Pajdak W, Radwan J, Guzik T J
Department of Clinical Immunochemistry, Jagiellonian University School of Medicine, Cracow, Poland.
J Physiol Pharmacol. 2004 Jun;55(2):477-84.
Thrombin plays a pivotal role in blood clotting as well as in the regulation of vascular remodeling and oxidative stress. Recent evidence suggests that auto-antibodies directed against prothrombin, may play an important role in the pathogenesis of atherosclerosis. It is however not clear, if prothrombin bound in an immune complex retains its clotting and regulatory properties or acts solely by increasing vascular inflammation. In order to answer this question, we used a newly developed stain for the detection of thrombin activity of such complexes. Plasma and serum samples were subjected to rocket immunoelectrophoresis in an anti-prothrombin antiserum containing agarose gel. Gel plates, covered with a nitrocellulose membrane were soaked with chromogenic thrombin substrate. The product of thrombin activity was diazotized to red azo dye bound to nitrocellulose. Activity stain revealed barely discernible rockets in plasma, but heavily stained ones in serum. Pre-incubation with trypsin enhanced activity of immunoprecipitates deriving from plasma, but not from serum. Densitometric analysis showed, that the trypsin-enhanced activity in plasma derived immune complexes was twice as high as in serum derived immunoprecipitates. Thrombin active centre is not blocked by anti-prothrombin antiserum allowing to retain thrombin activity. Moreover, prothrombin in immunoprecipitate is readily cleaved by proteolytic enzymes. This cleavage could potentially be enhanced by antibody binding, although these results need to be confirmed using different antibodies.
凝血酶在血液凝固以及血管重塑和氧化应激的调节中起着关键作用。最近的证据表明，针对凝血酶原的自身抗体可能在动脉粥样硬化的发病机制中起重要作用。然而，尚不清楚免疫复合物中结合的凝血酶原是否保留其凝血和调节特性，还是仅通过增加血管炎症起作用。为了回答这个问题，我们使用了一种新开发的染色剂来检测此类复合物的凝血酶活性。将血浆和血清样品在含有抗凝血酶原抗血清的琼脂糖凝胶中进行火箭免疫电泳。用发色凝血酶底物浸泡覆盖有硝酸纤维素膜的凝胶板。凝血酶活性产物重氮化后与结合在硝酸纤维素上的红色偶氮染料结合。活性染色显示血浆中几乎看不到火箭状条带，但血清中有明显染色的条带。用胰蛋白酶预孵育可增强源自血浆的免疫沉淀物的活性，但不能增强源自血清的免疫沉淀物的活性。光密度分析表明，血浆来源的免疫复合物中胰蛋白酶增强的活性是血清来源的免疫沉淀物中活性的两倍。抗凝血酶原抗血清不会阻断凝血酶活性中心，从而保留凝血酶活性。此外，免疫沉淀物中的凝血酶原很容易被蛋白水解酶切割。尽管这些结果需要使用不同的抗体进行证实，但抗体结合可能会增强这种切割作用。
