High postclopidogrel platelet reactivity in non-ST-elevation acute coronary syndrome treated with stenting: a clue for adverse prognosis?
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Acute Disease↗
Biomedical subjects
M Galvani
Publications and source records attributed to M Galvani.
Cardiac markers and risk stratification: an integrated approach.
Risk stratification of patients with acute coronary syndromes (ACS) is pivotal for correct allocation of health resources and for maximizing the benefit of available treatment modalities. However, clinical and electrocardiographic indicators of high risk lack sufficient sensitivity for the detection of major cardiac events. The complementary information provided by the measurement of different biomarkers is believed to be very useful. Specifically, elevations of cardiac troponin I (cTnI) and T (cTnT) are strongly associated with a high-risk profile both at short- and long-term. This has been definitely demonstrated in many studies as well as in cumulative meta-analysis. The role of different biomarkers, such as those reflecting activation of hemostasis and the presence of inflammation, is however less defined. At the moment, no study has prospectively evaluated these biomarkers in the whole spectrum of unselected patients with ACS. It is also unclear whether these biomarkers add independent prognostic value to the clinical and electrocardiographic indicators of adverse outcome and whether they offer additional information when compared to each other. The Early Prognostic Value of Biochemical Markers of Myocardial Damage, Activation of Hemostatic Mechanism and Inflammation in Acute Ischemic Syndromes (EMAI) study has been prospectively designed to solve these issues. In this study, we have evaluated the prognostic value of cTnI and cTnT, D-dimer, prothrombin fragment 1+2 (F1+2), thrombin-antithrombin complex (TAT) and C-reactive protein (CRP) in patients with ACS at the time of admission. We have enrolled in 31 Italian Coronary Care Units 1971 patients with rest anginal pain within 12 h from admission and electrocardiographic evidence of myocardial ischemia. Of these, 730 patients resulted to have ST-segment elevation myocardial infarction eligible for a reperfusion strategy and 1241, an acute coronary syndrome without persisting ST-segment elevation. Primary outcome measure of the study is the composite of death and non-fatal MI within 30 days from admission, which has occurred in 8.9% of the study population.
Animals↗
Prognostic role of hemostatic markers in acute coronary syndromes patients.
Coronary thrombosis is an important determinant of prognosis in patients with acute coronary syndromes (ACS). However, the identification of patients at high-risk for progression of coronary thrombosis is difficult in part because we currently lack clinically meaningful laboratory methods for its detection. The most promising approaches involve the measurement in plasma of markers of fibrin formation and degradation. Thrombin activity, as reflected by plasma or urine concentrations of fibrinopeptide A, is increased in patients with ACS and is associated with adverse outcome. However, the use of fibrinopeptide A as a marker of fibrin formation is limited by the very short half-life of the compound, by artifact due to sample acquisition, and by extremely long turnaround times. To overcome these limitations, measurement of soluble fibrin has been proposed. We have recently explored the prognostic value of a new fibrin-specific ELISA assay for soluble fibrin in patients with ACS and found that patients with highest levels had a twofold increased risk of early and late cardiac events. Increases in plasma concentrations of cross-linked fibrin degradation products (XL-FDPs), which reflect increased fibrin turn-over, are a marker of risk for complications of myocardial infarction. However, until recently, assays for XL-FDPs lacked specificity, because they did not distinguish between fibrin and fibrinogen degradation products. Recently, fibrin-specific ELISAs have been described and a rapid whole blood assay for D-dimer has been developed. We recently validated the prognostic value of this whole blood agglutination assay in patients with ACS. The results suggest that: (1) the detection of significant activation of the coagulation and/or fibrinolytic system may be important for rapid risk stratification of patients with ACS; (2) patients with biochemical evidence of ongoing coronary thrombosis may particularly benefit from aggressive antithrombotic strategies; (3) sequential measurements of these markers may be useful to guide antithrombotic treatment during the unstable phase of coronary artery disease.
Acute Disease↗
Investigating the reaction of a novel silica capillary coating compound with proteins/peptides by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry.
Quaternized piperazine ((N-methyl-N-omega-iodobutyl-N'-methyl)piperazine; QPzl) is a novel compound described as an ideal coating material for the silica capillaries that are commonly used for capillary zone electrophoresis. In the course of such analysis, contact between such coatings and biomolecules may result in certain modifications of the latter. To gain specific information on such potential modifications, solutions at pH 10.0 containing both QPzl and standard proteins/peptides were incubated for various periods and examined by matrix-assisted laser desorption/ionisation mass spectrometry. The reduction of the S-S bridges, denaturation in 8 M urea, the isoelectric point of the protein and the duration of the incubation had a profound influence on the investigated reaction. Analysis in reflectron mode and post source decay identified Cys as the likely site of interaction. The implications of the present measurements for proteome analysis using capillary and gel electrophoresis are discussed.
Animals↗
Protein alkylation by acrylamide, its N-substituted derivatives and cross-linkers and its relevance to proteomics: a matrix assisted laser desorption/ionization-time of flight-mass spectrometry study.
The present review highlights some important alkylation pathways of proteins, as measured by matrix assisted laser desorption/ionization-time of flight (MALDI-TOF)-mass spectrometric analysis, engendered by acrylamide and a number of its derivatives, including N-substituted acrylamides, cross-linkers and Immobilines (the acrylamido weak acids and bases used to create immobilized pH gradients). The present data are of relevance in two-dimensional maps and proteome analysis. It is shown that acrylamide can alkylate the -SH group of proteins even when engaged in disulfide bridges. An order of reactivity is obtained for a series of cross-linkers, which are shown to have an extremely reacting double bond, with the second one almost unreactive, originating "pendant, unreacted ends", which can subtract proteins migrating in a gel by covalently affixing them to it. An analogous reactivity scale is constructed also for the Immobiline chemicals, whose reactivity is shown to be linearly dependent on the pK values, the least reacting species being the acidic compounds. When analyzing real-life samples by two-dimensional (2-D) maps, like milk powders, a number of modifications can be detected by MALDI-TOF mass spectra of eluted spots, including variable phosphorylation sites (up to nine) and lactosyl moieties. If, for eluting such spots, formic acid is used, MALDI-TOF mass spectrometry (MS) reveals an incredible number of formylation sites, on Ser and Thr residues.
Acrylamide↗
Reduction and alkylation of proteins in preparation of two-dimensional map analysis: why, when, and how?
The standard procedure adopted up to the present in proteome analysis calls for just reduction prior to the isoelectric focusing/immobilized pH gradient (IEF/IPG) step, followed by a second reduction/alkylation step in between the first and second dimension, in preparation for the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) step. This protocol is far from being optimal. It is here demonstrated, by matrix assisted laser desorption/ionization-time of flight (MALDI-TOF)-mass spectrometry, that failure to reduce and alkylate proteins prior to any electrophoretic step (including the first dimension) results in a large number of spurious spots in the alkaline pH region, due to "scrambled" disulfide bridges among like and unlike chains. This series of artefactual spots comprises not only dimers, but an impressive series of oligomers (up to nonamers) in the case of simple polypeptides such as the human alpha- and beta-globin chains, which possess only one (alpha-) or two (beta-) -SH groups. As a result, misplaced spots are to be found in the resulting two-dimensional (2-D) map, if performed with the wrong protocol. The number of such artefactual spots can be impressively large. In the case of analysis of complex samples, such as human plasma, it is additionally shown that failure to alkylate proteins results in a substantial loss of spots in the alkaline gel region, possibly due to the fact that these proteins, at their pI, regenerate their disulfide bridges with concomitant formation of macroaggregates which become entangled with and trapped within the polyacrylamide gel fibers. This strongly quenches their transfer in the subsequent SDS-PAGE step.
Alkylation↗
Alkylation kinetics of proteins in preparation for two-dimensional maps: a matrix assisted laser desorption/ionization-mass spectrometry investigation.
All existing protocols for protein separation by two-dimensional (2-D) gel electrophoresis require the full reduction, denaturation, and alkylation as a precondition for an efficient and meaningful separation of such proteins. Existing literature provides a strong evidence to suggest that full reduction and denaturation can be achieved in a relatively short time; the same thing, however, can not be said for the alkylation process, which the present study shows that more than 6 h are required for a complete alkylation. We have used matrix assisted laser desorption/ionisation-time of flight-mass spectrometry (MALDI-TOF-MS) to monitor protein alkylation by iodoacetamide over the period 0-24 h at pH 9. The present, fast and specific MS method provided clear indication on the extent and speed of alkylation which reached approximately 70% in the first 2 min, yet the remaining 30% resisted complete alkylation up to 6 h. The use of sodium dodecyl sulfate (SDS) during the alkylation step resulted in a strong quenching of this reaction, whereas 2% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) exerted a much reduced inhibition. The implications of the present measurements on 2-D gel analysis in particular and proteomics in general are discussed.
Alkylation↗
Protein alkylation in the presence/absence of thiourea in proteome analysis: a matrix assisted laser desorption/ionization-time of flight-mass spectrometry investigation.
Although it is highly recommended that reduction and alkylation of free -SH groups in proteins should be performed prior to any electrophoretic step (including the first isoelectric focusing/immobilized pH gradient (IEF/IPG) dimension), it is here reported that one component of the sample solubilization cocktail adopted recently (namely thiourea) strongly quenches such alkylation process (as typically carried out with iodoacetamide, IAA). The present matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) analysis demonstrates that thiourea is an effective scavenger of IAA, since its sulfur atom reacts as efficiently as the ionized, free -SH group of Cys in proteins at alkaline pH values (pH 8.5-9.0). As a result of this reaction, free IAA is quickly depleted by thiourea, via the formation of an intermediate adduct, which is rapidly deamidated to form the cyclic compound thiazolinidone monoimine. This reaction strongly competes with the direct addition reaction of IAA onto the -SH group in proteins, resulting in poorly alkylated proteins. It is, therefore, recommended that, whenever possible and compatible with the type of sample, thiourea should be omitted from the solubilizing cocktail in proteome analysis. However, after proper sample reduction and alkylation, thiourea can be incorporated into the IEF/IPG gel, where it will have the beneficial effect of augmenting protein solubility at their pI values and scavenging the excess of free IAA.
Alkylation↗
Two-dimensional gel electrophoresis/matrix-assisted laser desorption/ionisation mass spectrometry of commercial bovine milk.
Proteins in commercial bovine milk have been separated by two-dimensional gel electrophoresis and examined by matrix-assisted laser desorption/ionisation mass spectrometry. Gel separation was conducted in two different pH gradients, 3-10 and 6-11; the latter range resulted in a higher spot resolution and favoured the basic proteins. We have limited the time-of-flight mass spectrometry analysis to the linear mode to examine the capability of reliable relative molecular masses of the intact proteins in their characterisation. The present study draws attention to the difficulty of identifying basic proteins with low molecular masses (below 12000 Da) that are commonly encountered in milk samples.
Animals↗
[Low-molecular weight heparin and acute coronary syndrome: theoretical background and its use in clinical practice].
Thrombosis is responsible for the acute manifestations of coronary artery disease. Intravenous heparin has been shown to be effective in reducing the risk of death or myocardial infarction in patients with acute coronary syndromes. Compared to standard heparin, low-molecular weight heparins (LMWHs) have improved pharmacological and pharmacokinetic properties. A number of LMWHs, such as nadroparin, dalteparin and enoxaparin, have been evaluated in patients with acute coronary syndromes. FRISC (Fragmin during Instability in Coronary Artery Disease) and FRIC (Fragmin in Unstable Coronary Artery Disease), evaluated dalteparin and found the LMWH to be more effective than aspirin alone (FRISC) and as effective as heparin in a direct comparison (FRIC). In a small trial, nadroparin was shown to significantly reduce the risk of ischemic outcomes compared with a combination of aspirin and heparin, but this effect was no longer significant in the large FRAX.I.S. trial (FRAXiparine in Ischaemic Syndrome). Enoxaparin resulted in a statistically significant reduction in the combined outcome of death, myocardial infarction and recurrent angina or of urgent revascularization when compared with heparin in the ESSENCE (Efficacy and Safety of Subcutaneous Enoxaparin in Non-Q-Wave Coronary Events) and TIMI 11B trials. Meta-analyzing of the data of these two trials revealed that even the combination of death and myocardial infarction was significantly reduced by the use of enoxaparin. There is accumulating evidence that LMWHs are safe and effective alternatives to standard heparin for the treatment of acute coronary syndromes and that they offer practical and therapeutic advantages.
Acute Disease↗
It's time for a change to a troponin standard.
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Biomarkers↗
Effect of experimental conditions on the analysis of sodium dodecyl sulphate polyacrylamide gel electrophoresis separated proteins by matrix-assisted laser desorption/ ionisation mass spectrometry.
Two mixtures of proteins having molecular weights in the range approximately 8-97 kDa were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and examined by delayed extraction matrix-assisted laser desorption/ionisation mass spectrometry (MALDI-MS). Part of our aim in this study is to gain more insight into the influence of the various experimental conditions on the overall quality of the acquired mass spectral data. Different protein extraction procedures, two staining agents, and extraction times, were among the parameters assessed. In terms of the overall quality of the acquired mass spectra and the speed of protein recovery, ultrasonic assisted passive elution, into a solvent mixture containing formic acid/acetonitrile/2-isopropanol/water, was found to be more efficient than other elution procedures. The higher resolution associated with the delayed extraction mode allowed the identification of a number of protein modifications, including multiple formylation provoked by formic acid, cysteine alkylation caused by unpolymerised acrylamide monomers, and complexation with the staining reagents. The detection of these modifications, however, was limited to proteins under 30 kDa. Analysis of a ubiquitin tryptic digest by reflectron MALDI time-of-flight (TOF) allowed reliable identification of a number of the formylation sites.
Animals↗
Electroelution and passive elution of gamma-globulins from sodium dodecyl sulphate polyacrylamide gel electrophoresis gels for matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry.
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Electrochemistry↗
Two-dimensional gel electrophoresis/matrix-assisted laser desorption/ionisation mass spectrometry of a milk powder.
Proteins in a commercial milk powder have been separated by two-dimensional gel electrophoresis and analysed by matrix-assisted laser desorption ionisation mass spectrometry. The mass spectrometric analyses were conducted in two steps: analysis of the intact proteins following their passive extraction into a suitable solvent mixture and analysis in reflectron mode of in situ digests of a number of gel spots. The combination of the two methods allowed a reliable identification of a number of proteins, including nine caseins as well as certain protein modifications including single/multiple phosphorylation, lactose-protein conjugates and Coomassie Brilliant Blue adducts. Analyses of the intact proteins prior to their in situ digestion contributed to a more efficient and reliable consultation of protein databases.
Animals↗
Probing protein unfolding through monitoring cysteine alkylation by matrix-assisted laser desorption/ionisation mass spectrometry.
Matrix-assisted laser desorption/ionisation mass spectrometry was used to monitor interaction between three proteins and two basic Immobiline chemicals (pK 10.3 and pK >12) commonly used in immobilised pH gradients (IPG). For two of the investigated proteins, the observed alkylation channels of the cysteine residues exhibited unmistakable response to their gradual denaturation following treatment with different concentrations (0-8 M) of two commonly used denaturants, urea and guanidine hydrochloride. Our assessment for protein unfolding is based on the number and relative intensity of the alkylation channels, yet the present mass spectrometry data are in good agreement with data based on optical rotatory dispersion, in which another approach was used to assess protein unfolding. Whether the present simple, fast and specific mass spectrometry method can be developed as a probe for monitoring folding/unfolding of cysteine-containing proteins can only be demonstrated by generating similar data for a larger number of proteins.
Alkylation↗
Investigating the reaction of a number of gel electrophoresis cross-linkers with beta-lactoglobulin by matrix assisted laser desorption/ionization-mass spectrometry.
A number of cross-linkers that are commonly used in polyacrylamide gels have been incubated with bovine beta-lactoglobulin B and the resulting reaction mixtures were examined by matrix assisted laser desorption/ionization-mass spectrometry. At concentrations of 0.1, 1, and 20 mM of each cross-linker incubated for 1 h with 50 pmol/microL of the protein, a reactivity scale can be expressed as polyethylene glycol diacrylate > N,N'-bisacrylylcystamine > bisacrylyl piperazine > N,N'-methylenebisacrylamide >> N,N'-diallyltartardiamide (PEGDA>BAC>BAP>Bis>>DATD). Relatively short incubation times indicated one of the five Cys residues as the target of reaction, which was confirmed by post-source decay measurements. Longer incubation times (24 h) with bisacrylamide extended the reaction to all five Cys residues and a number of Lys residues. A second consequence of longer reaction time is the involvement of both terminals of the cross-linker in the observed reaction. This experimental evidence is the first to demonstrate a different reactivity of both ends of one of the most commonly used cross-linkers. Investigation of solutions containing a cross-linker and acrylamide monomers provided useful information on the competition between the two identities for reaction with the protein. Possible implications of these experimental observations for isoelectric focusing separations in polyacrylamide gels are discussed.
Acrylamide↗
Soluble E-selectin is not a marker of unstable coronary plaque in serum of patients with ischemic heart disease.
Increased level of soluble cell adhesion molecules may be a marker for atherosclerosis and/or reflect complication of the atherosclerotic plaque. To test whether expression of cell adhesion molecules is more pronounced in unstable versus stable coronary plaques, we measured the serum level of soluble E-selectin (sE-selectin) in 99 consecutive patients admitted to the hospital for acute coronary syndromes (ACS) and in 61 patients with chronic coronary artery disease (CAD) using a commercially available ELISA kit. We also measured the sE-selectin concentration in 20 sex- and age-matched subjects without clinical evidence of atherosclerosis, who served as controls. The mean sE-selectin level was higher in both groups of patients compared with controls (ACS, 35.0 +/- 23.4 ng/mL; chronic CAD, 32.9 +/- 21.0 ng/mL; controls, 14.5 +/- 6.6 ng/mL; one-way ANOVA, P = 0.001), but there was no difference between patients with ACS and chronic CAD. Furthermore, there was a trend (P = 0.08) toward a decrease in sE-selectin with an increase in the extent and severity of CAD. In patients with ACS, the in-hospital cardiac event rate was 8%. Although mean sE-selectin concentration tended to be higher in patients with (49.2 +/- 42.1 ng/mL) than in those without (33.8 +/- 21.3 ng/mL) in-hospital cardiac events, the difference was not significant. In 53 patients with ACS, C-reactive protein was measured and showed no correlation with the sE-selectin concentration. These findings show that although sE-selectin concentration is elevated in the presence of clinically relevant atherosclerosis, it does not further increase during the unstable phase of the disease, indicating that sE-selectin is not a reliable indicator of a complicated atherosclerotic plaque.
Aged↗
Elevated cardiac troponin levels predict the risk of adverse outcome in patients with acute coronary syndromes.
BACKGROUND: Elevations of cardiac troponin T or I are predictive of adverse outcomes in patients with acute coronary syndromes. However, odds ratios (ORs) vary substantially between studies. This investigation refines these values by means of a meta-analysis. METHODS: Twenty-one studies were suitable. ORs were calculated for short-term (30 days) and long-term (5 months to 3 years) follow-up in patients with ST-segment elevation (ST upward arrow), in those without ST-segment elevation (no ST upward arrow), and in patients with unstable angina. The primary end point was a composite of death or nonfatal myocardial infarction. RESULTS: A total of 18,982 patients were included. At 30 days, the OR for death or myocardial infarction was 3.44 (95% confidence interval [CI], 2.94-4.03; P <. 00001) for patients with positive troponin. In the ST upward arrow group, troponin elevations carried a 2.86-fold (95% CI, 2.35-3.47; P <.0001) higher risk during short-term follow-up, which was maintained long term. The no-ST upward arrow patients with troponin elevations manifested a 4.93-fold (95% CI, 3.77-6.45; P <.0001) increase of adverse outcomes. The OR for patients with unstable angina and positive troponin was 9.39 (95% CI, 6.46-13.67; P <.0001). For cardiac death alone, the results were similar. CONCLUSIONS: Patients with acute coronary syndromes who have troponin elevations show a substantial increase in risk during short and long-term follow-up.
Angina, Unstable↗