Expression of t-PA receptor on the endothelial cell and analysis of its physiological function.

我们已证实组织型纤溶酶原激活剂(t-PA)与人脐静脉内皮细胞(HUVEC)上的组织型纤溶酶原激活物(t-PA)特异性受体(t-PAR)结合，t-PAR仅与t-PA相互作用形成t-PA/t-PAR复合体。为了研究t-PAR的功能，我们合成了重组t-PAR(RT-PAR)。进一步利用IAsys研究t-PA与t-PAR在血管内皮细胞上的结合能力。将t-PAR的编码区克隆到谷胱甘肽S转移酶表达载体中。目的蛋白以GST融合蛋白的形式表达。用酶切融合蛋白获得成熟的RT-PAR。配基印迹分析表明t-PA与RT-PAR特异结合，在IAsys的相互作用分析中，HUVEC与t-PA的结合信号随着细胞浓度的增加而增强。认为t-PAR可以使t-PA聚集在HUVEC表面并增强其周围的纤溶功能，当EACA作用于HUVEC时，HUVEC与t-PA的结合信号减弱，且呈EACA依赖性。提示t-PA与血管内皮细胞的结合是通过t-PA分子上的赖氨酸结合部位实现的。另一方面，用受体相关蛋白处理内皮细胞时，结合参数没有改变。提示低密度脂蛋白受体相关蛋白不参与t-PA与HUVEC的结合。抗Annexin II和抗αβ-enolase的多克隆抗体不干扰t-PA与人脐静脉内皮细胞的相互作用。结果表明，t-PAR是不同于Annexin II和αβ-enolase的新蛋白。

We demonstrated previously that tissue-type plasminogen activator (t-PA) bound to its specific receptor (t-PAR) on human umbilical vein endothlial cells (HUVEC) and that t-PAR interacted only with t-PA to form t-PA/ t-PAR complex. To investigate the function of t-PAR, we synthesized recombinant t-PAR (rt-PAR). Furthermore, the binding ability of t-PA to t-PAR on vascular endothelial cells was studied by using IAsys.The cDNA coding region for t-PAR was cloned into glutathione S-transferase (GST) expression vector. The target protein was expressed as the fusion protein with GST. The mature rt-PAR was obtained from cleaving fusion protein by protease. The ligand blot analysis revealed that t-PA specifically bound to rt-PAR.On the interaction analysis using IAsys, the binding signal of HUVEC to t-PA elevated in the cell concentration manner. It is considered that the t-PAR may concentrate t-PA on the surface of HUVEC and enhance the fibrinolytic properties around them.When HUVEC were treated with EACA, the binding signal of HUVEC to t-PA decreased in an EACA dependent manner. It was suggested that the binding of t-PA to endothelial cell was mediated via lysine binding site in t-PA molecule. On the other hand, when the endothelial cell was treated with receptor associated protein, the binding parameter did not change. This result indicated that low density lipoprotein receptor related protein was not involved in the binding of t-PA to HUVEC. Furthermore, polyclonal antibody to annexin II and that to α ^-enolase did not interfere the interaction between t-PA and HUVEC. It concluded that t-PAR is the novel protein which is different from annexin II and α ^-enolase.