Interaction of plasminogen activator inhibitor type-1 (PAI-1) with vitronectin - Characterization of different PAI-1 mutants

The serpin plasminogen activator inhibitor type-1 (PAI-1) is of importance in physiological processes such as fibrinolysis and thrombolysis as well as in pathophysiological processes like thrombosis, tumor cell adhesion or invasion, and metastasis. The interaction of PAI-1 with the extracellular matrix protein vitronectin (Vn) was implicated to play an important role in several of these processes and is therefore a possible target for therapeutic strategies. Understanding the PAI-1/Vn interaction is a substantial prerequisite for the development of therapeutically applicable compounds. Thus, in the present study, the Vn-binding behavior of PAI-1 was analyzed by characterizing twelve different PAI-1 variants with variations in regions previously implicated in Vn-binding of PAI-1 (i.e. mainly around alpha-helix E (hE) of PAI-1). For this, variant PAI-1 was expressed in E. coli and subsequently its inhibitory activity towards uPA was determined. Interaction with Vn was measured with four different techniques, including surface plasmon resonance. Moreover, several PAI-1 mutants were also characterized concerning their functional interaction with the extracellular matrix component heparin. On the one hand evidence is provided that the integrity of hE of PAI-1 is not essential for Vn-binding, on the other hand some cooperativity of hE and beta strand 1A of PAI-1 in Vn-binding may exist, as the mutant A114-118 (change of amino acids F114-R118 into AAAAA) shows reduced affinity towards Vn and the mutant Q123K only shows unspecific binding to multimeric Vn. Interestingly, the mutant Q123K which is now widely used as a non-Vn-binding PAI-1 variant in several in vitro and in vivo studies, only differs from wt-PAI-1 in binding to multimeric Vn, but not in binding to native Vn. Nevertheless, this mutant is not able to functionally interact with native Vn like wt-PAI-1 does. The biological relevance of PAI-1 and Vn in tumor cell invasion was studied in in vitro cell invasion assays. Here, the ovarian carcinoma cell line OV-MZ-19 displayed Vn-dependent invasiveness, which could be inhibited by addition of recombinant PAI-1.