A new platelet alloantigen, Swi(a) , located on glycoprotein Ia identified in a family with fetal and neonatal alloimmune thrombocytopenia.

BACKGROUND: Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is a bleeding disorder caused by transplacental passage of maternal antibodies to fetuses whose platelets (PLTs) express the corresponding human PLT antigen (HPA). STUDY DESIGNS AND METHODS: We observed a fetus with FNAIT who died from a severe intracranial hemorrhage. Analysis of maternal serum in antigen capture assay with paternal PLTs showed reactivity with PLT glycoprotein (GP)IIb/IIIa (α(IIb) ß(3) ) and GPIa/IIa (α(2) ß(1) integrin), indicating the presence of anti-HPA-1a and an additional alloantibody against GPIa (termed anti-Swi(a) ). RESULTS: By immunochemical studies, the localization of the Swi(a) antigen on GPIa/IIa could be confirmed. Analysis of paternal GPIa full-length cDNA showed a single-nucleotide substitution C(3347) T in Exon 28 resulting in a Thr(1087) Met amino acid substitution. Testing of family members by polymerase chain reaction-restriction fragment length polymorphism using MslI endonuclease showed perfect correlation with phenotyping. Extended family and population studies showed that 4 of 10 members of the paternal family but none of 500 unrelated blood donors were Swi(a) carriers. Expression studies on allele-specific transfected Chinese hamster ovary (CHO) cells confirmed that the single-amino-acid substitution Thr(1087) Met was responsible for the formation of the Swi(a) epitope. Adhesion of CHO cells expressing the Swi(a) alloantigen to immobilized collagens was not impaired compared to the wild-type control and was not inhibited by anti-Swi(a) alloantibodies. CONCLUSION: In this study we defined a new PLT alloantigen Swi(a) that was involved in a case of additional immunization against HPA-1a. Our observations demonstrate that combinations of PLT-specific alloantibodies may comprise low-frequency alloantigens.

What's happening--probing of HPA-1a antigen-antibody interaction by surface plasmon resonance technology.

This brief report summarizes the use of surface plasmon resonance technology (SPRT) in probing HPA-1a antigen-antibody interactions, based on a poster presented at the 60th meeting of the American Association of Blood Banks. It was concluded that the GP purification method could affect the performance of antigen in SPRT. It also highlighted that chips immobilised with Monoclonal antibody (Mab)-purified GP-IIb/IIIa work satisfactorily with both monoclonal and recombinant Abs with the appropriate concentration and binding affinity, while determination of the avidity and concentration of maternal polyclonal antibodies in respect to clinical severity on NAIT warrants further development.

Anti-Human Platelet Antigen-1a Immunoglobulin G Preparation Intended to Prevent Fetal and Neonatal Alloimmune Thrombocytopenia.

Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is a severe disease that is caused by maternal alloantibodies generated during pregnancy or at delivery as a result of incompatibility between maternal and fetal human platelet antigens (HPAs) inherited from the father. Antibody-mediated immune suppression using anti-HPA-1a immunoglobulins is thought to be able to prevent FNAIT caused by HPA-1a. A fractionation process to prepare anti-HPA-1a immunoglobulin (Ig) G (IgG) from human plasma was therefore developed. Anti-HPA-1a plasma was obtained from volunteer mothers who underwent alloimmunization against HPA-1a during a previous pregnancy. Plasma was cryoprecipitated and the supernatant treated with caprylic acid and solvent/detergent (S/D), purified by chromatography, nanofiltered, concentrated, and sterile-filtered. The anti-HPA-1a immunoglobulin fraction was characterized for purity and safety. PAK12 and quantitative monoclonal antibody immobilization of platelet antigen (MAIPA) assays were used to detect anti-HPA-1a IgG. Hepatitis C virus (HCV) removal during nanofiltration was assessed by spiking experiments, using cell culture-derived reporter HCV and luciferase analysis. The caprylic acid treatment precipitated non-Ig proteins yielding a 90% pure Ig supernatant. S-HyperCel chromatography of the S/D-treated supernatant followed by HyperCel STAR AX provided high IgG recovery (>80%) and purity (>99.5%), and efficient IgA and IgM removal. Concentrations of complement factors C3 and C4 were < 0.5 and < 0.4 mg/dL, respectively. The final IgG could be nanofiltered on Planova 20N under conditions removing more than 3 log HCV infectivity to baseline mock infection level, and concentrated to ca. 30 g/L. Proteolytic activity and thrombin generation were low in the final fraction. The Pak12 and MAIPA assays showed good recovery of anti-HPA-1a throughout the process. Clinical-grade HPA-1a IgG can be prepared using a process compliant with current quality requirements opening perspectives for the prevention of FNAIT.

Location of the quinine-dependent epitope on platelet glycoprotein IIIa.

Quinine-dependent (Q) IgG antibodies (Q.Ab) in drug-induced immune thrombocytopenia are heterogeneous and bind to different platelet surface glycoproteins (GP), namely GPIb, IX, IIb, IIIa and an unidentified 57-kDa membrane proteins. Although both the Q-dependent epitope on GPIIIa and the P1A1 antigen require intact disulphide bonds for their expression, they are distinct because Q.Ab bind to GPIIIa lacking P1A1. Epitopes for both antigens were examined on Western blots of either intact washed human platelets or purified GPIIIa. When intact platelets were digested with trypsin and washed and solubilised prior to electrophoresis, membrane-associated fragments of GPIIIa of 78 kDa were found to be reactive with both antibodies. In addition, 60- and 68-kDa fragments bound anti-P1A1 but not Q.Ab. Similar digestion with chymotrypsin produced only 60-kDa fragments containing both epitopes. Digestion of purified GPIIIa with chymotrypsin produced 60-kDa peptides reactive with Q.Ab and anti-P1A1 in immunoblotting studies. Similar digestion with elastase produced 58-kDa fragments also containing the epitopes for both antibodies. Longer digestion times or sequential digestion with different enzymes did not reveal extra fragments. However, immunoprecipitation of trypsin-digested 125I-labelled GPIIIa with affinity-purified Q.Ab produced a 17-kDa fragment containing the Q-dependent epitope.

A new private platelet antigen, Groa, localized on glycoprotein IIIa, involved in neonatal alloimmune thrombocytopenia.

The serum of a Caucasian woman who gave birth to a child with neonatal alloimmune thrombocytopenia contained antibodies directed against a platelet antigen of the newborn. There was no incompatibility for the known platelet alloantigens HPA-1 to HPA-7 or for the private or low-frequency antigens Sra and Vaa, between the platelets of the parents. However, crossmatching with the serum of the mother and the platelets of the child and the father was strongly positive, suggesting a new platelet antibody specificity. To investigate the inheritance of the 'Groa' antigen involved, the available family members were tested in the platelet immunofluorescence test (PIFT) and the monoclonal antibody-specific immobilization of platelet antigens (MAIPA) assay. The Groa antigen was found to be inherited in an autosomal-codominant fashion. In the MAIPA, we localized the Groa antigen on the glycoprotein IIb/IIIa complex (alpha IIb beta 3). The GP IIb/IIIa localization was confirmed in immunoprecipitation studies. In Western blotting experiments, we further localized the Groa antigen on the GP IIIa (beta 3) subunit of the GP IIb/IIIa complex. Until now we have tested approximately 400 unrelated donors. None of these appeared to be positive for the Groa antigen, suggesting a phenotype frequency in the Dutch population of less than 0.01.

Púrpura trombocitopênica aloimune neonatal: caracterizaçäo de aloanticorpo plaquetário anti-HPA-1A (Anti-PIA1) por radioimunoprecipitaçäo indireta / Alloimmune neonatal thrombocytopenic purpura: characterization of platelet alloantibody anti-HPA-1A (Anti-PIA1) by indirect radioimmunoprecipitation

A púrpura trombocitopênica alo-imune neonatal (PTAN) é uma doença grave na qual a hemorragia cerebral pode ser fatal ou levar a seqüelas cerebrais permanentes. Similarmente, à doença hemolítica do recém-nascido (RN), a PTAN ocorre devido a aloimunizaçäo materna por um alo-antigeno incompatível presente nas plaquetas fetais. A apresentaçäo clínica é de púrpura generalizada acompanhada de hemorragia gastrointestinal, urinária, e/ou intracranial. O alo-antigeno HPA-1 (PL(A)) é responsável por cerca de 70-80 por cento dos casos de PTAN. Nesse estudo, os autores descrevem o caso de uma gestante que deu a luz a um RN com plaquetopenia acentuada devido a trombocitopenia aloimune. A contagem plaquetária da mae e do RN era 173 x 10(9)/L e 15 x 10(9)/L, respectivamente. O estudo sorológico realizado com a técnica de radioimunoprecipitaçäo indireta demonstrou forte reatividade do soro materno com o complexo glicoprotético GPIIb/IIIa em plaquetas PI(A1)-positivas de doador normal. Assim, o soro materno continha forte atividade anti-HPA-1a (anti-PI(A1)). O RN foi tratado com corticosteróides e gamaglobulina intravenosa. Após o tratamento, o RN teve alta hospitalar com contagem plaquetária elevada para 70 x 10(9)/L.

Aloinmunización transplacentaria por antígenos plaquetarios específicos: prevalencia y características en una población chilena / Transplacental alloimmunization against platelet antigens: prevalence and features in a chilean population

Backgrour: Neonatal alloimmune thrombocytopenia (NAIT) is a result of fetomaternal incompatibility. Platelet destruction is caused by a maternal alntibody directed against a fetal platelet antigen inherited from the father and lacking on the mother's platelets. The incidence and features of transplacental alloimmunization depend on the frequency of expression of platelet specific antigens, which are highly variable among different populations. Aim: To determine the prevalence and characteristics of transplacental alloimmunization in a large, group of pregnant women in Chile. Material and methods: We, studied 3,041 samples obtained during the third trimester of gestation. In all samples, anti platelet antibodies were screened by ELISA with platelet membranes fixed to a microtiter plate. Positive samples were further studied for antigenic specificity with the monoclonal antibody specific immobilization of platelet antigens (MAIPA) test. Results: Anti platelet antibodies were found in 261 samples (8.5 percent). The MAIPA test identified 6 samples with antibodies directed against major platelet membrane glycoproteins, 2 anti GPIb, 2 anti GPIIb/IIIa and 2 anti GPIa/IIIa. In four cases, anti HLA antibodies coexisted. Two cases corresponded to well defined platelet antigen systems: one anti HPA-1a and one anti HPA-5b. No clinical evidence of thrombocytopenia of the newborn was detected in all these cases with anti GP antibodies. Conclusions: A prevalence of platelet specific antibodies of 0.2 por ciento with only one anti HPA-1a was detected. These findings are in contrast with those of other populations but in accordance with the low frequency of the HPA-1b/b phenotype in the Chilean population. The very low incidence of platelet specific antibodies and the lack of association with clinical thrombocytopenia in the newborn, do not support the recommendation of routine antenatal screening to all women in Chile