Characterization of the interaction of plasminogen activator inhibitor type 1 with vitronectin by surface plasmon resonance

Abstract

Abstract In plasma all plasminogen activator inhibitor type 1 (PAI-1) is found in complex with vitronectin (VN). We have investigated the interaction of human PAI-1 with monomeric native human VN by size exclusion chromatography with VN in solution and using the surface plasmon resonance technique (SPR) with immobilised VN. With these techniques PAI-1 is found to bind in a tight one-to-one complex. VN in solution competed with amine coupled VN in binding free active PAI-1 with a K D of 20 nM. A K D of 0.1 nM at 25°C was instead found for PAI-1 binding to immobilised VN. The extremely rapid binding of PAI-1 to immobilised VN had an association rate constant, κ on of 20 μ M –1 s –1 . By the immobilisation of VN the affinity for PAI-1 thus increased manifold. The stable substrate mutant PAI-1-Ala335Glu had similar binding characteristics for immobilised VN as active PAI-1. Upon injection of human α-thrombin onto PAI-1 captured on VN the ternary complex was formed and PAI-1 rapidly dissociated from immobilised VN, indicating that VN was not converted into the denatured form by amine coupling. Monoclonal antibodies against PAI-1, belonging to five different classes did all bind to PAI-1 captured on immobilised VN. There is thus no direct overlap of either of these five antibody binding epitopes on PAI-1 with the binding site for VN. PAI-1 was released from immobilised VN by addition of tissue plasminogen activator (tPA) (E.C. 3.4.21.68), with a dissociation rate depending on the tPA concentration. The preformed complex of tPA and PAI-1 did not bind to immobilised VN. An intermediate ternary complex with tPA and active PAI-1 captured on VN is not detected with SPR. On the contrary, a transient ternary complex was observed with tPA and PAI-1-Ala335Glu captured on VN. In conclusion, these results reject that VN is bound to any of the five antibody binding epitopes, and support the proposal that the binding surface for VN on PAI-1 is located near the epitope of tPA in the final complex, including parts of α-helices C, E and β-strand 1A.