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  • 1
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    Quantitation of membrane glycoprotein IIIa on intact human platelets using the monoclonal antibody, AP-3 (1985)
    American Society of Hematology
    Details
    Publication Date: 1985-01-01
    Description: A murine monoclonal antibody specific for glycoprotein (GP)IIIa was prepared by immunization with a GPIIb- and GPIIIa-enriched Triton X-114 extract of platelet membranes. This antibody, designated AP-3, was shown by indirect immunoprecipitation to react solely with GPIIIa derived from either P1A1-positive or -negative individuals. The epitope on GPIIIa recognized by AP-3 is expressed on dissociated GPIIIa as well as on Ca+2-dependent complexes of GPIIb and GPIIIa, as shown by crossed immunoelectrophoresis in the presence or absence of EDTA. A previously described monoclonal antibody specific for the GPIIb/IIIa complex (AP- 2) inhibited platelet aggregation induced by ADP, thrombin, collagen, or arachidonic acid (Pidard et al, J Biol Chem 258:12582–12586, 1983). In contrast, AP-3 had no effect on aggregation induced by any of these reagents, a finding similar to that previously reported for the GPIIb- specific monoclonal antibody, Tab (McEver et al, J Clin Invest 66:1311- 1318, 1980). At saturation, 40,200 AP-3 molecules were bound per platelet, a value similar to that obtained for AP-2 or Tab. Thus, data derived using AP-3 indicate that significant amounts of free GPIIIa are not present, thereby supporting the hypothesis that GPIIb and GPIIIa exist complexed in a 1:1 stoichiometry in the plasma membrane of intact, nonactivated platelets.
    Print ISSN: 0006-4971
    Electronic ISSN: 1528-0020
    Topics: Biology , Medicine
    Published by American Society of Hematology
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  • 2
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    Polymorphism of human platelet membrane glycoprotein IIb associated with the Baka/Bakb alloantigen system (1990)
    American Society of Hematology
    Details
    Publication Date: 1990-06-15
    Description: The human Baka/Bakb alloantigen system has been implicated in the pathogenesis of post-transfusion purpura and neonatal alloimmune thrombocytopenic purpura. Human alloantisera specific for either the Baka or Bakb allele have been shown to react exclusively with the heavy chain of membrane glycoprotein (GP) IIb. To investigate the structure of the Bak epitopes, we used the polymerase chain reaction (PCR) to amplify GPIIb cDNA synthesized from platelet RNA samples prepared from individuals of known serologic phenotype. Subsequent DNA sequence analysis of amplified GPIIb cDNAs derived from one Baka homozygous individual and one Bakb homozygous individual revealed a single nucleotide base difference near the 3′ end of the mRNA encoding the GPIIb heavy chain. Short 13 base allele-specific oligonucleotides (ASO) containing the putative phenotype-specific base in the middle were then synthesized, end-labeled with digoxigenin-11-dUTP using terminal transferase, and used as probes in subsequent dot-blot hybridization experiments. Platelet RNA was prepared from a panel made up of four Baka/a, three Bakb/b, and two Baka/b individuals, and the mRNA encoding GPIIb was amplified using PCR and spotted onto nylon membranes. ASO hybridization showed that the nucleotide base difference identified above segregated with Bak phenotype in all nine individuals examined (P = .002). The base pair substitution results in an amino acid polymorphism at residue 843 of the mature heavy chain. The Baka form of GPIIb encodes an isoleucine at this position, whereas the Bakb allele contains a serine. Identification of the polymorphism associated with this clinically important alloantigen system should permit new therapeutic and diagnostic approaches for treating and managing patients with alloimmune thrombocytopenic disorders.
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    Published by American Society of Hematology
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  • 3
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    A dinucleotide deletion in exon 4 of the PlA2 allelic form of glycoprotein IIIa: implications for the correlation of serologic versus genotypic analysis of human platelet alloantigens (1996)
    American Society of Hematology
    Details
    Publication Date: 1996-11-15
    Description: Platelets from a patient with a suspected case of posttransfusion purpura were subjected to alloantigen phenotyping and found to express the PlA1, but not the PlA2, allelic form of human platelet membrane glycoprotein (GP) IIIa on the platelet surface. However, genotyping showed unambiguously that the patient carried the genes for both of these GPIIa alleles. Based on these results, we postulated that the PlA2 allele was silent, ie, that this patient was a carrier for Glanzmann thrombasthenia (GT). Quantitative analysis of GPIIb-IIIa surface expression showed only 20,000 GPIIb-IIIa receptors/platelet, approximately half of the value obtained with control platelets. Southern blot analysis showed no large deletions or insertions within the GPIIIa gene, and amplification of all 14 exons encoding GPIIIa resulted in the production of normal sized polymerase chain reaction (PCR) products in all cases. DNA-sequence analysis showed an AG dinucleotide deletion affecting codons 210 and 211 within exon 4 of the GPIIIa gene, leading to a change in reading frame and the creation of a stop codon 38 nucleotides down-stream. The predicted truncated protein consists of only the first 223 of the normal 762 amino acids, thus accounting for the failure to express the PlA2 allele on the platelet surface. While encountered only rarely, carriers of either GT or Bernard Soulier syndrome that are at the same time heterozygous for human platelet alloantigenic epitopes found on GPIb, GPIIb, or GPIIIa have the possibility to give discrepant results when comparing genotypic versus phenotypic analysis. In such situations, the combination of serologic and DNA-based evaluation contributes complementary and beneficial diagnostic information than either one alone are able to provide.
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  • 4
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    Synergistic action of two murine monoclonal antibodies that inhibit ADP- induced platelet aggregation without blocking fibrinogen binding (1987)
    American Society of Hematology
    Details
    Publication Date: 1987-02-01
    Description: We have used two murine monoclonal antibodies, each directed against one component of the human platelet membrane glycoprotein (GP) IIb-IIIa complex, to examine further the molecular requirements for fibrinogen binding to the platelet surface and subsequent platelet-platelet cohesion (aggregation). Although neither AP3, which is directed against GPIIIa, nor Tab, which is specific for GPIIb, were individually able to inhibit adenosine diphosphate (ADP)-induced fibrinogen binding, platelet aggregation, or secretion, the combination of AP3 and Tab completely abolished platelet aggregation and the release reaction. Unexpectedly, this synergistic inhibition of platelet-platelet cohesion occurred in the presence of apparently normal fibrinogen binding. Both the number of fibrinogen molecules bound and the dissociation constant for fibrinogen binding remained essentially unchanged in the presence of these two antibodies. Inhibition of aggregation was dependent upon the divalency of both AP3 and Tab because substitution of Fab fragments of either antibody for the intact IgG resulted in a complete restoration of both aggregation and secretion. In contrast to ADP induction, thrombin-activated platelets neither aggregated nor bound fibrinogen in the presence of AP3 plus Tab but were fully capable of secretion, which illustrated the multiple mechanisms by which the platelet surface can respond to different agonists. These data demonstrate that fibrinogen binding to the platelet surface alone is not sufficient to support platelet-platelet cohesion and that an additional post-fibrinogen-binding event(s) that is inhibitable by these two monoclonal antibodies may be required.
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  • 5
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    Further characterization of the loop structure of platelet glycoprotein IIIa: partial mapping of functionally significant glycoprotein IIIa epitopes (1991)
    American Society of Hematology
    Details
    Publication Date: 1991-12-15
    Description: Glycoprotein (GP) IIb-IIIa serves as the platelet fibrinogen receptor. Studies of the tertiary structure of GPIIIa have shown that the protein has a large loop structure of at least 325 amino acids in length. To further characterize this loop structure, intact platelets were digested with alpha-chymotrypsin. Digestion products were examined using the anti-GPIIIa monoclonal antibodies (MoAbs) AP3, D3GP3, and C5GP3, as well as the human alloantibody, anti-PLA1. AP3 recognized GPIIIa digestion products of 109, 95, and 68 Kd. D3GP3 and C5GP3 recognized an additional band of 51 Kd. Time course digestions demonstrated that the 51-Kd fragment was generated by proteolysis of the 68-Kd peptide. Sequence analysis of the reduced 51-Kd peptide showed that this fragment began at amino acid 422. The nonreduced 51-Kd peptide was reactive with antibodies directed against the first 13 amino acids of GPIIIa, demonstrating the presence of a covalently attached N-terminal peptide. These data suggest that: (1) the minimum length of the loop structure is at least 384 amino acids; (2) the AP3 epitope is formed at least in part by a determinant contained within residues 348 to 421; and (3) the D3GP3 and C5GP3 epitopes are contained within amino acids 422 to 692 of GPIIIa, a region that may be flexible and involved in conformational changes that occur after ligand binding.
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  • 6
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    The molecular immunology of human platelet proteins (1992)
    American Society of Hematology
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    Publication Date: 1992-09-15
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  • 7
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    Characteristics of quinine- and quinidine-induced antibodies specific for platelet glycoproteins IIb and IIIa (1991)
    American Society of Hematology
    Details
    Publication Date: 1991-06-15
    Description: Recent studies have shown that antibodies characteristic of quinine- and quinidine-induced thrombocytopenia sometimes recognize the platelet membrane glycoprotein (GP) complex IIb/IIIa in addition to their well known target, GPIb/IX. We have investigated the frequency with which drug-induced antibodies bind to GPIIb/IIIa and the nature of their target epitopes. In studies of sera from 13 patients sensitive to quinidine or quinine, we found that 10 contained IgG antibodies specific for both GPIb/IX and GPIIb/IIIa, two reacted with GPIb/IX alone, and one reacted with GPIIb/IIIa alone. In all cases, the presence of drug was required for binding of IgG to target GPs. By immunoabsorption, we found that each of five polyspecific sera contained at least two different antibodies, one reactive with GPb/IX and the other with GPIIb/IIIa. Further studies with eight drug- dependent antibodies (DDAb) specific for GPIIb/IIIa showed that three recognized the GPIIb/IIIa complex only, one recognized GPIIb alone, and three recognized GPIIIa alone. The eighth serum appeared to bind to both GPIIIa alone and to an epitope determined by the GPIIb/IIIa complex. The three antibodies specific for GPIIIa alone also reacted with GPIIIa deglycosylated with endo-H, and with the major (61 Kd) fragment obtained by chymotryptic digestion of GPIIIa but failed to react with reduced GPIIIa. These findings demonstrate that, in drug- induced, immunologic thrombocytopenia, the anti-platelet immune response is typically directed against epitopes on both GPIb/IX and GPIIb/IIIa. The three DDAb we studied that were specific for GPIIIa alone recognize epitopes resistant to chymotrypsin and endo-H treatment that are dependent on intrachain disulfide bonding.
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  • 8
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    Unknown
    Synergistic action of two murine monoclonal antibodies that inhibit ADP- induced platelet aggregation without blocking fibrinogen binding (1987)
    American Society of Hematology
    Details
    Publication Date: 1987-02-01
    Description: We have used two murine monoclonal antibodies, each directed against one component of the human platelet membrane glycoprotein (GP) IIb-IIIa complex, to examine further the molecular requirements for fibrinogen binding to the platelet surface and subsequent platelet-platelet cohesion (aggregation). Although neither AP3, which is directed against GPIIIa, nor Tab, which is specific for GPIIb, were individually able to inhibit adenosine diphosphate (ADP)-induced fibrinogen binding, platelet aggregation, or secretion, the combination of AP3 and Tab completely abolished platelet aggregation and the release reaction. Unexpectedly, this synergistic inhibition of platelet-platelet cohesion occurred in the presence of apparently normal fibrinogen binding. Both the number of fibrinogen molecules bound and the dissociation constant for fibrinogen binding remained essentially unchanged in the presence of these two antibodies. Inhibition of aggregation was dependent upon the divalency of both AP3 and Tab because substitution of Fab fragments of either antibody for the intact IgG resulted in a complete restoration of both aggregation and secretion. In contrast to ADP induction, thrombin-activated platelets neither aggregated nor bound fibrinogen in the presence of AP3 plus Tab but were fully capable of secretion, which illustrated the multiple mechanisms by which the platelet surface can respond to different agonists. These data demonstrate that fibrinogen binding to the platelet surface alone is not sufficient to support platelet-platelet cohesion and that an additional post-fibrinogen-binding event(s) that is inhibitable by these two monoclonal antibodies may be required.
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    Electronic ISSN: 1528-0020
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  • 9
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    Neonatal alloimmune thrombocytopenia due to a new platelet-specific alloantibody (1993)
    American Society of Hematology
    Details
    Publication Date: 1993-06-15
    Description: An infant with severe neonatal alloimmune thrombocytopenia is described in whom an antibody directed at a new platelet-specific alloantigen, Ca (HPA-6b), is implicated. The new alloantigen is of low frequency in the population and was localized to platelet glycoprotein (GP) IIIa. Immunoprecipitation studies using murine monoclonal antibodies specific for the GP complex IIb-IIIa and GPIIIa alone (AP2 and AP3) suggest that the location of the Ca epitope on GPIIIa may be near the binding site for AP3. Neonatal alloimmune thrombocytopenia associated with Ca is likely to be as severe as that seen in cases due to incompatibilities for the HPA-1 (PIA) and HPA-4 (Pen) platelet alloantigen systems, because each is located on GPIIIa, a densely represented molecule on the platelet surface.
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  • 10
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    Polymorphism of human platelet membrane glycoprotein IIb associated with the Baka/Bakb alloantigen system (1990)
    American Society of Hematology
    Details
    Publication Date: 1990-06-15
    Description: The human Baka/Bakb alloantigen system has been implicated in the pathogenesis of post-transfusion purpura and neonatal alloimmune thrombocytopenic purpura. Human alloantisera specific for either the Baka or Bakb allele have been shown to react exclusively with the heavy chain of membrane glycoprotein (GP) IIb. To investigate the structure of the Bak epitopes, we used the polymerase chain reaction (PCR) to amplify GPIIb cDNA synthesized from platelet RNA samples prepared from individuals of known serologic phenotype. Subsequent DNA sequence analysis of amplified GPIIb cDNAs derived from one Baka homozygous individual and one Bakb homozygous individual revealed a single nucleotide base difference near the 3′ end of the mRNA encoding the GPIIb heavy chain. Short 13 base allele-specific oligonucleotides (ASO) containing the putative phenotype-specific base in the middle were then synthesized, end-labeled with digoxigenin-11-dUTP using terminal transferase, and used as probes in subsequent dot-blot hybridization experiments. Platelet RNA was prepared from a panel made up of four Baka/a, three Bakb/b, and two Baka/b individuals, and the mRNA encoding GPIIb was amplified using PCR and spotted onto nylon membranes. ASO hybridization showed that the nucleotide base difference identified above segregated with Bak phenotype in all nine individuals examined (P = .002). The base pair substitution results in an amino acid polymorphism at residue 843 of the mature heavy chain. The Baka form of GPIIb encodes an isoleucine at this position, whereas the Bakb allele contains a serine. Identification of the polymorphism associated with this clinically important alloantigen system should permit new therapeutic and diagnostic approaches for treating and managing patients with alloimmune thrombocytopenic disorders.
    Print ISSN: 0006-4971
    Electronic ISSN: 1528-0020
    Topics: Biology , Medicine
    Published by American Society of Hematology
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