Barron-Casella E A, Kickler T S, Rogers O C, Casella J F
Department of Pediatrics, Johns Hopkins University School of Medicine, Baltimore, MD 21205.
Blood. 1994 Aug 15;84(4):1157-63.
The platelet antigens, PlA1 and PlA2, are responsible for most cases of posttransfusion purpura (PTP) and neonatal alloimmune thrombocytopenia (NAIT) in the caucasian population and are determined by two allelic forms of the platelet glycoprotein GPIIIa gene. To study the interaction between these antigens and their respective antibodies, we inserted the sequence that encodes the signal peptide and the N-terminal 66 amino acids of the PlA1 form of GPIIIa into the expression vector pGEX1. To express the PlA2 antigen, nucleotide 196 of the PlA1 coding sequence was mutated to the PlA2 allelic form. When transformed and induced in Escherichia coli, the two constructs produce glutathione S-transferase (GST)/N-terminal GPIIIa fusion proteins, one containing leucine at position 33 (PlA1), the other proline (PlA2). These proteins are easily purified in milligram quantities using glutathione-Sepharose and react specifically with their respective antibodies by immunoblot and enzyme-linked immunosorbent assay. Antigenicity of the PlA1 fusion protein in reduced glutathione increases with time; moreover, the addition of oxidized glutathione accelerates this process, presumably because of formation of the native disulfide bonds. Neutralization assays indicate that the PlA1 fusion protein competes for all of the anti-PlA1 antibody in the serum of patients with PTP and NAIT that is capable of interacting with the surface of intact platelets. This study shows that the GST/N-terminal GPIIIa fusion proteins contain conformational epitopes that mimic those involved in alloimmunization, and that regions other than the amino terminal 66 amino acids of GPIIIa are not likely to contain or be required for the development of functional PlA1 epitopes. Furthermore, these recombinant proteins can be used for the affinity-purification of clinical anti-PlA1 antibodies and specific antibody identification by western blotting, making them useful in the diagnosis of patients alloimmunized to PlA1 alloantigens.
血小板抗原PlA1和PlA2是白种人群中大多数输血后紫癜（PTP）和新生儿同种免疫性血小板减少症（NAIT）的病因，由血小板糖蛋白GPIIIa基因的两种等位基因形式决定。为了研究这些抗原与其各自抗体之间的相互作用，我们将编码信号肽和GPIIIa的PlA1形式的N端66个氨基酸的序列插入表达载体pGEX1中。为了表达PlA2抗原，将PlA1编码序列的第196位核苷酸突变为PlA2等位基因形式。当在大肠杆菌中转化和诱导时，这两种构建体产生谷胱甘肽S-转移酶（GST）/N端GPIIIa融合蛋白，一种在第33位含有亮氨酸（PlA1），另一种含有脯氨酸（PlA2）。这些蛋白使用谷胱甘肽-琼脂糖很容易以毫克量进行纯化，并通过免疫印迹和酶联免疫吸附测定法与它们各自的抗体发生特异性反应。还原型谷胱甘肽中PlA1融合蛋白的抗原性随时间增加；此外，添加氧化型谷胱甘肽可加速此过程，推测是由于形成了天然二硫键。中和试验表明，PlA1融合蛋白与PTP和NAIT患者血清中所有能够与完整血小板表面相互作用的抗PlA1抗体竞争。本研究表明，GST/N端GPIIIa融合蛋白包含模拟同种免疫中涉及的构象表位，并且GPIIIa的N端66个氨基酸以外的区域不太可能包含功能性PlA1表位或其形成所必需。此外，这些重组蛋白可用于临床抗PlA1抗体亲和纯化和通过蛋白质印迹法进行特异性抗体鉴定，使其在诊断对PlA1同种抗原产生同种免疫的患者中有用。
