Fluorogenic Fibrinogen and Fibrin Facilitate Macromolecular Assembly and Dynamic Assay of Picomolar Levels of Plasminogen Activators under Well Mixed Conditions
Fluorogenic Fibrinogen and Fibrin Facilitate Macromolecular Assembly and Dynamic Assay of Picomolar Levels of Plasminogen Activators under Well Mixed Conditions
复制标题
荧光纤维蛋白原和纤维蛋白促进大分子组装以及充分混合条件下纤溶酶原激活剂皮摩尔水平的动态测定
DOI：
--
复制
发表时间：
1995
影响因子：
6.7
通讯作者：
S. Diamond
中科院分区：
文献类型：
--
作者：
Jung;S. Diamond
测试了用异硫氰酸荧光素标记的纤维蛋白原（FITC），其能够用作大分子组装的模板，并提供荧光信号，以连续监测纤溶酶原激活和纤维蛋白解。纤维蛋白纤维悬浮液在裂解过程中脱落，释放荧光片段消除了基于接近度的淬火，并在最终浓度为10 pm的纤维蛋白时分别增加了2.0或3.6倍的荧光强度。 NM产生了可检测的荧光去除水平。重组TPA和尿激酶的PM，使用10 nm FITC-纤维蛋白和10 nm和5 nm的纤溶酶原，分析对TPA的敏感性更高。 。凝血酶（1 U/mL）和纤溶酶（0.1 nm）首先在10 nm FitC-纤维蛋白原上产生荧光淬灭，这是由于纤维蛋白原聚合的作用，然后由于纤维蛋白溶解而导致的。纤溶酶可以在存在活性纤维蛋白的情况下对凝血酶活性进行淬灭评估。纤维蛋白是由血小板不良的纤维蛋白。在混合条件良好的条件下，在纤维蛋白表面上的结合和酶促事件的动态研究。
Summary Fibrinogen labeled with fluorescein isothiocyanate (FITC) was tested for its ability to serve as a template for macromolecular assembly as well as to provide a fluorogenic signal to allow continuous monitoring of plasminogen activation and fibrinolysis. As dilute solutions of FITC-fibrinogen or FITC-fibrin fiber suspension were degraded during lysis, release of fluorescent fragments abolished proximity- based quenching and resulted in a 2.0- or 3.6-fold increase in fluorescence intensity, respectively. Addition of plasmin at a final concentration of 10 pM to FITC-fibrinogen (10 nM) produced a detectable level of fluorescence dequenching. The assay had sufficient sensitivity to detect plasmin activity in the presence of excess antiplasmin activity, indicating the dissociation of a reversible antiplasmin-plasmin complex. The detection limit of the reaction assay was 20 pM and 200 pM of recombinant tPA and urokinase, using 10 nM FITC-fibrin and 10 nM and 5 nM plasminogen, respectively. The 10-fold greater sensitivity of the assay for tPA was likely due to the molecular assembly of tPA and plasminogen on the FITC-fibrin. Addition of thrombin (1 U/ml) and plasmin (0.1 nM) to 10 nM FITC-fibrinogen produced fluorescence quenching at first due to fibrinogen polymerization followed by dequenching due to fibrinolysis. Addition of 10 mM ∈-aminocaproic acid to mixtures of thrombin and plasmin allowed the quenching assay of thrombin activity in the presence of active plasmin. FITC-fibrinogen could be copolymerized with recalcified platelet poor plasma (isolated from citrated whole blood) to yield fibrin that was fluorogenic. Dequenching was observed when plasmin was used to degrade the fibrin formed from the platelet poor plasma. Given the large signal generated upon degradation of the fluorogenic fibrin(ogen), at least 105 determinations can be run from 100 mg of FITC-labeled fibrinogen using a standard fluorimeter and 0.1 to 3.0 ml reaction volumes. The versatility of the fluorogenic fibrinogen substrate allowed the configuration of assays to detect and measure the activity of thrombin, plasmin, tPA, uPA, and α2-antiplasmin. The ability to assemble blood proteins on a fluorogenic fibrinogen or fibrin template provides unique opportunities for the dynamic study of binding and enzymatic events on the fibrin surface under well mixed conditions.
组织纤溶酶原激活剂和尿激酶介导谷氨酸纤溶酶原与血浆纤维蛋白 I 的结合。纤溶酶降解的纤维蛋白 I 中新结合位点的证据。
DOI：
--
发表时间：
1985
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Harpel,PC;Chang,TS;Verderber,E
通讯作者：
Verderber,E
单链和双链组织纤溶酶原激活剂与非交联和交联纤维蛋白凝块的结合之间的差异。
影响因子：
20.3
作者：
Husain,SS;Hasan,AA;Budzynski,AZ
通讯作者：
Budzynski,AZ
纤维蛋白原的吸附后转变：聚合物特性的影响。
DOI：
10.1002/jbm.820241002
发表时间：
1990
期刊：
Journal of biomedical materials research
影响因子：
--
作者：
Rapoza,RJ;Horbett,TA
通讯作者：
Horbett,TA