重组apo(a) Kringle Ⅳ-10对纤溶酶原与内皮细胞结合的影响

通过研究重组apo(a)KringleⅣ 10 (KⅣ 10 )的赖氨酸结合能力对纤溶酶原与内皮细胞结合的影响 ,探讨apo(a)在抑制纤溶过程中的作用 ,为脂蛋白 (a) lipoprotein(a) ,Lp(a) ]致动脉粥样硬化机理研究提供依据 .将含apo(a)野生型KⅣ 10 ((wild typeKⅣ 10 Trp72 ,wt KⅣ 10 Trp72 )和突变型KⅣ 10 (mutate typeKⅣ 10 Trp72 ,mut KⅣ 10 Arg72 )基因片段重组质粒 ,分别转化至E .coliDH5α菌株中并表达含这 2个重组片段的融合蛋白 ,通过Glutathione Agarosebeads亲和层析柱进行分离和提纯 ,经L Lys Sepharose 4B亲和层析柱检测其赖氨酸结合能力 .再以异硫氰酸荧光素标记的纤溶酶原为配基 ,观察这 2种基因表达片段对纤溶酶原与人脐静脉内皮细胞 (humanumbilicalveinendothe lialcells ,HUVEC)结合的影响 .结果显示 :在E .coliDH 5α菌株中表达的野生型谷胱甘肽S 转移酶(glutathioneS transferase ,GST) KⅣ 10 Trp72 (GST wt KⅣ 10 Trp72 )融合蛋白和突变型谷胱甘肽S 转移酶 (GST mut KⅣ 10 Arg72 )融合蛋白在赖氨酸结合能力上存在明显差异 .其中GST wt KⅣ 10 Trp72能有效地抑制纤溶酶原与人脐静脉内皮细胞的结合 ;而GST mut KⅣ 10 Arg72 在任一浓度范围内均无这种抑制作用 .结果

The Effects of Recombinant Apolipoprotein (a) Kringle Ⅳ-10 on the Binding of Plasminogen to the Endothelial Cells

In order to elucidate the mechanisms of atherosclerosis which caused by lipoprotein(a) and discuss the inhibition of fibrinolysis by apolipoprotein(a) apo(a)], the effects of recombinant apo(a) Kringle Ⅳ 10(KⅣ 10) on the binding of plasminogen to endothelial cells by the capacity of lysine binding were studied. The recombinant plasmids designated pGEX KG/Trp 72 KⅣ 10 (wild type KⅣ 10/Trp 72 , wt KⅣ 10/Trp 72 ) and pGEX KG/Arg 72 KⅣ 10 (mutate type KⅣ 10/ Arg 72 , mut KIV 10/Arg 72 ) of apo(a) were transformed E.coli DH 5α and expressed as fusion GST proteins, following with isolation and purification by affinity chromatography on Glutathione Agarose. The lysine binding capacities of the GST KⅣ 10 fusion proteins were examined by affinity chromatography on Lys Sepharose. The effects of these two kinds of GST KⅣ 10 fusion proteins on the binding of plasminogen to human umbilical vein endothelial cells (HUVEC) were studied, using the plasminogen labeled with fluorescein isothiocyanate (FITC). The results indicated that the expressed GST wt KⅣ 10/Trp 72 fusion proteins bound strongly to Lys Sepharose, while the expressed GST mut KⅣ 10/Arg 72 fusion proteins had no affinity to Lys Sepharose at all. GST wt KⅣ 10/Trp 72 fusion proteins could effectively inhibit the binding of plasminogen to HUVEC, in contrast, GST mut KⅣ 10/Arg 72 fusion proteins could not even in higher concentration. The inhibition of fibrinolysis by apo(a) KⅣ 10 is associated with its capacity of lysine binding. The capacity of apo(a) KⅣ 10 is associated with Trp 72 in its lysine binding sites.