A functional platelet fibrinogen receptor with a deletion in the cysteine-rich repeat region of the β3 integrin: the Oea alloantigen in neonatal alloimmune thrombocytopenia

Blood ◽  
2002 ◽  
Vol 99 (4) ◽  
pp. 1205-1214 ◽  
Author(s):  
Sentot Santoso ◽  
Volker Kiefel ◽  
Ina G. Richter ◽  
Ulrich J. H. Sachs ◽  
Abdul Rahman ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Disulfide Bonds ◽  
Low Frequency ◽  
Platelet Glycoprotein ◽  
Mutational Event ◽  
Wild Type ◽  
Stable Transfectants ◽  
Fibrinogen Receptor ◽  
Β3 Integrin ◽  
Alloimmune Thrombocytopenia

This report describes a new low-frequency alloantigen, Oea, responsible for a case of neonatal alloimmune thrombocytopenia (NAIT). In a population study none of 600 unrelated blood donors was an Oea carrier. By immunochemical studies the Oea antigen could be assigned to platelet glycoprotein (GP) IIIa. Sequencing of GPIIIa complementary DNA from an Oea (+) individual showed deletion of a lysine residue at position 611 (ΔLys611). Analysis of 20 Oea(−) and 3 Oea (+) individuals showed that the ΔLys611 form of GPIIIa was related to the phenotype. Anti-Oea reacted with the ΔLys611, but not with the wild-type isoforms on stable transfectants expressing GPIIIa, indicating that ΔLys611 directly induces the expression of Oea epitopes. Under nonreducing conditions the Pro33ΔLys611 variant migrated with a slightly decreased molecular weight compared to the Pro33Lys611 isoform suggesting that ΔLys611 has an influence on the disulfide bonds of GPIIIa. The Pro33ΔLys611 GPIIIa could undergo conformational changes and bind to fibrinogen in a similar manner as the Pro33Lys611 isoform. No difference was found in the tyrosine phosphorylation of pp125FAK, suggesting that ΔLys611 has no effect on integrin function. In contrast to all other low-frequency antigens, the ΔLys611 isoform was associated with the HPA-1b, but not with the high frequency HPA-1a allele. Comparison with GPIIIa DNA from nonhuman primates indicated that the HPA-1a allele represents the ancestral form of GPIIIa. It can be assumed that the Oea form did arise as a result of a mutational event from an already mutated GPIIIa allele.

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

A Mutant (Arg327→His) GPIIb Associated to Thrombasthenia Exerts a Dominant Negative Effect in Stably Transfected CHO Cells

1996 ◽  
Vol 76 (03) ◽  
pp. 292-301 ◽  
Author(s):  
Milagros Ferrer ◽  
Marta Fernandez-Pinel ◽  
Consuelo Gonzalez-Manchon ◽  
Jose Gonzalez ◽  
Matilde S Ayuso ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Cho Cells ◽  
Functional Characterization ◽  
Surface Expression ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Platelet Glycoprotein ◽  
Wild Type ◽  
Glycoprotein Complex ◽  
Negative Effect

SummaryThis work reports the structural and functional characterization of the platelet glycoprotein complex GPIIb-IIIa (integrin αIIbβ3) in a patient of type II Glanzmann thrombasthenia, bearing a homozygous G→A base transition at position 1074 of GPIIb that results in an Arg327→His substitution.CHO cells stably transfected with cDNA encoding His327GPIIb showed a drastic reduction in the surface expression of αIIbβ3 complex relative to control cells transfected with wild type GPIIb. Immunopre-cipitation analysis demonstrated that GPIIb synthesis, heterodimeriza-tion, and short term maturation were not impeded, suggesting that conformational changes dependent on Arg327 of GPIIb may play an essential role in either the rate of maturation and/or transport of heterodimers to the cell surface.Cotransfection of CHO cells with equimolar amounts of cDNAs encoding wild type and mutant His327-GPIIb led to a marked reduction in the surface expression of αIIbβ3. This novel observation of a dominant-negative effect of the mutant His327αIIb subunit provides a molecular basis for the reduced platelet αIIbβ3 content observed in the heterozygous offspring.

Differential Pathogenesis of Bernard-Soulier Syndrome-Associated Mutations In the Platelet Glycoprotein Ibβ Ectodomain

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3202-3202
Author(s):  
Wenjun Yang ◽  
P.A McEwan ◽  
Xi Mo ◽  
Jonas Emsley ◽  
Renhao Li
Keyword(s):  
Cho Cells ◽  
Disulfide Bonds ◽  
Surface Expression ◽  
Full Length ◽  
Platelet Glycoprotein ◽  
Interfacial Region ◽  
Support Surface ◽  
Wild Type ◽  
Platelet Surface ◽  
Bernard Soulier Syndrome

Abstract Abstract 3202 Eight missense mutations in the ectodomain of glycoprotein (GP)Ibβ have been identified in patients with Bernard-Soulier syndrome (BSS) that is characterized by the deficiency of functional GPIb-IX complex on the platelet surface, clearly highlighting the importance of GPIbβ ectodomain in assembly of the GPIb-IX complex. To understand the molecular pathogenesis of these mutations, we have characterized their effects on the expression, secretion, folding of the isolated GPIbβ ectodomain as well as its interaction with GPIX ectodomain in the context of full-length complex. Each of the 8 mutations — C5Y, R17C, P29L, N64T, P74R, Y88C, P96S, and A108P — was constructed into genes encoding HA-tagged GPIbβ ectodomain or full-length GPIbβ subunit, and the mutant gene transfected transiently, along with GPIba and GPIX genes if desired, into Chinese hamster ovary (CHO) cells. Flow cytometry and Western blot analysis indicated that while all 8 mutations impeded formation of the disulfide bonds between GPIba and GPIbβ and significantly decreased the surface expression level of GPIb-IX complex comparing to the wild-type, the extent of disruption varies with each mutation. Further characterization in the context of isolated GPIbβ ectodomain revealed that the majority of 8 mutations — C5Y, R17C, P29L, N64T, Y88C, P96S — are detrimental to proper folding of the GPIbβ ectodomain, resulting in secretion defects and/or domain misfolding. In contrast, two mutations, P74R and A108P, preserved structural integrity of the GPIbβ ectodomain since the mutant ectodomains exhibited wild-type-like secretion levels and formed no inter-molecular disulfide bonds. However, neither of the two mutations, in the context of full-length GPIbβ, were able to support surface expression of GPIX in transfected CHO cells as the wild-type, indicating that P74R and A108P disrupt the interaction between GPIbβ and GPIX ectodomains. Thus, our results demonstrated although all 8 BSS mutations in GPIbβ share the same phenotype, they impair expression of the GPIb-IX complex by two different mechanisms — disrupting folding of the GPIbβ ectodomain or disrupting interactions between GPIb-IX subunits. Furthermore, our results suggest that Pro74 and Ala108 may be located in the interfacial region between GPIbβ and GPIX ectodomains, helping to shed light on the structure of GPIb-IX complex. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Tetrafibricin, a novel non-peptide fibrinogen receptor antagonist, induces conformational changes in glycoprotein IIb/IIIa

1994 ◽  
Vol 301 (3) ◽  
pp. 785-791 ◽  
Author(s):  
T Satoh ◽  
W C Kouns ◽  
Y Yamashita ◽  
T Kamiyama ◽  
B Steiner
Keyword(s):  
Conformational Changes ◽  
Human Platelets ◽  
Platelet Glycoprotein ◽  
Rgd Peptides ◽  
High Affinity ◽  
Fibrinogen Receptor ◽  
Fibrinogen Binding ◽  
Binding Function ◽  
High Affinity Receptor ◽  
Affinity Receptor

Arg-Gly-Asp (RGD) is an amino acid sequence in fibrinogen recognized by platelet glycoprotein (GP) IIb/IIIa. Recently, it was found that RGD peptide binding to GPIIb/IIIa leads to conformational changes in the complex that are associated with the acquisition of high-affinity fibrinogen-binding function. In this study, we found that tetrafibricin, a novel non-peptidic GPIIb/IIIa antagonist, induced similar conformational changes in GPIIb/IIIa as did RGD peptides. Tetrafibricin increased the binding of purified inactive GPIIb/IIIa to immobilized pl-80, a monoclonal antibody that preferentially recognizes ligand-occupied GPIIb/IIIa. Exposure of the pl-80 epitope by tetrafibricin was also observed on resting human platelets by flow cytometry. On intact platelets, the conformational changes transformed GPIIb/IIIa into a high-affinity receptor for fibrinogen and triggered subsequent platelet aggregation. Tetrafibricin is the first non-peptidic GPIIb/IIIa antagonist reported that has the capacity to induce conformational changes in GPIIb/IIIa.

scholarly journals New low-frequency platelet glycoprotein polymorphisms associated with neonatal alloimmune thrombocytopenia

Transfusion ◽  
2010 ◽  
Vol 50 (2) ◽  
pp. 324-333 ◽  
Author(s):  
Julie A. Peterson ◽  
Maria L. Gitter ◽  
Adam Kanack ◽  
Brian Curtis ◽  
Janice McFarland ◽  
...  
Keyword(s):  
Low Frequency ◽  
Platelet Glycoprotein ◽  
Alloimmune Thrombocytopenia

scholarly journals Neonatal alloimmune thrombocytopenia due to a new platelet-specific alloantibody

Blood ◽  
1993 ◽  
Vol 81 (12) ◽  
pp. 3318-3323 ◽  
Author(s):  
JG McFarland ◽  
V Blanchette ◽  
J Collins ◽  
PJ Newman ◽  
R Wang ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Binding Site ◽  
Low Frequency ◽  
Platelet Glycoprotein ◽  
Platelet Surface ◽  
Alloimmune Thrombocytopenia ◽  
Murine Monoclonal Antibodies

Abstract 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.

scholarly journals Neonatal alloimmune thrombocytopenia due to a new platelet-specific alloantibody

Blood ◽  
1993 ◽  
Vol 81 (12) ◽  
pp. 3318-3323
Author(s):  
JG McFarland ◽  
V Blanchette ◽  
J Collins ◽  
PJ Newman ◽  
R Wang ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Binding Site ◽  
Low Frequency ◽  
Platelet Glycoprotein ◽  
Platelet Surface ◽  
Alloimmune Thrombocytopenia ◽  
Murine Monoclonal Antibodies

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.

A point mutation in the cysteine-rich domain of glycoprotein (GP) IIIa results in the expression of a GPIIb-IIIa (αIIbβ3) integrin receptor locked in a high-affinity state and a Glanzmann thrombasthenia–like phenotype

Blood ◽  
2001 ◽  
Vol 98 (8) ◽  
pp. 2432-2441 ◽  
Author(s):  
Catherine Ruiz ◽  
Chao-Yan Liu ◽  
Qi-Hong Sun ◽  
Marianne Sigaud-Fiks ◽  
Edith Fressinaud ◽  
...  
Keyword(s):  
Point Mutation ◽  
Ex Vivo ◽  
Chinese Hamster ◽  
Platelet Glycoprotein ◽  
Glanzmann Thrombasthenia ◽  
Fibrinogen Receptor ◽  
Rich Domain ◽  
Aggregation Response ◽  
Fibrinogen Binding ◽  
Β3 Integrin

Abstract This article reports a Glanzmann thrombasthenia (GT) patient, N.M., with a point mutation in the third cysteine-rich repeat of β3-integrin or platelet glycoprotein (GP) IIIa, leading to the expression of a constitutively activated fibrinogen receptor. The diagnosis of GT was based on a severely reduced platelet-aggregation response to a series of agonists and approximately 20% of surface-expressed GPIIb-IIIa. The patient's GPIIb-IIIa constitutively expressed epitopes recognized by antibodies to ligand-induced binding sites (LIBS) and also spontaneously bound the fibrinogen-mimetic antibody, PAC-1. Furthermore, significant amounts of bound fibrinogen were detected on his platelets ex vivo. No signs of platelet activation were observed on sections of unstimulated platelets from N.M. by electron microscopy. Immunogold labeling highlighted the presence of surface-bound fibrinogen but revealed platelet heterogeneity with regard to the surface density. When the patient's platelets were stimulated by thrombin-receptor activating peptide, amounts of surface-expressed GPIIb-IIIa increased and the aggregation response improved, although it failed to normalize. Platelets from N.M. were able to adhere and spread on immobilized fibrinogen. Sequence analysis of genomic DNA from N.M. revealed a homozygous g1776T>C mutation in GPIIIa, leading to a Cys560Arg amino acid substitution. A stable Chinese hamster ovary (CHO) cell line was prepared expressing surface GPIIb-Arg560IIIa. Like platelets from the patient, GPIIb-Arg560IIIa–transfected CHO cells constitutively bound LIBS antibodies and PAC-1. They also showed an enhanced ability to adhere on surface-bound fibrinogen. Overall, these data demonstrate that a gain-of-function mutation can still be associated with a thrombasthenic phenotype even though platelets show spontaneous fibrinogen binding.

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