Caso R, Lane D A, Thompson E A, Olds R J, Thein S L, Panico M, Blench I, Morris H R, Freyssinet J M, Aiach M
Charing Cross and Westminster Medical School, London.
Br J Haematol. 1991 Jan;77(1):87-92. doi: 10.1111/j.1365-2141.1991.tb07953.x.
Antithrombin (AT) Vicenza has been previously identified as a functionally abnormal antithrombin associated with familial thrombosis (Finazzi et al, 1985). It binds normally to heparin, but loses its affinity following interaction with thrombin: it is a poor inhibitor of thrombin. AT Vicenza was isolated from plasma by heparin-Sepharose and thrombin-Sepharose chromatography, fragmented with cyanogen bromide (CNBr) and its tryptic peptides were analysed by fast atom bombardment mass spectrometry mapping. An abnormal peptide mass 1112 was identified. Edman degradation confirmed a substitution of Ala to Pro in the sequence Ala 383-Arg 393. Polymerase chain reaction amplification of exon 6 of the gene followed by genomic sequencing, localized the mutation to codon 384, GCA to CCA. The same mutation has recently been reported in AT Charleville (Mohlo-Sabatier et al, 1989). Sodium dodecyl-sulphate polyacrylamide gel electrophoresis of AT Vicenza (/Charleville) under non-reducing conditions revealed an apparent increase in mol. wt following interaction with thrombin: under reducing conditions the mol. wt was less than that of normal AT. This indicated cleavage and unfolding of the molecule. The site of cleavage was determined by incubation of AT Vicenza (/Charleville) with thrombin-Sepharose, reduction and S-carboxymethylation and reverse phase FPLC. A peptide was identified with the NH2-terminal sequence beginning Ser-Leu-Asn, demonstrating the cleavage had occurred at the reactive site of the variant. It is concluded that the Ala 384 to Pro substitution transforms AT Vicenza (/Charleville) from an inhibitor into a substrate.
抗凝血酶（AT）维琴察先前已被鉴定为一种与家族性血栓形成相关的功能异常抗凝血酶（菲纳齐等人，1985年）。它能正常与肝素结合，但与凝血酶相互作用后会失去亲和力：它是一种较差的凝血酶抑制剂。通过肝素-琼脂糖和凝血酶-琼脂糖色谱法从血浆中分离出AT维琴察，用溴化氰（CNBr）进行片段化，并通过快原子轰击质谱图谱分析其胰蛋白酶肽段。鉴定出一个异常肽质量为1112。埃德曼降解证实序列Ala 383 - Arg 393中Ala被Pro取代。对该基因的外显子6进行聚合酶链反应扩增，随后进行基因组测序，将突变定位到密码子384，即GCA变为CCA。最近在AT沙勒维尔也报道了相同的突变（莫洛-萨巴蒂埃等人，1989年）。在非还原条件下对AT维琴察（/沙勒维尔）进行十二烷基硫酸钠聚丙烯酰胺凝胶电泳，结果显示与凝血酶相互作用后分子量明显增加：在还原条件下，分子量小于正常AT。这表明分子发生了裂解和展开。通过将AT维琴察（/沙勒维尔）与凝血酶-琼脂糖一起孵育、还原和S-羧甲基化以及反相快速蛋白质液相色谱法确定裂解位点。鉴定出一个肽段，其NH2末端序列起始为Ser-Leu-Asn，表明裂解发生在变体的反应位点。得出结论，Ala 384到Pro的取代将AT维琴察（/沙勒维尔）从一种抑制剂转变为一种底物。
