Bruce D, Perry D J, Borg J Y, Carrell R W, Wardell M R
Department of Haematology, University of Cambridge, United Kingdom.
J Clin Invest. 1994 Dec;94(6):2265-74. doi: 10.1172/JCI117589.
A new variant of antithrombin (Rouen-VI, 187 Asn-->Asp) with increased heparin affinity was shown to have normal inhibitory activity which decreased slowly at 4 degrees C and rapidly at 41 degrees C. On electrophoresis the freshly isolated variant had an anodal shift relative to native antithrombin due to the mutation. A further anodal transition occurred after either prolonged storage at 4 degrees C or incubation at 41 degrees C due to the formation of a new inactive uncleaved component with properties characteristic of L-form (latent) antithrombin. At the same time, polymerization also occurred with a predominance of di-, tri-, and tetra-mers. These findings fit with the observed mutation of the conserved asparagine (187) in the F-helix destabilizing the underlying A-sheet of the molecule. Evidence of A-sheet perturbation is provided by the increased rate of peptide insertion into the A-sheet and by the decreased vulnerability of the reactive loop to proteolysis. The spontaneous formation of both L-antithrombin and polymers is consistent with our crystal structure of intact antithrombin where L-form and active antithrombin are linked together as dimers. The nature of this linkage favors a mechanism of polymerization whereby the opening of the A-sheet, to give incorporation of the reactive center loop, is accompanied by the bonding of the loop of one molecule to the C-sheet of the next. The accelerated lability of antithrombin Rouen-VI at 41 versus 37 degrees C provides an explanation for the clinical observation that episodes of thrombosis were preceded by unrelated pyrexias.
一种具有增强肝素亲和力的抗凝血酶新变体（鲁昂 - VI，187位天冬酰胺突变为天冬氨酸）显示具有正常的抑制活性，该活性在4℃时缓慢下降，在41℃时迅速下降。在电泳中，由于突变，新分离的变体相对于天然抗凝血酶有阳极迁移。在4℃长时间储存或在41℃孵育后，由于形成了一种具有L型（潜伏）抗凝血酶特性的新的无活性未切割成分，会发生进一步的阳极转变。与此同时，还发生了聚合反应，主要形成二聚体、三聚体和四聚体。这些发现与在F - 螺旋中保守天冬酰胺（187）的观察到的突变相符合，该突变使分子的基础A - 片层不稳定。肽插入A - 片层的速率增加以及反应环对蛋白水解的敏感性降低，提供了A - 片层扰动的证据。L - 抗凝血酶和聚合物的自发形成与我们完整抗凝血酶的晶体结构一致，其中L型和活性抗凝血酶以二聚体形式连接在一起。这种连接的性质有利于一种聚合机制，即A - 片层打开以纳入反应中心环时，一个分子的环与下一个分子的C - 片层结合。抗凝血酶鲁昂 - VI在41℃与37℃相比加速失活，为临床观察到的血栓形成发作之前出现无关发热提供了解释。
