PLASMINOGEN ACTIVATOR INHIBITORS 104 Plasminogen Activator Inhibitor 1 104 Plasminogen Activator Inhibitor 2 106 Protease Nexin I .... .... 108 PHYSIOLOGICAL FUNCTIONS OF PLASMINOGEN ACTIVATION ........ 109 Matrix Breakdown 109 Cell Migration 111 Ovulation 113 CONCLUDING REMARKS 115

Cell-associated plasminogen activation: regulation and physiological functions.

One-chain urokinase-type plasminogen activator from human sarcoma cells is a proenzyme with little or no intrinsic activity.

High resolution analysis of functional determinants on human tissue-type plasminogen activator

Isolation, characterization, and cDNA cloning of a vampire bat salivary plasminogen activator.

Strategies for the improvement of thrombolytic agents.

The effect of polymerised fibrin on the catalytic activities of one-chain tissue-type plasminogen activator as revealed by an analogue resistant to plasmin cleavage.

In contrast to most other serine proteases, tissue-type plasminogen activator (t-PA) possesses enzymatic activity as the one-chain zymogen form. The hypothesis that lysine residues 277 or 416 may be involved in stabilization of an active conformation of one-chain t-PA via salt-bridge formation with aspartic acid residue 477 was tested by site-directed mutagenesis. Four recombinant t-PA mutants were constructed. The amidolytic activities of these analogues were compared to that of authentic t-PA. Substitution of arginine-275 provided an analogue [( R275G]t-PA) resistant to plasmin cleavage. The amidolytic activity of [R275G]t-PA was comparable to that of authentic one-chain t-PA, and so was the activity of [R275L,K277L]t-PA, in which additional substitution of lysine residue 277 was carried out. This suggested that its presence was nonessential for obtaining one-chain t-PA activity. In contrast, substitution of lysine residue 416 to obtain [K416S]t-PA and [K416S,H417T]t-PA resulted in substantial quenching of amidolytic one-chain activity. As expected, the amidolytic activities of the two-chain forms were less affected by the substitution. Involvement of lysine residue 416 in one-chain t-PA activity was also indicated by decreased activities of [K416S]t-PA and [K416S,H417T]t-PA with plasminogen as the substrate. The one-chain activity of the lysine residue 416 substitution analogues was partially restored in the presence of fibrin. This could indicate that strong ligands such as fibrin might provide an alternative stabilization of the active conformation of one-chain t-PA.

Marie Johannessen

Papers

The effect of polymerised fibrin on the catalytic activities of one-chain tissue-type plasminogen activator as revealed by an analogue resistant to plasmin cleavage.

Quenching of the amidolytic activity of one-chain tissue-type plasminogen activator by mutation of lysine-416.