Xu H, Frojmovic M M, Wong T, Rauch J
Division of Rheumatology, Montreal General Hospital Research Institute, Quebec, Canada.
J Autoimmun. 1995 Feb;8(1):97-119. doi: 10.1006/jaut.1995.0008.
We have previously described an SLE-derived human hybridoma autoantibody, 9604, which binds to activated but not to resting platelets, inhibits platelet aggregation, and immunoprecipitates a surface-labelled poly-peptide of 32,000 molecular weight (MW) (p32). In the present study, using a murine monoclonal anti-p32 antibody (8E8), we show that p32 is responsible for the binding of 9604 to activated platelets. First, 8E8 bound to the surface of activated but not resting platelets, as detected by flow cytometry, showing saturation with 1900 antibody molecules bound/platelet and a Kd of 72 nM. Second, complete inhibition of the binding of 9604 to ADP-activated platelets by 8E8 and inhibition of the reactivity of 8E8 with p32 by 9604 demonstrated that 8E8 and 9604 recognize the same 32,000 MW platelet polypeptide. Isolation of platelet proteins, using an 8E8-affinity column, showed high MW bands corresponding to multimers of p32 under non-reducing conditions, and isolated p32 tended to self-associate even under reducing conditions. High performance liquid chromatography (HPLC) demonstrated that the native p32-containing protein has an approximate MW of 450,000. A comparison of the functional properties of murine monoclonal 8E8 and human monoclonal 9604 revealed that both antibodies are cytotoxic to platelets in 51Cr-release assays. In contrast, 8E8, unlike 9604, did not affect platelet aggregation, suggesting that different epitopes may be recognized by the two antibodies. These findings demonstrate that p32 is a subunit of an activation marker that is expressed on the surface of activated platelets and recognized by an SLE-derived anti-platelet autoantibody. Our data also indicate that murine monoclonal antibodies produced against platelet autoantigens may not mimic the functional activities of spontaneously occurring autoantibodies, probably due to the recognition of different epitopes.
我们之前描述过一种源自系统性红斑狼疮（SLE）的人杂交瘤自身抗体9604，它能与活化的血小板结合，但不与静息血小板结合，可抑制血小板聚集，并免疫沉淀出一种分子量为32,000（MW）的表面标记多肽（p32）。在本研究中，我们使用鼠单克隆抗p32抗体（8E8），证明p32是9604与活化血小板结合的原因。首先，通过流式细胞术检测发现，8E8能与活化血小板表面结合，而不与静息血小板表面结合，显示每个血小板结合1900个抗体分子达到饱和，解离常数（Kd）为72 nM。其次，8E8能完全抑制9604与ADP活化血小板的结合，而9604能抑制8E8与p32的反应性，这表明8E8和9604识别的是同一种分子量为32,000的血小板多肽。使用8E8亲和柱分离血小板蛋白，在非还原条件下显示出对应p32多聚体的高分子量条带，并且即使在还原条件下，分离出的p32也倾向于自我缔合。高效液相色谱（HPLC）表明，天然含p32的蛋白分子量约为450,000。对鼠单克隆8E8和人单克隆9604的功能特性进行比较发现，在51Cr释放试验中，这两种抗体对血小板都具有细胞毒性。相比之下，8E8与9604不同，它不影响血小板聚集，这表明这两种抗体可能识别不同的表位。这些发现表明，p32是一种活化标志物的亚基，该活化标志物在活化血小板表面表达，并被一种源自SLE的抗血小板自身抗体识别。我们的数据还表明，针对血小板自身抗原产生的鼠单克隆抗体可能无法模拟自发产生的自身抗体的功能活性，这可能是由于识别不同表位所致。
