RESUMO
BACKGROUND: Fetal and Neonatal Alloimmune Thrombocytopenia (FNAIT) results from maternal platelet alloimmunization against paternal antigens inherited by the fetus, most often due to the Human Platelet Antigen (HPA)-1 system in Caucasians. We investigated in 2023, a 30-year-old Caucasian woman Gravida 2 Para 1 who gave birth at 35 weeks of gestation to a male (body weight 2210 g) without signs of bleeding. A severe thrombocytopenia (platelet count at 3 G/L) was discovered incidentally a few hours after delivery in the context of the management of a respiratory distress. The newborn recovered after one platelet concentrate transfusion and normalized his platelet count at Day 5. STUDY DESIGN AND METHODS: FNAIT investigation was performed according to guideline recommendations. Platelet genotyping was carried out by multiplex PCR. Maternal serological investigation included Monoclonal Antibody-specific Immobilization of Platelet Antigens method (MAIPA) and Luminex technology. RESULTS: Parental and newborn genotyping pointed out an HPA-4 incompatibility between the mother and the newborn and the father. Serological investigation revealed an anti-HPA-4b alloantibody confirming the diagnosis of neonatal alloimmune thrombocytopenia. CONCLUSION: We described the third case of anti-HPA-4b alloantibody discovered in a Caucasian mother. This case strengthens the need for reference laboratory to genotype a panel of HPA alleles reflecting local genetic population diversity and for crossmatch of maternal serum with fresh paternal platelets in clinical suspected cases of neonatal alloimmune thrombocytopenia.
Assuntos
Antígenos de Plaquetas Humanas , Isoanticorpos , Trombocitopenia Neonatal Aloimune , Humanos , Isoanticorpos/sangue , Isoanticorpos/imunologia , Trombocitopenia Neonatal Aloimune/imunologia , Trombocitopenia Neonatal Aloimune/sangue , Feminino , Antígenos de Plaquetas Humanas/imunologia , Antígenos de Plaquetas Humanas/genética , Recém-Nascido , Adulto , Gravidez , Masculino , Transfusão de Plaquetas , População BrancaRESUMO
Integrin αIIbß3, a glycoprotein complex expressed at the platelet surface, is involved in platelet aggregation and contributes to primary haemostasis. Several integrin αIIbß3 polymorphisms prevent the aggregation that causes haemorrhagic syndromes, such as Glanzmann thrombasthenia (GT). Access to 3D structure allows understanding the structural effects of polymorphisms related to GT. In a previous analysis using Molecular Dynamics (MD) simulations of αIIbCalf-1 domain structure, it was observed that GT associated with single amino acid variation affects distant loops, but not the mutated position. In this study, experiments are extended to Calf-1, Thigh, and Calf-2 domains. Two loops in Calf-2 are unstructured and therefore are modelled expertly using biophysical restraints. Surprisingly, MD revealed the presence of rigid zones in these loops. Detailed analysis with structural alphabet, the Proteins Blocks (PBs), allowed observing local changes in highly flexible regions. The variant P741R located at C-terminal of Calf-1 revealed that the Calf-2 presence did not affect the results obtained with isolated Calf-1 domain. Simulations for Calf-1 + Calf-2, and Thigh + Calf-1 variant systems are designed to comprehend the impact of five single amino acid variations in these domains. Distant conformational changes are observed, thus highlighting the potential role of allostery in the structural basis of GT.
Assuntos
Mutação de Sentido Incorreto , Glicoproteína IIb da Membrana de Plaquetas/química , Glicoproteína IIb da Membrana de Plaquetas/genética , Domínios e Motivos de Interação entre Proteínas/genética , Sequência de Aminoácidos , Substituição de Aminoácidos , Modelos Moleculares , Glicoproteína IIb da Membrana de Plaquetas/metabolismo , Conformação Proteica , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Relação Estrutura-AtividadeRESUMO
This paper describes the development and application of a suite of tools, called PBxplore, to analyze the dynamics and deformability of protein structures using Protein Blocks (PBs). Proteins are highly dynamic macromolecules, and a classical way to analyze their inherent flexibility is to perform molecular dynamics simulations. The advantage of using small structural prototypes such as PBs is to give a good approximation of the local structure of the protein backbone. More importantly, by reducing the conformational complexity of protein structures, PBs allow analysis of local protein deformability which cannot be done with other methods and had been used efficiently in different applications. PBxplore is able to process large amounts of data such as those produced by molecular dynamics simulations. It produces frequencies, entropy and information logo outputs as text and graphics. PBxplore is available at https://github.com/pierrepo/PBxplore and is released under the open-source MIT license.
RESUMO
Integrin αIIbß3 mediates platelet aggregation and thrombus formation. In a rare hereditary bleeding disorder, Glanzmann thrombasthenia (GT), αIIbß3 expression / function are impaired. The impact of deleterious missense mutations on the complex structure remains unclear. Long independent molecular dynamics (MD) simulations were performed for 7 GT variants and reference structure of the Calf-1 domain of αIIb. Simulations were analysed using a structural alphabet to describe local protein conformations. Common and flexible regions as well as deformable zones were observed in all the structures. The most flexible region of Calf-1 (with highest B-factor) is rather a rigid region encompassed into two deformable zones. Each mutated structure barely showed any modifications at the mutation sites while distant conformational changes were observed. These unexpected results question the relationship between molecular dynamics and allostery; and the role of these long-range effects in the impaired αIIbß3 expression. This method is aimed at studying all αIIbß3 sub-domains and impact of missense mutations at local and global structural level.
Assuntos
Sítio Alostérico , Simulação de Dinâmica Molecular , Mutação , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/química , Trombastenia/genética , Regulação Alostérica , Humanos , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/genética , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismoRESUMO
Protein structures are valuable tools to understand protein function. Nonetheless, proteins are often considered as rigid macromolecules while their structures exhibit specific flexibility, which is essential to complete their functions. Analyses of protein structures and dynamics are often performed with a simplified three-state description, i.e., the classical secondary structures. More precise and complete description of protein backbone conformation can be obtained using libraries of small protein fragments that are able to approximate every part of protein structures. These libraries, called structural alphabets (SAs), have been widely used in structure analysis field, from definition of ligand binding sites to superimposition of protein structures. SAs are also well suited to analyze the dynamics of protein structures. Here, we review innovative approaches that investigate protein flexibility based on SAs description. Coupled to various sources of experimental data (e.g., B-factor) and computational methodology (e.g., Molecular Dynamic simulation), SAs turn out to be powerful tools to analyze protein dynamics, e.g., to examine allosteric mechanisms in large set of structures in complexes, to identify order/disorder transition. SAs were also shown to be quite efficient to predict protein flexibility from amino-acid sequence. Finally, in this review, we exemplify the interest of SAs for studying flexibility with different cases of proteins implicated in pathologies and diseases.
RESUMO
The human platelet alloantigen (HPA)-1 system, the first cause of alloimmune thrombocytopenia in Caucasians, results from leucine-to-proline substitution (alleles 1a and 1b) of residue 33 in ß3 subunit of the integrin αIIbß3. A third variant with a valine (V33) has been described. Although leucine and valine share similar physicochemical properties, sera containing alloantibodies to the HPA-1a antigen variably reacted with V33-ß3, suggesting structural alterations of ß3. To analyze the effect of the L33V transition, molecular dynamics simulations were performed on a 3D structural model of the V33 form of the whole ß3 extracellular domain (690 residues). Dynamics of the PSI (carrying residue 33), I-EGF-1, and I-EGF-2 domains of ß3 were compared to previously obtained dynamics of HPA-1a structure and HPA-1b structural model using classical and innovative developments (a structural alphabet). Clustering approach and local structure analysis showed that L33-ß3 and V33-ß3 mostly share common structures co-existing in different dynamic equilibria. The L33V substitution mainly displaces the equilibrium between common structures. These observations can explain the variable reactivity of anti-HPA-1a alloantibodies suggesting that molecular dynamic plays a key role in the binding of these alloantibodies. Unlike the L33P substitution, the L33V transition would not affect the structure flexibility of the ß3 knee, and consequently the functions of αIIbß3.
Assuntos
Antígenos de Plaquetas Humanas/química , Plaquetas/química , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/química , Antígenos de Plaquetas Humanas/genética , Epitopos/química , Epitopos/genética , Epitopos/imunologia , Humanos , Integrina beta3 , Isoanticorpos/imunologia , Modelos Moleculares , Simulação de Dinâmica Molecular , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/genética , TrombocitopeniaRESUMO
BACKGROUND: Fetal-neonatal alloimmune thrombocytopenia (FNAIT) diagnosis relies on maternofetal incompatibility and alloantibody identification. Genotyping for rare platelet (PLT) polymorphisms allowed the identification of three families with suspected or confirmed maternofetal incompatibility for the αIIb-c.2614C>A mutation (Halle et al., Transfusion 2008;48:14-15). STUDY DESIGN AND METHODS: A polymerase chain reaction-sequence-specific primers amplification assay was designed to genotype the αIIb-c.2614C>A mutation. HEK293 cells expressing αIIb-Leu841 or αIIb-Met841 αIIbß3 forms were used to probe the reactivity of maternal sera from these families and to study the effects of the substitution on αIIbß3 expression and functions. RESULTS: Tested by flow cytometry (FCM), one serum sample specifically reacted with αIIb-Met841 but not with αIIb-Leu841 αIIbß3. This specificity revealed the αIIb-Leu841 polymorphism as a new alloantigen named Cab3(a+) . Cross-match testing using FCM also showed the Cab3(a+) antigen to be expressed at the PLT surface. As for anti-human PLT alloantigen (HPA)-3a (or -3b) and anti-HPA-9bw, detection of anti-Cab3(a+) alloantibodies appeared difficult and required whole PLT assays when classical monoclonal antibody-specific immobilization of PLT antigen test failed. In our FNAIT set, the immune response to Cab3(a+) maternofetal incompatibility could induce severe thrombocytopenias and life-threatening hemorrhages. The p.Leu841Met substitution has limited effects, if any, on local αIIb structure, preserving both αIIbß3 expression and functions. CONCLUSION: The Cab3(a+) polymorphism is a new rare alloantigen (allelic frequency <1%) carried by αIIb that might result in severe life-threatening thrombocytopenias. In Sub-Saharan African populations, higher Cab3(a+) gene frequencies (up to 8.2%; Halle et al., Transfusion 2008;48:14-15) and homozygous people are observed.
Assuntos
Antígenos de Plaquetas Humanas/fisiologia , Glicoproteína IIb da Membrana de Plaquetas/genética , Trombocitopenia Neonatal Aloimune/genética , Adulto , Substituição de Aminoácidos/genética , Antígenos de Plaquetas Humanas/genética , Antígenos de Plaquetas Humanas/imunologia , Feminino , Morte Fetal/genética , Morte Fetal/imunologia , Células HEK293 , Humanos , Recém-Nascido , Doenças do Recém-Nascido/genética , Doenças do Recém-Nascido/imunologia , Leucina/genética , Masculino , Metionina/genética , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/genética , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/imunologia , Gravidez , Trombocitopenia Neonatal Aloimune/imunologiaRESUMO
BACKGROUND: The HPA-1 alloimmune system carried by the platelet integrin αIIbß3 is the primary cause of alloimmune thrombocytopenia in Caucasians and the HPA-1b allele might be a risk factor for thrombosis. HPA-1a and -1b alleles are defined by a leucine and a proline, respectively, at position 33 in the ß3 subunit. Although the structure of αIIbß3 is available, little is known about structural effects of the L33P substitution and its consequences on immune response and integrin functions. METHODOLOGY/PRINCIPAL FINDINGS: A complete 3D model of the L33-ß3 extracellular domain was built and a P33 model was obtained by in silico mutagenesis. We then performed molecular dynamics simulations. Analyses focused on the PSI, I-EGF-1, and I-EGF-2 domains and confirmed higher exposure of residue 33 in the L33 ß3 form. These analyses also showed major structural flexibility of all three domains in both forms, but increased flexibility in the P33 ß3 form. The L33P substitution does not alter the local structure (residues 33 to 35) of the PSI domain, but modifies the structural equilibrium of the three domains. CONCLUSIONS: These results provide a better understanding of HPA-1 epitopes complexity and alloimmunization prevalence of HPA-1a. P33 gain of structure flexibility in the ß3 knee may explain the increased adhesion capacity of HPA-1b platelets and the associated thrombotic risk. Our study provides important new insights into the relationship between HPA-1 variants and ß3 structure that suggest possible effects on the alloimmune response and platelet function.
Assuntos
Antígenos de Plaquetas Humanas/genética , Modelos Genéticos , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/genética , Alelos , Antígenos de Plaquetas Humanas/química , Epitopos/química , Epitopos/genética , Genótipo , Humanos , Integrina beta3 , Simulação de Dinâmica Molecular , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/química , Polimorfismo GenéticoRESUMO
BACKGROUND: A new ß3 allele was identified in a severe case of neonatal alloimmune thrombocytopenia (<7 × 10(9) /L). STUDY DESIGN AND METHODS: Diagnosis was done by use of monoclonal antibody-specific immobilization of platelet (PLT) antigen for serologic analyses and polymerase chain reaction (PCR)-sequence-specific primers (SSP) and PCR-restriction fragment length polymorphism (RFLP) for genotyping. Direct sequencing of PCR product was done and mutant αIIbß3 expressed in HEK-293 cells. RESULTS: Serologic analysis revealed in the maternal serum an anti-human PLT alloantigen (HPA)-1a alloantibody associated to an anti-α2ß1. Anti-HPA-1a alloimmunization diagnosis was confirmed by genotyping showing maternofetal incompatibility. However, investigation of rare HPA polymorphisms revealed discrepant HPA-16b assignation between PCR-RFLP and PCR-SSP. Sequencing revealed a new c.385C>A mutation in the ß3 coding sequence resulting in a false assignation of the HPA-16b allele by PCR-RFLP. This mutation leads to a Q103K substitution in mature ß3. The K103-ß3 form of the complex was expressed in HEK-293 cells but did not react with the maternal serum. CONCLUSION: We have characterized a new rare allele (frequency < 1%) of ß3 that yields false HPA-16b genotyping in PCR-RFLP. This new case of false typing assignation emphasizes the necessity to use two genotyping techniques in diagnosis. This particularly applies for rare HPA polymorphisms when PLT phenotyping cannot be used.
Assuntos
Complexo Glicoproteico GPIIb-IIIa de Plaquetas/genética , Trombocitopenia Neonatal Aloimune/genética , Adulto , Alelos , Antígenos de Plaquetas Humanas/genética , Feminino , Humanos , Integrina beta3 , Mutagênese Sítio-Dirigida , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição/genéticaRESUMO
BACKGROUND: A new platelet antigen, Cab2(a+), was identified in a case of severe neonatal alloimmune thrombocytopenia (<8 × 10(9)/L) in twins. STUDY DESIGN AND METHODS: Coding sequences of αIIb and ß3 genes from parents were amplified and sequenced. CHO cell lines expressing wild-type or mutated forms of the complex were established to study the role of the mutation in alloimmunization and in αIIbß3 functions. RESULTS: The father and twins were heterozygous for a single αIIb c.1508G>A mutation leading to a Ser472Asn substitution. Immunologic assays with transfected CHO cells revealed the Asn472 form of αIIbß3 responsible for the Cab2(a+) epitope but not an Ala472 form. Using these cells lines we demonstrated that both Ser472Asn and Ser472Ala substitutions produced limited structural alteration as revealed by the reactivity of a panel of anti-αIIbß3 monoclonal antibodies (MoAbs). Activated Asn472 and Ala472 forms of αIIbß3 supported 1) binding of soluble fibrinogen and of the ligand mimetic MoAb PAC-1, 2) ligand-induced binding site epitopes exposure (MoAbs AP-5 and D3GP3), and 3) cell aggregation. Adhesion onto adsorbed fibrinogen was conserved and was specifically inhibited by MoAb AP-2 or peptide RGDS. Finally outside-in signaling was not affected. CONCLUSION: We have characterized a new low-frequency alloantigen (<1%) resulting from the Ser472Asn substitution in αIIb and shown this polymorphism to have a limited effect, if any, on the αIIbß3 complex functions.
Assuntos
Doenças em Gêmeos/genética , Glicoproteína IIb da Membrana de Plaquetas/genética , Polimorfismo de Nucleotídeo Único , Trombocitopenia Neonatal Aloimune/genética , Adulto , Substituição de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Células CHO , Cricetinae , Cricetulus , Epitopos/imunologia , Feminino , Fibrinogênio/metabolismo , Heterozigoto , Humanos , Imunidade Materno-Adquirida , Recém-Nascido , Integrina beta3/química , Masculino , Oligopeptídeos/farmacologia , Adesividade Plaquetária/efeitos dos fármacos , Glicoproteína IIb da Membrana de Plaquetas/química , Glicoproteína IIb da Membrana de Plaquetas/imunologia , Mapeamento de Interação de Proteínas , Relação Estrutura-Atividade , TransfecçãoRESUMO
Glanzmann thrombasthenia (GT) is an autosomal recessive inherited bleeding disorder characterized by an impaired platelet aggregation due to defects in integrin alphaIIbbeta3 (ITGA2B, ITGB3), a fibrinogen receptor. Mutations from 24 GT patients and two carriers of various origins, Caucasian, North-African and Asian were characterized. Promoter and exon sequences of alphaIIb and beta3 genes were amplified and directly sequenced. Among 29 identified mutations, 17 new allelic variants resulting from nonsense, missense and deletion/insertion mutations were described. RNA alterations were evaluated by using Web servers. The alphaIIb p.S926L, p.V903F, and beta3 p.C38Y, p.M118R, p.G221D substitutions prevented complex expression at the surface of COS-7 cells by altering the alphaIIb or the beta3 subunit structure. As shown by free energy analyses applied on the resolved structure of alphaIIbbeta3 and structural modeling of the mutant, the p.K253M substitution of beta3 helped to define a key role of the K253 in the interaction of the alphaIIb beta-propeller and the beta3 beta-I domains. finally, the alphaIIb p.Q595H substitution allowed cell surface expression of the complex but its corresponding c.2800G>T mutation is predicted to alter normal RNA splicing. In conclusion, our study yielded the discovery of 17 new GT allelic variants, revealed the key role of K253 of alphaIIb for the alphaIIbbeta3 complex formation and provides an additional example of an apparently missense mutation causing a splicing defect.
Assuntos
Alelos , Processamento Alternativo , Integrina alfa2/genética , Integrina beta3/genética , Trombastenia/genética , Animais , Sequência de Bases , Células COS , Chlorocebus aethiops , Análise Mutacional de DNA , Regulação da Expressão Gênica , Humanos , Dados de Sequência Molecular , Receptores de Fibrinogênio/genética , Relação Estrutura-AtividadeRESUMO
BACKGROUND: Fetal/neonatal alloimmune thrombocytopenia (FNAIT) is caused by maternal alloimmunization against fetal platelet (PLT) antigens, inherited from the father and absent from maternal PLTs. STUDY DESIGN AND METHODS: A 29-year-old mother gave birth to a severely thrombocytopenic newborn (16 x 10(9) PLTs/L) leading to PLT transfusion therapy associated with intravenous immunoglobulins. The outcome was uneventful. Maternal serum showed a specific positive reaction with the antigen-capture assay (monoclonal antibody [MoAb]-specific immobilization of PLT antigens) only when it was tested with the paternal PLTs and a panel of MoAbs against glycoprotein (GP)Ia-IIa (alpha(2)beta(1) integrin) suggesting the implication of a new PLT antigen. RESULTS: Nucleotide sequence analysis of GPIa cDNA of the father and newborn showed a nucleotide substitution at position 2235 (2235G > T according to the international nomenclature). This substitution induces a Q716H amino acid change in the GPIa mature protein, located outside the I domain involved in cell adhesion for collagen. In vitro analysis of recombinant Chinese hamster ovary (CHO) cells expressing wild-type or mutant (Q716H) human GPIa allowed us to demonstrate that this single amino acid substitution is responsible and sufficient for inducing Cab(a) antigen expression. Adhesion of CHO cells to collagen was not modified by the Cab polymorphism, nor by the maternal anti-Cab(a) alloantibodies, indicating that the mutation does not affect the function of integrin alpha(2)beta(1). In a Caucasian population study, none of the 104 unrelated blood donors was found to be Cab(a)(+). CONCLUSION: We describe here a new PLT alloantigen Cab(a) involved in a severe case of FNAIT. Laboratory investigation for the "common" PLT alloantigens is no longer sufficient to evaluate neonatal alloimmune thrombocytopenia in suspected cases.
Assuntos
Antígenos de Plaquetas Humanas/genética , Integrina alfa2/genética , Mutação Puntual/genética , Trombocitopenia Neonatal Aloimune/genética , Adulto , Animais , Células CHO , Cricetinae , Cricetulus , Feminino , Citometria de Fluxo , Genótipo , Humanos , Recém-Nascido , Transfusão de Plaquetas , Gravidez , Trombocitopenia Neonatal Aloimune/imunologia , Trombocitopenia Neonatal Aloimune/terapiaRESUMO
BACKGROUND: The diagnosis of immune thrombocytopenic purpura (ITP) is a diagnosis of exclusion, as stated by international guidelines. Nevertheless, the assessment of platelet (PLT) antibodies has been reported as helpful for the diagnosis and the follow-up of ITP patients. PLT antibodies are detected by highly specialized assays, such as monoclonal antibody-specific immobilization of PLT antigen (MAIPA) test. Flow cytometry for PLT-associated immunoglobulin G (PAIgG) detection has been described more recently. This study was meant to evaluate the utility of flow cytometry to screen accurately patients needing further MAIPA testing. STUDY DESIGN AND METHODS: PAIgG, PAIgM, and PAIgA were determined in 107 consecutive patients and in 147 healthy controls in parallel. MAIPA testing was performed in all patients. The accuracy of flow cytometry was assessed with a receiver operating characteristics (ROC) curve analysis versus MAIPA. RESULTS: MAIPA assay found PLT-specific IgG in 27 patients (25%). The ROC curve analysis showed that no false-negative result in flow cytometry was obtained for a mean fluorescence intensity (MFI) cutoff of 0.2. With this cutoff, PAIgG were positive in 61 patients (57%). In this series, MAIPA was unnecessary in 42 percent of patients (corresponding to true-negative results). When MAIPA was positive, PAIgM values ranged from 0.1 to 1.0, and PAIgA from 0.1 to 2. CONCLUSION: Flow cytometry for PAIgG assessment may be used to accurately decide whether or not MAIPA must be subsequently performed. In this series, MAIPA was unnecessary in 42 percent of patients. Moreover, PAIgM results suggested that its determination combined with PAIgG may be of interest in ITP investigation.
Assuntos
Autoanticorpos/sangue , Plaquetas/imunologia , Citometria de Fluxo/métodos , Adulto , Idoso , Feminino , Humanos , Imunoglobulina G/sangue , Masculino , Pessoa de Meia-Idade , Reprodutibilidade dos TestesRESUMO
BACKGROUND: Fetal-neonatal alloimmune thrombocytopenia (FNAIT) linked to rare or private antigens is not a rare event. STUDY DESIGN AND METHODS: Such a case discovered during the follow-up of a second child with jaundice with mild thrombocytopenia is reported here. Platelet (PLT) genotyping was performed by polymerase chain reaction (PCR)-sequence-specific primers method and PCR-restriction fragment length polymorphism (RFLP) analysis. Serologic investigation was done with the monoclonal antibody-specific immobilization of PLT antigens technique. Glycoprotein Ia-specific amplification and sequencing were performed for the polymorphism 807 (exon 7). RESULTS: The mother was found to be HPA-13aaw, and the father HPA-13abw. A maternal alloantibody directed against HPA-13bw has been characterized, leading to the diagnosis of neonatal alloimmune thrombocytopenia. CONCLUSION: This report provides further evidence that NAIT associated with low-frequency antigens is not restricted to single families. Therefore, laboratory investigation of a suspected case should be carried out in a specialist laboratory well experienced in optimal testing to propose appropriate management for the index case and subsequent pregnancies.
Assuntos
Antígenos de Plaquetas Humanas/imunologia , Isoantígenos/imunologia , Trombocitopenia/imunologia , Adulto , Alelos , Plaquetas/imunologia , Éxons , Feminino , Humanos , Recém-Nascido , Integrina alfa2/genética , GravidezRESUMO
BACKGROUND: Fetal or neonatal alloimmune thrombocytopenia (FMAIT) results from a maternal alloimmunization against fetal platelet (PLT) antigens. In Caucasian persons, HPA-1a is the most frequently implicated antigen. During the past few years, FMAIT has been reported associated with rare or private antigens. STUDY DESIGN AND METHODS: Since the first documented case of FMAIT due to anti-HPA-9bw (Max(a)), no additional cases have been reported. Here a retrospective analysis is presented of the cases referred to our laboratories in recent years. The diagnosis was performed by genotyping and identification of the maternal alloantibody by the monoclonal antibody-specific immobilization of PLT antigens (MAIPA) technique. RESULTS: Parental genotyping showed HPA-9bw (Max(a)) mismatch as the sole antigenic incompatibility in seven of eight families. Because the father was found to be HPA-9bw (Max(a)) heterozygous in all the cases, the infant or fetus was genotyped to ascertain the diagnosis. The maternal alloantibody was identified in the MAIPA technique. These data strongly suggest, however, that recognition of the HPA-9bw (Max(a)) epitope is not uniform. The neonatal thrombocytopenia was severe in most cases with bleeding. The outcome was good in all the cases but one. CONCLUSION: This analysis confirms that anti-HPA-9bw (Max(a)) FMAIT is not uncommon and was found to be approximately 2 percent of our confirmed FMAIT cases. It is a clinically severe syndrome that requires prompt diagnosis, albeit difficult, and maternal PLT transfusion therapy. Laboratory investigation of a suspected FMAIT case should be carried out in a specialist laboratory well-experienced in optimal testing. Appropriate management and antenatal therapy should be considered for successive pregnancies to prevent fetal bleeding.
Assuntos
Antígenos de Plaquetas Humanas/imunologia , Doenças Fetais/etiologia , Doenças do Recém-Nascido/etiologia , Isoanticorpos/sangue , Troca Materno-Fetal/imunologia , Trombocitopenia/etiologia , Diagnóstico Diferencial , Feminino , Doenças Fetais/sangue , Doenças Fetais/diagnóstico , Doenças Fetais/terapia , Humanos , Imunoglobulinas Intravenosas/uso terapêutico , Recém-Nascido , Doenças do Recém-Nascido/diagnóstico , Doenças do Recém-Nascido/terapia , Contagem de Plaquetas , Transfusão de Plaquetas , Gravidez , Estudos Retrospectivos , Índice de Gravidade de Doença , Trombocitopenia/sangue , Trombocitopenia/diagnóstico , Trombocitopenia/terapia , Resultado do TratamentoRESUMO
Acquired Glanzmann's thrombasthenia is an uncommon event in association with leukemia. The authors describe a patient with acute lymphoblastic leukemia (ALL) who presented with severe hemorrhagic syndrome, without disseminated intravascular coagulation. The patient's course was complicated by the occurrence of severe hemorrhagic episodes, with a thrombasthenia-like profile, requiring multiple transfusions with packed red cells, platelets, and fresh-frozen plasma. Biological explorations detected anti-GPIIb/IIIa complex antibodies. The patient finally died with refractory disease and persistent bleeding. This case is the first reported of autoantibodies to GPIIb/IIIa in ALL. Such paraneoplastic syndrome is potentially responsible for severe life-threatening hemorrhage.
Assuntos
Leucemia-Linfoma Linfoblástico de Células Precursoras/complicações , Trombastenia/etiologia , Adolescente , Autoanticorpos/sangue , Transfusão de Componentes Sanguíneos , Evolução Fatal , Feminino , Citometria de Fluxo , Humanos , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/imunologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/imunologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia , Trombastenia/imunologia , Trombastenia/terapia , Falha de TratamentoRESUMO
BACKGROUND: Fetal-neonatal alloimmune thrombocytopenia results from a maternal alloimmunization against fetal platelet (PLT) antigens. In Caucasian persons, human PLT antigen-1a (HPA-1a) is the most frequently implicated antigen. Our aim was to develop a method supported by a mathematical approach to quantify HPA-1a antibodies. STUDY DESIGN AND METHODS: The monoclonal antibody immobilization of PLT antigens (MAIPA) protocol was applied on serial dilutions of a serum with high concentration of HPA-1a antibodies used as reference. Two mathematical procedures were used: the standard curve was constructed with a linear and a logistic regression. These regressions were used to calculate the amount of antibody in 14 test serum samples from mothers with detectable anti-HPA-1a. RESULTS: Similar quantifications were obtained with both mathematical procedures. In 11 serum samples, the results, expressed in arbitrary units (AU), ranged from 62 +/- 4 to 1096 +/- 19 with the linear regression and from 62 +/- 4 to 1117 +/- 38 with the logistic regression. In addition, the linear regression allowed us to measure 3 test serum samples with low antibody concentrations from 4 +/- 2 to 44 +/- 3 AU, whereas the logistic regression was not suitable for the quantification of antibodies in these serum samples. CONCLUSION: A simple quantification method was developed for anti-HPA-1a, based on the MAIPA procedure, allowing further studies concerning correlations between anti-HPA-1a quantification and clinical relevance.
Assuntos
Anticorpos Monoclonais/imunologia , Antígenos de Plaquetas Humanas/imunologia , Isoanticorpos/sangue , Feminino , Humanos , Integrina beta3 , Modelos Logísticos , Matemática , Gravidez , Análise de RegressãoRESUMO
BACKGROUND: Discrepancy between phenotyping and genotyping during the investigation of maternofetal alloimmunization leads to the identification of defects on genes encoding membrane glycoproteins (GPs) IIb and IIIa. STUDY DESIGN AND METHODS: Human platelet (PLT) alloantigen (HPA)-1 and -3 PLT phenotypes and genotypes were performed by use of, respectively, the monoclonal antibody-specific immobilization of PLT antigens and the polymerase chain reaction (PCR)-sequence-specific priming techniques for up to 2400 families with thrombocytopenic newborn. When a discrepancy was detected, genomic DNA from the mother was amplified for coding sequences of either the GPIIb or GPIIIa genes. The genetic abnormality responsible for the discrepancy was determined by direct sequencing of the PCR products. RESULTS: Two cases of discrepancy were identified among 2400 families tested. In the first case, Mother L, serologically assigned as HPA-1b-homozygous, was genotyped HPA-1a/1b. In the second case, Mother S, serogically defined HPA-3b-homozygous, was genotyped HPA-3a/3b. DNA sequence analysis revealed for Mother L a T(1447)-->C a point mutation within exon 10 of the GPIIIa gene and for Mother S a C(480)-->G point mutation within exon 4 of the GPIIb gene. These mutations reported in Glanzmann thrombasthenia (GT) patients account for nonexpression of the implicated allele on the PLT surface. Thus, the mothers are GT carriers. Alloantibodies, hallmarks of immunization, were not detected in the maternal serum samples. CONCLUSION: Although this situation is rarely encountered, it is important to combine phenotyping and genotyping to avoid false PLT typing assignation and erroneous diagnosis when alloimmunization might occur.
Assuntos
Trombastenia/genética , Trombastenia/imunologia , Trombocitopenia/genética , Trombocitopenia/imunologia , Antígenos de Plaquetas Humanas/genética , Antígenos de Plaquetas Humanas/imunologia , Erros de Diagnóstico , Eritroblastose Fetal/diagnóstico , Eritroblastose Fetal/genética , Eritroblastose Fetal/imunologia , Feminino , Genótipo , Heterozigoto , Humanos , Recém-Nascido , Fenótipo , Mutação Puntual , Gravidez , Complicações Hematológicas na Gravidez/diagnóstico , Complicações Hematológicas na Gravidez/imunologia , Trombastenia/diagnóstico , Trombocitopenia/diagnósticoRESUMO
ICAM-4 (LW blood group glycoprotein) is an erythroid-specific membrane component that belongs to the family of intercellular adhesion molecules and interacts in vitro with different members of the integrin family, suggesting a potential role in adhesion or cell interaction events, including hemostasis and thrombosis. To evaluate the capacity of ICAM-4 to interact with platelets, we have immobilized red blood cells (RBCs), platelets, and ICAM-Fc fusion proteins to a plastic surface and analyzed their interaction in cell adhesion assays with RBCs and platelets from normal individuals and patients, as well as with cell transfectants expressing the alpha(IIb)beta(3) integrin. The platelet fibrinogen receptor alpha(IIb)beta(3) (platelet GPIIb-IIIa) in a high affinity state following GRGDSP peptide activation was identified for the first time as the receptor for RBC ICAM-4. The specificity of the interaction was demonstrated by showing that: (i) activated platelets adhered less efficiently to immobilized ICAM-4-negative than to ICAM-4-positive RBCs, (ii) monoclonal antibodies specific for the beta(3)-chain alone and for a complex-specific epitope of the alpha(IIb)beta(3) integrin, and specific for ICAM-4 to a lesser extent, inhibited platelet adhesion, whereas monoclonal antibodies to GPIb, CD36, and CD47 did not, (iii) activated platelets from two unrelated type-I glanzmann's thrombasthenia patients did not bind to coated ICAM-4. Further support to RBC-platelet interaction was provided by showing that dithiothreitol-activated alpha(IIb)beta(3)-Chinese hamster ovary transfectants strongly adhere to coated ICAM-4-Fc protein but not to ICAM-1-Fc and was inhibitable by specific antibodies. Deletion of individual Ig domains of ICAM-4 and inhibition by synthetic peptides showed that the alpha(IIb)beta(3) integrin binding site encompassed the first and second Ig domains and that the G65-V74 sequence of domain D1 might play a role in this interaction. Although normal RBCs are considered passively entrapped in fibrin polymers during thrombus, these studies identify ICAM-4 as the first RBC protein ligand of platelets that may have relevant physiological significance.
Assuntos
Moléculas de Adesão Celular/metabolismo , Eritrócitos/metabolismo , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Humanos , Ligantes , Ativação PlaquetáriaRESUMO
We report here the identification and characterization of a new platelet alloantigen, Duv(a+), implicated in a case of neonatal thrombocytopenia. Immunochemical studies demonstrated that the epitope was localized on glycoprotein (GP) IIIa. Sequencing of the exons 2 to 15 of GP IIIa gene polymerase chain reaction products from both parents revealed a single base substitution 517C>T (complementary DNA) present in a heterozygous state in DNA from the father leading to amino acid substitution Thr140Ile (ACC>ATC) within the Arg-Gly-Asp binding domain of GP IIIa. Flow cytometry and immunoprecipitation studies of IIb-C517 or T517 IIIa transfected Cos cells allowed us to demonstrate this mutation was responsible for expression of the Duv(a+) epitope. By polymerase chain reaction-single-strand conformational-polymorphism analysis, the mutated allele could not be detected in a population of 100 healthy unrelated donors, indicating a low frequency of occurrence. The Thr140/Ile dimorphism, localized 3 amino acids upstream from the Arg143 involved in the expression of HPA-4a, did not interfere with the binding of an anti-HPA-4a antibody in flow cytometry. Results of functional analysis of wild-type or mutated transfected CHO cells-(1) aggregation in the presence of Ca(++) and soluble fibrinogen after complex activation by dithiothreitol, (2) adhesion on coated fibrinogen, (3) binding of monoclonal antibody PAC-1 or LIBS antibody D3, and (4) outside-in signaling-all suggest that the Thr140Ile polymorphism localized in the Arg-Gly-Asp binding domain of GP IIIa does not affect significantly, if at all, the integrin function. We have shown that the anti-Duv(a+) antibody may inhibit platelet GP IIb-IIIa function.