Departments of Medicine and Biochemistry and Biomedical Sciences, McMaster University and Thrombosis and Atherosclerosis Research Institute, Hamilton, Ontario L8L 2X2, Canada.
J Biol Chem. 2012 Feb 10;287(7):4652-61. doi: 10.1074/jbc.M111.317719. Epub 2011 Dec 20.
Fibrin (Fn) enhances plasminogen (Pg) activation by tissue-type plasminogen activator (tPA) by serving as a template onto which Pg and tPA assemble. To explore the contribution of the Pg/Fn interaction to Fn cofactor activity, Pg variants were generated and their affinities for Fn were determined using surface plasmon resonance (SPR). Glu-Pg, Lys-Pg (des(1-77)), and Mini-Pg (lacking kringles 1-4) bound Fn with K(d) values of 3.1, 0.21, and 24.5 μm, respectively, whereas Micro-Pg (lacking all kringles) did not bind. The kinetics of activation of the Pg variants by tPA were then examined in the absence or presence of Fn. Whereas Fn had no effect on Micro-Pg activation, the catalytic efficiencies of Glu-Pg, Lys-Pg, and Mini-Pg activation in the presence of Fn were 300- to 600-fold higher than in its absence. The retention of Fn cofactor activity with Mini-Pg, which has low affinity for Fn, suggests that Mini-Pg binds the tPA-Fn complex more tightly than tPA alone. To explore this possibility, SPR was used to examine the interaction of Mini-Pg with Fn in the absence or presence of tPA. There was 50% more Mini-Pg binding to Fn in the presence of tPA than in its absence, suggesting that formation of the tPA-Fn complex exposes a cryptic site that binds Mini-Pg. Thus, our data (a) indicate that high affinity binding of Pg to Fn is not essential for Fn cofactor activity, and (b) suggest that kringle 5 localizes and stabilizes Pg within the tPA-Fn complex and contributes to its efficient activation.
纤维蛋白（Fn）作为纤维蛋白原（Pg）和组织型纤溶酶原激活物（tPA）组装的模板，增强 tPA 对纤溶酶原的激活。为了探究 Pg/Fn 相互作用对 Fn 辅因子活性的贡献，生成了 Pg 变体，并使用表面等离子体共振（SPR）测定了它们与 Fn 的亲和力。Glu-Pg、Lys-Pg（des(1-77)）和 Mini-Pg（缺乏环 1-4）与 Fn 的 K(d)值分别为 3.1、0.21 和 24.5 μm，而 Micro-Pg（缺乏所有环）则不结合 Fn。然后在不存在或存在 Fn 的情况下，研究了 Pg 变体被 tPA 激活的动力学。尽管 Fn 对 Micro-Pg 激活没有影响，但在 Fn 存在的情况下，Glu-Pg、Lys-Pg 和 Mini-Pg 的催化效率比不存在 Fn 时高 300-600 倍。Mini-Pg 保留了 Fn 辅因子活性，尽管它与 Fn 的亲和力较低，但这表明 Mini-Pg 与 tPA-Fn 复合物的结合比 tPA 单独结合更紧密。为了探究这种可能性，使用 SPR 研究了 Mini-Pg 在不存在或存在 tPA 的情况下与 Fn 的相互作用。在存在 tPA 的情况下，Mini-Pg 与 Fn 的结合增加了 50%，这表明 tPA-Fn 复合物的形成暴露了一个结合 Mini-Pg 的隐蔽位点。因此，我们的数据表明：（a）Pg 与 Fn 的高亲和力结合对于 Fn 辅因子活性不是必需的；（b）kringle 5 定位并稳定 Pg 位于 tPA-Fn 复合物内，并有助于其有效的激活。
