Lindo V S, Kakkar V V, Learmonth M, Melissari E, Zappacosta F, Panico M, Morris H R
Thrombosis Research Institute, London.
Br J Haematol. 1995 Mar;89(3):589-601. doi: 10.1111/j.1365-2141.1995.tb08368.x.
An antithrombin (AT) variant Ala382 to Thr (AT-TRI) was identified by mass spectrometric techniques. The variant behaved as a substrate rather than a thrombin inhibitor, but, contrary to previously described P12 AT variants, AT-TRI, expressed as a heterozygous dominant trait, caused severe thromboembolic tendency beginning in their teens in affected members of an English family. In addition, it underwent the S-to-R conformational state transition as evidenced by an increased resistance to thermal denaturation on active centre cleavage, but did not react with a monoclonal antibody, 4C9, directed against a neoepitope that is present on complexed and cleaved normal AT. Antithrombin-TRI, in plasma, was also associated with an abnormal high molecular weight (M(r)) 194,000) component composed of non-covalently-linked antithrombin molecules. This component (D194) showed low affinity for heparin and was devoid of antithrombin progressive activity. D194, isolated by ammonium sulphate precipitation and three chromatographic steps (heparin Sepharose, ion exchange and immunoaffinity), migrated as a single band of M(r) 60,000 on SDS-PAGE under both reducing and non-reducing conditions and was recognized by monospecific anti-human antithrombin antibodies, but did not immunoreact with antibodies raised against a number of proteins including albumin and thrombin. The above data and the fact that the 15 N-terminal amino acids of this M(r) 60,000 band were identical to that of normal antithrombin indicated that the inactive D194 component was composed of aggregated antithrombin molecules, possibly antithrombin trimers. In conclusion, early adulthood severe thromboembolic tendency, failure to expose the 4C9 epitope, and presence of aggregated AT molecules in the plasma are characteristic features of AT-TRI not previously described in other ALA-382 THR mutations.
通过质谱技术鉴定出一种抗凝血酶（AT）变体，即Ala382突变为Thr（AT-TRI）。该变体表现为底物而非凝血酶抑制剂，但与先前描述的P12 AT变体不同，以杂合显性性状表达的AT-TRI在一个英国家族的受影响成员中，从十几岁起就引发了严重的血栓栓塞倾向。此外，它经历了从S构象状态到R构象状态的转变，这可通过活性中心裂解后对热变性的抵抗力增加来证明，但不与针对复合和裂解的正常AT上存在的新表位的单克隆抗体4C9发生反应。血浆中的抗凝血酶-TRI还与一种异常的高分子量（M(r) 194,000）成分相关，该成分由非共价连接的抗凝血酶分子组成。这种成分（D194）对肝素的亲和力较低，且缺乏抗凝血酶的渐进活性。通过硫酸铵沉淀和三步色谱法（肝素琼脂糖、离子交换和免疫亲和）分离得到的D194，在还原和非还原条件下的SDS-PAGE上均迁移为一条M(r) 60,000的单带，并被单特异性抗人抗凝血酶抗体识别，但不与针对包括白蛋白和凝血酶在内的多种蛋白质产生的抗体发生免疫反应。上述数据以及该M(r) 60,000条带的15个N端氨基酸与正常抗凝血酶相同这一事实表明，无活性的D194成分由聚集的抗凝血酶分子组成，可能是抗凝血酶三聚体。总之，成年早期严重的血栓栓塞倾向、未能暴露4C9表位以及血浆中存在聚集的AT分子是AT-TRI的特征，此前在其他ALA-382 THR突变中未描述过。
