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L Piérard

Publications and source records attributed to L Piérard.

At least 37 records · Page 2Linked 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↗
PubMed1997

[Detection of myocardial viability: role of stress echocardiography].

Identification of myocardial viability is of great clinical importance in patients with acute coronary events or chronic coronary artery disease. Stress echocardiography has emerged as a new accurate and cost-effective imaging modality to detect contractile reserve. Numerous studies have recently been published in this field. This article summarizes the main findings of these investigations and presents practical aspects of the method.

Dipyridamole↗
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↗