A new Ser472Asn (Cab2a+) polymorphism localized within the αIIb “thigh” domain is involved in neonatal thrombocytopenia
Vincent Jallu
VJ and MD contributed equally to this work. MD was recipient of a grant from Novo Nordisk. MD performed, analyzed, and interpreted experiments, VJ designed, performed, and analyzed the experiments; interpreted data; and drafted the manuscript. CK designed experiments, analyzed and interpreted data, and drafted the manuscript.
Search for more papers by this authorMathilde Dusseaux
VJ and MD contributed equally to this work. MD was recipient of a grant from Novo Nordisk. MD performed, analyzed, and interpreted experiments, VJ designed, performed, and analyzed the experiments; interpreted data; and drafted the manuscript. CK designed experiments, analyzed and interpreted data, and drafted the manuscript.
Search for more papers by this authorCecile Kaplan
From the Platelet Immunology Laboratory, INTS; and INSERM U932 Immunology Laboratory, Institut Curie, Paris, France.
Search for more papers by this authorVincent Jallu
VJ and MD contributed equally to this work. MD was recipient of a grant from Novo Nordisk. MD performed, analyzed, and interpreted experiments, VJ designed, performed, and analyzed the experiments; interpreted data; and drafted the manuscript. CK designed experiments, analyzed and interpreted data, and drafted the manuscript.
Search for more papers by this authorMathilde Dusseaux
VJ and MD contributed equally to this work. MD was recipient of a grant from Novo Nordisk. MD performed, analyzed, and interpreted experiments, VJ designed, performed, and analyzed the experiments; interpreted data; and drafted the manuscript. CK designed experiments, analyzed and interpreted data, and drafted the manuscript.
Search for more papers by this authorCecile Kaplan
From the Platelet Immunology Laboratory, INTS; and INSERM U932 Immunology Laboratory, Institut Curie, Paris, France.
Search for more papers by this authorAbstract
BACKGROUND: A new platelet antigen, Cab2a+, was identified in a case of severe neonatal alloimmune thrombocytopenia (<8 × 109/L) in twins.
STUDY DESIGN AND METHODS: Coding sequences of αIIb and β3 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 αIIbβ3 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 αIIbβ3 responsible for the Cab2a+ 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-αIIbβ3 monoclonal antibodies (MoAbs). Activated Asn472 and Ala472 forms of αIIbβ3 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 αIIbβ3 complex functions.
Supporting Information
Figure S1. Sequence analysis and RFLP profiles of amplified exon 15 of αIIb DNAs from the mother, the father and their twins. (A) The genomic DNA sequences corresponding to the region 1504 to 1511 of the αIIb cDNA are shown. The mother is homozygous for the 1508G and the father is heterozygous 1508G/A. The G to A change results in a Ser to Asn substitution at the amino acid 472 of the mature αIIb. The polymorphic position is indicated by an arrow. (B) Amplicons corresponding to the αIIb exon 15 were digested by the Alu I restriction enzyme. After electrophoresis on a 1% agarose gel, two restriction fragments of 59, 53 bp were obtained for the mother (M) and control (N) while a 71 bp fragment was observed for the father (F) and twins (Tw1 and 2). The 1508G to A mutation abolished one Alu I restriction site confirming the father and the twins heterozygous for the mutation.
Figure S2. Anti-αIIbβ3 Moabs binding to Asn472 and Ala472 forms of the complex expressed in CHO cells. CHO cell clones expressing Ser472 (white bar), Asn472 (black bar) and Ala472 (hatched bar) αIIbβ3 forms were incubated with saturating amount of Moab. Moabs were detected by using a polyclonal antibody to mouse IgG conjugated to FITC following flow cytometry analysis. Results were expressed as mean fluorescence intensity (MFI). Ser472Asn and Ser472Ala substitutions did not impaired Moabs binding to the complex. All Moabs bound significantly the three cells; however it should be noted a lower binding for all Moabs with the clone expressing the Asn472-αIIbβ3. These results were representative of three independent experiments.
Figure S3. Fibrinogen and PAC-1 binding to Asn472 and Ala472 forms of αIIbβ3. CHO cell clones expressing WT, Asn472 (Asn) and Ala472 (Ala) αIIbβ3 forms were incubated in buffer (black bar) or treated by 10 mM DTT (hatched bar) for 20 minutes at room temperature to activate the complex prior to incubation with 100 µg/mL fibrinogen-Alexa-488 (panel A) or 10 µg/mL PAC-1-FITC (panel B). Fibrinogen and PAC-1 binding was analyzed by flow cytometry and results expressed as mean fluorescence intensity (MFI). Ser472Asn and Ser472Ala substitutions did not impaired fibrinogen or PAC-1 binding to the integrin. The graph shown here represents the mean ± SD of 3 independent experiments.
Figure S4. Asn472 and Ala472 αIIbβ3 and LIBS epitopes expression. Stably transfected CHO cells expressing WT, Asn472 and Ala472 αIIbβ3 forms were treated by DTT (hatched bar) or not (black bar) prior to incubation with the anti-LIBS Moabs AP5 and D3GP3. Moabs binding was analyzed by flow cytometry. Despite low basal reactivity of Moabs AP5 and D3GP3 with αIIbβ3, complex activation by DTT led to the exposure of new LIBS epitopes whereas binding of Moabs P2 and PT25-2 did not. Untransfected cells did not bind any of the Moab tested. Histograms shown here represent the mean ± SD of 3 independent experiments.
Figure S5. Phosphorylation of pp125FAK: CHO cells expressing WT or Asn472 (Asn) or Ala472 (Ala) forms of αIIbβ3 were maintained in suspension (Non-Adherent) or layered onto immobilized fibrinogen (Adherent) for 2 hours at 37°C before the cells were lysed and the pp125FAK immunoprecipitated. Blots were performed to detect phosphotyrosine using the PY-20 Moab (pTyr) and the pp125FAK by using an anti-FAK Moab (FAK). Ser472Asn and Ser472Ala substitutions did not impair FAK phosphorylation. Results were representative of 3 independent experiments.
Figure S6. Thigh domain sequences comparison in different mammal species. The sequence corresponding to the thigh domain (Gln451 to Glu604) of human αIIb (NP_000410.2) was compared with corresponding sequences from ape (Gene Bank accession number XP_001150497.1), canine (NP_001003163.1), murine (NP_034705.2), equine (NP_001075262.1), and porcine (NP_999163.1). Color code indicated highly conserved amino acid position (red), lower conservation (bleu) and neutral (black). The arrow indicated the position of the Ser472 and its counter part Asn in species other than human and ape. Alignment was obtained by using the online software Multalin (http://multalin.toulouse.inra.fr/multalin/multalin.html; F. CORPET, 1988, Nucl. Acids Res., 16 (22), 10881-10890)
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