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J Amiral

Publications and source records attributed to J Amiral.

At least 109 records · Page 6Linked to original sources
PubMed1994

Laboratory diagnosis of heparin-associated thrombocytopenia and comparison of platelet aggregation test, heparin-induced platelet activation test, and platelet factor 4/heparin enzyme-linked immunosorbent assay.

BACKGROUND: As clinical diagnosis of heparin-associated thrombocytopenia (HAT) is often difficult, confirmation by sensitive laboratory assays is desirable. STUDY DESIGN AND METHODS: The sensitivity of the heparin-induced platelet activation (HIPA) test and the platelet aggregation test (PAT) was prospectively compared by using the sera of 209 patients with the putative diagnosis of HAT. Both assays were performed concomitantly with platelets of the same four donors using a different combination of donors from day to day. Further, all sera were assessed with a platelet factor 4 (PF4)/heparin enzyme-linked immunosorbent assay (ELISA). RESULTS: Positive results were obtained with 33 percent of sera in the PF4/heparin ELISA, with 33.5 percent of sera in the HIPA test, and with 11.5 percent of sera in the PAT. The PF4/heparin ELISA and the HIPA test showed no difference in sensitivity (p = 0.27 by McNemar's test) and were more sensitive than PAT (p < 10(-8) by McNemar's test). However, they recognized different patient cohorts. Nine HIPA-indeterminate and 12 HIPA-negative sera were positive in the PF4/heparin ELISA. Eight of the nine indeterminate sera caused platelet activation with high heparin concentrations in the HIPA test. Eleven of the 12 negative sera contained no IgG, but 9 contained IgM and 2 contained IgA HAT antibodies. Four sera that were indeterminate in the PF4/heparin ELISA and 18 sera that were negative were positive in the HIPA test. None of the sera that were positive in the PAT was missed in the HIPA test, but two of those were negative in the PF4/heparin ELISA. All sera were assessed with four low-molecular-weight heparins and a low-molecular-weight heparinoid in the HIPA test with platelets from the same four donors. Low-molecular-weight heparin caused platelet activation with positive sera in 98 percent of tests, and the heparinoid did so in 10 percent; in a further 12.8 percent, crossreactivity to the low-molecular-weight heparinoid could not be excluded. CONCLUSION: The majority of HAT antibodies react with a PF4/heparin complex, but there is strong evidence that other antigens are involved in some patients. The HIPA test and the PF4/heparin ELISA are sensitive for diagnosing HAT, and they complement one another.

Enzyme-Linked Immunosorbent Assay↗
PubMed1994

Design and validation of a new immunoassay for soluble forms of thrombomodulin and studies on plasma.

Thrombomodulin (TM), purified from human placental homogeneate by affinity chromatography on DIP-Thrombin agarose, was used to develop monoclonal antibodies (MAbs). Two of them, 3E2 and 24FM (both IgG1, K), which were not calcium-dependent, were found convenient for developing a two-site enzyme immunoassay. Testing of recombinant and truncated forms of TM26 demonstrated that the species containing the amino terminus including the lectin-like domain and the epidermal growth factor (EGF)-like domains 1-4 were fully measured. The working range was from 2 to 100 ng/ml with a detection threshold of 2 ng/ml. Intraassay and interassay reproducibilities were, respectively, below 7.4% and 8.6%, whereas recovery of purified TM was between 88 and 114% in plasma. Mean plasma concentration was 42.1 (+/- 11.3) ng/ml (males 51.8 +/- 7.9 ng/ml, females 34.8 +/- 7.8 ng/ml) and it was established on 62 normal individuals between the ages of 21 and 55 (28 males and 34 females). This new assay is a convenient tool for measuring plasma TM and establishing its diagnostic and predictive value in diseases associated to endothelial damage.

Adult↗
PubMed1994

New direct assay of free protein S antigen using two distinct monoclonal antibodies specific for the free form.

Monoclonal antibodies (mAbs) specific for free protein S and devoid of reactivity with protein S-C4b-BP complexes, have been produced. A one-step sandwich-type enzyme-linked immunoassay (ELISA) has been developed with two mAbs reacting with distinct epitopes of free protein S. F(ab')2 fragments from mAb 15C4 were coated on microplates and mAb 34G2 conjugated with horseradish peroxidase (HRP) was added immediately before diluted plasma. The presence of calcium in the sample diluent prevented dissociation of complexes during the assay. This assay was specific as demonstrated by good recovery of purified protein S added to plasma and the lack of influence of C4B-BP-protein S complexes. Thus, addition of increasing amounts of purified C4B-BP to human citrated plasma induced a dose-dependent decrease of free protein S. The assay was sensitive, allowing measurement of 5-500 ng/ml of free protein S, with a detection threshold of 2 ng/ml. It was also reproducible with inter-assay and intra-assay variation coefficients of 2.5-5.1% and 3.1-5.0%, respectively. Thus, this new ELISA of free protein S antigen in plasma has the advantages of being fast, accurate and reproducible. It appears to be extremely useful for routine studies as no preliminary treatment of plasma is required.

Antibodies, Monoclonal↗
PubMed1994

Clinical applications of a direct assay of free protein S antigen using monoclonal antibodies. A study of 59 cases.

A new one-step ELISA using two monoclonal antibodies specific for distinct epitopes of the free form of protein S (ELISA-m) has been developed for the direct measurement of free protein S in untreated plasma. This assay has been compared with the classic method using polyclonal antibodies to protein S (ELISA-p). The latter method has the drawback of requiring PEG precipitation of plasma which is time-consuming, difficult to perform with accuracy and therefore poorly reproducible in most laboratories. Results of both ELISAs were compared with those of a functional assay. In 30 normal subjects, there was an excellent correlation between ELISA-m and ELISA-p (r = 0.95) as well as between ELISA-m and the functional assay (r = 0.96). In twelve patients with a congenital deficiency, the levels of free protein S antigen were similarly decreased with ELISA-m and ELISA-p and in good agreement with those of protein S activity. In 20 patients with miscellaneous inflammatory diseases, the levels of free proteins S were normal with good correlation between both ELISAs and PS activity, despite high levels of C4bBP-protein S complexes. As expected, in 15 dicoumarol-treated patients, there was a significant and parallel decrease of free protein S antigen with both ELISAs, with even lower levels of protein S activity. In 14 patients with liver cirrhosis, the mean values for free protein S antigen were normal using both assays, but with wide extreme values, whereas protein S activity was significantly lower.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibodies, Monoclonal↗
PubMed1994

Elevated thrombomodulin plasma levels as a result of endothelial involvement in plasmodium falciparum malaria.

We used thrombomodulin (TM) to assess the participation of the vascular endothelium in human Plasmodium falciparum (P.F.) malaria. Before therapy TM plasma levels were elevated in P.F. malaria and fell to normal values during therapy. Parasitemia, TNF alpha, elastase and TAT levels correlated directly with TM. Elevated TM levels can not be explained by increased synthesis, since incubating HUVEC with pretherapy serum of patients with P.F. malaria, but not reconvalescence serum, suppressed TM transcription. This was partially prevented by adding a TNF alpha neutralizing antibody to patient serum before incubation with HUVEC. However, TNF alpha does not release TM from cultured HUVEC in vitro. Coincubation of HUVEC with pretherapy serum together with neutrophils resulted in endothelial cell destruction, which could be partly prevented by a TNF alpha neutralizing antibody. Hence the increase of TM during P.F. malaria might reflect the concerted action of cytokines and neutrophils on HUVEC.

Antimalarials↗
PubMed1994

Heparin-associated thrombocytopenia: isolation of the antibody and characterization of a multimolecular PF4-heparin complex as the major antigen.

Sera of 34 patients with heparin-associated thrombocytopenia (HAT), giving a positive result in the serotonin release assay (SRA), were assessed in a platelet factor 4 (PF4)/heparin ELISA. Three sera revealing indeterminate results in the SRA and 10 control sera were also investigated. Both tests correlated closely (Kappa 0.742; p = 2.67 x 10(-7)), but one positive serum in the SRA was negative in the pF4/heparin ELISA. We have isolated the HAT antibodies by absorbtion and elution of HAT sera using endothelial cells (HUVEC). Eluates gave similar results as the sera in the PF4/heparin ELISA (Kappa 0.837, p = 9.26 x 10(-9)), and they also correlated very closely with the SRA (Kappa 0.888; p = 8.89 x 10(-10)). This demonstrates that HAT antibodies bind to the same epitope on platelets and on endothelial cells. High heparin concentrations released PF4 in a dose dependent manner from microtiter plates if PF4/heparin, but not if PF4 alone, was covalently linked. Concomitant to the release of PF4, binding of HAT antibodies to PF4/heparin decreased, as indicated by the median optical density (OD) values of OD 0.88 in the presence of buffer compared to OD 0.181 in the presence of 100 IU/ml heparin. The latter values were similar to those obtained when plates were coated with PF4 alone (median OD 0.203). Binding of three eluates was not inhibited by high heparin concentrations and they reacted also with PF4 alone. We conclude that multimolecular PF4/heparin complexes represent the major antigen in HAT. These multimolecular complexes might present several epitopes and form immune complexes after HAT antibody binding which activate platelets via the FcRII. In a few cases, PF4 alone can be recognized by the antibody. However, there is also evidence that other molecules might be involved in some patients.

Antibodies↗
PubMed1993

The growth-supportive effect of thrombospondin (TSP1) and the expression of TSP1 by human MG-63 osteoblastic cells are both inhibited by dexamethasone.

Thrombospondin (TSP) is a 450-kDa extracellular matrix glycoprotein which supports the growth of human MG-63 osteoblastic cells [Abbadia et al., FEBS Lett., 329 (1993) 341-346]. In this study, we describe the effect of the glucocorticoid, dexamethasone, on cell proliferation and TSP expression by MG-63 cells. Using a serum-free mitogenesis assay, dexamethasone (25 to 500 nM) caused a dose-dependent decrease in [3H]thymidine incorporation by MG-63 cells in culture, reaching 40% inhibition of cell proliferation at a concentration of 250 nM. Similarly, the stimulatory effect of TSP (500 ng/ml) on proliferation of MG-63 cells was totally abolished in the presence of dexamethasone (250 nM). In situ hybridization indicated that TSP mRNA level in dexamethasone-treated MG-63 cells decreased compared to quiescent cells. As judged by fluorescence-activated cell sorting analysis, dexamethasone treatment of MG-63 cells resulted in a 50 to 70% decrease in TSP cell surface expression compared to quiescent cells. Secretion of TSP in the culture fluid of dexamethasone-treated MG-63 cells also decreased by 40% while, under similar experimental conditions, a 180% increase in alkaline phosphatase activity was observed in dexamethasone-treated cells. Because glucocorticoids induce osteoporosis in vivo and reduce proliferation of osteoblasts in vitro, our results argue for an important role of TSP during bone formation.

Alkaline Phosphatase↗
PubMed1993

Levels of plasma thrombomodulin are increased in atheromatous arterial disease.

The plasma thrombomodulin (TM) level depends on the integrity of the endothelium and the clearance of the molecule. In several different pathological conditions, plasma TM levels increase with damage to the endothelium. We studied plasma TM levels in patients with various localizations of atheromatous arterial disease who had normal serum creatinine levels. Two groups of patients had a single symptomatic localization, which was either peripheral occlusive arterial disease (POAD) or ischemic heart disease (IHD) and a third group of patients had multiple symptomatic localizations (polyvascular). We compared the plasma TM levels with the plasma levels of other specific markers of endothelial cell activation such as: prostacyclin (PGI2), tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor (PAI-1). Plasma TM levels were significantly increased in all three individual groups and when all patients were considered (total patients), as compared with normal controls. When all patients were considered, there was a significant positive correlation between plasma TM levels and t-PA and between plasma TM levels and PGI2. A significant positive correlation was also found between the plasma TM levels and PAI-1 for patients with POAD. Thus, our findings suggest that an increased influx of TM into the plasma may be caused by endothelial cell damage in patients with atheromatous arterial disease. However in our study, the plasma TM levels obtained were similar for all three types of atheromatous arterial disease. Though plasma thrombomodulin is a marker of endothelial cell injury, it cannot be of a clinical interest until its levels are related to the extend of the atheromatous lesions.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1993

Thrombospondin (TSP1) mediates in vitro proliferation of human MG-63 osteoblastic cells induced by alpha-thrombin.

Thrombospondin (TSP) is a 450-kDa glycoprotein synthesized and secreted by human MG-63 osteoblastic cells. In this study, we have first studied the effect of alpha-thrombin on TSP expression by human MG-63 cells. In situ hybridization indicated that TSP mRNA level in thrombin-treated MG-63 cells was increased when compared to unstimulated cells. As judged by immunofluorescence, thrombin-treatment of MG-63 cells resulted in increased cell surface expression of TSP when compared to quiescent cells. Because thrombin stimulates proliferation of osteoblastic cells, the involvement of TSP in proliferation of thrombin-stimulated osteoblastic cells was then investigated using a serum-free mitogenesis assay. Both alpha-thrombin (0.01 to 0.15 U/ml) and TSP (5 to 600 ng/ml) caused a dose-dependent increase in [3H]thymidine incorporation by MG-63 cells. Proliferation of osteoblastic cells induced by alpha-thrombin or TSP was specifically and totally inhibited by anti-TSP monoclonal antibodies (3-10 micrograms/ml) or by indomethacin (1 microM), an inhibitor of prostaglandin synthesis. Anti-TSP antibodies which inhibited cell proliferation also inhibit TSP expression to the surface of these cells. Our experiments support the existence of a mechanism whereby TSP bound to the cell surface of thrombin-treated MG-63 cells stimulates secretion of prostaglandins which, in turn, allow cell proliferation to proceed.

Cell Division↗
PubMed1993

Plasma thrombomodulin concentrations in relation to cardiovascular risk factors in a population sample.

Plasma thrombomodulin (TM) concentrations were studied in a population sample of 175 subjects aged 35-65 years. There was no age variation in males (mean value 65 +/- 15 micrograms/l) but females aged 35-45 years had lower values than women aged 55-65 years, suggesting an effect of the menopause. The TM levels were unrelated to body build measurements (body mass index, waist/hip ratio) and blood pressure, and also to lipid levels (triglycerides, total cholesterol) and insulin before and during an oral glucose tolerance test. There was no difference in mean TM levels in subjects with and without electrocardiographic signs of ischaemic heart disease. Tobacco smokers had 15% lower mean levels of plasma TM than non-smokers.

Adult↗
PubMed1993

Thrombomodulin is synthesized by osteoblasts, stimulated by 1,25-(OH)2D3 and activates protein C at their cell membrane.

We have previously shown that protein S, a vitamin K-dependent protein, is a bone matrix component synthesized and secreted by osteoblasts. Because protein S is a cofactor of protein C in inhibiting factor Va and VIIIa, we have looked for the presence of the proteins related to the anticoagulant protein C system in human MG 63 osteosarcoma cells and in human adult osteoblast-like cells. Using immunoblotting, we have shown that protein C, factor V, and C4b binding protein are not secreted by these cells. We have shown by enzyme-linked immunoassay, immunocytochemistry, and immunoprecipitation of labeled proteins that thrombomodulin, a transmembrane glycoprotein involved with thrombin in the activation of protein C, is present at the cell surface of osteoblasts. Moreover, using a protein C activation system where thrombin and protein C are added to the cells, we have shown that protein C could be activated at the osteoblast cell surface. This activation of exogenous protein C, reflecting the activity of thrombomodulin, as well as the expression of the thrombomodulin antigen, is regulated by some bone resorption-enhancing factors. 1,25-dihydroxyvitamin D3 and retinoic acid increase thrombomodulin expression and activity in a dose-dependent manner whereas tumor necrosis factor alpha and interleukin 1 decrease these parameters. Because thrombomodulin is known to inhibit single-chain urokinase-type plasminogen activator, a molecule present in the osteoblast microenvironment, these findings suggest that thrombomodulin could play a role in the regulation of bone resorption by modulating the plasmin system.

Calcitriol↗
PubMed1993

Measurement of beta-thromboglobulin and platelet factor 4 to follow up patients with artificial heart valves.

Efficient antithrombotic therapies are necessary for the prevention of thrombotic complications in patients with artificial heart valves. Antiplatelet drugs are commonly used since platelet activation is a feature frequently found in these pathologic conditions. The measurement of beta TG and PF4 is useful for the diagnosis of platelet activation, which may increase the thrombotic risk. Two commercially available ELISA kits for beta TG and PF4 were tested and compared with the corresponding RIA methods used previously. Coefficients of correlation were 0.976 for beta TG and 0.985 for PF4. Both methods gave identical results, although RIA was expressed in nanograms per milliliter, whereas ELISA results were in international units per milliliter by comparison with International Standards. For both parameters 1 IU/ml was found to be almost equal to 1 ng/ml. beta TG was elevated in the group of patients with heart valves (57.28 IU/ml); so was PF4 but to a lesser extent (11.28 IU/ml). The mean concentrations on a normal population were, respectively, 22.40 (+/- 5.15) IU/ml and 4.10 (+/- 2.02) IU/ml. The monitoring of patients with heart valves allowed the identification of two distinct groups: those who had persistent elevated levels of beta TG and PF4, and those whose levels of the same parameters were in the subnormal ranges. All the thrombotic complications were observed in the first group and were preceded by an elevation of platelet markers. beta TG was the most predictive and correlated closely with the evolution of the clinical state. Cerebrovascular accidents were the commonly observed complications. These patients were treated by hypocoagulant therapy and with antiplatelet drugs.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗
PubMed1993

Immunization by bovine thrombin used with fibrin glue during cardiovascular operations. Development of thrombin and factor V inhibitors.

Brief case histories of three patients aged 58, 38, and 44 years are reported. All underwent cardiovascular operations. Subsequently hemostasis test abnormalities developed between the seventh and eighth postoperative days after exposure to bovine thrombin used with fibrin glue. These were characterized by an increased activated partial thromboplastin time (64 to 147 seconds), prothrombin time (19 to 24 seconds), bovine thrombin time (> 120 seconds) and a markedly reduced factor V level (< 10% in two patients and 16% in the third patient). A patient plasma dilution of 1 in 200 with a normal plasma pool was necessary to correct bovine thrombin time. No fast-acting or progressive inhibitor against factor V could be detected by coagulation tests, and fresh frozen plasma perfusion had no effect. Plasmapheresis was performed preventatively to avoid bleeding, and factor V levels stabilized at around 50% after two to four exchanges. Immunologic studies showed that the inhibitors were directed not only against bovine factors but also against human ones. Therefore factor V decrease could have been the result of rapid clearance from the circulation of complexes formed with a nonneutralizing inhibitor that is not detected by clotting tests. These antibodies were purified by standard methods and immunoaffinity. Fast immunization could be explained by a prior sensitization to bovine thrombin exposure during previous operations. It is suggested that bovine thrombin used with fibrin glue contains small amounts of factor V and may be responsible for these abnormalities. This is in agreement with previous literature reports. However, these described neutralizing factor V inhibitors, which were easily detected.

Adult↗
PubMed1992

An enzyme-linked immunosorbent assay for heparin cofactor II (HCII). Application to the measurement of HCII in clinical materials.

Heparin cofactor II (HCII) is a thrombin inhibitor in human plasma, the activity of which is enhanced by heparin and dermatan sulfate. To measure the plasma antigen concentration of HCII, an enzyme-linked immunosorbent assay (ELISA) has been developed, and this is based on the use of polyclonal anti-HCII IgG, both as the antigen-capturing antibody and as the labelled antibody. The intra- and inter-assay reproducibilities have been calculated to be less than 6%. HCII antigen concentrations evaluated with this ELISA technique in clinical plasma samples correlated well with those determined by electroimmunodiffusion and with HCII activities evaluated using a functional assay (r = 0.909 and r = 0.930, P less than 0.0001). Since the correlation between HCII deficiency and thrombosis is still a controversial issue, the ELISA technique described in this report could be a useful tool for the large-scale studies needed to determine the prevalence of HCII deficiency in healthy individuals and in patients with a history of thrombosis.

Adult↗
PubMed1992

Study of the protein S system in HIV-infected patients: acquired protein S deficiency or unsuitable assays?

The plasma protein S (PS) system was studied in 120 HIV seropositive patients (CDC classification: group II: n = 35; group III: n = 6; groups IVA, IVC2 or IVE, n = 38; groups IVB, IVC1 or IVD, n = 41). Total PS antigen and C4b-binding protein (C4b-BP) values were not significantly different from control values. Free protein S levels assessed by an immunological method in the supernatant of PEG-precipitated plasma samples (PEG-fPS) were below the normal limit in 85 patients, lower values being found in patients with advanced HIV disease. No correlation was found between PEG-fPS levels and C4b-BP or total PS levels. At least 25 patients had a low functional PS value. Low functional levels of PS were found in each clinical group although there was no difference in the distribution of functional PS values among groups. Crossed immuno-electrophoresis showed an abnormal distribution of PS in some patients, but failed to confirm the marked decrease of free PS in patients with very low PEG-fPS. An impairment of the protein S system is observed in HIV-infected patients. However, the discrepancies found in some patients among the results of the various PS-related assays should lead to a cautious interpretation concerning the incidence of PS deficiency in HIV-infected patients.

Adult↗