Inactivation of receptor-bound pro-urokinase-type plasminogen activator (pro-uPA) by thrombin and thrombin/thrombomodulin complex.
Urokinase-type plasminogen activator (uPA) binds with high affinity to a specific cell surface glycosyl phosphatidyl-inositol (GPI)-anchored receptor, the urokinase receptor (uPAR). Pro-uPA, the enzymatically inactive single-chain form of uPA after having been activated by certain proteases, converts plasminogen into plasmin. This activation of pro-uPA to enzymatically active uPA can be prevented by the action of thrombin on pro-uPA. This inactivation process is accelerated in the presence of thrombomodulin (TM). The present study investigated whether pro-uPA bound to uPAR is still susceptible to inactivation by thrombin in the presence or absence of TM. A truncated soluble form of the uPAR lacking the GPI-anchor was cloned and expressed in CHO-cells (rec-uPAR277). Rec-uPAR277 efficiently inhibited the thrombin-mediated inactivation of pro-uPA up to 90% in a concentration dependent manner. The protective effect of rec-uPAR277 was far less pronounced when thrombin was complexed with TM. Enzyme kinetic experiments with varying concentrations of pro-uPA showed that in the presence of TM the catalytic efficiency (kcat/Km) of thrombin-mediated inactivation raised from 0.010 microM-1 s-1 to 0.50 microM-1 s-1 corresponding to a fifty-fold increase. In the presence of rec-uPAR277, however, the catalytic efficiency dropped by 4.1-fold (0.5 microM-1 s-1 to 0.122 microM-1 s-1). The inactivation kinetics of pro-uPA by thrombin (no TM added) in the presence of an excess of rec-uPAR277 could not be determined since virtually no inactivation occurred. Our data suggest that pro-uPA once bound to uPAR, is significantly protected from inactivation by thrombin.(ABSTRACT TRUNCATED AT 250 WORDS)
Wilhelm
S
S
Frauenklinik Technischen Universität München, Germany.
Wilhelm
O
O
Schmitt
M
M
Graeff
H
H
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D017261:Glycosylphosphatidylinositols / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D011485:Protein Binding; D011956:Receptors, Cell Surface / Q000302:isolation & purification / Q000378:metabolism*; D055293:Receptors, Urokinase Plasminogen Activator; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism; D013917:Thrombin / Q000378:metabolism / Q000494:pharmacology*; D018180:Thrombomodulin / Q000378:metabolism*; D014162:Transfection; D014568:Urokinase-Type Plasminogen Activator / Q000037:antagonists & inhibitors* / Q000378:metabolism
D017261:Glycosylphosphatidylinositols; C524898:PLAUR protein, human; D011956:Receptors, Cell Surface; D055293:Receptors, Urokinase Plasminogen Activator; D011994:Recombinant Proteins; D018180:Thrombomodulin; D013917:Thrombin; D014568:Urokinase-Type Plasminogen Activator; C067551:saruplase