抗凝血酶L99氨基酸位点突变对其功能的影响

The functional study of antithrombin L99 mutation

目的研究抗凝血酶（AT）L99氨基酸位点突变导致遗传性AT缺陷症的分子机制。方法利用果蝇细胞表达系统表达纯化野生型AT以及L99V、L99A、L99I和L99S等突变型AT蛋白；采用肝素结合实验检测重组AT蛋白与低分子量肝素钠的结合能力；采用表面等离子共振（SPR）技术检测重组AT蛋白与凝血酶（FⅡa）及活化凝血因子X（FXa）的结合能力；将野生型及各突变型AT蛋白的浓度调整至正常AT血浆浓度后，采用发色底物法检测重组AT蛋白活性。结果考马斯亮蓝染色及免疫印迹法显示各突变蛋白与野生型AT蛋白大小一致，未见明显杂带。肝素结合实验显示ATL99V、L99A、L99I和L99S突变蛋白与低分子量肝素钠的结合分别为相同浓度野生型AT蛋白的（44．8土3．6）％、（118．9~14．0）％、（15．2~8．8）％和（23．O士8．2）％。SPR检测显示ATL99V、L99A、L99I和L99S突变蛋白与F1Ia的结合分别为相同浓度野生型AT蛋白的13％、57％、3％和29％；ATL99V、L99A、L99I和L99S突变蛋白与FXa的结合分别为相同浓度野生型AT蛋白的7％、51％、1％和25％。发色底物法显示，野生型AT蛋白及L99V、L99A、L99I、L99S等突变型AT蛋白活性分别为146．5％、21．4％、120．9％、10.8％和39．0％。结论L99位点突变破坏了AT与肝素、FIIa及FXa的结合，使AT蛋白活性下降，从而导致遗传性AT缺陷症。

Objective To study the molecular mechanisms of inherited antithrombin （AT） defiency caused by AT L99 mutation. Methods Wild type （WT）, L99V, L99A, L99I and L99S AT were purified from drosophila expression system. The binding capacity of AT and the low molecular weight heparin sodium was analyzed by the heparin binding assay. Surface plasmon resonance （SPR） was used to detect the binding ability of AT to thrombin （F Ⅱ a） or AT to coagulation factor X a （F X a）. The activity of AT （AT： A） was detected by chromogenic assay. Results The purified WT and mutant AT were at the same size. No additional band was observed by coomassie blue staining and western blot assay. Compared to the WT AT, the binding abilities of the low molecular weight heparin sodium to the AT L99V, L99A, L99I and L99S were （44.8~3.6）%, （118.9~14.0）%, （15.2±8.8）%, and （23.0±8.2）%, respectively. The binding abilities of F 1I a to AT L99V, L99A, L99I and L99S were 13%, 57%, 3%, and 29%, while the binding of FXa to AT L99V, L99A, L99I and L99S were 7%, 51%, 1%, and 25%. The AT：A ofWT, L99V, L99A, L99I and L99S AT were 146.5%, 21.4%, 120.9%, 10.8%, and 39.0%, respectively. Conclusion The binding abilities of AT to heparin, F I1 a and F X a were damaged by the L99 mutation, which resulted in decreased AT： A and inherited AT deficiency.