New αIIbß3 variants in 28 Turkish Glanzmann patients; structural hypothesis for complex activation by residues variations in I-EGF domains.

Glanzmann thrombasthenia (GT) is a rare autosomal recessive bleeding disorder characterized by impaired platelet aggregation due to defects in integrin αIIbß3, a fibrinogen receptor. Platelet phenotypes and allelic variations in 28 Turkish GT patients are reported. Platelets αIIbß3 expression was evaluated by flow cytometry. Sequence analyzes of ITGA2B and ITGB3 genes allowed identifying nine variants. Non-sense variation effect on αIIbß3 expression was studied by using transfected cell lines. 3D molecular dynamics (MDs) simulations allowed characterizing structural alterations. Five new alleles were described. αIIb:p.Gly423Asp, p.Asp560Ala and p.Tyr784Cys substitutions impaired αIIbß3 expression. The αIIb:p.Gly128Val substitution allowed normal expression; however, the corresponding NM_000419.3:c.476G>T variation would create a cryptic donor splicing site altering mRNA processing. The ß3:p.Gly540Asp substitution allowed αIIbß3 expression in HEK-293 cells but induced its constitutive activation likely by impairing αIIb and ß3 legs interaction. The substitution alters the ß3 I-EGF-3 domain flexibility as shown by MDs simulations. GT variations are mostly unique although the NM_000419.3:c.1752 + 2 T > C and NM_000212.2:c.1697 G > A variations identified in 4 and 8 families, respectively, might be a current cause of GT in Turkey. MD simulations suggested how some subtle structural variations in the ß3 I-EGF domains might induce constitutive activation of αIIbß3 without altering the global domain structure.

Cab4b, the first human platelet antigen carried by glycoprotein IX discovered in a context of severe neonatal thrombocytopenia.

Essentials Life-threatening maternofetal thrombocytopenias mostly depend on αIIb ß3 antigens. We performed serological, genomic and in vitro studies of two life-threatening thrombocytopenias. Identification of a c.368C>T variation leading to Pro123Leu substitution in GPIX. A rare GPIX variant reported in a genomic database define a new alloantigen. SUMMARY: Background After three miscarriages, a 39-year-old woman gave birth, with a 1-year interval, to two severely thrombocytopenic neonates (4 ×109 L-1 and 33 ×109 L-1 ) with intracranial hemorrhages. Transfusion of platelet concentrates corrected the thrombocytopenia. The outcome was favorable for the first child, but the second one died 10 days after cesarean delivery (31 weeks of gestation + 6 days). Methods Serologic studies were performed with mAb-specific immobilization of platelet antigens and flow cytometry techniques. Human platelet alloantigen (HPA) genotyping was performed with the BioArray HPA BeadChip and PCR-sequence-specific primer techniques. Genomic DNA was studied by direct sequencing of PCR products. The mutant glycoprotein (GP) was expressed in transiently transfected HEK293 cells. Results In MAIPA assay, the maternal serum faintly reacted with GPIbIX from paternal and child 1 platelets, but not with maternal or panel platelets. No maternofetal incompatibility was found in the 22 known HPA systems, tested except for HPA-1b in child 2. A new alloantigen carried by GPIbIX was suspected. Genomic sequencing revealed a paternal GPIX variation (NM_000174.4:c.368C>T). The father and children were heterozygous and incompatible with the mother, who was NM_000174.4:c.368C homozygous. The maternal serum reacted with the GPIX NP_000165.1:p.Leu123 form coexpressed with GPIb in transfected HEK293 cells. The NM_000174.4:c.368T allele (rs202229101) has a minor allele frequency of 0.0002, and was not detected in 120 French subjects (families with fetal and neonatal alloimmune thrombocytopenia [FNAIT]), suggesting that it is rarely implicated in alloimmunization. Conclusion The NP_000165.1:p.Leu123 allele named Cab4b is the first platelet alloantigen described on GPIX. In the absence of other known maternofetal incompatibility, the child 1 case suggests that anti-Cab4b alloantibodies can induce severe thrombocytopenias.

HPA-5 typing discrepancy reveals an Ile503Leu substitution in platelet GPIa (α2 integrin).

BACKGROUND AND OBJECTIVES: In fetal/neonatal thrombocytopenia, maternal alloimmunization is diagnosed by the identification of the maternal alloantibody and the offending paternal antigen inherited by the foetus/neonate. Today, for practical reasons, most laboratories perform platelet genotyping instead of phenotyping. Here, we report the case of a human platelet antigen (HPA)-5 genotype/phenotype discrepancy observed in a mother who delivered a mildly thrombocytopenic newborn. MATERIALS AND METHODS: Platelet antibody detection and platelet phenotyping were performed using the MAIPA assay; platelet genotypes were determined using BeadChip technology (BioArray), PCR-SSP, PCR-RFLP and sequencing. RESULTS: Serological investigations revealed the presence of maternal anti-GPIIbIIIa autoantibodies. No alloantibodies were detected. No feto-maternal platelet incompatibility was observed for HPA-1 to -21. The mother and newborn were genotyped as HPA-5aa using BeadChips, but as HPA-5a (weak b) with PCR-SSP and HPA-5ab with PCR-RFLP. Mother's platelets were phenotyped as HPA-5b(+). GPIa exon 13 sequencing confirmed the HPA-5ab genotype of the mother and newborn, and revealed an NM_002203.3:c.1594A>C mutation near the HPA-5 polymorphism (5' side), leading to an I503L amino acid change. CONCLUSION: Feto-maternal alloimmunization was ruled out: the neonatal thrombocytopenia probably resulted from maternal anti-GPIIbIIIa autoantibodies. This case highlights that platelet typing should be performed using two different methods to avoid false diagnosis.

Expresión y serorreactividad de la lipoproteína recombinante de 43-kDa de Bartonella bacilliformis / Expresión and seroreactivity of the recombinant Bartonella bacilliformis 43-kDa lipoprotein

Objetivo: La lipoproteína de 43-kDa de Bartonella bacilliformis fue obtenida en su forma recombinante (rBbLppB) ypurificada para evaluar su serorreactividad mediante ELISA. Materiales y métodos: Los niveles de anticuerpos IgG eIgM humanos en los sueros de pacientes con Bartonelosis confirmada y sueros de otras enfermedades (salmonelosis,Brucelosis y leptospirosis) frente a rBbLppB fueron evaluados por ELISA, se utilizó sueros de personas sanas comocontroles. Resultados: La sensibilidad y la especificidad del ELISA IgG fueron 70,4 y 90 respectivamente. Asimismo,la sensibilidad y especificidad de ELISA IgM fueron 85,2 y 90 respectivamente. Conclusiones: Estos resultadosdemuestran que el ELISA usando rBbLppB tiene una buena sensibilidad y especificidad y puede ser consideradacomo un buen antígeno para el diagnóstico de Bartonelosis causada por B. bacilliformis.

Objective: The Bartonella bacilliformis 43-kDa lipoprotein was obtained from its recombinant form (rBbLppB) and then, purified to evaluate sero-reactivity through ELISA. Material and methods: IgG and IgM humanantibodies levels in the sera of patients with confirmed Bartonellosis and sera of patients with other diseases (salmonellosis, Brucellosis and Leptospirosis), when contrasted with rBbLppB were evaluated by ELISA. Sera from some healthy people were used for controls. Results: Sensitivity and specificity of the IgG ELISA were 70,4% and 90% respectively. Also, sensitivity and specificity of the IgM ELISA were 85,2% and 90%, respectively. Conclusions: These results show that ELISA using rBbLppB is highly sensitive and specific and may be considered a good antigen for the diagnosis of Bartonellosis caused by B. bacilliformis.