We describe a new principle for assessment of the activity of proteolytic enzymes of all classes and show the application of this principle for the quantitative assay of bacterial collagenase and human matrix metalloproteinases (MMPs). Central to this new principle is the presence of a proenzyme that can be activated into an active enzyme by a single proteolytic event. The regular activation sequence in the proenzyme is replaced using protein engineering by an artificial sequence recognized by the proteinase to be determined. The latter can act as an activator for the newly engineered proenzyme. In the present paper a simple colorimetric assay for the determination for MMPs is described based on this principle. With the aid of protein engineering, a modified pro-urokinase has been prepared in which the activation sequence normally recognized by plasmin (Pro-Arg-Phe-Lys upward arrowIle-Ile-Gly-Gly) has been replaced by a sequence expected to be recognized and hydrolysed by many MMPs (Arg-Pro-Leu-Gly upward arrowIle-Ile-Gly-Gly). The active urokinase resulting from activation of the modified pro-urokinase by a MMP could be measured either directly, using a specific chromogenic peptide substrate for urokinase, or indirectly via urokinase-catalysed plasminogen activation. The response of the assay to equal molar quantities of active MMPs decreases in the order MMP-2>MMP-9>MMP-1>MMP-3>MMP-7. The detection limit for MMP-9 was below 15 pM, corresponding to 3. 75x10(-15) mol per assay. Using the assay, increased MMP activity was detected in synovial tissue extracts from rheumatoid arthritis patients compared with those from osteoarthritis patients, and in stomach tumour extracts as compared with normal stomach tissue extracts.

SummaryThe plasminogen activation system is thought to be important in cell migration processes. A role for this system during smooth muscle cell migration after vascular injury has been suggested from several animal studies. However, not much is known about its involvement in human vascular remodelling. We studied the involvement of the plasminogen activation system in human smooth muscle cell migration in more detail using an in vitro wound assay and a matrix invasion assay. Inhibition of plasmin activity or inhibition of urokinase-type plasminogen activator (u-PA) activity resulted in approximately 40% reduction of migration after 24 h in the wound assay and an even stronger reduction (70-80%) in the matrix invasion assay. Migration of smooth muscle cells in the presence of inhibitory antibodies against tissue-type plasminogen activator (t-PA) was not significantly reduced after 24 h, but after 48 h a 30% reduction of migration was observed, whereas in the matrix invasion assay a 50% reduction in invasion was observed already after 24 h. Prevention of the interaction of u-PA with cell surface receptors by addition of soluble u-PA receptor or α2-macroglobulin receptor associated protein (RAP) to the culture medium, resulted in a similar inhibition of migration and invasion. From these results it can be concluded that both u-PA and t-PA mediated plasminogen activation can contribute to in vitro human smooth muscle cell migration and invasion. Furthermore, the interaction between u-PA and its cell surface receptor appears also to be involved in this migration and invasion process. The inhibitory effects on migration and invasion by the addition of RAP suggests an involvement of a RAP sensitive receptor of the LDL receptor family, possibly the LDL-receptor related protein (LRP) and/or the VLDL receptor.

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Fibrin-dependent plasminogen activation by tissue-type plasminogen activator (t-PA) is in part associated with the presence of the kringle 2 domain in t-PA. Within this kringle 2 domain a lysyl-binding site has been described. The plasminogen to plasmin conversion by urokinase-type plasminogen activator (u-PA), in contrast to that of t-PA, is not enhanced in the presence of fibrin. Within the u-PA kringle domain no lysyl-binding site is found. To study whether introduction of a lysyl-binding site in the u-PA kringle domain will make u-PA a fibrin-dependent plasminogen activator, three stretches of amino acid residues of the u-PA kringle domain (A28-Q33, D55-N57 and G67-V72) were substituted by three stretches of amino acids from the corresponding positions of the kringle 2 domain of t-PA (M28-K33, D55-D57 and N67-W72). These changes resulted in the creation of the lysyl-binding site consensus of the kringle 2 domain (K33, D55, D57, W62 and W72) in the u-PA kringle. However, the resulting u-PA mutant did not interact with lysyl-Sepharose, nor did it display fibrin-enhanced plasminogen activation in the presence of soluble fibrin mimic. When the kringle domain of u-PA was replaced by the kringle 2 domain of t-PA, similar results were obtained. The hybrid protein hardly interacted with lysyl-Sepharose and the plasminogen activation was not enhanced in the presence of fibrin mimic. However, the N-terminal fragment isolated from this hybrid molecule (consisting of growth factor domain and kringle 2 domain) did interact with lysyl-Sepharose, suggesting that in the hybrid molecule a functional lysyl-binding site is present but not operational. Indeed, lysine analogue (epsilon-amino-caproic acid) sensitive binding of isolated t-PA kringle 2 domain to u-PA could be observed. The modified u-PA kringle, the wild type u-PA kringle and the kringle 2 of the u-PA hybrid were also placed N-terminal of the protease domain of t-PA. As expected, the t-PA mutant consisting of the kringle 2 domain and the protease domain bound to lysyl-Sepharose and showed fibrin-dependent plasminogen activation. Further, the hybrid molecule consisting of the u-PA kringle placed N-terminal of the t-PA protease domain did not display these features. Introduction of the modified u-PA kringle N-terminal of the t-PA protease domain resulted in a very weak interaction with lysyl-Sepharose. Despite the high overall similarity in primary structure of the modified u-PA kringle and t-PA kringle 2 (68%), no fibrin-dependent plasminogen activation of this hybrid molecule was observed. The above-mentioned results question the concept that the structural auto-nomous domains within hybrid plasminogen activators t-PA and u-PA function as autonomous domains and suggest that interactions between the kringle and the protease domain in hybrid molecules strongly influences their functional features.