Lavie E, Bitton M, Shalev J, Weininger J, Abrashkin S, Varga E, Lubin E, Azoury R
Radiopharmaceuticals Department, Soreq Nuclear Research Center, Yavne, Israel.
Biochim Biophys Acta. 1989 Apr 25;991(1):62-7. doi: 10.1016/0304-4165(89)90029-9.
The clot uptake of labeled active and inhibited t-PA was compared. The most efficient inhibition was obtained with diisopropyl fluorophosphate (DFP) after 4 h incubation at room temperature. Enzyme activity was followed by fibrin-plate assay, radioactivity-release technique and proton magnetic resonance (PMR). The novel PMR method developed by us is sensitive to the effect of as low as nanogram amounts of t-PA on the interaction between the fibrin and the compartmentalized water trapped in the clot. Binding of labeled enzyme to fibrin-coated plates showed that the deactivation by DFP did not impair the affinity of t-PA for fibrin. A rapid binding of 125I-labeled t-PA to the clot occurred, which reached a maximum in 30 min and declined with time. This pattern was explained by consecutive clot binding and lysis. The binding of DFP-t-PA to the clot differed markedly from that of the active protein; 2 h post-incubation the uptake of DFP-t-PA was more than double that of the untreated t-PA. Parallel measurements in clots prepared from human blood showed a qualitatively similar trend. The biodistribution of radiolabeled t-PA in mice was similar for the active and inhibited forms. Blood activity reached 10% of the injected dose within 10 min. DFP-t-PA may prove to be a useful reagent for in-vivo localization of thrombi.
比较了标记的活性和抑制性组织型纤溶酶原激活剂（t-PA）的凝块摄取情况。在室温下孵育4小时后，用二异丙基氟磷酸酯（DFP）可获得最有效的抑制效果。通过纤维蛋白平板试验、放射性释放技术和质子磁共振（PMR）来跟踪酶活性。我们开发的新型PMR方法对低至纳克量的t-PA对纤维蛋白与凝块中截留的分隔水之间相互作用的影响很敏感。标记酶与纤维蛋白包被平板的结合表明，DFP使其失活并不损害t-PA对纤维蛋白的亲和力。125I标记的t-PA迅速与凝块结合，在30分钟时达到最大值，随后随时间下降。这种模式可以用连续的凝块结合和溶解来解释。DFP-t-PA与凝块的结合与活性蛋白明显不同；孵育2小时后，DFP-t-PA的摄取量是未处理t-PA的两倍多。用人血制备的凝块进行的平行测量显示出定性相似的趋势。放射性标记的t-PA在小鼠体内的生物分布对于活性形式和抑制形式是相似的。血液活性在10分钟内达到注射剂量的10%。DFP-t-PA可能被证明是一种用于血栓体内定位的有用试剂。
