Beauchamp N J, Pike R N, Daly M, Butler L, Makris M, Dafforn T R, Zhou A, Fitton H L, Preston F E, Peake I R, Carrell R W
Division of Molecular and Genetic Medicine, University of Sheffield, Royal Hallamshire Hospital, Sheffield, UK.
Blood. 1998 Oct 15;92(8):2696-706.
The inherent variability of conformational diseases is demonstrated by two families with different mutations of the same conserved aminoacid in antithrombin. Threonine 85 underlies the opening of the main beta-sheet of the molecule and its replacement, by the polar lysine, in antithrombin Wobble, resulted in a plasma deficiency of antithrombin with an uncharacteristically severe onset of thrombosis at 10 years of age, whereas the replacement of the same residue by a nonpolar methionine, antithrombin Wibble, gave near-normal levels of plasma antithrombin and more typical adult thromboembolic disease. Isolated antithrombin Wibble had a decreased thermal stability (Tm 56.2, normal 57.6 degreesC) but was fully stabilized by the heparin pentasaccharide (Tm 71.8, normal 71.0 degreesC), indicating that the prime abnormality is a laxity in the transition of the main sheet of the molecule from the 5- to 6-stranded form, as was confirmed by the ready conversion of antithrombin Wibble to the 6-stranded latent form on incubation. That this transition can occur in vivo was shown by the finding of nearly 10% of the proband's plasma antithrombin in the latent form and also, surprisingly, of small but definitive amounts of latent antithrombin in normal plasma. The latent transition will be predictably accelerated not only by gross mutations, as with antithrombin Wobble, to give severe episodic thrombosis, but also by milder mutations, as with antithrombin Wibble, to trigger thrombosis in the presence of other predisposing factors, including the conformational stress imposed by the raised body temperatures of fevers. Both antithrombin variants had an exceptional (25-fold) increase in heparin affinity and this, together with an increased inhibitory activity against factor Xa, provides evidence of the direct linkage of A-sheet opening to the conformational basis of heparin binding and activation.
抗凝血酶中同一保守氨基酸发生不同突变的两个家族证明了构象疾病的内在变异性。苏氨酸85是分子主要β-折叠打开的基础，在抗凝血酶Wobble中，它被极性赖氨酸取代，导致血浆中抗凝血酶缺乏，在10岁时出现异常严重的血栓形成发作，而用非极性甲硫氨酸取代同一残基的抗凝血酶Wibble，血浆抗凝血酶水平接近正常，且血栓栓塞疾病更具成人典型特征。分离出的抗凝血酶Wibble热稳定性降低（熔解温度56.2，正常为57.6℃），但被肝素五糖完全稳定（熔解温度71.8，正常为71.0℃），这表明主要异常是分子主折叠从5链形式向6链形式转变时的松弛，抗凝血酶Wibble在孵育时易于转化为6链潜伏形式证实了这一点。在体内可以发生这种转变，这一发现表明，先证者血浆中近10%的抗凝血酶呈潜伏形式，令人惊讶的是，正常血浆中也存在少量但确定量的潜伏抗凝血酶。可以预见，不仅像抗凝血酶Wobble那样的重大突变会加速潜伏转变，导致严重的发作性血栓形成，而且像抗凝血酶Wibble那样的较轻突变也会在存在其他诱发因素（包括发热引起的体温升高所施加的构象应激）时引发血栓形成。两种抗凝血酶变体的肝素亲和力都异常增加（25倍），这与对因子Xa的抑制活性增加一起，为A-折叠打开与肝素结合和激活的构象基础的直接联系提供了证据。
