Mapping of the epitope of a monoclonal antibody protecting plasminogen activator inhibitor-1 against inactivating agents Julie S. Bødker, Troels Wind, Jan K. Jensen, Martin Hansen, Katrine E. Pedersen and Peter A. Andreasen Laboratory of Cellular Protein Science, Department of Molecular Biology, University of Aarhus, Denmark Plasminogen activator inhibitor-1 (PAI-1) belongs to the serpin family of serine proteinase inhibitors. Serpins inhibit their target proteinases by an ester bond being formed between the active site serine of the proteinase and the P 1 residue of the reactive centre loop (RCL) of the serpin, fol- lowed by insertion of the RCL into b-sheet A of the serpin. Concomitantly, there are conformational changes in the flexible joint region lateral to b-sheet A. We have now, by site-directed mutagenesis, mapped the epitope for a mono- clonal antibody, which protects the inhibitory activity of PAI-1 against inactivation by a variety of agents acting on b-sheet A and the flexible joint region. Curiously, the epitope is localized in a-helix C and the loop connecting a-helix I and b-strand 5A, on the side of PAI-1 opposite to b-sheet A and distantly from the flexible joint region. By a combination of site-directed mutagenesis and antibody protection against an inactivating organochemical ligand, we were able to identify a residue involved in conferring the antibody-induced con- formational change from the epitope to the rest of the molecule. We have thus provided evidence for communi- cation between secondary structural elements not previously known to interact in serpins. Keywords: cancer; cardiovascular disease; monoclonal antibody; protease; serpin. The serpins constitute a protein family of which the best characterized members, including a 1 -proteinase inhibitor, antithrombin III, and plasminogen activator inhibitor-1 (PAI-1), are inhibitors of serine proteinases implicated in processes such as blood coagulation and turn-over of extracellular matrix. Of decisive importance for the inhibitory mechanism of serpins is the surface-exposed, approximately 20-amino acid long reactive centre loop (RCL) (see Fig. 1). Biochemical and biophysical evidence has shown that the reaction between a serpin and its target proteinase is initiated by formation of a reversible docking complex in which the P 1 –P 1 ¢ bond in the RCL interacts noncovalently with the active site of the proteinase [1]. In the locking step that follows, the P 1 – P 1 ¢ bond is cleaved [2,3] and the P 1 residue is coupled to the active site serine of the proteinase by an ester bond [4]. The N-terminal part of the RCL then becomes inserted as strand 4 in b-sheet A (s4A) [5]. Because of the covalent bond, the proteinase is translocated to the opposite pole of the serpin [6–8], the active site becoming distorted, the catalytic machinery inactivated, and the completion of the catalytic cycle disabled [8–16], resulting in formation of a stable covalently coupled complex of 1 : 1 stoichiometry (for reviews see [17–19]). The energy needed for the proteinase distortion comes from stabi- lization of the serpin in the ÔrelaxedÕ conformation by insertion of the RCL into b-sheet A, as opposed to the ÔstressedÕ, relatively unstable active conformation with a surface-exposed RCL. Under some conditions, proteinase distortion cannot keep pace with ester bond hydrolysis, resulting in abortive complex formation, full cleavage of the P 1 –P 1 ¢ bond, insertion of the RCL into b-sheet A and release of an active proteinase (for reviews see [17,20]). Serpins following this alternative path are said to exhibit substrate behaviour. Some serpins, including PAI-1 and antithrombin III spontaneously assume an inactive, relaxed, so-called latent state in which the intact RCL is inserted into b-sheet A, after passage through the so- called gate region between the s3C–s4C loop and the s3B–hG loop (Fig. 1) [21,22]. RCL insertion is coupled to conformational changes in the flexible joint region around a-helices D and E. The flexible joint region of stressed, but not relaxed PAI-1, binds to the N-terminal 44-amino acid long somatomedin B domain of the M r 70 000 glycoprotein vitronectin (VN) [23,24], which thereby delays the latency transition of PAI-1 (for a review see [20]). A few organochemical compounds able to inactivate PAI-1 have been indentified, including a group of negatively charged amphipathic compounds like bis-ANS (4,4¢-dianilino-1,1¢-bisnaphthyl- 5,5¢-disulfonic acid) [11,25] and the diketopiperazine derivative XR5118 ((3Z,6Z)-6-benzylidene-3-(5-((2-dimeth- ylaminoethyl-thio)-2-thienyl)methylene-2,5-piperazinedione Correspondence to J. S. Bødker, Department of Molecular Biology, University of Aarhus, Gustav Wied’s Vej 10C, 8000 C Aarhus, Denmark. Tel.: + 45 89425079, E-mail: jsb@mb.au.dk Abbreviations: bis-ANS, 4,4¢-dianilino-1,1¢-bisnaphtyl-5,5¢-disulfonic acid; h, a-helix; RCL, reactive centre loop; HBS, Hepes buffered saline; PAI-1, plasminogen activator inhibitor-1; s, b-strand; S-2444, pyro-Glu-Gly-Arg-p-nitroanilide; uPA, urokinase-type plasminogen activator; VN, vitronectin; wt, wild-type; XR5118, ((3Z,6Z)-6-benzylidene-3-(5-((2-dimethylaminoethyl-thio)- 2-thienyl)methylene-2,5-piperazinedione hydrochloride). (Received 3 December 2002, revised 5 February 2003, accepted 13 February 2003) Eur. J. Biochem. 270, 1672–1679 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03523.x hydrochloride) [11,26]. Their exact binding sites in PAI-1 remain to be established, but all available evidence is in agreement with these compounds having overlapping, but not identical, binding sites in the flexible joint region [27]. VN protects PAI-1 from inactivation by bis-ANS and XR5118 [11,24,28]. These compounds do not bind to relaxed PAI-1 [11]. Thus, there is bidirectional communi- cation between the flexible joint region and the move- ments of the RCL. Among a large number of monoclonal antibodies directed against PAI-1 raised since the mid-1980s, Mab-1 possesses a number of unique features. Mab-1 was raised against latent PAI-1 purified from HT-1080 cells [29]. It stabilizes PAI-1 against cold-induced substrate behaviour in buffers with nonionic detergents [30]. As monitored by proteolytic susceptibility, Mab-1 seems to induce con- formational changes of the RCL, s5A, and the flexible joint region [30,31]. In a recent study aimed at mapping molecular interactions of PAI-1 by random mutagenesis, Stoop et al. [32] identified residue Q58/74 as part of the epitope for Mab-1 (a double amino acid numbering system is used here, the first number following the numbering system of Andreasen et al. [33], the second number following the a 1 -antiproteinase inhibitor num- bering system of Huber and Carrell [34]). Q58/74 is localized in a-helix C (hC) (Fig. 1). Since it is localized distantly from the secondary structural elements affected by Mab-1, we hypothesized that a further characteriza- tion of the epitope for Mab-1 might yield important information about general aspects of serpin conforma- tional changes. Materials and methods PAI-1 The cDNAs for wild-type (wt) and substituted human PAI-1, extended at the N terminus with a His 6 -tag and a recognition motif for heart muscle kinase, were produced by standard methods in the Escherichia coli expression vector pT7-PL [35]. Transformed E. coli BL21(DE3)- pLysS cells from 1-L cultures, treated with 0.5 m M isopropyl thio-b- D -galactoside to induce PAI-1 expression, were harvested by centrifugation (7000 g, 30 min), resus- pended in 35 mL phosphate-buffered saline (10 m M Na 2 HPO 4 ,140 m M NaCl pH 7.4), and disrupted by soni- cation. The homogenates were centrifuged (10 000 g, 20 min), filtered (0.22 lm), supplemented with 2 M NaCl and 10 m M imidazole, and applied to a 5-mL Ni-NTA column equilibrated in the same buffer further supple- mented with 5% glycerol. PAI-1 was eluted with 200 m M imidazole. The eluted protein was subjected to gel filtration on a Superdex 75 column (1.6 · 60 cm) equi- librated in Hepes-buffered saline (HBS; 10 m M Hepes, 140 m M NaCl pH 7.4) supplemented with 5% glycerol andNaCltoafinalconcentrationof1 M . The procedure routinely gave 10–15 mg PAI-1 per litre bacterial culture. The preparations contained PAI-1 which was more than 95% pure as evaluated by SDS/PAGE and Coomassie blue staining. N-terminal sequencing showed the expected N terminus, i.e. (M)GSMGSHHHHHHGSRRASV HH…, missing only the initiating M indicated in paren- theses. The N-terminal extension did not affect the specific Fig. 1. Localization of the epitope for Mab-1 in the three-dimensional structure of PAI-1. (A and B) Ribbon presentations of PAI-1 in the active conformation in two different orientations. Relative to (A) the structure shown in (B) is turned approximately 180° around the y-axis and approximately 45° around the x-axis of the coordinate system shown in the figure. Relevant secondary structural elements are marked. Please note that T341/351 of the RCL is not visible in the structure. (C) Surface presentation of PAI-1 in the same orientation as in (B). Red residues were those implicated in the epitope for Mab-1. Blue residues (Q57/73, Q59/75, Q61/77, K67/83, K106/125, Q109/128, R302/313, F304/315, Q305/316, T309/ 319, D313/323, Q314/324, E315/325, P316/326, K325/335) were excluded from the epitope. D299/310 is indicated in yellow (see the text for details). Nearby alanine and glycine residues (G53/69, G54/70, A62/78, A63/79 and A306/317), not testable by alanine scanning mutagenesis, are indicated in cyan. These SWISS PDB VIEWER displays are based on the coordinates of Stout et al.[40]. Ó FEBS 2003 A PAI-1-protecting monoclonal antibody (Eur. J. Biochem. 270) 1673 inhibitory activity of PAI-1, its second-order rate constant for reaction with uPA, its VN binding, or its rate of latency transition. The specific inhibitory activity of wt PAI-1 was 50 ± 21% (n ¼ 17) of the theoretical maximum. Most of the mutants had a specific inhibitory activity indistinguish- able from that of wt PAI-1. The exceptions were D313/ 323A (112 ± 21%; n ¼ 17; P < 0.01) and Q58/74A- D307/318A (15 ± 3%; n ¼ 3; P < 0.01). Monoclonal antibodies Mab-1 was produced and purified as described previously [29]. Two other monoclonal antibodies against PAI-1, Mab- 2 [29,36,37] and Mab-5 [38], were produced and purified in the same way. Other proteins and miscellaneous materials Bis-ANS was from Molecular Probes. S-2444 (pyro-Glu- Gly-Arg-p-nitroanilide) was from Chromogenix (Mo ¨ lndal, Sweden). Human urokinase-type plasminogen activator (uPA) was from Wakamoto Pharmacautical Co. (Tokyo, Japan). XR5118 was a kind gift from Dr Thomas Frandsen, Finsen Laboratory, Copenhagen. The peptide TVASS, acetylated at the N terminus and amidated at the C terminus, was purchased from Eurogentec (Ougre ´ e, Belgium). ELISA To determine the relative affinity of Mab-1 for recombinant wt and mutant PAI-1, Mab-1 or Mab-2 was coated onto the solid phase of microtiter wells, using an antibody concen- tration of 2.5 lgÆmL )1 and a buffer of 50 m M NaHCO 3 , pH 9.6. After blocking with milk, dilution series of recom- binant PAI-1, spanning a concentration range from 0.1 ngÆmL )1 to 20 lgÆmL )1 , were applied to the wells. The bound PAI-1 was detected with a layer of rabbit polyclonal anti-PAI-1 Igs, a layer of peroxidase-conjugated swine anti- (rabbit IgG) Ig (DAKO), and a peroxidase reaction. The 50% effective concentrations (EC 50 ) for the binding of PAI-1 to the antibodies were defined as the amount of PAI-1 resulting in half-maximal binding. Measurements of the effects of Mab-1 or Mab-5 and neutralizers on the specific inhibitory activity of PAI-1 To measure the effects of antibodies and inactivators on the specific inhibitory activity of wt and substituted PAI-1, i.e. the fraction of inhibitor forming a stable complex with uPA, PAI-1 was serially diluted in HBS with 0.25% gelatine, resulting in PAI-1 concentrations between 0.01 and 20 lgÆmL )1 in a volume of 100 lL, with or without antibody (4 lgÆmL )1 Mab-1 or 80 lgÆmL )1 Mab-5). The dilution series were then incubated for 10 min at 37 °C to allow antibody–PAI-1 complex formation. Fifty-lL aliqu- ots of HBS with 0.25% gelatine with or without bis-ANS or XR5118 were added, the bis-ANS or XR5118 concentration varying between dilution series. The mixtures were incuba- ted for 10 min at 37 °C. Aliquots of 50 lLwith1lgÆmL )1 uPA were added. Incubation was continued for at least 5 min, sufficient for the process of inhibition of uPA to come to an end. The remaining uPA enzyme activity was determined by incubation with the substrate S-2444 and measurement of the increase in absorbance at 405 nm. The specific inhibitory activity of PAI-1 was calculated from the amount of PAI-1 that had to be added to inhibit 50% of the uPA (50% inhibitory concentrations; IC 50 ). The IC 50 for bis-ANS or XR5118 neutralization of PAI-1 were deter- mined as the neutralizer concentrations halving the PAI-1 specific inhibitory activity. The highest concentration of XR5118 and bis-ANS used in these assays were 250 l M and 80 l M , respectively, due to solubility limits. Measurements of the effects of Mab-1 and VN on PAI-1 latency transition and TVASS incorporation rate PAI-1 (20 lgÆmL )1 )wasincubatedat37°C in HBS supplemented with 0.25% gelatine, in the absence or presence of Mab-1 (100 lgÆmL )1 ), VN (30 lgÆmL )1 ), and/ or the TVASS pentapeptide (250 l M ). At regular time intervals, samples were withdrawn for measurement of the specific inhibitory activity of PAI-1. This was done by making serial dilution series at 37 °C with HBS supplemen- ted with 0.25% gelatine, resulting in PAI-1 concentrations between 0.01 and 20 lgÆmL )1 in a volume of 100 lL. Aliquots of 100 lLwith0.5lgÆmL )1 uPA were added. After incubation for at least 2 min, the remaining uPA enzyme activity was determined by incubation with the chromogenic substrate S-2444 and measurement of the increase in absorbance at 405 nm. The specific inhibitory activity of PAI-1 was calculated from the amount of PAI-1 that had to be added to inhibit 50% of the uPA. The half- lives of the functional activity were calculated from semi- logarithmic plots of the specific inhibitory activity vs. time. Statistical analysis Data were evaluated by Student’s t-test. Molecular graphics SWISS PDB VIEWER [39] was used to display the three- dimensional structure of active PAI-1 [40]. Results Epitope mapping To define in detail the epitope for Mab-1, we performed extensive alanine scanning mutagenesis around Q58/74, already identified as being part of the epitope by Stoop et al. [32]. Alanine-substituted and wt PAI-1s were tested in ELISA for their binding to the antibody. The substituted residues in variants with an EC 50 at least twofold higher than that for wt PAI-1 were considered to be part of the epitope. In this way, E55/71 and Q58/74 in hC and D307/318 in the hI/s5A loop were included in the epitope, while a number of adjacent residues were excluded from it (Table 1, Figs 1 and 2). Combining alanine substitutions of two of these three positions resulted in variants with more than 20 000-fold reduced EC 50 (Table 1). Attempts at expression of the mutant with 1674 J. S. Bødker et al. (Eur. J. Biochem. 270) Ó FEBS 2003 a triple substitution failed because of low yield. None of the variants with substitutions in the epitope had a specific inhibitory activity distinguishable from that of the wt. The substitutions had no or only minor effects on binding to a monoclonal antibody against PAI-1, Mab-2 (Table 1). Mab-2 has an epitope of residues in hF and its flanking sequences [37]. Mab-1 protection of PAI-1 against bis-ANS and XR5118 The IC 50 values for bis-ANS and XR5118 inactivation of wt PAI-1 were 0.62 ± 0.06 (n ¼ 6) and 10.1 ± 3.0 (n ¼ 11) l M , respectively, in agreement with previous reports [11,24,28]. We now found that the IC 50 values for bis- ANS and XR5118 inactivation of the Mab-1–PAI-1 complex were higher than 80 l M (n ¼ 3) and higher than 250 l M (n ¼ 12), respectively. Thus, Mab-1 protects wt PAI-1 against these neutralizers. Similar observations were done with two other neutralizers, 1-anilinonaphtalene-8- sulfonic acid and 1-dodecyl sulphuric acid (data not shown). In contrast, the monoclonal antibody against PAI-1 Mab-5, having an epitope not overlapping that of Mab-1 [38], did not protect PAI-1 against XR5118 and bis-ANS. The IC 50 values for bis-ANS and XR5118 inactivation of the Mab-5- PAI-1 complex were 0.57 ± 0.02 (n ¼ 3) and 9.8 ± 2.23 (n ¼ 6), respectively, not significantly different from the values without antibody (P < 0.01). In the absence of inactivators, neither antibody affected the specific inhibitory activity of PAI-1. To analyse the effect of the amino acid substitutions in and around the epitope of Mab-1 on the ability to protect against XR5118, we measured the specific inhibitory activity of each variant with alanine substitutions in the absence and presence of Mab-1, and in the presence of XR5118 at concentrations between 0 and 80 l M . In the absence of XR5118, Mab-1 did not affect the specific inhibitory activity of any of the variants, and all variants had IC 50 values for inactivation by XR5118 indistinguishable from that of wt (data not shown). Whereas wt PAI-1 was totally resistant to 80 l M XR5118inthepresenceofMab-1,someofthe mutants were only partially or not at all protected against XR5118 by Mab-1 (Fig. 3). As expected, Mab-1 gave little or no protection to the variants with substitutions in the epitope, i.e., E55/71A, Q58/74A, and D307/318A, and the double mutants E55/71A-Q58/74A, E55/71A-D307/318A and Q58/74A-D307/318A. In addition, D299/310A was incompletely protected by Mab-1. Mab-1 and PAI-1 latency transition and PAI-1 inactivation by an insertion peptide In the presence of Mab-1, the half-life for latency transition of PAI-1 was increased by a factor of  1.5. The half-lives in the presence of Mab-1 and in the presence of VN were indistinguishable. However, with the variant K325A, the effects of VN and Mab-1 were different. This variant has a twofold increased half-life as compared to wt, and the half- life is not increased, but decreased by VN. We found now that Mab-1 did not affect the latency transition rate of this variant (Table 2 and Fig. 4). Thus, Mab-1 and VN affect the latency transition rate by different mechanisms. Table 1. Effect of alanine substitutions on the affinity of PAI-1 to Mab-1. The EC 50 values for binding of each variant to Mab-1 or Mab-2 were determined in parallel with the EC 50 value for wt and expressed as a fraction of that. The means and standard deviations of triple determinations are indicated. Besides the results shown in the table, the following variants were tested, but found to be indistinguishable from wt with respect to the affinity to Mab-1: Q57/73A, Q59/75A, Q61/77A, K67/83A, K106/125A, Q109/128A, D299/310A, R302/313A, F304/315A, Q305/316A, T309/319A, D313/323A, Q314/324A, E315/325A, P316/326A, K325/335A. Substitution(s) Secondary structural element Amino acid in murine PAI-1 Mab-1 EC 50variant /EC 50wt Mab-2 EC 50variant /EC 50wt E55/71A hC K 22.7 ± 7.3 a 1.1 ± 0.1 Q58/74A hC R 6.6 ± 1.5 a 1.3 ± 0.2 D307/318A hI/s5A loop D 3.6 ± 0.7 a 1.8 ± 0.5 E55/71A-Q58/74A hC K and R >20 000 a 1.6 ± 0.3 E55/71A-D307/318A hC and hI/s5A loop K and D >20 000 a 3.6 ± 1.1 Q58/74A-D307/318A hC and hI/s5A loop R and D >20 000 a 2.6 ± 0.5 a a Significantly different from 1 (P < 0.025). Fig. 2. Localization of the amino acids in the epitope for Mab-1. The structure shown is a ribbon representation of the coordinates of Stout et al. [40]. The side chains of the amino acids in the epitope and D299/ 310 are displayed as sticks and CPK-coloured. The colours of the secondary structure elements are identical to those in Fig. 1 (see text for further details). Ó FEBS 2003 A PAI-1-protecting monoclonal antibody (Eur. J. Biochem. 270) 1675 Two molecules of the pentapeptide TVASS is able to insert between s3A and s5A in active PAI-1, mimicking the RCL of relaxed forms of PAI-1. The (TVASS) 2 –PAI-1 complex displays substrate behaviour, presumably due to a reduced rate of RCL insertion during the reaction with a target proteinase [41]. We measured the effect of Mab-1 on the rate of incorporation of TVASS into PAI-1 by measuring the specific inhibitory activity of PAI-1 after incubation with TVASS at 37 °C for different time periods in the absence or presence of Mab-1. However, Mab-1 did not affect the rate of TVASS-induced inactivation of PAI-1, the half-life for inactivation by 250 l M TVASS being 10.3 ± 2.7 min (n ¼ 3) in the absence of Mab-1 and 12.9 ± 0.9 min (n ¼ 3) in the presence of Mab-1. Discussion To the best of our knowledge, Mab-1 is the only mono- clonal antibody known to stabilize PAI-1 in an inhibitory active form. We previously reported that Mab-1 stabilizes PAI-1 against cold-induced substrate behaviour in buffers with nonionic detergents [30]. We report here that Mab-1 delays PAI-1 latency transition and protects PAI-1 against bis-ANS- and XR5118-induced inactivation. As monitored by proteolytic susceptibility, the protection by Mab-1 against cold-induced substrate behaviour in detergent-containing buffers is associated with conforma- tional changes of the RCL, the sequence Q321/331-K325/ 335 in s5A and of the flexible joint region [30,31]. Bis-ANS induces substrate behaviour and polymerization, and XR5118 induces conversion to an inert monomeric form [11]. Taken together, these observations show that Mab-1 stabilizes the inhibitory activity of PAI-1 against inactiva- tion by affecting the conformation of the flexible joint region, the central sequence of s5A, and/or the RCL. Stoop et al. [32] initially reported that Q58/74 is import- ant for binding of PAI-1 to Mab-1. We have demonstrated here that the epitope also includes E55/71 and D307/318A. In agreement with expectancies for a murine antibody against a human protein, two of the residues in the epitope are different in humans and mice (Table 1). The epitope spans residues in both hC and the loop connecting hI and s5A. It is thus obvious that Mab-1 affects interactions of residues of PAI-1 which are localized distantly from its epitope. We used a combination of site-directed mutagenesis and Mab-1 protection of PAI-1 against XR5118-induced inactivation to obtain information about how the conform- ational change initiated by the binding of Mab-1 spreads through the molecule. Generally, observation of a substi- tution having a different effect on XR5118 inactivation of PAI-1 in the absence and presence of Mab-1 shows that the Fig. 3. Effect of XR5118 on the specific inhibitory activities of wt PAI-1 and PAI-1 variants in the absence and the presence of Mab-1. The specific inhibitory activities of PAI-1 in the absence and presence of Mab-1 were measured in the presence of the indicated concentrations of XR5118 and expressed relative to the specific inhibitory activity in the absence of XR5118. Means and standard deviations are indicated. The four mutants shown are significantly different from wt with respect to residual inhibitory activity in the presence of Mab-1 and 80 l M XR5118 (P < 0.01). Besides the variants shown in the figure, we tested the following variants and found that they did not differ from wt with respect to the response of the specific inhibitory activity to Mab-1: Q57/73A, Q59/75A, Q61/77A, K67/83A, K106/125A, Q109/128A, R302/313A, F304/315A, Q305/316A, T309/319A, D313/323A, Q314/ 324A, E315/325A, P316/326A, K325/335A. 1676 J. S. Bødker et al. (Eur. J. Biochem. 270) Ó FEBS 2003 corresponding amino acid side chain is in different sur- roundings in the absence and presence of Mab-1. Accord- ingly, all the variants with substitutions in the Mab-1 epitope were susceptible to XR5118 in the presence of Mab-1. In addition, substitution of D299/310, localized in the hI-s5A loop (Fig. 2), but outside the epitope, also resulted in a reduced ability of Mab-1 to protect PAI-1 against XR5118. A Mab-1-induced reorientation of D299/ 310 may therefore be important for the transmission of a Mab-1-induced signal from the epitope to b-sheet A, the RCL, and the flexible joint region. It is interesting to note that whereas Mab-1 delayed the rate of latency transition, it did not measurably affect the rate of incorporation of TVASS into b-sheet A. This observation is in agreement with the notion that the rate- limiting step during latency transition is not insertion of the RCL into b-sheet A, but rather passage of the RCL through the gate region [42]. Anyway, the mechanism by which Mab-1 delays latency transition is different from that of VN, as we demonstrate here that the two have different effects on PAI-1 K325/335A. Conclusively, on the basis of the reported observations, we propose that the binding of Mab-1 to PAI-1 results in a conformational change of hC and the hI-s5A loop which spreads to the flexible joint region, the central portion of s5A, and the RCL, and thus affects the functional properties of PAI-1. PAI-1 is a potential target for antithrombotic and anticancer therapy (for a review see [20]). A variety of model systems is available for studying the effects of PAI-1 inactivators on thrombi and tumours (for reviews see [43–45]). By stabilizing PAI-1 against inactivation, Mab-1 may be used as a valuable reagent for controlling specificity for PAI-1 inactivators in such model systems. References 1. Ye, S., Cech, A.L., Belmares, R., Bergstro ¨ m, R.C., Tong, Y., Corey, D.R., Kanost, M.R. & Goldsmith, E.J. (2001) The struc- ture of a Michaelis serpin-protease complex. Nature Struct. Biol. 8, 979–983. 2. Wilczynska, M., Fa, M., Ohlsson, P.I. & Ny, T. (1995) The inhibition mechanism of serpins. Evidence that the mobile reactive center loop is cleaved in the native protease-inhibitor complex. J. Biol. Chem. 270, 29652–29655. 3. Lawrence, D.A., Ginsburg, D., Day, D.E., Berkenpas, M.B., Verhamme, I.M., Kvassman, J.O. & Shore, J.D. (1995) Serpin- protease complexes are trapped as stable acyl-enzyme inter- mediates. J. Biol. Chem. 270, 25309–25312. 4. Egelund, R., Rodenburg, K.W., Andreasen, P.A., Rasmussen, M.S., Guldberg, R.E. & Petersen, T.E. (1998) An ester bond linking a fragment of a serine proteinase to its serpin inhibitor. Biochemistry 37, 6375–6379. 5. Shore, J.D., Day, D.E., Francis-Chmura, A.M., Verhamme, I., Kvassman,J.,Lawrence,D.A.&Ginsburg,D.(1995)Afluor- escent probe study of plasminogen activator inhibitor-1. Evidence for reactive center loop insertion and its role in the inhibitory mechanism. J. Biol. Chem. 270, 5395–5398. 6. Stratikos, E. & Gettins, P.G. (1999) Formation of the covalent serpin-proteinase complex involves translocation of the proteinase by more than 70 A ˚ and full insertion of the reactive center loop into b-sheet A. Proc. Natl Acad. Sci. USA 96, 4808–4813. 7. Fa, M., Bergstro ¨ m, F., Hagglo ¨ f, P., Wilczynska, M., Johansson, L.B. & Ny, T. (2000) The structure of a serpin-protease complex revealed by intramolecular distance measurements using donor- donor energy migration and mapping of interaction sites. Struc- ture Fold. Des. 8, 397–405. 8. Huntington, J.A., Read, R.J. & Carrell, R.W. (2000) Structure of a serpin-protease complex shows inhibition by deformation. Nature 407, 923–926. Fig. 4. Effect of Mab-1 on the rate of latency transition of PAI-1 wt and K325/335A. PAI-1 wt and PAI-1 K523/335 A were incubated at 37 °C with or without Mab-1 or VN for various times, followed by meas- urements of the remaining specific inhibitory activity by titration against uPA. 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