Type‐1 Plasminogen‐Activator Inhibitor

We have analysed the susceptibility of latent, active, reactive-centre-cleaved and plasminogen-activator-complexed type-1 plasminogen-activator inhibitor (PAI-1) to the non-target proteinases trypsin, endoproteinase Asp-N, proteinase K and subtilisin. This analysis has allowed us to detect conformational differences between the different forms of PAI-1 outside the reactive-centre loop and β-sheet A. Proteinase-hypersensitive sites were clustered in three regions. Firstly, susceptibility was observed in the region around α-helix E, β-strand 1A, and the flanking loops, which are believed to form tlexible joints during movements of β-sheet A. Secondly, hypersensitive sites were observed in the loop between α-helix 1 and β-strand 5A. Thirdly, the gate region, encompassing β-strands 3C and 4C, was highly susceptible to trypsin in latent PAI-1, but not in the other conformations. The digestion patterns differed among all four forms of PAT-1, indicating that each represents a unique conformation. The differential proteolytic susceptibility of the flexible-joint region may be coupled to the differential affinity to vitronectin, binding in the same region. The analysis also allowed detection of conformational differences between reactive-centre-cleaved forms produced under different solvent conditions. The digestion pattern of plasminogen-activator-complexed PAI-1 was different from that of active PAI-1, but indistinguishable from that of one of the reactive-centre-cleaved forms, as the complexed and this particular cleaved PAI-1 were completely resistant to all the non-target proteinases tested. This observation is in agreement with the notion that complex formation involves reactive-centre cleavage and a large degree of insertion of the reactive-centre loop into β-sheet A. Our analysis has allowed the identification of some flexible regions that appear to be implicated in the conformational changes during the movements of β-sheet A and during the inhibitory reaction of serpins with their target proteinases.