Nesheim M, Fredenburgh J C, Larsen G R
Department of Biochemistry, Queen's University, Kingston, Ontario, Canada.
J Biol Chem. 1990 Dec 15;265(35):21541-8.
Active-site-blocked, fluorescent derivatives of tPA (Activase) and a variant (delta FEIX) which lacks the finger and epidermal growth factor-like domains and possesses Asn to Gln and Val to Met mutations at residues 117 and 245, respectively, were prepared. The binding of these to fibrin was studied by adding them at systematically varying concentrations to fibrinogen, at a fixed concentration, inducing clotting with thrombin, separating free and bound tPA or delta FEIX by centrifugation, and measuring the concentration of unbound material by extrinsic fluorescence. Similar studies were performed with Glu and Lys-plasminogen, using intrinsic fluorescence. epsilon-amino caproic acid (EACA) was utilized to distinguish kringle-dependent from finger-dependent binding. In the absence of EACA, delta FEIX-bound fibrin through a single class of sites with Kd = 0.69 microM and n = 1.34 delta FEIX/fibrin. The binding of delta FEIX was completely inhibited by EACA and 50% displacement occurred at [EACA] = 300 microM. Fibrin-bound tPA was only partially displaced with EACA. In the presence of 30 mM EACA, tPA binding reflected a single class of sites with Kd = 0.26 microM and n = 0.60 tPA/fibrin. In the absence of EACA, tPA binding was complex, typified by downwardly curved Scatchard plots, and was consistent with interactions of the two classes of sites, characterized by Kd = 0.13 microM, n = 0.60 and Kd = 0.61 microM, n = 1.23. These were attributed to finger and kringle-dependent interactions, respectively. Under the experimental conditions employed, Glu-plasminogen exhibited no binding to fibrin, whereas Lys-plasminogen bound to a single class of sites with Kd = 0.25 microM and n = 1.02 plasminogen/fibrin. This binding was completely inhibited by EACA and 50% displacement occurred at [EACA] = 28 microM. Competition experiments indicated that Lys-plasminogen does not displace either tPA or delta FEIX from fibrin. From these results the conclusions are drawn that tPA can interact with intact fibrin by two different and independent modes, involving, respectively, the finger and kringle 2 domains, and neither of these modes are competitive with the kringle-dependent binding of Lys-plasminogen.
制备了组织型纤溶酶原激活物（阿替普酶）的活性位点封闭的荧光衍生物，以及一种变体（δFEIX），该变体缺乏指状结构域和表皮生长因子样结构域，并且在第117位和第245位残基处分别存在天冬酰胺到谷氨酰胺以及缬氨酸到甲硫氨酸的突变。通过将它们以系统变化的浓度添加到固定浓度的纤维蛋白原中，用凝血酶诱导凝血，通过离心分离游离的和结合的组织型纤溶酶原激活物或δFEIX，并通过外在荧光测量未结合物质的浓度，来研究它们与纤维蛋白的结合。使用内在荧光对谷氨酸纤溶酶原和赖氨酸纤溶酶原进行了类似的研究。利用ε-氨基己酸（EACA）来区分依赖kringle结构域的结合和依赖指状结构域的结合。在没有EACA的情况下，δFEIX通过一类位点与纤维蛋白结合，解离常数Kd = 0.69μM，结合比n = 1.34δFEIX/纤维蛋白。EACA完全抑制了δFEIX的结合，在[EACA] = 300μM时发生50%的置换。纤维蛋白结合的组织型纤溶酶原激活物仅被EACA部分置换。在30 mM EACA存在的情况下，组织型纤溶酶原激活物的结合反映了一类位点，Kd = 0.26μM，n = 0.60组织型纤溶酶原激活物/纤维蛋白。在没有EACA的情况下，组织型纤溶酶原激活物的结合很复杂，以向下弯曲的Scatchard图为典型特征，并且与两类位点的相互作用一致，其特征分别为Kd = 0.13μM，n = 0.60和Kd = 0.61μM，n = 1.23。这些分别归因于依赖指状结构域的相互作用和依赖kringle结构域的相互作用。在所采用的实验条件下，谷氨酸纤溶酶原未表现出与纤维蛋白的结合，而赖氨酸纤溶酶原与一类位点结合，Kd = 0.25μM，n = 1.02纤溶酶原/纤维蛋白。这种结合被EACA完全抑制，在[EACA] = 28μM时发生50%的置换。竞争实验表明，赖氨酸纤溶酶原不会从纤维蛋白上置换组织型纤溶酶原激活物或δFEIX。从这些结果得出的结论是，组织型纤溶酶原激活物可以通过两种不同且独立的模式与完整的纤维蛋白相互作用，分别涉及指状结构域和kringle 2结构域，并且这些模式都不与赖氨酸纤溶酶原的kringle依赖性结合竞争。
