Type‐1 Plasminogen‐Activator Inhibitor

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Abstract
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.