ENHANCEMENT OF TISSUE-TYPE PLASMINOGEN-ACTIVATOR (T-PA) ACTIVITY BY PURIFIED T-PA RECEPTOR EXPRESSED IN HUMAN ENDOTHELIAL-CELLS

We demonstrated previously that tissue-type plasminogen activator (t-P A) bound to its specific receptor (t-PAR) on human umbilical vein endo thelial cells (HUVEC) in suspension and that t-PAR of mol wt. 20 kDa i nteracted only with t-PA to form 90 kDa complex (Fukao, H., Hagiya, Y. , Nonaka, T., Okada, K., and Matsuo, O. (1992) Biochem. Biophys. Res. Commun. 187, 956-962). In the present study, 20 kDa t-PAR was purified from HUVEC and the function of the t-PAR was investigated by analyzin g its effect on plasminogen activation by t-PA. About 2.2 E mu g t-PAR protein was purified from cell lysate of 1.0 x 10(9) HUVEC as a singl e band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (S DS-PAGE) by gel filtration with TSK-3000SW and reversed phase separati on with high performance liquid chromatography (HPLC). I-125-t-PA but not I-125-plasminogen specifically bound to the purified t-PAR in liga nd blot assay. Plasminogen activation by t-PA in the presence of purif ied t-PAR in solution was increased. Furthermore, t-PA bound to immobi lized t-PAR efficiently expressed its plasminogen activation activity. Kinetic analysis revealed that t-PA in the presence of soluble t-PAR and t-PA bound to immobilized t-PAR exhibited 34- and 90-fold increase in plasminogen activation, respectively. The t-PAR did not interact w ith anti-annexin II antibody. These findings indicate that the 20 kDa t-PAR is a novel molecule which immobilizes t-PA and enhances its prot eolytic activity on the cell surface of endothelial cells.