Katrine E. Pedersen

Plasminogen activator inhibitor-1 and tumour growth, invasion, and metastasis

In recent decades, evidence has been accumulating showing the important role of urokinase-type plasminogen activator (uPA) in growth, invasion, and metastasis of malignant tumours. The evidence comes from results with animal tumour models and from the observation that a high level of uPA in human tumours is associated with a poor patient prognosis. It therefore initially came as a surprise that a high tumour level of the uPA inhibitor plasminogen activator inhibitor-1 (PAI-1) is also associated with a poor prognosis, the PAI-1 level in fact being one of the most informative biochemical prognostic markers. We review here recent investigations into the possible tumour biological role of PAI-1, performed by animal tumour models, histological examination of human tumours, and new knowledge about the molecular interactions of PAI-1 possibly underlying its tumour biological functions. The exact tumour biological functions of PAI-1 remain uncertain but PAI-1 seems to be multifunctional as PAI-1 is expressed by multiple cell types and has multiple molecular interactions. The potential utilisation of PAI-1 as a target for anti-cancer therapy depends on further mapping of these functions.

Biochemical importance of glycosylation of plasminogen activator inhibitor-1

The serpin plasminogen activator inhibitor-1 (PAI-1) is a potential target for anti-thrombotic and anti-cancer therapy. PAI-1 has 3 potential sites for N-linked glycosylation. We demonstrate here that PAI-1 expressed recombinantly or naturally by human cell lines display a heterogeneous glycosylation pattern of the sites at N209 and N265, while that at N329 is not utilised. The IC(50)-values for inactivation of PAI-1 by 4 monoclonal antibodies differed strongly between glycosylated PAI-1 and non-glycosylated PAI-1 expressed in E. coli. For 3 antibodies, an overlap of the epitopes with the glycosylation sites could be excluded as explanation for the differential reactivity. The latency transition of non-glycosylated, but not of glycosylated PAI-1, was strongly accelerated by a non-ionic detergent. The different biochemical properties of glycosylated and non-glycosylated PAI-1 depended specifically on glycosylation of either one or the other of the utilised sites. The PAI-1-binding protein vitronectin reversed the changes associated with the lack of glycosylation at one of the sites. Our results stress the importance of the source of PAI-1 when studying the mechanisms of action of PAI-1-inactivating compounds of potential clinical importance.

Biochemical mechanism of action of a diketopiperazine inactivator of plasminogen activator inhibitor-1.

XR5118 [(3 Z,6 Z )-6-benzylidine-3-(5-(2-dimethylaminoethyl-thio-))-2-(thienyl)methylene-2,5-dipiperazinedione hydrochloride] can inactivate the anti-proteolytic activity of the serpin plasminogen activator inhibitor-1 (PAI-1), a potential therapeutic target in cancer and cardiovascular diseases. Serpins inhibit their target proteases by the P(1) residue of their reactive centre loop (RCL) forming an ester bond with the active-site serine residue of the protease, followed by insertion of the RCL into the serpins large central beta-sheet A. In the present study, we show that the RCL of XR5118-inactivated PAI-1 is inert to reaction with its target proteases and has a decreased susceptibility to non-target proteases, in spite of a generally increased proteolytic susceptibility of specific peptide bonds elsewhere in PAI-1. The properties of XR5118-inactivated PAI-1 were different from those of the so-called latent form of PAI-1. Alanine substitution of several individual residues decreased the susceptibility of PAI-1 to XR5118. The localization of these residues in the three-dimensional structure of PAI-1 suggested that the XR5118-induced inactivating conformational change requires mobility of alpha-helix F, situated above beta-sheet A, and is in agreement with the hypothesis that XR5118 binds laterally to beta-sheet A. These results improve our understanding of the unique conformational flexibility of serpins and the biochemical basis for using PAI-1 as a therapeutic target.

Binding areas of urokinase-type plasminogen activator-plasminogen activator inhibitor-1 complex for endocytosis receptors of the low-density lipoprotein receptor family, determined by site-directed mutagenesis.

Some endocytosis receptors related to the low‐density lipoprotein receptor, including low‐density lipoprotein receptor‐related protein‐1A, very‐low‐density lipoprotein receptor, and sorting protein‐related receptor, bind protease‐inhibitor complexes, including urokinase‐type plasminogen activator (uPA), plasminogen activator inhibitor‐1 (PAI‐1), and the uPA–PAI‐1 complex. The unique capacity of these receptors for high‐affinity binding of many structurally unrelated ligands renders mapping of receptor‐binding surfaces of serpin and serine protease ligands a special challenge. We have mapped the receptor‐binding area of the uPA–PAI‐1 complex by site‐directed mutagenesis. Substitution of a cluster of basic residues near the 37‐loop and 60‐loop of uPA reduced the receptor‐binding affinity of the uPA–PAI‐1 complex approximately twofold. Deletion of the N‐terminal growth factor domain of uPA reduced the affinity 2–4‐fold, depending on the receptor, and deletion of both the growth factor domain and the kringle reduced the affinity sevenfold. The binding affinity of the uPA–PAI‐1 complex to the receptors was greatly reduced by substitution of basic and hydrophobic residues in α‐helix D and α‐helix E of PAI‐1. The localization of the implicated residues in the 3D structures of uPA and PAI‐1 shows that they form a continuous receptor‐binding area spanning the serpin as well as the A‐chain and the serine protease domain of uPA. Our results suggest that the 10–100‐fold higher affinity of the uPA–PAI‐1 complex compared with the free components depends on the bonus effect of bringing the binding areas on uPA and PAI‐1 together on the same binding entity.

Plasminogen activator inhibitor-1 polymers, induced by inactivating amphipathic organochemical ligands.

Negatively charged organochemical inactivators of the anti-proteolytic activity of plasminogen activator inhibitor-1 (PAI-1) convert it to inactive polymers. As investigated by native gel electrophoresis, the size of the PAI-1 polymers ranged from dimers to multimers of more than 20 units. As compared with native PAI-1, the polymers exhibited an increased resistance to temperature-induced unfolding. Polymerization was associated with specific changes in patterns of digestion with non-target proteases. During incubation with urokinase-type plasminogen activator, the polymers were slowly converted to reactive centre-cleaved monomers, indicating substrate behaviour of the terminal PAI-1 molecules in the polymers. A quadruple mutant of PAI-1 with a retarded rate of latency transition also had a retarded rate of polymerization. Studying a number of serpins by native gel electrophoresis, ligand-induced polymerization was observed only with PAI-1 and heparin cofactor II, which were also able to copolymerize. On the basis of these results, we suggest that the binding of ligands in a specific region of PAI-1 leads to so-called loop-sheet polymerization, in which the reactive centre loop of one molecule binds to beta-sheet A in another molecule. Induction of serpin polymerization by small organochemical ligands is a novel finding and is of protein chemical interest in relation to pathological protein polymerization in general.

Inhibition of plasminogen activator inhibitor-1 binding to endocytosis receptors of the low-density-lipoprotein receptor family by a peptide isolated from a phage display library.

The functions of the serpin PAI-1 (plasminogen activator inhibitor-1) are based on molecular interactions with its target proteases uPA and tPA (urokinase-type and tissue-type plasminogen activator respectively), with vitronectin and with endocytosis receptors of the low-density-lipoprotein family. Understanding the significance of these interactions would be facilitated by the ability to block them individually. Using phage display, we have identified the disulfide-constrained peptide motif CFGWC with affinity for natural human PAI-1. The three-dimensional structure of a peptide containing this motif (DVPCFGWCQDA) was determined by liquid-state NMR spectroscopy. A binding site in the so-called flexible joint region of PAI-1 was suggested by molecular modelling and validated through binding studies with various competitors and site-directed mutagenesis of PAI-1. The peptide with an N-terminal biotin inhibited the binding of the uPA-PAI-1 complex to the endocytosis receptors low-density-lipoprotein-receptor-related protein 1A (LRP-1A) and very-low-density-lipoprotein receptor (VLDLR) in vitro and inhibited endocytosis of the uPA-PAI-1 complex in U937 cells. We conclude that the isolated peptide represents a novel approach to pharmacological interference with the functions of PAI-1 based on inhibition of one specific molecular interaction.

The role of β-strand 5A of plasminogen activator inhibitor-1 in regulation of its latency transition and inhibitory activity by vitronectin

Plasminogen activator inhibitor-1 (PAI-1) is a potential target for anti-thrombotic and anti-cancer therapy. It circulates in plasma in a complex with vitronectin (VN). We have studied biochemical mechanisms for PAI-1 neutralisation and its modulation by VN, using site-directed mutagenesis and limited proteolysis. We demonstrate that VN, besides delaying conversion of PAI-1 to the inactive latent form, also protects PAI-1 against cold- and detergent-induced substrate behaviour and counteracts conversion of PAI-1 to inert forms by certain amphipathic organochemical compounds. VN protection against cold- and detergent-induced substrate behaviour is associated with inhibition of the proteolytic susceptibility of beta-strand 5A. Alanine substitution of a lysine residue placed centrally in beta-strand 5A implied a VN-induced acceleration of latency transition, instead of the normal delay. This substitution not only protects PAI-1 against neutralisation, but also counteracts VN-induced protection against neutralisation. We conclude that beta-strand 5A plays a crucial role in VN-regulation of PAI-1 activity.