Distal hinge of plasminogen activator inhibitor-1 involves its latency transition and specificities toward serine proteases
© Wang and Shaltiel; licensee BioMed Central Ltd. 2003
Received: 17 January 2003
Accepted: 08 July 2003
Published: 08 July 2003
The plasminogen activator inhibitor-1 (PAI-1) spontaneously converts from an inhibitory into a latent form. Specificity of PAI-1 is mainly determined by its reactive site (Arg346-Met347), which interacts with serine residue of tissue-type plasminogen activator (tPA) with concomitant formation of SDS-stable complex. Other sites may also play roles in determining the specificity of PAI-1 toward serine proteases.
To understand more about the role of distal hinge for PAI-1 specificities towards serine proteases and for its conformational transition, wild type PAI-1 and its mutants were expressed in baculovirus system. WtPAI-1 was found to be about 12 fold more active than the fibrosarcoma PAI-1. Single site mutants within the Asp355-Arg356-Pro357 segment of PAI-1 yield guanidine activatable inhibitors (a) that can still form SDS stable complexes with tPA and urokinase plasminogen activator (uPA), and (b) that have inhibition rate constants towards plasminogen activators which resemble those of the fibrosarcoma inhibitor. More importantly, latency conversion rate of these mutants was found to be ~3–4 fold faster than that of wtPAI-1. We also tested if Glu351 is important for serine protease specificity. The functional stability of wtPAI-1, Glu351Ala, Glu351Arg was about 18 ± 5, 90 ± 8 and 14 ± 3 minutes, respectively, which correlated well with both their corresponding specific activities (84 ± 15 U/ug, 112 ± 18 U/ug and 68 ± 9 U/ug, respectively) and amount of SDS-stable complex formed with tPA after denatured by Guanidine-HCl and dialyzed against 50 mM sodium acetate at 4°C. The second-order rate constants of inhibition for uPA, plasmin and thrombin by Glu351Ala and Glu351Arg were increased about 2–10 folds compared to wtPAI-1, but there was no change for tPA.
The Asp355-Pro357 segment and Glu351 in distal hinge are involved in maintaining the inhibitory conformation of PAI-1. Glu351 is a specificity determinant of PAI-1 toward uPA, plasmin and thrombin, but not for tPA.
KeywordsPlasminogen-activator inhibitor-1 tissue-type plasminogen activator serine protease specificity latency transition serpin site mutation
Plasminogen activator inhibitor-1 (PAI-1) is a serine proteinase inhibitor in the serpin superfamily [1–3]. This 50 kDa glycoprotein is apparently the most important physiological inhibitor of tissue-type plasminogen activator (tPA) and of urokinase plasminogen activator (uPA). It was shown to play a crucial role in the regulation of vascular thrombosis, tumor invasion, neovascularization, inflammation and wound healing.[5, 6] Increased plasma levels of PAI-1 were found to be correlated with an increased risk for cardiovascular diseases. [6–8] The essential function of PAI-1 in the fibrinolytic system has been confirmed by studies with transgenic animals.[7, 9]
Amino acid sequence alignment at the carboxyl-terminal edges of various serpins
s1C s4B s5B
Comparison of variable region of protease domain in serine proteases
Constant region 1
Constant region 2
FAKHRR SPGER F
R TR FGMH
In this study, By producing wtPAI-1 and several single site mutations in the distal hinge of PAI-1 in a baculovirus expression system, we were able to find out the role of the distal hinge in conformational transition of PAI-1 and to elucidate the role of Glu351 in PAI-1 for serine protease specificities.
Characterization of wtPAI-1
Interestingly, the specific inhibitory activity of wtPAI was 63 ± 15 U/ug just after purification, and was increased to 84 ± 21 U/ug after denatured by 6 M Gdn-HCl and refolded in 50 mM sodium acetate (pH 5.6) at 4°C. In contrast, the specific activity of the fibrosarcoma PAI-1 was only 5 ± 3 U/ug before activation, and was only increased to 45 ± 16 U/μg after denaturing and refolding. The high specific inhibitory activity of wt-PAI-1 could be due to the fact that it was secreted from insect High-5 cells cultured at 27°C, while the fibrosarcoma preparation of PAI-1 was obtained from the medium of fibrosarcoma cells cultured at 37°C . Exposure to the higher temperature could accelerate the conversion of the inhibitor into its latent form.
The functional stability of wtPAI-1 and the fibrosarcoma PAI-1 in the absence or in presence of vitronectin are illustrated in Figure 1C. wtPAI-1 lost its inhibitory activity at the same pace as the fibrosarcoma PAI-1, indicating that its functional half life as an inhibitor and its ability to be stabilized by vitronectin are similar to those of the fibrosarcoma PAI-1.
Single site mutations within the distal hinge of PAI-1
Second-order rate constants of wtPAI-1, D355P, R356P, P357G toward tPA or uPA.
1.7 ± 0.6 × 107
4.4 ± 1.6 × 107
2.0 ± 0.7 × 107
5.1 ± 1.7 × 107
1.3 ± 0.5 × 107
4.7 ± 1.7 × 107
1.6 ± 0.7 × 107
4.9 ± 2.5 × 107
An interesting structural similarity among several serpins is known to occur at the position corresponding to 355 in PAI-1. While in PAI-1 this position accommodates a negatively charged aspartic acid residue, in other serpins it sometimes accommodates its non charged analog asparagine (Table 1). Asp355His, Asp355Lys, Asp355Glu, Asp355Gln and Asp355Asn were prepared and used to show that the negatively charged aspartic acid in PAI-1 is essential (though not sufficient) for its inhibitory activity. Mutation of Asp355 by asparagine (the non charged analog of aspartic acid), or by histidine or lysine (that have a positively charged side chain) or by alanine (neutral), inactivated PAI-1. Replacement of this aspartic acid residue even by the negatively charged glutamic acid (a very close analog of aspartic acid) did not significantly suffice to restore a full inhibitory activity of PAI-1 (Fig. 3B). After activation with 6 M Gnd-HCl, each of the six mutants exhibited a relatively high inhibitory activity, but their specific activities were still much lower than that of wtPAI-1 (Fig. 3B). Among them, Asp355Lys (5.1 ± 4.1 U/ug) had the lowest specific activity, while the Asp355Asn (20 ± 4.2 U/μg) had the highest specific activity (Fig. 3B). Although they had lower specific activities, all of the mutants mentioned above could still form SDS-stable complexes with tPA or uPA just as the wtPAI-1 (data not shown).
Two additional mutations, Arg356Ala and Arg356Glu, were prepared to further elucidate the role of Arg356. The specific inhibitory activity of Arg356Ala was 7.2 ± 2.5 U/μg, while Arg356Glu exhibited essentially no inhibitory activity. This specific activity was increased to 35.4 ± 8.7 U/ug for Arg356Ala and 6.4 ± 2.3 U/μg for Arg356Glu after denature and refolding. Just like ARG356Pro, Arg356Ala and Arg356Glu were much more labile than wtPAI-1. These results suggest that the role of the Asp355-Pro357 segment is to stabilize the inhibitory conformation of PAI-1.
Specific activity, functional stability and complex formation of Glu351Ala and Glu351Arg
Inhibition of thrombin by PAI-1s and Neutralization of PAI-1s by Thrombin
Inhibition of plasmin by PAI-1s
The second-order rate constants of wtPAI-1, E351A and E351R towards tPA, uPA, plasmin and thrombin.
tPA (107 M-1s-1)
uPA (107 M-1s-1)
Thrombin (103 M-1s-1)
Plasmin (104 M-1s-1)
2.11 ± 0.83
0.48 ± 0.27
0.72 ± 0.31
3.10 ± 1.15
1.25 ± 0.49
2.64 ± 0.53
1.74 ± 0.35
6.29 ± 1.83
1.52 ± 0.35
3.62 ± 0.74
3.30 ± 0.42
7.12 ± 1.96
The validity of human recombinant wtPAI-1 in representing the physiological form of the inhibitor is shown here by the following: (a) The inhibitory activity of this wtPAI-1 is actually higher than the inhibitory activity of the PAI-1 isolated from fibrosarcoma cells (12 fold more inhibitory prior to activation by 6 M Gnd HCl, and 2 fold more inhibitory after activation); (b) The inhibition rates of both tPA and uPA obtained with wtPAI-1 are essentially identical to those obtained with PAI-1 secreted by fibrosarcoma cells; (c) wtPAI-1 forms SDS-stable complexes with tPA and uPA that are similar to those obtained with the fibrosarcoma PAI-1; (d) The stabilization of the inhibitory conformation of wtPAI-1 by vitronectin is essentially identical to that of the fibrosarcoma PAI-1; (e) The molecular weight of the protein moiety of wtPAI-1 is similar to that of the fibrosarcoma PAI-1, if the two inhibitor preparations are deglycosylated with N-glycanase. In view of the above, we used mutated recombinant PAI-1s to assess the importance of specific amino acid residues in the inhibitory function of PAI-1.
This study focuses on the functional role of Glu351, Asp355, Arg356 and Pro357 within the distal hinge region of PAI-1. We suspected that the "bending" property of the prolyl residue at position 357 and the rigidity in the polypeptide backbone imposed by prolyl residues in general might have a role in maintaining the inhibitory conformation of this segment. Indeed, we found that a mutant of Pro357 in which the proline residue was replaced by glycine is non inhibitory. This mutant could, however, be activated by 6 M Gdn-HCl, supporting the notion that the lack of activity of Pro357Gly may indeed be due to a conformational effect on the structure of the inhibitor.
The structural importance of the local conformation of the Asp355-Pro357 segment is further illustrated by two single site mutants in which the original Pro357 was preserved, but an additional proline residue was introduced (by mutation) at positions 355 or 356. These two mutants were inactive but they were activated considerably by treatment with 6 M Gdn-HCl. The enhanced lability of these mutants was evident from a more rapid conversion into their latent structure. These mutants were found to form SDS-stable complexes with either tPA or uPA after treatment with 6 M Gdn-HCl. This treatment presumably allows them to restore (at least in part) their inhibitory structure, and their ability to inhibit plasminogen activators with second order rate inhibition constants similar to those obtained with the wtPAI-1. Since these mutants have faster latency transition time, we assume that these mutations do so by loosening the structural restrictions normally imposed on the mobility of the RCL.  However, we show here that in spite of the fact that these mutants are structurally more fragile, their second-order rate constants towards their target proteinases are very similar to those of wtPAI-1. This implies that a facilitated latency transition does not necessarily result in slow rate inhibition towards tPA or uPA. This is in line with the findings that stable PAI-1 mutants (72-fold or 245-fold longer half life than of wtPAI-1) have similar inhibition rate constants towards uPA. [22, 23] We therefore conclude that the segment Asp355-Pro357 is not involved directly in the complex formation, but rather in the latency transition.
The results presented here with the mutants expressed in High 5 cells show that indeed the conversion of the negative into a positive charge (Asp355His and Asp355Lys) yields an inactive inhibitor protein, but the preservation of the negative charge does not suffice by itself. Mutation of this aspartic acid residue into a negatively charged glutamic acid (also negatively charged, and differs from Asp355 by having an extra methylene group only) still yields a mutant PAI-1 that has a very low inhibitory activity. The fact that the negative charge is an important factor in the inhibitory activity is illustrated here by the finding that Asp355Asn and Asp355Gln have both a very low activity, yet they are more active than Asp355Lys and Asp355His. Generally speaking, it is clear from our studies that any mutation of Asp355, whether it preserves the negative charge or not, yields an inactive inhibitor.
The markedly enhanced functional stability of Glu351Ala (Fig. 4B) may be due to the unfavorable interactions resulted from Ala instead of Glu, which delays the latency conversion, while the decreased functional stability of Glu351Arg may be caused by positively charged Arg, which may facilitate the conformation transition. Here we show that different amino acid substitution at the same position result in opposite effect on the PAI-1 stability, suggesting that the network in which Glu351 is involving does provide PAI-1 stability. Moreover, the structural stabilization needs not necessarily affect its mechanism of reactive center loop insertion, but instead may occur through subtle optimization of interactions. Indeed, results of studies with other proteins have generally supported the notion that thermodynamic stabilization of a protein is global. This is also consistent with studies of mutant T4 lysozymes with multiple substitutions at one position demonstrated a direct relationship between the hydrophobicity of the substituted residue and the resulting increase in stability. 
Cognate protease specificity
Improvements in specificity of mutants for uPA indicates that Glu351 involve in the interaction with uPA. To the best of our knowledge, this is the first time that the specificity of PAI-1 toward uPA is partially mediated by the negatively charged residue located in the C-terminal of cleavage site in PAI-1. The interaction of Glu351 with uPA is much more important than that of Glu351 with tPA, which is demonstrated by improved reactivity of Glu351 mutants toward uPA, but not toward tPA, showing that tPA is not directly interact with Glu351 of PAI-1, whereas Glu351 is necessary for the optimal interaction with uPA. Our results are concordant with previous study,  which suggests that serpin body-protease body interactions play significant roles in determining serpin inhibitory activity against target proteases.
Substitution of Glu351 also altered the specificity toward non-cognate proteases: plasmin and thrombin. Glu at 351 may provide steric hindrance that slows the rate of thrombin or plasmin inhibition. Mutations at Glu351 may decrease the original repulsive interactions with variable region of thrombin, which may slow the rate of thrombin inhibition, or results in favorable interactions that may not exist when wtPAI-1 react with thrombin. So, both Glu351Arg and Glu351Arg are better thrombin inhibitors than wtPAI-1. Similar results were observed for plasmin. With single mutation at position Glu351, we improved reactivity of PAI-1 to thrombin and plasmin without significantly affecting its specificity to tPA. This substantiates previous mutagenesis studies, which demonstrated that repulsive interactions and/or lack of productive electrostatic interactions between Glu39 and Glu192 of thrombin and PAI-1 are responsible for the slow reaction of thrombin with this serpin. Furthermore, the rapid inhibition of plasmin requires a non-covalent interaction between an amino-terminal site of plasmin and a carboxyl-terminal on anti-plasmin, and a second site outside the RCL of PAI-1 contributes to its specific interaction with proteases.[15, 27] The specificity of the two mutants toward thrombin and plasmin implicates that Glu351 of PAI-1 is involved in its specificity toward thrombin and plasmin.
Single site mutations within the segment Asp355-Arg356-Pro357 of PAI-1 yield Gdn-HCl activatable inhibitors that can still form SDS stable complexes with plasminogen activators, with second order inhibition constants that are similar to the native inhibitor. Interestingly, the conversion of these mutants to latent forms was ~3–4 fold faster than wtPAI-1, suggesting that the Asp355-Pro357 segment is involved in maintaining the inhibitory conformation of PAI-1. Glu351 contributes to the optimal functional stability of PAI-1 and participates its conformational stability. More importantly, Glu351 of PAI-1 is a specificity determinant for uPA, thrombin and plasmin, but not for tPA.
The following materials were obtained from commercial sources: native PAI-1, secreted into the cell culture medium from human fibrosarcoma cell line HT 1080 (Biopool); single-chain tPA and two-chain tPA standard (Biopool); uPA, plasmin and thrombin were from Calbiochem; the chromogenic substrate for uPA, pGlu-Gly-Arg p-Nitroanilide (S-2444), the chromogenic substrate for tPA and H-D-Ile-Pro-Arg-pNA-2HCl (S-2288) from Chromgenix AB, chromogenic substrate for thrombin, H-D-Phe-pipecolyl-Arg-pNA(S-2238) and chromogenic substrate for plasmin, N-p-Tosyl-Gly-Pro-Lys-p-Nitroanilide from Sigma); heparin-Sepharose, monoclonal antibody against PAI-1 from American Diagnostica, Inc.; recombinant N-glycanase (Genzyme diagnostics); the pRSET(A) vector, baculovirus transfer vector pAcSG2 and BaculoGold transfection kit from Pharmgen; Sf9 cells (Spodoptera frugiperda), High Five cell (Trichoplusia ni) from Invitrogen; Restriction enzymes, T4 ligase and shrimp phosphatase were purchased from New England Biolab. BCA protein assay reagent from Pierce; A full-length cDNA of human PAI-1 (pPAI-1-AI)  was generously provided by Prof. Keld Danö, The Finsen Laboratory.
General DNA techniques
Plasmid DNA was purified using either Promega or QIAGEN miniprep kits. The DNA was sequenced using the di-deoxy chain termination reaction method using an ABI 373 DNA sequencer. Oligonucleotides were synthesized using a 380 B DNA synthesizer (ABI model). DNA manipulation techniques were carried out according to standard procedures .
Construction of wtPAI-1 and its mutants
The pPAI-1-AI was digested with EcoRI and BglII to release a 1426 basepair (bp) PAI-1 coding sequence, which was isolated, purified, and ligated into EcoRI and BglII digested pVL1393. The resultant pVL1393-PAI-1 was analyzed by restriction mapping and sequencing. The mutants at E351A, E351R, D355, R356, and P357 of PAI-1 were generated by PCR. The occurrence of the resulting mutations D355A, D355E, D355H, D355K, D355N, D355P, D355Q, R356P, P357G as well as E351A and E351R in pRSET was confirmed by sequencing. The PAI-1 sequence containing the desired mutations was digested with EcoR1 and BglII, and then subcloned into pVL1393. The recombinant baculoviruses containing either wtPAI-1, or one of the above mutants was obtained as deccribed by Kjoller et al. 
Expression and purification of wtPAI-1 and its mutants
High 5 cells, grown at 27°C in SF-900 serum-free medium, were infected with high-titer virus stocks (1–3 × 108/ml). The medium was collected 48 hrs after the infection (found to be the optimal harvesting time for wtPAI-1) and then centrifuged for 5 minutes (125 × g). The supernatents (50 ml) were collected and sodium chloride (final concentration 0.25 M) was added. They were then applied on a heparin-Sepharose column (bed volume 5 ml) that was equilibrated and run (flow rate 20 ml/h) with a buffer consisted of 50 mM sodium acetate, pH 5.6, containing 0.25 M NaCl and 0.01% Tween-20. The columns were washed and the PAI-1s were eluted with a 40 ml gradient of 0.25 – 1 M NaCl in the same buffer.
SDS-PAGE, immunoblotting and deglycosylation of PAI-1
The medium of the infected cells and the elutent from heparin-Sepharose were separated on 10% SDS-PAGE, and monitored by silver staining. Fibrosarcoma PAI-1 and wtPAI-1 were transferred onto nitrocellulose paper and the proteins were revealed with anti-human PAI-1 monoclonal antibodies, followed by horseradish peroxidase (HRP) conjugated rabbit anti-mouse antibody, and enhanced chemiluminescence (ECL). 5 ug of Fibrosarcoma PAI-1, or wtPAI-1 was denatured by boiling for 5 min in a buffer consisted of 0.5% SDS, 50 mM β-mercaptoethanol and 50 mM Tris-HCl, pH 7.4. They were then digested (18 hr, at 37°C) with 0.5 units of recombinant N-glycanase in the presence of 2.5% NP-40, and subjected to SDS-PAGE and silver staining. Protein concentrations were determined by the Bio-Rad protein assay reagent.
Functional stability of fibrosarcoma PAI-1, wtPAI-1 and its mutants
The inhibitory activities of fibrosarcoma PAI-1, wtPAI-1, and its mutants were measured by a direct chromogenic assay using uPA (100 U/ug) and its substrate S-2444 in a microtiter plate as described earlier . Briefly, fibrosarcoma PAI-1, wtPAI-1, or its mutants were diluted 1:10 in the activity measuring assay buffer (50 uM Tris-HCl, pH 7.5, containing 0.15 M NaCl and 100 ug/ml BSA). The PAI-1 samples were incubated in the absence or in the presence of a two molar excess of vitronectin (the fibrosarcoma PAI-1 and the wtPAI-1 were incubated with vitronectin) at 37°C. At the times indicated, aliquots were removed and analyzed immediately for their inhibitory activity. One unit of PAI-1 activity is defined as the amount that completely neutralizes 1 U of uPA. The specific activity obtained at time zero was assigned a value of 100%.
Activation of PAI-1 and kinetic analysis
Fibrosarcoma PAI-1, wtPAI-1 or its mutants were denatured with 6 M guanidine-hydrochloride (Gdn-HCl) (45 min at 37°C), followed by dialysis (4–16 hours at 4°C) against 50 mM sodium acetate, pH 5.6, containing 0.5 M NaCl and 0.01% Tween-20. This process which PAI-1 regained its activity is called activation by Gdn-HCl. The inhibitory activity of each of the PAI-1s was assayed immediately. The second-order rate constants for the interaction of the fibrosarcoma PAI-1, wtPAI-1 or the indicated mutants with single-chain tPA or uPA were determined in a single-step assay as previously described. 
Specific activity, functional stability and complex formation of PAI-1s
The inhibitory activities of wtPAI-1 and mutants were measured by a direct chromogenic assay using uPA (100 U/μg) and substrate S-2444.  Briefly, serially diluted wtPAI-1, or mutants were incubated with equal volume of uPA (50 μl, 50 U/ml) for 30 min at 37°C, followed by addition of 100 μl, 0.5 mM S-2444. Residual uPA activity was quantitated by measuring the change in absorbance at 405 nm with ELISA reader. The specific activity of PAI-1 was calculated based on the amount of uPA inhibited by PAI-1. The assay for PAI-1 inhibitory activity toward two-chain tPA was the same for uPA except that the samples were incubated with tPA and S-2288. PAI-1 inhibitory activity is expressed as the number of international units of PAs inhibited by PAI-1. Specific activity of uPA and two-chain tPA are 100 U/μg and 764 U/μg, respectively. For the functional stability, wtPAI-1, or mutants were incubated at 37°C. Aliquots were taken out at times indicated and analyzed immediately as described above. The specific activity obtained at time zero was assigned a value of 100%. To provide the evidence to support the conclusion obtained from enzyme assay, certain amount of wtPAI-1, E351R and E351A were incubated with equal amount of tPA at 37°C for 30 minutes before put on ice, at same time these PAI-1s were incubated in PBS at 37°C for 3 hours, respectively, before the PAI-1s were incubated with same amount of tPA at 37°C for 30 minutes, then the protein samples prepared at two time points were subjected to SDS-PAGE and western blot as described before.
Inhibition of thrombin and plasmin by PAI-1s
Thrombin (0.025 U), or plasmin (0.1 μg/ml) was incubated at 37°C for 30 min with same amount of wtPAI-1, E351A and E351R in term of inhibitory activity toward uPA (20 U in the concentration range of 4–10 μg/ml) in absence or presence of vitronectin (60 μg/ml). Linear increase of absorbance at 405 nm were recorded after adding S-2238 (final concentration 0.25 mM) for thrombin, or N-p-Tosyl-Gly-Pro-Lys-p-Nitroanilide (final concentration 0.5 mM) for plasmin. Thrombin, or plasmin activity without PAI-1 was taken as 100%.
Neutralization of PAI-1s by Thrombin
wtPAI-1 and mutants were incubated with four molar excess of thrombin (40 nM) in the absence or presence of vitronectin (30 nM). Control experiments were carried out in the absence of thrombin. After 20 min at 37°C, the activity of thrombin was quenched by the addition of hirudin (final concentration, 60 units /ml), resulting in inhibition of more than 95% of the amidolytic activity of thrombin. Subsequently, the residual PAI-1 inhibitory activity was determined by titration of aliquots on uPA, using S2444 as substrate. The increase of the optical density at 405 nm in the samples was corrected for the values measured with (hirudin-inactivated) thrombin alone, since the amidolytic activity of thrombin on S2444 was not completely blocked by hirudin,
Complex formation of PAI-1s with tPA or uPA
Complex formation of the fibrosarcoma PAI-1, wtPAI-1 or its mutants with single-chain tPA or uPA were performed as described earlier . Samples of PAI-1s (5 U) were incubated in the absence or in the presence of a 2-fold excess of uPA or tPA in the activity assay buffer. Following a 30 min incubation at room temperature, the samples were subjected to a 10% SDS-PAGE and immunoblotting as described above.
tissue-type plasminogen activator
urokinase-type plasminogen activator
plasminogen activator inhibitor type 1
serine protease inhibitor
reactive center loop
vitronectin, SDS, sodium dodecylsulfate
polyacrylamide gel electrophoresis. Gdn-HCl, guanidine-hydrochloride
bovine serum albumin.
This research was supported by the Israel Science Foundation, Award No. 415/99. S.SH was the Incumbent of the Kleeman Chair in Biochemistry. We are grateful to Prof. Keld Dano, The Finsen Laboratory, Rigshospitalet, Copenhagen, Denmark, for the gift of PAI-1 cDNA clone. We thank Professor Elizabeth Goldsmith, Dept. of Biochemistry and Internal Medicine, University of Texas Southwestern Medical Center, Texas, for providing the co-ordinates of latent PAI-1.
- Ny T, Sawdey M, Lawrence D, Millan JL, Loskutoff DJ: Cloning and sequence of a cDNA coding for the human beta-migrating endothelial-cell-type plasminogen activator inhibitor. Proc Natl Acad Sci USA. 1986, 83: 6776-80.PubMed CentralView ArticlePubMedGoogle Scholar
- Pannekoek H, Veerman H, Lambers H, Diergaarde P, Verweij CL, van Zonneveld AJ, van Mourik JA: Endothelial plasminogen activator inhibitor (PAI): a new member of the Serpin gene family. EMBO J. 1986, 5: 2539-44.PubMed CentralPubMedGoogle Scholar
- Bernd R. Binder Günter Christ, Florian Gruber, Nelly Grubic, Peter Hufnagl, Michael Krebs, Judit Mihaly and Gerald W. Prager: Plasminogen Activator Inhibitor 1: Physiological and Pathophysiological Roles. News Physiol Sci. 2002, 17: 56-61.PubMedGoogle Scholar
- Loskutoff DJ, Schleef RR: Plasminogen activators and their inhibitors. Methods Enzymol. 1988, 163: 293-302.View ArticlePubMedGoogle Scholar
- Andreasen PA Egelund R, Petersen HH: The plasminogen activation system in tumor growth, invasion, and metastasis. Cell Mol Life Sci. 2000, 57: 25-40. 10.1007/s000180050497.View ArticlePubMedGoogle Scholar
- Kohler HP Grant PJ: Plasminogen-activator inhibitor type 1 and coronary artery disease. N Engl J Med. 2000, 342: 1792-1801. 10.1056/NEJM200006153422406.View ArticlePubMedGoogle Scholar
- Erickson LA, Fici GJ, Lund JE, Boyle TP, Polites HG, Marotti KR: Development of venous occlusions in mice transgenic for the plasminogen activator inhibitor-1 gene. Nature. 1990, 346: 74-6 issn: 0028-0836. 10.1038/346074a0.View ArticlePubMedGoogle Scholar
- Nordt TK Peter K, Ruef J, Kubler W, Bode C: Plasminogen activator inhibitor type-1 (PAI-1) and its role in cardiovascular disease. Thromb Haemost. 1999, 82 Suppl 1: 14-18.PubMedGoogle Scholar
- Carmeliet P, Kieckens L, Schoonjans L, Ream B, van Nuffelen A., Prendergast G, Cole M, Bronson R, Collen D, Mulligan RC: Plasminogen activator inhibitor-1 gene-deficient mice. I. Generation by homologous recombination and characterization. J Clin Invest. 1993, 92: 2746-2755.PubMed CentralView ArticlePubMedGoogle Scholar
- Huber R, Carrell RW: Implications of the three-dimensional structure of alpha 1-antitrypsin for structure and function of serpins. Biochemistry. 1989, 28: 8951-8966. 10.1021/bi00449a001.View ArticlePubMedGoogle Scholar
- Sharp AM, Stein PE, Pannu NS, Carrell RW, Berkenpas MB, Ginsburg D, Lawrence DA, Read RJ: The active conformation of plasminogen activator inhibitor 1, a target for drugs to control fibrinolysis and cell adhesion. Structure Fold Des. 1999, 7: 111-118. 10.1016/S0969-2126(99)80018-5.View ArticlePubMedGoogle Scholar
- Stout TJ, Graham H, Buckley DI, Matthews DJ: Structures of active and latent PAI-1: a possible stabilizing role for chloride ions. Biochemistry. 2000, 39: 8460-8469. 10.1021/bi000290w.View ArticlePubMedGoogle Scholar
- Mottonen J, Strand A, Symersky J, Sweet RM, Danley DE, Geoghegan KF, Gerard RD, Goldsmith EJ: Structural basis of latency in plasminogen activator inhibitor-1. Nature. 1992, 355: 270-273. 10.1038/355270a0.View ArticlePubMedGoogle Scholar
- Sherman PM, Lawrence DA, Yang AY, Vandenberg ET, Paielli D, Olson ST, Shore JD, Ginsburg D: Saturation mutagenesis of the plasminogen activator inhibitor-1 reactive center. J Biol Chem. 1992, 267: 7588-7595.PubMedGoogle Scholar
- Sherman PM, Lawrence DA, Verhamme IM, Paielli D, Shore JD, Ginsburg D: Identification of tissue-type plasminogen activator-specific plasminogen activator inhibitor-1 mutants. Evidence that second sites of interaction contribute to target specificity. J Biol Chem. 1995, 270: 9301-9306. 10.1074/jbc.270.16.9301.View ArticlePubMedGoogle Scholar
- Keijer J, Linders M, van Zonneveld AJ, Ehrlich HJ, de Boer JP, Pannekoek H: The interaction of plasminogen activator inhibitor 1 with plasminogen activators (tissue-type and urokinase-type) and fibrin: localization of interaction sites and physiologic relevance. Blood. 1991, 78: 401-409.PubMedGoogle Scholar
- Madison EL, Goldsmith EJ, Gerard RD, Gething MJ, Sambrook JF, Bassel-Duby RS: Amino acid residues that affect interaction of tissue-type plasminogen activator with plasminogen activator inhibitor 1. Proc Natl Acad Sci U S A. 1990, 87: 3530-3533.PubMed CentralView ArticlePubMedGoogle Scholar
- Madison EL, Goldsmith EJ, Gething MJ, Sambrook JF, Gerard RD: Restoration of serine protease-inhibitor interaction by protein engineering. J-Biol-Chem. 1990, 265: 21423-6.PubMedGoogle Scholar
- Madison EL, Goldsmith EJ, Gerard RD, Gething MJ, Sambrook JF: Serpin-resistant mutants of human tissue-type plasminogen activator. Nature. 1989, 339: 721-724. 10.1038/339721a0.View ArticlePubMedGoogle Scholar
- Lawrence DL, Strandberg L, Grundstrom T, Ny T: Purification of active human plasminogen activator inhibitor-1 from Escherichia coli. Comparison with natural and recombinant forms purified from eucaryotic cells. Eur J Biochem. 1989, 186: 523-533. 10.1111/j.1432-1033.1989.tb15238.x.View ArticlePubMedGoogle Scholar
- Shore JD, Day DE, Francis-Chmura AM, Verhamme I, Kvassman J, Lawrence DA, Ginsburg D: A fluorescent probe study of plasminogen activator inhibitor-1. Evidence for reactive center loop insertion and its role in the inhibitory mechanism. J Biol Chem. 1995, 270: 5395-5398. 10.1074/jbc.270.10.5395.View ArticlePubMedGoogle Scholar
- Berkenpas MB Lawrence DA, Ginsburg D: Molecular evolution of plasminogen activator inhibitor-1 functional stability. EMBO J. 1995, 14: 2969-2977.PubMed CentralPubMedGoogle Scholar
- A. Allart Stoopa Eric Elderinga, Timothy R. Daffornb, Randy J. Readb and Hans Pannekoek: Different Structural Requirements for Plasminogen Activator Inhibitor 1 (PAI-1) during Latency Transition and Proteinase Inhibition as Evidenced by Phage-displayed Hypermutated PAI-1 Libraries. J Mol Biol. 2001, 305: 773-783. 10.1006/jmbi.2000.4356.View ArticleGoogle Scholar
- Matsumura M, Becktel WJ, Matthews BW: Hydrophobic stabilization in T4 lysozyme determined directly by multiple substitutions of Ile 3. Nature. 1988, 334: 406-410. 10.1038/334406a0.View ArticlePubMedGoogle Scholar
- Plotnick MI, Schechter NM, Wang ZM, Liu X, Rubin H: Role of the P6-P3' region of the serpin reactive loop in the formation and breakdown of the inhibitory complex. Biochemistry. 1997, 36: 14601-14608. 10.1021/bi971530j.View ArticlePubMedGoogle Scholar
- Rezaie AR: Elucidation of the structural basis for the slow reactivity of thrombin with plasminogen activator inhibitor-1. Biochemistry. 1998, 37: 13138-13142. 10.1021/bi9808518.View ArticlePubMedGoogle Scholar
- Lawrence DA, Strandberg L, Ericson J, Ny T: Structure-function studies of the SERPIN plasminogen activator inhibitor type 1. Analysis of chimeric strained loop mutants. J Biol Chem. 1990, 265: 20293-20301.PubMedGoogle Scholar
- Ginsburg D, Zeheb R, Yang AY, Rafferty UM, Andreasen PA, Nielsen L, Dano K, Lebo RV, Gelehrter TD: cDNA cloning of human plasminogen activator-inhibitor from endothelial cells. J Clin Invest. 1986, 78: 1673-80.PubMed CentralView ArticlePubMedGoogle Scholar
- Ausubel FM,R, Brent RE, Kingston DD, Moore JG, Seidman JA, Smith and, Struhl. K: Current protocols in molecular biology. 1996, John Wiley & Sons, IncGoogle Scholar
- Kjoller L, Martensen PM, Sottrup_Jensen L, Justesen J, Rodenburg KW, Andreasen PA: Conformational changes of the reactive-centre loop and beta-strand 5A accompany temperature-dependent inhibitor-substrate transition of plasminogen-activator inhibitor 1. Eur J Biochem. 1996, 241: 38-46. 10.1111/j.1432-1033.1996.0038t.x.View ArticlePubMedGoogle Scholar
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