Corral Javier, Rivera José, Martínez Constantino, González-Conejero Rocio, Miñano Antonia, Vicente Vicente
Hematology and Clinical Oncology Service, Department of Medicine, Hospital General Universitario, Centro Regional de Hemodonación, Universidad de Murcia, Spain.
J Lab Clin Med. 2003 Nov;142(5):298-305. doi: 10.1016/S0022-2143(03)00136-7.
The structural flexibility of antithrombin is essential for its molecular trapping mechanism but also makes it vulnerable to even minor changes affecting its conformational stability, which influences hemostasis significantly. The conformational transformation of this serpin has been poorly investigated in biologic samples because available immunologic methods hardly differentiate between different conformations of this protein. Crossed immunoelectrophoresis (CIE) in presence of heparin has been classically used to identify mutant antithrombins with low heparin affinity. We demonstrate that this method also separates native and relaxed antithrombin, permitting the analysis of conformational variations of this potent anticoagulant with just a few microliters of plasma. However, CIE does not distinguish between antithrombin conformations with reduced heparin affinity: latent, cleaved, thrombin-antithrombin complexes, or heparin-binding mutants. Therefore, clinical interpretation of CIE results should be examined with caution. Using this and other methods, and evaluating the functional activity of antithrombin, we analyzed the conformational transformation of antithrombin in biologic samples. We confirmed its transformation to the latent configuration by incubating it at 50 degrees C. This conformational change also occurs at 37 degrees C, supporting the idea that this process is involved in the senescence of antithrombin. However, fresh plasma contains only traces of latent antithrombin, suggesting that this conformation is rapidly cleared in vivo. Finally, small increases in temperature (to 40 degrees C) resulted in a faster conformational transformation of antithrombin. Fever has been suggested to have key structural, functional, and clinical consequences in patients with conformational mutations in antithrombin. Our results support a role for small changes in temperature in nonmutated antithrombin, suggesting that fever is a general risk factor for thrombosis.
抗凝血酶的结构灵活性对其分子捕获机制至关重要，但也使其容易受到影响其构象稳定性的微小变化的影响，而这会显著影响止血过程。由于现有的免疫学方法很难区分这种蛋白质的不同构象，因此在生物样品中对这种丝氨酸蛋白酶抑制剂的构象转变研究较少。在肝素存在下的交叉免疫电泳（CIE）传统上用于鉴定肝素亲和力低的突变抗凝血酶。我们证明该方法还可以分离天然和松弛的抗凝血酶，仅需几微升血浆就能分析这种强效抗凝剂的构象变化。然而，CIE无法区分肝素亲和力降低的抗凝血酶构象：潜在的、裂解的、凝血酶-抗凝血酶复合物或肝素结合突变体。因此，CIE结果的临床解释应谨慎审视。使用这种方法和其他方法，并评估抗凝血酶的功能活性，我们分析了生物样品中抗凝血酶的构象转变。我们通过在50摄氏度下孵育证实了它向潜在构象的转变。这种构象变化在37摄氏度时也会发生，支持了这一过程参与抗凝血酶衰老的观点。然而，新鲜血浆中仅含有微量的潜在抗凝血酶，这表明这种构象在体内会迅速被清除。最后，温度小幅升高（至40摄氏度）会导致抗凝血酶的构象转变加快。有人提出发热对具有抗凝血酶构象突变的患者具有关键的结构、功能和临床影响。我们的结果支持温度的微小变化在非突变抗凝血酶中的作用，表明发热是血栓形成的一个普遍危险因素。
