloprotease localized on the cell surface. One of the substrates that PAPP-A cleaves is the insulin-like growth factor binding protein-4 (IGFBP-4), a member of the family of proteins that bind insulin-like growth factor (IGF).

Myocardial Injury and the Release of Troponins I and T in the Blood of Patients.

Background: Cardiac troponin I (cTnI) and cTnT are the established biomarkers of cardiomyocyte damage and the recommended biomarkers for the diagnosis of acute myocardial infarction (MI). High-sensitivity immunochemical diagnostic systems are able to measure the cTn concentrations in the blood of a majority of healthy people. At the same time, the concentration of cTn may be increased not only after MI but also because of other pathologies that might affect myocardium.

CT-IGFBP-4 as a novel prognostic biomarker in acute heart failure.

Aims: Insulin-like growth factor binding protein-4 (IGFBP-4) fragments have been shown to predict the risk of major adverse cardiovascular events, including segment-elevation myocardial infarction, in patients with acute coronary syndrome. We evaluated the prognostic value of the carboxy-terminal fragment of IGFBP-4 (CT-IGFBP-4) for all-cause mortality in emergency room patients with acute heart failure (AHF).

Methods And Results: CT-IGFBP-4, N-terminal pro brain natriuretic peptide (NT-proBNP), and C-reactive protein (CRP) were measured at admission from the lithium-heparin plasma of 156 patients with AHF.

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.

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.

Full-Size Cardiac Troponin I and Its Proteolytic Fragments in Blood of Patients with Acute Myocardial Infarction: Antibody Selection for Assay Development.

Background: In the blood of patients with acute myocardial infarction (AMI), cardiac troponin I (cTnI) presents as an intact molecule with a repertoire of proteolytic fragments. The degradation of cTnI might negatively influence its precise immunodetection. In this study we identified cTnI fragments and calculated their ratio in the blood of patients at different times after AMI to discriminate the most stable part(s) of cTnI.

Glycosylated and non-glycosylated NT-IGFBP-4 in circulation of acute coronary syndrome patients.

Background: N-terminal and C-terminal proteolytic fragments of IGF binding protein 4 (NT-IGFBP-4 and CT-IGFBP-4) were recently shown to predict adverse cardiac events in acute coronary syndrome (ACS) patients. NT-IGFBP-4 and CT-IGFBP-4 are products of the pregnancy-associated plasma protein-A (PAPP-A)-mediated cleavage of IGFBP-4. It has been demonstrated that circulating IGFBP-4 is partially glycosylated in its N-terminal region, although the influence of this glycosylation on PAPP-A-mediated proteolysis and the ratio of glycosylated/non-glycosylated IGFBP-4 fragments in human blood remain unrevealed.

Thrombin-Mediated Degradation of Human Cardiac Troponin T.

Background: Cardiac troponin T (cTnT) is an acknowledged biomarker of acute myocardial infarction (AMI) that is known to be prone to proteolytic degradation in serum. Such degradation is usually explained by the action of μ-calpain, although there could be other candidates for that role. In the current study, we explored the hypothesis that thrombin-mediated cTnT cleavage occurs as a result of the serum sample preparation.

Searching for a BNP standard: Glycosylated proBNP as a common calibrator enables improved comparability of commercial BNP immunoassays.

Background: Circulating B-type natriuretic peptide (BNP) is widely accepted as a diagnostic and risk assessment biomarker of cardiac function. Studies suggest that there are significant differences in measured concentrations among different commercial BNP immunoassays. The purpose of our study was to compare BNP-related proteins to determine a form that could be used as a common calibrator to improve the comparability of commercial BNP immunoassay results.

Anti-Cardiac Troponin Autoantibodies Are Specific to the Conformational Epitopes Formed by Cardiac Troponin I and Troponin T in the Ternary Troponin Complex.

Background: Autoantibodies to cardiac troponins (TnAAbs) could negatively affect cardiac troponin I (cTnI) measurements by TnAAbs-sensitive immunoassays. We investigated the epitope specificity of TnAAbs and its influence on cTnI immunodetection in patients with acute myocardial infarction (AMI).

Methods: The specificity of TnAAbs was studied in immunoassays and gel-filtration experiments.

Analytical Issues with Natriuretic Peptides - has this been Overly Simplified?

Natriuretic peptides (NPs) were first described as cardiac biomarkers more than two decades ago. Since that time, numerous studies have confirmed NPs' diagnostic and prognostic utilities as biomarkers of myocardial function. However, we must now admit that despite the NPs' relatively long period of use in clinical practice, our understanding of the biochemistry and the variety of circulating forms of NPs, as well as of their potential as biomarkers, remains far from being complete and comprehensive.

Different Susceptibility of B-Type Natriuretic Peptide (BNP) and BNP Precursor (proBNP) to Cleavage by Neprilysin: The N-Terminal Part Does Matter.

Background: Protease neprilysin is known to be responsible for the degradation of natriuretic peptides. A recent heart failure (HF) drug, LCZ696 (Entresto(TM)), that combines a neprilysin inhibitor and an angiotensin II receptor inhibitor was suggested to augment circulating B-type natriuretic peptide (BNP) concentrations, making the results of BNP measurements diagnostically ambiguous. Because the main form of measured BNP in HF patients is represented by its uncleaved precursor, proBNP, it is important to know the susceptibility of proBNP to cleavage by neprilysin.

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.