CONFORMATIONAL-CHANGES OF THE REACTIVE-CENTER LOOP AND BETA-STRAND 5AACCOMPANY TEMPERATURE-DEPENDENT INHIBITOR-SUBSTRATE TRANSITION OF PLASMINOGEN-ACTIVATOR INHIBITOR-1

We have studied conformational changes of type-1 plasminogen-activator inhibitor (PAI-1) during a temperature-dependent inhibitor-substrate transition by measuring susceptibility of the molecule to non-target p roteinases. When incubated at 0 degrees C instead of the normally used 37 degrees C, a tenfold decrease in the specific inhibitory activity of active PAI-1 was observed. Accordingly, PAI-1 was recovered in a re active-centre-cleaved form from incubations with urokinase-type plasmi nogen activator (uPA) and tissue-type plasminogen activator (tPA) at 0 degrees C, but not at 37 degrees C. It thus behaved as a substrate fo r the target proteinases at the lower temperature. Active PAI-1 was ex posed to a variety of non-target proteinases, including elastase, papa in, thermolysin, trypsin, and V8 proteinase. It was found that specifi c peptide bonds in the reactive centre loop (RCL) and strand 5 in beta -sheet A (s5A) had a temperature-dependent proteolytic susceptibility, while the P-17-P-16 (E332-S333) bond, forming the hinge between s5A a nd the RCL, showed indistinguishable susceptibility to proteolysis by V8 proteinase at 0 degrees and 37 degrees C. In latent and reactive-ce ntre-cleaved PAI-1, all the bonds were resistant to proteolysis at the higher as well as the lower temperature. An anti-PAI-1 monoclonal ant ibody maintained the inhibitory activity of PAI-1 and prevented reacti ve centre cleavage at 0 degrees C, and thus prevented substrate behavi our. Concomitantly, it caused specific changes in proteolytic suscepti bility of s5A and the RCL, but it did not affect cleavage of the P-17- P-16 bond by V8 proteinase. Our observations suggest that temperature- dependent conformational changes of beta-sheet A and the RCL determine whether the serpin act as an inhibitor or a substrate. Furthermore th ey suggest that the RCL of PAI-1 is fully extracted from beta-sheet A in the inhibitory as well as in the substrate form, favoring a so-call ed induced conformational state model to explain why inhibitory activi ty requires partial insertion of the RCL into beta-sheet A.