Kassam G, Choi K S, Ghuman J, Kang H M, Fitzpatrick S L, Zackson T, Zackson S, Toba M, Shinomiya A, Waisman D M
Cancer Biology Research Group, Department of Medical Biochemistry, University of Calgary, Calgary, Alberta T2N 4N1, Canada.
J Biol Chem. 1998 Feb 20;273(8):4790-9. doi: 10.1074/jbc.273.8.4790.
Annexin II tetramer (AIIt) is a major Ca2+-binding protein of endothelial cells which has been shown to exist on both the intracellular and extracellular surfaces of the plasma membrane. In this report, we demonstrate that AIIt stimulates the activation of plasminogen by facilitating the tissue plasminogen activator (t-PA)-dependent conversion of plasminogen to plasmin. Fluid-phase AIIt stimulated the rate of activation of [Glu]plasminogen about 341-fold compared with an approximate 6-fold stimulation by annexin II. AIIt bound to [Glu]plasminogen(S741C-fluorescein) with a Kd of 1. 26 +/- 0.04 microM (mean +/- S.D., n = 3) and this interaction resulted in a large conformational change in [Glu]plasminogen. Kinetic analysis established that AIIt produces a large increase of about 190-fold in the kcat, app and a small increase in the Km,app which resulted in a 90-fold increase in the catalytic efficiency (kcat/Km) of t-PA for [Glu]plasminogen. AIIt also stimulated the t-PA-dependent activation of [Lys]plasminogen about 28-fold. Furthermore, other annexins such as annexin I, V, or VI did not produce comparable activation of t-PA-dependent conversion of [Glu]plasminogen to plasmin. The stimulation of the activation of [Glu]plasminogen by AIIt was Ca2+-independent and inhibited by epsilon-aminocaproic acid. AIIt bound to human 293 cells potentiated t-PA-dependent plasminogen activation. AIIt that was bound to phospholipid vesicles or heparin also stimulated the activation of [Glu]plasminogen 5- or 11-fold, respectively. Furthermore, immunofluorescence labeling of nonpermeabilized HUVEC revealed a punctated distribution of AIIt subunits on the cell surface. These results therefore identify AIIt as a potent in vitro activator of plasminogen.
膜联蛋白II四聚体（AIIt）是内皮细胞中一种主要的Ca2+结合蛋白，已证明其存在于质膜的细胞内和细胞外表面。在本报告中，我们证明AIIt通过促进组织纤溶酶原激活物（t-PA）依赖的纤溶酶原向纤溶酶的转化来刺激纤溶酶原的激活。与膜联蛋白II约6倍的刺激作用相比，液相AIIt刺激[Glu]纤溶酶原的激活速率约为341倍。AIIt与[Glu]纤溶酶原（S741C-荧光素）结合，解离常数（Kd）为1.26±0.04μM（平均值±标准差，n = 3），这种相互作用导致[Glu]纤溶酶原发生大的构象变化。动力学分析表明，AIIt使表观催化常数（kcat, app）大幅增加约190倍，而表观米氏常数（Km, app）略有增加，这导致t-PA对[Glu]纤溶酶原的催化效率（kcat/Km）提高了90倍。AIIt还刺激了[Lys]纤溶酶原的t-PA依赖激活约28倍。此外，其他膜联蛋白如膜联蛋白I、V或VI对[Glu]纤溶酶原的t-PA依赖转化为纤溶酶没有产生类似的激活作用。AIIt对[Glu]纤溶酶原激活的刺激作用不依赖Ca2+，并被ε-氨基己酸抑制。与人类293细胞结合的AIIt增强了t-PA依赖的纤溶酶原激活。与磷脂囊泡或肝素结合的AIIt也分别刺激了[Glu]纤溶酶原激活5倍或11倍。此外，对未通透的人脐静脉内皮细胞（HUVEC）进行免疫荧光标记显示，AIIt亚基在细胞表面呈点状分布分布。因此，这些结果表明AIIt是纤溶酶原的一种有效的体外激活剂。
