The role of h-strand 5A of plasminogen activator inhibitor-1 in regulation of its latency transition and inhibitory activity by vitronectin Signe Jensen a, * , Tove Kirkegaard a , Katrine E. Pedersen a , Marta Busse a , Klaus T. Preissner b , Kees W. Rodenburg a , Peter A. Andreasen a a Laboratory of Cellular Protein Science, Department of Molecular and Structural Biology, Aarhus University, Gustav Wieds vej 10C, DK-8000 Aarhus C, Denmark b Department of Biochemistry, Justus-Liebig-University, Giessen, Germany Received 17 December 2001; received in revised form 11 March 2002; accepted 4 April 2002 Abstract Plasminogen activator inhibitor-1 (PAI-1) is a potential target for anti-thrombotic and anti-cancer therapy. It circulates in plasma in a complex with vitronectin (VN). We have studied biochemical mechanisms for PAI-1 neutralisation and its modulation by VN, using site- directed mutagenesis and limited proteolysis. We demonstrate that VN, besides delaying conversion of PAI-1 to the inactive latent form, also protects PAI-1 against cold- and detergent-induced substrate behaviour and counteracts conversion of PAI-1 to inert forms by certain amphipathic organochemical compounds. VN protection against cold- and detergent-induced substrate behaviour is associated with inhibition of the proteolytic susceptibility of h-strand 5A. Alanine substitution of a lysine residue placed centrally in h-strand 5A implied a VN-induced acceleration of latency transition, instead of the normal delay. This substitution not only protects PAI-1 against neutralisation, but also counteracts VN-induced protection against neutralisation. We conclude that h-strand 5A plays a crucial role in VN-regulation of PAI-1 activity. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Cancer; Extracellular proteolysis; Plasminogen; Serpin; Thrombosis; Vitronectin 1. Introduction Plasminogen activator inhibitor-1 (PAI-1) is a fast and specific inhibitor of the plasminogen activating serine pro- teinases tissue-type plasminogen activator (tPA) and uroki- nase-type plasminogen activator (uPA) and, as such, an important regulator in turnover of extracellular matrix and in fibrinolysis (for reviews, see Refs. [1 –3]). The PAI-1 level in malignant tumours is a marker of a poor prognosis (for reviews, see Refs. [4,5]), and PAI-1 seems to be causally involved in tumour invasion and angiogenesis [6]. An impaired fibrinolysis in blood plasma due to a high level of PAI-1 is a risk factor for cardiovascular diseases (for review, see Ref. [7]). PAI-1 belongs to the serpin superfamily. Characteristi- cally, the serpins are able to undergo large conformational changes, consisting in insertion of a solvent-exposed, about 20-amino-acid-long peptide loop, the reactive centre loop (RCL) as strand 4 of the large central h-sheet A (s4A) (for reviews, see Refs. [3,4,8–10]). Upon interaction of a serpin with its target proteinase, the reactive centre peptide bond (P 1 –P 1 V ) becomes cleaved, but the P 1 residue remains linked by an ester bond to the active site Ser of the proteinase. Immediately after cleavage, the N-terminal part of RCL becomes inserted in h-sheet A, whereby the proteinase is translocated to the other pole of the serpin. This causes a distortion of the proteinase, for which reason it is unable to complete the catalytic cycle (for review, see Ref. [3]). It is thus the stabilisation of the serpin molecule in association with RCL insertion that drives the formation of a stable complex with the target proteinase. 0167-4838/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII:S0167-4838(02)00312-6 Abbreviations: a 1 PI, a 1 -proteinase inhibitor; ANS, 1-anilinonaphtalene- 8-sulfonic acid; bis-ANS, 4,4V -dianilino-1,1V -bisnaphtyl-5,5V -disulfonic acid; h, a-helix; hF/s3A-loop, the loop between a-helix F and h-strand 3A; IC 50 , concentration giving 50% inhibition; LMW-uPA, low molecular weight uPA; PAI-1, plasminogen activator inhibitor type-1; PBS, phosphate-buf- fered saline; RCL, reactive centre loop; s, h-strand; S-2444, L-pyroGlu-Gly- L-Arg-p-nitroaniline hydrochloride; tPA, tissue-type plasminogen activator; uPA, urokinase-type plasminogen activator; VN, vitronectin; wt, wild type; XR5118, (3Z,6Z)-6-benzylidene-3-(2-dimethyleaminoethyl-thio)-2-(thie- nyl)methylene-2,5-piperazinedione hydrochloride * Corresponding author. Tel.: +45-89422634; fax: +45-86196500. E-mail address: sgj@mbio.aau.dk (S. Jensen). URL: http://www.mbio.aau.dk/~pa/welcome.htm. www.bba-direct.com Biochimica et Biophysica Acta 1597 (2002) 301 – 310