BINDING OF TISSUE-TYPE PLASMINOGEN-ACTIVATOR TO FIBRINOGEN FRAGMENTS
BINDING OF TISSUE-TYPE PLASMINOGEN-ACTIVATOR TO FIBRINOGEN FRAGMENTS
复制标题
DOI：
10.1111/j.1432-1033.1988.tb13900.x
复制
发表时间：
1988-03-01
期刊：
影响因子：
--
通讯作者：
NIEUWENHUIZEN, W
中科院分区：
文献类型：
--
作者：
BOSMA, PJ;RIJKEN, DC;NIEUWENHUIZEN, W
为了定位纤维蛋白（原）分子中组织型纤溶酶原激活剂（t-PA）的结合位点，开发了以下结合测定法。双链 t-PA 固定在微量滴定板上。然后将t-PA包被的板与纤维蛋白原和各种纤维蛋白原片段一起孵育。用针对纤维蛋白（原）及其片段的酶标记抗体对结合程度进行定量。几乎没有观察到纤维蛋白原或片段 X、Y 和 E 与 t-PA 的任何结合；观察到与片段 Dcate 和 DEGTA 的中等结合，以及与溴化氰片段 FCB-2 的强结合（表观 Kd = 140 nM）。通过与结合测定对照相同的方法测量纤维蛋白原及其片段与固定的赖氨酸纤溶酶原的结合。结果与文献数据一致：与赖氨酸纤溶酶原和纤维蛋白原或片段 X 和 Y 几乎不结合，与片段 Dcate、DEGTA 和 E 中等结合，与 FCB-2 强结合（表观 Kd = 70 nM）。通过分光光度测定研究，各种片段对 t-PA 激活赖氨酸纤溶酶原的刺激能力被发现，片段 F 不存在，纤维蛋白原、X、Y、Dcate 和 DEGTA 片段较低，FCB-2 较高。结论是，t-PA 结合位点位于纤维蛋白原的 C 端球状结构域中，片段 D 和 FCB-2 源自该结构域。该位点隐藏在天然纤维蛋白原分子和早期纤维蛋白原降解产物中。 Lys-纤溶酶原和t-PA两者的结合似乎是纤溶酶原激活刺激剂所必需的，如片段E所示，其仅结合Lys-纤溶酶原并且没有刺激能力。
In order to localize the binding site(s) for tissue-type plasminogen activator (t-PA) in the fibrin(ogen) molecule, the following binding assay was developed. Two-chain t-PA was immobilized onto microtitration plates. The t-PA-coated plates were then incubated with fibrinogen and various fibrinogen fragments. The extent of binding was quantified with enzyme-labelled antibodies against fibrin(ogen) and its fragments. Hardly any binding to t-PA was observed with fibrinogen or fragments X, Y and E; a moderate binding was observed with fragments Dcate and DEGTA and a strong binding with the cyanogen bromide fragment FCB-2 (Kd apparent = 140 nM). The binding of fibrinogen and its fragments to immobilized Lys-plasminogen was measured by the same method as a control for the binding assay. Results were in line with literature data: virtually no binding to Lys-plasminogen and fibrinogen or fragments X and Y, a moderate binding with fragments Dcate, DEGTA and E and a strong binding with FCB-2 (Kd apparent = 70 nM). The stimulatory capacity of the various fragments on the Lys-plasminogen activation by t-PA, as studied in a spectrophotometric assay, was found to be absent for fragment F, low for fibrinogen, fragments X, Y, Dcate and DEGTA, and high for FCB-2. It is concluded that a t-PA-binding site resides in the C-terminal globular domains of fibrinogen from which fragments D and FCB-2 originate. The site is hidden in the native fibrinogen molecule and in early fibrinogen degradation products. Binding of both Lys-plasminogen and t-PA appears to be required for a stimulator of the plasminogen activation, as illustrated by fragment E which only binds Lys-plasminogen and has no stimulatory capacity.