20 research outputs found

    A myeloperoxidase precursor, promyeloperoxidase, is present in human plasma and elevated in cardiovascular disease patients

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    Myeloperoxidase (MPO)-derived oxidants have emerged as a key contributor to tissue damage in inflammatory conditions such as cardiovascular disease. Pro-myeloperoxidase (pro-MPO), an enzymatically active precursor of myeloperoxidase (MPO), is known to be secreted from cultured bone marrow and promyelocytic leukemia cells, but evidence for the presence of pro-MPO in circulation is lacking. In the present study, we used a LC-MS/MS in addition to immunoblot analyses to show that pro-MPO is present in human blood plasma. Furthermore, we found that pro-MPO was more frequently detected in plasma from patients with myocardial infarction compared to plasma from control donors. Our study suggests that in addition to mature MPO, circulating pro-MPO may cause oxidative modifications of proteins thereby contributing to cardiovascular disease

    Effects of nitric oxide synthase inhibition on Basal function and the force-frequency relationship in the normal and failing human heart in vivo.

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    BACKGROUND: Nitric oxide (NO) exerts autocrine/paracrine effects on cardiac function, including alterations of the inotropic state. In vitro studies suggest that NO modulates the myocardial force-frequency relationship. Basal left ventricular (LV) contractility is depressed and the force-frequency relationship is blunted in human heart failure, and it is speculated that an increase in NO production is involved. METHODS AND RESULTS: We compared the effects of intracoronary NO synthase inhibition with N(G)-monomethyl-L-arginine (L-NMMA; 25 micromol/min) on basal LV function and the response to incremental atrial pacing in patients with dilated cardiomyopathy (n=11; mean age, 51 years) and in control subjects with atypical chest pain and normal cardiac function (n=7; mean age, 54 years). In controls, L-NMMA significantly reduced basal LV dP/dt(max) (from 1826 to 1578 mm Hg/s; P<0.002), but had no effect on heart rate, mean aortic pressure, or right atrial pressure. Pacing-induced increases in LV dP/dt(max) were unaltered by L-NMMA. In patients with dilated cardiomyopathy, L-NMMA had no effect on baseline LV dP/dt(max) (from 1313 to 1337 mm Hg/s; P=NS). The blunted pacing-induced rise in LV dP/dt(max) in these patients was unaltered by L-NMMA. CONCLUSION: Endogenous NO has a small baseline positive inotropic effect in the normal human heart, which is lost in heart failure patients. NO does not significantly influence the force-frequency relationship in either the normal or failing human heart in vivo. Because this study was performed in patients with moderate heart failure, whether the findings apply to subjects with more severe heart failure requires further investigation

    Detection of mature MPO and pro-MPO in plasma using affinity-purification and immunblot analysis.

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    <p><b>A)</b> Plasma was spiked with MPO standard (10 nM) and HL60 cell lysate (containing 10 nM MPO) and subjected to affinity purification. Purified samples along with MPO, pro-MPO standards and HL-60 lysates were separated by 10% SDS/PAGE, transferred to PVDF and probed with MPO-specific antibody. <b>B)</b> Neutrophils (5x10<sup>6</sup> cells/ml) were added back into plasma and then stimulated with CytB and FMLP for 30 min at 37°C. Neutrophils were centrifuged and cell free plasma MPO was subjected to affinity purification and analyzed as described in A. After the ECL fluorescence of blots was developed, a photograph of the blot showing the molecular weight markers was taken and aligned with the fluorescence image as indicated by the black line.</p

    Halogenation activity of mature MPO and recombinant pro-MPO.

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    <p>Initial rates of NADH oxidation were determined with various concentrations of A) bromide or B) chloride, 20 nM mature MPO or recombinant pro-MPO and 100 μM NADH in 50 mM phosphate buffer (pH 7.4) at 21°C. The reaction was started by adding 50 μM H<sub>2</sub>O<sub>2</sub>. The kinetics of the reaction of myeloperoxidase-generated hypobromous and hypochlorous acids with NADH were monitored by measuring the bromohydrin and chlorohydrin product at 275 nm. Data are representative of two or more experiments.</p

    LC-MS/MS analysis of mutual, pro-MPO- and MPO- specific peptides.

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    <p><b>A)</b> Location of tryptic peptides used for LC-MS/MS analysis within the MPO sequence. The <i>N-</i>terminal signal peptide is shown in dark grey, the pro-peptide in bold light grey and the sequence of mature MPO in bold black. Highlighted by the black and grey boxes are tryptic peptides specific to pro-MPO or present in both mature MPO and pro-MPO, respectively. Twenty-five μg of recombinant pro-MPO or mature MPO were digested with trypsin and analyzed by LC-MS/MS. <b>B), D) +F)</b> Extracted ion chromatograms for SRM transitions specific for the mutual, pro-MPO and mature MPO-specific peptides (MS1/MS2 551.8/272.2, 1000.9/1033.5 and 648.8/935.5, respectively, MS1 = m/z for the doubly charged precursor species and MS2 = m/z for the singly charged y–ion fragment, c–carbamidomethyl-cysteine). <b>C), E) +G)</b> CID-MS/MS spectra confirming the sequence of the respective peptide. Representative chromatograms and spectra are shown.</p

    Increased detection of pro-MPO in plasma from patients with myocardial infarction (MI) compared to healthy controls.

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    <p>Plasma was purified by affinity chromatography with MPO-specific antibodies, subjected to digestion with trypsin and analyzed for the presence of pro-MPO and mature MPO-specific peptides by SRM-based LC-MS/MS. Samples that showed a peak are denoted by “+”, those that don’t by “-”.</p

    LC-MS/MS detection of pro-MPO in affinity-purified plasma separated by SDS/PAGE.

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    <p>Plasma was purified by affinity chromatography with an MPO-specific antibody and subjected to separation by SDS/PAGE. Bands with the molecular weight of pro-MPO (90 kDa) were subjected to in-gel tryptic digestion and analyzed by LC-MS/MS using SRM for a pro-MPO-specific peptide (1000.9->1033.5). <b>(A)</b> Extracted ion chromatogram for the SRM transition for the pro-MPO specific peptide. <b>(B)</b> CID-MS/MS spectrum confirming the sequence for the pro-MPO-specific peptide SSGcAYQDVGVTcPEQDK. Representative chromatograms and spectra are shown.</p
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