[Plasminogen activator-inhibitor 1: biochemical, structural and functional studies].
A large set of monoclonal antibodies has been raised against plasminogen activator inhibitor-1 (PAI-1) and have been applied (a) for the development of various enzyme-linked immunosorbent assays with various specificities towards the different forms of PAI-1; (b) for immunopurification purposes; (c) for immunoprecipitation and immunoblotting experiments. Data were provided showing that PAI-1 occurs in plasma (approximately 8% of total blood PAI-1 antigen) and in platelets (approximately 92% of total blood PAI-1 antigen); plasma was shown to contain mainly active PAI-1 while the majority of platelet PAI-1 occurs as an inactive form. In addition we obtained evidence for the existence of a mechanism responsible for reactivation of PAI-1 in vivo. Active PAI-1 in plasma is associated with a binding protein. This PAI-1 binding protein was isolated from human plasma and identified as vitronectin (S protein). It could be demonstrated that this PAI-1-binding protein had a stabilizing effect on the PAI-1 activity without interfering with the kinetics of the inhibition of t-PA by PAI-1. Subsequently, it was shown that vitronectin is also the major protein responsible for binding of active PAI-1 to the extracellular matrix, thereby suggesting that vitronectin may also play a role in the regulation of cell-associated fibrinolytic or proteolytic processes (e.g. cell migration, tumor metastasis...). Spectroscopic and functional studies revealed that conversion between active and latent PAI-1 is associated with conformational transitions mainly occurring at the level of the tertiary structure. In addition we have isolated and characterized a previously unrecognized conformation of PAI-1 (substrate PAI-1) acting exclusively as a non-inhibitory substrate for serine proteinases, indicating that the serpin PAI-1 has some unique conformational and functional properties. This observation may have implications for the regulation of the fibrinolytic system in vivo: inhibitory PAI-1 may not only convert to latent PAI-1, which is potentially reactivatable, but also to substrate PAI-1, which may be irreversibly degraded by target proteinases. Preliminary analysis of mutants of PAI-1 in the P12-P9 region revealed that this region in particular contributes significantly to the functional properties of PAI-1. More detailed structural and functional studies will be carried out to delineate the molecular determinants involved in the conformational changes associated with alterations of the functional properties of PAI-1.
Declerck
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Centrum voor Moleculaire en Vasculaire Biologie, K.U. Leuven-Gasthuisberg.
D001324:Autoantigens / Q000302:isolation & purification; D002352:Carrier Proteins / Q000302:isolation & purification; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D008968:Molecular Conformation; D017395:Plasminogen Activator Inhibitor 1 / Q000276:immunology / Q000302:isolation & purification*; D013329:Structure-Activity Relationship
D001324:Autoantigens; D002352:Carrier Proteins; D017395:Plasminogen Activator Inhibitor 1
Plasminogeen-activator-inhibitor-1: biochemische, structurele en functionele studies.