Ireland H, Lane D A, Thompson E, Walker I D, Blench I, Morris H R, Freyssinet J M, Grunebaum L, Olds R, Thein S L
Charing Cross and Westminster Medical School, London.
Br J Haematol. 1991 Sep;79(1):70-4. doi: 10.1111/j.1365-2141.1991.tb08009.x.
A female with recurrent thrombosis was found to have a functional abnormality of antithrombin, with a ratio of functional to immunological activity in plasma of approximately 50%. Crossed immunoelectrophoresis in the presence of heparin was normal, indicating an abnormality of the reactive site, rather than the heparin binding domain. Accordingly, the antithrombin was isolated by heparin-Sepharose chromatography: this produced a mixture of normal and variant antithrombin, as the patient was heterozygous for the abnormality. To remove the normal component, the antithrombin was passed through a column of thrombin-Sepharose. On sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), prior to its application to thrombin-Sepharose, the antithrombin migrated as a single band with identical mobility to that of normal antithrombin. After thrombin-Sepharose, the purified variant component was proteolysed, and migrated as two components, one with a reduced and one with enhanced mobility under non-reducing conditions. This demonstrated that the variant was unable to form stable inhibitor-thrombin complexes and was cleaved in a substrate reaction with thrombin. One site of cleavage was unambiguously ascertained to be the Arg 393-Ser 394 reactive site bond, by NH2 terminal sequencing of the cleaved variant antithrombin: 10 steps beginning at the P1' position, Ser-Leu-Asn-Pro-Asn-Arg,..., were clearly identified. The mutation responsible for this defect was studied by polymerase chain reaction (PCR) amplification of exon 6 of the antithrombin gene and direct sequencing of the amplified product. The presence of both a G and A in the first position of codon 382, identified the mutation GCA to ACA, which results in the substitution of Ala 382 to Thr. This is identical to that reported for antithrombin Hamilton (Devraj-Kizuk et al, 1988), although antithrombin gene polymorphism analysis suggests that the antithrombin Glasgow II mutation has arisen independently. We have recently shown (Caso et al, 1991) that mutation at a nearby position, Ala 384 to Pro, also transforms another variant, antithrombin Vicenza/Charleville, into a substrate for thrombin. The present results with antithrombin Glasgow II suggest that all the alanine residues at the base of the reactive site loop in positions P12-10 may be important for the formation of a stabilized inhibitor-thrombin complex.
一名患有复发性血栓形成的女性被发现抗凝血酶存在功能异常，血浆中功能活性与免疫活性的比率约为50%。在肝素存在下进行的交叉免疫电泳结果正常，表明是反应位点异常，而非肝素结合域异常。因此，通过肝素-琼脂糖凝胶层析法分离抗凝血酶：由于该患者为该异常的杂合子，所以得到了正常抗凝血酶和变异抗凝血酶的混合物。为去除正常成分，将抗凝血酶通过凝血酶-琼脂糖凝胶柱。在应用于凝血酶-琼脂糖凝胶柱之前，对其进行十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE），抗凝血酶迁移为单一泳带，其迁移率与正常抗凝血酶相同。经过凝血酶-琼脂糖凝胶柱后，纯化的变异成分被蛋白酶水解，在非还原条件下迁移为两个成分，一个迁移率降低，一个迁移率增加。这表明该变异体无法形成稳定的抑制剂-凝血酶复合物，并且在与凝血酶的底物反应中被切割。通过对切割后的变异抗凝血酶进行NH2末端测序，明确确定一个切割位点为精氨酸393-丝氨酸394反应位点键：从P1'位置开始的10个步骤，丝氨酸-亮氨酸-天冬酰胺-脯氨酸-天冬酰胺-精氨酸，……，被清晰鉴定。通过聚合酶链反应（PCR）扩增抗凝血酶基因的外显子6并对扩增产物进行直接测序，研究了导致该缺陷的突变。密码子382第一位同时存在G和A，确定了GCA突变为ACA，这导致丙氨酸382被苏氨酸取代。这与抗凝血酶汉密尔顿（Devraj-Kizuk等人，1988年）报道的相同，尽管抗凝血酶基因多态性分析表明抗凝血酶格拉斯哥II突变是独立发生的。我们最近表明（Caso等人，1991年），附近位置的突变，丙氨酸384突变为脯氨酸，也将另一个变异体抗凝血酶维琴察/沙勒维尔转变为凝血酶的底物。目前关于抗凝血酶格拉斯哥II的结果表明，反应位点环底部P12-10位置的所有丙氨酸残基对于形成稳定的抑制剂-凝血酶复合物可能都很重要。
