- Email: [email protected]
258 Role of human U937 monocytes in controlling single-chain urokinase-initated plasminogen activation
112
SESSION19: Pro-urokinase
257 PRO-u-PA REGULATION
ACTIVATION. A CRUCIAL STEP IN OF PERICELLULAR PROTEOLYSIS CELL MIGRATION? 1
DURING K. Dan d( & (l)Finsen Laboratory, Rigshospitalet, Strandboulevarden 49, 2100 Copenhagen Denmark. (2)Novo Research Institute, Alle, DK-2880 Bagsvierd, Denmark.
la., Novo
conversion assay and BY a 1251-plasminogen two types of coupled plasminogen activation assays we have found that the one-chain form of urokinase-type plasminogen activator (u-PA) produced by human HT-1080 fibrosarcoma cells is a genuine proenzyme with a capacity to activate plasminogen which is at least 250-fold lower than that of two-chain u-PA. These fibrosarcoma cells have at surface a their 55-60,000 Mr receptor protein that specifically binds both pro-u-PA and u-PA (Nielsen et al., J. Biol. Chem. 1988, in press). Pro-u-PA bound to this receptor can be converted to u-PA
258Role of human U937 monocytes single-chain urokinase-initiated activation. V. Ellis, MF Scully, King's Research Unit, & Dentistry, Denmark
in controlling plasminogen
W Kakkar, Thrombosis College School of Medicine Hill, London SE5 8RK.
Single-chain urokinase (scu-PA) is the major form of urokinase-type plasminogen activator found in It has been demonstrated that human plasma. various peripheral blood cells possess specific receptors for urokinase-type plasminogen actiand also for plasminogen. We have studied vators, the interaction of these components of the fibrinolytic system with the human monocyte-like cell line, U937, and its effect on the rate of Prior to their use in plasminogen activation. cultured U937 cells were washed in experiments, growth medium containing aprotinin, to remove The cells were traces of cell-bound plasmin. then incubated with varying concentrations of
and receptor bound u-PA is enzymatically active (see Blasi et al., J. Cell Biol. 1987). In addition 801-804, 104, studies have shown that cytochemical representing receptor immunoreactivity bound pro-u-PA or u-PA in I-IT-1080 Cells is discretely located at cell-cell and focal cell-substratum contact sites (PBllZnen et 1085-1096, 1987; Cell Biol. 104, al., J. Pollinen et al., J. Cell Biol. 1988, in press: Herbert and Baker, J. Cell Biol. Based on these findings it 1988, in press). activation of discussed that will be plasminogen by receptor bound u-PA may provide a localised directional pericellular proteolysis involved in breakdown of the contact sites during cell migration, and that activation of pro-u-PA to U-PA may this regulatory step in be a crucial process.
scu-PA and plasminogen. Plasmin generation was measured with the chromogenic substrate, S-2251. U937 cells were found to greatly enhance the rate of plasmin generation, with a maximum 20-fold acceleration observed. The potentiating effect of U937 cells was demonstrated to be primarily due to an increase in the rate of feedback activation of scu-PA to the more active two-chain urokinase by cell-bound plasmin, initially This effect was abolished generated by scu-PA. by the addition of either amino hexanoic acid or the purified amino-terminal fragment (residues l-135) of urokinase, suggesting that binding of both plasminogen and scu-PA to the U937 cells is necessary for the increased rate of plasminogen The enhanced proteolytic activity of activation. components of the fibrinolytic system when bound to these monocyte-like cells suggests a role for cell-associated proteolytic activity in a number of monocyte/macrophage functions.
SESSION19: Pro-urokinase
257 PRO-u-PA REGULATION
ACTIVATION. A CRUCIAL STEP IN OF PERICELLULAR PROTEOLYSIS CELL MIGRATION? 1
DURING K. Dan d( & (l)Finsen Laboratory, Rigshospitalet, Strandboulevarden 49, 2100 Copenhagen Denmark. (2)Novo Research Institute, Alle, DK-2880 Bagsvierd, Denmark.
la., Novo
conversion assay and BY a 1251-plasminogen two types of coupled plasminogen activation assays we have found that the one-chain form of urokinase-type plasminogen activator (u-PA) produced by human HT-1080 fibrosarcoma cells is a genuine proenzyme with a capacity to activate plasminogen which is at least 250-fold lower than that of two-chain u-PA. These fibrosarcoma cells have at surface a their 55-60,000 Mr receptor protein that specifically binds both pro-u-PA and u-PA (Nielsen et al., J. Biol. Chem. 1988, in press). Pro-u-PA bound to this receptor can be converted to u-PA
258Role of human U937 monocytes single-chain urokinase-initiated activation. V. Ellis, MF Scully, King's Research Unit, & Dentistry, Denmark
in controlling plasminogen
W Kakkar, Thrombosis College School of Medicine Hill, London SE5 8RK.
Single-chain urokinase (scu-PA) is the major form of urokinase-type plasminogen activator found in It has been demonstrated that human plasma. various peripheral blood cells possess specific receptors for urokinase-type plasminogen actiand also for plasminogen. We have studied vators, the interaction of these components of the fibrinolytic system with the human monocyte-like cell line, U937, and its effect on the rate of Prior to their use in plasminogen activation. cultured U937 cells were washed in experiments, growth medium containing aprotinin, to remove The cells were traces of cell-bound plasmin. then incubated with varying concentrations of
and receptor bound u-PA is enzymatically active (see Blasi et al., J. Cell Biol. 1987). In addition 801-804, 104, studies have shown that cytochemical representing receptor immunoreactivity bound pro-u-PA or u-PA in I-IT-1080 Cells is discretely located at cell-cell and focal cell-substratum contact sites (PBllZnen et 1085-1096, 1987; Cell Biol. 104, al., J. Pollinen et al., J. Cell Biol. 1988, in press: Herbert and Baker, J. Cell Biol. Based on these findings it 1988, in press). activation of discussed that will be plasminogen by receptor bound u-PA may provide a localised directional pericellular proteolysis involved in breakdown of the contact sites during cell migration, and that activation of pro-u-PA to U-PA may this regulatory step in be a crucial process.
scu-PA and plasminogen. Plasmin generation was measured with the chromogenic substrate, S-2251. U937 cells were found to greatly enhance the rate of plasmin generation, with a maximum 20-fold acceleration observed. The potentiating effect of U937 cells was demonstrated to be primarily due to an increase in the rate of feedback activation of scu-PA to the more active two-chain urokinase by cell-bound plasmin, initially This effect was abolished generated by scu-PA. by the addition of either amino hexanoic acid or the purified amino-terminal fragment (residues l-135) of urokinase, suggesting that binding of both plasminogen and scu-PA to the U937 cells is necessary for the increased rate of plasminogen The enhanced proteolytic activity of activation. components of the fibrinolytic system when bound to these monocyte-like cells suggests a role for cell-associated proteolytic activity in a number of monocyte/macrophage functions.