title: The kinetics of plasminogen activation by thrombin-cleaved pro-urokinase and promotion of its activity by fibrin fragment E-2 and by tissue plasminogen activator.
abstract: Thrombin hydrolyzes the Arg156-Phe157 bond in pro-urokinase (pro-UK), two residues from the activation site, generating a two-chain form (thromb-UK) believed to have little activity and that is resistant to plasmin activation. The kinetic constants for thromb-UK against synthetic substrate (S2444) were found to be essentially identical to pro-UK. Against native plasminogen, thromb-UK had a lower Michaelis constant (KM) and a higher (2-fold) catalytic efficiency. However, this difference with pro-UK was nullified by carboxypeptidase B (CpB) treatment of thromb-UK to remove the C-terminal arginine on the A-chain. Plasminogen activation by thromb-UK was substantially promoted by fibrin fragment E-2 but not by other fibrin derivatives, a phenomenon previously observed with pro-UK. Similarly, clot lysis by thromb-UK was promoted by tissue plasminogen activator because their combined effect was synergistic. Fibrinogenolysis in plasma occurred at 80-fold the concentration of thromb-UK as pro-UK, reflecting the 90-fold greater plasmin resistance of thromb-UK. Addition of a CpB inhibitor to the plasma enhanced fibrinogenolysis by thromb-UK and pro-UK by approximately 16%, consistent with the promotion of both forms by certain C-terminal lysines. In conclusion, CpB-thromb-UK corresponds functionally to a plasmin resistant form of pro-UK, indicating that the catalytic site of the single-chain pro-UK is unaffected by thrombin cleavage. The effect of CpB indicates that the C-terminal Arg of thromb-UK slightly enhances its affinity for plasminogen. Thromb-UK has potential plasminogen-activating activity at surfaces where C-terminal lysines, functionally comparable to fragment E-2, are found.
authors.affiliation: Vascular Research Laboratory, New England Deaconess Hospital, Harvard Medical School, Boston, MA 02215.
authors.forename: J N
authors.initials: JN
authors.lastname: Liu
authors.forename: V
authors.initials: V
authors.lastname: Gurewich
mesh_terms: D000818:Animals; D016466:CHO Cells; D043424:Carboxypeptidase B; D002268:Carboxypeptidases / Q000378:metabolism; D006224:Cricetinae; D004357:Drug Synergism; D004591:Electrophoresis, Polyacrylamide Gel; D005337:Fibrin / Q000378:metabolism; D005338:Fibrin Fibrinogen Degradation Products / Q000494:pharmacology*; D005340:Fibrinogen / Q000378:metabolism; D005341:Fibrinolysin / Q000378:metabolism; D006868:Hydrolysis; D007700:Kinetics; D009842:Oligopeptides / Q000378:metabolism; D010958:Plasminogen / Q000378:metabolism*; D011994:Recombinant Proteins; D013917:Thrombin / Q000378:metabolism*; D010959:Tissue Plasminogen Activator / Q000494:pharmacology*; D014568:Urokinase-Type Plasminogen Activator / Q000378:metabolism*
chemical_list: D005338:Fibrin Fibrinogen Degradation Products; D009842:Oligopeptides; D011994:Recombinant Proteins; C072054:fibrin fragment E-2; C028190:5-oxo-prolyl-glycyl-arginine-4-nitroanilide; D005337:Fibrin; D005340:Fibrinogen; D010958:Plasminogen; D002268:Carboxypeptidases; D043424:Carboxypeptidase B; D013917:Thrombin; D010959:Tissue Plasminogen Activator; D005341:Fibrinolysin; D014568:Urokinase-Type Plasminogen Activator; C067551:saruplase