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Biomedical subjects

L Piérard

Publications and source records attributed to L Piérard.

At least 19 recordsLinked to original sources
PubMed1997

Cardiac risk assessment before vascular surgery: a prospective study comparing clinical evaluation, dobutamine stress echocardiography, and dobutamine Tc-99m sestamibi tomoscintigraphy.

Preoperative evaluation for cardiac risk assessment before peripheral vascular surgery remains controversial. Between January and June 1994, a prospective open study was carried out in 156 patients scheduled for elective vascular procedures (63 carotid endarterectomies, 34 abdominal aortic aneurysms, 29 aortoiliac and 30 infrainguinal reconstructions) to compare the ability of clinical data, dobutamine stress echocardiography, and dobutamine Tc-99m sestamibi tomoscintigraphy to predict postoperative cardiac events. Pharmacological stress testing consisted of incremental dobutamine infusion (+/-1 mg atropine to achieve 85% of age-predicted maximal heart rate, with continuous echocardiographic monitoring, and injection of Tc-99m sestamibi after dobutamine infusion). Dobutamine echocardiography was abnormal in 36 patients (worsening resting wall motion abnormality in 11; new induced wall motion abnormality in 25). Dobutamine Tc-99m sestamibi tomoscintigraphy revealed a reversible perfusion defect in 34 patients, indicating the presence of myocardial ischaemia. As a result, eight patients underwent myocardial revascularization (n = 5) or the proposed operation was cancelled (n = 3). In the remaining 142 vascular procedures, there were eight (5.6%) adverse cardiac events: three myocardial infarctions (two fatal), three prolonged myocardial ischaemia, one acute congestive heart failure and one sustained ventricular arrhythmia in the post operative period. Univariate analysis selected unstable angina (relative risk (RR) 11.6), previous congestive heart failure (RR 6.4), Detsky's score of > or = 15 (RR 3.0), positive dobutamine stress echocardiography (RR 3.7), and positive dobutamine tomoscintigraphy (RR 7.4) as significant predictors of postoperative cardiac events. In patients without clinical markers of coronary artery disease (n = 66), non-invasive cardiac testing did not predict cardiac complications (n = 2; one prolonged myocardial ischaemia; one infarction). In the subset of 76 patients with definite clinical or electrocardiographic evidence of ischaemic heart disease, dobutamine stress testing provided additional information, and optimized risk stratification: five of six patients who suffered a cardiac complication had a pathologic dobutamine stress test. Furthermore, a negative dobutamine stress test was characterized by a high negative predictive value (0.96 for echocardiography; 0.97 for tomoscintigraphy). The study further demonstrated that the cardiac response (ischaemic versus non-ischaemic) to dobutamine stress was concordantly classified by echocardiographic and tomoscintigraphic techniques in 96% of cases. It is concluded that complementary non-invasive cardiac stress testing by dobutamine is indicated only in patients with clinically apparent coronary artery disease.

Aged↗
PubMed1991

Structural and biological properties of human recombinant myeloperoxidase produced by Chinese hamster ovary cell lines.

The cDNA encoding human myeloperoxidase carries three ATG codons in frame; 144, 111 and 66 bp upstream from the proprotein DNA sequence. In order to determine the most efficient signal sequence, three cDNA modules starting at each of the ATG were cloned into an eucaryotic expression vector and stably expressed in Chinese hamster ovary cell lines. In all three cases, recombinant human myeloperoxidase (recMPO) was secreted into the culture medium of transfected cells, indicating that each of the signal peptides functions efficiently. One of the recombinant cell lines, which was amplified using methotrexate, overexpresses enzymatically active recMPO up to 6 micrograms.ml-1.day-1. The recombinant product was purified by a combination of ion-exchange and metal-chelate chromatography, and characterized in terms of molecular mass, amino-terminal amino acid analysis, glycosylation, physicochemical properties and biological activity. The data show that recMPO is secreted essentially as a monomeric, heme-containing, single-chain precursor of 84 kDa which exhibits peroxidase activity. Amino-terminal analysis indicated that cleavage of the signal peptide occurs between amino acids 48 and 49. In addition, recMPO appeared to be glycosylated up to the last stage of sialylation, to an extent similar to that of the natural enzyme. Specific activity measurements as well as stability data, in various pH, temperature, ionic strength and reducing conditions, indicated that the recombinant single-chain enzyme behaves essentially in the same way as the natural two-chain molecule. Finally, recMPO was shown to exert potent cytotoxicity towards Escherichia coli when provided with its physiological substrates, i.e. hydrogen peroxide and chloride ions.

Amino Acid Sequence↗
PubMed1990

Development of new thrombolytic agents using recombinant DNA technology.

The increasing incidence of thromboembolic diseases has sustained the search for new agents able to stimulate the natural fibrinolytic system. The first generation of antithrombotic agents include bacterial streptokinase and human urine urokinase. Because these molecules lack specificity for the fibrin clot, important efforts have been made to produce, using recombinant DNA technology, agents presenting higher fibrin clot selectivity such as t-PA (tissue-type plasminogen activator) and scu-PA (single chain urokinase-type plasminogen activator). In parallel, several laboratories are presently attempting to create mutants and hybrids plasminogen activators displaying improved thrombolytic properties with respect to the natural molecules. In this paper, we describe briefly the mechanisms of fibrinolysis and the role of the different natural thrombolytic agents. In addition, we review the possibilities of genetic engineering for the production of natural and novel plasminogen activators.

Amino Acid Sequence↗
PubMed1989

Production in eukaryotic cells and characterization of four hybrids of tissue-type and urokinase-type plasminogen activators.

To investigate the structure-function relationship in tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA), four hybrid sequences were amplified and overexpressed in a mouse myeloma cell line. The following constructs were made starting from cDNA encoding human t-PA and u-PA: (i) a hybrid in which amino acids (AA) 1-262 of the A-chain of t-PA is fused to AA 139-411 of the B-chain of u-PA; (ii) a hybrid in which the kringle 2 region of t-PA (AA 173-262) is inserted between amino acids 130 and 139 of u-PA; (iii) hybrid #2 having amino acids 1 to 10 deleted and replaced by the finger region of t-PA (AA 1-50); and (iv) a chimera in which the finger region of t-PA is followed by amino acids 10-411 of u-PA and where the lysine residues at positions 135 and 136 of u-PA are replaced by glutamines. These four hybrids were efficiently secreted into the culture medium as single-chain polypeptides of the expected molecular weights and had fully functional catalytic activity. Replacement of the A-chain of u-PA by that of t-PA leads to increased fibrin binding, whereas additions of finger and kringle domains do not. These data suggest that structural domains in serine proteases may not fold and/or function autonomously.

Animals↗
PubMed1988

Characterization of a chimaeric plasminogen activator obtained by insertion of the second kringle structure of tissue-type plasminogen activator (amino acids 173 through 262) between residues Asp130 and Ser139 of urokinase-type plasminogen activator.

A chimaeric recombinant plasminogen activator (rscu-PA- K2) obtained by insertion of the second kringle (K2) of tissue-type plasminogen activator (t-PA) (amino acids 173-262) between residues Asp130 and Ser139 of single chain urokinase-type plasminogen activator (scu-PA) was purified from the conditioned medium of mouse myeloma cells transfected with the previously described plasmid pULB9137 (Piérard et al., J. Biol. Chem. 262, 11771-11778, 1987). Approximately 22 micrograms of purified protein was obtained per liter of conditioned medium with a yield of approximately 25 percent. On sodium dodecylsulfate gel electrophoresis under reducing conditions, rscu-PA- K2 migrated with an apparent Mr of 65,000. Plasmin caused a time- and concentration-dependent conversion to an amidolytically active two chain derivative (rtcu-PA- K2) with a specific activity of 45,000 IU/mg. Both rscu-PA- K2 and rtcu-PA- K2 activated plasminogen directly with Km = 2.0 microM and k2 = 0.00063 s-1 and Km = 100 microM and k2 = 4.1 s-1 respectively. rscu-PA- K2 did not bind extensively to fibrin. It caused concentration-dependent lysis of 125I-fibrin-labeled plasma clots immersed in human plasma with a comparable specific activity and fibrin-specificity as rscu-PA. It is concluded that insertion in scu-PA of the second kringle of t-PA, which is believed to be involved in its fibrin affinity, does not significantly alter the enzymatic properties of scu-PA, but does not confer marked fibrin-affinity to the molecule.

Binding Sites↗
PubMed1987

Mutant and chimeric recombinant plasminogen activators. Production in eukaryotic cells and preliminary characterization.

Mutant urokinase-type plasminogen activator (u-PA) genes and hybrid genes between tissue-type plasminogen activator (t-PA) and u-PA have been designed to direct the synthesis of new plasminogen activators and to investigate the structure-function relationship in these molecules. The following classes of constructs were made starting from cDNA encoding human t-PA or u-PA: 1) u-PA mutants in which the Arg156 and Lys158 were substituted with threonine, thus preventing cleavage by thrombin and plasmin; 2) hybrid molecules in which the NH2-terminal regions of t-PA (amino acid residues 1-67, 1-262, or 1-313) were fused with the COOH-terminal region of u-PA (amino acids 136-411, 139-411, or 195-411, respectively); and 3) a hybrid molecule in which the second kringle of t-PA (amino acids 173-262) was inserted between amino acids 130 and 139 of u-PA. In all cases but one, the recombinant proteins, produced by transfected eukaryotic cells, were efficiently secreted in the culture medium. The translation products have been tested for their ability to activate plasminogen after in situ binding to an insolubilized monoclonal antibody directed against urokinase. All recombinant enzymes were shown to be active, except those in which Lys158 of u-PA was substituted with threonine. Recombination of structural regions derived from t-PA, such as the finger, the kringle 2, or most of the A-chain sequences, with the protease part or the complete u-PA molecule did not impair the catalytic activity of the hybrid polypeptides. This observation supports the hypothesis that structural domains in t-PA and u-PA fold independently from one to another.

Animals↗
PubMed1987

Characterization of a recombinant fusion protein of the finger domain of tissue-type plasminogen activator with a truncated single chain urokinase-type plasminogen activator.

Human recombinant single chain urokinase-type plasminogen activator (recombinant scu-PA) and a hybrid between human tissue-type plasminogen activator (t-PA) and scu-PA, obtained by ligation of cDNA fragments encoding the NH2-terminal region (amino acids 1-67) of t-PA and the COOH-terminal region (amino acids 136-411) of scu-PA, were expressed in a mammalian cell system. The proteins were purified from conditioned culture media containing 2% fetal calf serum by chromatography on zinc chelate-Sepharose, immunoadsorption chromatography on an insolubilized murine monoclonal antibody directed against urokinase, benzamidine-Sepharose chromatography, and Ultrogel AcA 44 gel filtration. Between 180 and 230 micrograms of the purified proteins were obtained per liter of conditioned medium, with a yield of approximately 18% and a purification factor of 720-1900. On sodium dodecyl sulfate gel electrophoresis under reducing conditions, the proteins migrated as single bands with approximate Mr 50,000 for recombinant scu-PA and Mr 43,000 for the t-PA/scu-PA hybrid. Following conversion to urokinase with plasmin, the proteins had a specific amidolytic activity comparable to that of natural scu-PA. Both proteins activated plasminogen directly with Km = 0.53 and 1.4 microM and k2 = 0.0034 and 0.0027 s-1, respectively. Both proteins did not bind specifically to fibrin and had a comparable degree of fibrin selectivity as measured in a system composed of a whole human 125I-fibrin-labeled plasma clot suspended in human plasma. It is concluded that this chimeric protein, consisting of the NH2-terminal "finger-like" domain of t-PA and the COOH-terminal region of scu-PA, has very similar enzymatic properties as compared to scu-PA, but has not acquired the fibrin affinity of t-PA.

Adenocarcinoma↗
PubMed1986

Incidence, clinical significance and prognosis of ventricular fibrillation in the early phase of myocardial infarction.

Of 1265 patients admitted to the CCU with the diagnosis of acute MI, 96 (7.6%) developed ventricular fibrillation within 72 hours following admission. Of these 96, 35 (36.5%) had secondary VF associated with left ventricular failure; they had a high in-hospital mortality of 57.1%. The remaining 61 (63.5%) had primary VF, i.e. VF occurring in the absence of significant LV failure. Fourteen of these (23%) died in hospital: 9 due to PVF (3 during the first episode, 6 during a recurrence). This mortality figure was significantly higher (P less than 0.001) than the mortality of 10% seen among patients who did not experience VF. Primary VF showed a recurrence rate of 20%. Compared with the 1061 patients who left the hospital without primary VF, the 61 subjects with this rhythm disorder were older, had larger infarcts and more frequent complications, such as pericarditis, conduction abnormalities, frequent ventricular premature contractions and signs of right ventricular failure. These findings, in contrast with a widely held view, suggest that primary VF may carry a guarded prognosis.

Adult↗
PubMed1984

Two-dimensional echocardiographic guiding of endomyocardial biopsy.

Two-dimensional echocardiography provides adequate spatial orientation and anatomic definition. Eighteen consecutive patients undergoing myocardial biopsy had concomitant two-dimensional echocardiography during 22 biopsies of five to six samples each. The transducer was placed at the apex and in the subcostal area and four-chamber views were used. The bioptome was seen entering the right atrium and crossing the tricuspid valve to the right ventricle. The catheter was then manipulated under two-dimensional echocardiography and the tip's position was strictly adapted, using two different classic views before sampling. The success rate in visualizing the forceps and in defining the site of sampling was 100 percent, and no complication was noted. Radiation exposure was largely reduced. Two-dimensional echocardiography to guide endomyocardial biopsy may reduce the risk of perforation by a better anatomic definition of the sampling site. It permits sampling from different ventricular sites or from the same site during serial procedures for monitoring evolution or treatment.

Adult↗
PubMed1982

Videodensitometric processing of contrast two-dimensional echocardiographic data.

We developed a computer program to analyze videodensity changes due to contrast appearance within a given operator-designated rectangle using two-dimensional echocardiograms previously recorded on videotape. Videodensity curves have been obtained from two-dimensional echocardiographic recordings in 14 patients after a total of 32 injections of 5% dextrose solution into the left ventricle during cardiac catheterization. The resulting videodensity vs time curves have some characteristics of indicator-dilution curves. The decay phase of these curves is largely mono-exponential. Potential clinical applications of this technique in measurement of ejection fraction, cardiac output and shunt quantification are discussed, as well as some potential limitations.

Computers↗