We have now, by site-directed mutagenesis, mapped the epitope for a mono-clonal antibody, which protects the inhibitory activity of PAI-1 against inactivation by a variety of agents acti
Trang 1Mapping of the epitope of a monoclonal antibody protecting
plasminogen activator inhibitor-1 against inactivating agents
Julie S Bødker, Troels Wind, Jan K Jensen, Martin Hansen, Katrine E Pedersen and Peter A Andreasen Laboratory of Cellular Protein Science, Department of Molecular Biology, University of Aarhus, Denmark
Plasminogen activator inhibitor-1 (PAI-1) belongs to the
serpin family of serine proteinase inhibitors Serpins inhibit
their target proteinases by an ester bond being formed
between the active site serine of the proteinase and the P1
residue of the reactive centre loop (RCL) of the serpin,
fol-lowed by insertion of the RCL into b-sheet A of the serpin
Concomitantly, there are conformational changes in the
flexible joint region lateral to b-sheet A We have now, by
site-directed mutagenesis, mapped the epitope for a
mono-clonal antibody, which protects the inhibitory activity of
PAI-1 against inactivation by a variety of agents acting on
b-sheet A and the flexible joint region Curiously, the epitope
is localized in a-helix Cand the loop connecting a-helix I and
b-strand 5A, on the side of PAI-1 opposite to b-sheet A and distantly from the flexible joint region By a combination of site-directed mutagenesis and antibody protection against an inactivating organochemical ligand, we were able to identify
a residue involved in conferring the antibody-induced con-formational change from the epitope to the rest of the molecule We have thus provided evidence for communi-cation between secondary structural elements not previously known to interact in serpins
Keywords: cancer; cardiovascular disease; monoclonal antibody; protease; serpin
The serpins constitute a protein family of which the best
characterized members, including a1-proteinase inhibitor,
antithrombin III, and plasminogen activator inhibitor-1
(PAI-1), are inhibitors of serine proteinases implicated in
processes such as blood coagulation and turn-over of
extracellular matrix Of decisive importance for the
inhibitory mechanism of serpins is the surface-exposed,
approximately 20-amino acid long reactive centre loop
(RCL) (see Fig 1) Biochemical and biophysical evidence
has shown that the reaction between a serpin and its
target proteinase is initiated by formation of a reversible
docking complex in which the P1–P1¢ bond in the RCL
interacts noncovalently with the active site of the
proteinase [1] In the locking step that follows, the P1–
P1¢ bond is cleaved [2,3] and the P1 residue is coupled to
the active site serine of the proteinase by an ester bond
[4] The N-terminal part of the RCL then becomes
inserted as strand 4 in b-sheet A (s4A) [5] Because of the
covalent bond, the proteinase is translocated to the
opposite pole of the serpin [6–8], the active site becoming distorted, the catalytic machinery inactivated, and the completion of the catalytic cycle disabled [8–16], resulting
in formation of a stable covalently coupled complex of
1 : 1 stoichiometry (for reviews see [17–19]) The energy needed for the proteinase distortion comes from stabi-lization of the serpin in the relaxed conformation by insertion of the RCL into b-sheet A, as opposed to the
stressed, relatively unstable active conformation with a surface-exposed RCL Under some conditions, proteinase distortion cannot keep pace with ester bond hydrolysis, resulting in abortive complex formation, full cleavage of the P1–P1¢ bond, insertion of the RCL into b-sheet A and release of an active proteinase (for reviews see [17,20]) Serpins following this alternative path are said to exhibit substrate behaviour Some serpins, including PAI-1 and antithrombin III spontaneously assume an inactive, relaxed, so-called latent state in which the intact RCL is inserted into b-sheet A, after passage through the so-called gate region between the s3C–s4C loop and the s3B–hG loop (Fig 1) [21,22]
RCL insertion is coupled to conformational changes in the flexible joint region around a-helices D and E The flexible joint region of stressed, but not relaxed PAI-1, binds to the N-terminal 44-amino acid long somatomedin
B domain of the Mr70 000 glycoprotein vitronectin (VN) [23,24], which thereby delays the latency transition of PAI-1 (for a review see [20]) A few organochemical compounds able to inactivate PAI-1 have been indentified, including a group of negatively charged amphipathic compounds like bis-ANS (4,4¢-dianilino-1,1¢-bisnaphthyl-5,5¢-disulfonic acid) [11,25] and the diketopiperazine derivative XR5118 ((3Z,6Z)-6-benzylidene-3-(5-((2-dimeth-ylaminoethyl-thio)-2-thienyl)methylene-2,5-piperazinedione
Correspondence to J S Bødker, Department of Molecular Biology,
University of Aarhus, Gustav Wied’s Vej 10C, 8000 C Aarhus,
Denmark Tel.: + 45 89425079, E-mail: jsb@mb.au.dk
Abbreviations: bis-ANS, 4,4¢-dianilino-1,1¢-bisnaphtyl-5,5¢-disulfonic
acid; h, a-helix; RCL, reactive centre loop; HBS, Hepes buffered
saline; PAI-1, plasminogen activator inhibitor-1; s, b-strand;
S-2444, pyro-Glu-Gly-Arg-p-nitroanilide; uPA, urokinase-type
plasminogen activator; VN, vitronectin; wt, wild-type; XR5118,
((3Z,6Z)-6-benzylidene-3-(5-((2-dimethylaminoethyl-thio)-2-thienyl)methylene-2,5-piperazinedione hydrochloride).
(Received 3 December 2002, revised 5 February 2003,
accepted 13 February 2003)
Trang 2hydrochloride) [11,26] Their exact binding sites in PAI-1
remain to be established, but all available evidence is in
agreement with these compounds having overlapping, but
not identical, binding sites in the flexible joint region [27]
VN protects PAI-1 from inactivation by bis-ANS and
XR5118 [11,24,28] These compounds do not bind to
relaxed PAI-1 [11] Thus, there is bidirectional
communi-cation between the flexible joint region and the
move-ments of the RCL
Among a large number of monoclonal antibodies
directed against PAI-1 raised since the mid-1980s, Mab-1
possesses a number of unique features Mab-1 was raised
against latent PAI-1 purified from HT-1080 cells [29] It
stabilizes PAI-1 against cold-induced substrate behaviour
in buffers with nonionic detergents [30] As monitored by
proteolytic susceptibility, Mab-1 seems to induce
con-formational changes of the RCL, s5A, and the flexible
joint region [30,31] In a recent study aimed at mapping
molecular interactions of PAI-1 by random mutagenesis,
Stoop et al [32] identified residue Q58/74 as part of the
epitope for Mab-1 (a double amino acid numbering
system is used here, the first number following the
numbering system of Andreasen et al [33], the second
number following the a1-antiproteinase inhibitor
num-bering system of Huber and Carrell [34]) Q58/74 is
localized in a-helix C(hC) (Fig 1) Since it is localized
distantly from the secondary structural elements affected
by Mab-1, we hypothesized that a further
characteriza-tion of the epitope for Mab-1 might yield important
information about general aspects of serpin
conforma-tional changes
Materials and methods
PAI-1 The cDNAs for wild-type (wt) and substituted human PAI-1, extended at the N terminus with a His6-tag and a recognition motif for heart muscle kinase, were produced
by standard methods in the Escherichia coli expression vector pT7-PL [35] Transformed E coli BL21(DE3)-pLysS cells from 1-L cultures, treated with 0.5 mM
isopropyl thio-b-D-galactoside to induce PAI-1 expression, were harvested by centrifugation (7000 g, 30 min), resus-pended in 35 mL phosphate-buffered saline (10 mM
Na2HPO4,140 mM NaCl pH 7.4), and disrupted by soni-cation The homogenates were centrifuged (10 000 g,
20 min), filtered (0.22 lm), supplemented with 2M NaCl and 10 mM imidazole, and applied to a 5-mL Ni-NTA column equilibrated in the same buffer further supple-mented with 5% glycerol PAI-1 was eluted with 200 mM
imidazole The eluted protein was subjected to gel filtration on a Superdex 75 column (1.6· 60 cm) equi-librated in Hepes-buffered saline (HBS; 10 mM Hepes,
140 mM NaCl pH 7.4) supplemented with 5% glycerol and NaC l to a final concentration of 1M The procedure routinely gave 10–15 mg PAI-1 per litre bacterial culture The preparations contained PAI-1 which was more than 95% pure as evaluated by SDS/PAGE and Coomassie blue staining N-terminal sequencing showed the expected
HH…, missing only the initiating M indicated in paren-theses The N-terminal extension did not affect the specific
Fig 1 Localization of the epitope for Mab-1 in the three-dimensional structure of PAI-1 (A and B) Ribbon presentations of PAI-1 in the active conformation in two different orientations Relative to (A) the structure shown in (B) is turned approximately 180 around the y-axis and approximately 45 around the x-axis of the coordinate system shown in the figure Relevant secondary structural elements are marked Please note that T341/351 of the RCL is not visible in the structure (C) Surface presentation of PAI-1 in the same orientation as in (B) Red residues were those implicated in the epitope for Mab-1 Blue residues (Q57/73, Q59/75, Q61/77, K67/83, K106/125, Q109/128, R302/313, F304/315, Q305/316, T309/
319, D313/323, Q314/324, E315/325, P316/326, K325/335) were excluded from the epitope D299/310 is indicated in yellow (see the text for details) Nearby alanine and glycine residues (G53/69, G54/70, A62/78, A63/79 and A306/317), not testable by alanine scanning mutagenesis, are indicated
in cyan These SWISS PDB VIEWER displays are based on the coordinates of Stout et al [40].
Trang 3inhibitory activity of PAI-1, its second-order rate constant
for reaction with uPA, its VN binding, or its rate of
latency transition
The specific inhibitory activity of wt PAI-1 was
50 ± 21% (n¼ 17) of the theoretical maximum Most of
the mutants had a specific inhibitory activity
indistinguish-able from that of wt PAI-1 The exceptions were D313/
323A (112 ± 21%; n¼ 17; P < 0.01) and
Q58/74A-D307/318A (15 ± 3%; n¼ 3; P < 0.01)
Monoclonal antibodies
Mab-1 was produced and purified as described previously
[29] Two other monoclonal antibodies against PAI-1,
Mab-2 [Mab-29,36,37] and Mab-5 [38], were produced and purified in
the same way
Other proteins and miscellaneous materials
Bis-ANS was from Molecular Probes S-2444
(pyro-Glu-Gly-Arg-p-nitroanilide) was from Chromogenix (Mo¨lndal,
Sweden) Human urokinase-type plasminogen activator
(uPA) was from Wakamoto Pharmacautical Co (Tokyo,
Japan) XR5118 was a kind gift from Dr Thomas Frandsen,
Finsen Laboratory, Copenhagen The peptide TVASS,
acetylated at the N terminus and amidated at the
Cterminus, was purchased from Eurogentec (Ougre´e,
Belgium)
ELISA
To determine the relative affinity of Mab-1 for recombinant
wt and mutant PAI-1, Mab-1 or Mab-2 was coated onto the
solid phase of microtiter wells, using an antibody
concen-tration of 2.5 lgÆmL)1 and a buffer of 50 mM NaHCO3,
pH 9.6 After blocking with milk, dilution series of
recom-binant PAI-1, spanning a concentration range from
0.1 ngÆmL)1to 20 lgÆmL)1, were applied to the wells The
bound PAI-1 was detected with a layer of rabbit polyclonal
PAI-1 Igs, a layer of peroxidase-conjugated swine
anti-(rabbit IgG) Ig (DAKO), and a peroxidase reaction
The 50% effective concentrations (EC50) for the binding
of PAI-1 to the antibodies were defined as the amount of
PAI-1 resulting in half-maximal binding
Measurements of the effects of Mab-1 or Mab-5 and
neutralizers on the specific inhibitory activity of PAI-1
To measure the effects of antibodies and inactivators on the
specific inhibitory activity of wt and substituted PAI-1, i.e
the fraction of inhibitor forming a stable complex with uPA,
PAI-1 was serially diluted in HBS with 0.25% gelatine,
resulting in PAI-1 concentrations between 0.01 and
20 lgÆmL)1 in a volume of 100 lL, with or without
antibody (4 lgÆmL)1Mab-1 or 80 lgÆmL)1 Mab-5) The
dilution series were then incubated for 10 min at 37Cto
allow antibody–PAI-1 complex formation Fifty-lL
aliqu-ots of HBS with 0.25% gelatine with or without bis-ANS or
XR5118 were added, the bis-ANS or XR5118 concentration
varying between dilution series The mixtures were
incuba-ted for 10 min at 37C Aliquots of 50 lL with 1 lgÆmL)1
uPA were added Incubation was continued for at least
5 min, sufficient for the process of inhibition of uPA to come to an end The remaining uPA enzyme activity was determined by incubation with the substrate S-2444 and measurement of the increase in absorbance at 405 nm The specific inhibitory activity of PAI-1 was calculated from the amount of PAI-1 that had to be added to inhibit 50% of the uPA (50% inhibitory concentrations; IC50) The IC50for bis-ANS or XR5118 neutralization of PAI-1 were deter-mined as the neutralizer concentrations halving the PAI-1 specific inhibitory activity The highest concentration of XR5118 and bis-ANS used in these assays were 250 lMand
80 lM, respectively, due to solubility limits
Measurements of the effects of Mab-1 and VN on PAI-1 latency transition and TVASS incorporation rate PAI-1 (20 lgÆmL)1) was incubated at 37Cin HBS supplemented with 0.25% gelatine, in the absence or presence of Mab-1 (100 lgÆmL)1), VN (30 lgÆmL)1), and/
or the TVASS pentapeptide (250 lM) At regular time intervals, samples were withdrawn for measurement of the specific inhibitory activity of PAI-1 This was done by making serial dilution series at 37Cwith HBS supplemen-ted with 0.25% gelatine, resulting in PAI-1 concentrations between 0.01 and 20 lgÆmL)1 in a volume of 100 lL Aliquots of 100 lL with 0.5 lgÆmL)1 uPA were added After incubation for at least 2 min, the remaining uPA enzyme activity was determined by incubation with the chromogenic substrate S-2444 and measurement of the increase in absorbance at 405 nm The specific inhibitory activity of PAI-1 was calculated from the amount of PAI-1 that had to be added to inhibit 50% of the uPA The half-lives of the functional activity were calculated from semi-logarithmic plots of the specific inhibitory activity vs time
Statistical analysis Data were evaluated by Student’s t-test
Molecular graphics
SWISS PDB VIEWER [39] was used to display the three-dimensional structure of active PAI-1 [40]
Results
Epitope mapping
To define in detail the epitope for Mab-1, we performed extensive alanine scanning mutagenesis around Q58/74, already identified as being part of the epitope by Stoop
et al [32] Alanine-substituted and wt PAI-1s were tested
in ELISA for their binding to the antibody The substituted residues in variants with an EC50 at least twofold higher than that for wt PAI-1 were considered to
be part of the epitope In this way, E55/71 and Q58/74 in hCand D307/318 in the hI/s5A loop were included in the epitope, while a number of adjacent residues were excluded from it (Table 1, Figs 1 and 2) Combining alanine substitutions of two of these three positions resulted in variants with more than 20 000-fold reduced
EC (Table 1) Attempts at expression of the mutant with
Trang 4a triple substitution failed because of low yield None of
the variants with substitutions in the epitope had a specific
inhibitory activity distinguishable from that of the wt The
substitutions had no or only minor effects on binding to a
monoclonal antibody against PAI-1, Mab-2 (Table 1)
Mab-2 has an epitope of residues in hF and its flanking
sequences [37]
Mab-1 protection of PAI-1 against bis-ANS and XR5118
The IC50values for bis-ANS and XR5118 inactivation of wt
PAI-1 were 0.62 ± 0.06 (n¼ 6) and 10.1 ± 3.0 (n ¼ 11)
lM, respectively, in agreement with previous reports
[11,24,28] We now found that the IC50 values for
bis-ANS and XR5118 inactivation of the Mab-1–PAI-1
complex were higher than 80 lM(n¼ 3) and higher than
250 lM (n¼ 12), respectively Thus, Mab-1 protects wt
PAI-1 against these neutralizers Similar observations were done with two other neutralizers, 1-anilinonaphtalene-8-sulfonic acid and 1-dodecyl sulphuric acid (data not shown)
In contrast, the monoclonal antibody against PAI-1 Mab-5, having an epitope not overlapping that of Mab-1 [38], did not protect PAI-1 against XR5118 and bis-ANS The IC50 values for bis-ANS and XR5118 inactivation of the Mab-5-PAI-1 complex were 0.57 ± 0.02 (n¼ 3) and 9.8 ± 2.23 (n¼ 6), respectively, not significantly different from the values without antibody (P < 0.01) In the absence of inactivators, neither antibody affected the specific inhibitory activity of PAI-1
To analyse the effect of the amino acid substitutions in and around the epitope of Mab-1 on the ability to protect against XR5118, we measured the specific inhibitory activity
of each variant with alanine substitutions in the absence and presence of Mab-1, and in the presence of XR5118 at concentrations between 0 and 80 lM In the absence of XR5118, Mab-1 did not affect the specific inhibitory activity
of any of the variants, and all variants had IC50values for inactivation by XR5118 indistinguishable from that of wt (data not shown) Whereas wt PAI-1 was totally resistant to
80 lM XR5118 in the presence of Mab-1, some of the mutants were only partially or not at all protected against XR5118 by Mab-1 (Fig 3) As expected, Mab-1 gave little
or no protection to the variants with substitutions in the epitope, i.e., E55/71A, Q58/74A, and D307/318A, and the double mutants E55/71A-Q58/74A, E55/71A-D307/318A and Q58/74A-D307/318A In addition, D299/310A was incompletely protected by Mab-1
Mab-1 and PAI-1 latency transition and PAI-1 inactivation by an insertion peptide
In the presence of Mab-1, the half-life for latency transition
of PAI-1 was increased by a factor of 1.5 The half-lives in the presence of Mab-1 and in the presence of VN were indistinguishable However, with the variant K325A, the effects of VN and Mab-1 were different This variant has a twofold increased life as compared to wt, and the half-life is not increased, but decreased by VN We found now that Mab-1 did not affect the latency transition rate of this variant (Table 2 and Fig 4) Thus, Mab-1 and VN affect the latency transition rate by different mechanisms
Table 1 Effect of alanine substitutions on the affinity of PAI-1 to Mab-1 The EC 50 values for binding of each variant to Mab-1 or Mab-2 were determined in parallel with the EC 50 value for wt and expressed as a fraction of that The means and standard deviations of triple determinations are indicated Besides the results shown in the table, the following variants were tested, but found to be indistinguishable from wt with respect
to the affinity to Mab-1: Q57/73A, Q59/75A, Q61/77A, K67/83A, K106/125A, Q109/128A, D299/310A, R302/313A, F304/315A, Q305/316A, T309/319A, D313/323A, Q314/324A, E315/325A, P316/326A, K325/335A.
Substitution(s)
Secondary structural element
Amino acid in murine PAI-1
Mab-1
EC 50variant /EC 50wt
Mab-2
EC 50variant /EC 50wt
a Significantly different from 1 (P < 0.025).
Fig 2 Localization of the amino acids in the epitope for Mab-1 The
structure shown is a ribbon representation of the coordinates of Stout
et al [40] The side chains of the amino acids in the epitope and D299/
310 are displayed as sticks and CPK-coloured The colours of the
secondary structure elements are identical to those in Fig 1 (see text
for further details).
Trang 5Two molecules of the pentapeptide TVASS is able to
insert between s3A and s5A in active PAI-1, mimicking the
RCL of relaxed forms of PAI-1 The (TVASS)2–PAI-1
complex displays substrate behaviour, presumably due to a
reduced rate of RCL insertion during the reaction with a
target proteinase [41] We measured the effect of Mab-1 on
the rate of incorporation of TVASS into PAI-1 by measuring the specific inhibitory activity of PAI-1 after incubation with TVASS at 37Cfor different time periods
in the absence or presence of Mab-1 However, Mab-1 did not affect the rate of TVASS-induced inactivation of PAI-1, the half-life for inactivation by 250 lM TVASS being 10.3 ± 2.7 min (n¼ 3) in the absence of Mab-1 and 12.9 ± 0.9 min (n¼ 3) in the presence of Mab-1
Discussion
To the best of our knowledge, Mab-1 is the only mono-clonal antibody known to stabilize PAI-1 in an inhibitory active form We previously reported that Mab-1 stabilizes PAI-1 against cold-induced substrate behaviour in buffers with nonionic detergents [30] We report here that Mab-1 delays PAI-1 latency transition and protects PAI-1 against bis-ANS- and XR5118-induced inactivation
As monitored by proteolytic susceptibility, the protection
by Mab-1 against cold-induced substrate behaviour in detergent-containing buffers is associated with conforma-tional changes of the RCL, the sequence Q321/331-K325/
335 in s5A and of the flexible joint region [30,31] Bis-ANS induces substrate behaviour and polymerization, and XR5118 induces conversion to an inert monomeric form [11] Taken together, these observations show that Mab-1 stabilizes the inhibitory activity of PAI-1 against inactiva-tion by affecting the conformainactiva-tion of the flexible joint region, the central sequence of s5A, and/or the RCL Stoop et al [32] initially reported that Q58/74 is import-ant for binding of PAI-1 to Mab-1 We have demonstrated here that the epitope also includes E55/71 and D307/318A
In agreement with expectancies for a murine antibody against a human protein, two of the residues in the epitope are different in humans and mice (Table 1) The epitope spans residues in both hCand the loop connecting hI and s5A It is thus obvious that Mab-1 affects interactions of residues of PAI-1 which are localized distantly from its epitope We used a combination of site-directed mutagenesis and Mab-1 protection of PAI-1 against XR5118-induced inactivation to obtain information about how the conform-ational change initiated by the binding of Mab-1 spreads through the molecule Generally, observation of a substi-tution having a different effect on XR5118 inactivation of PAI-1 in the absence and presence of Mab-1 shows that the
Fig 3 Effect of XR5118 on the specific inhibitory activities of wt PAI-1 and PAI-1 variants in the absence and the presence of Mab-1 The specific inhibitory activities of PAI-1 in the absence and presence of Mab-1 were measured in the presence of the indicated concentrations
of XR5118 and expressed relative to the specific inhibitory activity in the absence of XR5118 Means and standard deviations are indicated The four mutants shown are significantly different from wt with respect
to residual inhibitory activity in the presence of Mab-1 and 80 l M
XR5118 (P < 0.01) Besides the variants shown in the figure, we tested the following variants and found that they did not differ from wt with respect to the response of the specific inhibitory activity to Mab-1: Q57/73A, Q59/75A, Q61/77A, K67/83A, K106/125A, Q109/128A, R302/313A, F304/315A, Q305/316A, T309/319A, D313/323A, Q314/ 324A, E315/325A, P316/326A, K325/335A.
Trang 6corresponding amino acid side chain is in different
sur-roundings in the absence and presence of Mab-1
Accord-ingly, all the variants with substitutions in the Mab-1
epitope were susceptible to XR5118 in the presence of
Mab-1 In addition, substitution of D299/310, localized in
the hI-s5A loop (Fig 2), but outside the epitope, also
resulted in a reduced ability of Mab-1 to protect PAI-1 against XR5118 A Mab-1-induced reorientation of D299/
310 may therefore be important for the transmission of a Mab-1-induced signal from the epitope to b-sheet A, the RCL, and the flexible joint region
It is interesting to note that whereas Mab-1 delayed the rate of latency transition, it did not measurably affect the rate of incorporation of TVASS into b-sheet A This observation is in agreement with the notion that the rate-limiting step during latency transition is not insertion of the RCL into b-sheet A, but rather passage of the RCL through the gate region [42] Anyway, the mechanism by which Mab-1 delays latency transition is different from that of
VN, as we demonstrate here that the two have different effects on PAI-1 K325/335A
Conclusively, on the basis of the reported observations,
we propose that the binding of Mab-1 to PAI-1 results in a conformational change of hCand the hI-s5A loop which spreads to the flexible joint region, the central portion of s5A, and the RCL, and thus affects the functional properties
of PAI-1
PAI-1 is a potential target for antithrombotic and anticancer therapy (for a review see [20]) A variety of model systems is available for studying the effects of PAI-1 inactivators on thrombi and tumours (for reviews see [43–45]) By stabilizing PAI-1 against inactivation, Mab-1 may be used as a valuable reagent for controlling specificity for PAI-1 inactivators in such model systems
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Fig 4 Effect of Mab-1 on the rate of latency transition of PAI-1 wt and
K325/335A PAI-1 wt and PAI-1 K523/335 A were incubated at 37 C
with or without Mab-1 or VN for various times, followed by
meas-urements of the remaining specific inhibitory activity by titration
against uPA The activities are given relative to the initial activity The
graphs show the results of representative experiments Data from all
determinations are shown in Table 2.
Table 2 Effect of Mab-1 on the rate of latency transition of PAI-1.
PAI-1 alone, with Mab-1, or with VN, was incubated at 37 C ; the
PAI-1 concentration was 20 lgÆmL)1, the Mab-1 concentration was
100 lgÆmL)1, and the VN concentration was 30 lgÆmL)1 After
vari-ous incubation times samples were taken for determination of the
specific inhibitory activity of PAI-1 The specific inhibitory activities
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the half-lives were calculated from the slopes of the lines by linear
regression analysis.
PAI-1
variant
Incubation condition
Half-life [mean ± SD (n)]
a
Significantly different from the corresponding value without
additions (P ¼ 0.01) b Significantly different from the
corres-ponding value for wt (P < 0.01).
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