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Biomedical subjects

J Amiral

Publications and source records attributed to J Amiral.

At least 127 records · Page 7Linked to original sources
PubMed1991

Functional assay of protein S in 70 patients with congenital and acquired disorders.

A functional assay for the selective measurement of the active form of protein S in plasma, based on the prolongation of an APTT, was previously developed. This assay is sensitive, reproducible and specific, not affected by other clotting factors including FVIII. This method was applied to the measurement of protein S activity in congenital and acquired disorders. Results of protein S activity were compared to those of total and free antigen measured by ELISA. In 30 controls, there was an excellent correlation between protein S activity and free antigen. In patients with inflammatory disease, protein S activity and free antigen were normal, despite high levels of both C4b-binding protein and total protein S antigen. In dicoumarol-treated patients, protein S activity was lower than free antigen due to the presence of acarboxylated forms. Surprisingly, in liver cirrhosis, free antigen was only slightly decreased whereas protein S activity was significantly reduced. In 23 patients with congenital deficiency, protein S activity was consistently decreased, from less than 5% to 60% and showed good correlation with the free antigen. This functional assay allows the rapid diagnosis of congenital or acquired deficiency of protein S.

Adult↗
PubMed1991

Involvement of heparin cofactor II in chymotrypsin neutralization and in the pancreatic proteinase-antiproteinase interaction during acute pancreatitis in man.

Heparin cofactor II is a proteinase inhibitor which inhibits both chymotrypsin and thrombin, and displays great similarities with antithrombin III, the main inhibitor of thrombin in human plasma. Since acute pancreatitis is known to be associated with modification of the proteinase-antiproteinase equilibrium, we studied heparin cofactor II and antithrombin III as well as other biochemical and haematological parameters in 10 patients experiencing attacks of acute pancreatitis. Heparin cofactor II activity decreased during the first week of illness, while its antigen concentration remained subnormal. This discrepancy between antigen concentration and activity which persisted during the first week of illness was due both to complex formation of heparin cofactor II with its target proteinases and to partial proteolysis of the inhibitor. Heparin cofactor II was shown to form a complex with chymotrypsin in the plasma of such patients. Antithrombin III levels remained unchanged throughout the study, with no discrepancy between its activity and antigen concentration. No modification of haemostasis was shown either, except for a rise in the fibrinogen level during the first days of illness. It is concluded that, unlike antithrombin III, heparin cofactor II is involved in the proteinase-inhibitor equilibrium in patients with acute pancreatitis, and that heparin cofactor II might react as an inhibitor of pancreatic proteinases rather than an inhibitor of thrombin.

Acute Disease↗
PubMed1990

Monoclonal antibodies to different neo-epitopes on fibrinogen and fibrin degradation products.

The measurement of fibrin or fibrinogen degradation products is widely used in clinical practice for the diagnosis and follow up of coagulolytic disturbances. Recently D-dimer assays have become very popular owing to their direct application to plasma. However, in some clinical situations there is a need to differentiate fibrin from fibrinogen degradation products. These are still routinely measured by conventional assays on serum. We tried to develop various monoclonal antibodies specific for the neo-epitopes unmasked during the degradation of fibrin or fibrinogen. Fifteen mice hybridomas producing the expected antibodies were obtained and ten were extensively characterized. They could be classified in three reactivity classes: D and D-dimer, D-dimer and early fibrinogen or fibrin degradation products. These monoclonal antibodies were used to develop latex slide assays and ELISA techniques. Two types of assays were obtained; those which were specific for fibrin-related products and those evaluating the totality of fibrin or fibrinogen degradation products. Assays discriminating the fibrinogen split products from those derived from fibrin, and performed directly on citrated plasma can be proposed. They provide complementary information in clinical states such as DIC, pulmonary embolism, leukaemias, thrombolysis, etc.

Animals↗
PubMed1990

Reactivity of D-dimer assays with the fibrinogen--fibrin split products generated by thrombolytic agents.

The reactivity of two D-dimer assays (a latex agglutination method, D-Di test and an ELISA procedure, Asserachrom D-Di) to the various fibrin or fibrinogen degradation products generated in plasma by three different thrombolytic agents was analysed, in the presence or absence of a fibrin clot. Other assays performed in parallel were an ELISA assay for (DD)E complexes and the conventional fibrinogen degradation products (FDP) latex test on serum. The thrombolytic agents urokinase, streptokinase or tPA were added at various concentrations and incubated for different times ranging from 10 min to 24 h. The data showed that the D-dimer latex assay was always negative provided there was no fibrin in plasma and despite the presence of high FDP levels (greater than 600 micrograms/ml) in serum. In contrast, D-dimer or (DD)E complexes were measured by ELISA, but up to a given concentration (15-20 micrograms/ml) which reached a plateau and remained stable irrespective of the thrombolytic concentrations or the degradation times. In the presence of fibrin clot, fibrinolysis was extremely fast with tPA and the FDP were generated at a much higher concentration that that expected from the size of the fibrin clot. This suggests the existence of fibrinogenolysis targeted by the presence of fibrin but negative in its absence. Urokinase and streptokinase generated FDP very quickly but a much slower degradation rate of fibrin was observed. The immunoblotting confirmed these data and showed that no late FDP were formed in plasma even at high thrombolytic concentrations except when fibrin was present.(ABSTRACT TRUNCATED AT 250 WORDS)

Enzyme-Linked Immunosorbent Assay↗
PubMed1990

Fibrin degradation products generation and fibrinopeptide A release in normal plasma incubated with thrombolytic agents: proposed mechanisms.

Clinical data have shown that the evaluation of fibrin degradation products (FbDP) does not reflect the efficiency of thrombolytic therapy in vivo. In this study, we found that the addition of plasminogen activators to normal plasma resulted in generation of FbDP and release of fibrinopeptide A (FpA) as shown by ELISA and HPLC. This FpA release was concomitant with fibrinogen degradation, and was not inhibited by thrombin inhibition or by prothrombin depletion in plasma. Thus, the increase in FpA did not result from coagulation activation and may result from the plasmin-induced release of FpA from fibrinogen degradation product E1. The generation of cross-linked FbDP after tPA addition occurred in normal plasma as well as in factor-XIII-deficient plasma and quickly reached a plateau. It was not inhibited by hirudin. Therefore FbDP in these plasmas probably derived from the plasmin degradation of cellular transglutaminase cross-linked fibrin/fibrinogen derivatives present in plasma.

Aprotinin↗
PubMed1990

Preparation of factor-VIII-depleted plasma with antibodies and its use for the assay of factor VIII.

Standardized factor VIII (FVIII)-deficient plasma is necessary for the clotting assay of this protein. Ethical considerations and prevalence of HIV seropositivity in hemophilia A patients have required the replacement of severe hemophilia A plasma by another specific substrate for FVIII clotting assays. FVIII-deficient plasma was prepared by immunodepletion using polyclonal anti-von-Willebrand factor (vWF) and monoclonal anti-FVIII antibodies coated to agarose beads. This plasma, deficient in both FVIII and vWF, allows the evaluation of FVIII activity with a detection limit of 1% as compared to normal plasma. This immunodepleted plasma was compared to plasma from severe hemophilia A for the FVIII determination in plasma from normal subjects, severe and mild hemophiliacs, patients with von Willebrand disease and dicoumarol-treated patients. In all cases, similar values were obtained with both reagents at various levels of FVIII, with a correlation coefficient of 0.994. Such a good correlation was also obtained for the assay of FVIII in concentrates and in plasma from hemophiliacs following infusion. This immunodeficient plasma thus represents a suitable reagent for the FVIII clotting assay and provides an accurate alternative to the use of hemophilic plasma.

Antibodies↗
PubMed1990

Study of quantitative modifications of the plasma protein S system components in non-acute inflammatory syndromes.

Thrombosis frequently accompanies inflammatory disease. There are numerous and frequent modifications of haemostasis parameters during inflammatory disease. Deficiencies in protein S, a protein C cofactor, are predisposing factors for thromboses. In 43 patients with biological inflammatory syndromes (ESR above 80 mm in the first hour found twice in a three-day period and disturbances of other biological inflammatory markers) we studied the variations of the protein S system: total antigenic protein S, free antigenic protein S, C4b BP, all antigenic fractions being assayed by electro-immuno diffusion. The results show an increase in free protein S (mean 114.3%, range 25%-180%, P less than 0.005), the biologically active fraction of protein S. They also evidence the expected increase in C4b BP (mean 188.3%, range 41%-335%, P less than 10(-5]. There is an increase in total protein S Ag (mean 145.3%, range 56%-220%, P less than 10(-5]. The results show a high positive correlation between the increased free protein S Ag and total protein S Ag (r' = 0.78, P less than 0.01), between total protein S and C4b BP (r' = 0.8, P less than 0.01) and between free protein S and C4b BP (r' = 0.73, P less than 0.01). C4b BP concentrations are correlated with haptoglobin (r' = 0.59, P less than 0.01), orosomucoid (r' = 0.53, P less than 0.01), C3 complement component (r' = 0.80, P less than 0.01), C4 (r' = 0.39, P less than 0.02) and platelet count (r' = 0.51, P less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1988

Binding of an anti D dimer monoclonal antibody to endotoxin-activated monocytes. Demonstration by immunogold-silver staining.

Blood monocytes exposed to a variety of stimuli, of which endotoxin is a potent one, produce and express on their membrane a procoagulant activity (PCA) which can trigger the formation of pericellular fibrin. We have developed an immunogold silver method using a monoclonal anti-D dimer antibody to detect the presence of crosslinked fibrin in derivatives on the monocyte membrane. The in vitro PCA of endotoxin-stimulated monocyte was shown to correlate significantly with the number of D dimer-positive monocytes. We suggest that this method could be used to identify activated monocytes expressing increased PCA.

Antibodies, Monoclonal↗
PubMed1988

A conserved epitope on several human vitamin K-dependent proteins. Location of the antigenic site and influence of metal ions on antibody binding.

A murine monoclonal antibody (designated H-11) produced by injecting mice with purified human protein C was found to bind several human vitamin K-dependent proteins. Using a solid-phase competitive radioimmunoassay with antibody immobilized onto microtiter plates, binding of 125I-labeled protein C to the antibody was inhibited by increasing amounts of protein C, prothrombin, and Factors X and VII over a concentration range of 1 X 10(-8) to 1 X 10(-6) M. Other vitamin K-dependent proteins including Factor IX and protein S did not inhibit or inhibited only at the highest concentration binding of radiolabeled protein C to the immobilized antibody. Chemical treatment of prothrombin with a variety of agents including denaturation by sodium dodecyl sulfate, reduction with mercaptoethanol followed by carboxymethylation with iodoacetic acid, citraconylation of lysine residues, removal of metal ion with EDTA, or heat decarboxylation did not destroy the antigenic site recognized by the antibody as measured by immunoblotting of prothrombin or prothrombin derivative immobilized onto nitrocellulose. Immunoblotting of purified vitamin K-dependent polypeptides with the monoclonal antibody following sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrophoretic transfer to nitrocellulose indicated that the antigenic site was found on the light chains of protein C and Factor X. Chymotrypsin digestion of prothrombin and isolation on QAE-Sephadex of the peptide representing amino-terminal residues 1-44 of prothrombin further localized the antigenic site recognized by the monoclonal antibody to the highly conserved gamma-carboxyglutamic acid-containing domain. The exact location of the antigenic determinant for antibody H-11 was established using synthetic peptides. Antibody H-11 bound specifically to synthetic peptides corresponding to residues 1-12 of Factor VII and 1-22 of protein C. Comparison of protein sequences of bovine and human vitamin K-dependent proteins suggests that the sequence Phe-Leu-Glu-Glu-Xaa-Arg/Lys is required for antibody binding. The glutamic acid residues in this peptide segment are the first 2 gamma-carboxyglutamic acid residues near the amino-terminal end in the native proteins. Increasing concentrations of Ca2+, Mg2+, or Mn2+ partially inhibited binding of 125I-protein C to the antibody in a solid-phase assay system with half-maximal binding observed at divalent metal ion concentrations of 2, 4, and 0.6 mM, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1988

Measurement of tPA and tPA-PAI-1 complexes by ELISA, using monoclonal antibodies: clinical relevance.

Two ELISA methods using monoclonal antibodies, are described for the measurement of tPA:Ag and tPA-PAI-1 complexes. On normal population tPA:Ag was found with a mean value of 5 ng/ml. Furthermore, despite that tPA activity was very low, only 50% (mean value) was measured as stable complexes with PAI-1. Important increase of tPA:Ag was observed in various pathologies (cardiac infarction, septicemia, respiratory distress syndrome). In liver disease, tPA:Ag reached high levels up to 100 ng/ml. Impaired liver clearance can potentiate the increased concentration which results from endothelial release. In all patients with elevated tPA:Ag level, 70 to 100% of tPA was complexed to PAI-1. Excess release of PAI-1 accompanys the increased release of tPA as it is proved by presence of high residual PAI-1 activity. Addition of exogeneous tPA to these pathological plasmas induced a high increase in tPA-PAI-1 complexes. Venous stasis in normal population resulted in a parallel increase of tPA:Ag and tPA-PAI-1 complexes. Although about a two fold increase was obtained for both parameters, post venous stasis plasma presented a much higher fibrinolytic activity while PAI-1 activity was moderately elevated. tPA:Ag and tPA-PAI-1 complexes have diagnosis and prognosis value in various pathologies as indicators of stimulated release of fibrinolysis activator and inhibitor.

Animals↗
PubMed1988

Association between coagulation factors VII and X with triglyceride rich lipoproteins.

The association between the concentration of different plasma lipoproteins and plasma factor VII (F VII) was analysed by isolating plasma very low density lipoprotein (VLDL), low density lipoprotein (LDL), and high density lipoprotein (HDL) lipoproteins and assessing their in vitro interaction with F VII by immunoenzyme assay using peroxidase labelled anti-factor VII immunoglobulins to determine whether F VII coagulant activity is prognostic for cardiovascular mortality. F VII bound to triglyceride rich lipoproteins, the fixation being stronger on chylomicrons and VLDL fractions than on LDL fractions. In our experiments HDL did not bind to F VII. The fixation of coagulation factor X (FX) tested by the same method is comparable with that of F VII. The nature of this fixation seemed to arise from hydrophobic interaction as calcium was not necessary and the use of Tween 20 inhibited the interaction. The binding of factors VII and X was increased when lipids were previously treated by phospholipase C and the interaction seemed to be completely dependent on the lipid part of the lipoproteins. Hyrophobic fixation is a possible mechanism of interaction of plasma lipoproteins and F VII and X, and it may be of importance in the covariance of triglyceride concentrations and the activity of vitamin K dependent coagulation factors.

Adult↗
PubMed1986

An enzyme immunoassay (ELISA) for the quantitation of human factor VII.

A new solid phase enzyme-linked immunosorbent assay (ELISA) was developed for the quantitation of human Factor VII antigen (F VII Ag), using a monospecific rabbit anti-F VII antiserum. Anti-F VII F(ab')2 fragments were adsorbed to polystyrene plates. The binding of serial dilutions of control or test plasma, containing F VII, was detected by incubation with peroxidase-labeled anti- FV II IgG followed by the addition of hydrogen peroxyde and O-phenylenediamine. This ELISA is specific, sensitive (detection limit: 0.05%) and accurate (coefficient of variation: 1.5-4% for within- and 1.6-9% for between-assays). F VII coagulant activity (F VII C) and F VII C) and F VII Ag were determined in large populations of controls and patients. In normal plasma (n = 38), F VII Ag ranged from 83 to 117% and the correlation coefficient between F VII Ag and F VII C was 0.94. In patients with severe (F VII C inf. 1%) congenital F VII deficiency (n = 5), F VII Ag was undetectable in two cases (inf. 0.05%) and markedly reduced (0.35 to 5.6%) in the three other cases. In patients with liver cirrhosis (n = 15), F VII Ag ranged from 21% to 59% and was in good correlation with F VII C (r = 0.84). In dicoumarol treated patients (n = 15), the levels of F VII Ag ranged from 51% to 79% and a poor correlation (r = 0.52) with F VII C was observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibody Specificity↗
PubMed1986

[Determination of factor X Ag by laser immunonephelemetry].

A method of dosage of X Ag factor by laser immunonephelemetry is proposed. This technique, carried out with a rabbit antiserum in the presence of PEG and EDTA, presents the advantage to be sensitive, reproducible and mostly faster than the other immunological methods, such as Electroimmunodiffusion (EID) and Enzyme Linked Immuno-Sorbent Assay (ELISA). It constitutes a simple tool, applicable in the demonstration of an enzyme dysfunction or liver pathology. Finally, it could bring a supplement of information in monitoring antivitamin K therapy.

Antigens↗
PubMed1985

Vitamin K dependent proteins in fetal hemostasis at mid trimester of pregnancy.

The vitamin K dependent coagulation factor activities were measured in 63 normal human fetuses from 19 to 28 weeks of pregnancy. These activities were included between 9 to 28 percent of normal adult levels. Prothrombin antigen, factor IX antigen and protein C were also measured. There is a good correlation between prothrombin procoagulant activity and antigenicity, suggesting that low level of these vitamin K dependent proteins in fetuses is probably a consequence of liver immaturity.

Antigens↗
PubMed1985

[Immunologic determination of the protein C of coagulation: detection of deficiencies].

Protein C, discovered by Stenflo in 1976, plays a major role in the regulation of thrombogenic processes. Even moderate deficiencies (0.40 to 0.60 U/ml) can be responsible for serious thromboembolic accidents. The authors evaluated the kits for the immunological assay of protein C, produced by Laboratoire Diagnostica Stago: a immuno-enzymatic method (ELISA) and Laurell's method. Despite the very different characteristics in terms of practicability, threshold of sensitivity and precision, the authors obtained an excellent correlation between these two techniques. The concentrations of protein C Ag obtained in 22 normal plasmas were 0.70 to 1.40 U/ml. In 32 patients with recurrent thrombosis with no apparent cause, 5 had a concentration of protein C Ag less than 0.70 U/ml. The identification of a deficiency of protein C Ag (Vitamin K depend protein) is often made difficulty by the fact that the patients are treated with anti-vitamin K drugs. The ratio between the vitamin K depend pro-coagulation factors and Protein C Ag may provide information about a possible deficiency.

4-Hydroxycoumarins↗