Extended genotyping of ?IIb and ?3 polymorphism ruled out the presence of rare HPAs on paternal platelets. These results indicated that the maternal serum contained an alloantibody against a brand new lowfrequency platelet alloantigen on ?IIb?3 heterodimer, which we termed Seca. Inside the loved ones on the father, two additional Secapositive men and women had been identified by the MAIPA assay . When immunoprecipitation evaluation of biotinlabelled platelets was performed with paternal platelets, maternal serum precipitated the ?IIb?3 complex . Inside the control experiment, antiSeca antibody failed to precipitate ?IIb?3 from maternal platelets. Genetic evaluation To ascertain the molecular genetic basis underlying the Seca antigen, paternal genomic DNA corresponding to the coding regions of ?IIb and ?three was sequentially amplified by PCR utilizing 28 sets of primers.
Nucleotide sequencing of ?three gene encompassing nucleotides 1 to 2367 showed one particular nucleotide substitution G>T at nt 1818 situated on exon 11 on the ?3 gene . This mutation predicted the amino acid Lys at position 580 in Secanegative and Asn in Secapositive men and women. This outcome was confirmed by nucleotide sequencing analysis of your child and other Secapositive family read the full info here members . Alignment analysis amongst human, mouse and canine genes showed that this mutation occurred inside the EGF4 conserved region of ?3, that is adjacent for the Cys residue at position 581 . To study the frequency of Seca, genotyping according to TaqMan approach was established. Among 300 unrelated Caucasian blood donors, no Secapositive person was identified.
Expression study on mammalian cells Allele specific constructs encoding wildtype ?3 or mutant Chrysin ?three had been transfected into CHO cells collectively with ?IIb construct to prove the effect of Lys580Asn mutation around the formation of Seca alloantigen. As shown in Inhibitor 4A, transfected cells expressing wildtype ?three didn’t show any reaction with antiSec alloantibody in flow cytometry. In contrast, antiSeca recognised the mutant ?three. These final results may be confirmed by immunoprecipitation analysis . Impact on the Lys580Asn on cell function To identify conceivable effects of your Lys580Asn substitution on platelet function, aggregation research with Secaphenotyped people have been performed. AntiSeca alloantibody was not able to inhibit platelet aggregation induced by ADP , and the platelet aggregation response of Secapositive and negative men and women was not various .
Unfortunately, all Seca constructive platelets identified so far are heterozygous, expressing both variants of ?3 integrin on their cell surface. Comparing the adhesion of platelets expressing homozygous Lys580/Lys580 and heterozygous Lys580/Asn580 ?three integrin onto immobilised fibrinogen didn’t reveal a considerable difference .