de Vries C, Veerman H, Koornneef E, Pannekoek H
Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, Department of Molecular Biology, Amsterdam.
J Biol Chem. 1990 Aug 15;265(23):13547-52.
The enzyme tissue-type plasminogen activator (t-PA) and its substrate Glu-plasminogen can both bind to fibrin. The assembly of these three components results in about a 1000-fold acceleration of the conversion of Glu-plasminogen into plasmin. Fibrin binding of t-PA is mediated both by its finger (F) domain and its kringle-2 domain. Fibrin binding of Glu-plasminogen involves its kringle structures (K1-K5). It has been suggested that particular kringles contain lysine-binding sites and/or aminohexyl-binding sites, exhibiting affinity for specific carboxyl-terminal lysines and intrachain lysines, respectively. We investigated the possibility that t-PA and Glu-plasminogen kringles share common binding sites in fibrin, limitedly digested with plasmin. For that purpose we performed competition experiments, using conditions that exclude plasmin formation, with Glu-plasminogen and either t-PA or two deletion mutants, lacking the F domain (t-PA del.F) or lacking the K2 domain (t-PA del.K2). Our data show that fibrin binding of t-PA, mediated by the F domain, is independent of Glu-plasminogen binding. In contrast, partial inhibition by Glu-plasminogen of t-PA K2 domain-mediated fibrin binding is observed that is dependent on carboxyl-terminal lysines, exposed in fibrin upon limited plasmin digestion. Half-maximal competition of fibrin binding of both t-PA and t-PA del.F is obtained at 3.3 microM Glu-plasminogen. The difference between this value and the apparent dissociation constant of Glu-plasminogen binding to limitedly digested fibrin (12.1 microM) under these conditions is attributed to multiple, simultaneous interactions, each having a separate affinity. It is concluded that t-PA and Glu-plasminogen can bind to the same carboxyl-terminal lysines in limitedly digested fibrin, whereas binding sites composed of intrachain lysines are unique both for the K2 domain of t-PA and the Glu-plasminogen kringles.
组织型纤溶酶原激活剂（t-PA）及其底物谷氨酸纤溶酶原均可与纤维蛋白结合。这三种成分的组装可使谷氨酸纤溶酶原转化为纤溶酶的速度加快约1000倍。t-PA与纤维蛋白的结合由其指状（F）结构域和kringle-2结构域介导。谷氨酸纤溶酶原与纤维蛋白的结合涉及其kringle结构（K1-K5）。有人提出，特定的kringle含有赖氨酸结合位点和/或氨基己基结合位点，分别对特定的羧基末端赖氨酸和链内赖氨酸表现出亲和力。我们研究了t-PA和谷氨酸纤溶酶原kringle在经纤溶酶有限消化的纤维蛋白中共享共同结合位点的可能性。为此，我们进行了竞争实验，使用排除纤溶酶形成的条件，用谷氨酸纤溶酶原和t-PA或两个缺失突变体（缺少F结构域的t-PA del.F或缺少K2结构域的t-PA del.K2）进行实验。我们的数据表明，由F结构域介导的t-PA与纤维蛋白的结合独立于谷氨酸纤溶酶原的结合。相反，观察到谷氨酸纤溶酶原对t-PA K2结构域介导的纤维蛋白结合有部分抑制作用，这种抑制作用依赖于在纤溶酶有限消化后暴露于纤维蛋白中的羧基末端赖氨酸。在3.3 microM谷氨酸纤溶酶原时，t-PA和t-PA del.F与纤维蛋白结合的半数最大竞争得以实现。在这些条件下，该值与谷氨酸纤溶酶原与经有限消化的纤维蛋白结合的表观解离常数（12.1 microM）之间的差异归因于多个同时发生的相互作用，每个相互作用都有单独的亲和力。得出的结论是，t-PA和谷氨酸纤溶酶原可在经有限消化的纤维蛋白中与相同的羧基末端赖氨酸结合，而由链内赖氨酸组成的结合位点对于t-PA的K2结构域和谷氨酸纤溶酶原kringle都是独特的。
