scholarly journals Single point mutation in human glycoprotein IIIa is associated with a new platelet-specific alloantigen (Mo) involved in neonatal alloimmune thrombocytopenia

Blood ◽  
1993 ◽  
Vol 81 (1) ◽  
pp. 70-76 ◽  
Author(s):  
RW Kuijpers ◽  
S Simsek ◽  
NM Faber ◽  
R Goldschmeding ◽  
RK van Wermerkerken ◽  
...  
Keyword(s):  
Dna Analysis ◽  
Single Point ◽  
Blot Hybridization ◽  
Platelet Glycoprotein ◽  
Single Point Mutation ◽  
Polymorphism Analysis ◽  
Coding Region ◽  
Dot Blot ◽  
Glycoprotein Iiia ◽  
Alloimmune Thrombocytopenia

Here we describe a new platelet-specific alloantigen that was identified in a case of neonatal alloimmune thrombocytopenia. This antigen has provisionally been called “Mo.” By studying the Mo family, it was shown to be inherited in an autosomal dominant manner. Immunoprecipitation and Western blot analysis showed that the antigen resides on platelet glycoprotein IIIa (GP IIIa). Genomic analysis, performed by applying polymerase chain reaction and sequencing, showed a C-->G substitution of base pair 1267 of the coding region of the DNA for GP IIIa, resulting in a substitution of Proline407 by Alanine407. That this substitution is associated with the antigen could be demonstrated by restriction fragment length polymorphism analysis of cDNA, prepared from platelet RNA, and of genomic DNA. It was confirmed by dot-blot hybridization with allele-specific oligonucleotides. All family members, also those being Mo antigen-positive, were healthy. None of them appeared to suffer from increased tendency of bleeding or thrombosis. Thus, the Mo mutation does not lead to significant platelet dysfunction in vivo with heterozygous carriers. One of 450 random healthy blood donors who were tested was positive for the Mo antigen. Typing was performed by the classical serologic methods as well as by DNA analysis.

scholarly journals Single point mutation in human glycoprotein IIIa is associated with a new platelet-specific alloantigen (Mo) involved in neonatal alloimmune thrombocytopenia

Blood ◽  
1993 ◽  
Vol 81 (1) ◽  
pp. 70-76 ◽  
Author(s):  
RW Kuijpers ◽  
S Simsek ◽  
NM Faber ◽  
R Goldschmeding ◽  
RK van Wermerkerken ◽  
...  
Keyword(s):  
Dna Analysis ◽  
Single Point ◽  
Blot Hybridization ◽  
Platelet Glycoprotein ◽  
Single Point Mutation ◽  
Polymorphism Analysis ◽  
Coding Region ◽  
Dot Blot ◽  
Glycoprotein Iiia ◽  
Alloimmune Thrombocytopenia

Abstract Here we describe a new platelet-specific alloantigen that was identified in a case of neonatal alloimmune thrombocytopenia. This antigen has provisionally been called “Mo.” By studying the Mo family, it was shown to be inherited in an autosomal dominant manner. Immunoprecipitation and Western blot analysis showed that the antigen resides on platelet glycoprotein IIIa (GP IIIa). Genomic analysis, performed by applying polymerase chain reaction and sequencing, showed a C-->G substitution of base pair 1267 of the coding region of the DNA for GP IIIa, resulting in a substitution of Proline407 by Alanine407. That this substitution is associated with the antigen could be demonstrated by restriction fragment length polymorphism analysis of cDNA, prepared from platelet RNA, and of genomic DNA. It was confirmed by dot-blot hybridization with allele-specific oligonucleotides. All family members, also those being Mo antigen-positive, were healthy. None of them appeared to suffer from increased tendency of bleeding or thrombosis. Thus, the Mo mutation does not lead to significant platelet dysfunction in vivo with heterozygous carriers. One of 450 random healthy blood donors who were tested was positive for the Mo antigen. Typing was performed by the classical serologic methods as well as by DNA analysis.

scholarly journals A novel murine model of fetal and neonatal alloimmune thrombocytopenia: response to intravenous IgG therapy

Blood ◽  
2006 ◽  
Vol 107 (7) ◽  
pp. 2976-2983 ◽  
Author(s):  
Heyu Ni ◽  
Pingguo Chen ◽  
Christopher M. Spring ◽  
Ebrahim Sayeh ◽  
John W. Semple ◽  
...  
Keyword(s):  
High Titer ◽  
Maternal Antibodies ◽  
Platelet Glycoprotein ◽  
Wild Type ◽  
Wild Type Male ◽  
Glycoprotein Iiia ◽  
Life Threatening ◽  
Β3 Integrin ◽  
Pathogenic Antibodies ◽  
Alloimmune Thrombocytopenia

AbstractFetal and neonatal alloimmune thrombo cytopenia (FNAITP) is a life-threatening bleeding disorder caused by maternal antibodies directed against fetal platelet antigens. The immunoreactive epitopes in FNAITP are primarily located in the extracellular regions of the platelet glycoprotein IIIa (β3 integrin). Here we have established a novel animal model of FNAITP using β3 integrin–deficient (β3-/-) mice. We demonstrated first that these mice are immunoresponsive to β3 integrin; β3-/- mice transfused with wild-type platelets generated specific anti–β3 antibodies which were able to induce thrombocytopenia in wild-type mice. Subsequently, β3-/- female mice (both naive and immunized) were bred with wild-type male mice to recapitulate the features of FNAITP. The titer of generated maternal antibodies correlated with the severity of FNAITP. High titer maternal anti–β3 anti-bodies caused severe fetal thrombocytopenia, intracranial hemorrhage, and even miscarriage. Furthermore, maternal administration of intravenous immunoglobulin G (IgG) ameliorated FNAITP and down-regulated pathogenic antibodies in both the maternal and fetal circulations.

scholarly journals A new low-frequency alloantigen (Khab) located on platelet glycoprotein IIIa as a cause of maternal sensitization leading to neonatal alloimmune thrombocytopenia

Transfusion ◽  
2014 ◽  
Vol 55 (6pt2) ◽  
pp. 1584-1585 ◽  
Author(s):  
Mia J. Sullivan ◽  
Julie Peterson ◽  
Janice G. McFarland ◽  
Daniel Bougie ◽  
Richard H. Aster ◽  
...  
Keyword(s):  
Low Frequency ◽  
Platelet Glycoprotein ◽  
Glycoprotein Iiia ◽  
Alloimmune Thrombocytopenia

HLA class II genotyping: two assay systems compared

1995 ◽  
Vol 41 (4) ◽  
pp. 553-556 ◽  
Author(s):  
J Thonnard ◽  
F Deldime ◽  
M Heusterspreute ◽  
B Delepaut ◽  
F Hanon ◽  
...  
Keyword(s):  
Large Scale ◽  
Clinical Laboratory ◽  
Blot Hybridization ◽  
Human Leukocyte ◽  
Hla Typing ◽  
High Rate ◽  
Polymorphism Analysis ◽  
Dot Blot ◽  
Pcr Products ◽  
Typing Methods

Abstract In the last few years, a variety of DNA-based human leukocyte antigen (HLA) typing methods have emerged, revealing the extreme polymorphism of HLA genes. This polymorphism makes it difficult for a clinical laboratory to establish the best HLA typing strategy. In this study we have compared two techniques for performing HLA-DRB typing: a commercial rapid assay based on the polymerase chain reaction (PCR) followed by reverse dot-blot hybridization of the PCR products (the Inno-LiPA assay), and a method based on PCR followed by restriction fragment length polymorphism analysis. We found that both methods provide reliable results with a high rate of concordance (97%) and that Inno-LiPA is convenient for large-scale routine typing. However, if a high-resolution allelic typing is required, each method lacks accuracy but using them in association improves the accuracy of the results.

scholarly journals Phenotypic and genotypic traits and epidemiological implication of Vibrio cholerae O1 and O139 strains in India during 2003

2007 ◽  
Vol 56 (6) ◽  
pp. 824-832 ◽  
Author(s):  
Souvik Chatterjee ◽  
Kausik Ghosh ◽  
Amit Raychoudhuri ◽  
Arpita Pan ◽  
Mihir Kumar Bhattacharya ◽  
...  
Keyword(s):  
Vibrio Cholerae ◽  
Point Mutation ◽  
Dna Analysis ◽  
Single Point ◽  
Striking Similarity ◽  
Single Point Mutation ◽  
Randomly Amplified Polymorphic Dna ◽  
Vibrio Cholerae O1 ◽  
Almost All ◽  
Pfge Profiles

During 2003, Vibrio cholerae O1 Ogawa was the predominant serotype among diarrhoeal patients admitted to different hospitals in India. With the exception of 3 strains from Kolkata, none of 172 strains examined exhibited resistance to tetracycline, but 45.7 % showed reduced susceptibility to ciprofloxacin. Extensive molecular characterization using randomly amplified polymorphic DNA analysis, ribotyping and PFGE revealed that almost all the strains within a serogroup were clonally related. Along with the H pulsotype, a newly described L pulsotype of recently emerged O1 Inaba strains was detected among the O1 Ogawa strains from 2003. The striking similarity in their molecular properties and antibiograms indicated that at least certain clones of recently emerged Inaba strains from 2004 may have evolved from O1 Ogawa strains. This view was further supported by the detection of a nearly identical wbeT region among the O1 Ogawa and recently emerged Inaba strains, the latter differing only by a single point mutation. Since 2003, a hiatus in the isolation of serogroup O139 was observed and these strains share the same PFGE profiles as those isolated during 2000. Organization of tandemly arranged CTXEl, CTXCal and truncated CTXCal (devoid of ctxAB) prophages was unique among the majority of these O139 strains.

C1GALT1C1 (COSMC) Mutations in Four Individuals with Tn+ Phenotype.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1727-1727
Author(s):  
Vanja Karamatic Crew ◽  
Carole Green ◽  
Stephen Parsons ◽  
Belinda K. Singleton ◽  
Geoff Daniels ◽  
...  
Keyword(s):  
Cell Line ◽  
Dna Analysis ◽  
Single Point ◽  
Jurkat Cells ◽  
Erythroid Cell ◽  
Single Point Mutation ◽  
Wild Type ◽  
Membrane Glycoproteins ◽  
Hemopoietic Cells ◽  
Elevated Expression

Abstract Tn is a cryptantigen located on O-linked oligosaccharides of cell membrane glycoproteins and is composed of N-acetylgalactosamine a -linked to serine or threonine of the protein backbone. Tn is expressed on hemopoietic cells of individuals with the rare idiopathic Tn syndrome, characterized by a variable pattern of Tn expression suggestive of its somatic and clonal origin. Tn is also associated with overexpression in several autoimmune diseases and malignancies. Tn is a bioprecursor of the T cryptantigen and subsequently the disialotetrasaccharide units typical of O-linked oligosaccharides of RBC sialoglycoproteins. Tn results from defective oligosaccharide biosynthesis caused by the malfunction of T-synthase, a b1,3-galactosyltransferase. Recent evidence suggested that the activity of T-synthase is dependent on a molecular chaperone, Cosmc (Ju and Cummings, PNAS2002;99:16613–18). In view of this evidence, we investigated whether Cosmc is required for T-synthase activity and ultimately for Tn phenotype by obtaining material from 4 apparently healthy, unrelated Caucasian individuals with Tn phenotype. Case 1 was 93.9% Tn+ on an EBV-transformed lymphoblastoma cell line. Analysis of the Cosmc gene, C1GALT1C1, showed a homozygous 428C>T, Ala143Val mutation and Case 1 showed a complete lack of expression of C1GALT1C1 cDNA. Case 2 revealed no C1GALT1C1 mutations in DNA extracted from plasma, but in DNA of lymphocyte origin an apparent heterozygous 454G>A, Glu152Lys change was observed, in agreement with Ju and Cummings (Nature2005, 437:1252). When Case 2 hemopoietic progenitor cells were expanded into the erythroid cell line, the mutation appeared homozygous. Tn expression varied from 76.1% in lymphocytes, 90.7% in RBCs to 96.9% in erythroblasts. Case 3 showed 19.4% Tn+ lymphocytes, compared to 97.0% Tn+ RBCs. In Case 3 we found a single point mutation 577T>C, Ser193Pro. Case 4 exhibited 46.5% Tn+ lymphocytes, 96.0% Tn+ RBCs and 90.2% Tn+ erythroblasts. DNA analysis revealed 3G>C, converting the translation-initiating methionine to isoleucine and predicting the loss of first 12 amino acids of the protein, potentially altering its morphology. The mutations found in Cases 2–4 reflected the clonal nature of Tn, appearing heterozygous in DNA of lymphocyte origin and homozygous in DNA of erythroid origin. To confirm that the observed mutations are indeed responsible for Tn phenotype, pBabe puro vector with Tn or wild type C1GALT1C1 inserts was transfected into Jurkat cells. Untransfected cells and cells transfected with bare vector expressed Tn. Jurkat cells transfected with wild-type C1GALT1C1 were Tn-negative while those transfected with C1GALT1C1 from Cases 2–4 expressed Tn. From this evidence we postulate that Cosmc is directly involved in the expression of Tn phenotype. To investigate the involvement of other genes, we performed expression profiling of 3 Tn and 4 control samples hybridized to HG-U133A arrays. A list of 100 up-regulated and 173 down-regulated genes, with 1.5× fold difference in expression, was obtained. Some genes, relating to erythrocyte development/heme biosynthesis were upregulated, while down-regulated genes were related to cholesterol/lipid metabolism. Real-time Q-PCR on six differentially expressed genes of interest, down-regulated FABP5, CYP1B1 and LRP8 and up-regulated AQP1, AQP3 and EPB42, confirmed the microarray results, and elevated expression of AQP3 on Tn + RBCs was detected serologically. The effects of C1GALT1C1 mutations are wider than Tn expression on hemopoietic cells.

scholarly journals Disruption of the Cys5-Cys7 disulfide bridge in the platelet glycoprotein Ibβ prevents the normal maturation and surface exposure of GPIb-IX complexes

2003 ◽  
Vol 90 (09) ◽  
pp. 456-464 ◽  
Author(s):  
Nora Butta ◽  
Gema Iruín ◽  
Sonia Alonso ◽  
Matilde Ayuso ◽  
Roberto Parrilla ◽  
...  
Keyword(s):  
Single Point ◽  
Molecular Genetic ◽  
Genetic Defect ◽  
Flow Cytometric Analysis ◽  
Surface Expression ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Platelet Glycoprotein ◽  
Single Point Mutation ◽  
Platelet Content

SummaryThis work aimed at elucidating the molecular genetic defect in two related patients with Bernard-Soulier syndrome (BSS) phenotype. Flow cytometric analysis revealed undetectable levels of platelet glycoproteins (GP), Ibα and IX, although plasma glycocalicin was detectable in both cases. The complete sequencing of GPIbα, GPIbβ, and GPIX revealed the presence of a single point mutation, a G to A substitution, in codon 30 of GPIbβ, that changes Cys5 to Tyr. The parents and sibling of the patients, heterozygotes for this mutation, were asymptomatic and they all showed a reduced platelet content of GPIbα and GPIX. Transient transfection of the mutant GPIbβ subunit failed to render surface expression of GPIbα and exerted a dominant-negative effect on the surface exposure of the GPIb-IX complex. Metabolic labelling and immunoprecipitation analysis of transfected cells indicated that [5Tyr]GPIbβ may associate with GPIX and GPIbα, but the maturation of the GPIb-IX complex is impaired. Substitution of either Cys5 or Cys7 by Ala failed to show surface expression of GPIb-IX, suggesting that the Cys5-Cys7 disulfide loop in GPIbβ is essential for the efficient processing and trafficking of GPIb-IX complexes toward the plasma membrane. Our findings indicate that the identified novel GPIbβ mutation is responsible for the BSS phenotype of the patients and provide an explanation for the molecular mechanism underlying the reduced platelet content of GPIb-IX complex in the heterozygous individuals.

Specific Cross-reaction of IgG Anti-phospholipid Antibody with Platelet Glycoprotein IIIa

1996 ◽  
Vol 75 (01) ◽  
pp. 168-174 ◽  
Author(s):  
Shigeru Tokita ◽  
Morio Arai ◽  
Naomasa Yamamoto ◽  
Yasuhiro Katagiri ◽  
Kenjiro Tanoue ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Heparan Sulfate ◽  
Disulfide Bonds ◽  
Dextran Sulfate ◽  
Human Platelets ◽  
Platelet Glycoprotein ◽  
Activated Platelets ◽  
Fibrinogen Binding ◽  
Dose Dependent Fashion ◽  
Glycoprotein Iiia

SummaryTo study the pathological functions of anti-phospholipid (anti-PL) antibodies, we have analyzed their effect on platelet function. We identified an IgG anti-PL mAb, designated PSG3, which cross-reacted specifically with glycoprotein (GP) IIIa in human platelets and inhibited platelet aggregation. PSG3 bound also to certain polyanionic substances, such as double-stranded DNA, heparan sulfate, dextran sulfate and acetylated-LDL, but not to other polyanionic substances. The binding of PSG3 to GPIIIa was completely inhibited by heparan sulfate and dextran sulfate, indicating that PSG3 recognizes a particular array of negative charges expressed on both GPIIIa and the specified polyanionic substances. Since neither neuraminidase- nor endoglycopeptidase F-treatment of GPIIIa had any significant effect on the binding of PSG3, this array must be located within the amino acid sequence of GPIIIa but not in the carbohydrate moiety. Reduction of the disulfide bonds in GPIIIa greatly reduced its reactivity, suggesting that the negative charges in the epitope are arranged in a particular conformation. PSG3 inhibited platelet aggregation induced by either ADP or collagen, it also inhibited fibrinogen binding to activated platelets in a dose-dependent fashion. PSG3, however, did not inhibit the binding of GRGDSP peptide to activated platelets. These results suggest that the PSG3 epitope on GPIIIa contains a particular array of negative charges, and possibly affects the fibrinogen binding to GPIIb/IIIa complex necessary for platelet aggregation.

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