Characterization of the Binding of Urokinase‐type Plasminogen Activator (u‐PA) to Plasminogen, to Plasminogen‐activator Inhibitor‐1 and to the u‐PA Receptor

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Abstract
Binding parameters [association‐rate (kass and dissociation‐rate (kdiss constants, and affinity constants (KA=kass/kdiss)] for the interaction between urokinase‐type plasminogen activator (u‐PA) and its substrate plasminogen, its inhibitor plasminogen activator inhibitor‐1 (PAI‐1) and its receptor (u‐PAR), were determined by real‐time biospecific interaction analysis (BIA). The KA values for the binding of [S741A]recombinant plasminogen (plasminogen with N‐terminal Glu and with the active site Ser741 mutagenized to Ala) or of active site‐blocked plasmin (d‐ValPheLysCH2‐plasmin) to the 54‐kDa or 32‐kDa molecular forms of recombinant single‐chain u‐PA (rscu‐PA) ranged between 0.57×106 M−1 and 1.7×106M‐1, compared to 14–22×106 M‐1 for binding to the corresponding active site‐blocked recombinant two‐chain u‐PA (rtcu‐PA) moieties. KA values for binding of these plasmin(ogen) moieties to [Ser356deHAla]rtcu‐PA (rtcu‐PA with the active site Ser356 converted to dehydroAla) were 81×106M‐1 and 670×106 M‐1 respectively. Binding of active site‐blocked LMM‐plasmin (a low‐molecular‐mass plasmin derivative lacking kringles 1–4) and of the plasmin B chain to [Ser356deHAla] rtcu‐PA occurred with KA values of 3.7×106M‐1 and 0.33×106 M‐1 compared to 670×106 M‐1 for the binding of intact d‐ValPheLysCH2‐plasmin to [Ser356deHala]rtcu‐PA. The KA values for binding of latent PAI‐1 to 54‐kDa or 32‐kDa molecular forms of rscu‐PA and rtcu‐PA were in the range 0.34–2.1×106 M‐1. Reactivated PAI‐1 bound to 54‐kDa and 32‐kDa rtcu‐PA moieties with KA values of 26×106M‐1 and 28×106 M‐1, compared to 0.77×106M‐1 and 3.2×106 M‐1 for binding to the corresponding single‐chain u‐PA species, and 450×106 M‐1 for binding to [Ser356deHAla]rtcu‐PA. KA values for binding of plasmin(ogen) to the covalent rtcu‐PA/PAI‐1 complex were similar or somewhat higher than those for binding to uncomplexed rtcu‐PA. Single‐chain and two‐chain 54‐kDa u‐PA moieties bound with a 1:1 stoichiometry and with very high affinity to u‐PAR (KA of 4.6–8.5×109 M‐1), whereas no significant binding of 32‐kDa u‐PA moieties was observed (KA≤0.2×106M‐1). Binding of the covalent rtcu‐PA/PAI‐1 complex to u‐PAR was indistinguishable from that of rtcu‐PA. These results indicate that plasminogen and plasmin bind with higher affinity to two‐chain u‐PA than to single‐chain u‐PA moieties, whereas binding requires the presence of both the A and the B chain; reactivated PAI‐1 binds to rtcu‐PA moieties with a higher affinity than latent PAI‐1, but has a very low affinity for rscu‐PA; 54‐kDa u‐PA moieties bind in a 1:1 stoichiometry to u‐PAR with high affinity which does not require the u‐PA active site; and the binding sites of u‐PA for PAI‐1 and for plasminogen are within the 32‐kDa u‐PA moiety (Leu144–Leu411), they do not overlap and are fully accessible when 54‐kDa u‐PA is bound to its receptor.