Enhancement of fibrinolytic activity in vascular endothelial cells by heterologous expression of adenine nucleotide translocase-1
Enhancement of fibrinolytic activity in vascular endothelial cells by heterologous expression of adenine nucleotide translocase-1
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DOI：
10.1097/mbc.0b013e328337b3dd
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发表时间：
2010-04-01
影响因子：
1.1
通讯作者：
Matsuo，Osamu
中科院分区：
文献类型：
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作者：
Ishida，Chikako;Ueshima，Shigeru;Matsuo，Osamu
血管内皮细胞表达组织型纤溶酶原激活物(t-PA)及其特异性抑制物-1纤溶酶原激活物抑制物(PAI-1)，调节血液纤溶活性。由于t-PA是血液中主要的纤溶酶原激活剂，因此认为t-PA结合蛋白存在于内皮细胞膜上，将t-PA固定在内皮细胞表面，增强其抗血栓性能。最近，我们在内皮细胞中发现了一种新的t-PA结合蛋白。它的氨基酸序列与人腺嘌呤核苷酸转位酶-1(ANT1)的氨基酸序列一致。本研究的目的是证实t-PA与ANT1的结合，并阐明ANT1对内皮细胞周围纤溶活性的影响。ANT1是由重组谷胱甘肽S转移酶-ANT1融合蛋白制备的，并在配体印迹实验中显示了t-PA结合活性。此外，ANT1通过pDisplay载体在血管内皮细胞膜上特异性表达。IAsys结合分析和显色分析证实了t-PA与ANT1的相互作用，ANT1表达于内皮细胞表面。ANT1在内皮细胞膜上的异源表达增强了内皮细胞与t-PA的结合能力，并通过增加t-PA催化的纤溶酶原激活来证明ANT1表达对纤溶活性的影响。这些结果表明，一种新的t-PA结合蛋白ANT1可以将t-PA浓缩在细胞表面，并增强内皮细胞周围的纤溶特性；因此，ANT1可以成为调节血管内纤溶酶原激活系统的有力工具。
The fibrinolytic activity of blood is regulated by expressing tissue-type plasminogen activator (t-PA) and its specific inhibitor, type-1 plasminogen activator inhibitor (PAI-1), from vascular endothelial cells. Since t-PA is a major plasminogen activator in blood, it is considered that the binding protein for t-PA, which exists on endothelial cell membrane, immobilizes t-PA on the surface of endothelial cells and enhances their antithrombotic property. Recently, we have found a new t-PA binding protein in endothelial cells. Its amino acid sequence has matched that of human adenine nucleotide translocase-1 (ANT1). The aims of this study are to confirm the binding of t-PA to ANT1, and to clarify the effect of ANT1 on fibrinolytic activity around endothelial cells. ANT1 is prepared from recombinant glutathione S-transferase (GST)-ANT1 fusion protein, and reveals t-PA binding activity in a ligand blot assay. In addition, ANT1 is exclusively expressed on endothelial cell membrane by using pDisplay vector. Interaction of t-PA with ANT1, which is expressed on the surface of endothelial cells, is confirmed by IAsys binding analysis and chromogenic assay. The heterologous expression of ANT1 on endothelial cell membrane enhances the t-PA binding ability of endothelial cells and the effect of ANT1 expression on fibrinolytic activity is demonstrated by increasing t-PA-catalyzed plasminogen activation. These results suggest that a novel t-PA-binding protein, ANT1, may concentrate t-PA on the surface of cells and enhance fibrinolytic properties around endothelial cells; therefore, ANT1 can be a powerful tool for regulating the plasminogen activation system in the vessel.