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-PA) bound to its specific receptor (t-PAR) on human umbilical vein endothelial cells (HUVEC) in suspension and that t-PAR of mol wt. 20 kDa interacted 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 analyzing its effect on plasminogen activation by t-PA. About 2.2 microg t-PAR protein was purified from cell lysate of 1.0 X 10(9) HUVEC as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) by gel filtration with TSK-3000SW and reversed phase separation with high performance liquid chromatography (HPLC). 125I-t-PA but not 125I-plasminogen specifically bound to the purified t-PAR in ligand blot assay. Plasminogen activation by t-PA in the presence of purified t-PAR in solution was increased. Furthermore, t-PA bound to immobilized 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 with anti-annexin II antibody. These findings indicate that the 20 kDa t-PAR is a novel molecule which immobilizes t-PA and enhances its proteolytic activity on the cell surface of endothelial cells.
Fukao
H
H
Department of Physiology, Kinki University School of Medicine, Osakasayama, Osaka, Japan.
Ueshima
S
S
Takaishi
T
T
Okada
K
K
Matsuo
O
O
D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008970:Molecular Weight; D010958:Plasminogen / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D010959:Tissue Plasminogen Activator / Q000378:metabolism*
D011956:Receptors, Cell Surface; D010958:Plasminogen; C067262:PLAT protein, human; D010959:Tissue Plasminogen Activator