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

Abstract

The Tn-syndrome is an acquired disorder characterized by the polyagglutination of blood cells and the pathological exposure of a-N-acetyl-D-galactosamine residues (Tn-antigen) at the cell surface. We now report studies on the platelets 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 twodimensional electrophoresis on sodium dodecyl sulfate (SDS)-polyacrylamide gels was followed by autoradiography that revealed normal '251-labeling of the major membrane glycoproteins (GP) but that GP lb had a faster than normal migration. The abnormal GP lb 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, PHAl, and quinidine-dependent antibody receptor activity were normally expressed by Ba. platelets, which also bound a monoclonal antibody (AN51) to GP lb. Analysis of Ba. platelets by crossed immunoelectrophoresis using a rabbit anti-human platelet antibody preparation revealed the presence of an immunoprecipitate in the GP lb 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 lb of Ba. platelets.Address reprint requests to Dr. Nurden, H6pital Lari-boisiere.

Thrombocytopenia and kidney disease in mice with a mutation in the C1galt1 gene

“…Mild hemolysis, thrombocytopenia, and͞or leucopenia is thought to ensue via reactivity of the Tn antigen with naturally occurring anti-Tn antibodies (7,21). Consistent with our findings in plt1͞plt1 mice, gpIb has been shown to be a major Tn-bearing protein in at least one Tn-syndrome patient (22). Because thrombocytopenia in plt1͞plt1 mice appears not to be caused by immune-mediated clearance, the phenotype of the plt1͞plt1 mice suggest that an intrinsic action of gp1b␣ is required for appropriate megakaryocyte maturation and platelet release and that thrombocytopenia of Tn syndrome might potentially also have a nonimmune, megakaryocyte-intrinsic component.…”

“…IgA levels in plt1͞plt1 mice were not elevated (data not shown), and the development of renal disease in rag1 Ϫ/Ϫ plt1͞plt1 mice excludes an Ig-mediated pathogenesis. Although reduced C1GalT1 activity has been shown clearly in B cells from IgA nephropathy patients (22), the phenotype of plt1͞plt1 mice suggests that reduced C1GalT1 activity in the kidney may cause renal pathology independent of immune effects and the potential contribution of intrinsic deficiency in C1GalT1 to renal disease may be worthy of closer examination in IgA nephropathy and other kidney disorders. In addition, mutations in several key kidney podocyte proteins have been implicated in congenital human diseases, including nephrin in congenital nephritic syndrome, podocin in steroid-resistant nephritic syndrome, and ␣-actinin-4 in focal segmental glomerular sclerosis (23).…”

Variations in glycosylation of von Willebrand factor with O-linked sialylated T antigen are associated with its plasma levels

“…36,37 Consequently, the O-linked carbohydrate structure consists predominantly of the Tn antigen (GalNAc␣1-Ser/ Thr) instead of the T antigen, and this Tn antigen is not recognized by PNA. Of interest, platelet VWF of patients with the Tn syndrome reacts differently with anti-VWF antibodies when assessed via crossed immunoelectophoresis, 38 underscoring the notion that O-linked glycosylation is an essential element of VWF biology. Of note, mice genetically deficient for T synthase indeed lack T-antigen-bearing proteins, and these knock-out mice display defective angiogenesis and fatal embryonic hemorrhage.…”

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

“…Appropriate controls were performed in the absence of added virus particles or V. cholerae neuraminidase. 3H-labeling of the surface glycoproteins oftreated platelets was performed according to the procedure of Nurden et al (31). In brief, suspensions of 109 platelets in I ml phosphate-buffered saline (PBS), pH 7.4, were first incubated with 10 U/ml of galactose oxidase (Kabi Diagnostics, Stockholm, Sweden) for 5 min at 37°C.…”

“…These included the high and low molecular weight standards used for molecular weight determination by SDS-PAGE. Samples (100 ug protein) were analyzed by electrophoresis on 7-12% gradient acrylamide slab gels (31,32). Proteins were detected by Coomassie blue staining and 3H-labeled glycoproteins by fluorography using EN3HANCE (New England Nuclear, Boston, MA) (31).…”