From the
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 P
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)
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
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
P
In the current study, we
report a detailed analysis of the structural requirements at P
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 P
While the M13PAI-1 proteins should contain seven
vector-derived amino acids at the NH
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 P
This report demonstrates that, unlike
uPA, tPA is remarkably tolerant of amino acid changes at the P
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
The effects of the
reactive center sequence and heparin on PAI-1 inhibitory activity
against the nontarget protease, thrombin, were also examined. wtPAI-1
(P
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
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
P
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.
The second-order rate constants
( k
The second-order rate
constants ( k
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.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
and 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
P
including 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 P
Tyr and P
His as
the most efficient tPA-specific inhibitors, with second-order rate
constants ( k
) of 4.0
10
M
s
and 3.6
10
M
s
, respectively. Additional PA-specific PAI-1
variants containing substitutions at P
through
P
` were constructed.
P
Tyr-P
Ser-P
Lys-P
`Trp
and
P
Tyr-P
Ser-P
Tyr-P
`Met
had k
values of 1.7
10
M
s
and 2.5
10
M
s
against tPA, respectively, but both were inactive against uPA. In
contrast, P
Arg-P
Lys-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
P
Arg 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.
(
)
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
( k
values
10
-10
M
s
) 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) .
-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 P
residue 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) .
for 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 P
positions 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.
and P
` for PAI-1 inhibition of tPA and thrombin.
Confirming our previous findings
(28) , all
P
-P
` variants with a basic residue at P
maintain tPA inhibitory activity with the exception of those
containing P
`Pro. However, in contrast to the absolute
requirement for a basic residue at P
for the inhibition of
uPA, a number of PAI-1 mutants with neutral or hydrophobic
substitutions at P
exhibit 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.
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
Met
of mature PAI-1 (corresponding to the P
and P
` residues, in the standard nomenclature of
Schechter and Berger
(30) ). 177 unique mutant sequences were
analyzed, with all possible amino acid substitutions at P
and P
` represented at least once.
were 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:
P
Arg-P
Lys-P
`Ala,
P
Tyr-P
Ser-P
Lys-P
`Trp,
and
P
Tyr-P
Ser-P
Tyr-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) . k
values 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
KH
PO
, 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
KH
PO
, 1.25
M NaCl, pH 6.0.
(NH
)
SO
was then added to 1
M, and the sample was applied to a phenyl Sepharose
(Pharmacia) column equilibrated with 50 m
M
KH
PO
, 1
M
(NH
)
SO
, pH 6.0, washed with the
same buffer, and step eluted with 50 m
M
KH
PO
, pH 6.0. The concentration of active PAI-1
was determined by titration with two-chain tPA of known concentration.
k
values 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.
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.
terminus, the variants
expressed from pSELPAI-1 begin at the native NH
terminus 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
M
s
(40) . Consistent with previous
results
(28) , all possible P
Arg and
P
Lys mutants except P
Arg-P
`Pro and
P
Lys-P
`Pro inhibited tPA (Fig. 1). In addition,
a number of variants with nonbasic amino acid substitutions at P
showed 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 P
variants containing the wild-type P
`Met that were
absent from the above P
-P
` library were
prepared. Of the 19 P
mutants, those containing Asn, Gln,
His, Leu, Lys, Met, Phe, Ser, Thr, Trp, or Tyr at P
showed
inhibitory activity against tPA (Fig. 1). All P
substitutions 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 P
variants, except for P
Lys,
maintained detectable inhibitory activity against uPA in a direct
chromogenic assay (data not shown), indicating that they have rate
constants of <10
M
s
against 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 P
resulted in
an approximately 25-240-fold reduction of
k
against 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
We previously
identified P -P
` Mutants
Lys-P
`Trp and
P
Lys-P
`Ala as relatively tPA- and uPA-specific
mutants, respectively
(28) . Similarly, York and co-workers
(24) reported that P
Tyr-P
Ser was
relatively specific for tPA and that P
Arg 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
P
Tyr-P
Ser-P
Lys-P
`Trp
(designated T1) and
P
Tyr-P
Ser-P
Tyr-P
`Met
(designated T2) were predicted to be tPA-specific, and the variant
P
Arg-P
Lys-P
`Ala (designated U1) was
predicted to be uPA-specific. The k
values for the interactions between these mutants and tPA or uPA
are shown in I, along with the corresponding
k
values 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
M
s
(40) .
In the absence of heparin, wtPAI-1 (P
Arg-P
`Met)
and six variants containing Arg at the P
position 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 P
and 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.
residue 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 P
position 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 P
and P
positions show considerable flexibility for the inhibition of
both uPA and tPA
(24) .
position of PAI-1. In addition to PAI-1 variants with basic
residues at P
, tPA was inhibited by mutants containing a
wide range of P
amino 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 P
residue 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 P
residues
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.
-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 P
residue 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.
Arg-P
`Met) has been reported to inhibit
thrombin with a k
of 1.1
10
M
s
(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
P
Arg by Lys
(53) , His
(54) , or Cys
(54) results in a loss of thrombin inhibitory activity.
Furthermore, the sole substitution of P
Met 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 P
position (Fig. 2), including
P
Arg-P
`Ser, which has the same reactive center
as antithrombin III, the main physiological inhibitor of thrombin.
10
M
s
(29, 55) . These values can be compared with 1.4
10
and 1.5-4
10
M
s
for
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 P
Arg 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 P
Arg PAI-1 variants with a positive
charge at P
` are inactive due to ionic repulsion at that
site.
Tyr-P
Ser-P
Tyr-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
Table:
Second-order rate constants for the inhibition
of tPA and uPA by PAI-1 P1 mutants
) for the interactions between PAI-1
mutants and two-chain tPA or uPA are shown. The ratio of the
k
value with tPA to the
k
value 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
) for the interactions
between PAI-1 variants and two-chain tPA or uPA are shown. The ratio of
the k
value with tPA to the
k
value with uPA (tPA/uPA) is given at
the right. k
values for mutants denoted
by * and ** were taken from Refs. 24 and 28, respectively. Amino acids
in boldface indicate mutated residues.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.