Noris P, Simsek S, de Bruijne-Admiraal L G, Porcelijn L, Huiskes E, van der Vlist G J, van Leeuwen E F, van der Schoot C E, von dem Borne A E
University of Pavia, IRCCS, Polyclinic S. Matteo, Italy.
Blood. 1995 Aug 1;86(3):1019-26.
We have identified a new platelet-specific alloantigen, Max(a), responsible for a typical case of neonatal alloimmune thrombocytopenic purpura. The maternal serum reacted strongly with paternal platelets in the platelet immunofluorescence test, whereas platelet alloantigen typing showed that no known human platelet antigen (HPA)-system was involved. In the monoclonal antibody (MoAb)-specific immobilization of platelet antigens (MAIPA) assay, the new antigen was located on the platelet membrane glycoprotein (GP) IIb-IIIa complex, but immunoprecipitation and immunoblot experiments to further localize the antigen failed. However, in the MAIPA assay, the binding of the anti-Max(a) antibodies from the maternal serum was blocked by two anti-GPIIb MoAbs. Thus, the antigen appeared to be located on GPIIb. Analysis of the family lead to the identification of six additional Max(a+) individuals. Three of these six individuals and the father were tested in the platelet aggregation test and were found to be normal. In the analysis of normal donors, three of 500 were typed positive for the new platelet-specific antigen, indicating a phenotype frequency of 0.6% in the normal population. Platelet RNA was isolated from the newborn's Max(a)+ father and from a healthy donor phenotyped as Max(a-), reverse-transcribed, and the entire GPIIb coding region was amplified by polymerase chain reaction. Subsequent nucleotide sequence analysis showed a single G-->A substitution at position 2,603, predicting a valine-->methionine amino acid substitution at position 837 of the mature glycoprotein. This mutation abolished a BsiYI restriction site at the cDNA level and a BstNI restriction site at genomic DNA level, respectively. The genetic association between the new antigen and this point mutation was confirmed by allele-specific restriction analysis on cDNA and on genomic DNA, as well as by allele-specific primer amplification on genomic DNA. The new mutation is 19 bp upstream of the mutation underlying the HPA-3 system. Therefore, we also evaluated the association between Mas and the HPA-3 polymorphism. So far, all Max(a+) individuals were also found to be HPA-3b, whereas 50 HPA-3a individuals were all Max(a-). This may indicate that Max(a) is a variant of the HPA-3 allele.
我们鉴定出一种新的血小板特异性同种抗原Max(a)，它导致了一例典型的新生儿同种免疫性血小板减少性紫癜。在血小板免疫荧光试验中，母亲的血清与父亲的血小板发生强烈反应，而血小板同种抗原分型显示未涉及已知的人类血小板抗原（HPA）系统。在单克隆抗体（MoAb）特异性血小板抗原固定（MAIPA）试验中，新抗原位于血小板膜糖蛋白（GP）IIb-IIIa复合物上，但进一步定位该抗原的免疫沉淀和免疫印迹实验未能成功。然而，在MAIPA试验中，母亲血清中抗Max(a)抗体的结合被两种抗GPIIb单克隆抗体阻断。因此，该抗原似乎位于GPIIb上。对该家系的分析又鉴定出另外6名Max(a+)个体。这6名个体中的3名以及父亲接受了血小板聚集试验，结果显示正常。在对正常供者的分析中，500名供者中有3名被检测为新的血小板特异性抗原阳性，表明该表型在正常人群中的频率为百分之0.6。从新生儿的Max(a+)父亲以及一名表型为Max(a-)的健康供者中分离血小板RNA，进行逆转录，然后通过聚合酶链反应扩增整个GPIIb编码区。随后的核苷酸序列分析显示在第2603位有一个单一的G→A替换，预测成熟糖蛋白的第837位氨基酸由缬氨酸替换为甲硫氨酸。该突变分别在cDNA水平消除了一个BsiYI限制性酶切位点，在基因组DNA水平消除了一个BstNI限制性酶切位点。通过对cDNA和基因组DNA进行等位基因特异性限制性分析，以及对基因组DNA进行等位基因特异性引物扩增，证实了新抗原与该点突变之间的遗传关联。该新突变位于HPA-3系统相关突变上游19个碱基对处。因此，我们还评估了Max(a)与HPA-3多态性之间的关联。到目前为止，所有Max(a+)个体也均为HPA-3b，而50名HPA-3a个体均为Max(a-)。这可能表明Max(a)是HPA-3等位基因的一个变体。
