Cheng X F, Bäck O, Nilsson T K, Nylander Lundqvist E, Pohl G, Wallén P
Department of Medical Biochemistry and Biophysics, Umeå University, Sweden.
Biochem J. 1992 Oct 15;287 ( Pt 2)(Pt 2):407-13. doi: 10.1042/bj2870407.
The aim of the present study was to investigate the binding of tissue plasminogen activator (tPA) to cultured endothelial cells and to characterize binding structures present in the cultures. Studies on the binding of 125I-tPA to cultured endothelial cells from human umbilical-cord veins (HUVEC) indicated that the number of sites for specific binding of tPA is 8 x 10(5) per cell. Treatment with an excess of antibodies against plasminogen-activator inhibitor type 1 (PAI-1) caused an 80% decrease in the binding, leaving about 1.6 x 10(5) unoccupied binding sites per cell, which appeared to be different from PAI-1. About 1.9 x 10(5) binding sites/cell for tPA were found on the surface of HUVEC that had been detached from the matrix. This indicates that only minor amounts of PAI-1 occur on the surface of the cells. In addition, immunocytochemical analysis showed that PAI-1 antigen is present almost exclusively in the cytoplasm but was not observed on the surface of the cells, whereas tPA antigen is abundant on the plasma membrane of tPA-treated cells as well as intracellularly. Competition studies using unlabelled compounds showed that native tPA and tPA B-chain (the proteinase domain), as well as the inactive derivatives, B-chain inactivated with D-Phe-Pro-Arg-chloromethane and tPA-PAI-1 complex, caused a considerable quenching of the binding of 125I-tPA to HUVEC, whereas the isolated A-chain had no demonstrable effect. Two components (apparent molecular masses 38 kDa and 56 kDa) reacting with tPA but lacking PAI-1 antigen determinants were identified. Thus the data suggest that tPA binds to HUVEC by two principally different mechanisms. One is mediated by PAI-1, which binds and inactivates tPA with a functional active site. The other binding is achieved by components which react with sites on the activator molecule other than structures of the A-chain or the active site.
本研究的目的是研究组织型纤溶酶原激活剂（tPA）与培养的内皮细胞的结合，并鉴定培养物中存在的结合结构。对125I-tPA与人脐静脉内皮细胞（HUVEC）结合的研究表明，tPA特异性结合位点的数量为每个细胞8×10⁵个。用过量的抗1型纤溶酶原激活剂抑制剂（PAI-1）抗体处理导致结合减少80%，每个细胞留下约1.6×10⁵个未占据的结合位点，这些位点似乎与PAI-1不同。在从基质上脱离的HUVEC表面发现每个细胞约有1.9×10⁵个tPA结合位点。这表明细胞表面仅存在少量的PAI-1。此外，免疫细胞化学分析表明，PAI-1抗原几乎仅存在于细胞质中，在细胞表面未观察到，而tPA抗原在tPA处理的细胞的质膜上以及细胞内都很丰富。使用未标记化合物的竞争研究表明，天然tPA和tPA B链（蛋白酶结构域），以及无活性衍生物，用D-苯丙氨酸-脯氨酸-精氨酸-氯甲基酮灭活的B链和tPA-PAI-1复合物，导致125I-tPA与HUVEC的结合显著淬灭，而分离的A链没有明显作用。鉴定出两种与tPA反应但缺乏PAI-1抗原决定簇的成分（表观分子量分别为38 kDa和56 kDa）。因此，数据表明tPA通过两种主要不同的机制与HUVEC结合。一种是由PAI-1介导的，它通过功能性活性位点结合并使tPA失活。另一种结合是由与激活剂分子上除A链结构或活性位点以外的位点反应的成分实现的。
