©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Identification of Tissue-type Plasminogen Activator-specific Plasminogen Activator Inhibitor-1 Mutants
EVIDENCE THAT SECOND SITES OF INTERACTION CONTRIBUTE TO TARGET SPECIFICITY (*)

Patti M. Sherman (1)(§), Daniel A. Lawrence (2), Ingrid M. Verhamme (4), Dell Paielli (4), Joseph D. Shore (4), David Ginsburg (1) (2) (3)(¶)

From the (1) Departments of Human Genetics and (2) Internal Medicine and the (3) Howard Hughes Medical Institute, University of Michigan, Ann Arbor, Michigan, 48109-0650 and the (4) Division of Biochemical Research, Henry Ford Hospital, Detroit, Michigan, 48202-2689

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Plasminogen activator inhibitor-1 (PAI-1) is the primary inhibitor of the plasminogen activators (PAs), tissue-type plasminogen activator (tPA), and urokinase-type plasminogen activator (uPA). A library of PAI-1 mutants containing substitutions at the Pand P` positions was screened for functional activity against tPA and thrombin. Several PAI-1 variants that were inactive against uPA in a previous study (Sherman, P. M., Lawrence, D. A., Yang, A. Y., Vandenberg, E. T., Paielli, D., Olson, S. T., Shore, J. D., and Ginsburg, D. (1992) J. Biol. Chem. 267, 7588-7595) had significant inhibitory activity toward tPA. This set of tPA-specific PAI-1 mutants contained a wide range of amino acid substitutions at Pincluding Asn, Gln, His, Ser, Thr, Leu, Met, and all the aromatic amino acids. This group of mutants also demonstrated a spectrum of substitutions at P`. Kinetic analyses of selected variants identified PTyr and PHis as the most efficient tPA-specific inhibitors, with second-order rate constants ( k) of 4.0 10 Msand 3.6 10 Ms, respectively. Additional PA-specific PAI-1 variants containing substitutions at Pthrough P` were constructed. PTyr-PSer-PLys-P`Trp and PTyr-PSer-PTyr-P`Met had kvalues of 1.7 10 Msand 2.5 10 Msagainst tPA, respectively, but both were inactive against uPA. In contrast, PArg-PLys-P`Ala inhibited uPA 74-fold more rapidly than tPA. The mutant PAI-1 library was also screened for inhibitory activity toward thrombin in the presence and absence of the cofactor heparin. While wild-type PAI-1 and several PArg variants inhibited thrombin in the absence of heparin, a number of variants were thrombin inhibitors only in the presence of heparin. These results demonstrate the importance of the reactive center residues in determining PAI-1 target specificity and suggest that second sites of interaction between inhibitors and proteases can also contribute to target specificity. Finally, the PA-specific mutants described here should provide novel reagents for dissecting the physiological role of PAI-1 both in vitro and in vivo.


INTRODUCTION

Plasminogen activation functions in a wide variety of physiological processes including fibrinolysis, ovulation, inflammation, tumor metastasis, embryonic development, and angiogenesis. The conversion of the inactive zymogen plasminogen into the broad specificity protease plasmin is catalyzed by the serine proteases tissue-type plasminogen activator (tPA)() and urokinase-type plasminogen activator (uPA). The activities of plasminogen activators (PAs) can be directly regulated by specific inhibitors. Of these, plasminogen activator inhibitor-1 (PAI-1) is the most efficient inhibitor of both tPA and uPA ( kvalues 10-10 Ms) and is considered a major regulator of fibrinolysis (for review, see Refs. 1 and 2). Deficiency of PAI-1 results in a hyperfibrinolytic state in animal models (3, 4) , and a mild to moderate bleeding disorder in humans (5) .

PAI-1 is a member of the serine protease inhibitor (serpin) superfamily. Serpins inactivate their target proteases by forming inactive, equimolar complexes that are stable in SDS. Models based on the three-dimensional structures of cleaved -antitrypsin (6, 7) , ovalbumin (8) , the latent conformation of PAI-1 (9) , and the recently reported intact -antichymotrypsin and antithrombin III structures (10, 11, 12) localize the P-P` reactive center of an active serpin to a mobile loop of amino acids protruding from the inhibitor. This exposed loop is thought to act as a ``bait'' for the target protease (13) . Although the Presidue is an important determinant of target specificity for serpins (14, 15, 16, 17, 18, 19, 20) , other amino acids near the reactive center (20, 21, 22, 23, 24) and regions outside the strained loop (25) also appear to play roles. In addition, cofactors such as heparin and vitronectin may contribute to a serpin's target specificity (26, 27) .

We previously reported functional analysis of a library of PAI-1 reactive center variants constructed by saturation mutagenesis (28) . Screening of 177 unique variants from this library indicated that a basic residue was required at Pfor significant inhibitory activity with uPA as the target protease. Several P-P` variants were identified with altered relative specificities for uPA and tPA. York and co-workers (24) studied the P, P, and Ppositions of PAI-1 by a similar approach and identified a number of amino acid substitutions that also resulted in mutants with altered relative specificities. Additional studies of PAI-1 variants containing substitutions of the entire strained loop (residues P-P`) (25) and of mutants with residues P-P` replaced by P-P` of antithrombin III (27) or -antiplasmin (29) , illustrated that amino acids around the reactive center can modestly influence PAI-1's relative specificity for uPA and tPA.

In the current study, we report a detailed analysis of the structural requirements at Pand P` for PAI-1 inhibition of tPA and thrombin. Confirming our previous findings (28) , all P-P` variants with a basic residue at Pmaintain tPA inhibitory activity with the exception of those containing P`Pro. However, in contrast to the absolute requirement for a basic residue at Pfor the inhibition of uPA, a number of PAI-1 mutants with neutral or hydrophobic substitutions at Pexhibit significant tPA inhibitory activity. Combining these data with the previous observations of our laboratory (28) and others (24) , novel PAI-1 variants with substitutions at P, P, P, and P` were designed that demonstrate either an absolute specificity for tPA or a marked relative specificity for uPA. Finally, the cofactor heparin was shown to modify the thrombin specificity of PAI-1 reactive center variants.


EXPERIMENTAL PROCEDURES

Construction of PAI-1 Mutants

The construction of the PAI-1 reactive center library in M13PAI-1 has been described previously (28) . Saturation mutagenesis was performed using a degenerate oligonucleotide that contained an equal mixture of all possible DNA sequences at the codons for Argand Metof mature PAI-1 (corresponding to the Pand P` residues, in the standard nomenclature of Schechter and Berger (30) ). 177 unique mutant sequences were analyzed, with all possible amino acid substitutions at Pand P` represented at least once.

Additional specific PAI-1 mutants were constructed using the Altered Sites in vitro mutagenesis system (Promega) as described (31) . Site-directed mutagenesis was performed on single-stranded pSELPAI-1 template using degenerate and specific oligonucleotides spanning the PAI-1 reactive center (, oligonucleotides A-H). For consistent numbering in this paper, the adenine of the PAI-1 translation start codon will be designated as nucleotide +1 (32) . Dideoxynucleotide sequence analysis (33) of PAI-1 nucleotides +1035 to +1140 was performed to identify the desired reactive center sequences, and to exclude other substitutions in this region. PAI-1 variants with the following single substitutions at Pwere prepared in the pSELPAI-1 vector: Asn, Asp, Cys, His, Ile, Met, Phe, Pro, Thr, Tyr, and Val. Data for all other variants shown (see Figs. 1-3) were derived from material expressed in M13PAI-1 (28) . In addition, the following compound mutants were constructed in pSELPAI-1: PArg-PLys-P`Ala, PTyr-PSer-PLys-P`Trp, and PTyr-PSer-PTyr-P`Met. These latter mutants are designated U1, T1, and T2, respectively.

Expression of PAI-1 Proteins

The expression of variant PAI-1 proteins from M13PAI-1 and the preparation of bacterial lysates containing the recombinant proteins were performed as described previously (28) . The expression of variant proteins from pSELPAI-1 was induced by the addition of 0.5 m M isopropyl-1-thio-- D-galactopyranoside to 100-ml cultures of log-phase ( A= 0.6) BL21(DE3) (34) transformed with the mutant pSELPAI-1 constructs. Cultures were grown an additional 2 h, harvested by centrifugation, and resuspended in 1.5 ml of lysis buffer (0.05 M Tris-HCl, pH 8.0, 1 m M EDTA, 0.1 M NaCl, 0.2 m M phenylmethanesulfonyl fluoride (Sigma)) containing 10 µg/ml RNase, 1 µg/ml soybean trypsin inhibitor (Sigma), 0.7 µg/ml pepstatin (Sigma), 0.5 µg/ml leupeptin (Boehringer Mannheim), and 0.2 µg/ml aprotinin (Sigma). 300 µg/ml lysozyme was added, and the cells were incubated on ice for 30 min. The suspension was subjected to three freeze/thaw cycles and incubated for 1 h at 25 °C after the addition of 10 µg/ml DNase I. Cell debris was removed by centrifugation at 16,000 g for 10 min, and the resulting crude lysates were used for all analyses. PAI-1 concentrations in the lysates were determined using a sandwich enzyme-linked immunosorbent assay (28) .

Assays for PAI-1 Activity

The mutant PAI-1 proteins were screened for their inhibitory activity against uPA and tPA using chromogenic assays as described previously (35, 28) . Briefly, crude lysates serially diluted in microtiter plates (Falcon Microtest III) were incubated with high molecular weight uPA (25 IU/ml (4.8 n M) final concentration; American Diagnostica) or single-chain tPA (150 IU/ml (4.6 n M) final concentration and >95% single-chain tPA; Genentech) for 30 min at 25 °C. The chromogenic substrate S-2444 (0.5 m M; KabiVitrum) for uPA, or Spectrozyme tPA (0.5 m M; American Diagnostica) for tPA, was added, and the change in absorbance at 410 nm was recorded. PAI-1 activity was quantitated from the amount of residual PA activity.

Kinetic Analyses

Second-order rate constants ( k) for the inhibition of high molecular weight uPA (Abbott Laboratories) by PAI-1 were determined as described previously (28) . kvalues for the inhibition of two-chain tPA were determined following partial purification of PAI-1. Escherichia coli lysates containing recombinant PAI-1 were desalted on a PD10 Sephadex G-25 (Pharmacia Biotech Inc.) column equilibrated in 0.1 M KHPO, 0.1 M NaCl, pH 6.0, and applied to a heparin-Sepharose (Pharmacia) column in this buffer. After washing with the same buffer, PAI-1 was eluted with 0.1 M KHPO, 1.25 M NaCl, pH 6.0. (NH)SOwas then added to 1 M, and the sample was applied to a phenyl Sepharose (Pharmacia) column equilibrated with 50 m M KHPO, 1 M (NH)SO, pH 6.0, washed with the same buffer, and step eluted with 50 m M KHPO, pH 6.0. The concentration of active PAI-1 was determined by titration with two-chain tPA of known concentration. kvalues were then determined under pseudo first-order conditions by continuous monitoring of two-chain tPA inactivation in the presence of competing chromogenic substrate (Spectrozyme tPA, American Diagnostica) in 0.1 M HEPES, 0.1 M NaCl, 1 m M EDTA, 0.1 % polyethylene glycol 6000, pH 7.4 (36) .

Assays for Inhibition of Thrombin

The PAI-1 variants were tested for thrombin inhibitory activity in the presence and absence of heparin using a chromogenic assay as described previously (25) , with modifications. Briefly, crude lysates were diluted to approximately 12.5 µg/ml (292 n M) PAI-1 (determined by enzyme-linked immunosorbent assay as described previously (28) ) and incubated with 7.5 ng/ml (0.2 n M) human thrombin (Sigma, T-8885) for 2 h at 25 °C. The chromogenic substrate S-2238 (KabiVitrum) was added to a final concentration of 0.25 m M, and the change in absorbance at 410 nm was recorded. PAI-1 activity was determined from the amount of residual thrombin activity. For assays performed in the presence of heparin, 5 units/ml of unfractionated heparin from porcine intestines (Elkins-Sinn) was added to the reaction buffer. This quantity of heparin was the amount that yielded maximal stimulation of wtPAI-1 activity against thrombin in a preliminary assay. For this optimization assay, 5 µg/ml (117 n M) or 10 µg/ml (234 n M) purified wtPAI-1 (28) was incubated with 7.5 ng/ml (0.2 n M) thrombin for 2 h at 25 °C in reaction buffer containing increasing amounts of heparin ranging from 0 to 20 units/ml. The change in absorbance was recorded after the addition of the substrate S-2238 to a final concentration of 0.25 m M, and PAI-1 activity was determined as above.


RESULTS

Expression of PAI-1 Variants

The variant PAI-1 proteins examined in this study were produced from two vectors. We previously reported the expression of 177 unique PAI-1 variants prepared by saturation mutagenesis at the Pand P` positions in an M13-based expression system (M13PAI-1) (28) . The remaining PAI-1 proteins were produced from a phagemid system (pSELPAI-1) derived from pSELECT (Promega) and pET3a (31) . Sequence analyses performed on a total of >2.5 kilobases of the PAI-1 coding sequences from these pSELPAI-1 variants and on the entire PAI-1 coding regions of eight independent clones derived from pSELPAI-1 (>10 kilobases) (31) detected no errors outside the reactive center region (<0.008% mutation frequency). In previous sequence analyses of the M13PAI-1 variants, 1 mutation/22.6 kilobases of PAI-1 coding region (0.004% mutation frequency) was observed outside the sequence spanned by the mutagenesis oligonucleotide. In addition, functional analyses of 48 independent duplicate clones yielded identical results (28) . Thus, although mutations outside of the sequenced areas of the pSELPAI-1 variants in this study cannot be excluded, the observed mutation rates suggest that they are unlikely.

While the M13PAI-1 proteins should contain seven vector-derived amino acids at the NHterminus, the variants expressed from pSELPAI-1 begin at the native NHterminus of mature PAI-1 (37) . Despite the additional NH-terminal sequence in M13PAI-1, the recombinant PAI-1 proteins produced in both systems have previously been demonstrated to be functionally equivalent (28) . Enzyme-linked immunosorbent assay analysis indicated that approximately 6 µg of PAI-1 antigen was produced from 1 ml of pSELPAI-1-transformed E. coli culture, and immunoblot analysis confirmed the presence of a single, 43-kDa protein product (data not shown). Since PAI-1 can exist in a noninhibitory latent conformation (38, 39) , titration of selected pSELPAI-1 proteins with uPA was performed, and indicated that approximately 50% of the PAI-1 protein was in the active conformation. This is similar to the specific activities obtained for the PAI-1 variants expressed in M13PAI-1 in our previous studies (28) .

Inhibitory Activity of Variants against tPA

The 177 novel PAI-1 proteins from the mutant P-P` library (28) were screened for their abilities to inhibit single-chain tPA using a direct chromogenic assay. The assay conditions employed should have detected inhibitors with bimolecular rate constants 10 Ms(40) . Consistent with previous results (28) , all possible PArg and PLys mutants except PArg-P`Pro and PLys-P`Pro inhibited tPA (Fig. 1). In addition, a number of variants with nonbasic amino acid substitutions at Pshowed inhibitory activity (Fig. 1). This latter group of active mutants contained a spectrum of amino acid substitutions at P`.


Figure 1: Inhibition of tPA by PAI-1 reactive center mutants. wtPAI-1 and P-P` mutants were screened for their inhibitory activity against single-chain tPA using a direct chromogenic assay. + and - indicate active and inactive, respectively. wt denotes wtPAI-1. Shaded boxes indicate tPA-specific variants that demonstrated no detectable inhibitory activity against uPA (see Ref. 28, ``Results''). * denotes variants containing an additional substitution(s) outside P-P` (as described in Ref. 28). Most of these additional mutations did not appear to affect PAI-1 function (28).



To allow for a direct comparison of the effects of specific Psubstitutions, all possible Pvariants containing the wild-type P`Met that were absent from the above P-P` library were prepared. Of the 19 Pmutants, those containing Asn, Gln, His, Leu, Lys, Met, Phe, Ser, Thr, Trp, or Tyr at Pshowed inhibitory activity against tPA (Fig. 1). All Psubstitutions associated with an active mutant in the original P-P` library were also active in combination with P`Met. Consistent with our previous data (28) , none of the Pvariants, except for PLys, maintained detectable inhibitory activity against uPA in a direct chromogenic assay (data not shown), indicating that they have rate constants of <10 Msagainst uPA.

Kinetic Analysis of tPA-specific Mutants

The second-order rate constants ( k) with two-chain tPA and uPA were determined for selected PAI-1 mutants identified as tPA-specific in the screening assay (). Substitution of Tyr, His, Asn, Thr, or Met at Presulted in an approximately 25-240-fold reduction of kagainst tPA as compared with wild-type (28) (). None of these mutants had detectable inhibitory activity against uPA, confirming their absolute specificity for tPA ().

Design and Analysis of PA-specific P -P ` Mutants

We previously identified PLys-P`Trp and PLys-P`Ala as relatively tPA- and uPA-specific mutants, respectively (28) . Similarly, York and co-workers (24) reported that PTyr-PSer was relatively specific for tPA and that PArg was relatively specific for uPA. By combining these observations with the current results, we constructed three additional PAI-1 variants designed to maximize specificity for tPA or uPA. The resulting variants PTyr-PSer-PLys-P`Trp (designated T1) and PTyr-PSer-PTyr-P`Met (designated T2) were predicted to be tPA-specific, and the variant PArg-PLys-P`Ala (designated U1) was predicted to be uPA-specific. The kvalues for the interactions between these mutants and tPA or uPA are shown in I, along with the corresponding kvalues for the ``parent'' mutants. Both compound mutants T1 and T2, demonstrated no detectable activity against uPA but maintained significant inhibitory activity against tPA. In contrast, while the U1 mutant had a slightly greater relative specificity for uPA than either of its contributing sequences or wtPAI-1, this variant was not absolutely uPA-specific.

Inhibition of Thrombin in the Absence and Presence of Heparin

The complete panel of PAI-1 variants containing substitutions at the Pand P` positions was screened for inhibitory activity against thrombin using a chromogenic assay. The assay conditions employed should have detected all inhibitors with second-order rate constants >3 10 Ms(40) . In the absence of heparin, wtPAI-1 (PArg-P`Met) and six variants containing Arg at the Pposition were active in this assay (Fig. 2). All other variants showed no detectable activity.


Figure 2: Inhibition of thrombin by PAI-1 mutants in the absence of heparin. Wild-type PAI-1 and P-P` mutants were screened for inhibitory activity against thrombin in the absence of heparin using a chromogenic assay. + and - indicate active and inactive, respectively. wt denotes wtPAI-1. Mutants marked * contain additional mutations as indicated in Fig. 1.



The inhibition of thrombin by wtPAI-1 and the six active variants from the above screen was enhanced in the presence of heparin (data not shown). In addition, a number of variants that were inactive against thrombin in the absence of heparin gained detectable thrombin inhibitory activity in the presence of heparin (Fig. 3). All PArg variants except those containing a basic residue, an acidic residue, or Pro at P` were active. The other functional variants contained a variety of substitutions at the Pand P` positions (Fig. 3). Maximal stimulation was observed between 5 and 10 units/ml heparin and was decreased in reactions containing 20 units/ml heparin (data not shown). These data suggest that heparin is acting as a template for PAI-1 and thrombin and are consistent with previous reports of the inhibition of thrombin by wtPAI-1 (41) .


Figure 3: Inhibition of thrombin by PAI-1 mutants in the presence of heparin. Wild-type PAI-1 and P-P` mutants were screened for their abilities to inhibit thrombin in the presence of heparin using a chromogenic assay. + and - indicate active and inactive, respectively. Shaded boxes indicate variants that are heparin-dependent thrombin inhibitors. wt denotes wtPAI-1. Mutants marked by * contain additional mutations, as described in Fig. 1.




DISCUSSION

Multiple structural components contribute to the determination of serpin target protease specificity including amino acids located on the exposed loop (21) , secondary sites outside the loop (25) , and sites of interaction with several specific ligands (7) . The central role of the Presidue in determining target protease specificity has been demonstrated for diverse members of the serpin family (14, 15, 16, 17, 18, 19, 20) , as well as for substrates (42) and non-serpin inhibitors (43) . We have previously demonstrated the absolute requirement for a basic residue at the Pposition of PAI-1 for maintenance of uPA inhibitory function (28) . In contrast, the P` position is tolerant of all amino acid substitutions except proline (28) , and the Pand Ppositions show considerable flexibility for the inhibition of both uPA and tPA (24) .

This report demonstrates that, unlike uPA, tPA is remarkably tolerant of amino acid changes at the Pposition of PAI-1. In addition to PAI-1 variants with basic residues at P, tPA was inhibited by mutants containing a wide range of Pamino acids including those with aromatic (Phe, Trp, Tyr), amide (Asn, Gln), small polar (Ser, Thr), and hydrophobic (Leu, Met) side-chains (Fig. 1). Serine proteases generally cleave substrates at a highly restricted set of peptide bonds, strongly influenced by the NH-terminal (S1) residue (42) . Similarly, the Presidue of a serine protease inhibitor usually corresponds to the substrate specificity of its target protease (13, 43) . This observation has been used to successfully predict the specificities of natural and engineered serpin variants (13) . Since both uPA and tPA are trypsin-like serine proteases, the unique ability of tPA to rapidly inhibit PAI-1 variants with a number of nonbasic Presidues was unexpected. The tPA-specific PAI-1 variants identified in this study are the first reported serpins to show a preference for tPA over uPA and would not have been predicted using currently understood structural principles. These results illustrate the power of a saturation mutagenesis approach.

Secondary interactions outside the active site and reactive center are necessary for the efficient reactions between some target proteases and serpins. The rapid inhibition of plasmin by -antiplasmin requires a noncovalent interaction between an amino-terminal site of plasmin and a carboxyl-terminal site on -antiplasmin (44) . Similarly, exosites on heparin cofactor II (45) and protease nexin-1 (46) interact with the anion binding site of thrombin, contributing to the specificity of these reactions. In addition, deletion of specific amino acids from the catalytic domains of tPA and uPA produces recombinant PAs that retain catalytic activity but are resistant to PAI-1 inhibition (47, 48, 49) . Others have suggested that a ``second site'' outside the reactive center of PAI-1 contributes to its specific interaction with tPA but not uPA (25, 50, 51) . This additional, noncovalent interaction may stabilize the initial, reversible complex between PAI-1 and a PA when the Presidue is not optimal. Absence of this interaction between uPA and PAI-1 rather than an inherent difference in the S1 subsites of uPA and tPA may explain the tPA-specificity of this subset of PAI-1 variants. Of note, PAI-1 is the only known serpin that is capable of rapidly inhibiting single-chain tPA (1) . This suggests that a noncovalent interaction may be critical for single-chain tPA inhibition.

The effects of the reactive center sequence and heparin on PAI-1 inhibitory activity against the nontarget protease, thrombin, were also examined. wtPAI-1 (PArg-P`Met) has been reported to inhibit thrombin with a kof 1.1 10 Ms(29) . Consistent with this observation, wtPAI-1 demonstrated significant inhibitory activity against thrombin in the screening assay (Fig. 2). Thrombin has been reported to have a narrow specificity for substrates and inhibitors, partly due to restriction of the active site cleft by insertion of a loop segment (52) . Previous studies have shown that substitution of antithrombin IIIs PArg by Lys (53) , His (54) , or Cys (54) results in a loss of thrombin inhibitory activity. Furthermore, the sole substitution of PMet by Arg in -antitrypsin (the Pittsburgh variant) changes -antitrypsin from an ineffective thrombin inhibitor to one that efficiently inhibits thrombin (14) . Our data in the absence of heparin are consistent with these reports. The PAI-1 variants that inhibited thrombin under these conditions all contained an Arg at the Pposition (Fig. 2), including PArg-P`Ser, which has the same reactive center as antithrombin III, the main physiological inhibitor of thrombin.

This study demonstrates that the specificity of PAI-1 reactive center mutants can also be modified by the cofactor heparin. Previous studies have shown that heparin increases the second-order rate constant for the interaction between wtPAI-1 and thrombin by approximately 100-fold, to 1.0 10 Ms(29, 55) . These values can be compared with 1.4 10and 1.5-4 10 Msfor antithrombin III in the presence and absence of heparin, respectively (56) . Detailed characterization of the interaction between PAI-1 and a variety of specific glycosaminoglycans has recently been reported (41, 57) . These studies demonstrate that optimal inhibition of thrombin by PAI-1 is obtained with high molecular weight heparin and that this inhibition most likely occurs via a template mechanism (41) . Consistent with these observations, several variants were active against thrombin only in the presence of heparin (Fig. 3). These included variants with non-Arg amino acids at P, an unexpected result given the narrow specificity of thrombin. In addition, all PArg mutants except those containing a charged residue or Pro at P` inhibited thrombin (Fig. 3). This contrasts with antithrombin III, which has size and hydrophobicity constraints at the P` position for thrombin inhibitory activity (58, 59) . It has been demonstrated that a Lys residue partially blocks the S1` subsite of thrombin (52) . It is possible that the PArg PAI-1 variants with a positive charge at P` are inactive due to ionic repulsion at that site.

Heparin has been shown to enhance PAI-1 inhibition of thrombin via a template mechanism, in which PAI-1 and thrombin are co-localized on the heparin surface, facilitating their interaction (41) . Other studies have also demonstrated that the cofactor vitronectin can enhance PAI-1 inhibition of thrombin, at least in part, by a template mechanism (60) . Our observation that high concentrations of heparin result in submaximal stimulation of PAI-1 inhibitory activity is characteristic of such a template mechanism. It has been proposed that cofactors such as heparin and vitronectin have their greatest effects on inhibition when the interactions between the protease and inhibitor are suboptimal (49, 61) . The detection of thrombin inhibition by a unique set of PAI-1 variants only in the presence of heparin is consistent with this hypothesis. The inability of heparin to significantly stimulate inhibition of thrombin by the inactive mutants is probably not due to defective heparin binding, since the heparin binding sites of both PAI-1 and antithrombin III have been localized to a region distant from the reactive center (62) . In addition, mutations at the reactive center of antithrombin III have been shown not to effect heparin binding (55, 58, 59) .

In conclusion, we have identified PAI-1 variants with marked specificities for tPA or uPA. Recombinant PAI-1 containing the reactive center sequence PTyr-PSer-PTyr-P-Met, has a second-order rate of inhibition for tPA that is nearly as rapid as that of wtPAI-1 but is completely inert toward uPA. These PA-specific variants should provide valuable tools for probing the relative importance of uPA and tPA inhibition by PAI-1 in vivo. In addition, several novel PAI-1 variants with heparin-dependent, thrombin inhibitory activity have been identified. Taken together, our data suggest that secondary sites of interaction between inhibitors and proteases can significantly enhance inhibitory function.

  
Table: Oligonucleotides for construction of PAI-1 variants

Sequences are shown 5` to 3`. ``N'' indicates incorporation of all four deoxynucleotides. Bases enclosed by parentheses are equally incorporated at that position. Underlined bases encode mutated residues. Location of the 5` nucleotide within PAI-1 cDNA is shown, with the adenine of the PAI-1 translational start codon designated as +1. The variant(s) constructed using each oligonucleotide are indicated.


  
Table: Second-order rate constants for the inhibition of tPA and uPA by PAI-1 P1 mutants

The second-order rate constants ( k) for the interactions between PAI-1 mutants and two-chain tPA or uPA are shown. The ratio of the kvalue with tPA to the kvalue with uPA (tPA/uPA) is given at the right.


  
Table: Second-order rate constants for the inhibition of tPA and uPA by PA-specific mutants

The second-order rate constants ( k) for the interactions between PAI-1 variants and two-chain tPA or uPA are shown. The ratio of the kvalue with tPA to the kvalue with uPA (tPA/uPA) is given at the right. kvalues for mutants denoted by * and ** were taken from Refs. 24 and 28, respectively. Amino acids in boldface indicate mutated residues.



FOOTNOTES

*
This work was supported in part by the National Institutes of Health Grants HL 08572 (to D. A. L.), HL 49184 (to D. G.), and HL 45930A (to J. D. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: The Johns Hopkins University School of Medicine, 804 PCTB, 725 N. Wolfe St., Baltimore, MD 21205-2185.

Howard Hughes Medical Inst. Investigator. To whom correspondence and reprint requests should be addressed: Howard Hughes Medical Inst., Rm. 4520 MSRB I, University of Michigan, 1150 W. Medical Center Dr., Ann Arbor, Michigan, 48109-0650. Tel.: 313-747-4808; Fax: 313-936-2888.

The abbreviations used are: tPA, tissue-type plasminogen activator; PAI-1, plasminogen activator inhibitor-1; PA, plasminogen activator; uPA, urokinase-type plasminogen activator; serpin, serine protease inhibitor supergene family; wtPAI-1, wild-type plasminogen activator inhibitor-1.


ACKNOWLEDGEMENTS

We thank J. Kvassman and D. Day for helpful discussions, Jack Henkin of Abbott Laboratories for recombinant uPA, and S. Labun for assistance in preparation of the manuscript.


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