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N Kieffer

Publications and source records attributed to N Kieffer.

66 records · Page 4Linked to original sources
PubMed1985

Aggregation of chymotrypsin-treated thrombasthenic platelets is mediated by fibrinogen binding to glycoproteins IIb and IIIa.

Previous experiments demonstrated that chymotrypsin, but not adenosine diphosphate (ADP), exposed fibrinogen binding sites on platelets from patients with Glanzmann's thrombasthenia. Three of these patients have been reexamined, and previous observations were confirmed. The quantity of iodine 125-labeled glycoprotein IIb (GPIIb) and glycoprotein IIIa (GPIIIa) on the platelets of these patients was considerably less than normal but was detectable by immunoprecipitation, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and autoradiography. The amount of residual GPIIb and GPIIIa as measured by binding studies with radiolabeled monoclonal antibodies was between 3% and 12% of the normal value. Platelet suspensions from these patients did not aggregate with fibrinogen and did not bind 125I-fibrinogen on stimulation with ADP. However, incubation of these platelets with chymotrypsin or pronase resulted in fibrinogen binding and platelet aggregation. Monoclonal antibodies specific for the GPIIb-GPIIIa complex blocked both the fibrinogen binding and the aggregation of enzyme-treated platelets. The treatment of washed platelets of a fourth thrombasthenic patient with ADP or with chymotrypsin failed to result in fibrinogen binding and aggregation. However, the level of GPIIb and GPIIIa on these platelets as measured by a Western blot technique and by monoclonal antibody binding amounted to less than 0.35% to 0.5% of normal values. In conclusion, fibrinogen binding sites exposed on thrombasthenic platelets by chymotrypsin are derived from GPIIb-GPIIIa molecules. Aggregation of chymotrypsin-treated thrombasthenic platelets by fibrinogen appears to represent a sensitive test for detection of functionally active GPIIb-GPIIIa complex on the platelet surface.

Antibodies, Monoclonal↗
PubMed1985

Residual amounts of glycoproteins IIb and IIIa may be present in the platelets of most patients with Glanzmann's thrombasthenia.

Glanzmann's thrombasthenia is an inherited bleeding disorder characterized by abnormalities of platelet membrane glycoproteins (GP) IIb and IIIa. Most patients, usually designated as type I, have been reported to have undetectable levels of GP IIb and GP IIIa with the assay used. We have used polyclonal rabbit antibodies against GP IIb and GP IIIa in a sensitive immunoblot procedure capable of revealing trace amounts of these glycoproteins. Platelets from nine thrombasthenic patients, including seven with type I disease, were studied. GP IIIa, although decreased, was clearly detectable in platelets of eight patients and GP IIb was identified in five. Our findings suggest that residual quantities of GP IIb and GP IIIa are present in most patients with thrombasthenia and therefore that major deletions in the gene or genes encoding these proteins are uncommon.

Animals↗
PubMed1984

Expression of platelet membrane glycoproteins and alpha-granule proteins by a human erythroleukemia cell line (HEL).

We demonstrate that HEL, a human erythroleukemic cell line, has numerous megakaryocytic markers which were markedly enhanced following the addition of the inducers dimethyl sulfoxide or 12-O-tetradecanoylphorbol-13-acetate to the culture medium. Ultrastructural and cytochemical studies showed: (i) the presence of organelles morphologically resembling the platelet alpha-granules; and (ii) a peroxidase activity with the same characteristics as that specifically found in platelets. The platelet alpha-granule proteins (von Willebrand factor, platelet factor-4 and beta-thromboglobulin) were immunologically detected in the HEL cell cytoplasm and their amounts increased after induction. Of particular interest was the presence of platelet membrane proteins. A monoclonal antibody specific for glycoprotein Ib bound to HEL cells. Platelet membrane glycoproteins IIb and IIIa were identified on intact cells using specific antibodies in a binding assay or in cell lysates using either crossed immunoelectrophoresis or an immunoblotting procedure following SDS-polyacrylamide gel electrophoresis. Most HEL cells also expressed the platelet alloantigen PIA1. All of the platelet membrane proteins were present in higher amounts after induction. Glycophorin A, specific for the erythroid lineage, was also detected on HEL cells. Thus, while confirming the presence of erythroid markers, our studies provide evidence that the HEL cell line also expresses platelet antigens. As such, HEL cells represent a unique system with which to study the biosynthesis of platelet-specific proteins and glycoproteins.

Beta-Globulins↗
PubMed1984

Membrane-bound hemagglutinin mediates antibody and complement-dependent lysis of influenza virus-treated human platelets in autologous serum.

Influenza A virus-treated human platelets were lyzed in autologous serum. Lysis required the presence of antibody and occurred predominantly through activation of the classical complement pathway. Binding of the virus followed by its elution at 37 degrees C resulted in a dose-dependent desialation of the cells with a maximal release of 45% of total platelet sialic acid. In contrast, platelets that had been treated with Vibrio cholerae neuraminidase and from which 55% of total sialic acid had been removed were not lyzed in autologous serum and did not bind C3 as shown in binding assays using radiolabeled monoclonal anti-C3 antibody. Thus, the immune-mediated lysis of virus-treated platelets in autologous serum did not involve neoantigens expressed by desialated cells. To assess the effect of viruses on the platelet surface, treated platelets were incubated with galactose oxidase and sodium [3H]borohydride prior to separation and analysis of the labeled glycoproteins by SDS-PAGE. Viral treatment resulted in a desialation of each of the surface glycoproteins. At the same time, a labeled component of Mr 72,000 (nonreduced) and Mr 55,000 (reduced) was observed that was not present when V. cholerae-desialated platelets were examined in the same way. Immunoblotting experiments performed using antiwhole virus and anti-hemagglutinin antibodies demonstrated this component to be viral hemagglutinin. Involvement of membrane-bound hemagglutinin in antibody and in complement-mediated lysis of virus-treated platelets in autologous serum was supported by the increased lytic activity of a postvaccinal serum containing an elevated titer of complement fixing anti-hemagglutinin antibodies. Binding of a viral protein to the platelet surface provides a model for immune thrombocytopenias occurring during acute viral infections at the time of the specific immune response.

Adult↗
PubMed1984

Immunochemical characterization of the platelet-specific alloantigen Leka: a comparative study with the PlA1 alloantigen.

We report the immunochemical characterization of a new platelet-specific alloantigen detected using an IgG antibody isolated from the serum of a patient with posttransfusion purpura (PTP). In indirect immunoprecipitation experiments, the antibody, termed anti-Leka, predominantly precipitated glycoprotein (GP) IIb from Triton X-100 lysates of normal human platelets. In an immunoblot procedure, which involved the transfer of platelet polypeptides separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to nitrocellulose membrane, anti-Leka bound exclusively to GP IIb. Under identical conditions, four anti-PlA1 antibodies each reacted with GP IIIa. No binding of anti-Leka IgG occurred to Leka (-) platelets or to their separated polypeptides although GP IIb was normally detected by Coomassie blue staining. After electrophoresis of reduced platelet proteins, the Leka determinant was localized to the IIb alpha chain. Thus, unlike the PlA1 antigen, the Leka determinant was not destroyed by disulfide reduction. Analysis of platelets from a patient with Glanzmann's thrombasthenia revealed little or no binding in the GP IIb position. Anti-Leka permitted the identification of 76,000 and 60,000 dalton fragments of GP IIb retained by the platelet following chymotrypsin treatment. Our results further highlight the immunogenicity of the GP IIb-IIIa complex. They also suggest that antibodies against GP IIb can cause the thrombocytopenia observed in PTP and that anti-PlA1 antibodies do not account exclusively for the pathophysiology of this immune disorder.

Adult↗
PubMed1984

The human platelet membrane glycoprotein complex GP IIb-IIIa expresses antigenic sites not exposed on the dissociated glycoproteins.

A number of recent reports have described murine monoclonal antibodies that react specifically with the complex formed by human platelet membrane glycoproteins (GP) IIb and IIIa. We show that the IgG L, a previously described human alloantibody isolated from a polytransfused thrombasthenia patient, has similar properties. When used in non-precipitating amounts in crossed immunoelectrophoresis (CIE), 125I-IgG L bound strongly to the IIb-IIIa complex. However, after dissociation of the complex with EDTA, only a weak binding to GP IIb and no binding to GP IIIa was detected. In further studies, increased amounts of IgG L were interacted with 125I-labeled membrane glycoproteins in (a) CIE and (b) classical indirect immunoprecipitation experiments. Although the antibody was able to quantitatively precipitate the IIb-IIIa complex from Triton X-100-soluble extracts of platelet membranes, no precipitation of GP IIb or GP IIIa was observed after divalent cation chelation. Addition of EDTA to immunoprecipitates containing GP IIb-IIIa resulted in dissociation and partial release of both glycoproteins. The interaction of the IgG L with electrophoretically separated GP IIb and GP IIIa was studied using a Western blot procedure in the presence of Ca2+, Mg2+, or EDTA. The presence of divalent cations did not increase the reactivity of the antibody with the individual glycoproteins. Overall, our results show that acquired antibodies to IIb-IIIa, such as the IgG L, may predominantly react with complex-dependent determinants.

Animals↗
PubMed1982

Surface modifications in the platelets of a patient with alpha-N-acetyl-D-galactosamine residues, the Tn-syndrome.

The Tn-syndrome is an acquired disorder characterized by the polyagglutination of blood cells and the pathological exposure of alpha-N-acetyl-D-galactosamine residues (Tn-antigen) at the cell surface. We now report studies on the platelet of a patient (Ba.) of which 81% reacted positively with a fluorescein conjugate of Helix pomatia agglutinin (HPA). The surface proteins of Ba. platelets were labeled with 125I by the lactoperoxidase-catalyzed procedure; single and two-dimensional electrophoresis on sodium dodecyl sulfate (SDS)-polyacrylamide gels was followed by autoradiography that revealed normal 125I-labeling of the major membrane glycoproteins (GP) but that GP Ib had a faster than normal migration. the abnormal GP Ib of Ba. platelets was strongly labeled when platelet suspensions were treated sequentially with neuraminidase, galactose oxidase, and sodium [3H]borohydride. Unlike the GP Ib of normal human platelets, it was also strongly labeled when Ba. platelets were treated with galactose oxidase and sodium [3H]borohydride alone. Both the alloantigen, PlA1, and quinidine-dependent antibody receptor activity were normally expressed by Ba. platelets, which also bound a monoclonal antibody (AN51) to GP Ib. Analysis of Ba. platelets by crossed immunoelectrophoresis using a rabbit anti-human platelet antibody preparation revealed the presence of an immunoprecipitate in the GP Ib position that had an abnormal appearance and migration in the second dimension. An altered position of the precipitate given by Factor VIIIR:Ag was also noted. Incorporation of HPA into the agarose gel during the first dimension electrophoresis resulted in the specific precipitation of the abnormal GP Ib of Ba. platelets. Our studies show that circulating Tn-platelets contain GP Ib with a modified oligosaccharide chain structure responsible for the platelet expression of Tn-antigen activity.

Acetylgalactosamine↗
PubMed1981

Monoclonal antibody to human platelet glycoprotein I. II. Effects on human platelet function.

The effect on platelet function of a monoclonal platelet antibody to platelet membrane glycoprotein I was tested. This antibody, AN51, inhibited ristocetin or bovine factor VIII-induced aggregation but did not modify ADP, collagen type I or type III, thrombin or arachidonic acid induced aggregations. Furthermore, the adhesion-aggregation of platelets induced by microfibrils was also inhibited by the antibody. Platelet adhesion to rabbit aorta subendothelium was impaired by the antibody. The persistent adhesion of platelets to collagenase-treated subendothelium was also inhibited. These findings strongly suggested that platelet membrane glycoprotein I could interact with a non-collagenic microfibrillar component of subendothelium. The binding of factor VIII/von Willebrand factor to platelet membrane in the presence of ristocetin was decreased in the binding site for factor VIII/von Willebrand factor to allow platelet adhesion to subendothelium.

Antibodies, Monoclonal↗
PubMed1977

[Schizophrenia: association of the paranoid form and antigens HLA-A9 and B5].

30 unrelated Schizophrenic patients (15 Hebephrenic and 15 Paranoïd forms) were Typed for 14 A and 16 B Specificities by microlymphocytotoxicity. We note: 10 (5H + 5P) HLA--A1 (33.3 % / controls 23.4 %) 5 (2H + 3P) HLA--A3 (16.6 % / controls 26.2 %) 6 (5H + 1P) HLA--B8, 0 HLA--B27, 0 HLA BW16 and more specially: 10 (3H + 7P) HLA--A9 (23 + 24) 8 (2H + 6P) HLA--B5 These two last antigens are more often found in the Paranoïd patients of whom 46.6% are A9 Tcontrols: 24 %) and 40 % are B5 (controls 13.2 %. The Pc values are not significant in these too short seria but the strongly increased frequencies of A9 and B5 in the Paranoïd group seem to open a new biological aspect in the nosographic distinction of the Schizophrenia and so confirm previous results of C. L. CAZZULLO et al.

Epitopes↗